Académique Documents
Professionnel Documents
Culture Documents
Roitt
Professor Roitt was born in 1927and educated
at King Edward's School, Birmingham and
Balliol College, Oxford. In 1956, together with
Deborah Doniach and Peter Campbell, he
made the classic discovery of thy~oglobulin
autoantibodies in Hashimoto's thvroiditis
J
Peter J. Delves
Dr Delves obtained his PhD from the
University of London in 1986and is currently
a Reader in Immunology at University
College London. His research focuses on
molecular aspectsof antigen recognition. He
has authored and edited a number of
immunology books, and teaches the subject at
a broad range of levels.
Essential
Immunology
lvan M. Roitt
MA, DSc(Oxon),FRDPath, Hon FRCP(Lond),FRS
Emeritus Professor, Department of Immunology and Molecular Pathology,
University College London,
London WIT 4JF
Peter J. Delves
PhD
Department of Immunology and Molecular Pathology,
University College London,
London WlT4JF
Blackwell
Science
O 1971, 1974, 1977, 1980, 1984, 1988, 1991, 1994, 1997,2001 by Blackwell Science Ltd
a Blackwell Publishing company
Blackwell Science, Inc., 350 Main Street, Malden, Massachusetts 02148-5018, USA
Blackwell Science Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK
Blackwell Science Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia
The right of the Authors to be identified as the Authors of this Work has been asserted in accordance with
the Copyright, Designs and Patents Act 1988.
All right reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any
form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the
UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher.
ISBN 0-632-05902-8
A catalogue record for this title is available from the British Library
The input of the editorial team of Nick Morgan, Meg 16.10; J. Horton for figure 12.20; G. Rook for figures
Barton and Fiona Goodgame at Blackwell Science, the 13.5 and 13.12; and J. Taverne for figure 13.21 and
illustrator Graeme Chambers and the indefatigable table 13.1.
secretarial assistance of Christine Griffin is warmly Every effort has been made by the authors and the
acknowledged. Wise counsel was provided by Kirsten publisher to contact all the copyrightholders to obtain
Fischer Lindahl, Jurg Tschopp and Helen Turner their permission to reproduce copyright material.
concerning aspects of MHC gene organization, ap- However, if any have been inadvertently overlooked,
optosis and IgE receptor signaling respectively. We the publisher will be pleased to make the necessary
are much indebted to the co-editors of Immunology, arrangements at the first opportunity.
J. Brostoff and D. Male, together with the publishers, A number of scientists very generously provided
Harcourt Health Sciences, and the following indi- illustrations for inclusion in this edition, and we have
viduals for permission to utilize or modify their acknowledged our gratitude to them in the relevant
figures: J. Brostoff and A. Hall for figures 1.15 and figure legends.
vii
It is now 30 years since the 1st Edition of Essential viral hijacking of host processes as evasion
Immunology appeared, and it seemed that the time was mechanisms
now appropriate for the task of producing the 10th DNA vaccines
Edition to be shared. The new co-author, Peter Delves, mucosal adjuvants
has been a close colleague of Professor Roitt for many 'shot gun' approach to identification of vaccine
years and is a highly experienced teacher. candidates
A wide range of subjects have been extensively primary immunodeficiencyincluding IL-7 receptor
revised, restructured or updated, and advanced mutation, and deficiency of VDJ recombination in
material is included in the figure legends to avoid severe combined immunodeficiency
disruption of the basic text. These subjects include: CCR5 CO-receptorfor HIV infection of cells
dendritic cells the importance of highly active anti-retroviral drug
intraepitheliallymphocytes therapy and of healthy CD8 response dependent on
NK-T and y6 T-cells robust CD4 Thl effectors in control of HIV infection
NK receptors pivotal role of IgE antibodies in pathogenesis
receptor editing relating to receptor diversity of asthma and atopic dermatitis, and remarkable
non-classical MHC and the presentation of non- therapeutic benefit of monoclonal anti-IgE
peptidic antigens the excessive hygiene hypothesis related to the
the role of chaperone proteins in antigen processing development of allergy
T-cell recognition of peptide-MHC reflecting the the role of Fcy receptors in the pathogenesis of type
latest crystallographic studies I1 and I11hypersensitivities
arrays for analysis of gene expression suppression of graft rejection by synergy between
tetramer evaluation of antigen-specificT-cells fungal metabolites and other drugs and by induc-
experimental genetic manipulation using con- tion of antigen-specific tolerance with high-dose
ditional 'knockouts' employing the Lox/Cre bone marrow transplantation combined with
system and 'knockins' to replace endogenous CO-stimulatory blockade by anti-CD40L and
genes CTLA-4-Ig
B- and T-cell signaling pathways and the role of engineeringgrafts from recipient cells
adaptor proteins the role of hsp70 and 90 in natural and induced
cytokine physiology tumor immunity
chemokines and their receptors peptide priming of dendritic cells to provoke anti-
memory cells cancer cytotoxic responses
intimate links of innate and adaptive immunity the avoidance of graft vs. host disease in allogeneic
the role of complement in modulating the adaptive bone marrow transplantation for leukemias
immune response inhibition of B-cell lymphomas and tumor angio-
regulatory T-cells genesis by radiolabeled monoclonals
activation-induced cell death thymic expression of some organ-specific antigens
neuroendocrineinfluences on the immune system role of autoimmunity to hsp65 in atherosclerosis
critical role of Pax 5 in B-cell differentiation autologous stem cell transplantation after cytotoxic
molecular basis of thymic development ablative therapy for some cases of SLE, scleroderma
signalingthrough pattern recognition systems and juvenile rheumatoid arthritis.
prions All in all, quite a mouthful!
viii
EXTERNAL B A R R I E R S A G A I N S T I N F E C T I O N
INTRODUCTION
The simplest way to avoid infection is to prevent the
We live in a potentially hostile world filled with a microorganisms from gaining access to the body (fig-
bewildering array of infectious agents (figure1.1) ure 1.2). The major line of defense is of course the skin
of diverseshape, size,compositionand subversive which, when intact, is impermeable to most infectious
character which would very happily use us as rich agents; when there is skin loss, as for examplein burns,
sanctuaries for propagating their 'selfish genes' infection becomes a major problem. Additionally,
had we not also developed a series of defense most bacteria fail to survive for long on the skin be-
mechanisms at least their equal in effectiveness cause of the direct inhibitory effects of lactic acid and
and ingenuity (exceptin the case of many parasitic fatty acids in sweat and sebaceous secretions and the
infections where the situation is best d e s c r i i as low pH which they generate.An exception is Staphylo-
an uneasy and often unsatisfactov truce). It is coccus aureus which often infects the relatively vulnera-
these defense mechanisms which can establish ble hair follicles and glands.
a state of inununity against infection ( L a k immu- Mucus, secreted by the membranes lining the inner
nitus, freedom from) and whose operation pm- surfacesof the body, acts as a protectivebarrier to block
vides the basis for the delightful subject called the adherence of bacteria to epithelial cells. Microbial
?mmunology'. and other foreign particles trapped within the adhe-
Aside from ill-understoodconstitutionalfactors sive mucus are removed by mechanical stratagems
which make one species innately susceptible and such as ciliary movement, coughing and sneezing.
another resistant to certaininfections,a number of Among other mechanical factors which help protect
relatively nonspecific antimicmbial systems (e.g. the epithelial surfaces, one should also include the
phagocytosis) have been recognized which are washing action of tears, saliva and urine. Many of the
innatein the sensethat they are not intrinsicallyaf- secreted body fluids contain bactericidal components,
fected by prior contact with the infectious agent. such as acid in gastric juice, spermine and zinc in
We shall discuss these systems and examine how, semen, lactoperoxidasein milk and lysozyme in tears,
in the state of specific acquired immunity, their nasal secretionsand saliva.
effectivenesscanbegreatlyincreased. A totally different mechanism is that of microbial
Figure 1.1. The formidable range of
infectious agents which confronts the
immune system.Although not normally
classified as such because of their lack of a
cell wall, the mycoplasmas are included
under bacteria for convenience.Fungi
adopt many forms and approximatevalues
for some of the smallest forms are given.
l,, range of sizes observed for the
organism(s)indicatedby the arrow; 4[, the
organisms listed have the size denoted by
the arrow.
oxidase and halide ions constitutes a potent halogenat- particularly macrophages and human neutrophils,
ing system capable of killing both bacteria and viruses thereby generating a powerful antimicrobial system
(figure 1.10a). Although H,O, and the halogenated (figure 1.lob). Whereas the NADPH oxidase is dedi-
compounds are not as active as the free radicals, they cated to the killing of extracellular organisms taken up
are more stable and therefore diffuse further, making by phagocytosis and cornered within the phagocytic
them toxic to microorganisms in the extracellular vacuole, the NO. mechanism can operate against mi-
vicinity. crobes which invade the cytosol; so, it is not surprising
that the majority of nonphagocytic cells which may be
infected by viruses and other parasites are endowed
Killing by reactive nitrogen intermediates
with an iNOS capability.The mechanism of action may
Nitric oxide surfaced prominently as a physiologic be through degradation of the Fe-S prosthetic groups
mediator when it was shown to be identical with en- of certain electron transport enzymes, depletion of
dothelium-derived relaxing factor. This has proved to iron and production of toxic -ONO0radicals. The N-
be just one of its many roles (includingthe mediation of ramp gene linked with resistance to microbes such
penile erection, would you believe it!),but of major in- as bacille Calmette-Gukrin (BCG), Salmonella and
terest in the present context is its formation by an in- Leishmania, which can live within an intracellular
ducible NO- synthase (iNOS) within most cells, but habitat, is now known to express a protein forming a
Figure 1.7. Phagocytosis and killing of a
bacterium. Stage 3/4, respiratoryburst
and activation of NADPH oxidase; stage 5,
damage by reactive oxygen intermediates;
stage 6/7, damageby peroxidase, cationic
proteins, antibiotic peptide defensins,
lysozyme and lactoferrin.
H, to form C3bH which is susceptible to attack by the change and its potentially reactive internal thiolester
C3b inactivator, factor I (figure 1.12; further discussed bond becomes exposed. Since the half-life of nascent
on p. 307).The inactivated iC3b is biologically inactive C3b is less than 100pec, it can only diffuse a short dis-
and undergoes further degradation by proteases in tance before reacting covalently with local hydroxyl
the body fluids. Other regulatory mechanisms are dis- or amino groups available at the microbial cell surface
cussed at a later stage (seep. 307). (figure 1.11). Each catalytic site thereby leads to the
clustering of large numbers of C3b molecules on the
microorganism. This series of reactions leading to C3
C3 convertase is stabilized on microbial surfaces
breakdown provoked directly by microbes has been
A number of microorganisms can activate the called the alternative pathway of complement activa-
C3bBb convertase to generate large amounts of C3 tion (figure 1.12).
cleavage products by stabilizing the enzyme on their
(carbohydrate) surfaces, thereby protecting the C3b
The post-C3 pathway generates a membrane
from factor H. Another protein, properdin, acts subse-
attack complex
quently on this bound convertase to stabilize it even
further,As C3 is split by the surface membrane-bound Recruitment of a further C3b molecule into the C3bBb
enzyme to nascent C3b, it undergoes conformational enzymic complex generates a C5 convertase which
activates C5 by proteolytic cleavage releasing a small lipid bilayer (cf. the colicins, p. 2) and polymerization
polypeptide, C5a, and leaving the large C5b fragment to an annular membrane attack complex (MAC; fig-
loosely bound to C3b. Sequential attachment of C6 and ures 1.13 and 2.4). This forms a transmembrane chan-
C7 to C5b forms a complex with a transient membrane- nel fully permeable to electrolytes and water, and due
binding site and an affinity for the P-peptide chain of to the high internal colloid osmotic pressure of cells,
C8. The C8a chain sits in the membrane and directs the there is a net influx of Na' and water frequently lead-
conformational changes in C9 which transform it into ing to lysis.
an amphipathic molecule capable of insertion into the
The next act sees C3a and C5a, together with the medi-
ators they trigger from the mast cell, acting to recruit
polymorphonuclear phagocytes and further plasma
complement components to the site of microbial inva-
sion. The relaxation induced in arteriolar walls causes
increased blood flow and dilatation of the small
vessels, while contraction of capillary endothelial
cells allows exudation of plasma proteins. Under the
influence of the chemotaxins, neutrophils slow down
and the surface adhesion molecules they are stimulat-
ed to express cause them to marginate to the walls of
the capillaries where they pass through gaps between
the endothelial cells (diapedesis) and move up the
concentration gradient of chemotactic factors until
they come face to face with the C3b-coated microbe.
Adherence to the neutrophil C3b receptors then
takes place, C3a and C5a at relatively high concentra-
tions in the chemotacticgradient activate the respirato-
ry burst and, hey presto, the slaughter of the last act
can begin!
The processes of capillary dilatation (redness),exu-
dation of plasma proteins and also of fluid (edema)
due to hydrostatic and osmotic pressure changes, and
accumulation of neutrophils are collectively termed
the acute inflammatory response.
Figure 1.14. The mast cell. (a)A resting cell with many membrane-
bound granules containing preformed mediators. (b) A triggered Althoughnot yet established with the same confidence
mast cell. Note that the granules have released their contents and are that surrounds the role of the mast cell in acute inflam-
morphologically altered, being larger and less electron dense. Al- mation, the concept seems to be emerging that the
though most of the altered granules remain within the circumfer-
ence of the cell, they are open to the extracellular space. (Electron
tissue macrophage may mediate a parallel series of
micrographs ~5400.)(Courtesy of Drs D. Lawson, C. Fewtrell, B. events with the same final end result. Nonspecific
Gomperts and M.C. Raff from (1975)Journalof Experimental Medicine phagocytic events and certain bacterial toxins such
142,391.) as the lipopolysaccharides (LPSs) can activate macro-
phages, but the phagocytosis of C3b-opsonized
microbes and the direct action of C5a generated
through complement activation are guaranteed to
COMPLEMENT CAN MEDIATE AN ACUTE
goad the cell into copious secretion of soluble
INFLAMMATORY REACTION
mediators of the acute inflammatory response
We can now put together an effectively orchestrated (figure 1.17).
defensive scenario initiated by activation of the alter- These upregulate the expression of adhesion mole-
native complement pathway (seefigure 1.16). cules for neutrophils on the surface of endothelial
In the first act, C3bBb is stabilized on the surface of cells, increase capillary permeability and promote the
the microbe and cleaves large amounts of C3. The C3a chemotaxis and activation of the polymorphonuclear
fragmentis released but C3b molecules bind copiously neutrophils themselves. Thus, under the stimulus of
to the microbe. These activate the next step in the se- complement activation, the macrophage provides
quence to generate C5a and the membrane attack com- a pattern of cellular events which reinforces the
Figure 1.15. Mast cell triggering leading
to release of mediatorsby two major
pathways: (i)release of preformed
mediators present in the granules, and (ii)
the metabolism of arachidonic acid
produced through activation of a
phospholipase. Intracellular Ca2+and
cyclic AMP are central to the initiation of
these events but details are still unclear.
Mast cell triggering may occur through
C3a, C5a and even by some
microorganismswhich can act directly on
cell surface receptors. Mast cell
heterogeneity is discussed on p. 323. ECF,
eosinophil chemotacticfactor; GM-CSF,
granulocyte-macrophage colony-
stimulating factor; NCF, neutrophil
chemotacticfactor. Chemotaxisrefers to
directed migration of granulocytes up the
pathway concentration gradient of the
mediator.
mast cell-mediated pathway leading to acute lysed by the insertion of the membrane attack com-
inflammation-yet another of the body's fail-safe plex. The spread of infection may be limited by en-
redundancy systems (often known as the 'belt and zymes released through tissue injury which activate
braces' principle). the clotting system. Of the soluble bactericidal sub-
stances elaborated by the body, perhaps the most
abundant and widespread is the enzyme lysozyme, a
HUMORAL MECHANISMS PROVIDE A
muramidase which splits the exposed peptidoglycan
SECOND DEFENSIVE STRATEGY
wall of susceptiblebacteria (cf.figure 13.5).
Like the a-defensins of the neutrophil granules,
the human P-defensins are peptides derived by pro-
Turning now to those defense systemswhich are medi- teolytic cleavage from larger precursors; they have
ated entirelyby soluble factors, we recollect that many P-sheet structures, 2 9 4 0 amino acids and three intra-
microbes activate the complement system and may be molecular disulfide bonds, although they differ from
Figure 1.16. The defensive strategy of the
acute inflammatory reaction initiated by
bacterial activation of the alternativeC
pathway. Directions: O start with the
activation of the ~3b- C3 convertaseby
the bacterium, C3 notice the generation of
C3b (O which binds to the bacterium), C3a
and C5a, @whichrecruit mast cell
mediators; C 3 follow their effect on
capillary dilatation and exudation of
plasma proteins and 8their chemotactic
attraction of neutrophils to the C3b-coated
bacterium and triumph in O the adherence
and final activation of neutrophils for
the kill.
EXTRACELLULAR KILLING
A wide range of innate immune mechanisms operate If penetration occurs, bacteria are destroyed by soluble
whichdonotimprovewithrepeatedexposure toinfection. factors such as lysozyme and by phagocytosis with intra-
cellular digestion.
Barriers against infection
Microorganismsarekeptoutofthebodyby theskin, the Phagocytic cells kill microorganisms
secretion of mucus, ciliary action, the lavaging action of The main phagocytic cells are polymorphonuclear
bactericidal fluids (e.g. tears), gastric acid and microbial neutrophils and macrophages.
antagonism. The phagocytic cells use their pattern recognition
(continuedp.20)
receptors (PRRs) to recognize and adhere to pathogen- face C3b receptors and may then ingest them. The influx
associated molecular patterns (PAWS) on the microbe of polymorphs and the increase in vascular permeability
surface. constitutethe potent antimicrobialacute inflammatoryre-
Organisms adhering to the phagocyte surface activate sponse (figure2.18).
the engulfmentprocess and are taken inside the cell where Inflammation can also be initiated by tissue
they fuse with cytoplasmicgranules. macrophages which subserve a similar role to the mast
A formidable array of microbicidal mechanisms then cell, sincesignalingby bacterial toxins, C5a or iC3b-coated
come into play: the conversion of O2to reactive oxygen in- bacteria adhering to surfacecomplement receptors causes
termediates,the synthesis of nitric oxide and the release of release of neutrophil chemotactic and activatingfactors.
multiple oxygen-independentfactorsfrom the granules.
Humoral mechanisms provide a second defensive strategy
Complement focilitates phagocytosis In addition to lysozyme, peptide defensins and the
The complement system, a multicomponent triggered complement system, other humoral defensesinvolve the
enzyme cascade, is used to attract phagocytic cells to the acute phase proteins, such as C-reactive and mannose-
microbes and engulf them. binding proteins, whose synthesis is greatly augmented
The most abundant component,C3, is split by a conver- by infection. Mannose-bindingprotein is a member of the
tase enzyme formed from its own cleavage product C3b collectin family including conglutinim and surfactants
and factor B and stabilized against breakdown caused by SP-A and SP-D, notable for their ability to distinguish
factorsHandI, throughassociationwiththemicrobialsur- microbial from 'self' surface carbohydrate groups by their
face.As it is formed, C3b becomes linked covalently to the pattern recognition molecules.
microorganism. Recovery from viral infections can be effectedby the in-
The next component, C5, is activated yielding a small terferonswhich block viral replication.
peptide, C5a; the residual C5b binds to the surface and as-
sembles the terminal components C6-9 into a membrane Extracellular killing
attack complex which is freely permeable to solutes and Virallyinfectedcellscanbekilledbylargegranularlym-
can lead to osmotic lysis. phocytes with NK activity through a perforin/granzyme
C5a is a potent chemotactic agent for neutrophils and and a separate Fas-mediated pathway, leading to pro-
greatly increases capillary permeability gramed cell death (apoptosis) mediated by activation of
C3a and C5a act on mast cells causing the release of the caspase protease cascade which fragments the nuclear
further mediators, such as histamine, leukotriene B, and DNA.
tumor necrosis factor (TNF), with effects on capillary per- Extracellularkilling by C3b-bound eosinophils may be
meability and adhesiveness, and neutrophil chemotaxis; responsible for the failure of many large parasites to estab-
they also activateneutrophils. lish a foothold in potential hosts.
compensatefor this, lymphocytes which are triggered lighted by the ability of antimitotic drugs to abolish
by contact with antigen undergo successive waves of antibody production to a given antigen stimulus
proliferation (figure 2.6b) to build up a large clone of completely.
plasma cells which will be making antibody of the kind Because it takes time for the proliferating clone to
for which the parent lymphocyte was programed. By build up its numbers sufficiently, it is usually several
this system of clonal selection, large enough con- days before antibodies are detectable in the serum
centrations of antibody can be produced to combat following primary contact with antigen. The newly
infection effectively (Milestone2.1; figure 2.11). formed antibodies are a consequence of antigen ex-
The importance of proliferation for the develop- posure and it is for this reason that we speak of the
ment of a significant antibody response is high- acquired immune response.
Figure 2.9. Plasma cell (X 10 000). Prominent rough-surfaced endo- Figure 2.10. Antigen activates those B-cells whose surface anti-
plasmic reticulum associated with the synthesis and secretion of Ig. body receptors it can combine with firmly.
Selectlon theories
The wheel turns full circle and we once more live with the
idea that, since different antibodies must be encoded by
separate genes, the informationfor making these antibod-
ies must pre-exist in the host DNA. In 1955,Nils Jeme per-
ceived that thiscould form the basis for a selective theory Figure M2.1.1. Ehrlichs side-chain theory of Ab production.
! of antibody production. He suggested that the complete (Reproducedfrom ProceedirigsoftlieRoynlSociety B (1900),66,424.)
I
Figure 2.13. Memory for a primary responsecan be transferredby donor cells to be followed. The reasons for the design of the experi-
small lymphocytes. Recipients are treated with a dose of X-rays ment are given in the text. In practice,because of the possibility of in-
which directly kill lymphocytes (highly sensitive to radiation) but terference between the two antigens, it would be wiser to split each
only affect other body cells when they divide; the recipient thus of the primary antigen-injected groups into two, giving a separate
functions as a living 'test-tubef which permits the function of the boosting antigen to each to avoid using a mixture.
from another goes even further. The individual must
also recognize what is foreign, i.e. what is 'nonself'.
The failure to discriminate between self and nonself
could lead to the synthesis of antibodies directed
against components of the subject's own body (auto-
antibodies), which in principle could prove to be
highly embarrassing. On purely theoretical grounds
it seemed to Burnet and Fenner that the body must
develop some mechanism whereby 'self' and 'nonself'
could be distinguished,and they postulated that those
circulatingbody componentswhich were able to reach
the developing lymphoid system in the perinatal peri-
od could in some way be 'learnt' as 'self '. Apermanent
unresponsiveness or tolerance would then be created
so that as immunologic maturity was reached there
would normally be an inability to respond to 'self'
components. At this stage it is salutory to note that
Burnet had the sagacity to realize that his clonal selec-
tion theory could readily provide the cellular basis for
Figure 2.14. The basis of vaccination illustrated by the response to such a mechanism to operate. He argued that if each
tetanus toxoid. Treatment of the bacterial toxin with formaldehyde lymphocyte were preoccupied with making its own
destroys its toxicity (associated with ) but retains antigenicity.
individual antibody, those cells programed to express
Exposure to toxin in a subsequent natural infection boosts the mem-
ory cells, producing high levels of neutralizing antibody which are antibodies reacting with circulating self components
protective. could be rendered unresponsive without affecting
those lymphocytes specific for foreign antigens. h
other words, self-reacting lymphocytescould be selec-
tively suppressed or tolerized without undermining
ACQUIRED IMMUNITY HAS ANTIGEN
the ability of the host to respond imunologically to
SPECIFICITY
infectious agents. As we shall see in Chapter 12, these
predictions have been amply verified, although we
will learn that, as new lymphocytes differentiate
The establishment of memory or immunity by one or- throughout life, they will all go through this self-
ganism does not confer protection against another tolerizing screeningprocess. However, self toleranceis
unrelated organism. After an attack of measles we not absolute and normally innocuous but potentially
are immune to further infection but are susceptible to harmful anti-self lymphocytes exist in all of us.
other agents such as the polio or mumps viruses. Ac-
quired immunity then shows specificity and the im-
VACCINATION DEPENDS ON
mune system can differentiatespecificallybetween the
ACQUIRED MEMORY
two organisms. A more formal experimental demon-
stration of this discriminatory power was seen in fig- Some 200 years ago, Edward Jenner carried out the
ure 2.13 where priming with tetanus toxoid evoked remarkable studies which mark the beginning of im-
memory for that antigen but not for influenza and munology as a systematic subject. Noting the pretty
vice versa. pox-free skin of the milkmaids, he reasoned that delib-
The basis for this lies of course in the ability of the erate exposure to the pox virus of the cow, which is
recognition sites of the antibody molecules to distin- not virulent for the human, might confer protection
guish between antigens; antibodies which react with against the related human smallpox organism. Ac-
the toxoid do not bind to influenza and, mutatis mutan- cordingly he inoculated a small boy with cowpox and
dis as they say, anti-influenza is not particularly smit- was delighted-and presumably breathed a sigh of
ten with the toxoid. relief-to observe that the boy was now protected
against a subsequent exposure to smallpox (what
would today's ethical committees have said about
that?!).By injecting a harmless form of a disease organ-
This ability to recognize one antigen and distinguish it ism, Jennerhad utilized the specificity and memory of
the acquired immune response to lay the foundations bility complex IMHC), identified originally through
for modern vaccination (Latinvacca, cow). their ability to evoke powerful transplantation reac-
The essential strategy is to prepare an innocuous tions in other members of the same species.Now naive
form of the infectious organism or its toxins which or virgin T-cells must be introduced to the antigen and
still substantially retains the antigens responsible for MHC by a special dendritic antigen-presenting cell
establishing protective immunity. This has been done (figures 2.6f and 8.13)before they can be initiated into
by using killed or live attenuated organisms, purified the rites of a primary response. However, once primed,
microbial components or chemically modified anti- they are activated by antigen and MHC present on the
gens (figure2.14). surface of other cell types such as macrophages as we
shall now see.
CELL-MEDIATED I M M U N I T Y PROTECTS
AGAINST INTRACELLULAR ORGANISMS
Many microorganismslive inside host cells where it is
impossible for humoral antibody to reach them. Oblig- These organisms only survive inside macrophages
ate intracellular parasites like viruses have to replicate through their ability to subvert the innate killing mech-
inside cells; facultative intracellular parasites like anisms of these cells. Nonetbeless, they cannotprevent
Mycobacteria and Leishmania can replicate within cells, the macrophage from processing small antigenic
particularly macrophages,but do not have to; they like fragments (possibly of organisms which have sponta-
the intracellular lifebecause of the protection it affords. neously died) and placing them on the host cell
A totally separate acquired immunity system has surface. A subpopulation of T-lymphocytes called T-
evolved to deal with this situation based on a distinct helper cells, if primed to that antigen, will recognize
lymphocyte subpopulation made up of T-cells, desig- and bind to the combination of antigen with so-called
nated thus because, unlike the B-lymphocytes, they class I1 MHC molecules on the rnacrophage surface
differentiate within the milieu of the thymus gland. and produce a variety of soluble factors termed cy-
Because they are specialized to operate against cells tokines which include the interleukins IL-2, etc. (p.
bearing intracellular organisms, T-cells only recognize 177). Different cytokines can be made by various cell
antigen when it is on the surface of a body cell.Accord- types and generally act at a short range on neighboring
ingly, the T-cell surface receptors, which are different cells. Some T-cell cytokines help B-cells to make anti-
from the antibody molecules used by B-lymphocytes, bodies, while others such as y-interferon (IFNy) act as
recognize antigen plus a surface marker which in- macrophage activating factors which switch on the
forms the T-lymphocyte that it is making contact with previously subverted microbicidal mechanisms of
another cell. These cell markersbelong to an important the macrophage and bring about the death of the in-
group of molecules known as the major histocompati- tracellular microorganisms (figure2.15).
Antibody -the specific adaptor ment through the classical pathway (binding C1 and gen-
-
The antibody molecule evolved as a specificadaptor to erating a C4b2a convertase to split C3) and phagocytes
attach to microorganisms which either fail to activate through their antibody receptors.
the alternativecomplementpathway or prevent activation This supplementary route into the acute inflammatory
of the phagocyticcells. reaction is enhanced by antibodies which sensitize mast
The antibody fixes to the antigenby its specific recogni- cellsand by immune complexeswhich stimulate mediator
tion site and its constant structureregions activatecomple- release from tissue macrophages (figure2.18).
Table 2.1. The major immunologic journals and their impact In-depth seriesfor the advanced reader
factors. Advances in Immunology (Annual).Academic Press, London.
Advances in Neuroimmunology (editedby G.B. Stefano& E.M.Smith).
Pergamon,Oxford.
Annual Review ofImmunology.Annual Reviews Inc., California.
Immunological Reviews (edited by P. Parham).Munksgaard, Copen-
hagen. (Specialized,authoritative and thoughtful.)
Progress in Allergy. Karger, Basle.
Seminars in Immunology. Academic Press, Cambridge. (In-depth
treatment of single subjects.)
Current information
Current Biology. Current Biology, London. (What the complete
biologist needs to know about significantcurrent advances.)
Current Opinion in Immunology. Current Science, London. (Impor-
tant personal opinions on focused highlights of the advances
made in the previous year; most valuable for the serious immu-
nologist.)
Trends in Molecular Medicine. Elsevier Science Publications,Amster-
dam. (Frequent articles of interest to immunologists with very
good perspective.)
The Immunologist. Hogrefe & Huber Publishers, Seattle. (Official
organ of the International Union of Immunological Societies-
IUIS. Excellent, didactic and compact articles on current trends in
immunology.)
Trends in Immunology. Elsevier Science Publications, Amsterdam.
(Theimmunologistsnewspaper Excellent.)
I.
Major journals
The major journals of interest and their impact factors are noted in
table 2.1.
N/ A, not available.
Antibodies
INTRODUCTION
The antibody molecule is made up of two identical
heavy and two identical light chains held together
by interchain disulfide bonds (figure M3.1.1).
These chains can be separated by reduction of the The two DNP groups are far enough apart not to
%S bonds and acidification.In the most abundant interfere with each other's combinationwith antibody
type of antibody in the circulation, immunoglobu- so that they can bring the antigen combining sites on
lin G , the exposed hinge region is extended in two different antibodies together end to end. When
structure due to the high proline content and is viewed by negative staining in the electron micro-
therefore vulnerable to proteolytic attack; thus the scope, a series of geometric forms is observed which
molecule can be easily split in the laboratory using represents the different structures to be expected if a
papain to yield two identical Fab fragments, each flexible Y-shaped hinged molecule with a combining
withasinglecombiningsite for antigen,anda third site at the end of each of the two arms of the Y were
fragment, Fc, which lacks the ability to bind anti- to complex with this divalent antigen. Triangular
gen. Pepsin strikes at a different point and cleaves trimers, square tetramers and pentagonal pentamers
the Fc from the remainder of the molecule to leave may be readily discerned (figure 3.1). The way in
a large 5s fragment which is designated F(ab), which these polymeric forms arise is indicated in
since it is still divalent with respect to antigenbind- figure 3.2. The position of the Fc fragment and its lack
ing just like the parent antibody (Milestone3.1). of involvement in the combination with antigen are
apparent from the shape of the polymers formed using
ANTIBODIES HAVE DISCREET SITES FOR the pepsin F(abf),fragment (figure3.1C).
BINDING ANTIGEN
The location of the antigen combining sites was ele-
AMINO ACID SEQUENCES REVEAL
VARIATIONS I N IMMUNOGLOBULIN
gantly demonstrated by a study of purified antibodies
STRUCTURE
to the dinitrophenyl (DNP) group mixed with the
compound: For good reasons, the antibody population in any
Early studies showed the bulk of the antibody activity in The clues to how the chains are assembled to form the
serum to be in the slow electrophoretic fraction termed y- antibody molecule came from selective cleavage using
globulin (subsequently immunoglobulin). The most proteolytic enzymes. Papain destroyed the precipitating
abundant antibodies were divalent, i.e. had two combin- power of the intact molecule but produced two univalent
ing sites for antigen and could thus form a precipitating Fab fragments still capable of binding to antigen (Fab =
complex (cf.figure6.2). fragment antigen binding); the remaining fragment had no
To Rodney Porter and Gerald Edelman must affinity for antigen and was termed Fc by Porter lfragment
go the credit for unlocking the secrets of thebasic structure crysfullizable). After digestion with pepsin a molecule
of the immunoglobulin molecule. If the internal disulfide called F(ab), was isolated; it still precipitated antigen and
bonds are reduced, thecomponentpolypeptidechainsstill so retained both binding sites, but the Fc portion was
hang together by strong noncovalent attractions. Howev- further degraded. The structural basis for these observa-
er, if the reduced molecule is held under acid conditions, tionsisclearlyevident fromfigureM3.l.l. Inessence, with
these attractive forces are lost as the chains become posi- minor changes, all immunoglobulin molecules are con-
tively charged and can now be separated by gel filtration structed from one or more of the basic four-chain
into larger so-called heavy chains of approximately 55 000 monomer units.
Da (for IgG, IgA and IgD) or 70 OOODa (for IgM and IgE)
and smaller light chains of about 24 000 Da.
Figure M3.1.1.The antibodybasic unit, consisting of two iden- with one. After pepsin digestion the pFc fragment representing
tical heavy and two identical light chainsheld together byinter- the C-terminal half of the Fc region is formed and is held together
chain disulfide bonds, can be broken down into its constituent by noncovalent bonds. The portion of the heavy chain in the Fab
polypeptide chains and to proteolytic fragments, the pepsin fragment is given the symbol Fd. The N-terminal residue is on the
F(ab), retaining two binding sites for antigen and the papain Fab left for eachchain.
given individual is just incredibly heterogeneous, and
this has meant that the determinationof amino acid se-
quences was utterly useless until it proved possible to
obtain the homogeneous product of a single clone. The
opporhnity to do this first came from the study of
myeloma proteins.
In the human disease known as multiple myeloma,
one cell making one particular individual im-
munoglobulin divides over and over again in the un-
controlled way a cancer cell does, without regard for
the overall requirement of the host. The patient then
possesses enormousnumbers of identical cells derived
as a clone from the original cell and they all synthe-
size the same immunoglobulin- the myeloma or M-
protein-which appears in the serum, sometimes in
very high concentrations.By purification of the myelo-
ma protein we can obtain a preparation of an im-
Figure 3.1. (A) and (B)Electron micrograph (~1000000)of com- munoglobulin having a unique structure.Monoelonal
plexes formed on mixing the divalent DNP hapten with rabbit
anti-DNP antibodies. The 'negative stain' phosphotungstic
antibodies can also be obtained by fusing individ-
acid is an electron-dense solution which penetrates into the spaces ual antibody-forming cells with a B-cell tumor to
between the protein molecules. Thus the protein stands out as a produce a constantly dividing clone of cells dedi-
'light' structure in the electron beam. The hapten links together the cated to making the one antibody (cf. figures 2.11 and
Y-shaped antibody molecules to form trimers (A) and pentamers (B)
(cf. figure 3.2). The flexibility of the molecule at the hinge region is
6.20).
evident from the variation in angle of the arms of the 'Y'. (C) As in The sequencing of a number of such proteins has
(A); but the trimers were formed using the F(ab'), antibody frag- revealed that the N-terminal portions of both heavy
ment from which the Fc structures have been digested by pepsin (X and light chains show considerablevariability, where-
500 000).The trimers can be seen to lack the Fc projectionsat eachcor-
ner evident in (A).(AfterValentine R.C. & Green N.M. (1967)Journal
as the remaining parts of the chains are relatively
ofMolecular B i o l o ~27,615; courtesy of Dr Green and with the per- constant, being grouped into a restricted number
mission of Academic Press, New York.) of structures. It is conventional to speak of variable
and constant regions of both heavy and light chains
(figure3.3).
Certain sequences in the variable regions show
quite remarkable diversity and systematic analysis
localizes these hypenrariable sequences to three seg-
ments on the light chain (figure 3.4) and three on the
heavy chain.
IMMUNOGLOBULIN GENES
segments, a large V, and a small f,,while a single gene Figure 3.4. Wu and Kabat plot of amino acid variability in the vari-
encodes the constant region (figure3.5).There is a clus- able region of immunoglobulin heavy and light chains. The se-
ter of -40 V, genes and just five functional J, genes. In quences of chains from a large number of myeloma monoclonal
the immature B-cell, a translocation event leads to the proteins are compared and the variability at each position is com-
puted as the number of different amino acids found divided by the
joining of one of the V, genes to one of the J, segments. frequency of the most common amino acid. Obviously, the higher
Each V segment has its own leader sequence and a the number the greater the variability; for a residue at which all
number of upstream promoter sites, including a char- 20 amino acids occur randomly, the number will be 400 (20+ 0.05),
acteristic octamer sequence, to which transcription and at a completely invariant residue, the figure will be 1 (1+ 1).The
three hypervariable regions (blue) in the (a) heavy and (b) light
factors bind (figure 3.6). When the Ig gene is tran- chains, usually referred to as complementarity determining
scribed, splicing of the nuclear RNA brings the V, f , regions (CDRs), are clearly defined. The intervening peptide
sequence into contiguity with the constant region C, sequences (gray) are termed framework regions (Frl-4). (Courtesy
sequence, the whole being read off as a continuous K of Professor E.A. Kabat.)
chainpolypeptidewithin the endoplasmicreticulum.
The same general principles apply to the arrange-
ment of h light chain and the heavy chain genes,
although the latter constellation shows additional
features:the differentconstant region genes form a sin- .
gle cluster and there is a group of around 25 highly In essence, the translocation involves the mutual
variable D segments located between the V and J re- recognition of conserved heptamer-spacer-nonamer
gions (figure 3.7). The D segment together with its recombination signal sequences which flank each
junctions to the V and J segments encodes almost the germ-line V, D andJ segment (figure3.8).The products
entire third hypervariable region, the first two hyper- of the recombination activating genes RAG-I and
variable regions being encoded entirely within the V RAG-2 catalyse the introduction of double-strand
sequence. breaks between the elements to be joined and their re-
spective flanking sequences. At this stage, nucleotides
STRUCTURAL VARIANTS OF THE BASIC
may either be deleted or inserted between the VD,
IMMUNOGLOBULIN MOLECULE
DJ or VJ joining elements before they are ultimately
ligated.
Figure 3.6. Genes controlling immunoglobulinheavy chain tran- teins, will increase the transcription rate to levels typical of actively
scription. Each VDJ segment encoding the variable region is associ- secreting B-cells.The enhancersare near to the regions which control
ated with a leader sequence. Closely upstream is the TATA box of the switching from one Ig class constant region to another, e.g. IgM to
promoter which binds RNA polymerase I1 and the octamer motif IgG. Primary transcripts are initiated 20 nucleotidesdownstream of
which is one of a number of short sequences which bind transacting the TATA box and extend beyond the end of the constant region.
regulatory transcription factors. The V-region promoters are rela- These are spliced, cleaved at the 3' end and polyadenylatedto gener-
tively inactive and only association with enhancers, which are also ate the translatablemRNA.
composites of short sequence motifs capable of binding nuclear pro-
Figure 3.7. Human V-region genes
shuffied by gene rearrangementto
generate the single heavy chain specificity
characteristic of each B-cell.The V, genes
can be grouped into seven different families
(VJ-V,7) based upon sequence homology
of > 80%, with VH3being by far the largest
family.Individual members of a family are
interspersed throughout the locus, i.e. there
is no significant grouping together of V,
family genes. Note the additional D
segment minigenes that are present in the
heavy chain locus.
(Gm = marker on IgG). Allotypic differences at a given example, the b4, b5 allotypes on rabbit light chains: an
Gm locus usually involve one or two amino acids in animal of b4b4genotype would express the b4 allotype,
the peptide chain. Take, for example, the Glm(a) whereas a rabbit of b4b5 genotype derived from b4b4
locus on IgGl (table 3.1). An individual with this and b5b5parents would express the b4 marker on one
allotype would have the peptide sequence fraction and b5 on another fraction of its immunoglob-
Asp.Glu.Leu.Thr.Lj..son each of his or her IgGl mole- ulin molecules.
cules. An individual possessing the a-negative allo-
type would have the sequence Glu.Glu.Met.Thr.Lys,
i.e. two amino acids different. To date, 20 Gm groups
have been found on the y heavy chains and a further We have seen that it is possible to obtain antibodies
three (the Km-previously Inv groups) on the K that recognize isotypic and allotypic variants; one can
constant region. also raise antiserumswhich are specific for individual
As in other allelic systems, individuals may be ho- antibody molecules and discriminate between one
mozygous or heterozygous for the genes encoding monoclonal antibody and another independently of
the markers; these are expressed codominantly and isotypic or allotypic structures (figure 3.9). Such anti-
are inherited in simple Mendelian fashion. Take, for serums define the individual determinants character-
Figure 3.8. The joining of V, D and J segments. Joining is master- The enzyme Ku (a dimer of Ku70 and Ku86) binds to the DNA ends
minded by the recombination activating genes RAG-1 and MG-2, and stimulatesDNA-dependentprotein kinase (DNA-PK,mutation
the products of which cleavethe DNAat the signalends. RAG-1 and of which gives rise to mice with severe combined imunodeficiency
RAG-2 together produce several thousand times more efficient VD] (SCID)) which facilitates the opening of the hairpin. Terminal de-
recombination than either alone. The introns adjoiningthe V, D and oxynucleotidyl transferase (TdT)adds nucleotides to the ends of the
J gene segments contain specialized recombination signal se- DNAstrands in order to generateN-region diversity.Unlike the pre-
quences (RSSs)which include conserved heptamers and nonamers cision of the signaljoint, the codingjoint is variablebecause it can in-
separated by spacers of either 12 or 23 base pairs. The two joining volve the addition of base pairs resulting from both the resolution of
segments, in this example V, and J,, are brought into proximity by in- the hairpin loop (P-elements)and the TdT-mediatedN-region diver-
teraction between their respective RSSs mediated by the DNA bend- sity. Nucleases remove any excess nucleotides and polymerases fill
ing and looping high mobility group-l and -2 proteins (HMG-l and in any gaps before the DNAligaseIVand XRCC4 enzymescarry out
HMG-2). RAG-1 and RAG-2 cleave the DNA to produce double- ligation of the two sequences.Sincethe codingelementsarejoined at
strand breaks at the border of the RSS. The excised signal sequences random with respect to the reading frames, two out of three events
are ligated to form the signal joint resulting in a piece of circular have two codingelementsout of frame. Although apparently waste-
DNA containingthe excised sequences.This is probably maintained ful, this is evolutionarilytolerated because it confers so much benefit
in the cell for a period of time before eventually being lost from the in the form of antigen receptor diversity. VD] recombination prod-
cell. The double strands of each coding segment form 'hairpin' ends. ucts define the major antigen-bindingdomains.
istic of each antibody,collectively termed the idiotype Anti-idiotypes which react with one antibody and
(Kunkel&Oudin). Not surprisingly it turns out that no other are said to recognize private idiotypes and
the idiotypic determinants are located in the variable provide further support for the idea that each antibody
part of the antibody associatedwith the hypervariable has a unique structure. Frequently, antibody mole-
regions (figure3.10). cules of closely similaramino acid structuremay, in ad-
Figure 3.9. How to use monoclonal myeloma proteins to produce iotypic determinants alone. (In an attempt to simplify, we have ig-
antibodiesspecific for different Ig structures.The rabbit makes an- nored subclasses and allotypes, but the same principles can be ex-
tibodies directed to different parts of the human IgG myeloma.Anti- tended to the generation of antisera specific for these variants.) The
bodies to those parts which are common to other Ig classes can be rabbit produces a mixture of polyclonal antibodies directed against
absorbed out with myelomas of those classesleaving other antibod- each structural site on the antigen, i.e. they are produced by clones
ies reacting with class-specific G and variable region-specific (idio- derived from a variety of antigen-specific parent cells which each
type=Id) structures on the original molecule. By the same token, react stereochemically in a slightly different way with the same
further absorption with other IgG myelomas will remove the com- structure (cf. p. 62).
mon IgG-specificantibodiesleaving an antiserum directed to the id-
dition, share idiotypes (e.g.M104 and HDEX 2 in figure demonstrating the same V region on different heavy
3.10) and we then speak of public or cross-reacting chains and on different cells, for identification of
idiotypes. specific immune complexes in patients' serums, for
Anti-idiotypic serums provide useful reagents for recognition of V, type amyloid in subjects excreting
Figure 3.10. Structural correlates of idiotopes (individual deter- tures of the CDR3 region in these antibodies. The presence of the
minants on an idiotype)on antidextranantibodies.Amino acid se- idiotopes on each antibody molecule is assessed by reaction with
quences of variable heavy chain regions of mouse monoclonal antisera specific for IdX, J558 Id1 and M104 Id1 (on the right). Cross-
antidextran antibodies are shown. All antibodies have h, L chains. reacting idiotopes may also be associated with the CDR3 region in
Horizontallines indicateidentity to the sequence of the first protein, other systems. The hatched boxes either side of the D segmentrepre-
J558; letters (Dayhoff code) show differences or regions correlated sent N-region diversity and P-elements. (Reproduced from Davie
with idiotopes (central boxed areas). The cross-reacting idiotope J.M. et al. (1986)AnnualReview of immunology 4,147 with permission.
(IdX) is associated with the second complementarity determining Oby Annual Reviews Inc.)
region (CDR.2)structures, while the private idiotopes (IdI) are fea-
Bence-Jones proteins, for detection of residual mono- That these variable regions of heavy and light chains
clonal protein after therapy and perhaps for selecting both contributeto antibody specificity is suggested by
lymphocytes with certain surface receptors. The read- experiments in which isolated chains were examined
er will (or should)be startled to learn that it is possible for their antigen combining power. In general,varying
to raise autoanti-idiotypic sera since this means that in- degrees of residual activity were associated with the
dividuals can make antibodies to their own idiotypes. heavy chains but relatively little with the light chains;
This has quite momentous consequences, as will be- on recombination, however, there was always a sig-
come apparent when we discuss the Jerne network nificant increase in antigen-binding capacity. Interest-
theory in Chapter 11. ingly though, the majority of immunoglobulins in
camels and llamas lack light chains, although this does
not seem to leave them unduly inconvenienced.
IMMUNOGLOBULINS ARE FOLDED INTO
GLOBULAR DOMAINS WHICH SUBSERVE
DIFFERENT FUNCTIONS
more readily than the other immunoglobulins into of C4b to the Cyl domain. Thereafter, one observes C3
the extravascular body spaces, it is the predominant convertase formation, covalent coupling of C3b to the
species in nonmucosal tissues, where it carries bacteria and release of C3a and C5a leading to the
the major burden of neutralizing bacterial toxins chemotactic attraction of our friendly p o l ~ o r p h o -
and of binding to microorganisms to enhance their nuclear phagocytic cells (cf. p. 2). These adhere to the
phagocytosis. bacteria through surface receptors for complement
and the Fc portion of IgG (Fcy)and then ingest the mi-
croorganisms through phagocytosis. In a similar way,
Activation ofthe classical campEementpathway
the extracellular killing of target cells coated with IgG
Complexes of bacteria with IgG antibody trigger the antibody is mediated largely through recognition of
C1 complex when a minimum of two Fcy regions in the the surface Fcy by NK (natural killer) cells bearing the
complex bind Clq (see figure 2.2). As shown by site- appropriate receptors (cf.p. 18).The thesis that the bio-
specific mutagenesis of the C@ domain, the common logical individuality of different immunoglobulin
core motif for binding to Clq is Glu.318-X-Lys.320-X- classes is dependent on the heavy chain constant re-
Lys.322 where X, the residue separating these charged gions, particularly the Fe, is amply borne out in rela-
amino acids, can vary. In keeping with this analysis, it tionship to activities such as transplacental passage,
is comforting to note that synthetic peptides with this complement fixationand binding to various cell types,
structure can block Clq binding to IgG. Activation of where function has been shown to be mediated by the
the next component, C4, tends to produce attachment Fc part of the molecule.
Figure 3.13. The disposition, interaction and biological proper- bined Cy2 and Cy3 domains bind to Fc receptors on phagocytic cells,
ties of the Ig domains in IgG. (a) Stylized representation of IgG in- NK cells and placental syncytiotrophoblast; also to staphylococcal
dicating typical positions of the intrachain and interchain disulfide protein A. (Notethe IgG heavy chain is designatedyand the constant
bonds and the location of the hinge region. The number of inter- region domains Cyl, Cy2 and C*.) (d) Diagram indicating IgG
heavy chain disulfide bonds varies with the subclass of IgG; al- flexibility (cf. Brekke O.H. et al. (1995)Immunology lbday 16,85)and
though for simplicity only one is shown, there are actually two in showing apposing domains making contact through hydrophobic
IgGl and IgG4, four in IgG2 and a massive 11 in IgG3. (b) regions (afterDr A. Feinstein).The structuresof these contact frame-
Computer-generatedmodel of IgG. One heavy chain is depicted in works are highly conserved, an essential feature if different V, and
light blue, the other in darker blue and the light chains in gray Car- V, domains are to associatein order to generatea wide variety of an-
bohydrate bound to and separating the Cy2 domains is in red. The tibody specificities. These hydrophobic regions on the two comple-
molecule analysed was a mutant with a truncated hinge region since ment-fixing CH2(C*) domains are partly masked by carbohydrate
the flexibility associated with the hinge (see (d) below) makes the and remain independent, so allowing the formation of a hinge re-
X-ray pictures fuzzy and difficult to interpret. (The structure was gion which is extremely flexible both with respect to variation in the
determined by Silverton E.W., Navia M.A. & Davies J.R. (1977) angle of the Fab fragments (waving) and their rotation about the
Proceedings ofthe Natio~zafAcadewry ofsciences ofthe U S A 74,5140, and hinge peptide chain. This flexibilityenables c o m b w g sites in IgG
the figure generated by computer graphics using R.J. Feldmann's to be readily adapted to spatial variations in the presentation of the
system (National Institute of Health).) (c) Diagram based on the antigenic epitopes; Fc 'wagging' permits optimal orientation to re-
model indicating domain location of biological function. The com- ceptors on effector cells.
Table 3.2. Physical properties of major
human immunoglobulin classes.
*7S monomer, 9s dimer, and 11s dimer in external secretions which carries secretory compo-
nent (S).
t IgAdimer and IgM containJ chain.
$ng = 10-~g.
Specific Fc receptors may exist for all the classes Fcy receptors (FcyRI, FcyRII, FcyRIII) utilize these
of antibody, although receptors for IgD and IgM are for binding Ig Fc regions, just as other molecules of
not particularly well characterized.For IgG a quite ex- related structure (the immunoglobulin 'superfamily',
tensive family of Fc receptors has emerged, the nine p. 245) are mutually attracted through domain
known genes encoding four major groups of receptors interactions.
FcyIU, FcyRII, FcyliIII and FcyRn (or, more simply, FcyRI (CD64) is constitutively present on mono-
FcRn).WiMn these groups there are anumber of splice cytes, macrophages and dendritic cells, and is induced
variants and inherited polymorphic forms. All the on neutrophils following their activation by IFNy and
Fcy receptors display immunoglobulin-like domains G-CSF (granulocyte colony-stimulating factor). Con-
(p. 45) on the extracellular surface and the leukocyte versely, FcyRI can be downregulated in response to IL-
4 and IL-13.Structurally,it consists of an IgG-binding a IgG on antibody production (cf.p. 201).Thus, whereas
chain which lacks cytoplasmic signaling domains and the isoforms on phagocytic cells are associated
a y chain homodimer containing immunoreceptor with ligand internalization, that on the B-cell fails to
tyrosine-based activation motifs (ITAMs) used to internalize but concentrates instead on lymphocyte
transmit an activation signal into the cell. It binds regulation.
monomeric IgG avidly to the surface of the cell thus The two Fc yR111 (CD26) genes encode the isoforms
sensitizing it for subsequent encounter with antigen. FcyRIIIA and FcyRIIIB which have a medium and
Its main roles are probably in facilitatingphagocytosis, low affinity for IgG, respectively. FcyRIIIA is found on
antigen presentation and in mediating extracellular most types of leukocyte, whereas FcyRIIIB is restricted
killing of target cells coated with IgG antibody, a mainly to neutrophils and is unique amongst the Fc
process referred to as antibody-dependentcellular cy- receptors in being attached to the cell membrane by
totoxicity (ADCC; p. 332). It might also be concerned a glycosylphosphatidylinositol (GPI) anchor rather
with the overall regulation of IgG levels in the body, than a transmembrane segment. FcyRIIIA is known
since the catabolic rate appears to depend directly upon to be associated with the y chain signaling dimer
the total IgG concentration and one might speculate on monocytes and macrophages, and with either c
that endocytosis of IgG attached to FcyRI, the only and/or y chain signaling molecules in NK cells,
leukocyte Fc receptor to have a high affinity for and its expression is upregulated by transform-
monomeric IgG, could contribute significantly to this ing growth factor P (TGFP) and downregulated by
degradation. On the other hand, synthesis is largely IL-4. With respect to their functions, FcyRIIIAis largely
governed by antigen stimulation, so that in germ-free responsible for mediating ADCC by NK cells and
animals, for example, IgG levels are extremely low but the clearance of immune complexes from the circula-
rise rapidly on transfer to a normal environment. tion by macrophages. For example, the clearance of
Unlike the single isoform of FcyRI, there are six ex- IgG-coated erythrocytes from the blood of chim-
pressed isoforms of FcyRII (CD32) which collectively panzees was essentially inhibited by the monovalent
are present on the surface of most types of leukocyte Fab fragment of a monoclonal anti-FcyIII (work out
(figure3.14).Binding of monomeric IgG to these recep- why the Fab fragment was used). FcyRIIIB cross-
tors is insignificant.This lends itself to a cheeky play of llnking stimulates the production of superoxide by
outstanding simplicity for the uptake of immune com- neutrophils.
plexes: because of the geometric increase in binding The fourth major type of FcyR is not present on
strength of polymeric vs monomeric ligands (cf. the leukocytes but instead is found on epithelia1 cells. It is
'bonus effect of multivalency', p. 87), complexes bind referred to as FcRn due to its original description as a
really well to these receptors and are selectively ad- neonatal receptor, although it is now known to be pre-
sorbed to the cell surface in the face of competition sent also in the adult. This molecule has a major histo-
from the dauntingly high concentrations of monomer- compatibility complex (MHC)-like structure and its a
ic IgG in the body fluids. Furthermore, because of their chain associateswith P,-microglobulin. It acts as an in-
fixed spatial relationship within the immune complex, tracellular trafficking receptor and at least four major
the bound IgG moleculesbring about the cross-linking activities have been proposed for this molecule. It is
of Fc receptors mandatory for cell signaling. Thus the fairly well established that it transfers maternal circu-
binding of IgG complexes triggers phagocytic cells lating IgG across the placenta to the fetus (figure3.15a)
and may provoke thrombosis through their reaction and, at least in rodents, colostral and maternal milk
with platelets. The FcyRIIAmediates phagocytosis and IgG from the intestine to the circulation of the neonate
ADCC whilst the FcyRIIB2 (and FcyRIII) efficientlyme- (figure 3.15b).IgG is alone amongst the Ig classes in its
diates endocytosis leading to antigen presentation. ability to cross the human placenta, thereby providing
FcyRIIBl on B-cells does not endocytose immune com- a major life-line of defense not only for the fetus in utero
plexes and therefore B-cells principally present only but also for the first few weeks of the newborn baby's
their cognate antigen following ligation and life. Transfer of IgG across the gut mucosa in the
endocytosis of the B-cell receptor (BCR). In fact, the neonate may further reinforce this protection. FcRn
FcyRIIB molecules have cytoplasmic domains which may additionally be involved in maintaining steady
contain immunoreceptor tyrosine-based inhibitory state levels of circulating IgG and in the bidirectional
motifs (ITIMs)and their occupation leads to downregu- transport of IgG across the adult intestinal epithelium
lation of cellular responsiveness. In the case of the and possibly across mucosal surfaces at other sites in
B-cell this mediates the negative-feedback effect of the body. Binding of IgG to FcRn is regulated by pH,
Figure 3.15. The epithelial cell surface receptor for IgG Fc regions. tion. FcRn has homology to MHC class I molecules and contains P,-
(a)The FcRn receptor is present in the placenta where it fulfilsthe im- microglobulin (cf. p. 70). Knockout mice lacking FcRn are incapable
portant task of transferringmaternal IgG to the fetal circulation.This of acquiring maternal Ig as neonates. Furthermore, they have a
will provide important protection prior to the generation of im- grossly shortened IgG half-life, suggestingthat FcRn may also serve
munocompetence in the fetus. Furthermore, it is self-evident that a role as a protective receptor which prevents degradation of IgG
any infectious agent which might reach the fetus in ufero will have and then recycles it to the circulation. The IgG half-life is unusually
had to have passed through the mother first, and the fetus will rely long compared with that of IgA and IgM and this enables the re-
upon the mother's immune system to have produced IgG with ap- sponse to antigen to be sustained for many months following infec-
propriate binding specificities. This maternal IgG also provides pro- tion. (c)An additional,and still to be proven, role of FcRn may be as a
tection for the neonate, because it takes some weeks following birth bidirectional shuttle receptor.IgG binding on the nonluminal side of
before the transferred IgG is eventually all catabolized. (b) It has the epithelial cell may occur, following endocytosis,within the more
been clearly demonstrated in rodents, although remains speculative favorable pH of acidic endosomes.This receptor may thus provide a
in humans, that there is epithelial transport of IgG from maternal mechanism for mucosal immunosurveillance, traveling back and
milk across the intestinal cells of the newborn. IgG binds to FcRn at forth across the epithelialcell, deliveringIgG to the intestinallumen
pH 6.0, is taken into the cell within a clathrin-coated vesicle and re- and then returning the same antibodiesin the form of immune com-
leased at the pH of the basal surface. The directional movement of plexes for the stimulationof B-cellsby follicular dendritic cells.
IgG is achieved by the asymmetric pH effect on Ig-receptor interac-
with high affinity binding at acidic pH but very little at tack by microorganisms. This responsibility is clearly
neutral or alkali pH. Unlike the transcytosis across ep- taken seriously since approximately 40 mg of secretory
ithelial cells mediated by the pIgA-R (seebelow), FcRn IgA/kg body weight is transported daily through the
does not undergo proteolytic cleavage and therefore human intestinal crypt epithelium to the mucosal sur-
has the potential for generating bidirectional traffic face as compared with a total daily production of IgG of
(figure3.15~). 30 mg/kg.
The IgA is synthesized locally by plasma cells and
dimerized intracellularly together with a cysteine-rich
Nonprecipitating 'univalent'antibodies
polypeptide called J chain, of molecular weight 15000.
IgG has two combining sites for antigen. However, The dimeric molecule is effectivelytetravalent and will
5-15% of the IgG in all antiserums appears to consist of have a much higher binding avidity for polymeric
nonprecipitating asymmetric molecules with a single antigens than the monomeric form due to the bonus
effective binding site. The other site is blocked stereo- effect of multivalency (cf. p. 87). If dimerization
chemically by a mannose-rich carbohydrate in the Fab occurred randomly after secretion, dimers of mixed
region. specificity would be formed which would be no
more effective in combining with antigen than the
monomers. The dimeric IgAbinds strongly through its
J chain to a receptor for polymeric Ig (poly-Igreceptor,
IgA appears selectively in the seromucous secretions, pIgR, which also binds polymeric IgM) present in the
such as saliva, tears, nasal fluids, sweat, colostrum, membrane of mucosal epithelial cells. The complex
and secretions of the lung, genitourinary and gastroin- is then actively endocytosed, transported across the
testinal tracts, where it clearly has the job of defending cytoplasm and secreted into the external body fluids
the exposed external surfaces of the body against at- after cleavage of the pIgR peptide chain. The fragment
Figure 3.16. Secretory IgA. (a) The mechanism of IgA secretion at which drives the transport of IgA dimers to the mucosal surface (in
the mucosal surface. The mucosal cell synthesizes a receptor for quite the opposite direction to the transcytosis of milk IgG in figure
polymeric Ig (pIgR) which is inserted into the basal membrane. 3.15).(b)Schematic view of the structure of secreted IgA. The J chain,
Dimeric IgAbinds to this receptor and is transported via an endocyt- which is an integral part of secreted polymeric Ig (IgA and IgM),
ic vacuole to the apical surface. Cleavage of the receptor releases se- forms disulfide bonds with the penultimate residue of the C& do-
cretory IgAstill attached to part of the receptor termed the secretory main which is a cysteine (Cys 495). Covalent linkage of the J chain
piece. Note how the receptor cleavage introduces an asymmetry seems to be criticalfor the initial binding to the polymeric Ig receptor.
of the receptor remaining bound to the IgA is termed coliform organisms, where it is supposed that com-
secretory component and the whole molecule, secre- plement-induced disruption of the outer surface
tory IgA (figure 3.16). The reader is strongly recom- permits access of the enzyme to the peptidoglycan
mended to turn to figure 8.9 (p. 156) for a dramatic wall.
demonstration of secretory IgA held in the surface Plasma IgA is predominantly monomeric and, since
mucus of intestinal mucosal epithelial cells. this form is a relatively poor activator of complement,
The function of the secretory piece may be to protect it seems likely that the body uses it for the direct
the IgA hinge from bacterial proteases. It would also neutralization of any antigens which breach the
be nice to think that it acted as a molecular Teflon to epithelial barrier to enter the circulation in appreci-
endow the IgA dimer with 'nonstick' potential, since able quantities. However, it has additional functions
IgA antibodies function by inhibiting the adherence of which are mediated through the FcaR (CD89) for
coated microorganisms to the surface of mucosal cells, IgA present on monocytes, macrophages, neutrophils
thereby preventing entry into the body tissues. They and subpopulations of both T- and B-lymphocytes.
will also combine with the myriad soluble antigens Structurally, this receptor most closely resembles
of dietary and microbial origin to block their access FcyRIIIA in that it has two immunoglobulin domains
to the body. Aggregated IgA binds to polymorphs and associates with a y chain signaling homodimer.
and can also activate the alternative (figure 2.3), as Following cross-linking, the receptor can activate
distinct from the classical, complement pathway, endocytosis, phagocytosis, inflammatory mediator
largely through its abundant carbohydrate groups. release and ADCC. Expression of the FcaR on
This may account for reports of a synergism between monocytes is strongly upregulated by bacterial
IgA, complement and lysozyme in the killing of certain lipopolysaccharide.
Figure 3.17. The structure of IgM. (a)The arrangement of domains 'starf-shaped configuration. (c)The structure as revealed in an elec-
in one of the five subunits showing how the pentamer is built up tron microscope preparation of specific sheep IgM antibody bound
through the disulfide linkages between the Cys 414 in the Cp3 do- to Salmonella paratyphi flagellum where the immunoglobulinhas as-
mains and between the Cys 575, the penultimate C-terminal residue sumed a 'crab-like' conformation in establishing its links with anti-
in the 19-aminoacid tailpiece which followsCp4 (after Hilschman & gen. With the F(abf),arms bent out of the plane of the central Fc,
Feinstein). Without too much aggravation, we hope the reader will region, the Cy3 complement binding domains are now readily ac-
appreciatethat the hinge region in IgG (cf.figure 3.13)is replaced by cessible to the first component of complement (cf.p. 22). The Fc, con-
a rigid pair of extra domains (Cy2), while CM and Cy4 domains in stellation obtained by papain cleavage can activate complement
IgM are structurally equivalent to the C$ and Cy3 regions, respec- directly. (Electron micrographs are negatively stained preparations
tively, in IgG. (b)The structure as shown by electron microscopy of a of magnification x2 000 000, i.e. 1mm represents 0.5nm; kindly pro-
human Waldenstrom's macroglobulin in free solution adopting a vided by Dr A. Feinstein and Dr E.A. Mum.)
Two subclassesof IgAhave alsobeen found, of which tween heavy and light chains.Class and subclassvaria-
IgAl constitutes 80-90% of the total. The IgA2 subclass tion is not restricted to human immunoglobulinsbut is
exists in two allotypic forms, one of which (IgA2m(l)) a feature of all the mammals so far studied: monkey,
is unusual in that it lacks interchain disulfidebonds be- sheep, rabbit, guinea-pig,rat and mouse.
The basicstructure is a four-peptide unit ingregions)on theheavy chainand threeonthelightchain
Immunoglobulins (Ig) have a basic four-peptide struc- form the antigen-binding site.
ture of two identical heavy and two identical light chains The constant region domains of the heavy chain (par-
joined by interchain disulfide links. ticularly the Fc) carry out a secondary biological function
Papain splits the molecule at the exposed flexible hinge after the binding of antigen, e.g. complement fixation and
region to give two identical univalent antigen-binding macrophage binding.
fragments (Fab) and a further fragment (Fc). Pepsin
proteolysis gives a divalent antigen-binding fragment lmmunoglobulinclasses and subclasses
F(ab), lacking the Fc. In the human there are five major types of heavy chain
giving five classes of Ig. IgG is the most abundant Ig in the
Amino acid sequences reveal variations in extravascular fluids where it neutralizes toxins and com-
immunoglobulinstructure bats microorganisms by fixing complement via the C1
There are perhaps 10*or more different Ig molecules in pathway and facilitatingthebinding tophagocyticcellsby
normal serum. receptors for C3b and Fcy. It crosses the placenta in late
Analysis of myelomaproteins, which are homogeneous pregnancy and the intestine in the neonate.
Ig produced by single clones of malignant plasma cells, Various Fcy receptors are specialized for different
hasshown theN-terminal regionofheavy and lightchains functions such as phagocytosis, antibody-dependent cel-
to have a variable amino acid structure and the remainder lular cytotoxicity, placental transport and B-lymphocyte
to be relatively constant in structure. regulation.
IgA exists mainly as a monomer (basic four-
lmrnunoglobulingenes polypeptide unit) in plasma, but in the seromucous secre-
Clusters of genes on three different chromosomes en- tions, where it is the major Ig concerned in the defense of
code K, hand heavy Ig chains, respectively. In each cluster the externalbody surfaces, it is present as a dimer linked to
in humans there are approximately 30-50 functional a secretory component.
variable region ( V )genes and around four to six small J IgM is most commonly a pentameric molecule although
minisegments. Heavy chain clusters in addition contain of a minor fraction ishexameric. It is essentially intravascular
the order of 25 D minigenes. There is a single gene encod- and is produced early in the immune response. Because of
ing each of the nine different class and subclass constant its high valency it is a very effective bacterial agglutinator
regions. and mediator of complement-dependent cytolysis and is
A special splicing mechanism involving mutual recog- therefore a powerful first-line defense against bacteremia.
nition of 5 and 3 flanking sequences, catalysed by recom- IgD is largely present on the lymphocyte and functions
binase enzymes, effects the DJ, VD and VJ translocations. together with IgM as the antigen receptor on naive B-cells.
IgE binds firmly to mast cells and contact with antigen
Structural variants of the basic lg molecule leads to local recruitment of antimicrobial agents through
Isotypes are Ig variants based on different heavy chain degranulation of the mast cells and release of inflammato-
constant structures, all of which are present in each indi- ry mediators. IgE is of importance in certain parasitic in-
vidual; examples are the Ig classes IgG, IgA, etc. fections and is responsible for the symptoms of atopic
Allotypes are heavy chain variants encoded by allelic allergy.
(alternative) genes at single loci and are therefore geneti- Further diversity of function is possible through the
cally distributed; examples are Gm groups. subdivision of classes into subclasses based on structural
An idiotype is the collection of antigenic determinants differences in heavy chains all present in each normal
on an antibody, usually associated with the hypervariable individual.
regions, recognized by other antigen-specific receptors,
either antibody (the anti-idiotype) or T-cell receptors.
The variable region domains bind antigen, and three
hypervariable loops (termed complementarity determin-
See the accompanying website (www.roin.corn)
for multiple choicequestions. I
FURTHER READING Dickinson B.L. et al. (1999) Bidirectional FcRn-dependent IgG trans-
port in a polarized human intestinal epithelia1 cell line. Journal of
Arnigorena S. & Bonnerot C. (1999) Fc receptor signaling and Clinical Investigation 104,903.
trafficking: a connection for antigen processing. Immunological Grawunder U . & Harfst E. (2001) How to make ends meet in V(D)J
Reviews 172,279. recombination. Current Opinion in Immunology 13,186.
Brown D.A. & London E. (1998)Functions of lipid rafts in biological Grawunder U., West R.B. & Lieber M.R. (1998) Antigen receptor
membranes. Annual Review of Cellular and Developmental Biology gene rearrangement. Current Opinion in Immunology 10,172.
14,111. Kinet J-P. (1999)The high-affinityIgE receptor (FCERI): from physiol-
Cedar H. & Bergrnan Y. (1999) Developmental regulation of im- ogy to pathology. Annual Review of Immunology 17,931.
mune system gene rearrangement. Current Opinion in Immunology Ravetch J.V. & Bolland S. (2001) IgG Fc receptors. Annual Review of
11,64. Immunology 19,275.
Daeron M. (1997) Fc receptor biology. Annual Review of Immunology Reddy P.S. & Corley R.B. (1999) The contribution of ER quality con-
15,203. trol to the biologic functions of secretory IgM. Immunology Today
Delves P.J. & Roitt I.M. (eds) (1998) Encyclopedia of Immunology, 2nd 20,582.
edn. Academic Press, London. (Articleson IgG, IgA, IgM, IgD and
IgE and immunoglobulin function and domains.)
Membrane receptors for antigen
Intmductlon, 59 NK recepton. 69
The B-cell surface receptorfor antigen (BCR), 59 The major histocompatibilitycomplex(MHC), 70
The B-cellinserts a transmembrane immunoglobulin into its Class I and class II molecules ore membrane-bound
surface, 59 heterodimers, 70
Thesurface immunoglobulin is compiexed with associated Several immune response-related genes contribute to the
membrane proteins, 59 remainingclass 111 region of Me MHC, 73
The T e l l surface receptor for antigen (TCR), 60 GenemapoftheMHC, 73
The receptorfor ontigen is o tronsrnembrane heterodimer, 60 Thegenesofthe MHCdisplayremarkablepolymorphism,75
Thereore two classes of T-cell receptors, 62 Nomenclature, 75
The encoding of T-cell receptors is similar to that of InheritonceoftheMHC, 76
immunoglobulins, 62 Thetissuedisfributionof MHC molecules, 77
TheCD3 complexisan integral portoftheFcell receptor, 63 MHCfunction, 77
The generation of diversity tor antigen recognition, 63 The nonclassicalMHC and closs I chain-related
lntrachoin amplification of diversity, 64 molecules, 77
lnterchoin amplification, 68 Summary, 78
Sornotic hypermutation, 68 Funher reading, 79
sequencehasbeenomitted. -
the membrane.For simplicity, the leader
=introns.
three amino acids long. In no way could this accommo- ITAMs) are present in the cytoplasmic domains of the
date the structural motifs required for interaction with Ig-a/P heterodimer (figure 4.3) and it is these that
intracellular protein tyrosine kinases which mediate undergo phosphorylationby tyrosine kinases.
the activation of signal transduction cascades. With
some difficulty, it should be said, it eventually proved
THE T-CELL SURFACE RECEPTOR FOR
possible to isolate a disulfide-linked heterodimer
ANTIGEN ( T C R )
which copurifies with the membrane Ig. This consists
of two glycoprotein chains called Ig-a (CD79a) and
Ig-P (CD79b) (figure 4.3). Both Ig-a and Ig-P have
an extracellular immunoglobulin-typedomain, but it
is their C-terminal cytoplasmic domains which are When it was eventually tracked down (Milestone 4.1),
obligatory for signaling and which become phospho- the antigen-specific T-cell receptor was identified as
rylated on cell activation by antigen-induced cross- a membrane-bound molecule composed of two
linking of the BCR, an event also associated with rapid disulfide-linked chains, a and P. Each chain folds into
Ca2+mobilization. Tyrosine-containingstructural mo- two Ig-like domains, one having a relatively invariant
tifs (immunoreceptortyrosine-basedactivation motifs, structure and the other exhibiting a high degree of
SinceT-lymphocytesrespond by activation and prolifera- that the structure permitting this selectivity would be
tion when they contact antigen presented by cells such as the combining site for antigen on the T-cell receptor.
macrophages, it seemed reasonable to postulate that they Immunoprecipitation with this antibody brought down
do so by receptors on their surface. In any case, it would be a disulfide-linked heterodimer composed of W k D a
difficult to fit T-cells into the clonal selection club if they subunits (figureM4.1.1).
lacked such receptors. Guided by Ockam's razor (the Larv The other approach went directlyfor the genes, arguing
ofPursimony, which contends that it is the aim of science as follows. The T-cell receptor should be an integral mem-
to present the facts of nature in the simplest and most brane protein not present in B-cells. Hence, T-ceU poly-
economical conceptual formulations),most investigators somal mRNA from the endoplasmic retidum, which
plumped for the hypothesis that nature would not indulge should provide an abundant source of the appropriate
in the extravaganceof evolving two utterly separate mole- transcript, was used to prepare cDNA from which genes
cularrecognitionspeciesforBandT-cells,andmanyfruit- common to 5 and T-cells were subtracted by hybridiza-
less years were spent looking for the Holy Grail of the tion to B-cell mRNA. The resulting T-specific clones were
T-cell receptor with anti-immunoglobulin serums or used to probe for a T-cell gene which is rearranged in all
monoclonal antibodies (cf. p. 120). Success only came functionally mature T-cells but is in its germ-line configu-
when a monodonal antibody directed to the idiotype ration in all other cell types (figure M4.1.2).In such a
of a T-cell was used to block the response to antigen. This way were the genes encoding the p u b u n i t of the T-cell
was identified by its ability to block one individual T-cell receptor uncoverrd.
clone out of a large number, and it was correctly assumed
Table 4.1. Calculations of human V gene diversity. It is known that the precise number of gene segments varies from one individual to
another, perhaps between 40 and 70 in the case of the V, genes for example, so that these calculations represent 'typical' numbers. The number of
specificities generated by straightforward random combination of germ-line segments is calculated. These will be increased by the further
mechanisms listed: *minimalassumption of approximately 10 variants for chains lacking D segments and 100 for chains with D segments.The
calculationfor the T-cellreceptor P chain requires further explanation.The first of the two D segments, DP,, can combinewith50 Vgenes and with
all 13JP, and JP, genes. DP2behaves similarly but can only combine with the seven downstreamJP,genes.
Figure 4.6. Junctional diversity between two germ-line segments
producing three variant protein sequences. The nucleotide triplet
which is spliced out is colored the darker blue.
Receptor editing
Figure 4.7. P-elements and N-region diversity. In addition to junc-
Recent observations have established that lympho-
tional diversity (cf. figure 4.6), additional nucleotides can be incor-
cytes are not necessarily stuck with the antigen recep- porated into the final sequence. (a)This can occur when hairpin loop
tor they initially make; if they don't like it they can structuresare created between the two strands of the DNA following
change it. The replacement of an undesired receptor cleavage at the RSS, and a subsequent cleavage of one strand creates
an overhangwhich acts as a template for the addition of nucleotides,
with one which has more acceptable characteristics
creating a Palindromic sequence (P-element,here GATC /CTAG and
is referred to as receptor editing. This process has TA/ AT, with the newly added sequence in bold). (b)Nucleotides can
been described for both imrnunoglobulins and for be added in a Nontemplated fashion (N-region diversity, indicated
TCR, allowingthe replacement of either nonfunctional by the red nucleotides) by the enzyme terminal deoxynucleotidyl
transferase (TdT).
rearrangements or autoreactive specificities. Further-
more, receptor editingin the periphery may rescue low
affinity B-cells from apoptotic cell death by replacing a ments, namely the immunoglobulin light chain and
low affinity receptor with a selectable one of higher the TCR a chain, a secondary rearrangement may
affinity. That this does indeed occur in the periphery is occur by a V gene segment upstream of the previously
strongly supported by the finding that mature B-cells rearranged VJ segment recombining to a 3' J gene
in germinal centers can express RAG-1 and RAG-2 sequence, both of these segments having intact RSSs
which mediate the rearrangement process. that are compatible (figure 4.8a). However, for im-
But how does this receptor editingwork?Well, in the munoglobulin heavy chains and TCR P chains the
case of the receptor chains which lack D gene seg- process of VDJ rearrangement deletes all of the D
Figure 4.8. Receptor editing. (a)For immunoglobulinlight chain or heptamer-nonamer sequences associated with a 23 base pair spacer
TCR a chain the recombination signal sequences (RSSs; heptamer- (colored violet) can only base pair with heptamer-nonamer se-
nonamer motifs) at the 3' of each variable (V) segment and the 5' of quences containing a 12 base pair spacer (colored red). The heavy
each joining U) segment are compatible with each other and there- chain Vand Jboth have an RSS with a 23 base pair spacer and so this
fore an entirelynew rearrangement can potentially occur as shown. is a nonstarter. Furthermore, all the unrearranged D segmentshave
This would result in a receptor with a different light chain variable been deleted so that there are no 12base pair spacersremaining. This
sequence (in this example VK~,JK~ replacing V K ~ ~ Jtogether
K ~ ) with apparent bar to secondary rearrangement is probably overcome by
the original heavy chain. (b) With respect to the immunoglobulin the presence of an RSS-like sequence near the 3' end of the V
heavy chain or TCR P chain the organizationof the heptamer-non- gene coding sequences, so that only the V gene segment is replaced
amer sequencesin the RSS precludes a Vsegment directly recombin- (in the example shown, the sequence VH48DH3JH2 replaces
ing with the J segment.This is the so-called 12/23 rule whereby the v~50D~J~2).
segment-associatedRSSs (figure4.8b).Because V, and near the 3' end of the V coding sequences that can func-
JH both have 23 base pair spacers in their RSSs, they tion as a surrogate RSS, such that the new V segment
cannot recombine: that would break the 12/23 rule. would simply replace the previously rearranged V,
This apparent obstacle to receptor editing of these maintaining the same D and J sequence (figure 4.8b).
chains may be overcome by the presence of a sequence This is probably a relatively inefficient process and
Figure 4.9. Mutations in a germ-line gene. The amino acid se- diversity being, as it is, encoded by the junctions
quences of the V,regions of five IgM and five IgG monoclonal phos- between three gene segments: V, D and J.
phorylcholine antibodies generated during an antipneumococcal
response in a single mouse are compared with the primary structure This random association between TCR y and 6
of the T15 germ-line sequence.Aline indicates identity with the T15 chains, TCR a and chains, and Ig heavy and light
prototype and an orange circle a single amino acid difference.Muta- chains yields a further geometric increase in diversity.
tions have only occurred in the IgG molecules and are seen in both From table 4.1 it can be seen that approximately
hypervariable and framework segments. (After Gearhart P.J. (1982)
Immunology Today 3,107.) Whilst in some other studies somatic hy- 230 functional T-cell receptor and 160 functional Ig
permutation has been seen in IgM antibodies, the amount of muta- germ-line segments can give rise to 4.5 million
tion usually greatly increases following class switching. and 2.4 million different combinations, respectively,
by straightforward associationswithout taking into ac-
count all of the fancy junctional mechanisms described
receptor editing may therefore occur more readily above. Hats off to evolution!
in immunoglobulin light chains and TCR a chains
than in immunoglobulin heavy chains and TCR
p chains. Indeed, it has been suggested that the
TCR a chain may undergo a series of rearrange- There is inescapable evidence that immunoglobulinV-
ment~,continuously deleting previously functionally region genes can undergo significant somatic hyper-
rearranged VJ segments until a selectable TCR is mutation.Analysis of 18murine h myelomas revealed
produced. 12 with identical structure, four showing just one
amino acid change, one with two changes and one
with four changes, all within the hypervariable re-
gions and indicative of somatic hypermutation of the
The immune system took an ingenious step forward single mouse h germ-line gene. In another study, fol-
when two different types of chain were utilized for the lowing immunization with pneumococcal antigen, a
recognition molecules because the combination pro- single germ-line T15 VHgene gave rise by mutation to
duces not only a larger combining site with potentially several different VH genes all encoding phosphoryl-
greater affinity, but also new variability. Heavy-light choline antibodies (figure4.9).
chain pairing amongst immunoglobulins appears to A number of features of this somatic diversification
be largely random and therefore two B-cells can em- phenomenon deserve a mention. The mutations are
ploy the same heavy chain but different light chains. the result of single nucleotide substitutions, they are
This route to producing antibodies of differing speci- restricted to the variable as distinct from the constant
ficity is easily seen in vitm where shuffling different re- region and occur in both framework and hypervari-
combinant light chains against the same heavy chain able regions. The mutation rate is remarkably high,
can be used to either fine tune or sometimes even alter approximately 10~-10" per base pair per generation,
the specificity of the final antibody. In general, the which is approximately a million times higher than for
available evidence suggests that in vivo the major con- other mammalian genes. In addition, the mutational
tribution to diversity and specificity comes from the mechanism is bound up in some way with class switch
heavy chain, perhaps not unrelated to the fact that the since hypermutation is more frequent in IgG and IgA
heavy chain CDR3 gets off to a head start in the race for than in IgM antibodies, affecting both heavy (figure
4.9) and light chains. However, VHgenes are on aver- tion may also be involved here. The sequences were
age more mutated than VLgenes. This might be a con- often a duplication of an adjacent sequence in the case
sequence of receptor editing acting more frequentlyon of insertions or a deletion of a known repeated se-
light chains, as this would have the effect of wiping the quence. This type of modification may like receptor
slate clean with respect to light chain Vgene mutations editing, play a major role in eliminating autoreactivity
whilst maintaining already accumulated heavy chain and also in enhancing antibody affinity.
Vgene point mutations. T-cell receptor genes, on the other hand, do not gen-
Somatic hypermutation does not appear to add sig- erally undergo somatic hypermutation. It has been ar-
nificantly to the repertoire available in the early phases gued that this would be a useful safety measure since
of the primary response, but occurs during the genera- T-cells are positively selected in the thymus for weak
tion of memory and is probably responsible for tuning reactions with self MHC (cf.p. 226), so that mutations
the response towards higher affinity. The mechanism could readily lead to the emergence of high affinity
behind the greatly enhanced mutation frequency in autoreactive receptors and autoimmunity.
immunoglobulingenes is unknown, but, intriguingly, One may ask how it is that this array of germ-line
it has recently been shown that the bcl-6 proto- genes is protected from genetic drift. With a library of
oncogene also undergoes somatic hypermutation in 390 or so functional V, D and J genes, selection would
around one-third of normal germinal center B-cells act only weakly on any single gene which had been
and memory B-cells. The rate of mutation is only functionally crippled by mutation and this implies
around 10%of that seen in the immunoglobulingenes, that a major part of the library could be lost before evo-
but nonetheless is way above that seen in other genes lutionary forces operated. One idea is that each sub-
in the B-cell. Furthermore,the types of mutation are ex- family of related V genes contains a prototype coding
tremely similar to those seen in the immunoglobulin V for an antibody indispensable for protection against
genes, suggesting a common mechanism. The present some common pathogen, so that mutation in this gene
favored idea is that somatic hypermutation is associat- would put the host at a disadvantage and would there-
ed with an error-prone DNA polymerase which is fore be selected against.If any of the other closely relat-
coupled to RNA transcription. ed genes in its set became defective through mutation,
Recently data have been put forward suggesting this indispensablegene could repair them by gene con-
that there is yet another mechanism for creating fur- version, a mechanism in which it will be remembered
ther diversity.This involves the insertion or deletion of that two genes interact in such a way that the nu-
short stretches of nucleotides within the immunoglob- cleotide sequence of part or all of one becomes identi-
ulin V gene sequence of both heavy and light chains. cal to that of the other. Although gene conversion has
This mechanism would have an intermediate effect on been invoked to account for the diversification of
antigen recognition, being more dramatic than single MHC genes, it can also act on other families of genes to
point mutation, but considerably more subtle than re- maintain a degree of sequence homogeneity.Certainly
ceptor editing. In one study, a reverse transcriptase- it is used extensivelyby, for example,chickens and rab-
polymerase chain reaction (RT-PCR)was employed to bits, in order to generate immunoglobulin diversity.
amplify the expressed VHand VLgenes from 365 IgG+ In the rabbit only a single germ-line VH gene is re-
B-cells and it was shown that 6.5% of the cells con- arranged in the majority of B-cells; this then becomes a
tained nucleotide insertions or deletions. The tran- substrate for gene conversion by one of the large num-
scripts were left in-frame and no stop codons were ber of VHpseudogenes.There are also large numbers of
introduced by these modifications. The percentage of V, pseudogenes and orphan genes (genes located out-
cells containing these alterations is likely to be an un- side the gene locus, often on a completely different
derestimate. All the insertions and deletions were in, chromosome) in humans which actually outnumber
or near to, CDRl and/or CDR2. N-region diversity of the functional genes, although there is no evidence to
the CDR3 meant that it was not possible to analyse the date that these are used in gene conversion processes.
third hypervariable region for insertions/deletions of
this type and therefore these would be missed in the
analysis. The fact that the alterations were associated
NK RECEPTORS
with CDRs does suggest that the B-cells had been sub- One of the main functions of natural killer (NK)cells is
jected to selection by antigen. It was also notable that to patrol the body looking for cells which have lost ex-
the insertions/deletions occurred at known hot spots pression of the normally ubiquitously present MHC
for somatic point mutation, and the same error-prone class I molecules. Such abnormal cells are usually ei-
DNA polymerase responsible for somatic hypermuta- ther malignant or infected with a microorganism that
interferes with class I expression. The NK cells carry
out their task using two sets of receptors, activating re-
ceptors that recognize molecules collectively present
on all cell surfaces and inhibitory receptors that recog-
nize MHC class I molecules.Upon ligation, the activat-
ing receptors signal the NK cell to kill the target cell
and/or to secrete cytokines. This potentially anarchic
situation in which the NK cells would attack all cells in
the body is normally prevented due to the recognition
of MHC class I by the inhibitory receptors (figure
4.10). Any nucleated cell lacking MHC class I will
not engage the inhibitory receptors and will only
trigger the activating receptors, resulting in its execu-
tion by the NK cell.
A second mode of killing which NK cells enjoy is
antibody-dependent cellular cytotoxicity (ADCC,
cf. p. 32) for which they are equipped with FcyRIII re-
ceptors in order to recognize antibody-coated target
cells (figure4.10).
Table M4.2.1. Identification of H-2 (antigen 11). *Atumor inoculum derived from A strainbearing antigen U is re-
jected by the C57host (+ = rejection;- = acceptance).
*Offspringof A x C57matingwerebackcrossedto the C57parent
and the resulting progeny tested for antigen I1 (Ag 11) and their
ability to reject the tumor. The figures in brackets =number ex-
pected if tumor growth is influenced by two dominant genes, one
of which determinesthe presence of antigen U.
extended a-helices above a floor created by strands class I structural amnesia. Another curious feature
held together in a P-pleated sheet, the whole forming emerged. The groove was occupied by a linear mole-
an undeniable groove (figure4.11b and c).The appear- cule, now known to be a peptide, which had cocrystal-
ance of these domains is so striking,we doubt whether lized with the class I protein. The significance of these
the reader needs the help of gastronomic analogies unique findings for T-cell recognition of antigen will be
such as 'two sausages on a barbecue' to prevent any revealed in the following chapter.
Figure 4.11. Class I and class I1 MHC molecules. (a) Diagram inside-facing surfaces of the two helices and the upper surface of the
showing domains and transmembrane segments; the a-helices and P-sheet form a cleft. The two black spheres represent an intrachain
P-sheets are viewed end on. (b) Schematic bird's eye representation disulfide bond. (c) Side view of the same molecule clearly showing
of the top surface of human class I molecule (HLA-A2)based on the the anatomy of the cleft and the typical Ig-type folding of the a,- and
X-ray crystallographicstructure. The strands making the P-pleated P,-microglobulin domains (four antiparallel P-strands on one face
sheet are shown as thick gray arrows in the amino to carboxy direc- and three on the other). (Reproduced from Bjorkman P.J.et al. (1987)
tion; a-helices are represented as dark red helical ribbons. The Nature329,506, withpermission.)
Figure 4.12. Genes encoding human HLA-DRa chain (darker immune defense functions. A notable cluster
blue) and their controlling elements (regulatory sequences in involves four genes coding for complement com-
light blue and TATAbox promoter in yellow). a, / a, encode the two
extracellulardomains; TM and CYT encode the transmembrane and ponents, two of which are for the C4 isotypes
cytoplasmic segments, respectively. 3' UT represents the 3' untrans- C4A and C4B and the other two for C2 and factor
lated sequence. Octamer motifs are also found in virtually all heavy B. The cytokines, tumor necrosis factor (TNF,
and light chain immunoglobulin V gene promoters (cf. figure 3.6) sometimes referred to as TNFa) and lympho-
and in the promoters of other B-cell-specific genes such as B29 and
CD20. toxin (LTa and LTP), are encoded under the class
I11 umbrella as are three members of the human
70 kDa heat-shock proteins. As ever, things don't
quite fit into the nice little boxes we would like to
MHC class II
put them in. Even if it were crystal clear where one
Class I1 MHC molecules are also transmembrane gly- region of the MHC ends and another begins (and it
coproteins, in this case consisting of a and P polypep- isn't), some genes located in the middle of the 'classi-
tide chains of molecular weight 34kDa and 29 kDa, cal' (cf. figure 4.13) class I or I1 regions should more
respectively. correctly be classified as part of the class I11 cohort.
There is considerable sequence homology with class For example, the LMP and TAP genes concerned
I and structural studies have shown that the a, and p, with the intracellular processing and transport of
domains, the ones nearest to the cell membrane, as- T-cell epitope peptides are found in the class I1
sume the characteristic Ig fold, while the a, and p, do- region (see below), but do not have the classical
mains mimic the class I a, and a, in forming a groove class I1 structure nor are they expressed on the cell
bounded by two a-helices and a P-pleated sheet floor surface.
(figure4.11a).
The organization of the genes encoding the a chain
of the human class I1 molecule HLA-DR and the main
regulatory sequenceswhich control their transcription The complete sequence of a human MHC was pub-
are shown in figure 4.12. lished at the very end of the last millennium after a
gargantuan collaborative effort involving groups in
England, France, Japan and the USA. The entire se-
quence, which represents a composite of different
MHC haplotypes, comprises 224 gene loci. Of the 128
of these genes which are predicted to be expressed, it is
A variety of other genes which congregate within estimated that about 40% of them have functions relat-
the MHC chromosome region are grouped under ed to the immune system. The region between class I1
the heading of class 111. Broadly, one could say and class I in the human contains 60 or so class I11
that many are directly or indirectly related to genes. An overall view of the main clusters of class I, I1
Figure 4.13. MHC class I gene map. The 'classical' polymorphic polymorphic or sometimes invariant, and many are silent or
class I genes, HLA-A, -B, -C in humans and H-2K, -D, -L in mice, are pseudogenes. In the mouse there are approximately 15 Q (also re-
highlighted with orange shading and encode peptide chains which, ferred to as &a)genes, 25 T (also referred to as TL or Tla) genes and 10
together with P,-microglobulin, form the complete class I molecules M genes. MICA and MICB are ligands for NK cell receptors. Tapasin
originally identified in earlier studies as antigens by the antibodies is involved in peptide transport (cf. p. 94). The gene encoding this
they evoked on grafting into another member of the same species. molecule is at the centromeric end of the MHC region and therefore
Note that only some strains of mice possess an H-2L gene. The genes is shown in this gene map with respect to the mouse, but in figure
expressed most abundantly are HLA-A and -B in the human and H- 4.14, the class I1 gene map with respect to the human -look at figure
2K and -D in the mouse. The other class I genes ('class Ib') are termed M4.2.1to see why.
'nonclassical' or 'class I chain-related'. They are oligo- rather than
Figure 4.14. MHC class I1 gene map with 'classical' HLA-DP, -DQ, class 11-associatedinvariant chain peptide (CLIP)from classical class
-DR in the human and H-2A (I-A) and H-2E (I-E)in mice more heav- 11 molecules to permit the binding of high affinity peptides. The
ily shaded. Both a and P chains of the class I1 heterodimer are tran- mouse H-2DM molecules are often referred to as H-2M1and H-2M2,
scribed from closely located genes. There are usually two expressed although this is a horribly confusing designation because the term
DRB genes, DRBl and one of either DRB3, DRB4 or DRB5. A similar H-2M is also used for a completely different set of genes which lie
situation of a single achainpairing with different P chains is found in distal to the H-2T region and encode members of the class Ib family
the mouse I-E molecule. The LMP2 and LMP7 genes encode part of (cf.figure 4.13).The HLA-DOA (alternativelycalled HLA-DNA)and
the proteasomecomplexwhich cleaves cytosolic proteins into small -DOB genes (H-20a and -0b in the mouse) also encode an ap het-
peptides which are transported by the TAP gene products into the erodimer which may play a role in peptide selection or exchange
endoplasmic reticulum. HLA-DMA and -DMB (mouse H-2DMa, with classical class I1molecules.
-DMbl and -DMb2) encode the DM ap heterodimer which removes
and I11 genes in the MHC of the mouse and human and H-2K, -D and -L in the mouse. These were defined
may be gained from figure M4.2.1 in Milestone 4.2. serologically by the antibodies arising in grafted indi-
More detailed maps of each region are provided in viduals using methods developed from Gorer's
figures 4.134.15. A number of pseudogenes have pioneering studies (Milestone 4.2). Other molecules,
been omitted from these gene maps in the interest of sometimes referred to as class Ib,have related struc-
simplicity. tures and are either encoded within the MHC locus it-
The cell surface class I molecule based on a trans- self ('nonclassical' MHC molecules, for example the
membrane chain with three extracellular domains as- human HLA-E, -F and -G, HFE, MICA and MICB, the
sociated with P2-microglobulinhas clearly proved to murine H-2T, -Q and -M), or elsewhere in the genome
be an advantageous model for evolution to mould as ('class I chain-related', including the CD1 family and
evidenced by the variety of molecular species which FcRn). Nonclassical MHC genes are far less polymor-
utilize this structure. It is helpful to subdivide them, phic than the classical MHC, are often invariant, and
first into the classical class I molecules (sometimesre- many are pseudogenes.
ferred to as class Ia), HLA-A, -B and -C in the human
Figure 4.15. MHC class I11 gene map. This region is somethingof a ities, and tenascin, an extracellular matrix protein. Of course many
'rag bag'. Aside from immunologically 'respectable' products like genes may have drifted to this location during the long passage of
C2, C4, factor B (encoded by the BF gene), tumor necrosis factor evolutionary time without necessarily having to act in concert with
(TNF), lymphotoxin-a and lymphotoxin-P (encoded by LTA and their neighbors to subservesome integrated defensive function.The
LTB, respectively) and three 70kDa heat-shock proteins (the 21-hydroxylases(210HA and B, encoded by CYP21A and CYP21B,
HSPAIA, HSPAl Band HSPAl Lgenes in humans, HSP70-1, HSP70-3 respectively) are concerned with the hydroxylationof steroids such
and Hsc70t genes in mice), genes not shown in this figure but as cortisone. Slp (sex-limitedprotein) encodes a murine allele of C4,
nonetheless present in this locus include those encoding valyl tRNA expressed under the influence of testosterone.
synthetase( G ~ u NOTCH4,
), which has a number of regulatoryactiv-
Table 4.2. The haplotypes of the H-2 complex of some commonly chromosomes. Thus, in the present context, the haplo-
used mouse strains and recombinantsderived from them.A/ J was type of the MHC derived from the mother will be iden-
derived by interbreeding (kxd) F1 mice, recombination occurring tical to that from the father; animals of the C57BL
between E (class11)and S (class111)regions*. strain, for example, will each bear two chromosomes
with the H-2bhaplotype (cf. table 4.2).
Let us see how the MHC behaves when we cross two
pure strains of haplotypes H-2kand H-24 respectively.
We find that the lymphocytes of the offspring (the F1
generation) all display both H-2k and H-2d molecules
on their surface, i.e. there is codominant expression
(figure 4.17). If we go further and breed Fls together,
the progeny have the genotypes k, k/d and d in the pro-
(figure4.17) is arbitrarily assigned the haplotype H-2a, portions to be expected if the haplotype segregates as
but table 4.2 shows that individual genes in the com- a single Mendelian trait. This happens because the
plex are identified by the haplotype symbol of the H-2 complex spans 0.5 centimorgans, equivalent to a
originalparents. recombination frequency between the K and D ends
of 0.5%, and the haplotype tends to be inherited
en bloc. Only the relatively infrequent recombinations
caused by meiotic cross-over events, as described
Pure strain mice derived by prolonged brother-sister for the A/J strain above, reveal the complexity of the
mating are homozygous for each pair of homologous system.
Figure 4.18. Comparison of the crystal structures of CD1 and However, when activated by agents such as y-interfer-
MHC class I. (a) Backbone ribbon diagram of mouse CDldl (red, a - on, capillary endothelia and many epithelia1 cells in
helices; blue, P-strands). (b) Ribbon diagram of the mouse MHC
class I molecule H-2Kb(cyan, a-helices; green, P-strands). (c)Super- tissues other than the thymus express surface class I1
position using alignment of P,-microglobulin highlights some of the and increased levels of class I.
differences between CDldl and H-2Kb.Note in particular the shift-
ing of the a-helices. This produces a deeper and more voluminous
groove in CDldl, which is narrower at its entrance compared with
H-2Kb. (Reprinted with permission from Porcelli S.A. et al. (1998)
Immunology Today 19,362.) Although originally discovered through transplanta-
tion reactions, as we will see in subsequent chapters,
the MHC molecules act as cell surface markers which
enable infected cells to signal cytotoxic and helper T-
Essentially, all nucleated cells carry classical class I cells. There is no doubt that this role in immune
molecules. These are abundantly expressed on both responsiveness is immensely important, and in this
lymphoid and myeloid cells, less so on liver, lung and respect the rich polymorphism of the MHC region
kidney and only sparsely on brain and skeletalmuscle. represents a species response to maximize protection
In the human, the surface of the placental extravillous against diverse microorganisms. An apparent exam-
cytotrophoblastlacks HLA-A and -B, although there is ple is the malaria-driven selection at the HLA-B locus
now some evidence that it may express HLA-C. What whereby resistance to severe malaria resulting from
is well established is that the extravillous cytotro- strains of Plasmodium falciparum in East Africa is
phoblast and other placental tissues bear HLA-G, a associated with HLA-DRB1*0101, whereas HLA-
molecule which generally lacks allodeterminants and DRB1*1302 confers resistance to West African strains
which does not appear on most other body cells, except of the parasite.
for medullary and subcapsular epithelium in the thy-
mus, and on blood monocytes following activation
with y-interferon. The role of HLA-G in the placenta
is unclear, but it may function as a replacement for
allodeterminant-bearing classical class I molecules These molecules include the CD1 family which utilize
and/or may play a role in shifting potentially harmful P,-microglobulin and have a similar overall structure
Thl responses towards a Th2-type response. Class I1 to the classical class I molecules (figure4.18).They are,
molecules, on the other hand, are highly restricted however, encoded by a set of genes on a different chro-
in their expression, being present only on B-cells, mosome to the MHC, namely on chromosome 1in hu-
dendritic cells, macrophages and thymic epithelium. mans and chromosome 3 in the mouse. Like its true
MHC counterparts, CD1 is involved in the presenta- -B, -C and -G molecules, and is recognized by the
tion of antigens to T-cells, but the antigen-binding CD94/NKG2 receptors on NK cells and cytotoxic T-
groove is to some extent covered over, contains m a d y cells, as well as by the ap TCR on some cytotoxic T-
hydrophobic amino acids, and is accessible only cells. HLA-E is upregulated when other HLA alleles
through a narrow entrance.Instead of binding peptide provide the appropriate leader peptides, thereby per-
antigens, the CD1 molecules generally present lipids haps allowing NK cells to monitor the expression of
or glycolipids. At least four different CD1 molecules polymorphic class I molecules using a single receptor.
are found expressed on human cells; CDla, b and c are The murine homolog, Qa-l, has a similar function.
present on cortical thymocytes, dendritic cells and a The stress-inducible MICA and MICB ( M C class I
subset of B-cells, whilst CDld is expressed on intesti- chain-related molecules)have the same domain struc-
nal epithelium, hepatocytes and all lymphoid and ture as classical class I and display a relatively high
myeloid cells. Mice appear to only express two differ- level of polymorphism. They are present on epithelia1
ent CD1 molecules which are both similar to the cells, mainly in the gastrointestinal tract and in the
human CDld in structure and tissue distribution and thymic cortex, and are recognized by the NKG2D acti-
are referred to as CDldl and CDld2 (or CD1.1 and vating molecule. One possible role for this interaction
CD1.2). is in the promotion of NK and T-cell antitumor
Genes in the MHC itself which encode nonclassical responses.
MHC molecules include the H-2T, -Q and -M loci in The function of HLA-F is unclear. In contrast, al-
mice, each of which encodes a number of differentmol- though HLA-G shows extremely limited polymor-
ecules. The T22 and T10 molecules, for example, are phism, it is known to bind a range of self peptides
induced by cellular activation and are recognized di- with a defined binding motif and there is evidence for
rectly by y6 TCR without a requirement for antigen, HLA-G restricted T-cells.
possibly suggesting that they are involved in trigger- HFE, previously referred to as HLA-H, possesses an
ing immunoregulatory y6 T-cells. Other nonclassical extremely narrow groove which is unable to bind pep-
class I molecules do bind peptides, such as H-2M3 tides, and it may serve no role in immune defense.
which presents N-formylated peptides produced However, it binds to the transferrin receptor and ap-
either in mitochondria or by bacteria. pears to be involved in iron uptake. A point mutation
In the human, HLA-E binds a nine-amino acid (C282Y) in HFE is found in 70-90% of patients with
peptide derived from the signal sequence of HLA-A, hereditary hemochromatosis.
The B-cell surface receptor for antigen The encoding of the TCR is similar to that of im-
The B-cell inserts its Ig gene product containing a trans- munoglobulins. The variable region coding sequence in
membrane segment into its surface where it acts as a the differentiating T-cell is formed by random transloca-
specificreceptor for antigen. tion from clusters of V, D (for p and 6 chains) and J seg-
The surface Ig is complexed with the membrane pro- ments to give a single recombinant V(D)J sequence for
teins Ig-a and Is-p which become phosphorylated on cell each chain.
activation and transduce signals received through the Ig Like the Ig chains, each variable region has three hyper-
antigen receptor. variable sequences which function in antigen recognition.
The CD3complex,composed of y, 6, E and either 5, <q or
The T-cell surface receptor for antigen q2covalently linked dimers, forms an intimate part of the
The receptor for antigen is a transmembrane dimer, receptor and has a signal transducing role following
each chain consisting of two Ig-likedomains. ligand binding by the TCR.
The outer domains are variable in structure, the inner
ones constant, rather like a membrane-bound Fab. The generation of antibody diversity for antigen recognition
Both chains are required for antigen recognition. Ig heavy and light chainsand TCR a and p chains gener-
Most T-cells express a receptor (TCR) with cc and p ally are represented in the germ-line by between 30 and
chains (TCRZ).Aseparate lineage (TCR1)bearing$ recep- 75 variable region genes, between 2 and 25 D segment
tors is transcribed strongly in early thymic ontogenybut is minigenes (Ig heavy and TCR p and 6 only) and 4-60 short
associated mainly with epithelia1tissues in the adult. Jsegments.
(continued)
TCRyand6chainsareencodedbyfarfewergenes. surface with P,-microglobulin. Class I1 molecules are
Random recombination of any single V, D and J from transmembrane heterodimers. Class 111products are het-
each gene cluster generates approximately 7.5 x 103 Ig erogeneous but include complement components linked
heavy chain VDJsequences,ZOOlight chains,4.5x103TCR to the formation of C3 convertases, heat-shock proteins
a,lxl@TCRP,butonly60TCRyand72TCR6. and tumor necrosis factors.
Random interchain combination produces roughly The genes display remarkable polymorphism. A given
1.5x1061g,4.5x106TCRaP a n d 4 . 3 103 ~ TCR y6receptors. MHC gene cluster is referred to as a 'haplotype' and is usu-
Further diversity is introduced at the junctionsbetween ally inherited en bloc as a single Mendelian trait, although
V, D and J segments by variable combination as they are its constituent genes have been revealed by cross-over
spliced together by recombinase enzymes and by the N- recombination events.
region insertion of random nontemplated nucleotide Classical class I molecules are present on virtually all
sequences. These mechanisms may be particularly impor- cells in the body and signal cytotoxicT-cells.
tant in augmenting the number of specificitieswhich can Class II molecules are particularly associated with
be squeezed out of the relatively smally6pool. B-cells, dendritic cells and macrophages but can be in-
Useless or self-reactive receptors can be replaced by duced on capillary endothelial cells and epithelia1 cells
receptor editing. by y-interferon. They signal T-helpers for B-cells and
In addition, after a primary response, B-cells but not T- macrophages.
cells undergo high rate somatic.mutationaffecting the V The two domains distal to the cell membrane form a
regions. peptide binding cavity bounded by two parallel a-helices
sitting on a floor of P-sheet strands; the walls and floor of
NK recepton the cavity and the upper surface of the helices are the sites
NK cells bear a number of receptors with Ig-type do- of maximum polymorphic amino acid substitutions.
mains and other receptors with C-type lectin domains. Silentclass I genes may increase polymorphism by gene
Members of both types of receptor family can function as conversion mechanisms.
inhibitory01activatoryreceptorstodetermineif the target NonclassicalMHC molecules and MHC-like molecules
cell is killed. have a number of functions, and include CD1 which pre-
sents lipid and glycolipid antigens to T-cells, and HLA-E
MHC which presents signal sequence peptides from classical
Each vertebrate species has an MHC identified ori- class I molecules to the CD94/NKG2 receptor of NK cells.
ginally through its ability to evoke very powerful trans-
plantation rejection. See the accompanyingwebsite (www.roin.com)
Each contains three classes of genes. Class I encodes for multiplechoice queslions.
44 kDa transmembrane polypeptides associated at the cell
INTRODUCTION
A man cannot be a husband without a wife and a
molecule cannot be an antigen without a corre-
sponding antiserum or antibody or T-cellreceptor.
The term antigen is used in two senses, the first to
describe a molecule which generates an immune
response (alsocalled an immunogen) and the sec-
ond, a molecule which reacts with antibodies or
primed T-cellsirrespectiveof its ability to generate
them. If this last situationsounds a trifle confusing,
an example may help. A mouse injected with its
own red cells, not too surprisingly,will not make
any antibodies; if it is now given rat erythrocytes,
r
Figure 5.1. A hapten on its own will not induce antibodies.
However,itwillreactin vitro withantibodiesformedtoaconju-
gate withanimmunogeniccarrier.
antibodies are formed to both rat and mouse red
cells and the latter bind to the animal's own cells in
vivo, i.e. the mouse red cell acts as antigen in bind-
ing antibodies even though unable to evoke their
formation. Similarly, haptens, which are small
well-defined chemical groupings such as dinitro-
phenyl (DNP; cf. p. 37) or m-aminobenzene sul-
fonate, are not immunogenic on their own but will
react with preformed antibodies induced by injec-
tion of the hapten linked to a 'carrier' molecule
whichis itself an immunogen (figure5.1).
The parts of the hypervariable regions on the
antibody which contact the antigen are termed the
paratope and the part of the antigen which is in
contact with the paratope is designated the epi-
Figure 5.2. Specificity and three-dimensional configuration in a
tope. To get some idea of size, if the antigen is a lin- globular protein, lysozyme. Antibodies to the whole molecule and
ear peptide or carbohydrate, the combining site to the isolated loop peptide do not react with the peptide after reduc-
can usually accommodate up to five or six amino tion of its disulfide bond, showing that the linear reduced peptide
acid residues or hexose units. With a globular has lost the antigenic configuration it had when held as a loop even
though the amino acid sequence is unchanged. (From Maron E.,
protein, typically 14-21 amino acid residues are Shiowa C., Arnon R. & Sela M. (1971)Biochemistry 10,763.)
in contact on both the antibody and the antigen
(cf. figure5.5).
combining site (figures 3.1 and 3.2). This means that where [Ab] is the concentration of free antibody com-
small haptens by themselves are monovalent with re- bining sitesand [Ag]is the concentrationof free antigen
spect to reaction with antibody. The experiment on at equilibrium. If the antibody and antigen fit together
mixing hapten with antibody in a dialysis bag (figure very closely, the equilibrium will lie well over to the
5.8) showed that the combination with antibody was right of the association reaction; we refer to such anti-
reversible and that the complex so formed could read- bodies which bind strongly to the antigen as high affin-
ily dissociate depending upon the strength of binding, ity antibodies. At a certain free antigen concentration
which we call affinity. In the simplisticsituationof one [Ag,] where half of the antibodysites are bound:
Fab arm binding in isolation to one epitope on the anti-
[AbAg]= [Abl and K, = l/[Ag,]
gen, the binding strength can be defined through the
equilibrium constant (K,)of the association reaction: i.e. the affinity constant K, is equal to the reciprocal of
the concentration of free antigen at the equilibrium
Ab + Ag =:AbAg
point where half the antibody sites are in the bound
form. In other words, when an antibody has a high
given by the mass action equation affinity constant and binds antigen strongly, it only
needs a low antigen concentration to half-saturate the
antibody. An individual epitope on the surface of a
complex antigen is by defmition monovalent, and the
where X is the universal gas constant, T is the absolute
temperature and ln is the natural logarithm.
One can study the interaction of hapten and anti-
body by the dialysis method described in figure 5.8
and use the data to calculate the affinity constant from
the mass action equation (figure 5.10). K, values may
sometimesbe as high as 1012M-'.
Analysis of the binding at different hapten concen-
trations generally shows a heterogeneity (figure 5.10)
which indicates that most antiserums, even those
raised against haptens with a simple structure, contain
a variety of different antibodieswith a range of binding
affinities which depend upon the area of contact
between the antibody and the hapten or epitope, the
closeness of fit, conformational changes necessitated
by electron-cloud overlap and the distribution of
charged and hydrophobic groups.
Figure 5.8. Reversibility of the interaction between antibody
( ) and hapten ( 1. Within the dialysis sac the hapten is partly in
the free form and partly bound to antibody according to the affinity
of the antibody.Only hapten can diffuse through the dialysis mem-
brane, and the external concentrationthen will equal the concentra- While the term affinity describes the binding of
tion of unbound hapten within the sac. Measurementof total hapten antibody to a monovalent hapten or single antigen
in the dialysis sac then enables the amount bound to antibody to be
calculated. Constant renewal of the external buffer will lead to total determinant, in most practical circumstances we are
dissociationand loss of hapten from inside the dialysis sac showing concerned with the interaction of an antiserum (i.e.the
the reversiblenature of the antigen-antibody bond. serum from an immunized individual) with a multiva-
lent antigen. The term employed to express this bind-
ing is avidity or functional affinity.
The factors which contribute to avidity are compli-
strength of its combination with one antigen-binding cated, including as they do the heterogeneity of anti-
arm (Fab) of an antibody is therefore defined by an bodies in a given serum which are directed against
affinity constant. each determinant on the antigen, and the heterogene-
Affinity can equally well be formulated in terms of ity of the determinants themselves (figures 5.4 and
the dissociation constant (Kd)of the reaction: 5.10). But yet a further factor must be considered. The
multivalency of most antigens leads to an interesting
AbAg =+
Ab + Ag bonus effect in which the binding of antigen to anti-
Expressing concentrations in moles per liter: body by multiple links is always greater, usually
many-fold greater, than the arithmeticsum of the indi-
[Ab moles/l][Ag moles/l] vidual antibodybonds. This is illustrated in figure 5.11.
Kd =
[ A b ~moles/l]
g The mechanism of this effect may be interpreted by
considering an analogy. Let us fabricate an unheard of
Clearly, Kd is the reciprocal of K,, i.e. l/&, and
disease in which we cannot stop our hands opening
has the units moles/l or M . Conversely, K, is ex-
and closing continuously. If we now try to hold an ob-
pressed in the units l/mole or M-' and has the ad-
ject in one hand, it will fall the moment we open that
vantage that the stronger the binding, the higher the
hand. However, if we use both hands to hold the object,
number.
provided that we open and close our hands at different
The value of K, is determined by the difference
times, there is much less chance of the object falling.
in free energy (AG) between the antigen and anti-
The reversible combination of antigen and antibody is
body in the free state on the one hand and in the
like the opening and closing of the hands; the more va-
complexed form on the other, according to the
lencies holding the antigen, the less likely it is to be lost
equation:
when the complex dissociates at any one binding site
(figure5.12).
Figure 5.9. Protein-protein interactions. (a) Coulombic attraction aqueous environment. The driving force for this hydrophobic inter-
between oppositely charged ionic groups on the two protein side- action derives from the fact that water in contact with hydrophobic
chains as illustratedby anionized amino group (NH,+)on a lysine of molecules ( ),with whichit cannot H bond, will associate with
one protein and an ionized carboxyl group (-COO-) of glutamate other water molecules, but the number of configurations which
on the other. The force of attraction is inversely proportional to the allow H bonds to form will not be as great as that occurring when
square of the distance between the charges. Thus, as the charges they are surrounded completely by other water molecules, i.e. the
come closer together, the attractive force increases considerably: if entropy is lower. The greater the area of contact between water and
we halve the distance apart, we quadruple the attraction. Further- hydrophobic surfaces, the lower the entropy and the higher the en-
more, since the dielectricconstant of water is extremelyhigh, the ex- ergy state. Thus, if hydrophobic groups on two proteins come to-
clusion of water molecules through the contiguity of the interacting gether so as to exclude water molecules between them ( the
residues would greatly increase the force of attraction. Dipoles on net surface in contact with water is reduced and the proteins
antigenand antibodycan also attract each other.In addition,electro- a lower energy state than when they are separated (in other words,
static forces may be generated by charge transfer reactions between there is a force of attraction between them). (d)Van der Waals force:
antibody and antigen; for example, an electron-donating protein the interaction between the electrons in the external orbitals of two
residue such as tryptophan could part with an electron to a group different macromolecules may be envisaged (for simplicity!) as the
such as dinitrophenyl (DNP) which is electron accepting, thereby attraction between induced oscillating dipoles in the two electron
creatinganeffective+lcharge on the antibodyand -1 on the antigen. clouds. The nature of this interaction is difficult to describe in non-
(b)Hydrogen bonding between two proteins involving the forma- mathematical terms, but it has been likened to a temporary pertur-
tion of reversible hydrogen bridges between hydrophilic groups, bation of electrons in one molecule effectively forming a dipole
such as .OH, -NH2and COOH, depends very much upon the close which induces a dipolar perturbation in the other molecule, the two
approach of the two molecules carrying these groups. Although H dipoles then having a force of attractionbetween them; as the dis-
bonds are relatively weak, because they are essentially electrostatic placed electrons swing back through the equilibrium position and
innature, exclusion of water between the reactingside-chainswould beyond, the dipoles oscillate. The force of attraction is inversely
greatly enhance the binding energy through the gross reduction in proportional to the seventh power of the distance and, as a result,
dielectric constant. (c) Nonpolar, hydrophobic groups such as the this rises very rapidly as the interacting molecules come closer
side-chains of valine, leucine and isoleucine tend to associate in an together.
Figure 5.10. Heterogeneity of IgG antihapten (dinitrophenyl, many different bands after separation of the individual antibodies
DNP) antibodies from the serum of an immunized animal contrast- by isoelectricfocusing of the serum. Extrapolation to r/ [H]=0 (at in-
ing with the homogeneity of a monoclonal IgG anti-DNP. (a) A finitely high concentration of antigen) gives the number of binding
Scatchard plot of hapten binding to antibodies purified from the sites on each IgG molecule as two (cf.figure 3.2).For IgM antibodies,
serum. If r represents the average number of DNP molecules bound the value would be 10.Because the slope of the Scatchardplot varies
to each antibodymolecule, of affinity constant k and number of bind- with r/2 (the fractional occupancy of the antibody combining sites
ing sites n, in the presence of a free hapten concentration [H], then for a bivalent Ab), the affinity (which=-slope) will vary depending
from the mass action equation of equilibriumrelationships (p. 86) it upon the range of values of r utilized in the experiments: thus affini-
can be shown that: ty must be defined in terms of standard conditionsof antibody dilu-
tion and concentration of hapten. (b) Histogram showing a typical
distribution of antibody affinities in the anti-DNP serum. Measur-
Thus, a Scatchardplot of r/ [H]against r for a single antibody species able affinitiestend to range between 104and 101or 1011M-' and have
will be a straight line of slope -k as seen for the monoclonal antibody; skewed and not necessarily unimodal distributions.The monoclon-
the deviation from a straight line given by anti-DNP from the anti- a1 antibody of course gives a single affinity value since it is a homo-
serum clearly indicates the existence of antibodies with different geneous protein.
affinities, as may be confirmed by the binding of labeled DNP to
The same considerations apply to the binding of reactivity of an antihapten for a series of structurally
antibody to a polymeric antigen with repeating deter- related molecules as described in table 5.1. Since the
minants, such as ovalbumin substilted by several strength of the reaction can be quantified by the affini-
DNP groups, or most bacterial polysaccharides or red ty or avidity, we would relate the specificity of an anti-
cells with repeating blood-group determinants.As one serum to its relative avidity for the antigens which are
moves from a univalent Fab fragment to a divalent IgG being discriminated.
to a pentameric IgM, the bonus effect of multivalency In so far as we recognize that an antiserum may have
produces striking increases in the strength of anti- a relatively greater avidity for one antigen rather
gen-antibody complex formation. than another, by the same token we are saying that the
Avidity, being a measure of the functional affinity of antiserum is displaying relative rather than absolute
an antiserum for the whole antigen, is of obvious rele- specificity; in practice we speak of degrees of cross-
vance to the reaction with antigen in the body. High reactivity.An antiserum raised against a given antigen
avidity is superior to low for a wide variety of func- can cross-react with a partially related antigen which
tions in vivo, e.g. immune elimination of antigen, virus bears one or more identical or similar determinants. In
neutralization, protective role against bacteria, and figure 5.13 it can be seen that an antiserum to antigen,
SO on. (Ag,) will react less stronglywith Ag,, which bears just
one identical determinant, because only certain of the
antibodiesin the serum can bind. Ag3,which possesses
a similar but not identical determinant, will not fit as
well with the antibody and the binding is even weaker.
Ag,, which has no structural similarity at all, will not
The ability of antibodies to discriminate between dif- react significantlywith the antibody.Thus,based upon
ferent antigens was well illustrated by the range of stereochemical considerations, we can see why the
90
promotion of assembly with P,-microglobulin. In the I molecule bound to these chaperones becomes linked
human, but not in the mouse, calnexin is then replaced to TAP1/2 by tapasin. Upon peptide loading, the class
by calreticulin. The ER-resident protein, Erp57, which I molecule can dissociate from the various accessory
has thiol reductase, cysteine protease and chaperone molecules, and the now stable peptide-class I heavy
functions, becomes associated with the complex of cal- chain-P,-microglobulin complex traverses the Golgi
reticulin-calnexinand class I heavy chain which now stack and reaches the surface where it is a sitting target
folds together with P,-microglobulin. The empty class for the cytotoxic T-cell.
PROCESSING OF ANTIGEN FOR CLASS II
MHC PRESENTATION FOLLOWS A
DIFFERENT PATHWAY
Class I1 MHC complexes with antigenic peptide are
generated by a fundamentally different intracellular
mechanism, since the antigen-presenting cells which
interact with T-helper cells need to sample the antigen
from both the exfracellular and infracellular com-
partments. In essence, a trans-Golgi vesicle containing
class I1has to intersectwith a late endosome containing
exogenous protein antigen taken into the cell by an
endocytic mechanism.
Regarding the class I1 molecules themselves, these
are assembled from a and P chains in the endoplasmic
reticulum in association with the transmembrane in-
variant chain (Ii) (figure 5.18) which has several func-
tions. Firstly, it acts as a dedicated chaperone to ensure
correct folding of the nascent class I1molecule. Second-
ly, an internal sequence of the lumina1 portion of Ii sits
in the MHC groove to inhibit the precocious binding of
peptides in the ER before the class I1 molecule reaches
the endocytic compartment containing antigen.
Additionally, combination of Ii with the ap class 11
heterodimer inactivates a retention signal and allows
transport to the Golgi. Finally, targeting motifs in the
N-terminal cytoplasmic region of Ii ensure delivery
of the class 11-containing vesicle to the endocytic
pathway.
Meanwhile, exogenousprotein is taken up by endo-
cytosis and, as the early endosome undergoes pro-
gressive acidification, is processed into peptides by
endosomal proteases such as asparaginyl endopepti-
dase. The late endosomes, which ultimately mature
into lysosomes, characteristically acquire lysosomal-
Figure 5.17. Processing and presentation of endogenous antigen associated membrane proteins (LAMPS),although the
by class I MHC. Cytosolic proteins are degraded by the proteasome function of these molecules is still unclear. These late
complex into peptides which are transported into the endoplasmic
endosomes fuse with the vacuole containing the class
reticulum (ER). TAP1 and TAP2 are members of the ABC family of
ATP-dependent transport proteins and, under the influence of these 11-Ii complex.Under the acidic conditionswithin these
transporters, the peptides are loaded into the groove of the mem- MHC class II-enriched compartments (MIICs), pro-
brane-bound class I MHC. The peptide-MHC complex is then re- teases degrade Ii except for the part sitting in the MHC
leased from all its associated transporters and chaperones, traverses
groove which, for the time being, remains there as a
the Golgi system, and appears on the cell surface ready for presenta-
tion to the T-cell receptor. Mutant cells deficient in TAP1/2 do not peptide referred to as CLIP (class 11-associated invari-
deliver peptides to class I and cannot functionas cytotoxic T-cell tar- ant chain peptide).An MHC-related dimeric molecule,
gets. However, if they are transfected with a gene encoding the anti- DM, then catalyses the removal of CLIP and keeps the
genic peptide linked to a cleavable signal sequence, the peptide is
groove open so that peptides generated in the endo-
delivered to the ER without the need for TAP1/2 and the cells once
again can become targets. some can be inserted (figure 5.19). This process may
be assisted by members of the hsp70 family which
promiscuouslybind unfolded peptides. Initial peptide
binding is determinedby the concentration of the pep-
tide and its on-rate,but DM may subsequently assist in
the removal of lower affinity peptides to allow their re-
placement by high affinity peptides, i.e. act as a pep-
tide editor permitting the incorporation of peptides the presentation of antigens internalized via the BCR
with the most stable binding characteristics, namely over those taken up by fluid phase endocytosis. The
those with a slow off-rate. Particularly in B-cells an tetraspanin family member CD82 is also present
additional MHC-related molecule, DO, associates in the MIIC, though its role is unclear at present.
with DM bound to class I1and modifies its function in a The class 11-peptide complexes are eventually trans-
pH-dependent fashion. Its effect may be to favor ported to the membrane for presentation to T-helper
cells.
Thus, in general, endogenous protein antigens are
processed for class I presentation whilst exogenous
protein antigens are processed for class I1presentation.
Processing of antigens for class I1 presentation is not,
however, confined to soluble proteins taken up from
the exterior, but can also encompass microorganisms
whose antigens reach the lysosomal structures, either
after direct phagocytosis or prolonged intracellular co-
habitation. Proteins and peptides within the ER are
also potential clients for the class I1 groove and could
also make the journey to the MIIC. Conversely, class
I-restricted responses can be generated against ex-
ogenous antigens, a process sometimes referred to
as cross-priming. This may occur either by a TAP-
dependent pathway in phagocytic cells where pro-
teins are transferred from the phagosome to the cy-
tosol, or by endocytosis of cell surface MHC class I
molecules which then arrive in the class 11-enriched
compartments where peptide exchange occurs with
sequences derived from the endocytic processing
pathway.
coding MHC molecules containing P2-microglobulin The best studied molecule encoded by the H-2M
with relatively nonpolymorphic heavy chains. These locus is H-2M3, which is unusual in its ability to pre-
are H-2M, Q and T in mice and HLA-E, F and G in sent bacterial N-formyl methionine peptides to T-cells.
Homo sapiens. Expression of H-2M3 is limited by the availability of
these peptides so that high levels are only seen during unlike class I-mediated presentation, is independent
prokaryotic infections. The demonstration of H-2M3- of TAP and DM.
restricted CD8-positive ap T-cells specific for Listeria
monocytogenes encourages the view that this class I-like
molecule could underwrite a physiological function in
infection. Discussion of the role of HLA-G expression NK T-cells possess the NKl .lf marker, characteristicof
in the human syncytiotrophoblast will arise in Chapter NK cells, together with a T-cell receptor. However, the
17 (p.369). TCR bears an invariant a chain (Va14-Ja281in mice,
Va24-JaQin humans) with no N-region modifications
and a limited P chain repertoire. These cells are a major
Thefamily of CD1 non-MHC class I-like molecules
component of the T-cell compartment, accounting for
can present exotic antigens
20-30% of T-cells in bone marrow and liver, and up
After MHC class I and class 11, the CD1 family (p. 77) to 1%of spleen cells. NK1.1+T-cells rapidly secrete in-
represents a third lineage of antigen-presenting terleukin-4 (IL-4)and IFNy following stimulation and
molecules recognized by T-lymphocytes. The CD1 therefore may have important regulatory functions.
polypeptide chain associateswith P2-microglobulinas Although substantial numbers of NK T-cells are CD1-
becomes an honest class I-like moiety, and the overall restricted, others are restricted by classical MHC
structure is similar to that of classical class I molecules, molecules.
although the topology of the binding groove is altered
(see figure 4.18).
CD1 molecules can act as restriction elements
for the presentation of lipid and glycolipid microbial Unlike ap T-cells, y6 T-cells recognize antigens directly
antigens to T-cells. A common structural motif without a requirement for antigen processing. Whilst
facilitates CD1-mediated antigen presentation and some T-cells bearing a y8 receptor are capable of
comprises a hydrophobic region of a branched or recognizing MHC molecules, neither the polymorphic
dual acyl chain and a hydrophilic portion formed residues associated with peptide binding nor the pep-
by the polar or charged groups of the lipid and/ tide itself are involved. Thus, a y6 T-cell clone specific
or its associated carbohydrate. The hydrophobic for the herpes simplex virus glycoprotein-l can be
regions are buried in the binding groove of CD1, stimulated by the native protein bound to plastic, sug-
whilst the hydrophilic regions, such as the carbo- gesting that the cells are triggered by cross-linking of
hydrate structures, are recognized by the TCR. their receptors by antigen which they recognize in the
Because antigen recognition by CD1-restricted T- intact native statejust as antibodiesdo. There are struc-
cells involves clonally diverse ap and y6 TCRs, tural arguments to give weight to this view. The CDR3
it is likely that CD1 can present a broad range of loops, which are critical for foreign antigen recognition
such antigens. One group of major ligands for by T-cells and antibodies, are comparable in length and
CDlb are glycophosphatidylinositols, such as the relatively constrained with respect to size in the a and
mycobacterial cell wall component lipoarabino- p chains of the ap TCR, presumablyreflecting a relative
mannan. constancy in the size of the MHC-peptide complexes
Just like their proteinaceous colleagues, exogenous- to which they bind. CDR3 regions in the immunoglob-
ly derived lipid and glycolipid antigens are delivered ulin light chains are short and similarly constrained in
to the acidic endosomal compartment. Localization of length, but in the heavy chains they are longer on aver-
CD1 molecules to the endocytic pathway is mediated age and more variable in length, related perhaps to
by a targeting sequence in the cytoplasmic tail. There is their need to recognize a wide range of epitopes. Quite
evidence that human CDla, which lacks the targeting strikingly,the y6 TCRs resemble antibodiesin that the y
motif, does not localize to this pathway and therefore chain CDR3 loops are short with a narrow length dis-
presumably must follow a different route for peptide tribution, while in the 6 chain they are long with a
loading. The acidic environment of the endosome broad length distribution. Therefore, in this respect,
induces a conformational change in CD1, thereby the y6 TCR resembles antibodymore than the ap TCR.
increasing accessibility to the lipid-binding site in The X-ray crystallographic structure of a TCR V6 do-
the hydrophobic groove of the molecule. Antigens de- main highlighted that the y6 TCR indeed incorporates
rived from endogenous pathogens can also be pre- key structural elements of both immunoglobulin and
sented by the CD1 pathway but in a process that, TCR V regions. Overall, the framework regions are
more like antibody VHthan TCR Va, whilst the confor- lation and enable them to function in the recognition
mations and relative positions of the CDRs share of microbial pathogens and of damaged or stressed
features with both Va and VH domains. The binding host cells.
site of the V6 in this particular crystal structure is
a relatively flat surface similar to that seen in ap
SUPERANTIGENS STIMULATE WHOLE
TCRs. However, the CDR3 loop of this V6 is 10 amino
FAMILIES OF LYMPHOCYTE RECEPTORS
acid residues long and, whilst this is approximately
the median length of V6 CDR3s, the broad length
distribution already alluded to means that many y6
receptors will bear longer or shorter V6 CDR3s.
These will have topographically more adventurous Whereas an individual peptide complexed to MHC
binding sites, thereby facilitating the ability of y6 will react with antigen-specificT-cells which represent
T-cells to interact with intact rather than processed a relatively small percentage of the T-cell pool because
antigen. of the requirement for specific binding to particular
More secrets are being teased out of the y6 T-cell CDR3 regions, a special class of molecule has been
sect. In the mouse, y6 T-cells have been isolated identified which stimulates the 5-20% of the total
which directly recognize the MHC class I molecule I-EL T-cell population expressing the same TCR VP family
and the nonclassical MHC molecules T10 and T22 in structure. These molecules do this irrespective of the
a peptide-independent fashion. T10 and T22 are antigen specificity of the receptor. They have been
expressed by ap T-cells following their activation, described as superantigens by Kappler and
and it has been suggested that y6 T-cells specific for Marrack.
these nonclassical MHC molecules may exert a regu- The pyogenic toxin superantigen family can cause
latory function. Stressed or damaged cells appear food poisoning, vomiting and diarrhea and includes
to be powerful activators of y6 cells, and there is Staphylococcus aureus enterotoxins (SEA, SEB and
evidence for molecules such as heat-shock proteins several others), staphylococcal toxic shock syndrome
as stimulators of y6 T-cells. Low molecular weight toxin-l (TSST-l), streptococcal superantigen (SSA)
phosphate-containing nonproteinaceous antigens, and several streptococcal pyogenic exotoxins (SPEs).
such as isopentenyl pyrophosphate and ethyl Although these molecules all have a similar structure,
phosphate, which occur in a range of microbial they stimulate T-cells bearing different VP sequences.
and mammalian cells, have been identified as potent They are strongly mitogenic for these T-cells in the
stimulators. presence of MHC class I1 accessory cells. SEA must be
A particular subset of y6 cells, which possess a di- one of the most potent T-cell mitogens known, causing
verse range of TCRs utilizing different D and gene marked proliferation in the concentration range 10-13
segments but always using the same Vgene segments, to 10-l6M. Like the other superantigens it can cause the
Vy2 and V32, expand in vivo to comprise a large pro- release of copious amounts of cytokines, including
portion (8-60%) of all peripheral blood T-cells during a IL-2 and lymphotoxin, and of mast cell leukotrienes,
diverse range of infections. These VPV62 T-cells rec- which probably form the basis for its ability to produce
ognize a newly appreciated group of antigens, the toxic shock syndrome. Other superantigens which do
alkylamines, which have chemical and biological not belong to the pyogenic toxin superantigen family
properties distinct from lipid and phosphate antigens, include staphylococcal exfoliative toxins (ETs),
further extending the variety of nonprotein antigens Mycoplasma arthritidis mitogen (MAM) and Yersinia
which can be recognized by T-cells. Anumber of alkyl- pseudotuberculosis mitogen.
amine antigens are produced by human pathogens, Superantigens are not processed by the antigen-
including Salmonella typhimurium, Listeria monocyto- presenting cell, but cross-link the class I1 and VP inde-
genes, Yersinia enterocolitica and Escherichia coli. Indi- pendently of direct interaction between MHC and
vidual Vy2V62 T-cells can recognize both positively TCR molecules (figure5.25).
charged alkylamines and negatively charged mole-
cules such as ethyl phosphate, but this should be fairly
straightforward for the receptor given the small
hapten-like size of these antigens.
The above characteristics provide the y6 cells with a Very many years ago, Festenstein made the curious ob-
distinctive role complementary to that of the ap popu- servation that B-cells from certain mouse strains could
produce powerful proliferative responses in roughly IgAand IgG F(ab'),, all of which belong to the V,3 fam-
20% of unprimed T-cells from another strain of identi- ily. This superantigen is mitogenic for B-cells through
cal MHC. The so-called Mls gene product responsible its recognition by a discontinuous binding sequence
for inciting proliferation turns out to be encoded by the composed of amino acid residues from FR1, CDR2 and
open reading frame (ORF) located in the 3' long ter- FR3 of the VHdomain. The human immunodeficiency
minal repeat of MMTV. They are type B retroviruses virus (HIV) glycoprotein gp120 also reacts with
transmitted as infectious agents in milk and are spe- immunoglobulinswhich utilize VH3family members.
cific for B-cells. They associatewith class I1MHC in the The binding site appears to partially overlap with that
B-cell membrane and act as superantigens through for protein A and utilizes amino acid residues from
their affinity for certain TCR VP families in a similar FR1, CDR1, CDR2 and FR3.
fashion to the bacterial toxins. Other proposed viral su-
perantigens capable of polyclonally activating T-cells
THE RECOGNITION OF DIFFERENT FORMS OF
include the nucleocapsid protein of rabies virus, and
ANTIGEN BY B- AND T-CELLS I S
antigens associated with cytomegalovirus and with
ADVANTAGEOUS TO THE HOST
Epstein-Barr virus.
It is our convictionthat this section deals with a subject
of the utmost importance, which is at the epicenter of
immunology.
Staphylococcal protein A reacts not only with the Fcy Antibodies combat microbes and their products in
region of IgG but also with 15-50% of polyclonal IgM, the extracellular body fluids where they exist essen-
SUMMARY
The nature of antigen recognition by ontibody The reaction of multivalent antigenswith the heteroge-
An antigen is defined by its antibody. The contact area neous mixture of antibodies in an antisemm is defined by
with an antibody is called an epitope and the correspond- avidity (functional affinity) and is usually much greater
ingarea on anantibody a paratope. than affinity due to the 'bonus effect of multivalency'.
Antiserarecognizeaseriesofdominantepitopechtem The spe&city of antibodies is not absolute &d they
on the surfaceof an antigen; each cluster called a may cross-react with other antigens to different extents,
determinant. measured by their relative avidities.
Most epitopes on globular proteins are discontinuous
rather than linear, involving amino adds far apart in the 1-cell recognition
primary sequence. up T-cells see antigen in association with MHC
The protruding regions and probably the 'flexible' molecules.
segments of globular proteins tend to be associated with They are restricted to the haplotype of the cell which
higher epitope densities. first primed theT-cell.
Protein antigens are processed by antigen-presenting
Antigens and antibodies Interact by spatial wmplementarily, cells to formsmalllinearpeptides which associatewith the
not by wvaient binding MHC molecules, binding to the central groove formed by
The forcesof interactionincludee l e c t r o s ~ the cc-helicesand the &sheet floor.
b o n d i i hydrophobic and van der Waals.
Theforcesbecomelargeastheseparationofantigenand Processing of antigen for presentation by class I MHC
antibody diminishes,especiallywhenwater moleculesare Endogenous cytosolic antigens such as viral proteins
excluded. are cleaved by immunopmteasomes and the peptides so
Antigen-antibody bonds are readily reversible. formed are transported to the ER by theTAPl/2system.
Antigens and antibodies aremutually deformable. The peptide then dissociatesfrom TAP1/2 and forms a
stable heterotrimer with newly synthesized class I MHC
Affinity heavy chain and µglobulii.
The strength of biding to a single antibody combining This peptide-MHC complex is then transported to the
site is measured by the affinity. surfacefor presentation to cytotoxicT-cells.
(continuedp. 106)
Processing of amlgen for presentation by class IIMHC tively nonpolymorphic and can present antigens such as
The ap class U molecule is synthesized in the ER and bacterialN-formylmethioninepeptides.
complexeswith membrane-bound invariant chain W. The CD1 family of non-MHC class I-like molecules can
Thisfadlitates transportof thevesiclescontainingclass present antigens such as lipid and glycolipid mycobac-
II across the Golgi and directs them to an acidified late en- terial antigens.
dosome or lysosome containing exogenous protein taken y6 T-cells resemble antibodies in recognizing whole
into thecell by endocytosisor phagocytosis. unprocessed molecules such as low molecular weight
Proteolytic degradation of Ii in the endosome leaves phosphatecontaining nonproteinaceousmolecules.
a peptide referred to as CLIP which protects the MHC
groove. Supemntlgens
Processing by endosomal proteases degrades the anti- These are potent mitogens which stimulate whole lym-
gen to peptides which replace the CLP. phocyte subpopulations sharing the same TCR VP or
The class 11-peptide complex now appears on the cell immunoglobulin VH family independently of antigen
surfacefor presentation to T-helper cells. specificity.
Staphylococcus aureus enterotoxins are powerful human
The nolureof lhe peptide superantigens which cause food poisoning and toxic
Class I peptides are held in extended conformation shocksyndrome.
within the MHC groove. T-cell superantigens are not processed but cross-link
They are usually 8-9 residues in length and have h MHC class II and TCR VP independently of their diiect
threekey anchor, relatively invariant residues which bind interaction.
to allel&specificpockets the MHC. Mouse mammary tumor viruses are B-cell retrovirust
Class I1 peptides are between 8 and 30 residues long, whichare superantigensin the mouse.
extend beyond the groove and usually have threeor four
anchor residues. Recognition of dlfteremformsof antlgen by B-ond
The other amino acid residues in the peptide are 1-cells is on advantage
greatly variable and are recognized by the T-cell receptor B-cells recognize epitopes on the native antigen; this
WR). important becauseantibodiesreact with native antigen in
the extracellularfluid.
Complex between TCR MHC and peptlde T-cells must contact infected cells and, to avoid confu-
The first and second hypervariable regions (CDR1 and sion between the two systems, the infected cell signals
CDR2) of each TCR chain mostly contact the MHC a- itself totheT-cellby thecombination of MHC and degrad-
helices, while the CDR3s, having the greatest variability, ed antigen.
interact with the antigenic peptide. Complexes of TCR,
with (MHC-pe~tide)~ are probably formed.
Seethe
See theaccompanying
occomponyingwebsite (www.roitI.com)
website(www.roitt.com)
Some 1-cells are Independentof claulcal MHC molecules formultiple
for multiplechoice
choicequestions.
questions.
MHC class I-like molecules, such as H-2M, are rela-
(continued)
Figure 6.1. Distribution of affinity and abundance of IgG Starting with the lowest affinity molecules in the s e m , we
molecules in an individual serum. (a)Distributionof affinities have charted the cumulative total of antibody bound for each
of IgG moleculesforagivenantigeninthesemof a hypotheti- antibody species up to and including the one being plotted. As
cal individual. There is a great deal of low affhty antibody might be expected, the very low affinity antibodies make no
which would be incapable of binding to antigeneffectively, and contributiontothetests. Serum2hasmorelow affinity antibody
much lower amounts of high affinity antibody whose skewed and virtually no high affinity, but it can produce just enough
distribution is assumed to arise kom exposure to infection. @) complex to react in the sensitive agglutination test although,
Relationshiv of affinitv distribution to vositivitv in tests for unlikesenunl, it forms insufficient togive apositiveprecipitin.
antigen binding. Rearranging the mass action equation, for all Because of its relatively high'content' of antibody, serum 1 can
moleculesof thesameaffinity . K,..andconcentrationof unbound be diluted to a much greater extent than serum 2 and yet still
antibody [Ab,]:
fixed [Agl.
-
the amount of complex formed [AgAbl Kx[Abxl for
give positive agglutination, i.e. it has a higher titer. Theprecip-
itin testis less sensitive.reauuinemorecom~lexformation.
. a "
serum 1cannot be diluted much before this test becomes nega-
and
tive, i.e. the precipitin titer will be far lower than the agglutina-
tion titer forthesame serum.
Figure 6.6. Surface plasmon resonance. (a) The principle: as anti- the association and dissociation rates. (Data kindly provided by Dr
gen binds to the antibody-coatedsensor chip it alters the angle of re- R. Karlsson, Biacore AB, and reproduced from Panayotou G. (1998)
flection. (b) This signals the rates of association during the antigen Surface plasmon resonance. In Delves P.J.& Roitt I.M. (eds)Encyclo-
pulse and dissociation. In this example, the same antigen was pedia of immunology, 2nd edn.Academic Press, with permission.)The
injected over three immobilized monoclonal antibodies.The arrows system canbe used with antigen immobilized on the sensor chip and
point to the beginning and end of the antigen injection, which is fol- antibody in the fluid phase, or can be applied to any other single
lowed by buffer flow. Note the differencesbetween the antibodiesin ligand-bindingassay.
Figure 6.7. Mechanism of agglutination of antigen-coated parti-
cles by antibody cross-linking to form large macroscopicaggregates.
If red cells are used, several cross-links are needed to overcome the
electrical charge at the cell surface. IgM is superior to IgG as an
agglutinator because of its multivalent binding and because the
charged cells are further apart.
paper disk, which is then treated with patient's serum. rently the most commonly used labels, with horserad-
The amount of specificIgE bound to the paper cannow ish peroxidase (HRP)and calf intestine alkaline phos-
be estimated by the addition of labeled anti-IgE. phatase (AP)being by far the most popular.Aspergillus
niger glucose oxidase, soy bean urease and Escherichia
A wide variety of labels are available coli P-galactosidase provide further alternatives. One
clever ploy for amplifying the phosphatase reaction is
whilst ~rovidingextremely good sensitivity, radio- to use nicotinamide adenine dinucleotide phosphate
labels have a number of disadvantages, including loss (NADP) as a substrate to generate NAD which now
of sensitivity during storage due to radioactive decay acts as a coenzyme for a second enzyme system (figure
the deterioration of the labeled reagent through radia- 6.11).
tion damage, and the precautions needed to minimize
human exposure to radioactivity. Therefore, other Other labels. Enzyme-labeled streptococcal protein G
types of label are often employed in immunoassays. or staphylococcal protein A will bind to IgG. Con-
jugation with the vitamin biotin is frequently used
ELISA (enzyme-linked immunosorbent assay). Enzymes since this can readily be detected by its reaction with
which give a colored soluble reaction product are cur- enzyme-linked avidin or streptavidin (the latter gives
2 binding of C3b-containing complexes to beads
coated with bovine conglutinin (cf.p. 17) and estima-
tion of the bound Ig with enzyme-labeled anti-Ig.
Other techniques include: (i)estimation of the bind-
ing of lZ51-Clqto complexes by coprecipitation with
polyethylene glycol, (ii) inhibition by complexes of
rheumatoid factor-induced aggregation of IgG-coated
particles, and (iii) detection with radiolabeled anti-Ig
of serum complexes capable of binding to the C3b (and
to a lesser extent the Fc) receptors on the Raji cell
line. Sera from patients with immune complex disease
often form a cryoprecipitate when allowed to stand at
4C. Measurement of serum C3 and its conversion
product C3c is sometimes useful.
Tissue-bound complexes are usually visualized by
the immunofluorescent staining of biopsies with
conjugated anti-immunoglobulins and anti-C3 (cf.
figure 16.17).
Figure 6.12. The principle of time-resolved fluorescence assay.
The problem with conventional methods of detection of low fluores- IDENTIFICATION AND MEASUREMENT
cent signalsis interferencefrom reflection of incident light and back- OF ANTIGEN
ground instrument fluorescence. By using just a short excitation
pulse and measuring the signal after background has fallen to zero
but before the europium with its long fluorescence half-life has de-
cayed completely, good discrimination between a weak signal and
background becomes possible. Characterization of antigens by electrophoresis
and immunofixation
lower background binding), both of which bind with This technique is most often applied to the detection of
ferocious specificity and affinity (K= 1015M-'). an abnormal protein in serum or urine, usually a
Chemiluminescent systems based on the HRP- monoclonal paraprotein secreted by a B-cell tumor.
catalysed enhanced luminolreaction, where light from The paraprotein localizes as a dense compact 'M' band
the oxidized luminol substrate is intensified and the of defined electrophoretic mobility and its antigenic
signal duration increased by the use of an enhancing identity is then revealed by immunofixation with spe-
reagent, provide increased sensitivity and dynamic cific precipitating antiserums applied in paper strips
range. Special mention should be made of time- overlying parallel lanes in the electrophoresis gel
resolved fluorescence assays based upon chelates of (figure 6.13).
rare earths such as europium 3+(figure 6.12),although
these have a more important role in antigen assays.
Quantification by single radial
immunodiflusion (SRID)
DETECTION OF I M M U N E COMPLEX When antigen diffuses from a well into agar containing
FORMATION
suitably diluted antiserum, initially it is present in a
Many techniques for detecting circulating complexes relatively high concentration and forms soluble com-
have been described and because of variations in the plexes; as the antigen diffuses further the concentra-
size, complement-fixingability and Ig class of different tion continuously falls until the point is reached at
complexes, it is useful to apply more than one method. which the reactants are nearer optimal proportions
Two fairly robust methods for general use are: and a ring of precipitate is formed. The higher the con-
1 precipitation of complexed IgG from serum at centration of antigen, the greater the diameter of this
concentrations of polyethylene glycol which do not ring (figure 6.14). By incorporating, say, three stan-
bring down significant amounts of IgG monomer, fol- dards of known antigen concentration in the plate, a
lowed by estimation of IgG in the precipitate by single calibration curve can be obtained and used to deter-
radial immunodiffusion (SRID) or laser nephelometry, mine the amount of antigen in the unknown samples
and tested (figure 6.15).The method was used routinely in
Figure 6.13. Electrophoresisand immunofixationof a paraprotein
in serum.The sample is separated into its componentbands by elec-
trophoresisin agarose gel and these are visualized by direct staining
after drying down the gel. The test sampleis also run on a parallel gel
which is then overlaid with strips of paper soaked in a specific anti-
serum. The antibodies diffuse into the gel and 'fixf the antigen by
precipitation; after washing to remove the nonprecipitated soluble
proteins, the gel is dried and stained to reveal the location of the
paraprotein-antibody complex.N, normal serum;P, patient's serum
showing compact paraprotein band; G, M, K and h represent im-
munofixations with antiserurn specific for each immunoglobulin
chain. In the example chosen, the paraprotein is an IgGh. (Material
Figure 6.15. Measurement of IgG concentrationin serum by sin-
kindly supplied by Mr T. Heys.)
gle radial immunodiffusion.The diameter of the standards ( ) en-
ables a calibration curve to be drawn and the concentration of IgG in
the serum under test can be read off:
T,+erum from patient with IgG myeloma; 15mg /ml;
T,+erum from patient with hypogammaglobulinemia; 2.6 mg/ml;
T3ilormal serum; 9.6 mg/ml.
(Courtesy of Professor F.C. Hay.)
OCCUPIED UNOCCUPIED
ANALME '11 1 Ii I
LIGAND BINDER
with antigen-specific T-cell lines or clones can be these positions. Thus, a positional scanning approach
deciphered to characterizethe active epitopes. employs a peptide library in which one amino acid at a
Dissecting out T-cell epitopes where the antigen has particular position is kept constant and all the different
not been characterized is a more daunting task. Ran- amino acids are used at the other positions.
domized peptide libraries can be produced but strat-
egies need to be devised in order to keep these within
B-cell epitopes
manageable numbers. Information from the accumu-
lated data deposited in various databanks can be used If they are linear protein epitopesformed directly from
to identify key anchor residues and libraries con- the primary amino acid sequence, then binding of anti-
structed that maintain the relevant amino acids at body to individual overlapping peptides synthesized
possible random hexapeptides.These are produced by
ligating degenerate oligonucleotideinserts (coding for
hexapeptides) to a bacteriophage coat protein in a
suitable vector; appropriate expression in E. coli can
provide up to 109different clones. The beauty of the
system is that a bacteriophage expressing a given hexa-
peptide on its external coat protein also bears the
sequence encoding the hexapeptide in its genome (cf.
p. 124).Accordingly, sequential rounds of selection, in
which the phages react with a biotinylated monoclon-
a1 antibody and are then panned on a streptavidin
plate, should isolate those bearing the peptides which
mimic the epitope recogruzed by the monoclonal;
nucleotide sequencing will then give the peptide
structure.
Even nonproteinaceous antigens can occasionally
be mimicked using peptide libraries, one example
being the use of a D-aminoacid hexapeptide library to
identify a mimotope for N-acetylglucosamine. Others
have used a single-chain Fv (scFv)library to isolate an
idiotypic mimic of a meningococcal carbohydrate.
First in rodents
A fantastic technological breakthrough was achieved
by Kohler and Milstein who devised a technique
for the production of 'immortal' clones of cells mak-
ing single antibody specificities by fusing normal
antibody-forming cells with an appropriate B-cell
tumor line. These so-called 'hybridomas' are selected
Figure 6.19. Synthesis of overlapping peptide sequences for
(PEPSCAN)epitope analysis. Aseries of pins which sit individually
out in a tissue culture medium which fails to support
in the wells of a 96-well microtiter plate each provide a site for solid- growth of the parental cell types, and by successive di-
phase synthesis of peptide. A sequence of such syntheses as shown lutions or by plating out, single clones can be estab-
in the figure provides the required nests of peptides. Incorporation lished (figure 6.20). These clones can be grown up in
of a readily cleavable linkage allows the soluble peptide to be re-
leased as the synthesis is terminated.
the ascitic form in mice when quite prodigious titers of
monoclonal antibody can be attained, but bearing in
mind the imperative to avoid using animals wherever
as described above will identify them. Unfortunately, feasible, propagation in large-scale culture is to be pre-
most epitopes on globular proteins recognized by anti- ferred.Remember that, even in a good antiserum, over
body are discontinuous and this makes the job rather 90% of the Ig molecules have little or no avidity for the
demanding, since one cannot predict which residues antigen, and the 'specific antibodies' themselves rep-
are likely to be brought together in space to form the resent a whole spectrum of molecules with different
epitope. To the extent that small linear sequences may avidities directed against different determinants on
contributeto a discontinuousepitope, the overlapping the antigen. What a contrast is provided by the mono-
peptide strategy may provide some clues. clonal antibodies, where all the molecules produced
A potentially promising approach to this problem by a given hybridoma are identical: they have the same
of mimicking the residues which constitute such epi- Ig class and allotype, the same variable region, struc-
topes (termed mimotopes by Geysen) is through the ture, idiotype, affinity and specificity for a given
production of libraries of bacteriophages bearing all epitope.
Figure 6.20. Production of monoclonal antibodies. Mice irnmu- complex mixtures of multiepitopic antigens. Fusions using rat cells
nized with an antigenbearing (shallwe say)two epitopes, a and b, instead of mouse may have certain advantages in giving a higher
develop spleen cells making anti-a and anti-b which appear as proportion of stable hybridomas, and monoclonals which are better
antibodiesin the serum. The spleenis removed and the individual at fixing human complement, a useful attribute in the context of
cells are fused in polyethylene glycol with constantly dividing (i.e. therapeutic applicationsto humans involvingcell depletion.
'immortal') B-tumor cells selected for a purine enzyme deficiency Naturally, for use in the human, the ideal solution is the produc-
and usually for their inability to secrete Ig. The resulting cells tion of purely human monoclonals. Human myeloma fusion part-
are distributed into microwell plates in HAT (hypoxanthine, ners have not found wide acceptance since they tend to have low
aminopterin, *ymidine) medium which kills off the fusion part- fusion efficiencies, poor growth and secretion of the myeloma Ig
ners. They are seeded at such a dilution that on average each well which dilutes the desired monoclonal. A nonsecreting heterohy-
will contain less than one hybridoma cell. Each hybridoma4he bridoma obtained by fusing a mouse myeloma with human B-cells
fusion product of a single antibody-forhg cell and a tumor can be used as a productive fusion partner for antibody-producing
cell-;will have the ability of the former to secretea single speciesof human B-cells. Other groups have turned to the well-characterized
antibody and the immortality of the latter enabling it to proliferate murine fusion partners, and the heterohybridomasso formed grow
continuously. Thus, clonal progeny can provide an unending well, clone easily and are productive. There is some instability from
supply of antibody with a single specificity-the monoclonal chromosome loss and it appears that antibody production is main-
antibody In this example, we considered the production of tained by translocation of human Ig genes to mouse chromosomes.
hybridomas with specificity for just two epitopes, but the same Fusion frequency is even better if Epstein-Barr virus (EBV)-
technique enables monoclonal antibodies to be raised against transformed lines are used instead of B-cells.
Whereas the large amount of nonspecific, relative to raised the pulse rate of the cognoscenti, since this is an
antigen-specific, Ig in an antiserum means that back- energetically difficult reaction which has an enormous
ground binding to antigen in any given immunologi- range of applications.Another innovative approach is
cal test may be uncomfortably high, the problem is to immunize with an antigen which is so highly reac-
greatly reduced with a monoclonal antibody prepara- tive that a chemical reaction occurs in the antibody
tion, since all the Ig is antibody, thus giving a much su- combining site. This recruits antibodies which are not
perior 'signal :noise' ratio. By being directed towards only complementary to the active chemical, but are
single epitopes on the antigen, monoclonal antibodies also likely to have some enzymic power over
frequently show high specificity in terms of their the immunogen-substrate complex. Thus, using this
low cross-reactivity with other antigens. Occasionally, strategy, an antibody with exceptionally broad sub-
however, one sees quite unexpected binding to mole- strate specificity for efficient catalysis of aldol and
cules which react poorly, if at all, with a specific anti- retro-aldol reactions was obtained. A key feature of
serum directed to the original antigen. The reason this antibody is a reactive lysine buried within a hy-
for this has alreadybeen discussed (seep. 89). Suffice it drophobic pocket in the binding site. The antibody
to say here that the problem can be circumvented remains catalytically active for several weeks follow-
by using a group of overlapping monoclonals react- ing i.v. injection into mice and has therapeutic
ing with the same determinant or a combination of potential for a version of antibody-directed enzyme
monoclonals to more than one determinant on the prodrug therapy (ADEPT, see p. 392), here with the
same antigen. enzyme component being a catalytic antibody.
An outstanding advantage of the monoclonal anti- Large combinatorial antibody libraries created by
body as a reagent is that it provides a single standard random association between pools of heavy and light
material for all laboratories throughout the world to chains and expressed on bacteriophages (see below)
use in an unending supply if the immortality and pu- can be screened for catalytic antibodies by using the
rity of the cell line are nurtured; antisera raised in dif- substrate in a solid-phase state. Cleavage by the cata-
ferent animals, on the other hand, may be as different lytic antibody leaves a solid-phase product which can
from each other as chalk and cheese. The monoclonal now be identified by a double antibody system using
approach again shows a clean pair of heels relative to antibodies specific for the product as distinct from the
conventionalstrategiesin the production of antibodies substrate.
specific for individual components in a complex mix- An area of great interest is the presence of catalytic
ture of antigens. The uses of monoclonal antibodies autoantibodies in certain groups of patients, with hy-
are truly legion and include: immunoassay, diagnosis drolytic antibodies against vasoactive intestinal pep-
of malignancies, tissue typing, serotyping of microor- tide, DNA and thyroglobulin having been described.
ganisms, the separation of individual cell types with Catalytic antibodies capable of factor VIII hydrolysis
specific surface markers (e.g. lymphocyte subpopula- have also recently been discovered in hemophiliacs
tions), therapeutic neutralization of inflammatory given this clottingfactor, the antibodies preventing the
cytokines and 'magic bullet' therapy with cytotoxic coagulation function of the factor VIII.
agents coupled to antitumor-specificantibody-these
and many other areas have been transformed by hy-
Human monoclonals can be made
bridoma technology.
Mouse monoclonals injected into human subjects
for therapeutic purposes are frightfully immunogenic
Catalytic antibodies
and the human anti-mouse antibodies (HAMA in the
An especially interesting development with tremen- trade) so formed are a wretched nuisance, accelerating
dous potential is the recognition that a monoclonal clearance of the monoclonal from the blood and pos-
antibody to a stable analog of the transition state of sibly causing hrpersensitivity reactions; they also pre-
a given reaction can act as an enzyme ('abzymef)in vent the mouse antibody from reaching its target and,
catalysing that reaction. The possibility of generating in some cases, block its binding to antigen. In some cir-
enzymes to order promises a very attractive future, cumstances it is conceivable that a mouse monoclonal
and some exceedingly adroit chemical maneuvers taken up by a tumor cell could be processed and be-
have already extended the range of reactions which come the MHC-linked target of cytotoxic T-cells or
can be catalysed in this way. A recent demonstrationof help to boost the response to a weakly immunogenic
sequence-specific peptide cleavage with an antibody antigen on the tumor cell surface.In general, however,
which incorporates a metal complex cofactor has logic points to removal of the xenogeneic (foreign)
portions of the monoclonal antibody and their replace- myeloma cell line to produce hybridomas, or the genes
ment by human Ig structuresusing recombinant DNA can be isolated and used to produce a recombinant
technology. Chimeric constructs, in which the V, and antibody.
V Lmouse domains are spliced onto human C, and C, A radically different approach involves the pro-
genes (figure 6.21a), are far less immunogenic in duction of transgenic xenomouse strains in which
humans. megabase-sized unrearranged human Ig H and light
A more refined approach is to graft the six comple- chain loci have been introduced into mice whose
mentarity determining regions (CDRs) of a high af- endogenous murine Zg genes have been inactivated.
finity rodent monoclonal onto a completely human Ig Immunization of these mice yields high affinity
framework without loss of specific reactivity (figure ( 1 0 - ~ ~ - 1 0 - lhuman
~ ~ ) antibodies which can then be
6.21b). This is not a trivial exercise, however, and the isolated using hybridoma or recombinant approaches.
objective of fusing human B-cells to make hybridomas Potent anti-inflammatory (anti-IL-8) and anti-tumor
is still appealing, taking into account not only the gross (anti-epidermal growth factor receptor) therapeutic
reduction in immunogenicity, but also the fact that, agents have already been obtained using such mice.
within a species, antibodies can be made to subtle dif- There is still a snag in that even human antibodies
ferences such as major histocompatibility complex can provoke anti-idiotype responses; these may have
(MHC) polymorphic molecules and tumor-associated to be circumvented by using engineered antibodies
antigens on other individuals. In contrast, xenogeneic bearing different idiotypes for subsequent injections.
responses are more directed to immunodominant Even more desirable would be if the prospective
structures common to most subjects, making the pro- recipients could be first made tolerant to the idiotype,
duction of variant-specific antibodies more difficult. perhaps by coadministering the therapeutic antibody
Notwithstanding the difficulties in finding good together with a nondepleting anti-CD4.
fusion partners, large numbers of human monoclonals Many human monoclonals are awaiting the go-
have been established. A further restriction arises be- ahead for clinical use; one can cite IgG anti-RhD for the
cause the peripheral blood B-cells, which are the only prevention of rhesus disease of the newborn (see p.
B-cells readily availablein the human, are not normally 334), and highly potent monoclonals for protection
regarded as a good sourceof antibody-formingcells. against varicella zoster, cytomegalovirus, group B
Immortalized Epstein-Barr virus-transformed B- streptococci and lipopolysaccharide endotoxins of
cell lines have also been used as a source of human Gram-negativebacteria.
monoclonal antibodies.Although these often produce
relatively low affinity IgM antibodies, some useful
higher affinity IgG antibodies can occasionally be ob-
tained. The cell lines frequently lose their ability to There are other ways around the problems associated
secrete antibody if cultured for long periods of time, al- with the production of human monoclonals which ex-
though they can sometimes be rescued by fusionwith a ploit the wiles of modern molecular biology. Reference
has already been made to the 'humanizing' of rodent
antibodies (figure6.21),but an important new strategy
based upon bacteriophage expression and selection
has achieved a prominent position. In essence, mRNA
from primed human B-cells is converted to cDNA and
the antibody genes, or fragments therefrom, expanded
by the polymerase chain reaction (PCR). Single con-
structs are then made in which the light and heavy
chain genes are allowed to combine randomly in tan-
dem with the gene encoding bacteriophage coat pro-
tein I11 (pIII) (figure 6.22). This combinatorial library
containing most random pairings of heavy and light
chain genes encodes a huge repertoire of antibodies
(or their fragments) expressed as fusion proteins with
Figure 6.21. Genetically engineering rodent antibody specific- p111 on the bacteriophage surface. The extremely high
ities into the human. (a) Chimeric antibody with mouse variable
regions fused to human Ig constant regions. (b) 'Humanized' rat number of phages produced by E. coEi infection can
monoclonal in which gene segments coding for all six CDRs are now be panned on solid-phase antigen to select those
grafted onto a human Ig framework. bearing the highest affinitv antibodies attached to their
Figure 6.22. Selection of antibody genes from a combinatorial li- ly combined in constructs fused to the bacteriophage p111 coat pro-
brary. B-cells from an immunized donor (in one important experi- tein gene as shown. These were incorporated into phagemids such
ment, human memory peripheral blood cells were boosted with as pHENl and expanded in E. coli. After infectionwith helper phage,
tetanus toxoid antigen after transfer to SCID mice; Duchosal M.A. the recombinant phages bearing the highest affinitywere selectedby
et al. (1992)Nature 355,258) are used for the extraction of IgG mRNA rounds of panning on solid-phaseantigenso that the genes encoding
and the light chain (VLCL)and VHCHIgenes (encodingFab) random- the Fabs could be cloned. L =bacterialleader sequence.
surface (figure 6.22). Because the genes which encode duced by these techniques, antigen can be used to
these highest affinity antibodies are already present select higher affinity mutants produced by random
within the selected phage, they can readily be cloned mutagenesis or even more effectively by site-directed
and the antibody expressed in bulk. It should be recog- replacements at mutational hotspots (figure 6.23b),
nized that this selection procedure has an enormous again mimicking the natural immune response which
advantage over techniques which employ screening involves random mutation and antigen selection (see
because the number of phages which can be examined p. 192).Affinity has also been improved by gene 'shuf-
is severallogs higher. fling' in which a VHgene encodinga reasonable affinity
Combinatorial libraries have also been established antibody is randomly combined with a pool of
using mRNA from unimmunized human donors. VH' V, genes and subjected to antigen selection. The
V, and VAgenes are expanded by PCR and randomly process can be further extended by mixing the VLfrom
recombined to form single-chain Fv (scFv) constructs this combination with a pool of VH genes. It has
(figure 6.23a) fused to phage pIII. Soluble fragments also proved possible to shuffle individual CDRs
binding to a variety of antigenshave been obtained. Of between variable regions of moderate affinity anti-
special interest are those which are autoantibodies to bodies obtained by panning on antigen, thereby creat-
molecules with therapeutic potential such as CD4 and ing antibodies of high affinity from relatively small
tumor necrosis factor-a (TNFa);lymphocytes express- libraries.
ing such autoantibodies could not be obtained by Other novel antibodies have been created. In one
normal immunization since they would probably be construct, two scFv fragments associate to form
tolerized, but the random recombination of VH and V, an antibody with two different specificities (figure
can produce entirely new specificities under condi- 6.23~).Another consists of a single heavy chain
tions in vitro where tolerance mechanisms do not variable region domain (DAB) whose affinity can
operate. be surprisingly high-of the order of 2 0 ~ If. it
Although a 'test-tube' operation, this approach to were possible to overcome the 'stickiness' of these
the generation of specific antibodies does resemble the miniantibodies, their small size could be exploited
affinity maturation of the immune response in vivo for tissue penetration. The design of potential 'magic
(see p. 195) in the sense that antigen is the deter- bullets' for immunotherapy can be based on fusion
mining factor in selecting out the highest affinity of a toxin (e.g. ricin) to an antibody Fab (figure
responders. 6.23d).
In order to increase the affinities of antibodies pro-
Figure 6.23. Other engineered antibodies. (a)A single gene encod- associate to form a 'diabody' with two specificities.These bispecific
ing VHand V, joined by a sequence of suitable length gives rise to a antibodies have a number of uses. Note that such a bispecific anti-
single-chain Fv (scFv)antigen-bindingfragment. (b) By site-specific body directed to two different epitopes on the same antigen will
mutagenesisof residues in or adjacent to the complementarity deter- have a much higher affinity due to the 'bonus effect' of cooperation
mining region (CDR), it is possible to increase the affinity of the anti- between the two binding sites (cf.p. 90). (d) Potential 'magic bullets'
body. (c) Two scFv constructs expressed simultaneously will canbe constructedby fusing the gene for a toxin ( g .ricin) to the Fab.
I M M U N O H I S T O C H E M I S T R Y -LOCALIZATION
OF ANTIGENS I N CELLS AND TISSUES
mmd
ngrucm1.i.i. The~~pieormcr~w~orflowcytofluorime-can ue acovated to separate cells of high from low fluo-
try of the fluoRscRKp on stained cells (green rimmed drcles) and, using light scatter, of large from small size and dead fm
and physical sqwation from unstained cells. The charge si& living.
I
biotin to the antiserum and then finally staining with anti-human immunoglobulin; if antibodies were pre-
fluorescent avidin is often employed. sent, there would be staining of the thyroid epithelial
cells.
This technique has several advantages. In the first
Indirect testfor antibody
place the fluorescence is brighter than with the direct
In this double-layer technique, the unlabeled antibody test since several fluorescent anti-immunoglobulins
is applied directly to the tissue substrate and visual- bind on to each of the antibody molecules present in
ized by treatment with a fluorochrome-conjugated the first layer (figure 7.3b). Second, even when many
anti-immunoglobulin serum (figure 7.3b).In this case, sera have to be screened for specific antibodies it is
in order to find out whether or not the serum of a only necessary to prepare (or, more usually the case,
patient has antibodies to thyroid epithelial cells, we purchase) a single labeled reagent, viz. the anti-
would first treat a thyroid section with the serum, immunoglobulin. Furthermore, the method has great
wash well and then apply a fluorescein-labeledrabbit flexibility. For example,by using conjugates of antisera
Figure 7.2. Separation of activated peripheral blood memory (b) negative (CD45RA) and (c) positive (CD45RO) populations,
T-cells (CD45RO positive) from naive T-cells (CD45RO negative; which were each tested for their proliferative response to a mixture
but positive for the CD45RA isoform) in the FACS after staining the of two anti-CD2 monoclonals (OKT11 and GT2) in the presence of
surface of the living cells in the cold with a fluorescent monoclonal 10% antigen-presenting cells (d). 3H-Thymidinewas added after 3
antibody to the CD45RO (seep. 196).The unsorted cells showed two days and the cells counted after 15h. Clearly the memory cell popu-
peaks (a); cells with fluorescence intensity lower than the arbitrary lation proliferated, whereas the naive population did not. (Data
gate were separated from those with higher intensity giving kindly provided by D. Wallace and R. Hicks.)
Table 7.1. Evaluation of neutrophil function. Figure 7.8. Gene expressionduring lymphocyte developmentand
activation. The data were generated from over 3.8 million measure-
ments of gene expression made on 13637 genes using 243 microar-
rays. Each experiment represents a different cell population. For
Phagocyiasis Measure the upme 01 palticies such OS
latex or bocterla by counting or by example, experiment 1 utilized polyclonally activated fetal CD4+
chemiluminescence thymic cells, whereas experiment 2 shows the same population prior
to stimulation.Overexpressed or induced genes are colored red, un-
1 Respiratory burst 1 Measure reductionof nltmblue tetrazolium derexpressed or repressed genes green. Certain gene expression sig-
natures become apparent in the different cell populations, indicated
intracellular killing Micmbicidal test using viable on the right. For example, the T-cell gene expression signature in-
Saphylococcus aureus
cludes CD2, TCR, TCR signaling molecules and many cytokines.
D rectlono migror'on Movemenl inrough fliers JP concenirohon (Reproduced with permission of the authors and the publishers
grad ent oi cnemoiactlc ogent S L C as
~ from Alizadeh A.A. & Staudt L.M. (2000) Current Opinion in Im-
I I formvl.Met.Leu.Phe munology 12,219.)
Surface LFA-1 ond CR3
upreguiotion I Ascertained with monoclonal antibody
staining
p. 167) and this can be readily revealed with a fluores- measure extracellularkilling of antibody-coated or un-
cent or chromogenicenzyme substrate. coated targets by NK cells. Now a word of caution re-
The ability of cytotoxic T-cells to kill their cell targets garding the interpretation of in vitro assays. Since one
extracellularly is usually evaluated by a chromium canmanipulate the cultureconditions within wide lim-
release assay. Target cells are labeled with 51Crand the its, it is possible to achieve a result that might not be at-
release of radioactive protein into the medium over tainable in vivo. Let us illustrate this point by reference
and above that seen in the controls is the index of cyto- to cytotoxicity for murine cells infected with lympho-
toxicity. The test is repeated at different ratios of effec- cytic choriomeningitis virus (LCMV) or vesicular
tor to target cells. A similar technique is used to stomatitisvirus (VSV).The most sensitive in vitro tech-
nique proved to be chromium release from target cells tain an average of one precursor cell per aliquot, and
after secondary stimulation of the lymphocytes. How- one can therefore calculate the precursor frequency in
ever, this needs 5 days, during which time a relatively the original cell suspension. An example is shown in
small number of memory CD8 cytotoxic T-cell precur- some detail in figure 7.9.
sors can replicate and surpass the threshold required to It has been argued that limiting dilution analysis
produce a measurable assay. Nonetheless, a weak cyto- often underestimates the true precursor frequency. An
toxicity assay under these conditionswas not reflected accurate measure of the percentage of lymphocytes
by any of the in vivo assessments of antiviral function bearing a specific antigen receptor can be obtained by
implyingthat they had no biological relevance. flow cytometry of cells stained with labeled antigen. In
the case of B-cells this is fairly straightforward given
that their antigen receptors recognize native antigen.
However, it is only recently that technical finesse, in
Programed cell death occurs frequentlyin the immune the form of peptide-MHC tetramers, has brought this
system in a number of different situationsand a variety technique to T-cells (figure 7.10). This approach over-
of approaches can be used to measure the apoptotic comes the problem of the relatively weak intrinsic
cells. Gel electrophoresis will detect the characteristic affinity of TCR for peptide-MHC by presenting a
laddering pattern of the DNA fragments generated tagged peptide-MHC as a multivalent tetramer, there-
due to internucleosomal cleavage (see figure 1.19). by exploiting the bonus effect of multivalency (cf. p.
An alternative way to detect this DNA fragmenta- 90). Peptide-MHC complexes are produced by per-
tion, known as the TUNEL (TdT-mediated dUTP (de- mitting recombinant MHC molecules to refold with
oxyuridinetriphosphate) nick end labeling)technique, the appropriate synthetic peptide. The recombinant
utilizes the enzyme terminal deoxynucleotidyl trans- MHC molecules are biotinylated on a special carboxy-
ferase (TdT)to add labeled nucleotides to the 3' ends of terminal extension, which ensures that the biotin is in-
the fragments.An earlier event in apoptosis is the loss corporated at a distance from the site to which the TCR
of membrane symmetry resulting in the expression binds, and mixed with fluorescently labeled strept-
of phosphatidylserine on the cell surface -readily de- avidin, which not only binds biotin with a very high
tected by flow cytometry using a labeled version of affinity but also has a valency of four with respect to the
its ligand Annexin V. For sophisticates, more detailed biotin -hence the formation of tetramers.
analyses can be undertaken in which individual Numerous adaptations of this technology are ap-
components of the apoptotic pathways, such as Bcl-2, pearing. For example, incubation at 37C of tetramers
Bcl-xv Bax, PARP (poly(ADP-ribose)polymerase) or bound to their cognate TCR leads to internalization;by
specific procaspases and caspases, are measured in tagging them with a toxin individual T-lymphocytes
fluorimetric or colorimetricassays. of a single specificity can be eliminated. Another ap-
proach is to use the FACS to directly sort stained cells
into an ELISPOT microtiter plate in which cytokine
secretion is measured, providing a functional analysis
The magnitude of lymphocyte responses in culture of the cells.
is closely related to the number of antigen-specific
lymphocytes capable of responding. Because of the
clonality of the responses, it is possible to estimate
the frequency of these antigen-specificprecursors by
The immunofluorescence sandwich test
limiting dilution analysis. In essence, the method de-
pends upon the fact that, if one takes several replicate This is a double-layer procedure designed to visualize
aliquots of a given cell suspension which would be ex- specific intracellular antibody. If, for example, we
pected to contain on average one precursor per aliquot, wished to see how many cells in a preparation of lym-
then Poisson distribution analysis shows that 37% of phoid tissue were synthesizing antibody to Pneumo-
the aliquotswill contain no precursor cells (through the coccus polysaccharide, we would first fix the cells with
randomness of the sampling). Thus, if aliquots are ethanol to prevent the antibody being washed away
made from a series of dilutions of a cell suspension during the test, and then treat with a solution of the
and incubated under conditions which allow the pre- polysaccharide antigen. After washing, a fluorescein-
cursors to mature and be recognized through some labeled antibody to the polysaccharide would then be
amplification scheme, the dilution at which 37% of the added to locate those cells which had specifically
aliquots give negative responses will be known to con- bound the antigen.
Figure 7.9. Limiting dilution analysis of cytotoxicT-cell precursor and each point above that line is counted as positive for cytotoxicity.
frequency in spleen cells from a BALB/c mouse stimulated with ir- (b) The data replotted in terms of the percentage of negative wells at
radiated C57BL/6 spleen cells as antigen. BALB/c splenic respon- each concentration of responder cells over the range in which the
der cells were set up in 24 replicates at each concentration tested data titrated ( 5 ~ 1 0 - ~ / w eto
l l 0.625xlO-~/well).The dashed line is
together with antigen and an excess of T-helper factors. The genera- drawn at 37% negative wells and this intersects the regression line to
tion of cytotoxicity in each well was looked for by adding 51Cr- give a precursor (Tcp)frequency of 1in 2327 responder cells. The re-
labeled tumor cells (EL-4) of the C57BL/6 haplotype; cytotoxicity gression line has an r2value of 1O . O in this experiment. (Reproduced
was then revealed by measuring the release of soluble 51Cr-labeled with permission from Simpson E. &Chandler P. (1986)In Weir D.M.
intracellular material into the medium. (a) The points show the per- (ed.) Handbook of Experimental Immunology, figure 68.2. Blackwell
centage of specific lysis of individual wells. The dashed line indi- ScientificPublications,Oxford.)
cates three standard deviations above the medium release control,
The name of the test derives from the fact that anti-
gen is sandwiched between the antibody present in
the cell substrate and that added as the second layer
(figure 7.3~).
Plaque techniques
Antibody-secreting cells can be counted by diluting
them in an environment in which the antibody formed
by each individual cell produces a readily observable
effect. In one technique, developed from the original
method of Jerne and Nordin, the cells from an animal
immunized with sheep erythrocytes are suspended
together with an excess of sheep red cells and com-
plement within a shallow chamber formed between
two microscope slides. On incubation, the antibody-
forming cells release their immunoglobulin which
coats the surrounding erythrocytes. The complement
Figure 7.10. Peptide-MHC tetramer. A single fluorochrome- will then cause lysis of the coated cells and a plaque
labeled peptide-MHC complex (top right) has only a low affinityfor
the TCR and therefore provides a very insensitive probe for its
clear of red cells will be seen around each antibody-
cognate receptor.However, by biotinylating ( ) the MHC molecules forming cell (figure 7.11). Direct plaques obtained in
and then mixing them with streptavidin, which has a valency of four this way largely reveal IgM producers since this anti-
with respect to biotin binding, a tetrameric complex is formed which body has a high hemolytic efficiency. To demonstrate
has a much higher functional affinity (avidity) when used as a probe
IgG synthesizing cells it is necessary to increase the
for the specific TCRs on the T-cell surface.
complement binding of the erythrocyte-IgG antibody
complex by adding a rabbit anti-IgG serum; the 'indi-
Figure 7.11. Jerne plaque technique for enumerating antibody- ing cells by capturing secreted Ig on red cells coated with anti-Ig.
forming cells (Cunninghammodification).(a)The direct technique Multiple plaque assays can be carried out by a modification using
for cells synthesizing IgM hemolysin is shown. The indirect tech- microtiter plates. (b) Photograph of plaques which show as circular
nique for visualizing cells producing IgG hemolysins requires the dark areas (some of which are arrowed) under dark-ground illumi-
addition of anti-IgG to the system. The difference between the nation. They vary in size depending upon the antibody affinity and
plaques obtained by direct and indirect methods gives the number of the rate of secretion by the antibody-forming cell. (Courtesy of C.
'IgG' plaques. The veverse plaque assay enumerates total Ig-produc- Shapland, P. Hutchings and Professor D. Male.)
Figure 7.18. Conditional knockout. The endogenous gene that is mice which contain a transgene for the Cre recombinase under the
under study (hereB7.2)is homologously replaced in ES cells with an control of specific regulatory elements, only those cells in which the
identical gene, as in figure 7.17, but here flanked by loxP sequences promoter is active will produce the Cre enzyme necessary to delete
(brownboxes) and with the neoRgene incorporated in a nondisrup- the sequence flanked by 1oxP.The example givenwould represent an
tive manner purely for selection purposes. Nonhomologous recom- experiment aimed at investigathg the effect of specifically knocking
binants will containthe tk gene and are eliminatedusing ganciclovir. out B7.2 in B-cells whilst maintaining its expression in, for example,
Transgenic animals are then generated from ES cells which are resis- dendritic cells.
tant to G418. If homozygous B7.2-loxP transgenics are mated with
when and where Cre is expressed (figure 7.18). The replaced by a functional gene, be it a modified version
Cre/ EoxP systemcan also be organized in such a way as of the original gene or an entirely different gene, are re-
to turn on expression of a gene by incorporatinga stop ferred to as 'knocked in mice'. Hence, in the example
sequenceflanked by loxP sites. above, knocking in a EoxP flanked gene leads eventual-
Mice in which an endogenous gene is purposefully ly to a knocked out gene in a selected cell type. Another
Table 7.2. Some gene 'knockouts' and their effects. example of a knocked in gene was seen on p. 133with
the replacement of endogenous RAG-2 with a RAG-
2-GFP fusion gene.
(continued)
Gene expression irradiation of chromophore-conjugated specific anti-
mRNAexpression can be localizedby in situ hybridiza- bodies which localize to the target area by penetrating
tion using a complementaryoligonucleotideprobe. permeabilized cells.
A complete picture of cellular gene expression is now
attainableby hybridization to microarraychips. Genetic engineering of cells
Genes canbe inserted into mammaliancells by transfec-
Assessment of functional activity tion using calcium phosphate precipitates, electropora-
Neutrophil chemotaxis, phagoeytosis,NADH (reduced tion, liposomes and microinjection.
nicotinamide adenine dinucleotide) oxidase activity and Genes can also be taken into a cell after incorporation
microbicidal potency can all be studied, almost on a into vaccinia or retroviruses.
routinebasis. Endogenous gene function can be inhibited by anti-
Lymphocyteresponsestoantigen aremonitoredbypro- sense RNA or by homologous recombination with a
liferation and/or cytokine release. Individual cells secret- disrupted gene.
ing cytokines can be identified by the ELISPOT technique Transgenic mice bearing an entirely new gene intro-
in which the secreted product is captured by a solid-phase ducedinto the fertilized eggby microinjectionof DNAcan
antibody and then stained with a second labeled antibody. be established as inbred lines.
Extracellular killing by cytotoxic T-cells, and NK cells, Genes can be introduced into embryonic stem cells;
can be measured by the release of radioactive 51Cr from thesemodified stem cells are injectedbackinto ablastocyst
prelabeled target cells. andcandevelop intofoundermicefromwhichpure trans-
The precursor frequency of effector T-cells can be mea- genic animalscanbebred.0neveryimportantapplication
sured by staining the cells with peptide-MHC tetramers of this techniqueinvolves the disruptionof a targeted gene
or by limiting dilution analysis. in the embryonic stem cellby homologous recombination,
Antibody-forming cells can be enumerated, either by producing 'knockout' mice lacking a specific gene. Condi-
an immunofluorescence sandwich test or by plaque tional knockouts employ recombinase systems such as
techniques in which the antibody secreted by the Cre/loxP in order to control the deletion either temporally
cells causes complement-mediated lysis of adjacent red or in a tissue-specificmanner.
cells, or is captured by solid-phase antigen in an ELISPOT 'Knock in' mice have a specified endogenous gene
assay. homologously replaced with either a variant of that gene
9 Functional activity can be assessed by cellular reconsti- or an entirelydifferent gene.
tution experiments in which leukocyte sets and selected Human gene therapy promises an exciting future.
lymphoid tissue can be transplanted into unresponsive Delivery of genes by vectors based on retroviruses or
hosts such as X-irradiated recipients or K I D mice. De- adeno-relatedvirus is under intensiveinvestigation.
fined cell populations can also be separated and selec-
tivelyrecombined in vitro.
Antibodies can be used to probe cellular function by See the accompanying website (www.roin.com)
cross-liiking cell surface components or by selective fnr multiple choice questions.
destruction of particular intracellular sites by laser
THE NEED FOR ORGANIZED immmity. In addition, memory cells for secondary
LYMPHOtD TISSUE responses must be formed and the whole response
controlled so that it is adequate but not excessive and
For an effective immune response, an intricate series is appropriate to the type of infection being dealt
of cellular events must occur. Antigen must bind and with. By working hard, we can isolate component cells
if necessary be processed by antigen-presenting cells, of the immune system and persuade them to carry out
whichmust then make contactwith and activateT- and a number of responses to antigen in the test-tube, but
B-cells; T-helpers must assist B-cells and cytotoxic compared with the efficacy of the overall development
T-cell precursors, and there have to be mechanisms of immunity in the body, our efforts still leave much to
which amplify the numbers of potential effector cells be desired.In vivo the integration of the complex cellu-
by proliferation and then bring about differentiation lar interactions which form the basis of the immune
to generate the mediators of humoral and cellular response takes place within the organized architecture
Table 8.1. Some of the major clusters of differentiation (CD)
markers on human cells.
*, activated; B, B-lymphocytes; FDC, follicular dendritic cells; from bone marrow stem cells, the T-cells first dif-
G, granulocytes; IDC, interdigitating dendritic cells; Mast, mast ferentiating into immunocompetent cells by a high-
cells; M@,macrophages; MO,monocytes; NK, natural killer cells; T,
T-lymphocytes. pressure training period in the thymus, the B-cells
undergoing their education in the bone marrow itself
of peripheral, or secondary, lymphoid tissue which (figure 8.1).In essence, the lymph nodes filter off and,
includes the lymph nodes, spleen and unencapsulated if necessary, respond to foreign material draining
tissue lining the respiratory, gastrointestinal and body tissues, the spleen monitors the blood and the
genitourinarytracts. unencapsulated lymphoid tissue is strategically inte-
These tissues become populated by cells of reticular grated into mucosal surfaces of the body as a forward
origin and by macrophages and lymphocytes derived defensive systembased on IgA secretion.
The anatomical disposition of these lymphoid Communication between these tissues and the
tissues is illustrated in figure 8.2. The lymphatics and rest of the body is maintained by a pool of recirculat-
associated lymph nodes form an impressive network, ing lymphocytes which pass from the blood into the
draining the viscera and the more superficial body lymph nodes, spleen and other tissues and back to
structures before returning to the blood by way of the the blood by the major lymphatic channels such as the
thoracic duct (figure8.3). thoracic duct (figures8.4 and 8.10).
*Inlymph node and Peyer 'S patches the molecules are present on the specializedHEV.
CCR7, CC subgroupchemokinereceptor-7;ESL, E-selectinligand; GlyCAM, glycosylation-dependentcell adhesion molecule; PSGL, P-selectin
glycoproteinligand; SLC, secondary lymphoid tissue chemokine.
antigenic challenge they form secondary follicles of T-cells, scattered conventional reticular macro-
(figure8.7f)which consist of a corona or mantle of con- phages containing 'tingible bodies' of phagocytosed
centrically packed, resting, small B-lymphocytes pos- lymphocytes,and a tight network of specializedfollic-
sessingboth IgM and IgD on their surface surrounding ular dendritic cells (FDCs) with elongated cytoplas-
a pale-staining germinal center (figure8.21).This con- mic processes and few, if any, lysosomes. Germinal
tains large, usually proliferating, B-blasts, a minority centers are greatly enlarged in secondary antibody
Figure 8.7. Lymph node. (a) Diagrammatic representation of nized with the thymus-independent antigen, Pneumococcus polysac-
section through a whole node. (b) Diagram showing differentia- charide SIII, revealing prominent stimulation of secondary follicles
tion of B-cells during passage through different regions of an with germinal centers. (h) Methyl green/pyronin stain of lymph
active germinal center. FDC, follicular dendritic cell; M@,macro- node draining site of skin painted with the contact sensitizer oxa-
phage; X, apoptotic B-cell. (c) Human lymph node, low-power zolone, highlightingthe generalized expansion and activation of the
view. (d) Medulla stained with methyl green (DNA)/pyronin paracortical T-cells, the T-blasts being strongly basophilic. (i) The.
(RNA) to show the basophilic (pink) cytoplasm of the plasma same study in a neonatally thymectomized mouse shows a lonely
cells with their abundant ribosomes. (e) Medullary sinus of primary nodule (follicle) with complete lack of cellular response in
lymph node draining site of lithium carmine injection showing the paracortical area. GC, germinal center; LM, lymphocytemantle
macrophages which have phagocytosed the colloidal dye (one of secondary follicle; MC, medullary cords; MS, medullary sinus;
is arrowed). (f) Secondary lymphoid follicle showing germinal PA, paracortical area; PC, plasma cell; PF, primary follicle; SF,
center surrounded by a mantle of small B-lyrnphocytes stained secondary follicle; SM, sinusoidal macrophage; SS, subcapsular
by anti-human IgD labeled with horseradish peroxidase (brown sinus. ((c)Photographedby Professor P.M. Lydyard; (d-f) by Dr K.A.
color). There are few IgD-positive cells in the center but both areas MacLeman; (g-i) courtesy of Dr M. de Sousa and Professor D.M.V.
contain IgM-positive B-lymphocytes. (g) Node from mouse immu- Parrott.)
responses during which they constitute sites of B-cell tion of cells in the thymus-dependent area and typical
maturation and the generation of B-cell memory. lymphoblasts are evident (figure 8.7h). In contrast,
In the absence of antigen drive, the primary follicles stimulation of antibody formation by the thymus-
are composed of a mesh of FDCs whose spaces independent antigen, Pneumococcus polysaccharide
are filled with recirculating, but resting, small B- SIII, leads to proliferation in the cortical lymphoid
lymphocytes. On priming with a single dose of a T- follicles with the development of germinal centers,
dependent antigen (i.e. antigen for which the B-cells while the paracortical region remains inactive, re-
require cooperation from T-helper cells; cf. p. 171),the flecting the inability to develop cellular hypersensitiv-
FDC network can be colonized by as few as three pri- ity to the polysaccharide (figure 8.7g). As expected,
mary B-blasts which undergo exponential growth, nodes taken from children with congenital hypogam-
producing around lo4 so-called centroblasts and dis- maglobulinemia associated with failure of B-cell de-
placing the original resting B-cells which now form the velopment conspicuously lack primary and secondary
follicular mantle. These highly mitotic centroblasts, follicles.
with no surface IgD (sIgD) and very little sIgM, then
differentiate into light zone centrocyteswhich are non-
SPLEEN
cycling and begin to upregulate their expression of sIg.
At this stage there is very extensive apoptotic cell On a fresh section of spleen, the lymphoid tissue form-
death, giving rise to DNA fragments which are visible ing the white pulp is seen as circular or elongated gray
as 'tingible bodies' within the macrophages, the final areas (figure 8.8b) within the erythrocyte-filled red
resting place of the dead cells. The survivors undergo pulp which consists of splenic cords lined with
their final training in the apical light zone. A macrophages and venous sinusoids. As in the lymph
proportion of those which are shunted down the node, T- and B-cell areas are segregated (figure 8.8a).
memory cell pathway take up residence in the mantle The spleen is a very effective blood filter removing
zone population, the remainder joining the recirculat- effete red and white cells and responding actively to
ing B-cell pool. Other cells differentiate into plas- blood-borne antigens, the more so if they are particu-
mablasts with a well-defined endoplasmic reticulum, late. Plasmablasts and mature plasma cells are present
prominent Golgi apparatus and cytoplasmic Ig; these in the marginal zone extending into the red pulp
migrate to become plasma cells in the medullary cords (figure8.8~).
which project between the medullary sinuses (figure
8.7d). This maturation of antibody-forming cells at a
MUCOSAL-ASSOCIATED LYMPHOID
site distant from that at which antigen triggering has
TISSUE (MALT)
occurred is also seen in the spleen, where plasma cells
are found predominantly in the marginal zone. One's The respiratory, gastrointestinal and genitouri-
guess is that this movement of cells acts to prevent the nary tracts are guarded immunologically by sub-
generation of high local concentrations of antibody epithelial accumulations of lymphoid tissue which are
within the germinal center, so avoiding neutralization not constrained by a connective tissue capsule (figure
of the antigen on the FDCs and premature shutting off 8.9).These may occur as diffuse collections of lympho-
of the immune response. cytes, plasma cells and phagocytes throughout the
The remainder of the outer cortex is also essentially a lung and the lamina propria of the intestinal wall (fig-
B-cell area with scattered T-cells. ure 8.9a and b), or as more clearly organized tissue
with well-formed follicles. In humans, the latter in-
cludes the lingual, palatine and pharyngeal tonsils
(figure 8 . 9 ~the
) ~ Peyer's patches of the small intestine
T-cells are mainly confined to a region referred to as the (figure 8.9d) and the appendix. MALT forms an
paracortical (or thymus-dependent) area (figure 8.7a); interconnected secretory system within which cells
in nodes taken from children with selective T-cell committed to IgA or IgE synthesis may circulate
deficiency (figure 15.5), or from neonatally thymec- (figure 8.10).
tomized mice (figure 8.7i), the paracortical region is In the gut, antigen enters the Peyer 'S patches (figure
seen to be virtually devoid of lymphocytes. Further- 8.9d) across specialized epithelial cells (cf. figure 8.15)
more, when a T-cell-mediated response is elicited in a and stimulates the antigen-sensitive lymphocytes.
normal animal, say by a skin graft or by painting After activation these drain into the lymph and, after a
chemicals such as picryl chloride on the skin to induce journey through the mesenteric lymph nodes and the
contact hypersensitivity, there is a marked prolifera- thoracic duct, they pass from the bloodstream into the
155
Figure 8.8. Spleen. (a) Diagrammatic representation. (b) Low- cells. Note that the marginal zone is only present above the sec-
power view showing lymphoid white pulp (WP) and red pulp (RP). ondary follicle.(d)Localization of the thymus-independent antigen,
(c) High-power view of germinal center (GC) and lymphocyte ficoll, on the marginal zone macrophages. The ficoll is visualized by
mantle (M) surrounded by marginal zone (MZ) and red pulp (RP). labeling with the red fluorescent dye tetramethyl-rhodamine. ((b)
Adjacent to the follicle, an arteriole (A) is surrounded by the peri- Photographed by Professor P.M. Lydyard; (c) by Professor I.C.M.
arteriolar lymphoid sheath (PALS) predominantly consisting of T- MacLennan; (d)kindly provided by Professor J.H.Humphrey.)
lamina propria (figure 8.10) where they become IgA- integrin (table 8.2), the ligand for MAdCAM-1 on the
forming cells which, because they are now broadly lamina propria postcapillary venules (figure 8.11).
distributed, protect a wide area of the bowel with These T-cells bear a phenotype roughly comparable to
protective antibody. The cells also appear in the lym- that of peripheral blood lymphocytes:viz. > 95% T-cell
phoid tissue of the lung and in other mucosal sites receptor (TCR)ap and a CD4 :CD8 ratio of 7 :3. Within
guided by the interactions of specifichoming receptors the lamina propria there is also a generous sprinkling
with appropriate HEV addressins as discussed earlier. of activated B-blasts and plasma cells secreting IgA
Similarly, intranasal immunization is particularly for transport by the poly-Ig receptor to the intestinal
effective at generating antibody production in the lumen (cf. p. 53).
genitourinary tract. Intestinal intraepithelial lymphocytes (IELs) are
quite a different kettle of fish. They are also mostly T-
cells, 10-40/~of which have a yS TCR. Of those bearing
Intestinal 1ymphocytes
an ap TCR, most are CD8 positive and can be divided
The intestinal lamina propria is home to a predomi- into two populations. One-third of them possess the
nantly activated T-cell population rich in the LPAM-1 conventional form of CD8, which is a heterodimer
Figure 8.9. The IgA secretory immune system (MALT).(a)Section mucus; altogether a super picture! (c) Low-power view of human
of lung showing a diffuse accumulation of lymphocytes (Ly) in the tonsil showing the MALT with numerous secondary follicles (SF)
bronchial wall. (b) Section of human jejunum showing lymphoid containing germinal centers. (d) Peyer's patches (PP) in mouse
cells (Ly), stained green by a fluorescent antileukocyte monoclonal ileum. The T-cell areas are stained brown by a peroxidase-labeled
antibody, in the mucosal epithelium (ME) and in the lamina propria monoclonal antibody to Thy 1. ((a) Kindly provided by Professor
(LP). A red fluorescent anti-IgA conjugate stains the cytoplasm of P. Lydyard; (b) by Professor G. Jannosy; (c)by Mr C. Symes; and (d)
plasma cells (PC)in the lamina propria and detectsIgAin the surface by Dr E. Andrew.)
composed of a CD8 a chain and a CD8 P chain. cells have been found to have specificityfor heat-shock
However, two-thirds of them instead express a CD8 aa proteins, which are widely distributed in nature and
homodimer which is almost exclusively found only on usually highly immunogenic, it has been postulated
IELs. Whilst the CD8 ap IELs are restricted by classical that they act as a relatively primitive first line of
MHC class I molecules, the CD8 aa IELs appear to defense at the outer surfaces of the body.
recognize nonclassical MHC molecules (cf. p. 77) Reflect for a moment on the fact that roughly
perhaps including TL and Qal but not, appar- 1014 bacteria reside in the intestinal lumen of the
ently, CD1. The antigen specificity of most IELs is un- normal adult human. That is a pretty impressive
known. Intraepithelial lymphocytes and intraepithe- number of 'noughts' to swallow. Yet combined with
lial dendritic cells expresshigh levels of the aJ3,integrin the barrier of mucins produced by goblet cells and the
whichbinds E-cadherinonintestinalepithelia1cells. protective zone of secreted IgA antibodies, these col-
The relatively high proportion of TCR y6 cells is also lections of intestinal lymphocytes represent a crucial
unusual and most of these also express the CD8 aa line of defense.
characteristic of IELs. Since a number of cloned y6 T- It is not only the intestine that has a localized
157
Figure 8.13. Dendritic antigen-presenting cell. (a) Interdigi- teristic of the Langerhans' cell), although some IDCs in lymph
tating dendritic cell (IDC) in the thymus-dependent area of the nodes do possess these granules, perhaps after antigenic stimula-
rat lymph node. This is thought to be an antigen-presenting tion (~2000).(b) Scanning electron micrograph of a veiled cell. In
cell derived from the Langerhans' cell in the skin and dendritic contrast with these dendritic cells which present antigen to
cells in other tissues, which travels to the node in the afferent T-cells, the follicular dendritic cells in germinal centers stimulate B-
lymph as a 'veiled' cell bearing antigen on its profuse surface cells. ((a)Reproduced with permission of the authors and publishers
processes. Intimate contacts are made with the surface membranes from Kamperdijk E.W.A., Hoefsmit E.Ch.H., Drexhage H.A. & Bal-
(arrows) of the surrounding T-lymphocytes (TL). The cytoplasm four B.H. (1980) In Van Furth R. (ed.) Mononucleav Phagocytes, 3rd
of the IDC contains relatively few organelles and does not show edn. Rijhoff Publishers, The Hague. (b) Courtesy of Dr G.G.
Birbeck granules (racket-shaped cytoplasmic organelles, charac- MacPherson.)
Figure 8.14. Migration and maturation of
interdigitating dendritic cells. The
precursors of the IDCs are derived from
bone marrow stem cells. They travel via
the blood to nonlymphoid tissues. These
immature IDCs, e.g. Langerhans' cells in
skin, are specialized for antigen uptake.
Subsequently they travel via the afferent
lymphatics as veiled cells (cf.figure 8.13b)
to take up residence within secondary
lymphoid tissues (cf.figure 8.13a)where
they express high levels of MHC class I1 and
costimulatory molecules such as B7. These
cells are highly specialized for the activation
of naive T-cells.
Figure 8.15. M-cell within Peyer's patch epithelium. (a) Scanning lymph nodes. E, enterocyte; L, lymphocyte; M@,macrophage. (c)
electron micrograph of the surface of the Peyer 'S patch epithelium. Electron photomicrograph of an M-cell (M in nucleus) with adjacent
The antigen-sampling M-cell in the center is surrounded by absorp- lymphocyte (Lin nucleus). Note the flanking epithelia1cells are both
tive enterocytes covered by closely packed, regular microvilli. Note absorptive enterocytes with a typical brush border. In some cases,
the irregular and short microfolds of the M-cell. (Reproduced with proteases on the surface of the M-cells modify the pathogen so that it
permission of the authors and publishers from Kato T. & Owen R.L. canadhere and be takenup. Pathogenic Salmonella caninvade and de-
(1999)In Ogra R. et al. (eds)Mucosal Immunology, 2nd edn. Academic stroy M-cells, making a hole through which other bacteria can invade
Press, San Diego.) (b)After uptake and transcellular transport by the the underlying tissue. (Lead citrate and uranyl acetate, X 1600.)((b)
M-cell (M),antigen is processed by macrophages and thence by IDCs BasedonSminiaT.&KraalG. (1998)InDelvesP.J.&RoittI.M.(eds)En-
which present antigen to T-cells in Peyer's patches and mesenteric cyclopediaof immunology, 2ndedn,p. 188.AcademicPress, London.)
follicular dendritic cells, nor generate memory B-cells drites on the follicular cells, to which the immune com-
in response to T-dependent antigens (see p. 171). plexes are bound, form into beads which break off
Classically,a secondary response would be initiated as structures called 'iccosomes' (immune complex-
at the T-helper level by antigen, alone or as a complex, coated bodies). These bind to germinal center B-cells
being taken up by IDCs and macrophages. However, which then endocytose and process the antigen for
the capture of immune complexes on the surface of presentation by the B-cell MHC class 11, and sub-
FDCs opens up an alternative pathway. One to three sequent stimulation of T-helper cells to kick off the
days after secondary challenge, the filamentous den- secondary response.
cialized antigen-transporting cells with short, irregu-
lar microvillae, strong nonspecific esterase activity
and no MHC class 11. They overlay intraepithelial
The mucosal surface is in the front line facing an un- lymphocytes and macrophages (figure 8.15b and c).
friendly sea of microbes and, generally, antigens are Foreign material, including bacteria, is taken up
excluded by the epithelium with its tight junctions and by M-cells and passed on to the underlying antigen-
mucous layer. Gut lymphoid tissue is separated from presenting cells which, in turn, migrate to the
the lumen by a layer of columnar epithelium inter- local lymphoid tissue to activate the appropriate
spersed with microfold (M)-cells (figure 8.15a); spe- lymphocytes.
@her sires
Bonemarrow isa majorsiteofantibodyproduction
The brain, anterior chamber of the eye and testis eethe occomponying website (www.roln.corn)
are privileged sites in which antigens can be safely ir multiplechoice questions.
sequestered.
FURTHER READING Cyster J.G. (1999) Chemokines and cell migration in secondary
lymphoid organs. Science 286,2098.
Barclay A.N. et al. (1997) The Leucocyte Antigen Factsbook, 2nd edn. Grabbe S., Kampgen, E. & Schuler G. (2000) Dendritic cells: multi-
Academic Press, London. lineal and multi-functional. Immunology Today 21,431.
Bell D.,Young J.W. & Banchereau J. (1999)Dendritic cells.Advances in Pringault E. (ed.) (1999)M cell development and function in physiol-
Immunology 72,255. ogy and disease. Seminars in Immunology 11,155.
Butcher E.C. et al. (1999) Lymphocyte trafficking and regional Shaw S., Turni L.A. & Katz K.S. (eds) Protein Reviews on the Web
immunity.Advances in Immunology 72,209. (PROW).http:/ /www.ncbi.nlm.nih.gov/prow.
Caligaris-Cappio F. (1998) Germinal centers. In Delves P.J. & Roitt Shimizu Y., Rose D.M. & Ginsberg M.H. (1999) Integrins in the
I.M. (eds) Encyclopedia of immunology, 2nd edn, p. 992. Academic immune system. Advances in Immunology 72,325.
Press, London.
CHAPTERQ
Following TCR signaling, there is an early increase in Within 15 seconds of TCR stimulation, the yl isoform
the level of active Ras-GTP (guanosine triphosphate) of phospholipase C (PLCyl), an enzyme which (like
complexes which regulate pivotal mitogen-activated the isoform) activates the phosphatidylinositol
protein kinases (MAPK),such as JNK or ERK, through pathway, is phosphorylated and its catalytic activity
sequential kinase cascades. Thus, in one of the phos- increased. This early increase in phospholipase C ac-
phorylation amplifying cascades, MEK (MAP-ERK tivity accelerates the hydrolysis of phosphatidylinosi-
kinase) acts as a MAP kinase kinase and Raf as a to1 4,5-biphosphate (PIP,) to diacylglycerol (DAG)
MAP kinase kinase kinase or MAPKKK (figure9.6)!As and inositol 1,4,5-triphosphate (IP,) (figure 9.7). The
illustrated in figure 9.7, this is just one of a number of triphosphate binds to specific receptors on specialized
calcium storage vesicles and triggers the release of
Ca2+into the cytosol; this is supplemented by an influx
from the external milieu. The raised ca2+level has at
least two consequences. First, it synergizes with di-
acylglycerol to activate protein kinase C (PKC); sec-
ond, it acts together with calmodulin to increase the
activity of calcineurin.
Figure M9.1.2.The antibody response to a thymus-dependent which fails to givearespnme in neonatally thyrnectomizedmice;
antigen requires two different cell populations. Different figure M9.1.1) and examined for the production of antibody after
populations of cells from a normal mouse histocompatible with Zweeks. The small amount of antibody synthesized by animals
the recipient (i.e. of the same H-2 haplotype) were injected into receiving bone marrow alone is due to the presence of thymocyte
recipients which had been X-irradiated to destroy their own prrcursors in the cell inoculum which differentiate in the intact
lymphocyte responses. They were then primed with a thymus- thymusglandoftherecipient.
dependent antigen such a s sheep red blood cells (i.e. an antigen
lmmunocompetentT- and B-cells differ in many respects the MAP kinase kinase MEK and the MAP kinase ERK.
The antigen-specific receptors, TCR/CD3 on T-cells CD28 through PI3 kinase can also influence MAP kinase.
and surface Ig on B-cells, provide a clear distinction The transcription factors Fos and Jun, NFAT and NFKB
between these two cell types. are activated by MAP kinase, calcineurinand PKC, respec-
T- and B-cells differ in their receptors for C3d, IgG and tively, and bind to regulatory sites in the IL-2 promoter
certain viruses. region.
There are distinct polyclonal activatorsof T-cells (PHA, A small number of MHC-peptide complexes can
anti-CD3) and of B-cells (anti-Ig,Epstein-Barr virus). serially trigger a much larger number of TCRs thereby
providing the sustained signal required for activation.
T-lymphocytes and antigen-presentingcells interact through Initial binding of integrins facilitates the formation of
pairs of accessory molecules an immunological synapse, the core of which exchanges
The docking of T-cells and APCs depends upon strong integrins for TCRinteractingwith MHC-peptide.
mutual interactions between complementary molecular B7 delivers a negative signal through CTLA-4, and Cbl
pairs on their surfaces: MHC II-CD4, MHC I-CD8, family adaptor molecules are also involved in negative
VCAM-1-VLA-4, ICAM-1-LFA-1, LFA-3-CD2, B7-CD28 signalingpathways.
(and CTLA-4). The phosphatase domains on CD45 are required to
remove phosphates at inhibitorysites on kinases.
Activation of T-cells requires two signals
Two signals activate T-cells, but one alone produces B-cells respond to three different types of antigen
unresponsiveness(anergy). Type 1thymus-independent antigens are polyclonal ac-
One signal is provided by the low affinity cognate tivators focused onto the specific B-cells by sIg receptors.
TCR-MHC plus peptide interaction. Type 2 thymus-independent antigens are polymeric
The second costimulatory signal is mediated through molecules which cross-link many sIg receptors and, be-
ligation of CD28 by 87. cause of their long half-lives,provide a persistent signal to
the B-cell.
Protein tyrosine phosphorylation is an early event in Thymus-dependentantigens requirethe cooperationof
T-cell signaling helper T-cells to stimulate antibody production by B-cells.
The TCR signal is transduced and amplified through a Antigen captured by specific sIg receptors is taken into
protein tyrosine kinase (PTK)enzymic cascade. the B-cell, processed and expressed on the surface as a
CD4/TCR colocation leads to phosphorylation of peptide in associationwith MHC 11.
ITAM sequences on CD3-associated 6 chains by the CD4- This complex is recognized by the T-helper cell which
associated Lck PTK. The phosphorylated ITAMs bind and activates the resting B-cell.
then activate the ZAP-70 kinase. The ability of protein carriers to enable the antibody
response to haptens is explained by T-B collaboration,
Downstreamevents following TCR signaling with T-cells recognizing the carrier and B-cells the hapten.
Nonenzymic adaptor proteins form multimeric com-
plexes with kinases and guanine nucleotide exchange The nature of B-cell activation
factors (GEFs). Cross-linking of surface Ig receptors (e.g. by type 2
Hydrolysis of phosphatidylinositol diphosphate by thymus-independent antigens) activatesB-cells.
phospholipase Cyl or C P produces inositol triphosphate T-helper cells activate resting B-cells through TCR
(IP,) and diacylglycerol(DAG). recognition of MHC II-carrier peptide complexes and co-
IP, mobilizes intracellular calcium. stimulation through CD4OL-CD40 interactions (analo-
DAG and increased calcium activate protein kinase C. gous to the B7-CD28 second signal for T-cell activation).
The raised calcium together with calmodulin also
stimulates calcineurin activity.
Activation of Ras by the guanine nucleotide exchange
factor SOSsets off a kinase cascade operating through Raf,
Myung P.S., Boerthe N.J. & Koretzky G.A. (2000) Adaptor proteins
FURTHER READING in lymphocyte antigen-receptor signaling. Current Opinion in
Acuto 0 . & Cantrell D. (2000) T cell activation and the cytoskeleton. Immunology 12,256.
Annual Reviews in Immunology 18,165. Olson M.F. & Marais R. (2000) Ras protein signalling. Seminars in
Bromley S.K., Burack W.R., Johnson K.G. et al. (2001) The immuno- Immunology 12/63.
logical synapse. Annual Review of Immunology 19,375. Oosterwegel A. et al. (1999) CTLA-4 and T cell activation. Current
Grakoui A. et al. (1999) The immunological synapse: a molecular Opinion in Immunology 11,294.
machine controlling T cell activation. Science 285,221. Schraven B. et al. (1999) Integration of receptor-mediated signals
Jenkins M.K., Khoruts A., Ingulli E. et al. (2001) In vivo activation of in T cells by transmembrane adaptor proteins. Immunology Today
antigen-specificCD4 T cells. Annual Review of Immunology 19/23. 20,431.
Michel G. (ed.) (1999) Biochemical Pathways: An Atlas of Biochemistry
and Molecular Biology.John Wiley & Sons, New York.
The production of effectors
Interferon receptors
including IL-1 and tumor necrosis factor (TNF), also These also consist of two polypeptide chains and, in
exist in membrane forms which can exert their stimu- addition to the IFNa, IFNP and IFNy receptors (figure
latory effects without becoming soluble. 10.2b),this family includes the IL-10 receptor.
T N F receptors
Cytokines are highly potent, often acting at femto- The receptor polypeptides are cysteine rich and
molar (10-l5M ) concentrations, combining with small trimerize following cytokine binding. They include
numbers of high affinity cell surface receptors to pro- the tumor necrosis factor (TNF)receptor (figure 1 0 . 2 ~ ) ~
duce changes in the pattern of RNA and protein lymphotoxin (LT) and nerve growth factor (NGF) re-
synthesis. A common feature in the triggering of the ceptors, and the cell surface-associated molecules
cytokine receptors is the ligand-induced associationof CD40 and CD95 (Fas).
receptor subunits which allows signal transduction
through the interplay of their juxtaposed cytoplasmic
&SF cytokine receptors
domains. There are six major cytokine receptor struc-
tural families (figure 10.2). Immunoglobulin superfamily members are broadly
utilized in many aspects of cell biology (cf.p. 245) and
include the IL-1 receptor (figure 10.2d), and the
Hematopoietin receptors
macrophage colony-stimulating factor (M-CSF) and
These are the largest family, sometimes referred to stem cell factor (SCF/c-kit)receptors.
simply as the cytokine receptor superfamily. These
receptors generally consist of one or two polypeptide
Chemokine receptors
chains responsible for cytokine binding and an addi-
tional shared (common or 'c') chain involved in signal As we shall discover shortly, these comprise a family
transduction. The yc (CD132)chain is used by the IL-2 of approximately 20 different G-protein-coupled,
Table 10.1. Cytokines:their originand function.
APP, acute phase proteins; B, B-cell; baso, basophil; BM, bone marrow; Endo, endothelium; eosino, eosinophil; Epith, epithelium;Fibro, fibro-
blast; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LIF, leukemia inhibitory factor; M$, macrophage; MC,
mast cell; Mono, monocyte; neutro, neutrophil;NK, natural killer; SLF, steel locus factor;T, T-cell; TGFp ,transforminggrowth factor-p.Note that
there is not an interleukin-14.This designationwas given to an activity that, upon further investigation, could not be unambiguously assigned
to a single cytokine. IL-8 is a member of the chemokine family.These cytolines are listed separatelyin table 10.3.
Figure 10.2. Cytokine receptor families. One example is shown for tor, the a chain binds the IL-2 rapidly and facilitates its binding to a
each family. (a) The hematopoietin receptors operate through a separate site on the p chain from which it can only dissociate slowly.
common subunit (yc, pc or gp130, depending on the subfamily) Since the final affinity (Kd) is based on the ratio of dissociation
which transduces the signal to the interior of the cell. In essence, to association rate constants, then Kd = ~ o ~ s ~ c - ~ / ~ o ~ M - ~ s ~ c - ~
binding of the cytokine to its receptor must initiate the signaling 10-l1M,which is a very high affinity. The y chain does not itself bind
process by mediating hetero- or homodimer formation involving IL-2 but contributestowards signal transduction. (b)The interferon
the common subunit. In some cases the cytokine is active when receptor family consists of heterodimeric molecules each of which
bound to the receptor either in soluble or membrane-bound form bears two fibronectintype I11domains. (c)The receptors for TNF and
(e.g. IL-6). The IL-2 receptor is interesting with respect to its ligand related molecules consist of a single polypeptide with four TNFR
binding. The a chain (CD25, reacting with the Tac monoclonal)of the domains. The receptor trimerizes upon ligand binding and, in com-
receptor possesses two complement control protein structural do- mon with a number of other receptors, is also found in a soluble form
mains and binds IL-2 with a low affinity; the P chain (CD122)has a which, when released from a cell following activation, can act as an
membrane proximal fibronectin type I11 structural domain and a antagonist. (d) Another group of receptors contains varying num-
membrane distal cytokine receptor structural domain, and associ- bers of Ig superfamily domains, whereas (e) chemokine receptors
ates with the common ychain (CD132)which has a similar structural are members of the G-protein-coupled receptor superfamily and
organization.The P chain binds IL-2 with intermediate affinity. IL-2 have seven hydrophobictransmembranedomains. (f)The final fam-
binds to and dissociates from the a chain very rapidly but the same ily illustrated are the TGF receptors which require association be-
processes involving the p chain occur at two or three orders of mag- tween two molecules, referred to as TGFR type I and TGFR type 11,
nitude more slowly. When the a, P and y chains form a single recep- for signalingto occur.
seven transmembrane segment, receptors (figure and the Ras-MAP kinase pathways. Members of the
10.2e). cytokine receptor superfamily (hematopoietin re-
ceptors)lack catalytic domains and therefore associate
with JAKs including JAK1, JAK2, JAK3 and Tyk2.
TGF recep tors
Upon cytokine-induced receptor dimerization, the
Receptors for transforming growth factors such as JAKs reciprocally phosphorylate, and thereby acti-
the TGFP receptor (figure 10.2f) possess cytoplasmic vate, each other. Once activated, they can phosphory-
portions with serine/ threonine kinase activity. late the cytoplasmic domains of the receptors to create
binding sites for SH2-containing signaling proteins.
These include one or more of the group of transcription
factors termed STATs (signal transducers and activa-
The ligand-induced homo- or heterodimerization of tors of transcription),which also become phosphory-
cytokine receptor subunits represents a common lated by the workaholic JAKs, an event which causes
theme for signalingby cytokines.The two major routes the STATsto dissociatefrom the receptor and dimerize.
that are utilized are the Janus kinase (JAK)-STAT The dimerized STATs then translocate to the nucleus
and there is considerable overlap and redundancy
between them with respect to individual functions,
partially accounted for by the sharing of receptor com-
ponents and the utilization of common transcription
factors. For example, many of the biological activities
of IL-4 overlap with those of IL-13. However, it should
be pointed out that virtually all cytokines have at least
some unique properties.
Their roles in the generation of T- and B-cell effec-
tors, and in the regulation of chronic inflammatory
reactions (figure 10.4a and b), will be discussed
at length later in this chapter. We should note here
the important role of cytokines in the control of
hematopoiesis (figure 10.4~).The differentiation of
stem cells to become the formed elements of blood
within the environment of the bone marrow is care-
fully nurtured through the production of cytokines
by the stromal cells. These include GM-CSF, G-
CSF (granulocyte colony-stimulating factor), M-CSF,
IL-6 and -7 and LIF (see table 10.1),and many of them
are also derived from T-cells and macrophages. It is not
surprising therefore that, during a period of chronic
inflammation, the cytokines that are produced recruit
new precursors into the hematopoietic differentiation
Figure 10.3. Cytokine receptor-mediated pathways for gene tran-
pathway-a useful exercise in the circumstances.
scription. Cytokine-induced receptor oligomerization activates the
associated JAK kinases. These phosphorylate the STAT DNA- One of the cytokines, IL-3, should be highlighted for
binding transcription factors which dimerize and translocate to the its exceptional ability to support the early cells in
nucleus. Cytokine receptors can also activate src family kinases this pathway, particularly in synergy with IL-6 and
which, via adaptor proteins such as She, Grb2 and SHP-2, could
G-CSF.
generate transcription factors via the two routes shown. MAPK,
mitogen-activated protein kinases (see figure 9.7); MEK, kinase
activator of MAPK; PI-3 kinase, phosphatidylinositol3-kinase.
In general, cytokines are pleiotropic, i.e. exhibit Helper T-cell clones can be divided into two main
multiple effects on a variety of cell types (table 10.1), types with distinct cytokine secretion phenotypes
Table 10.2. Cytokine patterns of helper T-cell clones. Interleukin-
10is not listed in the table. Although classed as a Th2 cytokine in the
mouse, it is produced by both Thl and Th2 cells in the human.
with clones which had been maintained in culture for ticularly important for the production of Thl cells and
long periods and might have been artifacts of condi- IL-4 for the production of Th2 cells. Invasion of phago-
tions in vitro. The use of cytokine-specific monoclonal cytic cells by intracellular pathogens induces copious
antibodiesfor intracellular fluorescent staining,and of secretion of IL-12, which in turn stimulates IFNy pro-
ELISPOT assays (cf. p. 140) for the detection of the se- duction by NK cells. These two cytokines selectively
creted molecules, has demonstrated that the Thl /Th2 drive differentiation of Thl development and inhibit
dichotomy is also apparent in freshly sampled cells Th2 responses (figure 10.6). However, IL-4 effects
and thus also applies in vivo. Nonetheless, it is perhaps appear to be dominant over IL-12 and therefore the
best not to be too rigidly constrained in one's thinking amounts of IL-4 relative to the amounts of IL-12 and
by the Thl/Th2 paradigm, but rather to look upon IFNy will be of paramount importance in determining
T-cells as potentially producing a whole spectrum of the differentiation of ThO cells into Thl or Th2 (figure
cytokine profiles (ThO, figure 10.6), with possible 10.7).IL-4 downregulates the expression of the IL-12R
skewingof the responses towards the extreme Thl and p, subunit necessary for responsiveness to IL-12,
Th2 patterns depending on the nature of the antigen further polarizing the 73.2dominance.
stimulus. Thus, other subsets may also exist, in partic- A special cell population, the NK-T cells bearing the
ular the transforming growth factor-p (TGFP)and IL- NK1.1+ marker, rapidly releases an IL-4-dominated
10-producing Th3/Trl (T-regulatory 1) cells, which pattern of cytokines on stimulation. These cells have
are of interest because these cytokines can mediate im- many unusual features. They may be CD4-8- or
munosuppressive effects and may be involved in the CD4'8- and express low levels of T-cell ap receptors
induction of mucosally induced tolerance (cf.p. 444). with an invariant a chain and very restricted P, many
of these receptors recognizing the nonclassical MHC-
like CD1 molecule. Their morphology and granule
content are intermediatebetween T-cells and NK cells.
Although they express TCRaP, there is an inclination
Antigen-presenting cells, and in particular dendritic to classify them on the fringe of the 'innate' immune
cells, appear to be pivotal in driving differentiation to- system with regard to their primitive characteristics
wards a Thl or Th2 phenotype. IL-12 seems to be par- and possession of the lectin-likeNK1. l receptor which
populations, this is an area that is still under intensive
investigation. However, it should be obvious from the
above discussion that the cytokines produced in the
immediate vicinity of the T-cell will be important. To
give one recent example, Cantor and colleagues
homologously deleted the gene for the cytokine Eta-l
(osteopontin) in mice and found that these animals
had severely impaired immunity to infection with
herpes simplex virus and to the intracellularbacterium
Listeria monocytogenes. This was due to a deficient Thl
immunity caused by reduced IL-12 and IFNy and en-
hanced IL-10 production. It appears that Eta-l produc-
tion by activated T-cells stimulates IL-12 production
by macrophage lineage cells and downregulates IL-10
production. Interestingly, both serine phosphorylated
and nonphosphorylated forms of Eta-l are secreted
by T-cells, and the IL-12 effect is phosphorylation-
dependent whereas the IL-10 effect is not, indicating
that phosphorylation can regulate the activity of
secreted proteins.
Figure 10.6. The generation of Thl and Th2 CD4 subsets. It is en-
In so far as T-cells are concerned, amplification follow-
visaged that, following the initial stimulation of T-cells, a range of ing activation is critically dependent upon IL-2 (figure
cells producing a spectrumof cytokinepatterns emerges. Under dif- 10.8). This cytokine is a single peptide of molecular
ferent conditions, the resulting population can be biased towards weight 15.5kDa which acts only on cells which express
two extremes.IL-12, possibly produced through an 'innate1-type ef-
fect of an intracellular infectionon macrophages,encourages the de- high affinity IL-2 receptors (figure 10.2).These recep-
velopment of Thl cells which produce the cytokinescharacteristicof tors are not present on resting cells, but are synthesized
cell-mediated immunity. IL-4, possibly produced by interaction of mi- within a few hours after activation.
croorganisms with the lectin-like NK1.1+ receptor on NK-T cells, Separation of an activated T-cell population into
skews the development to the production of Th2 cells whose cy-
tokines assist the progression of B-cells to antibodysecretionand the
those with high and low affinity IL-2 receptors showed
provision of humoral immunity. Cytokines produced by polarized clearly that an adequate number of high affinity re-
Thl and Th2 subpopulations are mutually inhibitory. LT, lyrnpho- ceptors were mandatory for the mitogenic action of
toxin (TNFP);Thp, T-helper precursor; ThO, early helper cell produc- IL-2. The numbers of these receptors on the cell in-
ing a spectrumof cytokines; other abbreviations as in table 10.1.
crease under the action of antigen and of IL-2 and, as
antigen is cleared, so the receptor numbers decline
may be involved in the recognition of microbial and, with that, the responsiveness to IL-2. It should be
carbohydrates. appreciated that, although IL-2 is an immunologically
Whilst there is a certain amount of evidence indicat- nonspecific T-cell growth factor, it only functions
ing the existence of subpopulations of dendritic cells appropriately in specific responses because unstimu-
specialized for the stimulation of either Thl or Th2 lated T-cells do not express IL-2 receptors.
Figure 10.7. Polarization of T-helper responses is driven by den- by the presence of IL-12 and IFNy. The ThO cells express both the P,
dritic cells. Initial activation of the ThO cell is dependent upon pre- and P2chains of the IL-12 receptor, but IL-4 causes loss of expression
sentation of peptide-MHC to the TCR, together with costimulatory of the P, chain. Whilst IL-12 is predominantly a product of the
signals such as that provided by CD80 and CD86 (B7.1 and 87.2 re- macrophage/dendritic cell lineage, IL-4 is produced by a number of
spectively) on the dendritic cell. Subsequently,the T-cells are driven cell types including mast cells, yS T-cells, NK-T cells, ThO and Th2
towards a Th2 phenotype by the presence of IL-4 or a Thl phenotype cells.
Early events
The initiating event is probably a local inflammatory
Figure 10.8. Activated T-blasts expressing surface receptors for
response to tissue injury caused by the infectious agent
IL-2 proliferatein response to IL-2 produced by itself or by another which would upregulate the synthesis of adhesion
~-celisubset. ~ x ~ a n s i oisncontrolled through downregilation of molecules such as VCAM-1 (vascular cell adhesion
the IL-2 receptoi by IL-2 itself. The expandid populati& secretes
a wide variety of biologically active cytokines of which IL-4 also
molecule)and ICAM-1 on adjacent
vascular endothe-
enhances T-cell proliferation.
lial cells. These would permit entry of memory T-cells
to the infected site through their VLA-4 and LFA-1
homing receptors (cf. p. 152). Contact with processed
Figure 10.9. Cytokines controlling the antibody and T-cell- location, whereas C chemokines lack cysteines 1 and
mediated inflammatory responses.Abbreviationsas in table 10.1. 3 found in other chemokines. Chemokines bind to
Gprotein-coupled seven transmembrane receptors
antigen derived from the intracellular parasite will (figure 10.2).Despite the fact that a single chemokine
activate the specific T-cell and induce the release of can sometimes bind to more than one receptor, and a
secreted cytokines. TNF will further enhance the single receptor can bind several chemokines, many
expression of endothelial accessory molecules and chemokines exhibit a strong tissue and receptor
increase the chances of other memory cells in the cir- specificity.They play important roles in inflammation,
culation homing in to meet the antigen provoking lymphoid organ development, cell trafficking, cell
inflammation. compartrnentalizationwithin lymphoid tissues, Thl /
Th2 development,angiogenesisand wound healing.
Chemotaxis
Macvophage activation
The recruitment of T-cells and macrophages to the in-
flammatory site (figure 10.9)is greatly enhancedby the Macrophages with intracellular organisms are ac-
action of chemotactic cytokines termed chemokines tivated by agents such as IFNy, GM-CSF, IL-2 and TNF
(chemoattractant cytokine). These can be potentially and should become endowed with microbicidal
produced by a variety of cell types and are divided into powers. During this process, some macrophages may
four families based on the disposition of the first (N- die (probablyhelped alongby cytotoxic T-cells)and re-
terminal) two of the four canonical cysteine residues lease living parasites, but these will be dealt with by
(table10.3).CXC chemokineshave one amino acid and fresh macrophages brought to the site by chemotaxis
CX3C have three amino acids between the two cys- and newly activated by local cytokines so that they
teines. CC chemokines have adjacent cysteines at this have passed the stage of differentiation at which the
Table 10.3. Chemokines and their receptors.The chemokines are grouped according to the arrangement of their cysteines (see text).The letter
L designates ligand (i.e. the individual chemokine), whereas the letter R designates receptors. Names in parentheses refer to the murine ho-
mologs of the human chemokinewhere the names of these differ, or the murine chemokine alone if no human equivalenthas been described.
B, B-cell; Baso, basophil; DC, dendritic cell; Eosino, eosinophil; Mono, monocyte; Neutro, neutrophil; NK, natural killer; T, T-cell.
intracellular parasites can subvert their killing mecha- induce 2'-5' (A) synthetase, a protein which is in-
nisms (cf.p. 263). volved in viral protection. TNF has another string to its
bow in its ability to kill certain cells, since death of an
infected cell before viral replication has occurred is
Combating viral infection
obviously beneficial to the host. Its cytotoxic potential
Virally infected cells require a different strategy and was first recognized using tumor cells as targets (hence
one strand of that strategy exploits the innate inter- the name), and IFNy and lymphotoxin can act syner-
feron mechanism to deny the virus access to the cell's gistically, with IFNy setting up the cell for destruction
replicative machinery. IFNy, TNF and lymphotoxinall by inducing the formation of TNF receptors.
Thegeneration of cytotoxic T-cells
Cytotoxic T-cells (Tc), also referred to as cytotoxic T-
lymphocytes (CTLs),represent the other major arm of
the cell-mediated immune response and are of strate-
gic importance in the killing of virally infected cells
and possibly in contributing to the postulated surveil-
lance mechanisms against cancer cells.
The cytotoxic cell precursors recognize antigen on
the surface of cells in association with class I major his-
tocompatibility complex (MHC) molecules and, like
B-cells, they usually require help from T-cells. The
mechanism by which help is proffered may, however,
be quite different. As explained earlier (see p. 173),
effective T-B collaboration is usually 'cognate' in that
the collaborating cells recognize two epitopes which
are physically linked (usuallyon the same molecule).If
we may remind the reader without causing offense, the
reason for this is that the surface Ig receptors on the
B-cell capture native antigen, process it internally and
present it to the Th as a peptide in association with
MHC class 11.Although it has been shown that linked
epitopes on the antigen are also necessary for coopera-
tion between Th and the cytotoxic T-cell precursor
(Tcp), the nature of T-cell recognition prevents native
antigen being focused onto the Tcp by its receptor for
subsequent processing,even if that cell were to express Figure 10.10. T-helper cell activation of cytotoxic T-cells. Activa-
tion of the CD4+helper T-cells (Th)by the dendritic cell involves a
MHC 11, which in its resting state it does not. It seems CD40-CD40 ligand (CD154)costimulatory signal and recognitionof
most likely that Th and Tcp bind to the same APC, for an MHC class I1 peptide presented by the T-cell receptor. (a) If both
example a dendritic cell, which has processed viral the Th and the cytotoxic T-lymphocyte (Tc) are present at the same
antigen and displays processed viral peptides in asso- time, the release of cytokines from the activated Th cells stimulates
the differentiation of the CD8+ precursor into an activated, MHC
ciation with both class I1 (for the Th cell) and class I (for class I-restricted Tc. However, as shown in (b), the Th and the Tc do
the Tcp) on its surface; one cannot exclude the possibil- not need to interact with the APC at the same time. In this case, the Th
ity that the APC could be the virally infected cell itself. cell 'licenses' the dendritic cell for future interaction with a Tc cell.
Cytokines from the triggered Th will be released in Thus the Th cell, by engaging CD40, drives the dendritic cell from a
resting state into an activated state with upregulation of costimula-
close proximity to the Tcp which is engaging the tory molecules such as B7.1 and B7.2 (CD80 and CD86, respectively)
antigen-MHC signal and will be stimulated to prolif- and increased cytokine production, particularly of IL-12.
erate and differentiate into a Tc under the influence of
IL-2 and -6 (figure 10.10a).However, interaction of the
APC with the Th and the Tc cell can be temporally sep- possibly involving adjuvant-induced production of
arated and, in this case, it appears that the helper T-cell proinflammatory cytokines and cell surface costimu-
'licenses' the dendritic cell for future interaction with latory molecules.
the cytotoxic T-cell. It does this by activating the den-
dritic cell through CD40, thereby upregulating co-
The lethal process
stimulatory molecules and cytokine production, in
particular IL-12, by the dendritic cell (figure 10.10b). Cytotoxic T-cells (Tc) are generally of the CD8 subset,
An entirely Th-independent mechanism of Tc activa- and their binding to the target cell through T-cell recep-
tion is also thought to occur. This has been demon- tor recognition of antigen plus class I MHC is assisted
strated in, for example, the response to protein by association between CD8 and class I and by other
antigens given with potent adjuvants such as im- accessory moleculessuch as LFA-1and CD2 (seefigure
munostimulatory DNA sequences (ISSs), in this case 9.3).
Tc are unusual secretory cells which use a modified granules provide the dominant attack on virally in-
lysosome to secrete their lytic proteins. Following de- fected cells. Videomicroscopy shows that Tc are
livery of the TCR/CD3 signal, the lytic granules are serial killers. After the 'kiss of death', the T-cell can
driven at a rare old speed (up to 1.2pm/sec) along the disengage and seek a further victim, there being
microtubule system and delivered to the point of con- rapid synthesisof new granules.
tact between the Tc and its target (figure 10.11).This One should also not lose sight of the fact that CD8
guarantees the specificity of killing dictated by TCR cells synthesize other cytokines such as IFNy which
recognition of the target and limits any collateral dam- also have antiviral potential.
age to surrounding cells. As with NK cells, which have
comparable granules (cf. p. l$), exocytosis of the
granule contents, including perforins, granzymes and
TNF, causes lesions in the target cell membrane and Once the inflammatory process has cleared the inciting
death by inducing apoptosis. Tc are endowed with a agent, the body needs to switch it off. IL-10 has pro-
second killing mechanism involvingFas and its ligand found anti-inflammatory and immunoregulatory ef-
(cf.p. l9),but the inability of perforin knockout mice to fects, acting on macrophages and Thl cells to inhibit
clear viruses effectively suggests that the secretory release of factors such as IL-1 and TNF. It induces the
release of soluble TNF receptors which are endoge-
nous inhibitors of TNF, and downregulates surface
TNF receptor. Soluble IL-1 receptors released during
inflammation can act to 'decoy' IL-1 itself.IL-4 not only
acts to constrainThl cells but also upregulates produc-
tion of the natural inhibitor of IL-1, the IL-1 receptor
antagonist (IL-1Ra).The role of TGFP is more difficult
to tease out because it has some pro- and other
anti-inflammatory effects, although it undoubtedly
promotes tissue repair after resolution of the
inflammation.
P R O L I F E R A T I O N A N D M A T U R A T I O N OF
B-CELL R E S P O N S E S ARE M E D I A T E D
BY C Y T O K I N E S
The activation of B-cells by Th, through the TCR
recognition of MHC-linked antigenic peptide plus
the costimulatory CD40L-CD40 interaction, leads to
upregulation of the surface receptor for IL-4. Copious
local release of this cytokine from the Th then drives
powerful clonal proliferation and expansion of the
activated B-cell population. IL-2 and IL-13 also con-
tribute to this process (figure 10.12).
Under the influence of IL-4 and IL-13, the expanded
clones can differentiate and mature into IgE synthesiz-
ing cells. TGFP and IL-5 encourage cells to switch their
Ig class to IgA. IgM plasma cells emerge under the
tutelage of IL-4 plus -5, and IgG producers result
from the combined influence of IL-4, -5, -6, -13 and
Figure 10.11. Conjugation of a cytotoxic T-cell (on left) to its target,
here a mouse mastocytoma, showing polarization of the granules to-
IFNy (figure 10.12).
wards the target at the point of contact. The cytoskeletons of both Type 2 thymus-independent antigens can activate
cells are revealed by imrnunofluorescent staining with an antibody B-cells directly (cf. p. 171) but nonetheless still
to tubulin (green) and the lytic granules with an antibody to need cytokines for efficient proliferation and Ig
granzyme A (red). Twenty minutes after conjugation the target cell
cytoskeleton may still be intact (above), but this rapidly becomes
production. These may come from accessory cells
disrupted (below). (Photographs kindly provided by Dr Gillian such as NK and NK-T cells which bear lectin-like
Griffiths.) receptors.
migrate to the germinal center. There they divide in
response to powerful stimuli from complexes on
follicular dendritic cells (cf.p. 160)and from cytokines
released by T-cells in response to antigen-presentingB-
cells. During this particularly freneticbout of cell divi-
sion, somatic hypermutation of B-cell Ig genes occurs.
The cells also undergo Ig class switching. Thereafter,
as they transform to centrocytes, they are vulnerable
and die readily, whence they are taken up as the 'tingi-
ble bodies1by macrophages, unless rescued by associ-
ation with antigen on a follicular dendritic cell. This
could result from cross-linking of surface Ig receptors
and is accompanied by expression of bcl-2 which pro-
tects against apoptosis. Signaling through CD40, by
presentation of antigen to Th cells, would also prolong
the life of the centrocyte.In either case, the interactions
will only occur if the mutated surface Ig receptor still
binds antigen and, as the concentration of antigen
gradually falls, only if the receptor is of high affinity.
In other words, the system can deliver high affinity
antibody by a Darwinian process of high frequency
mutation of the Ig genes and selection by antigen of
the cells bearing the antibody which binds most
Figure 10.12. B-cell response to thymus-dependent(TD) antigen:
clonal expansionand maturation of activatedB-cells under the in- strongly (figure 10.14).This increase of affinity as the
fluence of T-cell-derived soluble factors. Costimulation through antibody level falls late in the response is of obvious
the CD40L-CD40 interactionis essential for primary and secondary benefit, since a small amount of high affinity antibody
immune responses to TD antigens and for the formationof germinal can do the job of a large amount of low affinity (as in
centers and memory. c-myc expression, which is maximal 2hours
after antigen or anti-y stimulation, parallels sensitivity to growth boxing, a small 'goodun' will generally be a match for a
factors; transfection with c-myc substitutes for anti-p. mediocre 'bigun').
Further differentiation now occurs. The cells either
migrate to the sites of plasma cell activity (e.g. lymph
WHAT I S GOING ON I N THE node medulla) or go to expand the memory B-cell pool
GERMINAL CENTER?
depending upon the cytokine and other signals re-
The secondary follicle with its corona or mantle of ceived. CD40 engagement by CD40 ligand on a T-cell
small lymphocytes surrounding the pale germinal guides the B-cell into the memory compartment.
center is a striking and unique cellular structure. First,
let us recall the overall events described in Chapter 8.
THE SYNTHESIS OF ANTIBODY
Secondary challenge with antigen or immune com-
plexes induces enlargement of germinal centers, for- The sequential processes by which secreted Ig arises
mation of new ones, appearance of memory B-cells are illustrated in figure 10.15. In the normal antibody-
and development of Ig-producingcells of higher affin- forming cell there is a rapid turnover of light chains
ity. B-cells entering the germinal center become cen- which are present in slight excess.Defective control oc-
troblasts which divide with a very short cycle time of curs in many myeloma cells and one may see excessive
6hours, and then become nondividing centrocytes in production of light chains or complete suppression of
the basal light zone, many of which die from apoptosis heavy chain synthesis.
(figure 10.13). As the surviving centrocytes mature, The variable and constant regions are spliced to-
they differentiate either into immunoblast plasma cell gether in the mRNAbefore leaving the nucleus. Differ-
precursors, which secrete Ig in the absence of antigen, ential splicing mechanisms also provide a rational
or into memory B-cells. explanation for the coexpression of surface IgM and
What then is the underlying scenario? Following IgD with identicalV regions on a single cell, and for the
secondary antigen challenge, primed B-cells may be switch from production of membrane-bound IgM
activated by paracortical Th cells in association with receptor to secretory IgM in the antibody-forming cell
interdigitating dendritic cells or macrophages, and (cf. figures 4.1 and 4.2).
Figure 10.13. The events occurring in lymphoid germinal centers. Throughtheir surface receptors, FDCs bind immune complexescon-
Germinal center B-cells can be enriched through their affinity for the taining antigen and C3which, in turn, are very effective B-cell stimu-
peanut agglutinin lectin. They show numerous mutations in the an- lators since coligation of the surface receptors for antigen and C3
tibody genes. Expression of LFA-1and ICAM-1 on B-cellsand follic- (CR2)lowers their threshold for activation. The costimulatory mole-
ular dendritic cells (FDCs) in the germinal center makes them cules CD40 and B7 play pivotal roles. Antibodies to CD40 prevent
'sticky'. Centroblasts at the base of the follicleare stronglyCD77pos- formation of germinal centers and anti-CD40L can disrupt estab-
itive. The Th cells bear the unusual CD57 marker. The FDCs all ex- lished germinalcenters within 12hours. Anti-B7.2, given early in the
press CD21 and CD54; those in the apical light zone are strongly immune response, prevents germinal center formation and, when
CD23 positive, those in the basal light zone express little CD23. given at the onset of hypermutation, suppresses that process.
Figure 10.15. Synthesis of immunoglobulin. As rnRNAis translat- BiP (heavy chain-binding protein) which binds to the heavy chain
ed on the ribosome, the N-terminal signal sequence (SS)is bound by C,1 and VL domains to control protein folding. The unassembled
a signal recognition particle (SW) which docks onto a receptor on chains oxidize and dissociate as the full H2L, Ig molecule. The as-
the outer membrane of the endoplasmic reticulum (ER) and facili- sembled H2L2molecules can now leave the ER for terminal glycosy-
tates entry of the nascent Ig chain into the ER lumen. The SS associ- lation in the Golgi and final secretion. Surface receptor Ig would be
ateswith a specificmembrane receptor and is cleaved; the remainder inserted by its hydrophobic sequences into the membrane of the
of the chain, as it elongates, complexeswith the molecular chaperone endoplasmicreticulum as it was synthesized.
(seep. 189).Let us take another look at the stimulation These cytokines induce the formation of germ-line
of small, surface IgM-positive, B-cells by LPS. As we sterile transcriptswhich start at the I (initiation)exon 5'
noted, on its own, the nonspecific mitogen evokes the of the switch region for the antibody class to which
synthesisof IgM, IgG3 and some IgG2b. Following ad- switching will occur and terminate at the polyadenyla-
dition of IL-4 to the system, there is class switching tion site 3' of the relevant CH gene (figure 10.17).The
from IgM to IgE and IgGl production, whereas IFNy transcripts are not translated but instead remain asso-
stimulates class switching from IgM to IgG2a and ciated with the template DNA, forming RNA-DNA
TGFP induces switching from IgM to IgA or IgG2b. hybrids within the S regions of the DNA which might
act as targets for enzymes involved in the recombina-
tion process. Under the influence of the recombinase, a
given VDJ gene segment is transferred from p6 to the
new constant region gene (figure 10.17), so yielding
antibodiesof the same specificitybut of different class.
Figure 10.19. An 'antigen's eye view' of sequence diversity Somatic hypermutation is therefore complementary to germ-
in human antibodies. The sequence diversity has been plotted line diversity. The V, CDR3, which lies at the center of the antigen-
on a scale of blue (more conserved) to red (more diverse). The V, binding site, was not included in this analysis and therefore is
domain is on the right and the V, domain on the left in both pictures. shown in gray as a loop structure. The end of the V, CDR3 (also
(a) Germ-line diversity prior to somatic hypermutation is focused excluded) lies at the center of the binding site and is not visible in
at the center of the antigen-bindingsite. (b)Somatic hypermutation this representation. (Reproduced with kind permission from
spreads diversity to regions at the periphery of the binding site Tomlinson I.M. et al. (1996) Journal of Molecular Biology 256,
that are highly conserved in the germ-line V gene repertoire. 813.)
FACTORS AFFECTING ANTIBODY AFFINITY
I N THE I M M U N E RESPONSE
memory cells provide a clonally expanded pool of markers has greatly facilitated the separation of B- and
antigen-primed cells which can travel to secondary T-cells into naive and memory populations for further
lymphoid organs under the influence of the CCL21 study. The costimulatory molecules B7.1 (CD80) and
(SLC) chemokine (cf. table 10.3) and, following re- B7.2 (CD86) are rapidly upregulated on memory B-
encounter with antigen, can stimulate dendritic cells, cells, and the potent ability of these cells to present
help B-cells and generate effector cells. In contrast, ef- antigen to T-cells could well account for the brisk and
fector memory T-cells possess CCRI, CCR3 and CCR5 robust nature of secondary responses. A scheme simi-
receptors for proinflammatory chemokines and con- lar to that outlined in figure 10.21 for T-cells may also
stitute tissue-homing cells which mediate inflamma- exist for the B-lymphocyte compartment, with an ini-
tory reactions or cytotoxicity. tial population of memory cells possessing the B220
Virgin B-cells lose their surface IgM and IgD and marker developing into B220- memory B-cells which
switch receptor isotype on becoming memory cells, then go on to generate antibody-secreting effector
and the differential expression of these surface cells.
Asuceessionof genes are upregulated byl-cell aaivatlon although circulating IL-1 and IL-6 can mediate release of
9 Within 15-30minutes, genes for tra-ption factors acute phase proteins from the liver.
concernedin theprogmsionGOtoG1 andinthecontrolof They act through surface receptors belonging to six
L-Zareexpressed. structural families.
Up to 14hours. cytokines and their receptors are Cytokine-induced dimerization of individual subunits
expmsed. of the main (hematopoietin) receptor family activates
Later, a variety of genes related to cell division and protein tyrosine kinases, including JAG, and leads to
adhesion are upregulated. phosphorylation and activation of STAT transcription
factors.
Cytokinesaa as lnteroellularmessengers Cytokines are pleiotropic, i.e. have multiple effects
Cytokines act transiently and usually at short range, in the general areas of (i) control of lymphocyte
(continuedp. 198)
growth, (ii) activation of innate immune mechanisms Prollfemrionof Bcell responses Is medioredby cytokines
(includmginflammation),and(i) controlofbonemarrow Early proliferationis mediated by IL-4 which also aid
hematopoiesis(cf.figure10.4). IgE synthesis.
Cytokines may act sequentially, through one cytokine IgAproducersarr!drivenbyTGFpandIL-5.
inducing production of another or by transmodulationof IL-4plus IL-5promote IgM and IL-4, -5, -6 and -13 plu
the receptor for anothercytokine; they can also act syner- IFNystimulateIgG synthesis.
gisticallyor antagonistically.
Their roles in vim can be assessed by gene 'knockout', Events In the germlnolcenter
transfection or inhibitionby specificantibdies. There is clonal expansion,isotype switchand mutatio
in the darkzonecentroblasts.
DifferentT-cell subserscon make different cytoklnes TheB-cellcentroblastsdiethroughapoptosisunlessm
Asimmunizationproceeds,Thtendtodevelopintotwo cued by certain signals which upregulate bcJ-2. These ir
subsets: Thl cells concerned in inflammatory processes, cludecrosslinkingofsurfaceIgbycomplexesonfollicula
macrophageactivationand delayed sensitivitymake IL-2 dendritic cells and engagement of the CD40 e p t o
and -3, IFNT TNF, lymphotoxh and GMCSF; Th2 cells which drivesthe cellto the memory compartment.
help B-cells to synthesizeantibody and secrete IL-3,-4, -5, Theselectionofmutantsbyantigenguidesthedev~oF
-6 and -13,TNFand GMCSF. E-10 is secretedby ThZ cells ment of high affinity B-cells.
inmicebutbybothThlandTh2subsetsinhumans.
Early interaction of antigen with macmphages or The synthesis of antibody
dendritic cells producing E-12or with a variety of cells RNA for variable and constant regions is spliced
secreting IL-4 will skew the responses to Thl or Th2, together beforeleaving the nucleus.
IWpXtiVely. Differential splicing allows coexpression of IgM and
Other subsetsmay exist, includingTGF~-secretingTh3 IgDwith identicalV regions on a singlecelland the switch
(Trl)regulatorycells. frommembrane-boundto secreted IgM.
Tcl ( M y ) and Tc2 (IL-4) populations can also be
distinguished. clossswltching occurs in individual B-cells
I#
IgM produced early in the response switches to IgG,
Aaivored T-cells prolitem in response to cytoklnes particularlywith thymus-depdent antigens.Theswitch
IL-2acts as an autocrine growth factor for Thl and is largely under T-cell control.
paracrine for Th2 cells which have upregulated their IL-2 I@, but not IgM, respnses improve on secondat
receptors. challenge.
Cytokines act on cells which express the appropriate
cytokme receptor. Antibody a~nityduringrheimmune response
Low doses of antigen tend to selecthigh affinity B-cells
T-cell elfecton in cell-mediored immunity and hence antibodiessinceonly thesecan be rescued in the
Cytokines mediate chronic inflammatory responses germinalcenter.
and induce the expressionof MHC class 11on endothelial For the same reasons, amity matures as antigen
cells,avarietyofepithelialceUsandmanytumorcelllines, concentrationfalls duringan immuneresponse.
so facilitating interactions between T-cells and nonlym-
phoid cells. Memorycells
Differentialexpressionof chemokinereceptorspermits Memory T-cells can be maintained in the absence of
selective recruitment of neutrophils, macrophages, den- antigen.
driticcellsand T- and B-cells. However, immune complexes on the surfaceof follicu-
TNF synergizeswith IFNyin killingcells. lar dendriticcells in the germinal centers provide a long-
CytotoxicT-cells are generated againstcells (e.g. virally termsourceof antigen.
infected) which have intracellularly derived peptide Memorycellshavehigheraffinitythannaivecells,inthe
associated with surfaceMHC class I. They kill using lytic caseof B-cellsthrou~somaticmutation,andinthecaseof
granulescontainingperforin, granzymesand TNF. Teells through selekve proliferationof cells with higher ,
T-cell-mediated inflammation is strongly down- affinity receptors and through upregulated expressionof
regulatedby IL-4 and IL-10. associated molecules such as CD2 and LFA-I, which in-
crease the avidity (fUndi0~1affinity) for the antigen- High levels of CDQa expression are also characteristic
pmentingcell. of memory T-cells, low level expressionbeing associated
Activated memory and naive T-cells are distinguished WithnaiveT-ceb.
by the expressionofCD45isoforms, the formerhaving the
CD45RO phenotype, the latter CD45RA. It seems likely
that a proportion of the CD45RO population reverts to a
CD45RApoolof restingmemorycells.cD45RA-memory
cells can be divided into CCW+ central memory and
CCRTeffector memory cells.
Control mechanisms
Introduction, 200 The influence of geneticfactors, 21 1
Antigens can interferewith each other, 200 Some genes affect general responsiveness, 2 1 1
Complement and antibody also play a role, 201 Antigen receptor genes are linked to the immune response, 2 1 1
Activation-induced cell death, 201 lmmuneresponsecan beinfiuenced bytheMHC, 212
T-cell regulation, 203 Regulatory immunoneuroendocrine networks, 214
T-helpercells, 203 A neuroendocrinefeedback loop affectingimmune
T-cell suppression, 203 responses, 21 5
Effector T-cells are guided to the appropriate target by MHC sulface Sex hormones come into the picture, 2 16
molecules, 206 Psychoimmunolagy, 21 6
ldiotype networks, 207 Effectsof diet, exercise, trauma and age on immunity, 21 7
Jernes network hypothesis, 207 Malnutrition diminishes the effectiveness ofthe immune response,
Evidencefor idiotypic networks, 207 217
Preoccupation of networks with self, 208 Otherfactors, 218
ldiotypic regulation of immune responses, 209 Summary, 218
Monipulotion of the immune response through idiotypes, 21 1 Fuilher reading, 220
balance between caspase 8 and FLIP levels can deter- for induction of the class switch to IgA, since Peyer's
mine the fate of the cell when the death receptor is patch T-cells do not markedly enhance IgA production
engaged by its ligand, but does not affect apoptosis by splenic B-cells. Evidence for the production of an
induced by the stress-activated mitochondrial path- IgE-binding factor from Fce. receptor-bearing T-cells,
way (figure 11.6). which enhances B-cell secretion of IgE, has been ob-
tained; intriguingly, a 13kDa T-cell-derived cytokine
called glycosylation inhibitingfactor (GIF) inhibits N-
T-CELL REGULATION
glycosylation of the IgE-binding factor so that it now
becomes a suppressor of IgE synthesis. It should also
be evident from the previous discussion of AICD that
There is abundant evidence to suggest that different Th cells will not be around to expand B-cell and Tc
populations of Th cells are specialized for different clone sizes indefinitely.
helper functions.With respect to help for antibodypro-
duction, T-cell lines derived from Peyer's patches are
much better at helping IgA-producing B-cells from
Peyer 'S patch precursors than are splenic T-cell lines. It is perhaps inevitable that nature, having evolved
The help is for IgA-precommitted B-cells rather than a functional set of T-cells which promote immune
Figure 11.6. Life and death decisions. (a)
The relative amounts of anti-apoptotic
FLIP and pro-apoptotic caspase 8 can
determine the fate of the cell. (b)
Experimentsinvolving overexpression of
FLIP in a transgenic mouse model show
that this protein protects T-cells from AICD
stimulated through the death receptor
pathway by Fas-ligand, but not from cell
death triggered via the mitochondrial
pathway using the drug staurosporine.
(Based on data obtained by J. Tschopp and
colleagues.)
Figure 19.12. T-helper bypass through new carrier epitope ( ) eliminated from the body and with it the T-cell epi-
generates autoimmunity.For simplicity, processing for MHC asso- tope, the only way that the autoimmunity can be sus-
ciation has been omitted from the diagram, but is elaborated in fig-
ure 19.13. (a) The pivotal autoreactive T-helper is unresponsive
tained is for the activated B-cell to capture circulating
autoantigen and, Figure 11.7.processing,
after Demonstration of T- it to the T-
present
either through tolerance or inability to see a cryptic epitope. (b) Dif- suppressorcells. Amouse of an appropriate
ferent mechanisms providing a new carrier epitope. helper (figurestrain
19.13~). This is
immunized with not possible for cell-
an immunogenic
associated antigens but their
dose of sheep special
erythrocytes linka strong
makes with T-cell
recognition puts antibody
themresponse. However,
in a totally if spleenballpark.
different cells
frominfecting
In this case, if an a donor of the samemimics
agent strain previously
an autoanti-
injected with a high dose of antigen are first
detail in figure 19.13a. Two low molecular weight en- gen by producing a cross-reacting
transferred to the syngeneic T-cell
animal, epitope,
they the
velope proteins of Yersinia enterolytica share epitopes resulting T-celldepress
autoimmunity
the antibodycould responsetheoretically
to a normallyper-
with the extracellular domain of the human thyroid- sist even afterimmunogenic
eliminationdose of ofthe
the antigen.
infection. The effect
The au-
is lost if the spleen cells are first treated with
stimulating hormone (TSH) receptor; in rheumatic toantigen will normally be presented to the resting
a T-cell-specific antiserum (anti-Thy-l) plus
fever, antibodies produced to the Streptococcus also autoreactive T-cell as a cryptic
complement, showing epitope and by defini-
that the suppressors
react with heart, and the sera of 50% of children with tion will be unable to provide an activating
are T-cells. (After Gershon R.K. & Kondo signal.
K. The
the disease who develop Sydenham's chorea give (1971) Immunology 21,903.)
cross-reactinginfectious agent will provide abundant
neuronal immunofluorescent staining which can be antigen on professional APCs which can prime the T-
absorbed out with streptococcal membranes. Colon cell and upregulate its adhesion molecules so that it
antibodies
responses, shouldpresent in ulcerative
also develop colitis
a regulatory sethave
whosebeen now has the
all surprising, but at thattotime
avidity bindantigen-induced
to and be persistently toler-acti-
found be
job would to cross-react
to modulate the Escherichia
with coli suppres-
helpers. T-cell 014. There is ancevated by the cryptic
was regarded self-epitope
essentially presented
as a negative on the tar-
phenome-
sionalso
wassome evidence
first brought tofor
thethe viewattention
serious that antigens
of thecommon
im- non get tissue cell
involving theprovided
depletionthat or itsilencing
is associated with the
of clones
to Trypanosoma cruzi and cardiac muscle
munological fraternity by a phenomenon colorfully and peripher- rather than a state of active suppression. OverRemem-
appropriate MHC molecule (figure 19.13b). the
al nervous
named system provoke
by its discoverer, some of
'infectious the immunopatho-
tolerance'. Quite berT-cell
years, the transgenic
suppression cytotoxic
has beenT-cellsshown(Tc) which could
to modulate
logical lesions
surprisingly it wasseen in Chagas'
shown that, ifdisease.
mice were made only of
a variety destroy
humoral theand pancreatic p-cells bearing
cellular responses, a viral
the latter
unresponsive by injection of a high dose of sheep transgene
including when they were
delayed-type primed by a real
hypersensitivity, viral infec-
cytotoxic
red 3blood cells (SRBC), tion and
(cf. figure 12.10).Recall T-cell also theproliferation.
tumor cells that
Molecular mimicytheir T-cells
of T-cell would suppress
epitopes T-cells antigen-specific
specific antibody formation in normal recipients to could only
However, be recognized
the existence by primed
of dedicated not resting T-cells
professional T-
Thethey
which drawback
had beenwith the Allison-Weigle
transferred model
(figure 11.7). of cross-
It may suppressor cells is a question which has generated could
(cf. figure 18.9). Theoretically, the resting T-cell a
reaction of B-cell epitopes and the provision
not be apparent to the reader why this result was of a new
at T- greatalso
dealbeofprimed
heat. in a nonantigen-specific manner by a
cell carrier is that, once the cross-reacting agent is microbial superantigen.
Suppressor and helper epitopes can be discrete Characteristics of suppression
Detailed analysis of murine responses to antigenssuch What of the effectors of suppression? In a number of
as hen egg-white lysozyme tells us that certain deter- experimental systems the cells which mediate sup-
minants can evoke very strong suppressor rather than pression are far more vulnerable than helpers to adult
helper responses depending on the mouse strain, and thymectomy, X-irradiation and cyclophosphamide.
also that T-suppressors directed to one determinant For example, adult thymectomy in the mouse has little
can switch off helper and antibody responses to other effect on the Th population, but leads to a fall in the T-
determinants on the same molecule. Thus mice of H-2b suppressors, thereby increasing the response to T-
haplotype respond poorly to lysozyme because they independent antigens and preventing the fall-off in
develop dominant suppression; however, if the three IgE antibody to haptens coupled with Ascaris extracts
N-terminal amino acids are removed from the antigen, which occurs in intact animals.
these mice now make a splendid response, showing Classically, suppressor T-cells have been described
that the T-suppression directed against the deter- as CD8+.For example, the BIO.A (2R)mouse strain has
minant associated with the N-terminal region has a low immune response to lactate dehydrogenase P
switched off the response to the remaining determi- (LDHB) associated with the possession of the H-2EP
nants on the antigen. Similar results have been ob- gene of k rather than b haplotype. Lymphoid cells taken
tained in several other systems. This must imply that from these animals after immunization with LDHP
the antigen itself acts as a form of bridge to allow com- proliferate poorly in vitro in the presence of antigen,
munication between T-suppressor and cells reacting to but if CD8+cells are depleted, the remaining CD4+cells
the other determinants, as might occur through these give a much higher response. Adding back the CD8+
cells binding to an antigen-presentingcell expressing cells reimposes the active suppression. Human sup-
several different processed determinants of the same pressor T-cells can also belong to the CD8 subset.Thus,
antigen on its surface (figure 11.8). CD8' CD28- cells can prevent antigen-presenting B-
cells from upregulating costimulatory B7 molecules in
response to CD40-mediated signals from the CD40 lig-
and on Th cells. Following interaction with the CD8 T-
cells, the APCs are then capable of inducing anergy in
Th cells. This effect on B7 is mediated by inhibition of
NFKB activation in the APC, an event necessary for
transcription of both the 87.1 (CD80)and B7.2 (CD86)
molecules.
More recently, it has become appreciated that regu-
latory CD4+cells can also be major effectors of suppres-
sion. In a model of inflammatory bowel disease (IBD),
in which transfer of C D ~ ~ R B ~ T-cells ~ ~ ~ C from
D ~ +
normal donor mice into mice with severe combined
immunodeficiency (SCID)leads to the development of
IBD, the disease can be prevented if C D ~ ~ R B ~ O ~ C D ~ +
regulatory T-cells (Tr)are transferred at the same time.
This immunoregulatory effect could be blocked by
antibodies to the IL-10 receptor or to TGFP in mice, or
to IL-4 and TGFP in rats.
Suppressor cells of the CD4 phenotype have also
been identified within the minor population which
express the IL-2 receptor a chain (CD25). These sup-
pressors are able to abrogate the responses of CD4+
CD25- T-cells by an as yet undefined cell contact-
Figure 11.8. Possiblemechanism to explain the need for a physical dependent, cytokine-independentmechanism.
linkage between suppressor and helper epitopes. The helper and
putative suppressor cells can interact by binding close together on
the surface of an antigen-presentingcell, which processes the anti- Suppression is a regulated phenomenon
gen and displays the different epitopes on separate MHC molecules
on its surface. Let us look in a little more depth at some potential
just as IgG holds back the B-cells by feedback control.
There is indeed abundant evidence for such a regula-
tory mechanism whereby suppressors can be induced
by Thl cells.
The nature of the antigen-presenting cell, upon
whose surface these interactions are thought to
occur, may influence the type of cells involved and
the outcome. It may be recalled that dendritic cells,
macrophages, B-cells, activated T-cells and even
epithelia1 cells with upregulated class I1 can all act
as antigen presenters. Of interest is the finding that
irradiation with UVB light inactivates dendritic
cells and stimulates T-suppression; IL-2 and IFNy are
downregulated and IL-4 production is increased.
Other issues which require solutions are the mecha-
nism by which high-dose antigen induces suppres-
sion, the role of cytotoxic CD4 and CD8 T-cells in these
phenomena and the extent to which idiotype-specific
T-suppressors contribute.
It is important not to lose track of the so-called 'nat-
ural suppressor cells', such as those provoked by total
Figure 11.9. Mutual antagonisms between T-cell subsets linked in-
directly by processed antigen on an antigen-presentingcell lead to
lymph node irradiation, and we would like to draw
functionally distinct modes of suppression. (Leaning heavily on attention to a report that natural killer (NK) cells can
Bloom B.R., Salgame P. & Diamond B. (1992)Immunology Today 13, inhibit the one-way mixed lymphocyte reaction (see
131.)Yet another mechanism may prove to be important. Unlike the p. 352) or the primary IgM response to sheep cells
mouse, many other mammalian species can express MHC class I1on
a proportion of their activated T-cells; presentation of processed
in vitra, by suppressing dendritic cells which have
peptide by these cells can induce CD4-positive cytotoxic cells with already taken up the antigen. This offers a further
suppressor potential. We also need to know more about the circurn- opportunity for feedback control since IFNy and IL-2
stances leading to the production of TGFP by suppressors since this produced by Th can activate NK cells.
cytokine inhibits T-cell proliferation.
Overall, suppression may be most commonly
mediated by secretion of cytokines such as TGFP, by
secretion of yet to be defined suppressor molecules
mechanisms of suppression. We have already enter- and by inactivation of APC function. Make no mis-
tained the idea that antigen-linked T-T interactions take, suppression is still a murky area, and is not for the
can occur on the surface of an antigen-presentingcell unwary, but the light at the end of the tunnel is
(figure 11.8)and the concept of T1 and T2 CD8 subsets beckoning.
paralleling the Thl /Th2 dichotomy. Furthermore, the
evidence for mutual antagonism (suppression) be-
tween Thl and Th2 cells is very strong indeed. One
could postulate downregulation of Thl cells by type 2
IL-4-producingCD8 cells, and suppression of Th2 cells Not only do MHC molecules bearing a tell-talepeptide
by type 1IFNy-producing CD8 cells, interacting on the signal the presence of an intracellular precursor
surface of an antigen-presenting cell (figure 11.9). In (cf. p. 95, Chapter 5), they also ensure that the T-cell
this model, when the immune response has locked makes contact with the surface of the appropriate
onto a particular mode, e.g. Thl-mediated cellular target cell.
immunity, other types of response, such as T-B collab- Let us explore this point by looking at the role of
oration, are restricted through a cytokine inhibitory ef- Tc in viral infection. When a cell is first infected
fect. Although these cells mediate T-suppression, they with virus, there is an eclipse phase during which
would not be called dedicated professional suppres- the machinery of the cell is being switched for viral
sors since, in a sense, their suppressive powers are a replication and the only marker of the complete
by-product of their main defensive function. Perhaps microbe is the processed viral antigen peptide on the
we need these cytokine-secreting Tc cells to prevent cell surface. At this stage, killing of the cell by a cyto-
Th cells getting out of hand by excessive proliferation, toxic T-cell will prevent viral replication. The killer
T-cell knows it has reached its target when it recog- IDIOTYPE NETWORKS
nizes the surface viral peptide in associationwith class
I MHC molecules which are present on nearly every
cell in the body. Thus the killer cell operates on the
basis that processed viral antigen is the code for 'viral The hypervariable loops on the immunoglobulin
infection' and class I molecules are the code for 'cell' molecule which form the antigen combining site have
(cf.figure 5.26). individual characteristic shapes which can be recog-
The situation is quite different with intracellular nized by the appropriate antibodies as idiotypic deter-
bacteria and protozoa which do not go through an minants (cf.p. 42). There are hundreds of thousands, if
eclipse phase after phagocytosis by macrophages, but not more, different idiotypes in one individual.
are held as infectious entities; lysis by cytotoxic T-cells Jerne reasoned brilliantly that the great diversity of
will merely release the organisms, not kill them. A sep- idiotypes would to a considerable extent mirror the di-
arate strategy is required and, in this case, the effector versity of antigenic shapes in the externalworld. Thus,
Thl lymphocyte recognizes the infected macrophage he said, if lymphocytes can recognize a whole range of
by the presence of microbial antigen on the surface in foreign antigenic determinants, they should be able to
association with a class I1 molecule which is now a recognize the idiotypes on the antigen receptors of
code for 'macrophage'. This interaction triggers the re- other lymphocytes. They would therefore form a large
lease of macrophage-stimulating cytokines, dominant network or series of networks depending upon idio-
among which is IFNy. Multiple microbicidal me- type-anti-idiotype recognition between T- and B-
chanisms are triggered in the activated macrophages, cells (figure 11.10), and the response to an external
including the formation of reactive oxygen intermedi- antigen perturbing this network would be condi-
ates and the synthesis of NO., to the detriment of the tioned by the state of the idiotypic interactions.
intracellular parasites (seep. 265).Similarly,in T-B co-
operation, the B-cell is recognized through its class
I1 molecule associated with the foreign antigen, al-
though in this case costimulatory signals through
Anti-idiotype can be induced by autologous idiotypes
CD40L-CD40 interactions are required for activation.
T-cells mediating suppression also utilize MHC mole- There is no doubt that the elements which can form
cules, but the mechanisms are unclear. In summary, an idiotypic network are present in the body, and
each antigen-specificT-lymphocyte subset has to com- autoanti-idiotypesoccur during the course of antigen-
municate with a particular cell type in order to make induced responses. For example, certain strains of
the appropriate immune response, and it does so by mice injected with pneumococcal vaccines make an
recognizing not only processed foreign antigen but antibody response to the phosphorylcholinegroups in
also the particular MHC molecule used as a marker of which the germ-line-encodedidiotype T15 dominates.
that cell (table11.l). If the individual antibody-formingcells are examined
Some types of immunoregulatory cells, such as cer- by plaque assays at different times after immuniza-
tain y6 T-cells, may recognize activation-induced, non- tion, waves of T15+ and of anti-T15 (i.e. autoanti-
classical MHC molecules, as appears to be the case idiotype)cells are demonstrable.Similarly,irnrnuniza-
with the recognition of the class Ib molecules T22 and tion with the acetylcholine agonist BISQ, followed
T10 in the mouse, although the precise role of such cells by fusion of the spleen cells to produce hybridomas,
is still being elucidated. yielded a series of anti-BISQ monoclonals (idiotypes)
and a smaller number of anti-idiotypic monoclonals,
of which a surprising proportion behaved as internal
Table 11.1. Guidance of T-subpopulations to appropriate target images of BISQ in their ability to stimulate acetyl-
cell by MHC molecules. choline receptors (figure 11.11).Anti-idiotypic reactiv-
ity has also been demonstrated in T-cell populations
using various experimental systems. Indeed, anti-
idiotypic T-cells recognizing peptide derived from the
CDR2, CDR3 and framework regions of other TCRs
appear to form a part of the normal human T-cell reper-
toire. Immunizationof multiple sclerosis patients with
myelin basic protein reactive T-cell clones induces
Tostimulatory molecules and their ligand. CD8+ cytotoxic T-cells which recognize a peptide
Figure 11.10. Elements in an idiotypic network in which the antigen. The same idiotype ( ) may be shared by receptors of
antigen receptors on one lymphocyte reciprocally recognize an different specificity, the nonspecific parallel set (since the several
idiotype on the receptors of another. T-helper, T-suppressor and B- hypervariable regions provide a number of potential idiotypic
lymphocytesinteract through idiotype-anti-idiotype reactions pro- determinants and a given idiotype does not always form part of the
ducing either stimulation or suppression. T-T interactions could epitope-binding site, i.e. the paratope), so that the anti-(anti-Idl)
occur through direct recognition of one T-cell receptor (TCR)by the does not necessarilybind the original antigen. (Thefollowing abbre-
other, or more usually by recognition of a processed TCR peptide as- viations are often employed:a as a prefix = anti; Id = idiotype; Ab, =
sociated with MHC. One of the anti-idiotypesets, Ab2P,may bear an Id; Ab2a = aId not involving the paratope; Ab2P= internal image
idiotype of similar shape to (i.e.provides an internal image of) the aId involvingthe paratope;Ab3 = a(aId).)
derived from a TCR CDR3-associated idiotype pre- bodies' which appear spontaneouslyin germ-free ani-
sented by MHC class I. mals not exposed to exogenous antigens. Not only are
many of them directed to polysaccharide antigens of
common pathogens, e.g. phosphorylcholine, but a
A network is evident in early life
number of them have low affinity for self-components
If the spleens of fetal mice which are just beginning to such as DNA, IgG, heat-shock proteins and cytoskele-
secrete immunoglobulin are used to produce hybrido- tal elements. The concept of a largely CD5 B-l cell pop-
mas, an unusually high proportion are interrelated as ulation forming an inward-looking world in which the
idiotype-anti-idiotype pairs. This high level of idio- component cells recognize and stimulate each other
type connectivity is not seen in later life and suggests ceaselessly through their idiotypic receptor interac-
that these early cells, largely the CD5+B-l subset (cf. tions to produce a range of IgM antibodies which
p. 236), are programed to synthesize germ-line gene provide an early defense against infection is most
specificities which have network relationships. plausible. But the self-reactivityof many of these cells
is enigmatic. Preset regulatory T-cell networks may
also involve certain dominant autoantigens, such as
heat-shock protein hsp65 and myelin basic protein
Paradoxically, there is increasing evidence that pre- from nervous tissue. Is recognition of self as important
formed idiotype networks have what might seem at as nonself?
first sight a somewhat unhealthy interest in self- Irun Cohen has proposed the intriguing notion of an
antigens. The CD5 IgM hybridomas produced from immunological homunculus (little man) in which a
fetal mouse spleen with high idiotype connectivity functional picture of the body is encoded within the
have specificities similar to those of the 'natural anti- immune system by regulatory network committees of
Figure 11.11. The spontaneous production of autoanti-idiotype
during immunization with a hormone agonist. Hybridomas ob-
tained from the immunized mouse secrete anti-hormone and anti-
idiotype which reacts with the hormone receptor; this indicates a
close relationship between hormone, receptor, anti-hormone and
anti-idiotype, implying some connection between autoantibodies
Figure 11.12. Speculation on the role of self-reacting CD5 B-l cells
within the idiotype network and hormone systems.
in microbial infection. The surface immunoglobulin (sIg)receptor
selectively captures the cross-reacting conserved bacterial antigen
(in this case, heat-shock protein hsp65) and, after processing, pre-
B- and T-cells which recognize certain dominant self- sents self and microbe-specific epitopes associated with MHC class
antigens representing the major organs in the body. 11.The autoreactive T-cell is heavily controlled by the idiotype regu-
latory network since (ex hypothesi)important self-antigens are dom-
(The analogy is with the neurological homunculus, a inantly encoded within the 'immunological homunculus', but the
functional picture of the body in which the space occu- antimicrobial T-cell is free to mount a vigorous response. This ac-
pied by a given neural network is directly related to the countsfor the dominance of conserved antigens.(Based upon Cohen
neurological importance of the organ it encodes, e.g. I.R. &YoungD.B. (1991)Immunology Today 12,105.)
human visual and speech organs and canine olfactory
organs are prominently represented.)The relevance of
these ideas to the control of autoimmunity will be dis- following cascade protocol. Antigen is injected into
cussed in Chapter 19, but here we can speculate on animal, and the antibody produced, Ab, (idiotype),is
how they might also relate to the response to infection. purified and injected into animal2.Ab2 (anti-idiotype)
Consider a dominant microbial antigen such as hsp65 so formed is purified and used to immunize animal,
which is highly conserved in nature and bears several and so on (figure 11.13).Consistently, it is found that
potential epitopes identical with the self-homolog. In Ab2 (anti-Id,) recognizes an idiotype (Id,) on Ab,
an infection, any B-l cells which recognize selfhsp65 which is also strongly present in Ab,. Ab, behaves like
will selectively focus the bacterial hsp65 onto their Ab, in seeing the common idiotype on Ab, and Ab,.
surface receptors (makingit dominant over other bac- Nonetheless, although Ab, and Ab, share idiotypes,
terial antigens) and process it. The self-epitopes will only a small fraction of Ab, reacts with the original
be recognized by autoreactive T-cells which are highly antigen. This is the result one would expect if the
regulated within the homunculus, whereas T-cells idiotype was a cross-reactingId (publicId) present on
specific for the nonself hsp epitopes are not so con- a variety of antibodies (and thus B-cell receptors) of
strained and will generate an effective immune re- different specificities. The anti-Id, (Ab,), when inject-
sponse (figure 11.12). ed into animal,, would react with all B-cellsbearing Id,
(figure 11.13)and presumably trigger them to produce
Id, antibodies, only a fraction of which have specificity
for the original antigen.
There have been a series of investigations based on the Such frequently occurring and usually germ-line-
Figure 11.13. An idiotype cascade.Ab, produced by the antigen is mon or cross-reacting idiotype. On this basis, one can understand
injected into a second animal to produce Ab,; this in turn is purified the paradoxical finding of Oudin and Cazenave that not all the Ig
and injected into animal3and so on. Ab, and Ab, both react with an molecules bearing a given Id in response to an antigen can function
idiotype ( ) on Ab, and Ab3, but only a fraction of Ab3reacts with as specific antibody since they belong to the nonspecific parallel set.
the original antigen. Bona and Paul (ImmunologyToday 1982,3,230) The presence of large amounts of Id, in Ab, also suggests that the lin-
interpret the results in terms of a common regulatory idiotype Id, ear relationship through the cross-reactingId, is dominant, with rel-
shared by many antibodies other than those reacting with the origi- atively insignificant branching through the variety of 'private'
nal antigen, but recruited by the injection of anti-Idl (Ab,) which idiotypes on Ab, molecules (cf. figure 11.10)because of the low fre-
stimulates the range of lymphocytes whose receptors bear this com- quency of such idiotypes and their anti-idiotypes.
encoded idiotypes seem to be provoked fairly readily with Id-positive receptors. We can now see how the
with anti-Id and are therefore candidates for regu- antigen- and idiotype-specificTh synergize in the anti-
latory Id which can be under some degree of control body response, the latter expanding Id-positive clones
by a limited idiotypic network. Germane to this idea induced by the former.
are the observations that, late in immunization, anti- The phenomenon of 'original antigenic sin' occurs
bodies with utterly distinct specificities, directed when the immune response becomes 'locked in' to
against totally different epitopes on the same antigen, particular epitopes originally encountered on a
often bear a common or cross-reacting idiotype. microorganism, such that it largely ignores even
Presumably, the first clone of B-cells to be expanded normally immunodominant epitopes during a sub-
which bears a dominant cross-reactingId can generate sequent encounter with an antigenically related
a population of regulatory Th cells which recognize but nonidentical microorganism. Although competi-
this Id as well as antigen. Processing of internalized tion for antigen by the expanded population which
Ig receptor plus antigen leads to the expression of forms the memory B-cells plays a major role, idiotype-
peptides derived from the idiotype and antigen in specific memory Th cells could also contribute to this
association with MHC class 11; these B-cells can phenomenon.
then access the full repertoire of antigen-specific and Idiotype networks may also allow the immune re-
idiotype-specific Th cells. The latter may be of two sponse to 'tick over' for extended periods and main-
types, one recognizing the native receptor idiotype tain the memory cell population, while the presence of
(non-MHC-restricted) and the other, processed idio- primed Th cells directed against a common Id on the
type (MHC-restricted).From the complex mixture of various memory B-cells specific for a given antigen
B-lymphocytes activated by the other epitopes on the would increase their rate of mobilization during a sec-
antigen, these Th cells will selectively recruit those ondary response.
set (figure 11.10) to stimulate antigen-specific T-
suppressors capable of turning off B-cells directed to
other epitopes on the antigen through bridging by the
Quite low doses of anti-idiotype, of the order of antigen itself (cf. figure 11.8).
nanograms, can greatly enhance the expression of the Since we know that under suitable conditions anti-
idiotype in the response to a given antigen, whereas Id can also stimulate antibody production, it might be
doses in the microgram range lead to a suppression possible to use 'internal image' monoclonal anti-Ids as
(figure 11.14). Thus the idiotypic network provides 'surrogate' antigens for immunization in cases where
interesting opportunities to manipulate the immune the antigen is difficult to obtain in bulk -for example,
response, particularly in hypersensitivity states antigens from parasites such as filaria or the weak
such as autoimmune disease, allergy and graft rejec- embryonic antigens associated with some cancers.
tion. However, the B-cell response is normally so di- Another example is where protein antigens obtained
verse, suppressionby anti-Id is likely to prove difficult; by chemical synthesis or gene cloning fail to fold
even when the response is dominated by a public Id into the configuration of the native molecule; this is
and that Id is suppressed, compensatory expansion not a problem with the anti-Id which by definition
of clones bearing other idiotypes ensures that the has been selected to have the shape of the antigenic
fall in the total antibody titer is relatively undra- epitope.
matic (cf. figure 11.14). Conceivably, Th cells may At this stage, if the reader is feelinga little groggy, try
express a narrower spectrum of idiotypes, thereby a glance at figure 11.15 which attempts to summarize
being more susceptible to suppression by Id auto- the main factors currently thought to modulate the
immunization. Reports that 'vaccination' with irra- immune response.
diated lines of Th cells specific for brain or thyroid
antigens prevents the induction of experimental
autoimmunity against the relevant organ are encour- THE INFLUENCE OF GENETIC FACTORS
aging. A totally different approach would be to use
monoclonal anti-Id of the 'antigen internal image'
Mice can be selectively bred for high or low antibody
responses through several generations to yield two
lines, one of which consistently produces high-titer
antibodies to a variety of antigens, and the other, anti-
bodies of relatively low titer (figure 11.16; Biozzi and
colleagues). Out of the ten or so different genetic loci
involved, some give rise to a higher rate of B-cell prolif-
eration and differentiation, while one or more affect
macrophage behavior.
Figure 11.16. Selectivebreedingof high and low antibody respon- of other antigens. The same was done for the poorest responders
ders (after Biozzi and colleagues).A foundation population of wild ( ), yielding a strain of low responder animals. The two lines are
mice (with crazy mixed-up genes and great variability in antibody comparable in their ability to clear carbon particles or sheep erythro-
response)is immunized with sheep red blood cells (SRBC),a multi- cytes from the blood by phagocytosis, but macrophages from the
determinant antigen. The antibody titer of each individual mouse is high responders present antigen more efficiently (cf.p. 158).On the
shown by a circle. The male and female giving the highest titer anti- other hand, the low responders survive infection by Salmonella
bodies ( ) were bred and their litter challenged with antigen. fyphimurium better and their macrophages support much slower
Again, the best responders were bred together and so on for 20 gen- replication of Listeria (cf. p. 262), indicative of an inherently more
erations when all mice were high responders to SRBC and a variety aggressive microbicidalability.
REGULATORY IMMUNONEUROENDOCRINE
NETWORKS
Figure 11.17. Different mechanisms can account for low T-cell There is a danger' as One more and On the
response to antigen in associationwith MHC class 11. antics of the immune system, of looking at the body as
a collection of myeloid and lymphoid cells roaming
around in a big sack and of having no regard to the
recognize A69 cells infected with influenza A virus. integrated physiology of the organism. Within the
Interestingly, individuals with the HLA-A69 class I wider physiological context, attention has been drawn
MHC develop immunity to a different epitope on the increasingly to interactions between immunological
same protein. and neuroendocrinesystems.
Figure 11.18. Genetic control of the immune response.
strain unlimited clonal expansion by a process referred to IFNycapable of suppressing Th2 cells.
as activation-induced cell death (AICD).
ldiotype networks I1 locus and control the interactions required for T-B
Antigen-specific receptors on lymphocytescan interact collaboration
with the idiotypes on the receptors of other lymphocytes Class JI-linkedhigh and low responsivenessmaybe due
to form a network (Jerne). to defectivepresentationby MHC, a defective T-cellreper-
Anti-idiotypescan be induced by autologousidiotypes. toire caused by tolerance to MHC+self-peptides and T-
An idiotype network involving mostly CD5 B-1 cells is suppression.
evident in early life.
T-cell idiotypic interactions can also be demon- lmmunoneuroendocrinenetworks
strated. Immunological,neurological and endocrinologicalsys-
Preset idiotypicnetworks involve a number of lympho- tems interact, forming regulatory circuits.
cytes with self-reactivity for dominant autoantigens. Feedback by cytokines augments the production of cor-
It is speculated that this preoccupation with self helps to ticosteroids and is importantbecause this shuts down Th1
regulate unwanted autoimmune reactions and may and macrophageactivity
help to target the response to infectious agents on domi- Estrogens may be largely responsible for the more ac-
nant conserved antigens like hsp65 which cross-reactwith tive immune responses in females relative to males. The
self. male hormone, DHEA, prolongs life in females with the
Idiotypes which occur frequently and are shared by a murine equivalent of the human autoimmune disease
multiplicity of antibodies (public or cross-reactingId) are SLE.
targets for regulation by anti-idiotypes in the network,
thus providing a further mechanism for control of the im- Effectsofdietandotherfoctorson immunity
Protein-caloriemalnutrition grossly impairs cell-medi-
The network offers the potential for therapeutic inter- ated immunity and phagocyte microbicidalpotency.
vention to manipulate immunity. Exercise, trauma, age and environmental pollution can
all act to impair immune mechanisms. The pattern of cy-
Genetic factors influence the immune response tokines produced by peripheral blood cells changes with
Approximately 10genescontroltheoverallantibodyre- age, IL-2 decreasing and TNF, IL-1 and IL-6 increasing; the
sponse to complex antigens: some affect macrophageanti- latter is associated with a lowered DHEAlevel.
genprocessingandmicrobicidalactivity andsome the rate
of proliferation of differentiatingB-cells. Foclors influencingthe bias between Thl and Th2 subsets
ImmunoglobulinandTCRgenes arevery adaptablebe- These have figured with some prominence in this chap-
cause they rearrange to create the antigen receptors, but ter and a summary of some of the major influences on the
holesin the repertoire can occur. balance between Thl and ThZ responses is presented in
Immune response genes are located in the MHC class figure 11.22.
Introduction, 221 B-1 and 8-2 cells represent two distinct populations, 236
Hemotopoieticstem cells, 221 Development of B-cell specificity, 237
The thymus provides theenvironment forT-cell The sequence of immunoglobulin gene rearrangements, 237
differentiation, 223 The importanceofallelic exclusion, 240
Bone marrow stem cells became immunocompetent T-cells Differentspecific responses can appear sequentially, 240
in the thymus, 224 The induction of tolerance in B-lymphocytes, 240
T-cell ontogeny, 225 Tolerance can be caused by clonal deletion and clonal
Differentiation isaccompanied by changes in surtace anergy, 240
markers, 225 Tolerance may result from helpless B-ceiis, 241
Receptor rearrangement, 227 Notural killer (NK) cell ontogeny, 242
Cells are positively selectedfor self-MHC restriction in The overall response in the neonate, 242
thethymus, 228 The evolution of the immune response, 242
T-cell tolerance, 229 Invertebrates have microbial defense mechanisms, 242
The induction of immunological tolerance is necessary to avoid Adaptive immune responses appearwith the
self-reactivity, 229 vertebrates, 243
Self-tolerancecan be induced in the thymus, 231 The evolution of distinct B- and T-cell lineages was
lntrathymic clonal deletion leads to self-tolerance, 231 accompanied by the development of separate sites for
T-cell tolerance can also be due to clonal anergy, 233 differentiation, 244
Lack of communication can cause unresponsiveness, 234 Cellular recognition molecules exploit the immunoglobulin
B-cells differentiate in the fetal liver and then in bone genesuperfamily, 245
marrow, 234 Summary, 246
Pax5is a major determining factor in B-cell differentiation, 235 Further reading, 247
Hematopoiesis originates in the early yolk sac but, We have come a long way towards the goal of isolating
as embryogenesis proceeds, this function is taken highly purified populations of hematopoietic stem
over by the fetal liver and finally by the bone cells, although not all agree that we have yet achieved
marrow where it continues throughout life. The it. In the mouse, the most likely candidate, at least a
hematopoietic stem cell which gives rise to the very early progenitor, is the cell with the following sur-
formed elements of the blood (figure 12.1)can be face phenotype: high expression of MHC, low positiv-
shown to be multipotent, to seed other organs and ity for Thy-l, clearly positive for Sca-1,AA4.1and c-kit,
to have a relatively unlimited capacity to renew it- and for the adhesion molecule PGP-1, negative or only
self through the creation of further stem cells. Thus weakly positive for Lin, and negative for B220, Mac-l,
an animal can be completely protected against the Gr-l and CD8 (the last four being markers for B-cells,
lethal effects of high doses of irradiation by injec- macrophages, granulocytes and cytotoxic T-cells, re-
tion of bone marrow cells which will repopulate its spectively).Impressively,less than 100of such cells can
lymphoid and myeloid systems. The capacity for
prevent death in a lethally irradiated animal. Recently,
a 'molecular phenotype' of the mouse stem cell has
self-renewal is not absolute and declines with age
in parallel with a shortening of the telomeres and a been obtained by analysis of subtracted cDNA li-
reduction in telomerase, the enzyme which repairs braries from highly purified fetal liver stem cells.It was
the shortening of the ends of chromosomes which revealed that there is a very high degree of precision in
would otherwise occur at every round of cell the control of transcription at each stage of the
division. hematopoietic hierarchy. Certain molecules are more
predominant in either fetal or adult hematopoietic
Figure 12.1. The multipotential hematopoietic stem cell and its of platelets, erythrocytes, all the types of myeloid cells, and also
progeny which differentiateunder the influenceof a seriesof soluble the progenitors of B-, but not T-, cells; GM-CSF, granulocyte-
growth factors within the microenvironment of the bone marrow. macrophage colony-stimulating factor, so-called because it pro-
The expression of various nuclear transcription factors directs the motes the formation of mixed colonies of these two cell types
differentiation process. For example, the h r o s gene encodes a zinc- from bone marrow progenitors either in tissue culture or on
fingered transcription factor critical for driving the development transfer to an irradiated recipient where they appear in the spleen;
of a common myeloid/lyrnphoid precursor into a lymphoid- G-CSF, granulocyte colony-stimulating factor; M-CSF, monocyte
restricted progenitor giving rise to T-, B- and NK cells. SCF, stem colony-stimulating factor; EPO ,erythropoietin; TPO, thrombopoi-
cell factor; LIF, leukemia inhibitory factor; IL-3, interleukin-3, etin; TNF, hunor necrosis factor; TGFP, transforming growth
often termed the multi-CSF because it stimulates progenitors factor p.
stem cells, and evidence was obtained to support so on. The importance of this interaction between un-
an important role for TGFP in the control of differentiated stem cells and the microenvironment
hematopoiesis. Similarly, the expression of Notch-l, which guides their differentiation is clearly shown by
our old friend NFKB and the wonderfully named studies on mice homozygous for mutations at the W or
Manic Fringe and Dishevelled-l genes underscores the sl loci which, amongst other defects, have severe
the importance of the Notch signaling pathway in macrocytic anemia. Bone marrow stromal cells pro-
hematopoietic development. Finally, a large number duce stem cell factor (SCF) which remains associated
of genes for cell adhesion molecules were present with the extracellular matrix and acts on primitive
in the cDNA libraries, including several that implicate stem cells through a tyrosine kinase membrane recep-
members of the semaphorin family in stem cell tor, c-kit (CD117).sl/sl mutants have normal stem cells
homing. In the human, CD34 is a marker of an ex- but defective stromal production of SCF which can be
tremely early cell but, again, there is some debate as to corrected by transplantation of a normal spleen frag-
whether this identifies the holy pluripotent stem ment; w/w mutant myeloid progenitors lack the c-kit
cell itself. surface receptor for SCF, and so can be restored by
The stem cells differentiate within the microenvi- injection of normal bone marrow cells (figure 12.2).
ronment of sessilestromalcells which produce various Mice with severe combined immunodeficiency
growth factors such as IL-3, -4, -6 and -7, GM-CSF, and (SCID)provide a happy environment for fragments of
Figure 12.2. Hematopoiesis requires normal bone marrow stem anemia which can be corrected by transplantation of appropriate
cells differentiating in a normal microenvironment. The W locus normal cells. The experiments show that the w/w mutant lacks
codes for c-kit, a stem cell tyrosine kinase membrane receptor for the normal stem cells and the sl/sl mutant lacks the environmental fac-
stem cell factor (SCF) encoded by the sl locus. Mice which are ho- tor needed for their development.
mozygous for mutant alleles at these loci develop severe macrocytic
human fetal liver and thymus which, if implanted con- ologically from an outpushing of the gut endoderm of
tiguously, will produce formed elements of the blood the third pharyngeal pouch and which form well-
for 6-12 months. Fetal liver provides a source of hema- defined cortical and medullary zones (figure 12.3).
topoietic stemcells;but what is the role of the thymus? This framework of epithelial cells provides the mi-
croenvironrnent for T-cell differentiation. There are
subtle interactions between the extracellular matrix
THE THYMUS PROVIDES THE ENVIRONMENT
proteins and a variety of integrins on different lym-
FOR T-CELL DIFFERENTIATION
phocyte subpopulations produced by differential
The thymus is organized into a series of lobules based splicing and post-translational glycosylation; current
upon meshworks of epithelial cells derived embry- musings are that the expression of these integrins, to-
gether with chemokine and chemokine receptor ex- A fairly complex relationship with the nervous sys-
pression (cf. table 10.3), plays a role in the homing of tem awaits discovery; the thymus is richly innervated
progenitors to, and their subsequent migration within, with both adrenergic and cholinergic fibers, while the
the thymus. In addition, the epithelial cells produce neurotransmitters oxytocin, vasopressin and neuro-
a series of peptide hormones which mostly seem ca- physin are synthesized endogenouslyby subcapsular,
pable of promoting the appearance of T-cell differen- perivascular and medullary epithelial cells and nurse
tiation markers and a variety of T-cell functions on cells. Acute stress leads to an indecently rapid loss of
culture with bone marrow cells in vitro. Several have cortical thymocytes and an increase in epithelial cells
been well characterized and sequenced, including thy- expressing both cortical and medullary markers-
mulin, thymosin a,, thymic humoralfactor (THF) and surely intrathymic epithelial stem cells? The destruc-
thymopoietin (and its active pentapeptide thymopon- tion of cortical thymocytes is at least partly due to the
tin, TP-5). Of these, only thymulin is of exclusively cytolytic action of steroids, the relative invulnerability
thymic origin. This zinc-dependent nonapeptide of the medullary lymphocytes being attributable to
tends to normalize the balance of immune responses: their possession of a 20a-hydroxyl steroid dehydroge-
it restores antibody avidity and antibody production nase. The distinctive nature of the two main compart-
in aged mice and yet stimulates suppressor activity in ments in the gland is emphasized by the selective
animals with autoimmune hemolytic anemia induced atrophy induced by a number of agents; thus the pri-
by cross-reactive rat red cells (cf. p. 416). Thymulin mary target of organotin is the immature cortical thy-
may be looked upon as a true hormone, secretedby the mocyte. Dioxin interacts with a receptor on cortical
thymus in a regulated fashion and acting at a distance epithelial cells, while the immunosuppressive drug
from the thymus as a fine physiological immunoregu- cyclosporin A causes atrophy of all the medullary ele-
lator contributing to the maintenance of T-cell subset ments, thereby blocking differentiation of cortical to
homeostasis. medullary thymocytes.
Specialized large epithelial cells in the outer cortex, In the human, thymic involution commences within
known as 'nurse' cells, are associated with large the first 12months of life, reducingby around 3%a year
numbers of lymphocytes which lie within pockets to middle age and by 1% thereafter. The size of the
produced by the long membrane extensions of these organ gives no clue to these changesbecause there is re-
epithelial cells. The epithelial cells of the deep cortex placement by adipose tissue. In a sense, the thymus is
have branched dendritic processes, rich in class I1 progressively disposable because, as we shall see, it es-
MHC, and connect through desmosome cell junctions tablishes a long-lasting peripheral T-cell pool which
to form a network through which corticallymphocytes enables the host to withstand loss of the gland without
must pass on their way to the medulla (figure12.3).The catastrophic failure of immunological function, wit-
cortical lymphocytes are densely packed compared ness the minimal effects of thymectomy in the adult
with those in the medulla, many are in division and compared with the dramatic influence in the neonate
large numbers of them are undergoing apoptosis. On (Milestone 12.1). Nevertheless, the adult thymus
their way to the medulla, the lymphocytes pass a cor- retains a residue of corticomedullary tissue containing
don of 'sentinel' macrophages at the corticomedullary a normal range of thymocyte subsetswith a broad spec-
junction. Anumber of bone marrow-derivedinterdigi- trum of TCR gene rearrangements.Adult patients re-
tating dendritic cells are present in the medulla and the ceiving either T-cell-depleted bone marrow or
epithelial cells have broader processes than their corti- peripheral blood hematopoietic stem cells following
cal counterparts and express high levels of both class I ablative therapy are able to generate new naive T-cells
and class I1MHC. Whorled keratinized epithelial cells at a rate that is inversely related to the age of the indi-
in the medulla form the highly characteristic Hassall's vidual. These observations establish that new T-cells
corpuscles beloved of histopathology examiners. can be generated in adult life, either in the thymus or in
These structures may serve as a disposal system for the still mysterious 'extrathymic' sites that have
dying thymocytes and are the only location where been proposed as additional locations for T-cell
apoptotic cells are found in the medulla. The presence differentiation.
of HLA-DO and HLA-DM, molecules which mediate
intracellular peptide exchange, in the epithelial cells
which ring the Hassall's corpuscles suggest that they
may also play a role in activation and/or tolerance
of mature thymocytes, particularly given that these The evidence for this comes from experiments on the
epithelial cells also express the peptide-presenting reconstitution of irradiated hosts. An irradiated ani-
HLA-DR molecule. mal is restored by bone marrow grafts through the
Ludwig Gross had found that a form of mouse leukemia Laiicet ii, 748),Miller opined that during embryogenesis
could be induced in low-leukemia strains by inoculating the thymus would produce the originators of immunolog-
filtered leukemic tissue from high-leukemia strains pro- ically competent cells, many of which would have migrat-
videdthatthiswasdoneintheimmediateneonatalperiod. ed to other sites at about the time of birth. All in all a
Since the thymus was known to be involved in the superb example of the scientific method and its applica-
j leukemic process, Jacques Miller decided to test the hy- tionbya top-flight scientist.
pothesis that the Gross virus could only multiply in the
neonatal thymus by infecting neonatally thymectomized
mice of low-leukemia strains. The results were consistent
with this hypothesis but, strangely, animals of one strain
.. I
immediate restitution of granulocyte precursors; in to the thymus by one or more of the numerous
the longer term, also through reconstitution of the T- chemokines secreted by the thymic stromal cells-
and B-cells destroyed by irradiation. However, if the which are the critical ones is yet to be established.
animal is thymectomized before irradiation, bone These progenitors express CD34 and the enzyme ter-
marrow cells will not reconstitute the T-lymphocyte minal deoxynucleotidyl transferase (TdT) (figure
population (cf.figure 7.13). 12.4),which is involved in the insertion of nucleotide
By day 11-12 in the mouse embryo, lymphoblastoid sequences at the N-terminal region of D and J variable
stem cells from the bone marrow begin to colonize the region segments to increase diversity of the T-cell re-
periphery of the epithelia1 thymus rudiment. If the ceptors (TCRs)(cf.p. 65).They also express high levels
thymus is removed at this stage and incubated in organ of the adhesion molecule CD44 and the stem cell factor
culture, a whole variety of mature T-lymphocytes will receptor (c-kit, CD117) (p. 222), but their lack of both
be generated. This is not seen if 10-day thymuses are CD4 and CD8 designates them as double-negative
cultured and shows that the lymphoblastoid coloniz- thymocytes. They express high levels of Notch mole-
ers give rise to the immunocompetent small lympho- cules. These cell surface proteins provide signals at
cyte progeny. several key points during thymocyte differentiation.
Indeed, they are thought to be necessary for commit-
ment to the T-cell lineage, T-cell developmentbeing se-
T-CELL O N T O G E N Y
verely impaired in ~otch-l-knockout mice. Under the
influence of IL-1 and TNF, the progenitors differentiate
into prothymocytes, committed to the T-lineage, and
these now undergo IL-7-mediated proliferation to
The incoming thymic lymphoid progenitors express form a population of CD44- CD117-pre-T-cells. At this
a number of chemokine receptors and are attracted stage, the cells begin to express various TCR chains
and are then expanded, ultimately synthesizing CD3, immunocompetent populations of single-positive
the invariant signal transducing complex of the TCR, CD4+T-helpers and CDB+cytotoxic T-cell precursors
and becoming double positive for CD4+, CD8+, the (figure 12.4). Notch-l signaling is involved in the
markers of the helper and cytotoxic subsets respect- maturation of both of these subsets, with the Notch-l
ively. Finally, again under the guiding hand of ligands Jagged-l, Jagged-2 and &like-l being ex-
chemokines, the cells traverse the corticomedullary pressed on thymic epithelia1cells in a highly regulated
junction to the medulla as the CD4 and CD8 markers way. The y6 cells remain double negative, i.e. CD4-8-,
segregate in parallel with differentiation into separate except for a small subset which express CD8.
Figure 12.4. Differentiation of T-cells within the thymus. Num- mainly appear to recognize antigen directly, in a manner analogous
bers refer to CD designation. TdT, terminal deoxynucleotidyl trans- to the antibody molecule on B-cells, although some may be restricted
ferase. Negatively selected cells in gray. The diagram is partly by nonclassical MHC class Ib or by classical MHC class 11. The rela-
simplified for the sake of clarity. Autoreactive cells with specificity tively primitive NK-T cells bearing an ap TCR and the NK1.l
for self-antigensnot expressed in the thymus may be tolerized by ex- marker (cf. p. 102)are often restricted by CD1, although some are re-
trathymic peripheral contact with antigen (dashed circles). *yti cells stricted by classical MHC molecules.
The factors which determine whether the double-
The development o f @ receptors
positive cells become CD4 or CD8 cells in the thymus
are still not fully established.Two major scenarioshave The Vp is first rearranged in the double-negative
been put forward. The stochastic hypothesis suggests CD4-8- cells and associates with an invariant pre-a
that expression of either CD4 or CD8 is randomly chain, pTa, to form a single 'pre-TCR' (figure 12.4).Al-
switched off, whereas the instructive hypothesis de- though the pre-TCR mediates feedback inhibition on
clares that interaction of the TCR with MHC-peptide further TCR Vp gene rearrangement, the extracellular
results in signals which instruct the T-cells to become domain of the pTa is not required for this inhibition,
either CD4+ class 11-restricted cells or CD8+ class I- suggesting that there may not be an extracellular lig-
restricted cells. The weight of the evidence currently and for pTa, as had previously been assumed. A cys-
available would seem to support the instructive teine just inside the membrane on the cytoplasmic tail
model, whilst not entirely excluding the possibility of a becomes palmitoylated and it may be this process that
contribution from stochastic events. Thus it appears recruits pTa into lipid rafts containing the p56lCk
that the strength of the p56lCksignal (see p. 166)corre- signalingmolecule. The pre-T-cells undergo a frenetic
lates with lineage choice and is determined by the dur- burst of proliferation controlled by thymic epithelia1
ation of TCR engagement during the double-positive cells and fibroblasts, producing double-positive
stage of thymocyte differentiation, a stronger cumula- CD4+8+ cells. Further development requires rear-
tive signal favoring the generation of CD4 cells (figure rangement of the Va gene segments so allowing for-
12.5). mation of the mature ap TCR. The cells are now ready
for subsequentbouts of positive and negative receptor
editing as will be discussed shortly.
Rearrangement of the Vp genes on the sister chro-
The rearrangement of V-, D- and J-region genes re- matid is suppressed following the expression of the
quired to generate the TCR (seep. 62) has not yet taken pre-TCR (remember each cell contains two chromo-
place at the prothymocyte stage. The gene for TdT and somes for each a and p cluster).Thus each cell only ex-
the recombinase activator genes, RAG-1 and RAG-2, presses a single TCR P chain and the process by which
are transcribed at the pre-T stage and, by day 15, the homologous genes on the sister chromatid are sup-
cells with the y6 TCR can be detected in the mouse pressed is called allelic exclusion (cf. p. 240). The a
thymus followed soon by the appearance of a 'pre- chains appear not to always be allelically excluded, so
TCR' version of the ap TCR. Notch-l signals (again!) that some T-cells may have two antigen-specificrecep-
appear to play a role in ap versus y6 lineage com- tors, each with their own a chain but sharing a com-
mitment, although the details are still being worked mon p chain. It is not clear, however, whether both
out. receptors can be functional.
-
tificially induced in this way. Thus neonatal injection of NEWBORN
A STRAIN
CBAmousecells into newborn A strain animals suppress- LITTER-MATES
-
es their ability to reject a CBA graft immunologically in
adult life (figure M12.2.1).Tolerance can also be induced
with soluble antigens; for example, rabbits injected with Maturation
bovine serum albumin without adjuvant at birth fail to
make antibodies on later challenge with this protein. ADULT
culeswhich must be discriminated by the immune sys- mechanism which functionally deletes self-reacting
tern if potentially disastrous autoreactivity is to be cells and leaves the remainder of the repertoire un-
avoided.The restriction of each lymphocyte to a single scathed, The most radical difference between self and
specificity makes the job of establishing self-tolerance nonself molecules lies in the fact that, in early life, the
that much easier, simply because it just requires a developing lymphocytes are surrounded by self and
normally only meet nonself antigens at a later stage antigen in the thymus, they are deleted. In other
and then within the context of the adjuventicityand cy- words, self-reactive cells undergo a negative selection
tokine release usually associated with infection. With process in the thymus. A similar phenomenon is seen
its customary efficiency, the blind force of evolution when the thymic cells bear certain self-components
has exploited these differences to establish the mecha- which act as superantigens (cf. p. 103) by reacting
nisms of immunological tolerance to host con- with a whole family of VP receptors through recogni-
stituents (Milestone 12.2). tion of nonvariable structures on a VP segment. An
example is the H-2E molecule which reacts with
receptorsbelonging to the VPl7a family; strains which
cannot express H-2E because of a defect in the Ea gene
Since developingTT-cells are to be fourzd in the thymus, possess mature T-cells utilizing V@%, whereas
one might expect &is to be the milieu in which expo- strains which express H-2E normally delete their
sure to self-antigens on the surrounding cells would VP17a-positive T-cells. Likewise, mice of the MEsa
induce tolerance, The expectation is reasonable. If genotype delete VP6-bearing cells, the Mls being a
stem cells in bone marrow of H-2k haplotype are cul- locus encoding a B-cell superantigen which induces
tured with fetal thymus of H-2d origin, the maturing strongproliferation in VP6 T-cells from a strain bearing
cellsbecome tolerant to H-2d,as shownby their inabil- a different Mls allele (cf. p. 104).Even exogenous su-
ity to give a mixed lymphocyte proliferative response perantigens, such as staphylococcal enterotoxin B
when cultured with stimulators of H-2d phenotype; which activates the VP3 and VP8 T-cell families in the
third-party responsiveness is not affected. Further ex- adult, will eliminate these cells when incubated with
periments with deoxyguanosine-treated thymuses early immature thymocytes. Even more enlightening
showed that the cells responsible for tolerance induc- is the fact that, under these circumstances, the VP3
tion were deoxyguanosine-sensitive, bone marrow- and I438 thymocytes can actually be seen to undergo
derived macrophages ox dendritic cells which are apoptosis (cf.p. 19).
abundant at the corticomedullaryjunction (table 12.2).
Factors aflectingpositise or negative selection
in the thymus
There seemslittle doubt that self-reactiveT-cellscan be It is established that engagement of TCR by the
physically deleted within the thymus. If we look at the MHC-peptide complex on some type of antigen-
experiment in table 12.lb, we can see that SCID males presenting cell underlies both positive and negative
bearing the rearranged transgenes coding for the ap selection. But how can the same MHC-peptide signal
receptor reacting with the male H-Y antigen do not have two totally different outcomes? Well, positive
possess any immunocompetent thymic cells express- and negative selection may occur at low and high de-
ing this receptor, whereas the females which lack H-Y grees of TCR ligation, respectively. For example, high
do. Thus, when the developing T-cells react with self- concentrations of antibody to the TCR induce apopto-
sis in thymocytes (figure 12.7),whereas low concentra-
tions of anti-TCR do not. Furthermre, many examples
Table12.2, Induction of tolerance in bone marrowstem cellsby h- have been published showing that the same peptide
cubation with deoxyguanosine (dGuo)-sensitive macrophages or will induce positive selectionat low concentration and
dendritic cells in the thymus. Clearly,the bone marrow cells induce negative selectionat high concentration (see legend to
tolerance to their own haplotype. Thus the thymic tolerance-
inducing cells can be replaced by progenitors in the bone marrow
figure 12.8). This has led to the avidity model, which
inoculum (JenkinsonE.J., Jhittayl?., Kingston R. & Owen J.J. (1985) postulates that a functionally low avidity interaction
Transplantation39,331) or by adult dendritic cellsfrom spleen,show- between T-cell and peptide-MHC involving a rela-
ing that it is the stage of differentiationof the immaktre T-cell rather tively low number of TCRs will positively select dou-
than any special nature of the thymic antigen-presentingcell which
leads to tolerance (Matzinger F. & Guerder S. (1989)Nature 338,74).
ble-positive CD4+8+thymocytes, while a high avidity
interaction will lead to clonal deletion (figure 12.8).
Since the overall avidity of the T-cell interaction will be
inter alia a function of ligand density X TCR density X
affinity, an increase in peptide concentration will in-
crease ligand density and hence avidity. One problem
will be immediately apparent to the discerning reader
in that a given peptide ligand, giving a low avidity
initial stimulus for positive selection, should give a protects the cells from a signal that would otherwise
negative signal as the thymocyte differentiatesand the result in activation-inducedcell death.
density of TCRs increases with the change from dou- To pause for a moment, we seem to be saying that
ble- to single-positivecells. This has led to the sugges- engagement of the TCR of differentiating double-
tion that thymic cortical cells progressivelydesensitize positive CD4+8+thymocyteswith self-MHCon cortical
the maturing thymocyte so that it resists the more pow- epithelia1 cells leads to expansion and positive selec-
erful stimulus of the macrophages and medullary den- tion for clones which recognize self-MHC, perhaps
dritic cells, which would otherwise induce apoptosis. with a whole range of affinities, but that engagement
Evidence is also accumulating that thymic epithelium of the TCR with high affinity for self-MHC (+self-
can synthesize glucocorticoids, hormones classically peptide) on bone marrow-derived medullary cells will
associated with the adrenal gland. Maybe the gluco- lead to elimination and hence negative selection. Al-
corticoid-induced leucine zipper (GILZ) (cf. p. 215) though still not fully worked out, there are also obvi-
ous differences in the biochemical pathways used
for positive and negative signaling. Positive selection
is cyclosporin A-sensitive and dependent on the
Ras-MEK-ERK pathway (cf.p. 167),whereas negative
selection is cyclosporin A-resistant and independent
of this pathway. Different intensities of signaling from
the TCR, and/or the types of coreceptor used, may in-
fluence which pathway is utilized. Let us finish on a
cautionary note: the avidity model may be substantial-
ly correct but it could be an oversimplification. For in-
stance, certain superantigens, which can cause clonal
deletion of certain VD families, fail to expand them
even at very low concentrations when the model
Figure 12.7. Electron micrograph of cells induced to undergo would have indicated positive selection. This has
apoptosis in intact fetal thymus lobes after short-term exposure to spawned other models involving conformational
anti-CD3. A and N indicate representative apoptotic and normal changes, and given the complex interactions of pep-
lymphocytes, respectively. Note the highly condensed state of the
nuclei of the apoptotic lymphocytes. (Photograph kindly donated
tides behaving as agonists, partial agonists and antag-
by Professor J.J.T.Owen, from Smith et al. (1989)Nature 337,181. Re- onists (cf.p. 170);the last word has not yet been spoken
produced by permissionfrom MacmillanJournalsLtd, London.) (not that it ever is in science!).
Figure 12.8. The aviditymodel of thymic positive and negative se- LCM virus peptide, the positive selection of the transgenic T-cells is
lection. It is postulated that a low avidity interactionbetween the T- impaired because of lack of MHC-peptide. However, low concen-
cell and antigen-presenting cell (APC) will give positive selection trations of the peptide added to fetal organ cultures of these mice se-
and that high avidity will give deletion. DP, double-positive CD4+8+; lected the transgenic T-cells positively; while higher concentrations
SP, single-positive CD4+or CD8+;"refersto affinity of peptide for the gave negative selection (LjunggrenH.G. &van Kaer L. (1995)T h e h -
MHC or of the MHC-peptide complex for the TCR. munologist 3,136). 'Cryptic selfr-peptides(cf.p. 200) are presented at
When Tap-l mutant mice (cf. p. 93) are mated with mice bearing very low concentrations and will not delete potentially autoreactive
the transgenes for the TCR specific for a complex of H-2Dbwith an clones, which may therefore escape to the periphery.
Looking again at figure 12.8,the specificitiesof the T- single transgenic animals lacking IKb. If lCb is expressed
cells entering the periphery from the thymus must on cells of neuroectodermal origin or hepatocytes,
be moulded by the self-peptides, which drive positive again the double transgenic mice are tolerant but there
selection, since normally the only peptides around is dramatic downregulation of TCR and CD8 mole-
must be derived from self. It is satisfying to note cules; in the formerbut not the latter case, downregula-
therefore that the T-cell repertoire tends to be biased tion of TCR could be reversed by exposureto antigen in
towards peptides from extrinsic antigens which vitm. In some experimental models, tolerance can be
resemble self; thus, T-cell epitopes recognized on im- abrogated by IL-2. To recapitulate, autoreactiveT-cells
munization with xenogeneic lysozyme corresponded leaving the thymus can be rendered anergic in the
with sequences having the highest homology to the periphery and can display different degrees of poten-
syngeneicprotein. tially reversible unresponsiveness.
Infectious anergy
We have already entertained the idea that engage- If a clone of T-helpers is subject to a limiting dilution
ment of the TCR plus a costimulatory signal from an experiment (p. 137),the minimal unit of proliferation
antigen-presenting cell are both required for T-cell in response to peptide on an APC is usually several
stimulation,but, when the costimulatory signalis lack- cells not just one. This implies that triggering only oc-
ing, the T-cell becomes tolerized or anergic, or, if you curs in small groups or clusters of cells and suggests
prefer, paralysed. that paracrine or multicellular interactions between
Thus, anergy can be induced in extrathymic T-cells potential respondersbound to a singleAPC are needed
by peripheral antigens in viva when presented by cells to drive the cells into division (figure 12.9a).It will be
lacking costimulatory molecules. If a transgene con- appreciated that, if a newly arising extrathymic naive
struct of H-2Ebattached to an insulin promoter is intro- T-cell binds to its antigen, even on a professional APC,
duced into a mouse which normally fails to express it will not be stimulated if its neighbors in the cluster
H-2E, the H-2Eb transgene product appears on the P- have already been made anergic. Indeed, instead of
cells of the pancreas and induces tolerance to itself. being triggered, it will itself become anergic, so perpet-
Whereas the expression of H-2E on bone marrow- uating the infectious anergic process (figure 12.9b).
derived cells in the thymic medulla deletes T-cells Recent evidence suggeststhat anergic T-cellscan act as
bearing Vp17a receptors, these cells are not lost in the immunoregulatory cells by causing the downregula-
tolerant transgenic mouse expressing pancreatic H-2E, tion of MHC class I1and the costimulatoryCD80 (87.1)
i.e. there is a state of clonal anergy, not deletion. The al- and CD86 (B7.2) molecules on the APC, generating an
tered immunologicalstatus of these cells is revealed by infectious anergy which does not require the simulta-
their inability to proliferate when their receptors are neous presence of the regulatory anergic cells and the
cross-linkedby an antibody to VP17a. cells which will themselves be made anergic (figure
It is unlikely that these results are due to low level ex- 12.9~). We shall see later in Chapter 17 that the induc-
pression of antigen in the thymus. Similar experiments tion of transplantation immunosuppression with a
showed that mice expressing influenza hemagglutinin nondepleting anti-CD4 can be long-lasting because
on the pancreatic p-islets also became tolerant irre- the production of anergic cells prevents the priming
spective of whether the transgenic thymus was re- of newly immunocompetent T-lymphocytes by the
placed by a normal gland or not. Nonetheless, anergic transplantation antigen(s).
cells can also be generated within the thymic popula- These anergic cells are really acting as suppressors.
tion as seen in mice transgenic for both an anti-lCbTCR So far in our discussions, we have not asked the
and a IKb gene controlled by a truncated fragment of a question: do dedicated T-suppressors contribute to
keratin IV promoter which allowed expression on self-tolerance?Frankly, another gray area, but experi-
thymic medullary cells. mentally we can demonstrate that, if autoimmunity is
Peripheral T-cell anergy can occur at different levels induced in a normal animal, either actively, by injec-
depending upon the circumstances of antigen expo- tion of an antigen cross-reactingwith self, or passively,
sure. If the above double transgenic experiment is re- by injection of autoreactive T-cells (cf. p. 416 and
peated with a full keratin IV promoter, the Kbantigen is p. 433), the self-reactingclones are usually quashed by
expressed on keratinocytes and induces full tolerance, idiotype- or antigen-specific T-suppression. In a nut-
even though the same high frequency of cytotoxic T- shell, suppressors probably do not prevent autoimmu-
cell precursors with the transgene TCR is seen as in nity but they may reverse it.
Figure 12.9. Infectious anergy.(a)
Clusters of normal T-cells (green)around
newly immunocompetent cells (gray)
reacting with the same APC mutually
support activation and proliferation.
(b)Newly immunocompetent cells
surrounded by anergic T-cells (red)
receive no stimulatory signals from their
neighbors and are themselves rendered
anergic. (c)Upon being made anergic, T-
cells can assume an immunoregulatory
function whereby they downregulate
expression of MHC class 11, CD80 (B7.1)
and CD86 (B7.2)molecules on the APC.
This effect requires cell-cell contact
between the anergic T-cells and the APC, is
not blocked by neutralizing antibodies to
the cytokines IL-4, IL-10 or TGFP, and
would exhibit linked suppression to other
epitopes on the antigen presented by the
APC. Subsequent encounter of newly
immunocompetent cells with this APC
will lead to anergy.
der of life. Using the modified cultureconditions intro- pro-B-cells progressing to the pre-B-cell stage (figure
duced by Whitlock and Witte, it is now possible to 12.12). Also required is expression of the Pax5 gene
grow bone marrow cells in vitro and achieve the differ- which encodes the BSAP (B-cell-specificactivator pro-
entiation of B-cells and their precursors. Stromal tein) transcription factor. Thus, in Pax5-/- knockout
reticular cells, which express adhesion molecules and mice, early pre-B-cells (containing partially re-
secrete IL-7, extend long dendritic processes making arranged immunoglobulin heavy chain genes) fail to
intimate contact with IL-7 receptor-positive B-cell differentiate into mature, surface Ig+, B-cells (figure
progenitors. Although early B-cells comprise only a 12.12).However, if the pre-B-cells from Pax5-l- knock-
minor subpopulation of the cells in those cultures, it is out mice are provided with the appropriatecytokines
possible to analyse the different stages in their devel- in vitro, they can be driven to produce T-cells, NK cells,
opment by rescue with the Abelson murine leukemia macrophages, dendritic cells, granulocytes and even
virus (A-MuLV),a replication-defectiveretrovirus ca- osteoclasts! These unexpected findings clearly show
pable of transforming pre-B-cells at various points in that the early pre-B-cell has the potential to be diverted
their development into clones. A series of differentia- from its chosen path and instead provide a source of
tion markers associated with B-cell maturation have cells for many other hematopoieticlineages. However,
now been established (figure 12.11). these pre-B-cells are not pluripotent as, unlike bone
marrow stem cells, they are unable to rescue lethally ir-
radiated mice. In the light of these findings, it has been
proposed that Pax5 acts as a master gene critical for B-
cell development and functions by inhibiting, rather
Development of hematopoietic cells along the B-cell than activating, the expression of a set of genes. Pax5
lineage requires expression of E2A and of early B-cell expression thereby suppresses alternative lineage
factor (EBF); the absence of either of these prevents choice in early B-cells.
Table 12.3. Comparison of two mouse B-cell subsets. (Developed
from Herzenberg L.A., StallA., Melchers F. et al. (eds) (1989)Progress
in Immunology 7, p. 409. Springer-Verlag, Berlin.)
tain their numbers by self-replenishment and limit down self-components, as envisaged by dear Pierre
their de novo production from progenitors by feedback Grabar many years ago when he thought of them as
regulation. They can express both CD5 and its ligand globulines transporteurs.
CIX'2 on their surface, which should encourage mutu- Other functions of B-l cells may be the generation of
al interaction, but a major factor influencing self- an idiotype network concerned in self-tolerance, the
renewal could be the constitutive production of IL-10, response to conserved microbial antigens, and pos-
since treatment of mice with anti-IL-l0 from birth vir- sibly the idiotypic regulation of B-2 responses. They
tually wipes out the B-l subset. The predisposition for are certainly the source of 'natural antibodiesfwhich
self-renewal may underlie their undue susceptibility provide a pre-existing first line of IgM defense against
to become leukemic, with the malignant cells in common microbes. Up to 50% of the IgA-producing
chronic lymphocytic leukernia being almost invari- cells in the lamina propria are derived from peritonea1
ably CD5+. cavity B-l cells. These cells are therefore an important
B-l cells tend to use particular germ-line V genes, source of the mucosal IgA which coats the normal
and the autoantibody response to bromelain-treated microflora of the gut.
erythrocytes is restricted to this subset which utilizes
the rather diminutive VH21and V,12 families. Clonal
DEVELOPMENT OF B-CELL SPECIFICITY
expansion seems to be driven by reaction with self-
antigens (see legend to figure 12.13).They tend to re-
spond to type 2 thymus-independent antigens (cf. p.
171)and, unlike the B-2 population, they do not enter
into liaisons with thymus-dependent antigens, do not By analysis of A-MuLV-transformed clones of pre-B-
enter germinal centers and hence do not undergo so- cells, it has proved possible to unravel the orderly cas-
matic mutation or form high affinity antibodies. This cade of Ig gene rearrangements which occur during
may be just as well if the harmless low affinity autoan- differentiation.
tibodies which are produced by many B-l cells are not Stage 1. Initially, the D-J segments on both heavy
automatically driven to high affinity pathogenic au- chain coding regions (one from each parent) rearrange
toantibodies. In a weak moment one sometimes hears (figure 12.14).
of 'good' and 'bad' autoantibodies, with the 'good Stage 2. A V-DJrecombinational event now occurs on
guys' possibly having the job of sweeping up broken one heavy chain. If this proves to be a nonproductive re-
scribed to form a 'pseudo-light chain' which associates
with the p chains to generate a surface surrogate 'IgMr
receptor, together with the Ig-a (CD79a) and Ig-P
(CD79b) chains conventionally required to form a
functionalB-cell receptor. Expression of this receptor is
absolutely essential for further differentiationof the B-
lymphocytes since disruption of the membrane exon
of the p chain or of the A5 gene by homologous recom-
bination of embryonic stem cells (cf.p. 141)arrests de-
velopment at the pre-B stage and the animal is devoid
of mature B-cells. This surrogate receptor closely par-
allels the pre-TalP receptor on pre-T-cell precursors of
ap TCR-bearing cells.
Stage 4. The surface receptor is signaled, perhaps by a
stromal cell, to suppress any further rearrangement of
heavy chain genes on a sister chromatid. This is termed
allelic exclusion and was first discussed in relation to
the rearrangement of TCR P chains (see p. 227).
Stage 5. It is presumed that the surface receptor now
initiates the next set of gene rearrangements which
occur on the K light chain gene loci. These involve V-J
recombinations on first one and then the other K allele
until a productive V,-J rearrangement is accom-
plished. Were that to fail, an attempt would be made
Figure 12.13. The development of separate B-cell subpopulations.
It is presumed that 23-1cells of sufficientlyhigh avidity for, say, self- to achieve productive rearrangement of the h alleles.
surface antigens are eliminated, leaving positively selected lower Synthesisof conventional sIgM now proceeds.
affinity specificities for soluble self-antigens and the spectrum of Stage 6. The sIgM molecule now prohibits any further
'natural antibody'-producingB-1 cells. In contrast, 8-2 cells undergo gene shufflingby allelic exclusion of any unrearranged
negative, rather than positive, selection by self-antigens and the
surviving B-2 cells give rise to the higher affinity IgG antibody light chain genes.
produced by helper T-cell-dependent class-switched B-cells. It is At the next stage of differentiation,the cell develops
thought that, although these subsetsmightbe able to give rise to each a commitmentto producinga particular antibody class
other under some circumstances, generally they are maintained as and either bears surface IgM alone or in combination
separate lineages. Direct evidence that self-antigenspositively select
B-l cells is provided by mice made transgenic for the VH3609heavy with IgA or IgG. The further addition of surface IgD
chain gene. The transgene-encoded heavy chain pairs with endoge- now marks the readiness of the virgin B-cell for prim-
nous Vrr21C light chain to produce an anti-thymocyte autoantibody ing by antigen. Some cells, therefore, bear surface Ig of
associated with CD5+ B-cells and which recognizes a Thy-l- three different classes: M, G and D or M, A and D; but
associated carbohydrate epitope. High levels of the transgenic B-
cells and of the serum autoantibody were found only in the presence all Ig molecules on a single cell have the same idiotype
of the autoantigen, being absent in Thy-l knockout mice. SC, stem and therefore are derived from the same VH and VL
cell; CD23, FceRII; CD43, leukosialin. genes, presumably by splicing of a long RNA tran-
script. IgD is lost on antigenicstimulationso that mem-
ory cells lack this Ig. At the terminal stages in the life of
arrangement (i.e.adjacent segments arejoined in anin- a fully mature plasma cell, virtually all surface Ig is
correct reading frame or in such a way as to generate a shed. Injection of anti-p (anti-IgM heavy chain) into
termination codon downstream from the splice point), chick embryos prevents the subsequent maturation of
then a second V-DJ rearrangement will occur on the IgM and IgG antibody-producing cells, whereas anti-y
sister heavy chain region. If a productive rearrange- inhibits only IgG development. Although we have
ment is not achieved, we can wave the pre-B-cell a fond seen earlier that T-helpers can induce class switching,
farewell. it is also the case that some isotype switching probably
Stage 3. Assuming that a productiverearrangement is occursindependently of antigen as a result of microen-
made, the pre-B-cell can now synthesize p chains. At vironmental factors. In the embryonicchickenbursa, a
around the same time, two genes, V p B(CD179a)and A5 regular switch from IgM to IgG is observed and it
(CD179b),with homology for the VLand CLsegments seems possible that local influences in the gut will
of h light chains respectively, are temporarily tran- prove to be responsible for the predominant
Figure 12.14. Sequence of B-cell gene rearrangementsand postulated mechanism of allelic exclusion (see text).
development of IgA-bearing cells. These cells are gen-
THE INDUCTION OF TOLERANCE I N
erated in Peyer's patches, pass into the blood via the
B-LYMPHOCYTES
thoracic duct and return to populate the diffuse lym-
phoid tissue in the lamina pr>$a of the gut.
gene transcription. The E-selectin engages the glyco- the important role of chemokines (see table 10.3) in
protein E-selectin ligand-l (ESL-l)on the neutrophil. selectively attracting multiple types of leukocytes to
Other later acting components are the chemokines inflamatory foci. Inflammatory chemokines are
(chemotactic cytokines) IL-8 (CXCL8) and epithelial- typically induced by microbial products such as
derived neutrophil attractant-78 (ENA-78, CXCL5) lipopolysaccharide (LPS) and by proinflammatory
which are highly effective neutrophil chemoattrac- cytokines including IL-1, TNF and IFNy. As a very
tants. IL-1 and TNF also act on endothelial cells, fibro- broad generalization, chemokines of the CXC subfam-
blasts and epithelial cells to stimulate secretion of ily, such as IL-8, are specific for neutrophils and, to
another chemokine, MCP-1 (CCL2), a chemotactic varying extents, lymphocytes, whereas chemokines
protein for several different cell types which is particu- with the CC motif are chemotactic for T-cells, mono-
larly potent at attracting mononuclear phagocytes to cytes, dendritic cells, and variably for natural killer
the inflammatory site to strengthen and maintain the (NK)cells, basophils and eosinophils. Eotaxin (CCL11)
defensive reaction to infection. is chemotactic for eosinophils, and the presence of
Perhaps this is a good time to remind ourselves of significant concentrations of this mediator together
Figure 13.3. Early events in inflammation affecting neutrophil along the extracellular matrix vitronectin is dependent upon very
marginationand diapedesis. Induced upregulation of P-selectinon rapid cycles of integrin-dependent adhesion and detachment regu-
the vessel walls plays the major role in the initial leukocyteeen- lated by calcineurin. The cytokine-induced expression of E-selectin,
dothelialinteraction (rolling)by interactionwith ligands on the neu- which is recognized by the glycoprotein E-selectin ligand-l (ESL-1)
trophil such as the mucin-like P-selectin glycoprotein ligand-l on theneutrophil, occursas a later event. Chemotacticfactors such as
(PSGL-1, CD162). Recognition of extracellular gradients of the IL-8, which is secreted by a number of cell types including the en-
chemotacticmediators by receptors on the polymorphonuclearneu- dothelium itself, are important mediators of the inflammatory
trophil (PMN) surface triggers intracellular signals which generate process. (Compareevents involved in homing and transmigrationof
motion. The neutrophils crawl rather than swim and migration lymphocytes, figure 8.6.)
with RANTES (regulated upon activation normal T- responses and hyped-up killing powers, adding up to
cell expressed and secreted, CCL5) in mucosal sur- bad news for the bugs.
faces could account for the enhanced population of Of course it is beneficial to recruit lymphocytes
eosinophilsin those tissues. The different chemokines to sites of infection and we should remember that
bind to particular heparin and heparan sulfate gly- endothelial cells in these areas express VCAM-1 (cf.
cosaminoglycans so that, after secretion, the chemo- p. 151) which acts as a homing receptor for VLA-
tactic gradient can be maintained by attachment to the 4-positive activated memory T-cells, while many
extracellular matrix as a form of scaffolding. chemokines are chemotacticfor lymphocytes.
Clearly, this whole operation serves to focus the im-
mune defenses around the invading microorganisms.
These become coated with antibody, C3b and certain
acute phase proteins and are ripe for phagocytosis With its customary prudence, evolution has estab-
by the granulocytes and macrophages; under the lished regulatory mechanisms to prevent inflamma-
influence of the inflammatory mediators these have tion from getting out of hand. At the humoral level we
upregulated C3 and Ig receptors, enhanced phagocytic have a series of complement regulatory proteins: C1
Figure 13.4. Events initiated by damaged vascular endotheliurn. cytokines and adhesion molecules, and the i~duction
Note that thrombin can also release platelet activating factor (PAX;) of the inflammation inhibitors lipocortin-l, secre-
which induces thrombus formation.
tory leukocyte proteinase inhibitor and IL-1 receptor
antagonist. IL-10 inhibits antigen presentation,
inhibitor, C4b-binding protein, the C3 control protein cytokine production and nitric oxide (NO) killing by
factors H and I, complement receptor CR1, decayuccel- macrophages, the latter inhibition being greatly en-
erating factor (DAF), membrane cofactor protein hanced by synergistic action with IL-4 and TGFP.
(MCP) and immunoconglutinin, and finally the pro- Once the inflammatory agent has been cleared, these
teins which block the membrane attack complex, regulatory processes will normalize the site. When
homologous restriction factor and CD59 (discussed the inflammation traumatizes tissues through its in-
further on p. 307). Some of the acute phase proteins tensity and extent, TGFP plays a major role in the sub-
derived from the plasma transudate would be expect- sequent wound healing by stimulating fibroblast
ed to act as protease inhibitors. division and the laying down of new extracellular ma-
At the cellular level, PGE,, transforming growth trix elements.
factor-P (TGFP) and glucocorticoids are powerful
regulators. PGE, is a potent inhibitor of lymphocyte
proliferation and cytokine production by T-cells and
macrophages. TGFP deactivates macrophages by in- If an inflammatory agent persists, either because of its
hibiting the production of reactive oxygen intermedi- resistance to metabolic breakdown or through the in-
ates and downregulating MHC class I1 expression; ability of a deficient immune system to clear an infec-
it also quells the cytotoxic enthusiasm of both tious microbe, the character of the cellular response
macrophages and y-interferon (1FNy)-activated NK changes.The sitebecomes dominated by macrophages
cells. Endogenous glucocorticoids produced via the with varying morphology: many have an activated ap-
hypothalamic-pituitary-adrenal axis exert their anti- pearance, some form arrays of what are termed 'ep-
inflammatory effects both through the repression of a ithelioid' cells and others fuse to form giant cells. If an
number of genes, includingthose for proinflammatory adaptive immune response is involved, lymphocytes
in various guises will also be present. This characteris- toxins, which actually poison the leukocytes (figure
tic granuloma walls off the persisting agent from the 13.6b).Yet another devious ruse is to exploitbinding to
remainder of the body (see section on type IV hyper- the surface of a nonphagocytic cell so gaining entry
sensitivity in Chapter 16,p. 343). into a shelter from the depredationsof the professional
phagocyte (figure13.6~). Presumably, some organisms
try to avoid undue provocation of phagocytic cells by
EXTRACELLULAR BACTERIA SUSCEPTIBLE
adhering to and colonizing the external mucosal surfaces
TO KILLING BY PHAGOCYTOSIS AND
of the intestine.
COMPLEMENT
Figure 13.5. The structure of bacterial cell walls. M types have an oligosaccharideside-chainsattached to a basal core polysaccharide,
inner cell membrane and a peptidoglycan wall which can be cleaved itself linked to the rnitogenic moiety, lipid A; 1480 antigen variants
by lysozyme and lysosomal enzymes. The outer lipid bilayer of of Escherichia coli are known). The mycobacterial cell wall is highly
Gram-negative bacteria, which is susceptible to the action of com- resistant to breakdown. When present, outer capsules may protect
plement or cationic proteins, sometimes contains lipopolysaccha- the bacteria from phagocytosis.
ride (LPS; also known as endotoxin; composed of 0-specific
Figure 13.6. Avoidance strategies by extracellular bacteria. (a) for alternative pathway convertase. (e) Accelerating breakdown of
Capsule gives poor phagocyte adherence. (b) Exotoxin poisons complement by action of microbial products. (f) Complementeffec-
phagocyte. (c) Microbe attaches to surface component to enter tors are deviated from the microbial cell wall. (g) Cell wall impervi-
nonphagocytic cell. (d) Capsule provides nonstabilizing surface ous to complement membrane attack complex (MAC).
C3b by the serine protease factor I (cf. p. 11). This is evolved thick peptidoglycan layers which prevent the
seen, for example, with Neisseria gonorrhoeae.Similarly, insertion of the lytic C5b-9 membrane attack complex
the hypervariable regions of the M-proteins of certain into the bacterial cell membrane (figure 13.6g).Many
Streptococcus pyogenes (group A streptococcus)strains capsules do the same.
are able to bind FHL-1, whilst other strains downregu-
late complement activation by interacting with C4BP,
Interfering with internal events in the rnacrophage
this time acting as a cofactor for factor I-mediated
degradation of the C4b component of the classical Enteric Gram-negativebacteria in the gut have devel-
pathway C3 convertase C 4b2a. There is evidence that oped a number of ways of influencing macrophage
C4BP can also inhibit activationof the alternative path- activity, including inducing apoptosis, enhancing
way. Certain strains of group B streptococci produce the production of IL-1, preventing phagosome-
a C5a-ase which may act as a virulence factor by lysosome fusion and affecting the actin cytoskeleton
proteolytically cleaving and thereby inactivating (figure13.7).
C5a (figure 13.6e).
Antigenie variation
Complement deviation. Some species manage to avoid
lysis by deviating the complement activation site Although the strategy of varying individual antigens
either to a secreted decoy protein or to a position on in the face of a determined host antibody response is
the bacterial surface distant from the cell membrane more usually associated with viruses and parasites,
(figure13.6f). there are a few well-defined examples in bacteria.
These include variation of surface lipoproteins in the
Resistance to insertion of terminal complement compo- lyme disease spirochete Borrelia burgdorfEri,alterations
nents. Gram-positive organisms (cf. figure 13.5) have in enzymes involved in synthesizing surface struc-
tures in Campylobacter jejuni and antigenic variation
of the pili in Neisseria meningitidis. In addition,
new strains can arise, as has occurred with the life-
threatening E. coli 0157: H7 which can cause hemoly-
tic uremic syndrome and appears to have emerged
about 50 years ago by incorporation of Shigella toxin
genes into the E. coli 055 genome.
(continued)
Figure M13.1.3. Sir Almroth Wright. (Slide kindly supplied E
Figure M13.1.1. Emil von Behring (1854-1917). The Wellcome Centre Medical PhotographicLibrary,London.)
alerting service for the immune system, which detects from the circulation. Binding of LPS to the CD14 PRR
their presence using a number of pattern recognition on monocytes, macrophages, dendritic cells and
receptors (PRRs) expressed on the surface of antigen- B-cells leads to the recruitment of the toll-like receptor
presenting cells. Such receptors include the mannose 4 (TLR4) molecule which is able to mediate signals
receptor (CD206) which facilitates phagocytosis of activating the expression of a broad range of pro-
microorganisms by macrophages and the scavenger inflammatory genes, including those for IL-1, IL-6,
receptor (CD204)whichmediatesclearanceofbacteria IL-12 and TNF and for the B17.1 (CD80) and B7.2
(CD86) costimulatory molecules. A related receptor,
TLR2, recognizes Gram-positive bacterial cell wall
components.
Toxin neutralization
Circulating antibodies act to neutralize the soluble
antiphagocytic molecules and other exotoxins (e.g.
phospholipase C of Clostridium welchii) released by
bacteria. Combination near the biologically active site
of the toxin would stereochemically block reaction
with the substrate, particularly if it were macromole-
cular; combination distant from the active site may
also cause inhibition through allosteric conforma-
tional changes. In its complex with antibody, the toxin
may be unable to diffuse away rapidly and will be
susceptible to phagocytosis, especially if the complex
can be increased in size by the action of naturally
occurring autoantibodies to complexed IgG (anti-
globulin factors) and C3b (immunoconglutinin,not to
be confused with bovine conglutinin, a nonantibody
molecule which combines with the carbohydrate
portion of C3b). Figure 13.8. Effect of opsonizing antibody and complement on
rate of clearance of virulent bacteria from the blood. The uncoated
bacteria are phagocytosed rather slowly (innate immunity) but, on
Opsovlizatiotz ofbacteria coating with antibody, adherence to phagocytes is increased many-
fold (acquired immunity).The adherence is less effective in animals
temporarily depleted of complement.This is a hypotheticalbut real-
independently ofantibody. Differences between the car- istic situation; the natural proliferation of the bacteria has been
bohydrate structures on bacteria and self are exploited ignored.
by the collectins, a series of molecules with similar ul-
trastructure to Clq and which bear C-terminal lectin
domains. These include mannose-binding protein through specific high affinity receptors for IgG and
(MBP; also referred to as mannose-binding lectin, C3b on the phagocyte surface (figure 13.9).It is clearly
MBL), lung surfactantproteins SP-A and SP-D and, in advantageous that the subclasseswhich bind strongly
cattle, conglutinin, which all recognize carbohydrate to these Fc receptors (e.g.IgGl and IgG3 in the human)
ligands. Mannose-binding protein can bind to termi- also fix complement well, it being appreciated that the
nal mannose on the bacterial surfaceand then interacts heterodimer of C3b bound to IgG is a very efficient
with MBP-associated serine protease (MASP)which is opsonin because it engages two receptors simultane-
homologous in structure to Clr and Cls. Thus the in- ously. Complexes containing C3b and C4b may show
teraction is closely similar to that of Clq with Clr and s immune adherence to the CR1 complement receptors
(cf. p. 22) and, in this way, leads to the antibody- on erythrocytesto provide aggregateswhich are trans-
independent activation of the classical pathway. ported to the liver and spleen for phagocytosis.
SP-A, SP-D and conglutinin can all act as opsonins Some elaboration on complement receptors may be
(see Milestone 13.1) and can mediate phagocytosis pertinent at this stage. The CR1 receptor (CD35) for
by virtue of their binding to the Clq receptor. C3b is also present on neutrophils, eosinophils,mono-
cytes, B-cells and lymph node follicular dendriticcells.
Augmented by antibody. Encapsulated bacteria which Together with the CR3 receptor (CDllb/CD18), it has
resist phagocytosis become extremely attractive to the main responsibilityfor clearance of complexescon-
neutrophils and macrophages when coated with anti- taining C3. The CRI gene is linked in a cluster with
body and their rate of clearance from the bloodstream C4b-binding protein and factor H, all of which sub-
is strikingly enhanced (figure 13.8).The less effective serve a regulatory functionby binding to C3b or C4b to
removal of coated bacteria in complement-depleted disassemble the C3/C5 convertases, and act as cofac-
animals emphasizes the synergism between antibody tors for the proteolytic inactivation of C3b and C4b by
and complement for opsonization which is mediated factor I.
Figure 13.9. Imrnunoglobulin and complement coats greatly menting effect of complement is due to the fact that two adjacent
increase the adherence of bacteria (and other antigens) to IgG molecules can fix many C3b molecules, thereby increasing
macrophages and neutrophils. Uncoated bacteria adhere to lectin- the number of links to the macrophage (cf. 'bonus effect of mul-
like sites, including the mannose-binding receptor. There are tivalency'; p. 87). Although IgM does not bind specifically to
no specific binding sites for IgM ( ), but there are high affinity the macrophage, it promotes adherence through complement
receptors for IgG (Fc) ( ) and iC3b ( ) on the macrophage surface fixation.Specific receptors for the Fca domains of IgAhave also been
which considerably enhance the strength of binding. The aug- defined.
high percentage react to the 65 kDa heat-shock protein. to tuberculosis I ) and Nrampl (natural resistance-
A vital role for these cells in murine TB is indicated by associated macrophage protein 1).At least 11 poly-
the death of y6 TCR deletion mutants after an infection morphisms have been identified in the human Nrampl
with M. tuberculosis which was still tolerated by im- homolog and studies are underway to link indivi-
munocompetent animals. dual polymorphisms to susceptibility.
Given these potential CM1 defenses, why do some Where the host has difficulty in effectively eliminat-
individuals fail to eradicate their infections with my- ing these organisms, the chronic CM1 response to
cobacteria and other intracellular facultative bacteria local antigen leads to the accumulation of densely
and so suffer from diseases such as TB and leprosy packed macrophages which release angiogenic and
even though they have established Thl responses? fibrogenic factors and stimulate the formation of gran-
One important clue is provided by the demonstration ulation tissue and ultimately fibrosis. The activated
that inbred strains of mice differ dramatically in their macrophages, perhaps under the stimulus of IL-4,
susceptibility to infection by various mycobacteria, transform to epithelioid cells and fuse to become giant
Salmonella typhimurium and Leishmania donovani. Re- cells. As suggested earlier, the resulting granuloma
sistance is associated with a T-cell-independent en- represents an attempt by the body to isolate a site of
hanced state of macrophage priming for bactericidal persistent infection.
activity involving oxygen and nitrogen radicals.
Moreover, macrophages from resistant strains
Leprosy
have increased MHC class I1 expression and a higher
respiratory burst, are more readily activated by IFNy, In human leprosy, the disease presents as a spectrum
and induce better stimulation of T-cells, whereas ranging from the tuberculoid form, with lesions
macrophages from susceptible strains tend to have containing small numbers of viable organisms, to the
suppressor effects on T-cell proliferation to mycobac- lepromatous form, characterized by an abundance
terial antigens. Susceptibility and resistance to My- of M. leprae within the macrophages. The tuberculoid
cobacterium tuberculosis in murine models depend state is associated with good cell-mediated dermal
upon a number of genes, including sstl (susceptibility hypersensitivity reactions and a bias towards Thl-
type responses, although these are still not good
enough to eradicate the bacilli completely.In the lepro-
matous form, there is poor T-cell reactivity to whole
bacilli and poor lepromin dermal responses, although
there are numerous plasma cells which contribute to a
high level of circulating antibody and indicate a more
prominent Th2 activity. Clearly, CM1 rather than hu-
moral immunity is important for the control of the lep-
rosy bacillus, but reasons for the inadequate responses
of the lepromatous patients are still uncertain.
I M M U N I T Y TO V I R A L INFECTION
Genetically controlled constitutional factors which
render a host or certain of their cells nonpermissive
(i.e.resistant to takeover of their replicative machinery
by virus) play a dominant role in influencing the
vulnerability of a given individual to infection. Figure 13.18. Antigenic drift and shift in influenza virus. The
Macrophages may readily take up viruses nonspecifi- changes in hemagglutinin structure caused by drift may be small
cally and kill them. However, in some instances, the enough to allow protection by immunity to earlier strains. This may
not happen with radical changes in the antigen associated with anti-
macrophages allow replication and, if the virus is genic shift and so new virus epidemics break out. There have been 31
capable of producing cytopathic effects in various documented influenza pandemics (widespread epidemics occur-
organs, the infection may be lethal; with noncyto- ring throughout the population) since the first well-described pan-
pathic agents, such as lymphocytic choriomeningitis, demic of 1580. In the last century there were three, associated with
the emergence by antigenic shift of the Spanish flu in 1918 with the
Aleutian mink disease and equine infectious anemia structure HINl (the official nomenclature assigns numbers to each
viruses, a persistent infection may result. Viruses can hemagglutinin and neuraminidase major variant),Asian flu in 1957
avoid recognition by the host's immune system by la- (H,N2) and Hong Kong flu in 1968 (H3N2);note that each new epi-
tency or by shelteringin privileged sites, but they have demic was associated with a fundamental change in the hemagglu-
tinin. The pandemic in 1918killed an estimated 40 million people.
also evolved a maliciously cunning series of evasive
strategies.
lar infective agents and CM1 with intracellular ones. threatening viral infections, including EBV, varicella
The same holds true for viruses which try to shelter and cytomegaloviruses (CMVs). The surface of a
from antibody in an intracellular habitat. Local or sys- virally infected cell undergoes modification, probably
temic antibodies can block the spread of cytolytic in its surface carbohydrate structures, making it an at-
viruses which are released from the host cell they have tractive target for NK cells, which can be shown to be
just killed, but alone they are usually inadequate to cytotoxic in vitvo to cells infected with a number of dif-
control those viruses which bud off from the surface as ferent viruses. The NK cell possesses two families of
infectious particles because they are also capable of surface receptors. One, killer activating receptors,
spreading to adjacent cells without becoming exposed binds to carbohydrate and other structures expressed
to antibody (figure 13.20). The importance of CM1 for collectively by all cells; the other, killer inhibitory re-
recovery from infection with these agents is under- ceptors, recognizes MHC class I molecules and over-
lined by the inability of children with primary T-cell rules the signal from the activating receptor. Thus, the
immunodeficiencyto cope with such viruses, whereas sensitivity of the target cell is closely related to self-
patients with Ig deficiency but intact CM1 are not MHC class I expression; sensitive targets have low
troubled in this way. class 1, but transfection with the self-MHC molecule
will generally protect them. Karrer has suggested that,
whereas T-cells search for the presence of foreign
Nf(cells caH kill virally iafected targets
shapes, NK cells survey tissues for the absence of self
In earlier chapters, we have explained how early as indicated by aberrant or absent expression of MHC
recognition and killing of a virally infected cell before class I, which might occur in tumorigenesis or in cer-
replication occurs is of obvious benefit to the host. The tain viral infections. The ability of EBV to downregu-
importance of the NK cell in this role as an agent of pre- late class I would explain the drastic nature of this
formed innate immunity can be gauged from observa- infection in NK-deficient patients mentioned above.
tions on the exceedingly rare patients with complete The production of IFNa during viral infectionnot only
absence of these cells who suffer recurrent life- protects surrounding cells, but also activates NK cells
and upregulates MHC expression on the adjacent cells, rather than CD4 T-cells as the major defensive force.
making them more resistant to cytotoxicity. The knee-jerk response would be to implicate cytotox-
icity, but remember that CD8 cells also produce cy-
tokines. This may well be crucial when viruses escape
Cytotoxic T-cells (Tc)are cmcial elements in
the cytotoxic mechanism and manage to sidle laterally
immunity t o iurfection by budding viruses
into an adjacent cell. CM1 can now play some new
T-lymphocytes from a sensitized host are directly cyto- cards: if T-cells stimulated by viral antigen release cy-
toxic to cells infected with viruses, the new MHC- tokines such as IFNy and macrophage or monocyte
associated peptide antigens on the target cell surface chemokines,the mononuclear phagocytes attracted to
being recognized by specific ap receptors on the ag- the site will be activated to secreteTNF, which will syn-
gressor lymphocytes. There is a quite surprising fre- ergize with the IFNy to render the contiguous cells
quency of dual specificitiesin target cell recognitionby nonpermissive for the replication of any virus ac-
virus-specific Tc clones, which can lyse uninfected al- quired by intercellular transfer (figure 13.20). In this
logeneic cells or targets expressing peptides with little way, the site of infection can be surrounded by a cor-
homology from different regions of the same viral pro- don of resistant cells. Like IFNa, IFNy increases the
tein, from different proteins of the same virus, or even nonspecific cytotoxicity of NK cells (see p. 18) for in-
from different unrelated viruses. Thus activation by a fected cells. This generation of 'immune interferon'
second virus may help to maintain memory and there (IFNy) and TNF in response to non-nucleic acid viral
may be a spontaneous immunity to an unrelated virus components provides a valuable back-up mechanism
after initial infection with a cross-reacting strain. when dealing with viruses which are intrinsically poor
Downregulationof MHC class I poses no problems for stimulators of interferon synthesis.
TCRl y6 Tc which recognize native viral coat protein
(e.g. herpes simplex virus glycoprotein) on the cell
Antibody has a part too
surface (figure 13-20).
Tc cells can usually be detected in the peripheral The neutralization of free virus particles by antibody
blood lymphocytes of individuals who have recovered is relatively straightforward but the interaction with
from infection with influenza, CMV or EBV by re- infected cells is rather more complex. Access to the
exposure in uitro to appropriately infected cells. In the surface antigensby ap T-cells cannotbe blocked by an-
case of CMV, for example, the targets are cells with the tibody since these cells recognize processed antigen,
'early antigen' on their surface expressed within 6 whereas antibody binds native antigen. Antibodies
hours of infection. As discussed above, it is clearly can, however, block y6 Tc by reacting with surface anti-
advantageous for the cytotoxic cell to strike so soon gen on incipiently budding virions, but should be able
after infection. Studies on volunteers, showing that to initiate ADCC (p. 32) as has been reported with
high levels of cytotoxic activity before challenge with herpes-, vaccinia- and mumps-infected target cells.
live influenza correlated with low or absent shedding Do not forget the importanceof antibody in prevent-
of virus, speak in favor of the importance of Tc in ing reinfectionwith most viruses.
human viral infection.
After a natural infection, both antibody and Tc cells
I M M U N I T Y TO FUNGI
are generated; subsequent protection is long-lived
without reinfection, possibly being reinforced by by- Not too much is known about this subject. Many fun-
stander activation through cytokines released from gal infections become established in immunocompro-
other stimulated T-cells, or perhaps by random trig- mised hosts or when the normal commensal flora are
gering with unrelated viruses based on the dual speci- upset by prolonged administration of broad-spectrum
ficity described earlier. By contrast, injection of killed antibiotics. T-cells are important in defense and can
influenza produces antibodies but no Tc and protec- recognize antigens from organisms such as Cryp-
tion is only short term. tococcus neoformans and Candida albicans, killing
them by the granzyme system. NK cells can also lyse C.
neofarmans.
Cytokines recmit eflectors and provide
a 'cordon sanitaire '
I M M U N I T Y TO PARASITIC INFECTIONS
A number of studies on the transfer of protection to
influenza, lymphocytic choriomeningitis, vaccinia, The diverse organisms responsible for the major para-
ectromelia and CMV infections have focused on CD8 sitic diseases are listed in figure 13.21.The numbers af-
Figure 13.21. The major parasites in
humans and the sheer enormity of the
numbers of people infected. (Data from
World Health Organization,1990.)
fected are truly horrifying and the sum of misery these that a humoral response developswhen the organisms
organisms engender is too large to comprehend. The invade the bloodstream (malaria, trypanosomiasis),
consequences of parasitism could be, at one extreme, a whereas parasites which grow within the tissues (e.g.
lack of immune response leading to overwhelming cutaneous leishmaniasis) usually elicit CM1 (table
superinfection, and, at the other, an exaggerated 13.1).Often, a chronicallyinfected host will be resistant
life-threatening immunopathologic response. To be to reinfection with fresh organisms, a situation termed
successful, a parasite must steer a course between concomitant immunity. This is seen particularly in
these extremes, avoiding wholesale killing of the schistosomiasisbut also in malaria, where historically
human host and yet at the same time escaping the phenomenon was called 'premunition'. The resi-
destruction by the immune system. In practice, each dent and the infective forms must differ in some way
type of parasite is virtually a world unto itself in the yet to be pinpointed.
complexity of the mechanisms by which this is
achieved.
merozoite stages of malaria. Thus, individuals receiv- the eosinophilic granules is released onto the parasite
ing IgG from solidly immune adults in malaria endem- and brings about its destruction.A contribution to this
ic areas are themselves temporarily protected against process from CM1 is emerging, since eosinophils can
infection, the effector mechanisms being opsonization express class I1 MHC and their IgG-mediated ADCC
and phagocytosis, and complement-dependentlysis. is strongly enhanced by granulocyte-macrophage
A marked feature of the immune reaction to colony-stimulating factor (GM-CSF)and TNF. Further
helminthic infections, such as Tvichinella spivalis in evidence for an involvement of this cell comes from
humans and Nippostrongylus bvasiliensis in the rat, is the experiment in which the protection afforded by
the eosinophilia and the high level of IgE antibody passive transfer of antiserum in vivo was blocked by
produced. In humans, serum levels of IgE can rise pretreatment of the recipient with an anti-eosinophil
from normal values of around 100ng/ml to as high as serum. It has also been found that eosinophilscan kill
10000ng/ml. These changes have all the hallmarks IgE-coated schistosomula, but the mechanism is dif-
of response to Th2-type cytokines (cf. p. 181) and it ferent because activation of the IgE (FceRI) receptors
is notable that, in animals infected with helminths, in- now triggers release of platelet activating factors and
jection of anti-IL-4 greatly reduces IgE production the eosinophil peroxidase. This dichotomy in Fcy and
and anti-IL-5 suppresses the eosinophilia. This excep- FCEreceptor pathways is also evident from reports that
tional increase in IgE has encouraged the view that it IgE but not IgG can mediate schistosome killing by
represents an important line of defense. One can see macrophages or platelets.
that antigen-specific triggering of IgE-coated mast Two further points are in order. The IgE-mediated
cells would lead to exudation of serum proteins con- reactions may be vital for recovery from infection,
taining high concentrations of protective antibodiesin whereas the resistance in vaccinated hosts may be
all the major Ig classes and the release of eosinophil more dependent upon preformed IgG and IgA
chemotacticfactor. It is relevant to note that schistoso- antibodies.
mula, the early immature form of the schistosome,
have been killed in cultures containing both specific
Cell-mediated immunity
IgG and eosinophils,which induce a form of ADCC by
binding through their FcyRII receptors to the IgG-coat- Just like mycobacteria, many parasites have adapted
ed organism (figure 13.22); after 12hours or so, the to life within the macrophage despite the possession
major basic protein forming the electron-dense core of by that cell of potent microbicidal mechanisms includ-
In vivo, the balance of cytokines produced is of the
utmost importance. Infection of mice with Leishmania
major is instructive in this respect; the organism pro-
duces fatal disease in susceptible mice but other strains
are resistant. This is partly controlled by alleles of the
Nramp-l gene (cf. p. 266) but, as discussed earlier in
Chapter 10, in susceptible mice there is excessive
stimulation of Th2 cells producing IL-4 which do not
help to eliminate the infection, whereas resistant
strains are characterized by the expansion of Thl cells
which secrete IFNy in response to antigen presented by
macrophages harboring living protozoa. Combined
therapy of susceptible strains with the leishmanicidal
drug, Pentostam, plus IL-12, which recruits Thl cells,
provides promise that Th2 activities which exacerbate
disease can be switched to protective Thl responses.
CD4 clones which recognize only lysates of the organ-
ism do not confer protection even though they pro-
Figure 13.22. Electron micrograph showing an eosinophil (E) at- duce IFNy, a point to be borne in mind in designing
tached to the surface of a schistosomulum (S) in the presence of vaccines. Experiments in knockout mice suggest that
specific antibody.The cell develops large vacuoles (V) which appear perforins, granzyme B and FasL are not necessary for
to release their contents on to the parasite (X 16500).(Courtesy of Drs the protection afforded to mice by vaccination with
D.J.McLaren and C.D. Mackenzie.)
irradiated sporozoites. Of particular importance for
protection, however, is the induction of IFNy and
CD8' T-cells. Interleukin-12 and nitric oxide are also
required, and NK cells may play a subsidiary role.
Organisms such as malarial plasmodia, and inciden-
tally rickettsiae and chlamydiae, that live in cells
which are not professional phagocytes, may be elimi-
nated through activation of intracellular defense
mechanisms by IFNy released from CD8+ T-cells or
even by direct cytotoxicity.This is very much the case
in hepatic cells harboring malarial sporozoites, and it
is pertinent to note that, followingthe recognition of an
association between HLA-B53 and protection against
severe malaria, B53-restricted Tc reacting with a con-
served nonamer from a liver stage-specific antigen
Figure 13.23. Role of NO. in macrophage leishmanicidal activity. were demonstrated in the peripheral blood of resistant
The NO. synthase inhibitor L-NMMA ( 5 0 m ) inhibits the ability individuals. A large case control study of malaria
of macrophages to kill intracellular Leishmania where growth is in Gambian children showed that the protective B53
monitored by [3H]thymidine incorporation (a) and also blocks NO
production measured by accumulation of NO, in the culture super- class I antigen is common in West African children but
natant at 72 hours (b).The D-isomer,D-NMMA,used as a control does rare in other racial groups, lending further credence to
not inhibit the enzyme. (Data taken from Liew F.Y. & Cox F.E.G. the hypothesis that MHC polymorphism has evolved
(1991)Immunology Today 12,Al7.) primarily through natural selection by infectious
pathogens.
ing NO-(figure 13.23).Intracellular organisms, such as Eliminating worm infestations of the gut is a more
Toxoplasma gondii, Trypanosoma cruzi and Leishmania tricky operation and the combined forces of cellular
spp., use a variety of ploys to subvert the macrophage and humoral immunity are required to expel the un-
killing systems (see below) but again, as with wanted guest. One of the models studied is the re-
mycobacterial infections, cytokine-producing T-cells sponse to Nippostrongylus brasiliensis; transfer studies
are crucially important for the stimulation of in rats showed that, although antibody produces some
macrophages to release their killing power and damage to the worms, T-cells from immune donors are
dispose of the unwanted intruders. also required for vigorous expulsion, which is proba-
Figure 13.24. The expulsion of nematode
worms from the gut. The parasite is first
damaged by IgG antibody passing into the
gut lumen, perhaps as a consequence of
IgE-mediatedinflammation and possibly
aided by accessory ADCC cells. Cytokines
released by antigen-specific triggering of T-
cells stimulate proliferation of goblet cells
and secretionof mucous materials, which
coat the damaged worm and facilitateits
expulsion from the body by increased gut
motility induced by mast cell mediators,
such as leukotriene-D4, and diarrhea
resulting from inhibition of glucose-
dependent sodium absorption by mast
cell-derivedhistamine and PGE,.
bly achieved through a combination of mast cell- plement directly, but eject the bound C3 by shedding
mediated stimulation of intestinal motility and cyto- their glycocalyx. The Plasmodium falciparum protein
kine activation of the innumerable intestinal goblet PfEMPl is expressed on the surface of infected ery-
cells. These secrete a complex mixture of densely gly- throcytes and can bind to CR1 (CD35)on other infected
cosylated high molecular weight molecules which erythrocytes leading to rosette formation, which may
form a viscoelastic gel around the worm, so protecting facilitatespread of the parasite with minimal exposure
the colonic and intestinal surfaces from invasion (fig- to the host immune system.In a similar fashion, malar-
ure 13.24). Another model, this time of Trichinella ial sporozoites shed their circumsporozoite antigen
spiralis infection in mice, again hints at a duality of T- when it binds antibody, and T'ypanosoma bvucei
subset cytokine responses. One strain, which expels releases its surface antigens into solution to act as
adult worms rapidly, makes large amounts of IFNy decoy proteins (p. 255). In each case, these shedding
and IgG2a antibody, while, in contrast, more suscepti- and decoy systems are well suited to parasites or
ble mice make miserly amounts of IFNy and favor stages in the parasite life cycle which are only briefly in
IgG1, IgA and IgE antibody classes. Clearly, the protec- contact with the immune system.
tive strategy varies with the infection. We have already mentioned the way in which differ-
ent protozoa1 parasites hide away from the effects of
antibody by using the interior of a macrophage as a
sanctuary. To do this they must block the normal mi-
crobicidal mechanisms and they use similar methods
Resistance to efSectov mechanisms
to those deployed by intracellular obligate and faculta-
Some tricks to pre-empt the complement defenses are tive bacteria (cf. p. 263). Toxoplasma gondii inhibits
of interest. T. cruzi has elegantly created a DAF-like phagosome-lysosome fusion by lining up host cell
molecule (cf. p. 307) which accelerates the decay of mitochondria along the phagosome membrane.
C3b. The cercariae of Schistosoma mansoni activate com- Trypanosoma cruzi escapes from the confines of the
phagosome into the cytoplasm, while Leishmania to-blood contact. The same phenomenon has been
parasites are surrounded by a lipophosphoglycan observed with Plasmodium spp. and this may explain
which protects them from the oxidative burst by why in hyperendemic areas, children are subjected to
scavenging oxygen radicals. They also downregulate repeated attacks of malaria for their first few years and
expression of MHC and B7 so diminishing T-cell are then solidly immune to further infection. Immuni-
stimulation. ty must presumably be developed against all the anti-
genic variants before full protection can be attained,
and indeed it is known that IgG from individuals with
Avoiding antigen recognition by the host
solid immunity can effectively terminate malaria
Some parasites disguise themselves to look like the infectionsin young children.
host. This can be achieved by molecular mimicry as
demonstrated by cross-reactivitybetweenAscaris anti-
Deviation o f the host immune response
gens and human collagen. Another way is to cover the
surface with host protein. Schistosomesare very good Immunosuppression has been found in most of the
at that; the adult worm takes up host red-cell glycopro- parasite infections studied. During infection by try-
teins, MHC molecules and IgG and lives happily in the panosomes, for example, antibody and CM1 are only
mesenteric vessels of the host, despite the fact that the 5-10% of the normal values while T-suppressor activi-
blood which bathes it contains antibodies which can ty mediated by IL-10 and IFNy is prominent, presum-
prevent reinfection. ably related to an excessiveload of antigen. Schistosoma
Another very crafty ruse, rather akin to moving the mnsoni possesses a gene with homology for proopio-
goalposts in football, is antigenic variation, in which melanocortin which, in the human, is a prohormone
the parasites escape from the cytocidal action of hu- that is cleaved to generate adrenocorticotropic hor-
moral antibody on their cycling blood forms by the in- mone (ACTH), melanocyte-stimulating hormone
genious trick of altering their antigenic constitution. (MSH) and the opioid P-endorphin. All have im-
Figure 13.25 illustrates how the trypanosome contin- munomodulatory properties and both ACTH and P-
ues to infect the host, even after fully protective anti- endorphin have been demonstrated in culture when
bodies appear, by switching to the expression of a new adult worms were incubated at 37C in minimum
antigenic variant which these antibodiescannot inacti- essential medium; if neutrophils are added to the
vate; as antibodiesto the new antigens are synthesized, system, a-MSH, derived from ACTH by the cell's neu-
the parasite escapes again by changing to yet a further tral endopeptidase (CD10; CALLA, p. 387), is also
variant and so on. In this way, the parasite can remain formed.
in the bloodstreamlong enough to allow an opportuni- Parasites may also manipulate T-cell subsets to their
ty for transmissionby blood-sucking insects or blood- own advantage. Filariasis provides a case in point: it
Figure 13.25. Antigenic variation during chronic trypanosomein- response and is succeeded by variant C. At any time, only one of
fection. As antibody to the initial variant A is formed, the blood try- the variant surface glycoproteins (VSGs) is expressed and covers
panosomes become complexed prior to phagocjrtosis and are no the surface of the protozoan to the exclusion of all other antigens.
longer infective, leaving a small number of viable parasites which Nearly 9% of the genome (approximately1000 genes) is devoted to
have acquired a new antigenic constitution. This new variant (B) generation of VSGs. Switching occurs by insertion of a duplicate
now multiplies until it, too, is neutralized by the primary antibody gene into a new genomic location in proximity to the promoter.
has been suggested that individuals with persistent to the TSE agent.B-lymphocytes may play a subsidiary
microfilariae fail to mount presumably protective im- role via their production of lymphotoxin, a cytokine
mediate hypersensitivityresponses, including IgE and necessary for the maintenance of a differentiated
eosinophilia, as a result of active suppression of Th2 state in the FDCs. Furthermore, in addition to FDCs,
cells. macrophages have been proposed as providing a
Epidemiological surveys accord with a protective reservoir of infectivity.
role for IgE antibodiesin schistosomiasis, but they also
reveal a susceptible population producing IgM and
IgG4 antibodies which can block ADCC dependent
upon IgE. The ability of certain helminths to activate Where parasites persist chronicallyin the face of an im-
IgE-producing B-cells polyclonally is good for the par- mune response, the interaction with foreign antigen
asite and correspondingly not so good for the host, frequently produces tissue-damaging reactions. One
since a high concentration of irrelevant IgE binding to example is the immune complex-induced nephrotic
a mast cell will crowd out the parasite-specificIgE mol- syndrome of Nigerian children associated with quar-
ecules and diminish the possibility of triggering the tan malaria. Increased levels of TNF are responsible for
mast cell by specific antigen to initiate a protective pulmonary changes in acute malaria, cerebral malaria
defensive reaction. in mice and severe wasting of cattle with trypanosomi-
asis. Another example is the liver damage resulting
from IL-4-mediated granuloma formation around
schistosomeeggs (cf. figure 16.28);one of the egg anti-
New variant Creutzfeldt-Jakob disease (nvCJD) was gens directly induces IL-10 production in B-cells,
first described in 1996 and, in common with sheep thereby contributing to Th2 dominance. Remarkably,
scrapie and bovine spongiform encephalopathy (BSE), the hypersensitivity reaction helps the eggs to escape
is classed as a transmissible spongiform encephalopa- from the intestinalblood capillaries into the gut lumen
thy (TSE) caused by prions. The role of the immune to continue the cycle outside the body, an effect medi-
system in prion diseases seems to be one of helping the ated by TNFa.
disease rather than combating it. These infectious dis- Cross-reaction between parasite and self may give
eases lead to abnormally folded, relatively protease- rise to autoimmunity, and this has been proposed
resistant forms of host prion protein (PrP).Infectivity as the basis for the cardiomyopathyin Chagas' disease.
usually replicates to high levels in lymphoid tissues It is also pertinent that the nonspecific immuno-
before spreading to the central nervous system, and suppression which is so widespread in parasitic dis-
there is evidence to suggest that follicular dendritic eases tends to increase susceptibility to bacterial and
cells (FDCs)in spleen, lymph node and Peyer 'S patch- viral infections and, in this context, the association
es may be involved in this replication. This may be be- between Burkitt's lymphoma and malaria has been
cause FDCs naturally express high levels of the normal ascribed to an inadequate host response to the
PrP which then becomes abnormalfollowingexposure Epstein-Barr virus.
~ SUMMARY
Immunity to infection involves a constant battle between and then passage across the blood vessel up the chemotac-
thehostdefensesandthemutantmicrobestryingtoevolve tic gradient to the site of inflammation.
evasive strategies. IL-1, TNF and chemokines such as IL-8 are involved in
maintaining the inflammatoryprocess.
-
Inflornmation revisited
Mammation is a major defensive reaction initiated by
infection or tissue injury.
Inflammation is controlled by complement regulatory
proteins, PGE,, TGFP, glucocorticoidsand IL-10.
LPS is bound by LBP which transfers the L E to the
The mediators released upregulate adhesion molecules CDIPTLR4 complex, thereby activatinggenes in the APC
on endothelial cells and leukocytes, which pair together which encodeproinflammatorymolecules.
causing, first, rolling of leukocytes along the vessel wall Inability to eliminate the initiating agent leads to a
(continued p. 278)
chronic inflammatory response dominated by macm Infected cellsexpressa processed viral antigen peptide on
phagesoftenforminggranulomas. their surface in associationw t h MHC class I a short time
afterentryof thevirus,andrapidkilgof thecell bycyto-
UmPcellularbacIeriasusceplibletakilling by toxic af3 T-cells prevents viral multiplication which de-
phogoeyloslsondcomplement pends upon the rephcahve machinery of the intact host
Bacteria try to avoid phagocytosis by surrounding cell. y8 Tc m g r u z e native viral coat protein on the target
themselveswith capsules, secreting exotoxins which kill cell surface. NK cells are also cytotoxic.
phagocytes or impede inflammatoryreactions, deviating T-cells and macrophages producing IFNy and TNF
complementtoinoffensivesitesorbycolonizingrelatively bathe the contiguouscells and prevent them from becom-
inaccgsiblelocations. ing infectedby lateral spread of virus.
Antibody combats these tricks by neutralizing the tox-
ins,makingcomplementdepositionmoreevenonthebac- lmmunitytaparasiticinfections
terial surface, and overcomingthe antiphagocyticnature Diseases involving pmtoml purasrtes and helrninfhsaf-
of the capsulesbyopsonizing themwith Ig andC3b. fecthundredsofnullionsofpeople.Anhbodiesareusually
The secretory immune system protecti the external effeetiveagainstthe blood-borne forms. IgE production is
muCOsal surfaces. IgA inhibits adhemce of bacteria and notoriously incwasedinworminfstationsandcanleadto
canopsonizethem. IgEboundtomastcellscaninitiatethe mast fen-mediated influx of Ig and eosinophils; schisto
influx of protective IgG, complement and polymorphs to somes coated with IgG or IgE are kiUed by adherent
thesitebyaminiatureacuteineammatoryresponse. eosinophils through extracellular mechanisms lnvolving
therelease of cationicprotem and peroxldase.
BPdeda which gmw in an immcellulot bobaot Organisms such as Leishinania spp., Tiypanosoma cruzi
Intmcellular bacteria such as tubede and leprosy and Tomplasm gondn hide from antibodies inside
baciUi grow within mamphages. They defy killing macmphages,use the same strategiesas intracellular par-
mechanisms by blocking macrophage activation, scav- asiticbacteriatosurmve,andhkethemarekilledwhenthe
enging oxygen radicals, inhibiting lysosome fusiin, mamphages are activated by Thl cytokines produced
havingstrongoutercoatsandbyescapingfromthepha~ duringcell-mediatedimmunerrsponses.NO-isanimpor-
some into the cytoplasm. tant W i g agent.
TheyarekjlledbyCMI:specEcallysensitizedT-helpers
release cytokines on contact with infeaed mamphages
which powerfully activate the formation of nitric oxide
- CDScellsalsohave aprotectiverole.
Expulsion of intestinal worms usually dependsheavily
on Th2 responzes and requires the coordinated action of
(NO.), reactive oxygen intermediates (ROIS) and other antibody, the release of much by cytokinestimulated
mimbicidal mechanisms. goblet cells and the production of intestinal contraction
lmmunilytovim1 infeuion
V i hy to avoid the immune system by changes in
-
anddiarrheabymast cellmediators.
Some parasites avoid recognitionby disguising them-
selvesas the host, either through molecularmimicry or by
the antigeniatyof theiisurface antigens. Point mutations absorbinghost proteinsto their surface.
bring about minor changes (antigenic drift), but radical OtherorganismssuchasTrypanosom bruceiandvarious
changes leading to endemics can result from wholesale malarial species have the extraordinary abfity to cover
swapping of genetic material with different viruses in their swfacewith a dominantantigenwhch is changed by
otheranimalhosts(antigenicshift). geneticswi~hmechaniSmstoadifferentmoleculeasanti-
Some virws subvert the function of the complement bodyisformedtothefirstvariant
system to their own advantage. Most parasites also tend to produce nonspecific s u p
Antibody neutralizes free virus and is paaicularly ef- pression of host nsponses.
fective when the virus has to travel through the blood- Chronicpersistence of parasite antigen in the face of an
sixearnbeforereadringitsfinal target. immune response often produces tissuedamaging im-
Where the tuget is the same as the portal of enhy, e.g. munopathologid reactions such as immune complex
the lungs, IFNis dominantinrecoveryf m m i . nephrotic syndrome, liver granulomas and autoim-
Antibodyisimportantinpreventingreinfection mune lesions of the heart. Generalized immunmuppres-
Budding viruses which can invade lateral & with- sion increase$ susceptibility to bacterial and viral
out becoming exposed to antibwiy are combatedby CMI. infeCtiOIlS.
Figure 13.26. Simplified scheme to
emphasize the interactions between
innate and acquired immunity
mechanisms.The dendritie cell which
presents antigen to E-cellsin the form
of m u n e complexes is the follicular
dendritic cell in germinal centers,
whereasthe MHC class 11-positive
interdigitating dendritic cell presents
antigen to T-cells. (Developedfrom
PlayfaYJ.H L. (1974)BritcshMedicnl
Bullehn 30,24.)
Asthe featuresoftheresponsetoinfectionareanalysed, 9 Abnormally folded, protease-resistant forms of host
we see more clearly how the specfic acquired response prionprotein(PrP) develop.
operates to a m p m and enhance m a t e immune mecha- FDCs in lymphoid tissues become infected prior to
nisms; the mteractionsare summarized in figure 13.26. spread of the infectious agent to the CNS
I
Prion diseases See the accompanying website (www.roiH.com)
Scrapie, BSE and nvCJD are transmissible spongiform
encephalopathiescaused by prions.
Prophylaxis
Table 14.1. Passive immunotherapywith antibody. cases of infectious hepatitis may also be afforded pro-
tection by y-globulin. Human antitetanus irnmuno-
globulin is preferable to horse antitoxin serum, which
may cause hypersensitivity reactions. Curiously,
pooled y-globulin is being increasingly used as a treat-
ment for autoimmune diseases such as idiopathic
thrombocytopenic purpura, possibly acting through
anti-idiotypic mechanisms.
Isolated y-globulin preparations tend to form small
aggregates spontaneously and these can lead to severe
anaphylactic reactions when administered intra-
venously, on account of their ability to aggregate
platelets and to activate complement and generate C3a
and C5a anaphylatoxins.For this reason, the material
is always injected intramuscularly. Preparations free
of aggregates are available, and separate pools with
raised antibody titers to selected organisms such as
vaccinia, herpes zoster, tetanus and perhaps rubella
would be welcome. This need may ultimately be
antigens, migrate and colonize breast tissue (as part satisfied as it becomes possible to produce human
of the MALT immune system; see p. 154), where the monoclonal antibodieson demand.
antibodies they produce appear in the milk. The case
for mucosal vaccination of future mothers against
selected infections is inescapable.
The techniques for producing human monoclonal anti-
bodies to predetermined specificities from hybridoma
cells (cf. p. 122)still leave something to be desired and
Regular injection of pooled human adult y-globulin is restlessly we look to recombinant DNA technology to
an essential treatment for patients with long-standing satisfy the need (figure 14.1). There are several ap-
immunodeficiency. The preparations are also of value proaches to the production of antibodies which do not
to modify the effects of chickenpox or measles in other depend upon the human immune system, such as the
individuals with defective immune responses, such Fab and single chain Fv (VH-vd constructs (cf.p. 124)
as premature infants, children with protein malnutri- selected from phage libraries. Single VHdomain anti-
tion or patients on steroid treatment. Contacts with bodies, being so small, may well be capable of reaching
cell receptors on viruses which are tucked away at the
bottom of protein canyons where they might be inac-
cessible to the Fv of an intact antibody, but the sticky
nature of these V, domains has to be addressed. Some
antibodies possess peptidase activity, particularly
where the light chains have homology to serum pro-
teases, and this could lead to enhanced degradation of
the antigen. Expression of antibody genes in plants
is going to be big business; the technology is now in
place to produce recombinant IgA antibodies coupled
to secretory pieces in plants, and these should pro-
vide an invaluable supplement to dried cows' milk
baby food in cases where the mother's milk is of poor
Figure 14.2. Notification of diphtheria in England and Wales per
quality. 100 000 population showing dramatic fall after immunization. (Re-
Never neglect innate immune mechanisms. produced from Dick G. (1978) Immunisation.Update Books; with
Defensins, the broad-range antimicrobial peptides kind permission of the author and publishers.)
present in polymorphonuclear neutrophil (PMN)
granules (cf. p. 9), are now being engineered in to-
bacco plants and it is planned to use them for fungal
and bacterial infections which become refractory to
conventional antibiotics. A good example of lateral this figure must be maintained; there is no room for
thinking by the project leaders. complacency. In contrast, focal outbreaks of measles
have occurred in communities which object to immu-
nization on religious grounds, raising an important
point for parents in general. Each individual must
This is a labor-intensive operation and will be restrict- compare any perceived disadvantage associated with
ed to instances where the donor shares an MHC class I vaccination in relation to the increased risk of disease
allele. To give one example, up to 30% of recipients of in their unprotected child.
bone marrow allografts from mismatched family
members or matched unrelated donors develop
Epstein-Barr virus (EBV) lymphoma. Pilot studies
aimed at potential prophylaxis showed that EBV- The objective of vaccination is to provide effective im-
induced cytotoxic T-cell (Tc) lines transferred to the munity by establishing adequate levels of antibody
bone marrow recipients reconstituted the patients' and a primed population of memory cells which can
immune responses to EBV for at least 18months. rapidly expand on renewed contact with antigen and
so provide protection against infection. Sometimes, as
with polio infection, a high blood titer of antibody is re-
VACCINATION quired; in mycobacterialdiseases, such as tuberculosis
(TB), a macrophage-activating cell-mediated immun-
ity (CMI) is most effective, whereas with influenza
In the case of tetanus, active immunization is of benefit virus infection, cytotoxic T-cells probably play a sig-
to the individual but not to the community since it will nificant role. The site of the immune response evoked
not eliminate the organism which is found in the feces by vaccination may also be most important. For exam-
of domestic animals and persists in the soil as highly ple, in cholera, antibodies need to be in the gut lumen
resistant spores. Where a disease depends on human to inhibit adherence to and colonization of the intesti-
transmission, immunity in just a proportion of the nal wall.
population can help the whole community if it leads to Eradication of the infectious agent is not always the
a fall in the reproduction rate (i.e.the number of further most practical goal. To take the example of malaria, the
cases produced by each infected individual) to less blood-borne form releases molecules which trigger
than one; under these circumstances the disease will tumor necrosis factor (TNF) and other cytokines from
die out: witness, for example, the disappearance of monocytes, and the secretion of these mediators is re-
diphtheria from communities in which around 75% of sponsible for the unpleasant effects of the disease. Ac-
the children have been immunized (figure 14.2). But cordingly an antibody response targeted to these
Table 14.2. Factors requiredfor a successful vaccine.
released antigens with structurally conserved epi- Figure 14.3. Notifications of paralytic poliomyelitis in England
and Wales showing the beneficial effects of community immuniza-
topes may be a realistic holding strategy, while the tion with killed and live vaccines. (Reproduced from Dick G. (1978)
search for a global vaccine aimed at the more elusive Immunisation. Update Books; with kind permission of the author and
antigen-swapping parasite itself is grinding forward. publishers.)
Under these circumstanceslife with the parasite might
be acceptable.
In addition to an ability to engender effective immu-
nity, a number of mundane but nonetheless crucial tive antigens are not destroyed in the inactivation
conditionsmust be satisfied for a vaccine to be consid- process. During the production of an early killed
ered successful (table14.2).The antigensmust be read- measles vaccine, the fusion antigen, which permits cel-
ily available, and the preparation should be stable, lular spread of virus, was inactivated; as a result,
cheap and certainly, safe, bearing in mind that the re- incomplete immunity was produced and this left
cipients are most often healthy children. Clearly, the the individual susceptible to the development of
first contact with antigen during vaccination should immunopathological complications on subsequent
not be injurious and the maneuver is to avoid the path- natural infection. The dangers of incomplete im-
ogenic effects of infection, while maintaining protec- munity are especiallyworrying in areas where measles
tive immunogens. is endemic and the immune response is relatively
enfeebled due to protein malnutrition. Since the wide-
spread correction of this dietary deficiency is unlikely
KILLED ORGANISMS AS VACCINES
in the near future, it is worth considering whether non-
The simplest way to destroy the ability of microbes to specific stimulation by immunopotentiating drugs or
cause disease, yet maintain their antigenic constitu- thymus hormones at the time of vaccination might
tion, is to prevent their replication by killing in an ap- provide a feasible solution.
propriate manner. Parasitic worms and, to a lesser
extent, protozoa are extremely difficult to grow up in
LIVE ATTENUATED ORGANISMS HAVE MANY
bulk to manufacture killed vaccines. This problem
ADVANTAGES AS VACCINES
does not arise for many bacteria and viruses and, in
these cases, the inactivated microorganismshave gen- The objective of attenuation is to produce a modified
erally provided safe antigens for immunization. Ex- organism which mimics the natural behavior of the
amples are typhoid (in combinationwith the relatively original microbe without causing significant disease.
ineffective paratyphoid A and B), cholera and killed In many instances the immunity conferred by killed
poliomyelitis (Salk) vaccines. The success of the Salk vaccines, even when given with adjuvant (seebelow),
vaccine was slightly marred by a small rise in the inci- is often inferior to that resulting from infection with
dence of deaths from poliomyelitis in 1960-61 (figure live organisms. This must be partly because the repli-
14.3), but this has now been attributed to poor anti- cation of the living microbes confronts the host with a
genicity of one of the three different strains of virus larger and more sustained dose of antigen and that,
used, and present-day vaccines are far more potent. with budding viruses, Infected cells are required for
Care has to be taken to ensure that important protec- the establishment of good cytotoxic T-cell memory.
best example of this was Jenner's seminal demonstra-
tion that cowpox would protect against smallpox.
Since then, a truly remarkable global effort by the
World Health Organization (WHO), combining ex-
tensive vaccination and selective epidemiological
control methods, has completely eradicated the
human disease -a wonderful achievement. Embold-
ened by this success, the WHO embarked upon a pro-
gram to eradicate polio and, despite setbacks caused
by armed conflict limiting access to local populations,
it is hoped to declare the world free of polio by 2005.
One can even follow the progress of this campaign on
http:/ /www.polioeradication.org.
Attenuation itself was originally achieved by em-
pirical modification of the conditions under which an
Figure 14.4. Local IgA response to polio vaccine. Local secretory
antibody synthesis is confined to the specific anatomical sites which
organismgrows. Pasteur first achieved the production
have been directly stimulated by contact with antigen. (Data from of live but nonvirulent forms of chicken cholera bacil-
Ogra P.L. et al. (1975) In Notkins A.L. (ed.) Viral Immunology and lus and anthrax (cf. Milestone 14.1)by such artifices as
Immunopathology,p. 67.Academic Press, New York.) culture at higher temperatures and under anerobic
conditions, and was able to confer immunity by in-
fection with the attenuated organisms. A virulent
strain of Mycobacterium tuberculosisbecame attenuated
Another significantadvantage of using live organisms by chance in 1908 when Calmette and GuQin at the
is that the immune response takes place largely at the Institut Pasteur, Lille, added bile to the culture me-
site of the natural infection.This is well illustrated by dium in an attempt to achieve dispersed growth. After
the nasopharyngeal IgA response to immunization 13 years of culture in bile-containing medium, the
with polio vaccine. In contrast with the ineffectiveness strain remained attenuated and was used successfully
of parenteral injection of killed vaccine, intranasal ad- to vaccinate children against tuberculosis. The same
ministration evoked a good local antibody response; organism, BCG (bacille Calmette-Guerin), is widely
however, whereas this declined over a period of 2 used today for the immunization of tuberculin-
months or so, per oral immunization with live attenu- negative individuals. Attenuation by cold adaptation
ated virus established a persistently high IgA antibody of influenza and other respiratory viruses seems
level (figure 14.4). hopeful; the organism can grow at the lower tem-
There is in fact a strong upsurge of interest in strat- peratures (32-34C) of the upper respiratory tract, but
egies for mucosal immunization. Remember, the fails to produce clinical disease because of its inability
MALT system involves mucous membranes covering to replicate in the lower respiratory tract (37OC).
the aerodigestive and urogenital tracts as well as the
conjunctiva, the ear and the ducts of all exocrine
glands which are essentially protected by sIgA anti-
bodies. Resident T-cells in these tissues produce large It must be said that many of the classical methods of at-
amounts of transforming growth factor-p (TGFP),and tenuation are somewhat empirical and the outcome is
the interleukins IL-10 and IL-4, which promote the B- difficult to control or predict. With knowledge of the
cell switch to IgA, and note also that human intestinal genetic makeup of these microorganisms, we can
epithelia1 cells themselves are major sources of TGFP apply the molecular biologist's delicate scalpel to de-
and IL-10. liberately target the alterations in life-style which are
needed for successful attenuation. Thus genetic re-
combination is being used to develop various attenu-
ated strains of viruses, such as influenza, with lower
The objective of attenuation, that of producing an or- virulence for humans and some with an increased
ganism which causes only a very mild form of the multiplication rate in eggs (enabling newly endemic
natural disease, can be equally well attained if one strains of influenza to be adapted for rapid vaccine
can identify heterologous strains which are virulent production). Not surprisingly, strains of HIV-1, with
for another species, but avirulent in humans. The vicious deletions of the regulatory genes, are being
investigated as protective vaccines. The potential is
clearly quite enormous.
The tropism of attenuated organisms for the site at
which natural infection occurs is likely to be exploited
dramatically in the near future to establish gut im-
munity to typhoid and cholera using attenuated forms
of Salmonella strains and Vibrio ckolerae in which the
virulence genes have been identified and modified by
genetic engineering.
The persistent but low level of expression of the pro- There is a lot going for DNA vaccines. Considering
tein antigen by naked DNAvaccines establishes a pool influenza virus, for example, the DNA needed to pre-
of relatively high affinity memory B-cells which can pare a vaccine can be obtained directly from current
readily be revealed by boosting with protein antigen clinical material without having to select specific mu-
(figure 14.9).This has given rise to a rather impressive tant strains.Altogether, the speed and simplicity mean
'prime and boost' strategy where these memory that the 2 years previously needed to make a recombi-
cells are expanded by boosting with a nonreplicating nant vaccine can be reduced to months. DNA vaccines
viral vector, such as fowlpox virus or Ankara strain- do not need the cumbersome and costly protein syn-
modified vaccinia virus, bearing a gene encoding the thesis and purification procedures that subunit formu-
antigen. Mice immunized in this fashion with in- lations require; almost identical production facilities
fluenza virus hemagglutinin produced satisfyingly can be used for totally different vaccine candidates;
high levels of IgG2a antibody and were protected and they can be prepared in a highly stable powder
against challenge with live virus. Remarkably, up to form which does not depend on the cold complex
30% of circulating CD8 T-cells were specific for the chain logistically needed for heat-sensitive vaccines,
immunizing epitope as shown by MHC class I such as the oral polio vaccine in tropical countries.But,
tetramer binding (cf. p. 138).A similar strategy with above all, they are incredibly cheap. The $3 required
Plasmodium berghei produced high levels of peptide- for an injection of recombinant hepatitis B represents
specific CD8 cells secreting IFNy which protected the whole health budget for a single individual in some
against challengeby sporozoites. countries, whereas the single shot of DNA vaccine
Anew generation of genetic vaccines overcomes the would be a tiny fraction of that. Current trials are ad-
poor efficacy of some DNA- and RNA-based vaccines dressing a number of safety considerations, such as the
by incorporating the replicating machinery used by possibility of permanent incorporation of a plasmid
members of the alphavirus genus, which includes into the host genome. The widespread use of DNAvac-
the Sindbis and Semliki Forest viruses, into the DNA cines in humans will depend upon the results of such
plasmid. The alphaviral genome consists of a single trials, although observations to date have generally
positive-strandedRNAencoding structural genes, one been very encouraging.
of which can be substituted by the antigen gene, and an
RNA replicase. Cells transfected with the plasmid
EPITOPE-SPECIFIC VACCINES
replicon become loaded with alphavirus and antigen
MAY BE NEEDED
and meet a sticky apoptotic end whereupon they are
taken up by antigen-presentingcells to initiate a pow- Most immunogens, especially proteins, present a
erful immune response. variety of B- and T-cell epitopes to the immune system.
Most, if not all, will elicit protective responses, but generate effective humoral immunity. Nonetheless,
some may have undesirable characteristics.For exam- in many of these infections, a deliberately selected
ple, if there is cross-reaction with a self-epitope, as be- monoclonal antibody specific for more weakly im-
tween Typanosoma cruzi and heart muscle, potentially munogenic conserved regions of the microbe can be
pathogenic autoimmune reactions may result. Some- protective, as has been demonstrated in models of HIV,
times an irnmunodominantregion, such as the V3 loop Ebola virus and Candida infections.In other words, the
in HIV gp120, hogs the antibody response at the ex- epitope giving rise to the protective monoclonal anti-
pense of the more weakly immunogenic conserved re- body is subdued as an immunogen when present as a
gions, but continually escapes from capture by a high component of the infectious microbe, but potentially
mutation rate. Similar escape mutants crop up in the might be exploited to stimulate high levels of protec-
Tc response to highly mutating dominant T-cell epi- tive antibodiesif it could be extracted from the molecu-
topes in malaria and various viral protein antigens. lar 'woodwork' so to speak.
Unwanted epitope effects are seen in the phenomenon Bearing in mind the many different circum-
of 'original antigenic sin', in which a second infection stances we have discussed, the requirement may
with influenza virus involvingan antigenically related arise for a vaccine in which the good protective
but not identical strain generates antibodies with a epitopes can be brought into the front line and disso-
higher titer for the strain which produced the first in- ciated from the molecular environment of the 'bad
fection. It is conceivable also that certain epitopes may guys' which compromise the protective responses. In
bias T-helpers towards an inappropriate subset or may other words, we need to construct epitope-specific
engender a predominantly suppressive, antagonistic vaccines, preferably based on conserved regions,
or anergic response which could downregulate T-cell which provide broad defense. Several approaches are
reactivity to linked epitopes on the same immunogen possible.
(figure 14.10).
Frequently,a natural infection gives rise to a poorly
functional protective antibody response and discour-
ages the search for vaccine candidates which could
B-cell epitopes
Small peptide sequences corresponding to impor-
tant epitopes on a microbial antigen can be syn-
thesized readily and economically; long ones are
rather expensive to manufacture. One might predict
that, although the synthetic peptide has the correct
linear sequence of amino acids, its random structure
would make it a poor model for the conformation of the
parent antigen and hence a poor vaccine for evoking
humoral immunity. Curiously, this does not always
seem to be a seriousdrawback.The 20-amino acid pep-
tide derived from the foot and mouth virus-specific
protein (VP1) evokes a good neutralizing response.
The explanation has been forthcoming from X-ray
structural analysis which shows the peptide sequence
to be in a 'loop' region with blurred electron density in-
dicative of dramatic disorder. In this case, the epitope
is linear and evidently the flexibility of the loop struc-
ture may approach that of the free peptide which can
thus mimic the epitope on the native VP1 molecule and
Figure 14.10. Desirable (green)and unwanted (red)epitopes on a
stimulate a protective antibody response when used as
protein immunogen. Conserved epitopes give broader protection
against mutant variant strains although they may sometimes be a vaccine (figure14.11aand b).Where the epitope is lin-
weakly immunogenic when present in the whole microbe. Epitopes ear but is restricted in conformation by adjacent struc-
may be unwanted because: (i)they are immunodominant and attract tures in the intact protein, immunization with free
the main immune response but continually escape by mutation; (ii)
peptide tends to produce antibodies of disappointing
they cross-react with self-epitopes and can trigger autoimmunity;
(iii) they are responsible for 'original antigenic sin' (see text); or (iv) affinity for the protein itself for the reasons outlined in
they downregulate or antagonizeT-cell-mediated immunity. figure 14.11~.
Figure 14.11. Structural basis for peptide mimicry of protein tein has been denatured after sodium dodecyl sulfate (SDS)
epitopes. (a) The free peptide is very flexible and can adopt a treatment and the peptide structure is relatively free.) Preformed
large number of structures in solution. (b) If the peptide sequence antibodies to the protein would react with the peptide, albeit
is present as a linear epitope on a part of a protein which is a with lower affinity, because energy must be used to constrain the
flexible loop or chain, this will also exist in a variety of structures peptide to the one structure which fits the antibody-just like the
resembling the free peptide to a fair extent, and will behave com- force used to restrain a madman in a strait-jacket. Where the se-
parably as an antigen and as an immunogen (vaccine) so that quence has a comparable degree of constraint in both peptide and
the peptide will raise antibodies which react well with the native protein, as in the disulfide-bondedloops in diphtheria toxin and he-
protein. (c) If the linear epitope on the protein is structurally patitis Bsurface antigen, antipeptide sera react reasonably well with
constrained (i.e. inflexible), it represents only one of the many the native protein. (d)Most commonly, the epitopes are discontinu-
structures adopted by the free peptide; thus if this peptide is ous and, even if with difficulty we can predict the contact residues,
used for immunization, only a minority of the B-cells stimulated the techniques for designing a peptide with appropriate structure
will be complementary in shape to the native protein, so the are not robust, although some progress is being made using anti-
peptide would be a poor vaccine for humoral immunity to microbes body to select from a random bacteriophage library in which the
containing the protein antigen. (Note, however, that the antibodies peptides are constrained on a structural scaffold, such as that sup-
produced would be good for Western immunoblots where the pro- porting the Ig CDR3 loop.
Figure 14.12. Derivation of anti-idiotypicmimics of a B-cell epi- screened with the original monoclonal Id to select the best fitting
tope. A high affinity monoclonal antibody (idiotype; Id) specific for anti-Id monoclonals or antibody fragments. These, in turn, are
the a epitope on the antigen is injected to generate an anti-Id re- monitored for behavior as internal image of a through ability
sponse. Spleen cells from the immunized mice are used (1)to make a to block binding of the original Id to a, to bind to other anti-a mono-
range of hybridomas,each secreting an individual monoclonal anti- clonals and to be recognized by a polyclonal antiserum raised
body, a small proportion of which will be internal image anti-Id, or against the antigen in other species.A successfulcandidate can then
(2) a phage library expressing surface antibody fragments.These are be produced in bulk to provide a surrogate vaccine for epitope a.
Figure 14.13. (a)Selective mutation of unwanted (dark blue) with prevent successfulpregnancy. However, the production of antibod-
retention of desirable (green) epitopes in a protein vaccine. Suc- ies to hLH is undesirable since this hormone is present all the time,
cess of the mutation strategy can be monitored by reactivity with not just during pregnancy as is essentially the case for hCG (cf.figure
monoclonal antibodies (B-epitopes)and T-cell clones (T-epitopes). 17.21).The mutant epitopes (orange)avoid this unwanted LH cross-
Our immunoprotein engineering group at University College reactivity. The C-terminal end of the construct can be extended to
London has used this strategy to mutate human chorionic go- encode suitable T-helper carrier epitopes. For simplicity, the T-cell
nadotropin-P (P-hCG)so that it still retains P-hCG-specific epitopes recognition of MHC-peptide has been ignored. (b) FACS analysis
but has lost those shared with human luteinizinghormone (hLH).P- of cells transfected to express P-hCG on their surface, showing a
hCG has been investigated as a contraceptive vaccine to aid popula- mutant which retains the P-hCG-specific epitope but has lost the
tion control, since antibodies to P-hCG neutralize the hormone and cross-reactiveepitopes characteristic of the wild-type protein.
Likewise, the isolated 19kDa C-terminal fragment culty in identifying high-risk individuals have led to
of the merozoite-specific protein MSP-1 confers recommendations for vaccination in the 6-18-month
antibody-mediated protection on monkeys to chal- age group.
lenge with the blood stage of the malaria parasite. Maternally derived IgG antibody can inhibit de novo
immune responses through feedback control (cf. p.
201). Preliminary results suggest that infants of 4-6
CURRENT VACCINES
months can be seroconverted by inhaled aerosol
The established vaccines in current use and the sched- measles vaccine which presumably evades the ma-
ules for their administration are set out in tables 14.3 ternal antibody; this will have singular relevance in
and 14.4. endemic measles areas, where almost split-second
Because of the pyrogenic reactions and worries timing is required with conventional immunization as
about possible hypersensitivity responses to the passively acquired antibody wanes. Naked DNA vac-
whole-cell component of the conventional pertussis cines may also have a role to play here.
vaccine, a new generation of vaccines containing one Tuberculosis is the largest cause of death in the
or more purified components of Bordetella pertussis, world from a single infectious disease and, while
and therefore termed 'acellular' vaccines, has been li- it remains a truly major problem in developing coun-
censed in the USA. The combination of diphtheria and tries, cases have also increased by around one-third in
tetanus toxoids with acellular pertussis (DTP) is rec- Western countries. There is an alarmingly heightened
ommended for the later fourth and fifth 'shots' and for susceptibility to TB of individuals with HIV, and
children at increased risk for seizures. Children under worldwide multidrug-resistant strains are appearing.
2 years of age make inadequate responses to the T-in- Thus, although BCG has been in use for 70 years and is
dependent H. influenzae capsular polysaccharide, so reasonably efficacious and safe in healthy non-T-cell-
they are now routinely immunized with the antigen deficient subjects, there is certainly a vital need for the
conjugated with tetanus or diphtheria toxoids. development of new drugs and vaccines. It remains to
A resurgence of measles outbreaks in recent years be seenwhether the disappointing degree of protection
has prompted recommendations for the reimmuniza- againstTB andMycobacterium leprae found with BCG in
tion of children, at the age of school entry or at the field trials in certain parts of the globe is due to the use
change to middle school, with measles. The consider- of high doses, deficient strains or subversion of the
able morbidity and mortality associated with hepatitis response to group i cross-reactingantigensby suppres-
B infection, its complex epidemiology and the diffi- sive mycobacterialspeciesin the local environment.
Table 14.3. Current and experimental vaccines. Table 14.4. Current vaccination practice.
EXPERIMENTAL VACCINES I N
DEVELOPMENT
malaria field have turned their attention to the invari-
ant antigens of the sporozoite, which is the form with
which the host is first infected; this rapidly reaches the
Don't sneer at low technology. The major advance in liver to emerge later as merozoites which infect the red
malaria control has been the finding that the impreg- cells (figure 14.14). Because the sporozoite only takes
nation of bed nets with the insecticide pyrethroid 30 minutes to reach the liver, the antibody has to act
reduces Plasmodium falciparum deaths by 40%. How- fast, so that inactivation is limited by diffusion events
ever, with the emergence of drug-resistant strains of and hence dependent on the concentration of antibody
mosquito, vaccines must be developed. The goal is molecules. The sporozoite has a characteristic antigen
achievable since, although children are very suscep- with multiple tetrapeptide repeats. Plasmodium falci-
tible, adults resident in highly endemic areas acquire parum, for example, has 37 repeats of NANP. Field tri-
a protective but nonsterilizing immunity probably als of vaccines with polymers of NANP and similar
mediated by antibodies specific for the blood stages. tetrapeptides from other species have not so far been
Antigen variation poses a big problem for vaccine spectacularly successful, but building in powerful
development and a number of investigators in the T-cell help in the form of the promiscuous 378-398
Figure 14.14. The life cycle of the malaria parasite.
heat-labile enterotoxin (LT) and cholera toxin to target plex. The differing pathways for processing peptides
intestinalcells. LT is very immunogenic when givenby within antigen-presenting cells can be turned to ad-
the oral route and a nontoxicmutant of comparable po- vantage by encapsulating antigen in acid-resistant
tency as an adjuvant (figure 14.16)has been developed liposomes so that they can only enter the MHC class I
for ultimate use in humans. Couplinga protein antigen route and stimulate CD8 T-cells. Antigens within acid-
to cholera toxin B subunit targets the vaccine to the sensitive liposomes become associated with both class
epithelia1 cells of the intestinal tract and usually pro- I and class I1 molecules. Proteins anchored in the lipid
duces good IgG and IgA antibodies which appear membrane by hydrophobic means give augmented
also in the saliva, tears and milk, indicating that the CMI. Short synthetic peptides coupled covalently to
antigen-specific IgA precursor cells become dissemi- monophosphoryl lipid A or tripalmitoyl-S-glyceryl-
nated throughout the MALT system. Whether the im- cysteinyl-seryl-serine(P3CSS)have high priming effi-
mune response to the cholera precludes its use as a ciency. One can readily envisage the possibility of
carrier for immunization with further antigens re- a single-shot liposome vaccine with multiple poten-
mains an open question. Speaking of cholera toxin, it tialities which incorporate several antigens, different
was reported that a soluble antigen, such as diphtheria adjuvants and specialized targeting molecules (figure
toxoid, applied to the skin of mice together with 14.17).
cholera toxin produced persistent high levels of IgG Another innovation is the Iscom (immunostimulat-
antibodies, a not entirely predictable outcome. ing complex), a hydrophobic matrix of the surfactant
saponin, with antigen, cholesterol and phosphatidyl-
choline.Antigens with a transmembranehydrophobic
region, such as surface molecules of lipid-containing
Recent interest has centered on the use of small lipid viruses, are powerfully immunogenic in this vehicle
membrane vesicles (liposomes) as agents for the and may engender cytotoxic T-cell responses. Iscoms
presentation of antigen to the immune system. It may are extremely resistant to acid and bile salts and are
be that the liposome acts as a storage vacuole within immunogenic by the oral route, producing systemic
the macrophage or perhaps fuses with the macrophage immunity and good local secretory IgA. Intranasal ex-
membrane to provide a suitably immunogenic com- posure established protective immunity to influenza
oxypropylene core linked to hydrophilic polymers of
polyoxyethylene, are likely to be acceptable for use in
humans.
It may be useful to focus on the notion floated earlier
that polymeric antigens tend to be more immuno-
genic, and to note a novel form of solid matrix of the
Cowan strain of S. aureus which can bind several mol-
ecules of monoclonal antibody which can, in turn, im-
mobilize several molecules of antigen. Using a variety
of monoclonals, one can purify onto their binding sites
appropriate antigens from a mixture to give a multiva-
lent subunit vaccine. One could also incorporate the E.
coli enterotoxin mutants mentioned above to give
good mucosal immunity.
With respect to the practicability and convenience
of administration of vaccines, we should not ignore
the use of the biolistics gun (cf. p. 141) to introduce
gold microspheres coated with plasmids of the
gene encoding the vaccine antigen or antigens (cf.
Figure 14.17. The 'do-it-all-in-one' omnipotent liposome particle
p. 290).
illustrating some possible ways to build immunogenic flexibility There have been some very important advances in
into a single-shot liposome vaccine.The interior of the liposome may the design of controlled-release systems for antigen
also contain depots of certain components. MDP, muramyl dipep- delivery in vivo. Polymers of polylactic-polyglycolic
tide; MLA, monophosphoryl lipid A; hsp70, mycobacterial heat-
shock protein 70.
esters are nontoxic biodegradable vehicles which can
be prepared in a mixture of different formulations to
provide slow release of an antigen (or any active drug
in mice. Saponin itself, initially purified from the bark or hormone) for periods up to several months.
of the tree Quillaja saponaria, is too toxic for human use. There are exciting times ahead for vaccine develop-
A less toxic derivative, Quil A, is widely used for ment, but it should always be borne in mind that no
veterinary vaccines, but an even less toxic derivative, matter how successful these clever strategies prove to
QS21, is being evaluated for safety and efficacy in be in experimental animals, the acid test which de-
clinical trials. Another group of surfactants, the non- stroys so many promising approachesis the long-term
ionic block copolymers having a hydrophobic poly- effect in the human.
I SUMMARY I
I
Passively acquired immunity easily available, cheap, stable under extreme climati
Passive immunity can be acquired by maternal anti- conditionsand nonpathogenic.
bodies or from homologouspooled y-globulin.
Horseantisera aremorerestrictedbecauseof thedanger Killed organisms as vaccines
of serum sickness. Killed bacteria and viruses have been widely used
Antibodies are being constructed to order using recom-
binant DNA technology and can be produced in bulk in Live attenuated organisms
plants. The advantages are: replication gives a bigger dose, the
immune response is produced at the site of the natural
Vaccination infection.
Active immunization provides a protective state Attenuatedvaccinia orpoliocanprovidea'piggy-back'
through contact with a harmless form of the disease carrier for genes from other organisms which are difficult
organism. to attenuate.
Agood vaccine should be based on antigens which are ECG is a good vehicle for antigens requiring CD4 T-cell
(continued)
immunity and salmonella constn~ctsmay give oral and
systemic immunity. Intranasal immunization is fast gain-
ing popularity.
The risk with live attenuated organisms is reversion
to the virulent form and danger to immunocompromised
individuals.
Subunitvocclnes
Whole organisms have a multiplicity of antigens, some
of which are not protective, may induce hype-itivity
or might even be frankly immunosuppressive.
Itmakessenseinthesecasestousepurifiedcomponents.
There is greatly increased use of recombmant DNA
technology to produce these antigens. Expression in ba- Figure 14.18. Strategies forvaccination.
nanas provides a very cheap way of achievingoral immu-
nization in the developingworld.
Naked DNAencodmgthe vaccinesubunitcanbeinject- Vnccines In development
ed directly into muscle, where it expressesthe protein and In malaria, the experimentalvaccinesare targeted at th,
produces immune lesponses. The advantagesare stability, sporozoiteund blood stages, the toxinproducing seriousside-
ease of productionand cheapness. effects, thegametesand the insectgut trypsin.
Epitope-specific vaccines based on conserved struc- Recombinant glutathione-S-trferase is a promisimg
tures have the advantagethat they can providebroad pro- candidatefor a vaccineagainst schistosomiasis.
tection and may avoid the possible deleterious effects of AnoralvaccinecomposedofcholeratoxinBsubunitand
other epitopes (autoimmunity, T-downregulation, origi- killedvibriosinducedgoodmucosalimmunitytocholera.
nal antigenicsin,escapebymutation of immunodominant
epitopes) when certain whole antigens are used for Adjuvonls
immunization. Adjuvants work by producing depots of antigen, and
Epitopespecific vacdnes can be based on peptides, byactivatingmacrophages;theysometimeshavedireftef-
internalimage anti-idiotypes or epitope-lossmutants. fects onlymphocytes.
Peptidesmayonlyusefullymimicthenativeproteinfor Adjuvants such as the muramyl dipeptide analogs de-
vaccinationto produceantibodyif the epitopeis linearand rived from mycobacterial cell walls and the monophos-
relatively unconstrained in structure. Carriers such as phoryl lipid A derivative from Gram-negative LPS may
tetanus toxoid or mycobacterial heat-shock proteins are soonbeingeneraluse.
needed to make the peptide immunogenic. Linear pep- New methods of delivery include lmking the antigen
tidescanmimicT-cellepitopesinthewholeprotein. to small lipid membrane vesicles (liposomes)or a special
Epitope-loss mutants have undesirable epitopes re- glycoside matrix (Iscom). These delivery particles can
placed but still fold correctly to produce the wanted dis- be fumished with many factors which improve their
continuoush e l l epitope(s). immunogenicity and flexibility.One can build in several
antigens into the same particle, adjuvants such as MDP
Currenl voccines and MLA, cytokines to influence lymphocyte subset re-
Children in the USA and UK are routinely immunized sponses and molecules such as cholera toxin Band E. cdi
with diphtheria and tetanus toxoids and pertussis (Dl" enterotoxinto target particular sites in the body.
triple vaccine) and attenuated strains of measles, mumps Antigensbuilt into biodegradablepolymers of varying
andrubella(MMR)andpolio.BCGisgivenatlC-l4years. half-life can provide single-shot vaccines which mimic a
Subunit formsofpertussislackmgside-effectsarebeing conventional course of immunization requiring several
introduced. injections,
The capsular polysaccharide of H.mflumzue has to be Theoverallstrategiesforvaccination aresummarized 1
linked to a carrier. figure 14.18.
The elderly receive vaccines of influenza and Pneumo-
coccus polysaccharides.
Vaccine forhepahtisA and B,meningitis,yellow fever,
typhoid, cholera and rabies are availablefor travelers and
high-risk groups.
Nicholson B.H. (ed.) (1994) Synthetic Vaccines. Blackwell Science,
FURTHER READING Oxford.
Del Giudice G., Covacci A., Telford J.L., Montecucco C. & Rappuoli Nossal G.J.V. (2000)The global alliance for vaccines and immuniza-
R. (2001) The design of vaccines against Heliobacter pylori tion -a millennia1challenge.Nature Immunology 1,5.
and their development. Annual Review of Immunology 19, Peter G. (1992) Current concepts: childhood immunizations. New
523. England Journal of Medicine 327,1794.
Featherstone C. (1996) Vaccines by agriculture. Molecular Medicine Powell M.F. & Newman M.J. (eds) (1995) Vaccine Design. Plenum
Today 2,278. Publishing, New York.
Harding C.V., Collins D.S., Kanagawa 0 . & Unanue E.R. (1991) Sherwood J.K. et al. (1996) Controlled release of antibodies for
Liposome-encapsulated antigens engender liposomal processing long-term topical passive immunoprotection of female mice
for class I1 MHC presentation and cytosolic processing for class I against genital herpes. Nature Biotechnology 14,468.
presentation. Journalof Immunology 147,2860. Whitturn-Hudson J.A. et al. (1996) Oral immunisation with anti-
Kumar V. & Sercarz E. (1996) Geneticvaccination:the advantages of idiotype to exoglycolipid antigen protects against Chlamydia tra-
going naked. Nature Medicine 2,857. chomatis infection. NatureMedicine 2,1116.
Lambert P.H. (1993) New vaccines for the world-needs and Yap P.L. (ed.) (1992) Clinical Applications of Intravenous Immunoglobu-
prospects. The immunologist 1/50. lin Therapy. Churchill Livingstone,Edinburgh.
Mims C.A., Playfair J.H.L., Roitt I.M., Wakelin D. & Williams R. Zinkernagel R. &Lambert P.H. (1992)Immunity to infection.Current
(1998)Medical Microbiology,2nd edn. Mosby, London. Opinion in Immunology 4,385 (also 1993,5,issue 4).
I CHAPTER 15
I
Immunodef iciency
may be overwhelmed by vaccinia (figure 15.6) or infection and the disease is usually fatal unless a bone
measles, or by bacille Calmette-Guerin (BCG)if given marrow transplant life-line is offered.
by mistake. Humoral antibodiescan be elicited, but the Mutations in the recombinase enzymes, RAG-1 and
responseis subnormal,presumably reflecting the need RAG-2 (cf. figure 3.8), which initiate V D ] recombina-
for the cooperative involvement of T-cells. (The simi- tion events, usually result in complete failure to gener-
larity of this condition to neonatal thymectomy and of ate mature antigen-specific lymphocyte receptors and
B-cell deficiency to neonatalbursectomy in the chicken give rise to the severe combined immunodeficiency
should not go unmentioned.) Treatment by grafting (SCID)phenotype (seebelow). In Omenn's syndrome,
neonatal thymus leads to restoration of immunocom- the particular RAG mutants allow some T-cells, appar-
petence, but some matching between the major histo- ently of Th2 phenotype, to sneak through, although
compatibility antigens on the nonlymphocytic thymus they are not capable of preventing a failure to thrive
cells and peripheral cells is essential for the proper and relatively early death.
functioning of the T-lymphocytes (p. 228). Complete MHC class I1 deficiency, previously known by its
absence of the thymus is pretty rare and more often one more sleazy appellation,'bare lymphocyte syndrome',
is dealing with a 'partial DiGeorge' in which the T-cells is associated with recurrent bronchopulmonary infec-
may rise from 6% at birth to around 30%of the total cir- tions and chronic diarrhea occurring within the first
culating lymphocytes by the end of the first year; anti- year of life, with death from overwhelming viral infec-
body responses are adequate. tions at a mean age of 4 years. The conditionarises from
Mutation of the purine degradation enzyme, purine mutationsaffectingthe RFX-B, C11TA, RFX-5 and RFXAP
nucleoside phosphorylase, results in the accumu- promoter proteins that bind to the 5'-untranslated re-
lation of the metabolite deoxy-GTP which is toxic to gion of the class I1 genes. Feeble expression of class I1
T-cell precursors through its ability to inhibit ribonu- molecules on thymic epithelia1 cells grossly impedes
cleotide reductase, an enzyme required for DNA syn- the positive selection of CD4 T-helpers, and those that
thesis and hence cell replication. Targeting of the T-cell do leak through will not be encouraged by the lack of
lineage by this deficiency could well be linked to a rela- class I1on antigen-presentingcells and B-lymphocytes.
tively low level of 5'-nucleotidase. Some T-cells 'leak Note also that rare patients with mutations in TAP-1 or
through' but they give inadequate protection against TAP-2have an MHC classIbare lymphocytesyndrome.
ing T-B collaboration. Poor cell-mediated immunity
and impaired antibody production in affected boys are
hardly surprising consequences.
Ataxia telangiectasia is a human autosomal reces-
sive disorder of childhood which has been recognized
as a chromosomal breakage syndrome characterized
by progressive cerebellar ataxia with degeneration of
Purkinje cells, a hypersensitivity to X-rays and an
unduly high incidence of cancer. The mutated gene,
ATM, encodes a protein kinase (Atm)which is a mem-
ber of the phosphatidylinositol 3-kinase family. Fol-
lowing ultraviolet or ionizing radiation, the normal
gene acts through the tumor suppressor protein p53
and thence p21 to arrest the cell cycle at the Gl-S bor-
der, and through the Chk2 kinase and Cdc25 to block
the G2-M transition. Presumably, the cellular DNA
repair mechanisms now have a chance to operate.
Regrettably, the relationship of the Atm defect to
immunodeficiency is not clear. Another disease char-
acterized by immune dysfunction, radiation sensitivi-
ty and increased incidence of cancer is the Nijmegen
breakage syndrome where a mutation in the NBSl
gene leads to a defective protein, nibrin, which nor-
mally functions as a component of a double-stranded
DNA repair complex.
It is exciting to see the molecular basis of diseases
being unraveled and an excellent example of nature
yielding its secretshas been provided by studies on the
XL hyper-IgM syndrome, a rare disorder character-
Figure 15.6. A child with severe combined immunodeficiency ized by recurrent bacterial infections, very low levels
showing skin lesions due to infection with vaccinia gangrenosum or absence of IgG, IgA and IgE and normal to raised
resulting from smallpox immunization. Lesions were widespread
over the whole body. (Reproduced by kind permission of Professor concentrations of serum IgM and IgD. It transpires that
R.J. Levinsky and the Medical Illustration Department of the Hospi- point mutations and deletions in the T-cell CD4OL
tal for Sick Children, Great Ormond Street,London.) (CD154)map to the part of the molecule thought to be
concerned in the interaction with B-cell CD40 (cf. p.
174), thereby rendering the T-cells incapable of trans-
mitting the signals needed for Ig class switching in B-
cells. Maybe a note of caution regarding gene therapy
would not be amiss at this point. CD4OL knockout mice
mimicking human X-linked hyper-IgM syndrome
Cell-mediated immunity (CMI) is depressed in im- were injected with transduced BM or thymic cells
munodeficient patients with ataxia telangiectasia using a retroviral vector containing the CD40L gene.
or with thrombocytopenia and eczema (Wiskott- Low level constitutiveexpression stimulated humoral
Aldrich syndrome). The Wiskott-Aldrich syndrome and cellular immune functions, but 12 of 19 mice de-
protein (WASP) plays a critical role in signal trans- veloped T-lymphoproliferative disorders, suggesting
duction by interacting with Src-homology 3 (SH3) that current methods of gene therapy may be inappro-
domains and in the regulation of cytoskeletalreorgan- priate for disorders involving highly regulated genes
ization. WASP clusters physically with actin through in essential positions in proliferative cascades.
the GTPase Cdc42 and possibly other molecules which Also for the collector are the rare cases of severe T-
regulate actin polymerization.Mutations in the WASP cell deficiency arising from mutation in the E and y
gene thus adversely affect cell motility, phagocyte chains of the CD3 complex and the ZAP-70 kinase
chemotaxis, dendritic cell trafficking and the polariza- (p. 166)which generate dysfunctionalT-cells and pref-
tion of the T-cell cytoskeleton towards the B-cells dur- erentially impair the differentiation of CD8 T-cells.
Omenn's syndrome, grossly dysfunctional mutations
COMBINED IMMUNODEFICIENCY
in the recombinase enzymes, which catalyse the intro-
Severe combined immunodeficiency disease (SCID) duction of the double-stranded breaks permitting
involving B-, T- and NK cells represents the most se- subsequent recombination of the V, D and J segments,
vere form of primary imrnunodeficiency,affecting one prevent the emergence of any mature lymphocytes.
child in approximately every 80 000 live births. These Failure of the VDJ recombination mechanism is also a
Infants exhibit profound defects in cellular and hu- feature of the radiosensitive cells from SCID patients
moral immunity, with death occurring within the first with defective DNA-dependent protein kinase, a com-
year of life due to severe and recurrent opportunistic ponent of the complex which realigns and repairs the
infections. Prolonged diarrhea resulting from gas- free coding ends created by the RAG enzymes. Just
trointestinal infections and pneumonia due to Pneu- a snippet-naturally occurring SCID with a similar
mocystis cavinii are common; Candida albicans grows phenotype has been identified in Arabian foals.
vigorously in the mouth or on the skin. If vaccinated
with attenuated organisms (figure l5.6), these infants
usually die of progressive infection. SCIDinfants must
be rescued by a bone marrow transplant if they are to
survive. Many SCID patients have a genetic deficiency of the
Reticular dysgenesis is a rapidly fatal variant of se- purine degradation enzyme, adenosine deaminase
vere combined immunodeficiency associated with a (ADA), which results in the accumulation of the
lack of both myeloid and lymphoid cell precursors, but metabolite, dGTP, which is toxic to early lymphoid
SCID can arise from a variety of circumstances which stem cells. The comparable immunodeficiency seen in
se1ectivel.ytarget lymphoid cell differentiation. acute lymphocytic leukemia patients treated with the
ADA inhibitor deoxycoformacin attests to the validity
of this analysis. Half the ADA-deficient SCID patients
do reasonably well on transfusions of normal red cells
containing the enzyme, whereas others with a longer
Over half of the cases of severe combined immunodefi- standing more severe deficiency,which might have af-
ciency (SCID), which affects both B- and T-cell devel- fected the thymus epithelium, also require treatment
opment, derive from mutations in the yc chain of the with the enzyme modified by polyethylene glycol
receptors for interleukins IL-2, -4, -7, -9 and -15. Of which extends its half-life. These patients are excellent
these, IL-7R is the most crucial for lymphocyte differ- candidates for gene therapy. Children have been treat-
entiation, and mutations in the interleukin-specific IL- ed with periodic infusion of their own T-cells corrected
7R a chain, or in JAK3which transduces the yc signal, by transfection with the ADA gene linked to a retro-
produce similar phenotypes. Mutations in the yc cy- viral vector. Significant reconstitution of antibody re-
tokine receptor gene were completely rescued with sponses and delayed-type skin tests to environmental
autologous bone marrow stem cells cultured with antigens has been achieved without apparent compli-
growth factors on fibronectin fragments (the magic cations. Hematopoietic stem cells from umbilical cord
ingredient needed for success) and transfected with a blood transfected with the ADA gene could be detect-
retroviral vector carrying the normal gene. While the ed up to 18months of age, but the level of expression
T-cell defects can be corrected by transplantation of was very low and, until the technology is improved,
haplotype-matched allogeneic marrow within the first such patients must be maintained on enzyme replace-
3 months of life without the need for myeloablative ment therapy.
pretreatment, and with survival rates of as high as
96%,donors may not always be readily accessible and,
in a proportion of cases, the B- and NK cell defects are
not corrected; these would benefit from the transfected
autologous cell strategy. X-linked lymphopvolifevative disease (XLP), previ-
ously known as Duncan's syndrome, is a progressive
immunodeficiency disorder characterized by fever,
pharyngitis, lymphadenopathy and dysgammaglo-
bulinemia, with a particular vulnerability to Epstein-
Unlike the sneak through of immunocompetent cells Barr viral infection. Mutations have been uncovered in
which accompanies the partial RAG deficiency in the SH2DIA/SAP gene encoding SAP (signaling lym-
phocytic activation molecule (SLAM)-associated pro- and other functions of polymorphs are available,while
tein) which binds to SLAM through its SH2 domain. the reduction of nitroblue tetrazolium (NBT) or the
Since triggering of SLAM leads to strong induction stimulation of superoxide production provides a
of IFNy in both Thl and Th2 cells and acts on B-cells to measure of the oxidative enzymes associated with
enhance proliferation and increase susceptibility to active phagocytosis.
apoptosis, mutations in SAP which adversely affect
the activation of SLAM will weaken the immune re-
SECONDARY IMMUNODEFICIENCY
sponse, especially with regard to EBV infection where
viral replication with B-cells is heavily controlled by Immune responsiveness can be depressed nonspecifi-
host T-cells. Mutation in another signal regulatory pro- cally by many factors. CM1 in particular may be im-
tein, the CD45 protein tyrosine phosphatase, gave rise paired in a state of malnutrition, even of the degree
to the SCID seen in a child with feeble lymphocyte which may be encountered in urban areas of the more
responses at 2 months of age. affluent regions of the world. Iron deficiency is par-
Mice with the lpr gene exhibit a progressive lym- ticularly important in this respect, as are zinc and se-
phoproliferative syndrome reflecting an accum- lenium deficiencies.
ulation of abnormal CD4-8- T-cells with variable Viral infections are not infrequently immunosup-
autoimmune features (cf. table 19.4). The mutation pressive, and the profound fall in cell-mediated immu-
encodes an incompetent Fas (CD95) molecule and nity which accompanies measles infection has been
thereby defective Fas-triggered lymphocyte apop- attributed to specific suppression of IL-12 production
tosis. The homozygous deficiency is extremely rare by viral cross-linking of monocyte surface CD46 (the
in humans. Impaired T- and B-cell development complement regulator also known as membrane co-
and excessive production of autoantibodies by B-l factor protein; cf. p. 307).The most notorious immuno-
cells are the consequences of mutations in moth- suppressive virus, human immunodeficiency virus
eaten mice (cf. p. 417), affecting the hematopoietic (HIV), will be elaborated upon in the next major sec-
cell phosphatase (HCP) which regulates phosphory- tion. In lepromatous leprosy and malarial infection
lation events triggered by engagement of antigen there is evidence for a constraint on immune respon-
receptors. siveness imposed by distortion of the normal lym-
phoid traffic pathways and, additionally, in the latter
instance, macrophage function appears to be aberrant.
RECOGNITION OF IMMUNODEFICIENCIES
Skewing of the balance between Thl and Th2 cells as
Defects in immunoglobulinscan be assessed by quan- a result of infection may also depress the subset most
titative estimations; levels of 2g/l arbitrarily define appropriate for immune protection.
the practical lower limit of normal. The humoral im- Many therapeutic agents, such as X-rays, cytotoxic
mune response can be examined by first screening the drugs and corticosteroids, although often used in a
serum for natural antibodies (A and B isohemagglu- nonimmunological context, can nonetheless have
tinins, heteroantibody to sheep red cells, bactericidins dire effects on the immune system (see p. 382). B-
against E. coli) and then attempting to induce active lymphoproliferative disorders, such as chronic
immunization with diphtheria, tetanus, pertussis and lymphatic leukemia, myeloma and Waldenstrom's
killed poliomyelitis-but no live vaccines. CD19, 20 macroglobulinemia, are associated with varying de-
and 22 are the main markers used to enumerate B-cells grees of hypo-y-globulinemia and impaired antibody
by immunofluorescence. responses. Their common infections with pyogenic
Patients with T-cell deficiency will be hypo- or unre- bacteria contrast with the situation in Hodgkin's
active in skin tests to such antigens as tuberculin, disease where the patients display all the hallmarks
Candida, trichophytin, streptokinase/streptodornase of defective CM1-susceptibility to tubercle bacillus,
and mumps. Active skin sensitization with dini- Brucella, Cryptococcus and herpes zoster virus.
trochlorobenzene may be undertaken. The reactivity
of peripheral blood mononuclear cells to phytohemag-
ACQUIRED IMMUNODEFICIENCY
glutinin is a good indicator of T-lymphocyte reactivity
SYNDROME (AIDS)
as is also the one-way mixed lymphocyte reaction (see
Chapter 17). Enumeration of T-cells is most readily
achieved by cytofluorimetry using CD3 monoclonal
antibody. AIDS is a particularly unpleasant fatal disease caused
In vitro tests for complement and for the bactericidal by infection with the human immunodeficiency virus
HIV-1/2 are members of the lentivirus group, which
produce disease with a long latency and are adept at
evading the immune system. They are RNA retro-
viruses with a relatively complex and tightly com-
pressed genome (figure 15.8a) and the many virion
proteins are generated by RNA splicing and cleavage
by the viral protease (figure 15.8~).
cytomegalovirus-specific cytotoxic T-cells (Tc), be- By looking for correlations between the speed with
come quite devastating. The patient is now wide open which infected individuals develop disease and the
to life-threatening infections caused by normally non- various parameters of the immune response, one
pathogenic (i.e.opportunistic)agents such as Pneumo- hopes to gain insight into those elements which afford
Figure 15.13. Factors affecting HIV pro-
gression. "Neutralizingantibody more ef-
fective against HN-2. HAART, highly
active antiretroviraldrug therapy.
some degree of protection and could guide the at- Enumeration with HLA class I/peptide tetramers
tempts to produce effective vaccines. Bearing in has revealed that up to 5%of total CD8 cells in the acute
mind the discovery of the importance of CCR5 as a phase are specificfor a single epitope and the numbers
chemokine coreceptor for the infection of cells with inversely correlate with viral load. As time goes on,
NSI HIV clades, it is gratifying to note the 10-fold re- however, the protective power of the cells is not com-
duced risk of HIV infection in individuals homo- mensurate with their numbers; in boxing jargon, they
zygous for the CCR5A32 allele, where the 32 base 'punch below their weight'. Whether they are purely
deletion encodes a nonfunctional product. Homozy- dysfunctional or represent anergicor regulatory IL-10-
gotes represent 1-3% of northern Europeans, declin- producing cells is not clear, but on one point there is a
ing as one goes south, and absent in African, Asiatic general consensus: a sustained healthy CD8 response
and Oceanic populations. It has been speculated that depends heavily on robust CD4 Thl effectors con-
the mutation may havebeen fixed innorthwestEurope tributing IFNy, chemokines and useful cell to cell inter-
in the 14th century to provide a selective advantage actions. And here is the root of the problem since,
against bubonic plague. The progression of HIV infec- initially, although more than 0.5%of total CD4 cells are
tion is delayed even in the heterozygotes. specific for viral proteins, these are deleted soon after
As mentioned above, a powerful early CD8 CTL re- infection. There is no shortage of hypotheses to ac-
sponse can drastically reduce the viral load with bene- count for this depletion, but money tends to be riding
ficial effects long term. An important role for CTLs is on the induction of apoptosis in proliferating antigen-
implied by the extended survival of 28-40% of HIV-1- specific CD4 cells infected either directly or through
infected Caucasians who were fully heterozygous at the intimacy of syncytium formation with dendritic
HLA class I loci lacking B35 or CW4 alleles or both. Fur- cells harboring the virus.
thermore, viral load increased dramatically following The horrendous fall in overall numbers of CD4 cells
CD8 depletion in macaque monkeys infected with associated with the onset of clinical AIDS may be pre-
simian immunodeficiency virus (SW)which causes an cipitated by the evolutionof T-cell syncytiuminducing
AIDS-like syndrome. Being an RNA retrovirus, HIV is (SI) strains as the disease progresses. The ability of S1
subject to a ferociously high mutation rate, and longi- virus to infect thymocytesmight exacerbate an already
tudinal studies in this model showed that CTL evasion precarious situation by cutting off the admittedly low
by epitope mutation is an important escape strategy volume supply of new T-cells still generated by an
for compromisingthe antiviral immune response. aged thymus. (Enthusiasticreaders can ferret out even
In addition to their capacity as CTL effectors, CD8 more hypothetical mechanisms for HIV-mediated
cells from infected patients produce a number of fac- T-depletion, e.g. in Essential Immunology, 9th edn,
tors which suppress HIV replication in cultured CD4 pp. 323-324, and Hazenberg M.O. et al. (2000)Nature
cells. One of these factors is K-16 which cross-links Immunology 1,285.)
the membrane-proximalD4 domainof CD4 and blocks Antibodies to variable loop 3 (V3) of the envelope
infection by the virus. It is relevant that high serum gp120 protein arise during disease and are frequently
levels of K-16 correlate well with slow progression of capable of preventing viral infection of CD4 T-cells
the disease. in vitro. Nonetheless, they are rarely protective in
vivo, partly because monkey experiments showed spired that the most promising containment of chal-
that very high titers are required and masking of lenge by a highly virulent virus with an envelope
epitopes by overhanging carbohydrate groups may heterologous to the immunizing strain was achieved
blunt the immunogenicity, perhaps because virus by intradermal priming with envelopegag, p01 and Nef
may be directly transmitted from follicular dendritic DNA, followed b y boosting with recombinant fowl-
cells to CD4 T-cells without exposure to antibody, and pox virus. Oral immunization with an attenuated re-
certainly because the V3 sequencesundergo extensive combined SIV/HIV vaccine in which the Vpu and Nef
mutation. genes were deleted led to protection against vaginally
Neutralizing antibodies are more effective in HIV-2 transmitted infection, but there was a disturbing ten-
infection where progression to death is much slower dency for even the attenuated strains to be pathogenic.
than with HIV-1, and asymptomatic individuals com- This underscores the importance of mucosal defense
monly reveal deletions in the Nef gene, a slow or negli- against initial infection and must give some credence
gible decline in CD4 cells and strong CTL responses. to the idea that the presence of antiviral antibodies in
mucosal secretions could be a significant factor in pri-
mary defense. In fact, despite the discouraging com-
ments raised earlier, the identification of a strongly
Highly active antiretroviral drug therapy (HAART) neutralizing monoclonal antibody directed to a con-
is very effective when given early in the disease and served region of the gp160 envelope precursor sug-
can reduce plasma viral load to undetectable levels. gests that, in a normal infection, antibodies to this
But the virus persists in a latent form in resting CD4 T- potentially protective epitope might be overwhelmed
cells with a decay half-life of 40 months. Supposing by non-neutralizing responses to other adjacent B-cell
that the reservoir of latent cells is as small as 1x105 epitopes; if this were so, an epitope which mimicked
cells, effective eradication could take as long as 60 this conserved epitope could be an additional valuable
years and, in any case, the treatment is very costly and vaccine candidate.
not without side-effects. The sensible strategy is to Some other approaches, still in their formative
keep this residual virus under control by the immune stages, are in the wings. Now that techniques of trans-
system. One approach is to use an intermittent drug fecting bone marrow cells are more successful, one
regimen. This allows a re-emergence of virus capable looks for advances in 'intracellular immunization'
of boosting reasonably robust immune responses through the introduction of genes encoding poten-
which, in turn, would synergize with the reinstalla- tially anti-HIVmolecules which will protect the future
tion of combination drug therapy. The goal, though, is differentiated CD4 T-cell from infection. In one ap-
to develop a vaccine which could contain the infection proach, bone marrow cells are transfected with a se-
following the initial HAART without the need for fur- quence of the CD4 gene coding for an HIV-blocking
ther antiviral drugs. peptide; on regrafting, some of the stem cells will be-
Given the relative efficacy of CD8 T-cells early in in- come CD4+T-cells, secreting a surrounding barrier of
fection and their dependence on robust CD4 helpers, the blocking peptide which protects the lymphocyte
together with the evidence for the protective role of from infectionby the virus. Another group have trans-
cell-mediated immunity in the SIV rhesus monkey fected the stem cells with an scFv (cf. p. 125) gene
model, effort is being directed towards Thl-biased encoding a single HIV-specific antibody combining
vaccines. Logically, this should go hand-in-hand with site which disrupts the viral replication cycle. Using
the eradication of any concurrent worm infestation a similar system, one could also envisage the trans-
which would favor Th2 responses. Trials are in fection of antisense genes to block the expression of
progress with vaccines incorporating a large number HIV regulatory genes. Despite this frantic activity, a
of T-cell epitope peptide sequences to minimize the change in socialbehavior patterns would have a major
chance of the infecting strains evading immune recog- impact on the relentless spread of AIDS. One cannot
nition by mutation and, where prophylactic immu- help recalling the aggrieved request by God to the sup-
nization is contemplated, to take advantage of the fair plicant, frustrated by the failure of his prayers to win
degree of cross-clade recognition of defined CTL epi- him a lottery prize, to help Him by buying a lottery
topes. In a series of studies in rhesus macaques, it tran- ticket.
I SUMMARY I
Primary immunodeficiencystales (table15.1) or the B-cell system lead in particular to infection with
These OCN in the human, albeit somewhat rarely, as a bacteria which are disposed of by opsonization and
result of a defect in almost any stage of dfiexentiation in phagocytosis.
the whole immune system. Patients with T-cell deficiencies are susceptible to
Rare X-linked mutationsproduce disease in males. viruses and moulds which are normally eradicated by
Defects in phagocyhc cells, the complement pathways CMI.
precursors
iL-7Ra. ye, Jak3 WO due to defecfne 11-7 signaling
RAG1/2 Cmplete failure of VDJrecombination
AD/ Adenosine deamimse deficiency; toxic to early lymphoic :ells
X-linked h/mphoprcliferotivedisease; defectivecell signal
(continued)
Secondary immunodeficiency threatening infections through opportunist organisms
Immunodeficiency may arise as a secondary con- such as Pnearrnocystis cariprii and cytomegalovirus.
sequence of malnutrition, lymphoproliferativedisorders, There is a tremendous battle between the immune
agents such as X-rays and cytotoxic drugs, and viral system and the virus, with extremely high rates of viral
infections. destruction and CD4 T-cell replacement.
0 CD4 T-cell depletion may eventually occur as a result of
Ho D.D., Neumann A.U., Perelson A.S., Chen W., Leonard J.M. &
FURTHER READING
Markowitz M. (1995) Nature 373, 123; and Wei X., Ghosh S.K.,
Austen K.F., Burakoff S.J., Rosen F.S. & Strom T.B. (eds) (1996) Taylor M.E. et al. (1995) Nature 373,117. (Both study the effect of
TherapeuticImmunology. Blackwell Science, Oxford. initial treatment with antiviral drugs and show the very high rate
Brostoff J., Scadding G.K., Male D. & Roitt I.M. (eds) (1991) of viral replication and CD4 repletion.)
Clinical hmunology, Chapters 23-25. Gower Medical Publishing, Huss R. (1996) Inhibition of cyclophilin function in HIV-1 infection
London. by cyclosporin A. Immunology Today 17,259.
Buckley R.H. (2000)Advances in immunology: primary immunode- Leonard W.J. (ed.) (2000)Genetic effects on immunity. Current Opin-
ficiency diseases due to defects in lymphocytes. New England ion in Immunology 12,465. (Criticalreviews appearing annually.)
Journalof Medicine 343,1313. Lokki M.-L. & Colten H.R. (1995) Genetic deficiencies of comple-
Burton D.R. & Parren P.W. (2000)Vaccines and the induction of func- ment. Annals of Medicine 27,451.
tional antibodies: time to look beyond the molecules of natural McMichael A. (ed.) (2000) HIV. Currenf Opinion in Imnzunology 12,
infection? Nature Medicine 6,123. 367. (Criticalreviews appearing annually.)
Chapel H., Haeney M., Misbah S. & Snowden N. (1999) Essen- Ochs H.D., Smith C.I.E. & Puck J.M. (eds)(2000)Prima y Immunodefi-
tials of Clinical Immunology, 4th edn. Blackwell Science, ciency Diseases-A Molecular and GeneticApproach. Oxford Univer-
Oxford. sity Press, New York and Oxford.
Fauci A.S. (1996) Host factors and the pathogenesis of HIV-induced Poignard P., Klasse P.J. & Sattentau Q.J. (1996)Antibody neutraliza-
disease. Nature 384,529. tion of HIV-1. Immunology Today 17,239.
Fischer A. et al. (1997) Naturally occurring primary deficiencies of Rosen F.S. et al. (1997) Primary immunodeficiency diseases: report
the immune system. Annual Reviews of Immunology 15,93. of WHO Scientific Group. Clinical and Experimental Immunology
Hammarstrom L., Vorechovsky I. & Webster D. (2000) Selective 109, S1.
IgA deficiency (SigAD)and common variable immunodeficiency Stiehm E.R. (ed.) (1989) Immunological Disorders in Infants and
(CVID).Clinical and Experimental Immunology 120,225. Children,3rd edn. W.B. Saunders, Philadelphia.
~~
CHAPTER16
Hypersensit ivity
ANAPHYLACTIC HYPERSENSlTlVlTY
(TYPE I )
Two main types of mast cell have been recognized,ex-
emplified in the rat by those in the intestinal mucosa
The earliest accounts of inappropriate responses to and those in the peritoneum and other connective tis-
foreign antigens relate to anaphylaxis (Milestone sue sites. They differ in a number of respects, for exam-
16.1).The phenomenon can be readily reproduced in ple in the type of protease and proteoglycan in their
guinea-pigs which, like humans, are a highly suscep- granules, and in the proliferative response of the mu-
tible species. A single injection of 1 mg of an antigen cosal mast cell to the T-cell cytokine IL-3 (table 16.1).
such as egg albumin into a guinea-pig has no obvious This last point is made rather tellingly by the striking
effect. Repeat the injection 2-3 weeks later and the sen- proliferation of mast cells in the intestinalmucosa dur-
People are not equal. Idiosyncratic responses to given Onre~gtoFrance,IcouldnotobtainPhpZiuandde
stimuli have been recognized from time immemorialand aded to study comparativelythetentaclesofActinsria(sea
hypersensitive reactions in some individuals to normally anemone). While endeavouring to determine the toxic
innocuousenvironmentalagentshavebeen frequentlyob- dose (of extracts), we soon discoverad that some days
served. As Lucrefius remarked two thousand years ago, must elapse before h g it; for several dogs did not die
Differences are so great that one mans meat is another until the fourth or fifth day after administration 01even
mans poison. Sii Thorns More records that the future later. We kept those that had been given insuffxient to kill,
King Richard III was aware that strawberries gave him in order to carry out a second investigation upon these
urticaria and arranged to be served a bowl of the fruit at when they had recovered. At this point an unforeseen
a banquet attended by an arch-enemy. When he broke out event occurred. The dogs which had m v e r e d were in-
in a spectacular rash, he accused his guest of attempted tenselysensitiveanddiedafewminutesaftertheadminis-
poisoningandhadhimsummarily executed. trationofsmalldoses.Themosttypicalexperiment,thatin
Scientificinterestinhypersensitivitywasarousedby the which the result was indisputable, was carried out on a
observations of Rchet and Portier. During a South Sea particularly healthy dog. It was given at first 0.1 ml of the
cruise on Prince Albert of Monacos yacht, the Prince, pre- glycerin extract without becoming ill: 22 days later, as it
sumably smarting from an encounter with Physalin (the was in perfect health, I gave it a second injection of the
jelly-fish known as the PortugeseMan-of-Warwith very sameamount.Inafew secondsitwasextreme1yill;breath-
nasty tentacles), suggested that toxin production by the ing became distressful; it could scmely drag itself along,
fish might be of interest. Let Rchet and Portier take up the lay on its side, was seized with diarrhea, vomited blood
story in their own words (1902). anddiedin25minutes.
Onboard the Princes yacht, experimentswere carried The development of sensitivity to relatively harmless
out proving that an aqueous glycerin extract of the fila- substances was termed by these authors anaphylaxis, in
ments of P h p h is extremely toxic to ducks and rabbits. contrast to prophylaxis.
Table 16.1. Comparison of two types of mast cell. ing infection with certain parasites in intact, but not in
T-depleted, rodents, the effect being mediated by a
combination of IL-3 and IL-4. The two types have com-
mon precursors and are interconvertible depending
upon the environmental conditions, with mucosal
MC, (tryptase)phenotype favored by IL-3 and connec-
tive tissue MC, (tryptase chymotryptase)being pro-
moted by a fibroblast factor. However, both types
display a high affinity receptor for IgE (FceRI: cf.
figure 3.18), a property shared with their circulating
counterpart, the basophil. The strength of binding to
the mast cell is evident from the retention of IgE anti-
bodies at a site of intradermal injection for several
weeks; IgG4 in the human also binds to the mast cell re-
ceptor, but more weakly, and disperses from the injec-
tion site within a day or so. It has long been established
that the anaphylactic antibodies in the human are
mainly of the IgE class.
*Basedon protease in granules. Anaphylaxis is mediated by the reaction of allergen
**Predominatein normal skin and intestinal submucosa.
with IgE antibodiesbound strongly through their Cc3
domains to the a chain of the high affinity receptor
(FcERI;Kd = loP9to 10-l0M) on the surface of the mast
cell (figure 16.1). Cross-linking of these IgE anti-
bodies by a multivalent hapten will trigger media-
Figure 16.1. The structural basis of the
binding of IgE to the high affinitymast cell
receptor FceRI. Side view of the complex with
the two Fc chains in yellow and red and the
FccRIa chain in blue; carbohydrate residues
are shown as sticks.The two C&3domains
of the heavy chain dimer of IgE bind
asymmetrically to two distinct interaction
sites on the a chain of the receptor. The p-turn
loop on one C Ebinds
~ along one side of the
d2 domain, while surface loops plus the
C&2-C&3linker region on the other Cc3
interact with the top of the dl-d2 interface.
The 1 : 1stoichiometry of this asymmetric
binding precludes the linkage of one IgE to
two receptor molecules and ensures that
triggering due to a-a aggregation only
occurs through multivalent binding to
surface IgE (seefigure 16.2).The cleft between
the d2 and d l domains facing the plasma
membrane is probably connected in some
way to the receptor p chain to provide
information on the aggregation status of the
a chain. (Photograph kindly provided by Dr
Ted Jardetzky and reproduced by permission
of the Nature Publishing Group.)
formed by the cyclo-oxygenase and lipoxygenase get that complement fragments C3a and C5a can also
pathways (cf. figure 1.15). To recapitulate, the pre- trigger mast cells, although not through IgE receptors).
formed mediators released from the granules include When there is a massive release of these mediators
histamine, heparin, neutral protease, eosinophil and under abnormal conditions, as in atopic disease, their
neutrophil chemotactic factors and platelet activating bronchoconstrictive and vasodilatory effects pre-
factor, while leukotrienes LTB4, LTC4 and LTD4, the dominate and become distinctly threatening.
prostaglandin PGD2 and thromboxanes are all newly
synthesized.We now know that IL-3, IL-4, IL-5 and IL-
6 and granulocyte-macrophage colony-stimulating
factor (GM-CSF), a typical Th2 pattern of cytokines
Clinical responses t o extrinsic allergens
cells, are also released.
Under normal circumstances, these mediators help At least 10% of the population suffer to a greater or
to orchestrate the development of a defensive acute in- lesser degree with allergies involving localized IgE-
flammatory reaction (and in this context let us not for- mediated anaphylactic reactions to allergens such
Figure 16.4. House dust mite-a major cause of allergic disease. Figure 16.5. Atopic allergy. Skin prick tests with grass pollen aller-
gen in a patient with typical summer hay fever. Skin tests were per-
The electronmicrograph shows the rather nasty looking mite graced
formed 5 hours (left) and 20 minutes (right)before the photograph
by the name Dermatophagoides pteryonyssinus and fecal pellets on the
was taken. The tests on the right show a typical end-point titration of
bottom left which are the major source of allergen. The biconcave
a type I immediatewheal and flare reaction. The late phase skin reac-
pollen grains (top left) shown for comparison indicate the size of
particle which can become airborne and reach the lungs. The mite tion (left) can be clearly seen at 5 hours, especially where a large
itself is much too large for that. (Reproducedby courtesy of Dr E. immediate response has preceded it. Figures for allergen dilution
are given.
Tovey.)
as grass pollens, animal danders, the feces from mites ence late phase responses after bronchial challenge
in house dust (figure 16.4) and so on. An increasing with allergen eventually develop chronic asthma.This
number of allergens have now been cloned and ex- disease affects 155million individuals worldwide and
pressed including Der p1 from mites and Lol PI-V costs $6 billion a year to treat in the USA alone. Patients
from rye grass pollen. Der p1 proves to be a protease fall into three main categories.
which increases the permeability of the bronchial 1 The majority who are extrinsic asthmatics associ-
mucosa, thereby facilitating its own passage along ated with atopy, i.e. the genetic predisposition to syn-
with other allergens across the epithelium and allow- thesize inappropriate levels of IgE specific for external
ing access to and sensitization of cells of the immune allergens.
system. It splits the low affinity IgE receptor, CD23, on 2 Nonatopic intrinsic asthmatics.
B-cells so reducing its negative impact on IgE synthesis 3 Occupational asthmatics exposed to specific pro-
when occupied by IgE; it also cleaves the IL-2 receptor teins or small molecular weight chemicals.
a chain which biases the immune response to Th2- Bronchial biopsy and lavage of asthmatic patients
dependent IgE production. Short cuts to allergen puri- reveal an unequivocal involvement of mast cells and
fication can be achieved by screening cDNA libraries eosinophils as the major mediators secreting effector
for IgE-binding proteins using immunoblotting tech- cells, while T-cells provide the microenvironment re-
niques. This was a godsend for the purification of the quired to sustain the chronic inflammatory response
allergen from the venom of the Australianjumper ant, which is an essential feature of the histopathology in
Myrmecia pilosula; just think of trying to accumulate each category (figure 16.6).The resulting variable air-
ants by the kilogram to isolate the allergen using flow obstruction and bronchial hyper-responsiveness
conventional protein fractionation. are the cardinal clinical and physiological features of
The local anaphylactic reaction to injection of anti- the disease.
gen into the skin of atopic patients is manifest as a The atopic trait can also manifest itself as an atopic
wheal and flare (figure 16.5) which is short lived and dermatitis (figure 16.7),with house dust mite, domes-
resolves within an hour or so; it may be succeeded by a tic cats and German cockroaches often proving to be
late phase response involving eosinophil infiltrates the environmental offenders. Recalling the inflamma-
which peak at around 5 hours. Contact of the allergen tion in asthma, skin patch tests with Der p1 in these
with cell-bound IgE in the bronchial tree, the nasal mu- eczema patients produce an infiltrate of eosinophils, T-
cosa and the conjunctival tissues releases mediators of cells, mast cells and basophils. The number of individ-
anaphylaxis and produces the symptoms of asthma or uals affected is comparable to the number affected by
allergic rhinitis and conjunctivitis (hay fever) as the asthma which may be a surprise to some.
case may be. A proportion of the patients who experi- Awareness of the importance of IgE sensitizationto
Figure 16.7. An atopic eczema reaction on the back of a knee of
a child allergic to rice and eggs. (Kindly provided by Professor
J. Brostoff.)
which generate the chronicity of asthma. Remember by eotaxin. Since the T-cell response is heavily skewed
that there is an early phase bronchial response to in- towards the Th2 subset in asthma (figure 16.9), en-
haled antigen essentially involving mast cell media- counter with the allergen on antigen-presenting cells
tors, and an inflammatory late phase dominated by will promote the synthesis of IL-4, -5 and -13. IL-4
eosinophils. Both phases are IgE-dependentas shown stimulates further eotaxin release, while IL-5 upregu-
by their marked attenuation in the great majority of lates chemokine receptors on eosinophils, maintains
asthmatics treated for around 9 weeks with a human- their survival through an inhibitory effect on natural
ized monoclonal antibody (RhuMAb-E25)specific for apoptosis and is involved in their longer term recruit-
the binding site of human IgE to its high affinity recep- ment from bone marrow.
tor, which reduces IgE to almost undetectable levels Things now look bad for the bronchial tissues and a
and downregulates FcERIexpression. Activated mast multitude of factors contribute to allergen-induced
cells make some contribution to eosinophil recruit- airway dysfunction: (i) a virtual soup of bronchocon-
ment by secretion of an eosinophil chemoattractant strictors, the leukotrienes being especially important,
and tryptase, which can switch on a protease-activated bathe the smooth muscle cells, (ii)edema of the airway
receptor (PAR-2)on the surface of endothelial and ep- wall, (iii) altered neural regulation of airway tone
ithelial cells, leading to cytokine production and the through binding of eosinophil major basic protein
expression of adhesion molecules which selectively re- (MBP)to M2 autoreceptors on the nerve endings with
cruit eosinophils and basophils. The most important increased release of acetylcholine, (iv) airway epithe-
trigger of the late phase reaction is now thought to be lial cell desquamation due to the toxic action of MBP,
activation of alveolar macrophages through the inter- there being a strong correlation between the number
action of allergen with IgE bound to the low affinity re- of desquamated cells in bronchoalveolar lavage fluid
ceptors (FceRII;CD23),leading to a significantincrease and the concentration of MBP, (v) mucus hyper-
in the production of TNF and IL-1P. These cytokines secretion due to IL-13 and, to a lesser extent, IL-4,
then stimulate the release of the powerful eosinophil leukotrienes and platelet activating factor acting on
chemoattractants:eotaxin (CCLll), RANTES (CCL5) submucosal glands and their controlling neural ele-
and MCP5 (CCL12) (cf. p. 187) from bronchial epithe- ments, and finally (vi) a repair-type response involv-
lial cells and fibroblasts. Note also that eotaxin and ing the production of fibroblast growth factor, TGFP
RANTES can contribute directly to local inflammation and platelet-derivedgrowth factor, the laying down of
by IgE-independentdegranulation of basophils. collagen, scar and fibrous tissue and hypertrophy of
A new player now enters the field: primed T-cells smooth muscle, leading to an exaggerated narrowing
traffic into the inflamed site and are strongly attracted of the airways in response to a variety of environmen-
tal stimuli (figure 16.6). The wide range of cytokines turn, promote the release of the eosinophil chemoat-
and mediators produced by lung epithelia1 and en- tractants, RANTES and eotaxin, from keratinocytes
dothelial cells, fibroblasts and smooth muscle cells and fibroblasts. A newly identified keratinocyte CC
may account for the persistence of airway inflamma- chemokine, CTACK (CCL27; cf. p. 187),preferentially
tion and the permanent structural changes in chronic attractsskin-homingCLA+memory T-cells;thesemake
disease sufferers, even in the absence or apparent up 80-90% of the T-cells in the infiltrate and account
absence of ongoing exposure to inhalant allergens to for the specificresponse to the offending allergen.
which subjects are sensitized, a state where conven-
tional immunotherapy might not be expected to be
Etiologicalfactors in the development of
beneficial.
atopic allergy
Unlike atopic asthmatics, intrinsic asthmatics have
negative skin tests to common aeroallergens, no clini- There is a strong familial predisposition to the devel-
cal or family history of allergy, normal levels of serum opment of atopic allergy (figure 16.10).One factor is
IgE and no detectable specific IgE antibodies to com- undoubtedly the overall ability to synthesize the IgE
mon allergens. Nonetheless, they resemble the atopics isotype-the higher the level of IgE in the blood, the
in important respects: bronchial biopsies show en- greater the likelihood of becoming atopic (figure
hanced expression of IL-4, IL-13, RANTES and 16.10).Genetic studies show this to be linked to chro-
eotaxin, and of the mRNA for the E germ line trans- mosome 13q. Linkages have also been established for
cript and the E heavy chain, suggestive of local IgE other factors contributingto atopy:to chromosome l l q
synthesis. Is there a role for virus-specific IgE or for involving the FcERIPchain, to 2q containing the IL-1
IgE autoantibodiesto the FcERI? cluster, and to 5q within the IL-4, -9, -13, GM-CSF and
The inflammatory infiltrate in atopic dermatitis CD14 gene cluster.A C + T base change 159bases up-
closely resembles that in asthma. The epidermal stream of the transcription start site for CD14, the high
dendritic cells have markedly upregulated FcERI affinity receptor for bacterial LPS endotoxin, is signifi-
expression, and an incoming allergen will activate cantly associated with high levels of soluble CD14 and
them either directly or as allergen-IgE complexes; the low levels of IgE. What relevance might this have for
TNF, IL-IP and GM-CSF which they produce will, in atopic disease? Well, excessive exposure of babies to
Figure 16.12. Antibody-dependent cytotoxic hypersensitivity rected to tumors or to glomerular basement membrane are 10-100
(type 11). Antibodies directed against cell surface antigens cause cell times more pathogenic in animals deficient in FcyRIIB relative to
death not only by C-dependent lysis but also by Fcy and C3b adher- their wild-type controls, pointing to a balance of activating and in-
ence reactions leading to phagocytosis, or through nonphagocytic hibitory signals controlling the dominant effector pathway in these
extracellular killing by certain lymphoid and myeloid cells (anti- responses, and implicating the macrophage as the critical cell type
body-dependent cellular cytotoxicity).IgG cytotoxic antibodies di- involved.
Figure 16.13. Killing of Ab-coated target by
antibody-dependentcellularcytotoxicity
(ADCC).Fcyreceptorsbind theeffector to the
target which is killed by an extracellularmech-
anism. Human monocytesand IFNy-activated
neutrophils kill Ab-coated tumor cellsusing
their FcyRIreceptors;lymphocytes (NK cells)
kill hybridoma targets through FcyRIIIrecep-
tors. (a)Diagram of effectorand target cells.
(b)Electronmicrograph of attack on Ab-coated
chick red cell by a mouse large granular lym-
phocyte showing close appositionof effector
and target and vacuolationin the cytoplasm of
the latter. ((b)Courtesy of P. Penfold.)
movement, and by aggregated IgG which binds firmly of glycosyltransferases encoded by A or B genes, re-
to the Fc receptors and blocks their ability to interact spectively. Individuals with both genes (group AB)
with antibody on the surface of the target. have the two antigens on their red cells, while those
ADCC can be readily observed as a phenomenon lacking these genes (group 0)synthesize H substance
in vitro; to give examples, human K-cells have been only. Antibodies to A or B occur spontaneously when
shown to be strikingly unpleasant to chicken red cells the antigen is absent from the red cell surface; thus a
coated with rabbit antibody, while schistosomules person of blood group A will possess anti-B and so on.
coated with either IgG or IgE can be killed by These isohemagglutinins are usually IgM and prob-
eosinophils (cf. figure 13.22). Whether ADCC plays a ably belong to the class of 'natural antibodies'; they
positive role in viva remains a tricky question, but func- would be boosted through contact with antigens of the
tionally this extracellular cytotoxic mechanism would gut flora which are structurally similar to the blood
be expected to be of significance where the target is too group carbohydrates, so that the antibodies formed
large for ingestion by phagocytosis, e.g. large parasites cross-react with the appropriate red cell type. If an in-
and solid tumors. It could also act as a back-up system dividual is blood group A, he/she would be tolerant
for T-cell killing. to antigens closely similar to A and would only form
cross-reacting antibodies capable of agglutinating B
red cells; similarly an 0 individual would make anti-A
and anti-B (table 16.2). On transfusion, mismatched
red cells will be coated by the isohemagglutinins and
will cause severe complement-mediated intravascular
Transfusion reactions
hemolysis.
Of the many different polymorphic constituents of the Clinical refractoriness to platelet transfusions is
human red cell membrane, A B 0 blood groups form frequently due to HLA alloimmunization, but one can
the dominant system. The antigenic groups Aand B are usually circumvent this problem by depleting the
derived from H substance (figure 16.14) by the action platelets of leukocytes.
Figure 16.15. Hemolytic disease of the
newborn due to rhesus incompatibility.
(a) RhD+vered cells from the first baby
sensitize the RhD-ve mother. (b)The
mother's IgG anti-D crosses the placenta
and coats the erythrocytes of the
second RhD+vebaby causing type I1
hypersensitivity hemolytic disease. (c)IgG
anti-D given prophylactically at the first
birth removes the baby's red cells through
phagocytosis and prevents sensitization of
the mother.
Table 16.2. A B 0 blood groups and serum antibodies. opsonic adherence, giving hemolytic disease of the
newborn (figure 16.15).
These anti-D antibodies fail to agglutinate RhD+ve
red cells in vitro ('incomplete antibodies') because the
low density of antigenic sites does not allow sufficient
antibody bridges to be formed between the negatively
charged erythrocytes to overcome the electrostatic
repulsive forces. Erythrocytes coated with anti-D can
be made to agglutinate by addition of albumin or of
an anti-immunoglobulin serum (Coombs' reagent;
figure 16.16).
Rhesus incompatibility
If a mother has natural isohemagglutinins which
The rhesus (Rh) blood groups form the other major can react with any fetal erythrocytes reaching her cir-
antigenic system, the RhD antigen being of the most culation, sensitization to the D-antigens is less likely
consequence for isoimmune reactions. A mother with due to 'deviation' of the red cells away from the
an RhD-ve blood group (i.e. dd genotype) can readily antigen-sensitive cells. For example, a group 0
be sensitized by red cells from a baby carrying RhD RhD-ve mother with a group A RhD+ve baby would
antigens (DD or Dd genotype).This occurs most often destroy any fetal erythrocytes with her anti-A before
at the birth of the first child when a placental bleed can they could immunize to produce anti-D. In an exten-
release a large number of the baby's erythrocytes into sion of this principle, RhD-ve mothers are now
the mother. The antibodies formed are predominantly treated prophylactically with small amounts of avid
of the IgG class and are able to cross the placenta in any IgG anti-D at the time of birth of the first child, and
subsequent pregnancy. Reaction with the D-antigen this greatly reduces the risk of sensitization. Another
on the fetal red cells leads to their destruction through success for immunology.
Another example of disease resulting from transpla-
cental passage of maternal antibodies is neonatal
alloimmune thrombocytopenia. The fall in platelet
numbers is greatly amelioratedby high-dose i.v. injec-
tions of pooled human IgG, thought by some to in-
volve anti-idiotypenetworks (cf. p. 443), although the
efficacy of Fcy fragments, anti-RhD and anti-FcyR
rather point the finger at blockade of the Fcy receptors.
Organ transplants
Along-standingallograft which has withstood the first
onslaught of the cell-mediated reaction can evoke hu-
moral antibodies in the host directed against surface
transplantation antigens on the graft.These may be di-
rectly cytotoxic or cause adherence of phagocytic cells
or 'nonspecific' attack by K-cells.They may also lead to
platelet adherence when they combine with antigens
on the surface of the vascular endothelium (figure17.6,
p. 354). Hyperacute rejection is mediated by pre-
formed antibodiesin the graft recipient.
Figure 16.19. Histology of acute inflammatory reaction in pol- the renal artery of a patient with chronic hepatitis B infection
yarteritis nodosa associated with immune complex formation stained with fluoresceinated antihepatitis B antigen (left) and rho-
with hepatitis B surface (HBs)antigen. (a)Avessel showingthrom- daminated anti-IgM (right).The presence of both antigen and anti-
bus (Thr) formation and fibrinoid necrosis (FN) is surrounded by a body in the intima and media of the arterial wall indicates the
mixed inflammatory infiltrate, largely polymorphs. (b)High-power deposition of the complexesat this site. IgG and C3 deposits are also
view of acute inflammatory response in loose connective tissue of detectable with the same distribution. ((a)and (b)provided by cour-
patient with polyarteritis nodosa-polymorphs (PMN) are pro- tesy of Professor N. Woolf; (c) kindly provided by Professor A.
minent. (c) Immunofluorescence studies of immune complexes in Nowoslowski.)
Figure 16.20. Extrinsic allergic alveolitis due to rat serum proteins
in a research assistant handling rats (type I11 hypersensitivity).
Typical systemic and pulmonary reactions on inhalation and
positive prick tests were elicitedby rat serum proteins; precipitins
against serum proteins in rat urine were present in the patient's
serum. (a) Bilateral micronodular shadowing during acute
episodes. (b)Marked clearing within 11days after cessation of
exposure to rats. (c)Temporaryfall in pulmonary gas exchange
measured by DL,, (gas transfer, single breath) following a 3-day
exposure to rats at work (arrowed). (FromCarroll K.B., Pepys J.,
LongbottomJ.L., Hughes D.T.D. & Benson H.G. (1975)Clinical
Allergy 5,443; figures by courtesy of Professor J. Pepys.)
Figure 16.22. Deposition of immune complexes in the kidney cally attracted neutrophils release granule contents in 'frustrated
glomerulus. (1) Complexes induce release of vasoactive mediators phagocytosis' to damage basement membrane and cause leakage of
from basophils and platelets which cause (2) separation of endothe- serum proteins. Complex deposition is favored in the glomerular
lial cells. (3) Attachment of larger complexes to exposed basement capillary because it is a major filtrationsite and has a high hydrody-
membrane, with smaller complexes passing through to the epithe- namic pressure.Deposition is greatly reduced inanimals depleted of
lial side. (4) Complexesinduce platelet aggregation.(5)Chemotacti- platelets or treated with vasoactive amine antagonists.
minor degrees of glomerular injury (figure 16.23, plexes with antigens of the infecting organism have
lane 3). been implicated. Immune complex nephritis can arise
Many cases of glomerulonephritis are associated in the course of chronic viral infections; for example,
with circulating complexes, and biopsies give a mice infected with lymphocytic choriomeningitis
fluorescent staining pattern similar to that of figure virus develop a glomerulonephritis associated with
l6.l7b, which depicts DNA/ anti-DNA / complement circulating complexes of virus and antibody. This may
deposits in the kidney of a patient with SLE (cf. p. well represent a model for many cases of glomeru-
427). Well known is the disease which can follow infec- lonephritis in humans.
tion with certain strains of so-called 'nephritogenic'
streptococci and the nephrotic syndrome of Nigerian
Deposition of immune complexes at other sites
children associated with quartan malaria, where com-
The choroid plexus, being a major filtration site, is also
favored for immune complex deposition and this
could account for the frequency of central nervous dis-
orders in SLE. Neurologically affected patients tend to
have depressed C4 in the cerebrospinal fluid (CSF)
and, at post-mortem, SLE patients with neurologic dis-
turbances and high titer anti-DNA were shown to have
scattered deposits of immunoglobulinand DNA in the
choroid plexus. Subacute sclerosingpanencephalitisis
associated with a high CSF to serum ratio of measles
antibody, and deposits containing Ig and measles Ag
may be found in neural tissue.
Vasculitic skin rashes are also characteristic of sys-
temic and discoid lupus erythematosus (figure 16.24a
and b), and biopsies of the lesions reveal amorphous
deposits of Ig and C3 at the basement membrane of the
dermal-epidermal junction (cf.figure 20.6).
Figure 16.23. Proteinuria demonstrated by electrophoresis. Lane Another example of immune complex hypersen-
1:Normal serum as reference. The major band nearest to the cathode
is albumin.Lane 2: Normal urine showing a trace of albumin.Lane 3:
sitivity is the hemorrhagic shock syndrome found
Glomerular proteinuria showing a major albumin component.Lane with some frequency in South-EastAsia during a sec-
4: Proteinuria resulting from tubular damage with a totally different ond infectionwith a dengue virus. There are four types
electrophoretic pattern. Lane 5: Bence-Jones proteinuria represent- of virus, and antibodies to one type produced during a
ing excreted paraprotein light chains (cf.p. 385).Lane 6: Bence-Jones
proteinuria with a trace of the intact paraprotein. Some of the sam-
first infection may not neutralize a second strain but
ples have been concentrated. (Electropherograms kindly supplied rather facilitate its entry into, and replication within,
by T. Heys.) human monocytes by attachment of the complex to
Figure 16.25. Cell-mediated (type IV) hypersensitivity reactions. to nickel caused by the clasp of a necklace. ((a) Kindly provided by
(a)Mantoux test showing cell-mediated hypersensitivityreaction to Professor J. Brostoff and (b) by Professor R. Barnetson; (c) repro-
tuberculin, characterized by induration and erythema. (b) Chronic duced from the British Society for Immunology teaching slides with
type IV inflammatory lesion in tuberculous lung showing caseous permission of the Society and the Dermatology Department,
necrosis (CN),epithelioid cells (E), giant cells (G) and mononuclear London Hospital.)
inflammatory cells (M).(c)Type I Vcontact hypersensitivityreaction
(whichlacks the mycobacteria),the delayed hypersen- conferring antigen-specific reactivity is still a highly
sitivity state is of shorter duration and the dermal re- contentious issue.
sponse more transient. This is known as 'Jones-Motef It cannotbe stressed too often that the hypersensitiv-
sensitivity but has recently been termed cutaneous ity lesion results from an exaggerated interaction be-
basophil hypersensitivity on account of the high tween antigen and the normal cell-mediated immune
proportion of basophils infiltrating the skin lesion. mechanisms (cf. p. 185). Following earlier priming,
memory T-cells recognize the antigen together with
class II major histocompatibilitycomplex (MHC)mole-
cules on an antigen-presenting cell and are stimulated
Unlike the other forrns of hypersensitivity which we into blast cell transformation and proliferation. The
have discussed, delayed-type reactivity cannot be stimulatedT-cells release a number of cytokines which
transferred from a sensitized to a nonsensitized indi- function as mediators of the ensuing hypersensitivity
vidual with serum antibody; lymphoid cells, in par- response, particularly by attracting and activating
ticular the T-lymphocytes, are required. Transfer has macrophages if they belong to the Thl subset, or
been achieved in the human using viable white blood eosinophilsif they are Th2; they also help Tc precursors
cells and, interestingly, by a low molecular weight to become killer cells which can cause damage to
material extracted from them (Lawrence's transfer virally infected target cells (figure 16.26), the CD8
factor). The nature of this substance is, however, a TCRaP cytotoxic cells being activated by recognition
mystery. The extracts contain a variety of factors of MHC class I complexes with processed viral pro-
which appear capable of stimulating precommitted teins and TCRyG killers operating through binding to
T-cells mediating delayed hypersensitivity, but native viral proteins on the surface of the infected
whether there is also an informational molecule cells.
Th2-mediated hypersensitivity
The examples of type IV hypersensitivity we have Figure 16.28. Th2-mediated response to schistosome egg. Th2-
type hypersensitivity lesion of inflammatory cells (M) around a
discussed so far are centered essentially on the Thl
schistosome egg (SE) within the liver parenchyma (LP). (Photo-
macrophage pathway, but it is now clear that excessive graph by courtesy of Professor M. Doenhoff.)
responses by Th2 cells can damage tissues through
activation of eosinophils (figure 16.26).As recounted
earlier, T-cells synthesizingIL-5 are largely responsible by.exposure to dietary wheat gliadin. The disorder in-
for the sustained influx of eosinophils in asthma and volves what is probably a geneticallyrelated increased
atopic dermatitis (cf. p. 328). They also essentially ac- mucosal activity of transglutaminase (the target of
count for the liver pathology in schistosomiasiswhich anti-endomysium autoantibodies; cf. p. 423). This en-
has been attributed to a reaction against soluble zyme deamidates the glutamine residues in gliadin
enzymes derived from the eggs which lodge in the and createsa new T-cell epitope that binds efficientlyto
capillaries (figure 16.28). DQ2 and is recognized by gut-derived CD4 and y6
T-cells.
Psoriasis involves marked proliferation of epider-
Other examples
mal keratinocytes and accelerated incomplete epi-
Delayed hypersensitivity contributes significantly to dermal differentiation. There is inflammation in the
the prolonged reactions which result from insect bites. skin with pockets of microabscesses containing
The possible implication of allograft rejection by Tc neutrophils and, in all instances, CD4 cells in the psori-
cells as a mechanism for the control of cancer cells atic dermis and CD8 in the lesional epidermis.A rever-
is discussed in Chapter 18. In certain organ-specific sal of epidermal dysfunction and a marked reduction
autoimrnune diseases, such as type I diabetes, cell- in intraepidermal CD8 T-cells were achieved by sys-
mediated hypersensitivity reactions undoubtedly temic administration of a fusion protein of IL-2 and
provide the major engine for tissue destruction. fragments of diphtheria toxin, which selectively
The intestinal inflammation in celiac disease, an blocks the growth of activated lymphocytes but
HLA-DQ2/8-associated enteropathy, is precipitated not keratinocytes. This shows that the disorder is
primarily immunological rather than a dysfunction (cf. figure 1.6). Normally this would enhance host
of keratinocytes which secondarily activate T-cells defenses, aiding the recruitment of phagocytes by
through, say, TGFP. The party line now would be that promoting adherence to endothelium, priming
CD8 T-cells attack the skin, activating injury repair neutrophils for subsequent release of reactive oxygen
programs associated with wound healing, while intermediates, inducing febrile responses (immune
cytokines act directly as mitogens for epidermal responses improve steadily from 33 to 44OC), and so
keratinocytes. on. Unfortunately,the excess of circulating LPS and the
cytokines released by it lead to unwanted pathophy-
siology at distant sites, such as the adult respiratory
STIMULATORY HYPERSENSlTlVlTY (TYPE V) distress syndrome brought about by an overwhelm-
Many cells are signaled by agents such as hormones ing invasion of the lung by neutrophils. There is a pro-
through surface receptors to which they specifically longed pathologically high concentration of nitric
bind the external agent, presumably through comple- oxidebut, additionally,LPS can activate the alternative
mentarity of structure. For example, when thyroid- complement pathway and this may be linked to its
stimulating hormone (TSH) of pituitary origin binds ability to induce the release of thromboxane A2 and
to the thyroid cell receptors, adenyl cyclase is acti- prostaglandin from platelets leading to disseminated
vated, and the CAMP 'second messenger' which is intravascular coagulation.
generated acts to stimulate the thyroid cell. The Whereas the major culprit in Gram-negative sepsis
thyroid-stimulating antibody present in the sera of is LPS, Gram-positive organisms possess a variety of
thyrotoxic patients (cf. p. 422) is an autoantibody componentswhich act on host defense elements to ini-
directed against a site on the TSH receptor which tiate septic shock. Thus adherence of Staphylococcus
produces the changes required for adenyl cyclase acti- aureus to macrophages induces TNFa synthesis, and
vation. Intriguingly, but not much more at present, peptidoglycan-mediated aggregation of platelets by
there are indications that cimetidine-resistant duode- the same organism leads to disseminated intravascu-
nal ulcer patients might have stimulatory antibodies lar coagulation. The staphylococcal and streptococcal
directed to histamine receptors. enterotoxins induce toxic shock syndrome by quite
Experimentalexamplesof stimulationby antibodies different means. By functioning as superantigens (cf.
to cell surface antigens may be cited: the activation of p. 103),they react directly with particular T-cell recep-
B- and T-lymphocytes by F(ab'), fragments directed to tor families and give rise to massive cytokine release,
their antigen-specificreceptors; the production of cell including TNF and macrophage migration inhibitory
division in thyroid cells by 'growth' autoantibodies; factor (MIF),which is detected in high concentrations
the induction of pinocytosis by antimacrophage in the plasma of patients with septic shock. Various
serum; and the mitogenic effect of antibodies to sea- treatments are under investigation. Pentoxifylline
urchin eggs. It is worthy of note that, although anti- blocks TNF production by macrophages. Experimen-
bodies to enzymes directed against determinants near tal models of septic shock can be blocked by anti-MIF
to the active site can exert a blocking effect, combina- and by a peptide derived from the natural sequence
tion with more distant determinants can sometimes 150-161 of staphylococcalenterotoxin B, which is part
bring about allosteric conformational changes which of a domain crucial for T-cell activation.
are associated with a considerable increase in enzymic Germ-linemutations in the extracellular domains of
activity, as has been described for certain variants of the 55 kDa TNF receptor, TNFRI, define a family of
penicillinase and P-galactosidase. dominantly inherited autoinflammatory syndromes
involvingunpredictable periodic fever episodes.
The reader 'S attention has alreadybeen drawn to the
'INNATE' HYPERSENSlTlVlTY REACTIONS
unusual susceptibility of erythrocytes to lysis in par-
Many infections provoke a 'toxic shock syndrome' oxysmal nocturnal hemoglobinuria resulting from
characterized by hypotension, hypoxia, oliguria and deficiency in complement control proteins on the red
microvascular abnormalities and mediated by ele- cell surface (see p. 307). Undue C3 consumption is
ments of the innate immune system independently of associated with mesangiocapillary glomerulonephri-
the operation of acquired immune responses. tis and partial lipodystrophy in patients with the
Septicemia associated with Gram-negative bacteria so-called C3 nephritic factor, which appears to be an
results in excessive release of TNF, IL-1 and IL-6 IgG autoantibody capable of activating the alterna-
through stimulation of macrophages and endothelial tive pathway by combining with and stabilizing the
cells by the lipopolysaccharide (LPS) endotoxin C3bBb; convertase.
We should include idiopathic pulmonary fibrosis disease are extracellularplaques and intracellular neu-
in this section. It is a chronic fatal disorder character- rofibrillary tangles. One of the constituents of senile
ized by diffuse fibrosis of the alveolar walls in which plaques is P-amyloid composed of a hydrophobic frag-
local macrophages play a central role. On activation, ment of amyloid precursor protein (APP). Normally
they produce an early G1 competence growth signal, APP is cleaved by an a-secretase into soluble products
such as platelet-derivedgrowth factor and fibronectin, which cannot form P-amyloid, but members of a
and then a late G1 progression growth signal, such as family diagnosed with Alzheimer's disease, and
insulin-like growth factor 1. As a result, the fibroblasts having a pathogenic mutation at residues 670-671 of
multiply and become embedded in a collagen matrix APP, had very low levels of this soluble cleaved form
to the respiratory detriment of the host. compared with noncarriers. This could also represent
The neuropathological hallmarks of Alzheimer's a new and promising diagnostic marker.
Excessive stimulation of the normal effector mecha- cruitment of tissue-damaging eosinophils through the re-
nisms of theinununesystem canlead totissuedamageand lease of IL-5. The soup of powerful bronchoconstrictors,
we speak of hypersensitivity reactions, several types of the injurious effect of eosinophil major basic protein
which canbe distinguished. and reactive oxygen intermediatesand the mucus hyper-
secretion stimulated by IL-13 and IL-4, all contribute to
Anaphylactic hypersensitivity(type I) the drastic airway damage characteristic of chronic
Anaphylaxis involves contraction of smooth muscle asthma.
and dilatation of capillaries. Many food allergies involvetypeI hypersensitivity.
This depends upon the reaction of antigen with speeific Strong genetic factors include linkage to the propensity
IgE antibody bound through its Fc to the mast cell high to make the IgE isotype and to genes involving the IL-
affinity receptorFc&I. 4/9/13,GM-CSFandCD14cluster.
C r o s s - l i i g and clustering of the IgE receptors Exposure to certain foods and to Thl-stimulating infec-
activates the Lyn protein tyrosine kinase, recruits other tions may strongly influence the 'immunostat' setting of
kinassandleads toreleasefromthegranulesofmediators the tendency to either Thl or Th2 responses, the latter
including histamine, leukotrienes and platelet activating increasing the risk of allergy through promotion of IgE
factor, plus eosinophiland neutrophil chemotacticfactors synthesisand eosinophilrecruitment
and the cytokines IL-3,4, -5and GMCSF. The offending antigenis identifiedbyintradermalprick
tests, giving immediate wheal and erythema reactions, by
Atopic allergy provocationtesting and by RAST.
* Atopy stems from an excessive IgE response to extrinsic Where possible, allergen avoidance is the best
antigens (allergens)whichleads tolocalanaphylactic reac- treatment
tions at sites of contact with allergen. A monoclonal antibody directed to the receptor-
* Hay fever and extrinsicasthma represent the most com- binding domain of IgE dramatically reduces IgE levels
mon atopic allergic disorders resulting from exposure to and synthesis, and decreases mast cell responsiveness.
inhaled allergens. Atopic dermatitis is also extremely Symptomatic treatment involves the use of long-acting
common. p,-agonists andnewly developed leukotriene antagonists.
Whereas the immediate reaction to extrinsic allergen Chromones, such as sodium cromoglycate,block chloride
(maximum at 30 minutes) is due to mast cell triggering, a channel activity thereby stabilizing mast cells and in-
late phase reaction peaking at 5 hours, involving heavy hibiting bronchoconstriction. Theophylliie, the single
eosinophil infiltration, is initiated by the activation of most prescribed drug for asthma, is a phosphodiesterase
alveolar and other macrophages through surface-bound inhibitor which raises intracellular calcium; this causes
IgE; secreted TNF and IL-lp now act upon epithelia1cells bronchodilatation and inhibition of IG5 effects on
and fibroblasts to release powerful eosinophil chemoat- eosinophils. ChmNc asthma is dominated by activated
tractants such as RANTES and eotaxin. Th2cells and is treated with topicalsteroidswhichdisplay
In asthma, serious prolongation of the response to aller- a wide range of anti-inflammatory actions, including
gen is caused by T-cells of Th2 type which sustain the re- the ability to block the production of mediators by
. .
(cont i m e d )
stimulated macrophages or Th2 cells. These are supple- of polymorphonucleax leukocytes which release tissue-
mented where necessary by long-acting B2-agonists and damaging mediators on contact with the complex, (ii)
theophylline. stimulation of macmphages to release proinaammatory
Courses of antigen injectionmay desensitizeby the for- cytokines, and (iii) aggregation of platelets to cause
mation of blocking,or possibly regulatory, IgG antibodies microthmmbi and vasoactiveamine release.
or through T-cell regulation. T e l l epitope peptides may Where Circutating antibody levels are high, the antigen
bemanipulated tomodulate the atopic state. isprecipitatednearthesiteofentryintothe body. Thereac-
tion in the skin is characterizedby polymorph infiltration,
Amibody-dependent cytotoxic hypemensitlvity(iype 11) edema and erythema maximal at 3-8 hours (Arthus
This involves the death of cells bearing antibody reaction).
attached to a surfaceanfigen. Examples are farmers lung, pigeon-fanciers disease
The cells may be taken up by phagocytic cells to which and pulmonary aspergillosis where inhaled antigens
they adhere through their coating of IgG 01C3b or lysed by provoke high antibody levels, reactions to an abrupt in-
theoperation of the fullcomplement system. crease in antigen caused by micmbial cell death during
Cells bearing IgG may also be killed by polymorphs chemotherapyfor leprosyor syphilis,polyartdtis nodosa
and macrophages or by K-cells through an extracellular linked to complexeswith hepatitis B virus and an element
mechanism (antibody-dependentcellular cytotoxicity). of the synoviallesioninrheumatoidarthritis.
Examples are: transfusion reactions,hemolytic dlsease In relative mtigm excss, soluble complexes are formed
of the newborn through rhesus incompatibility,antibody- which are removedbybinding to the CR1C?,b reeeptors on
mediated graft destruction, autoimmune reactions dir- red cells. If this system is overloaded or if the classical
ected against the formed elementsof the blood and kidney complement components are deficient, the complexes
glomerular basement membranes, and hypersensitivity circulate in the free state and are deposited under cir-
resulting from the coating of erythrocytesor platelets by a cumstances of increased vascular permeability at certain
drug. preferred sites: the kidney glomerulus, the joints, the skin
and the choroid plexus.
Complex-mediated hypemenslllvlty(iype Ill) Examples are: glomerulonephritis associated with
This results fmm the effects of antigen-tibody
com- systemic lupus erythematosus (SLE) or infections with
plexesthrough(i)activationofcomplementandattraction streptococci,malaria and otherparasites,neUgicaldis-
(continuedp.348)
turbancesinSLE and subacute sclerosingpanencephalitis, reactions to intestinal bacteria or certain foods such as
and hemorrhagicshock in dengue viral infection. wheat gliadin.
Silmulatory hypersensitlviiy(type V)
Cell-medlated or delayed-type hypenensitivily(lype IV) The antibody reacts with a key surface componentsuch
This is based upon the interaction of antigen with as a hormone -tor and 'switches on' the cell.
primed T-cells and represents tissue damage resulting An example is the thyroid hyper-reactivity in Graves'
from inappropriatecell-mediated immunity reactions. disease due to a thyroid-stimulating autoantibody.
A number of soluble cytokines including IFN'y are re- Features of these five types of acquired hypersensitivity
leased, which activate maaophages and account for the are compared in table 16.3.
events that occur in a typical delayed hype-itivity re- 'Innate' hypenensitivilyreactions
sponse such as the Mantoux reaction to tuberculin,that is, Many infections provoke a 'toxic shock syndrome'
the delayed appearance of an indurated and erythema- involving excessive release of TNF, L-1 and K-6 and
tous reaction which reaches a maximum at 24-48 hours intravascular activationof complement.
and is characterized histologically by infiltration with Septic shodc associated with Gram-negativebacteria is
mononuclear phagocytesand lymphocytes. primarily due to the lipopolysaccharide( L E ) endotoxin.
Continuing provocation of delayed hypersensitivity Gram-positive organisms cause release of TNF and
by persisting antigen leads to the formation of chronic macrophagemigrationinhibitoryfactor (MIF) throughdi-
granulomas. rect action on maaophages and stimulation of selected
Tk2-typecellsproducingIL4andIL-5canalsoproduce T-cell familiesby the enterotoxin superantigens.Aggrega-
tissue damage through their ability to recruit eosinophils. tion of platelets by S. nureus induces disseminated in-
CD8 T-cells are activatedby class I majorhistocompati- travascular coagulation.
bility antigens to become directly cytotoxic to target cells Aberration of innate mechanisms may underlie idio-
bearing the appropriate antigen. pathic pulmonary fibrosis and contribute to the Qamyloid
Examples are: tissue damage occurring in bacterial (tu- plaques inkheimer's disease.
berculosis, leprosy), viral (measles, herpes), fungal (can-
didiasis, histoplasmosis) and parasitic (leishmaniasis,
schistosomiasis)infections, contact dermatitis from expo- ee the accompanying website (www.roln.com)
sure to chromates and poison ivy, insect bites and psoria- ir multiplechoice questions.
sis. Mammatory bowel disease can result from Thl-type I
Transplantation
(continued)
pendentlybyCalneandZukowskiin1960totheprolonga- readersofahistoricalbentwillgainfwtherinsightintothe
tion of renal allografts in dogs. This was followed very development of this field and the minds of the scientists
rapidlyby Murray's successfulgrafting in 1962of a n w e - who gave medicine this wonderful prize in Terasaki P.I.
lated cadaveric kidney under the immunwuppressive (ed.) (1991) History of Transplantation; Thirty-Fiue Recollec-
umbrella of azathioprine, themore effective derivative of fwns, UCLA Tissue Typing Laboratory, Los Angeles, CA
6-mercaptopueinedevised by Hutchings and Elion. and,subsequently, h n t L. (1996) AHistory ofTransphtu-
This story is studded with Nobel Prize winners and tionImmunology,AcademicPress,London.
Figure 17.9. Polymorphic HLA specificitiesand their inheritance. at each locus, the probability of a random pair of subjects from the
The complex lies on chromosome 6, the DP locus being closest to the general population having identical HLA specificities is very low.
centromere. The numbers at the A and B loci do not overlap. The However, there is a 1 :4 chance that two siblings will be identical in
nomenclature for the DR alleles is set out in Bodmer J.G. et al. (1994) this respect because each group of specificities on a single chromo-
Tissue Antigens 44,l. DR11/12(5) shows that the 'old pre-Bodmer' some forms a haplotype which will be inherited en bloc, giving four
nomenclature DR5 can now be split by DNA analysis into two new possible combinations of paternal and maternal chromosomes.Par-
specificities, DRll and DR12. This list includes the most common ent and offspring can only be identical (1:2 chance)if the mother and
alleles but is not complete! Since there are several possible alleles father have one haplotype in common.
available organs can be increased through the devel-
The value of matching tissue types
opment of long-term tissue storage banks, but tech-
Improvements in operative techniques and the use of niques are not good enough for this at present, except
drugs such as cyclosporin A have greatly diminished in the case of bone marrow cells which can be kept
the effects of mismatching HLA specificities on solid viable even after freezing and thawing. With a paired
graft survival but, nevertheless, most transplanters organ such as the kidney, living donors may be used;
favor a reasonable degree of matching (see figure siblings provide the best chance of a good match (cf.
17.17).The consensus is that matching at the DR loci is figure 17.9).However, the use of living donors poses
of greater benefit than at the B loci, which in turn are of difficult ethicalproblems and there has been encourag-
more relevance to graft survival than the A loci. In ing progress in the use of cadaver material.There is ac-
addition, the need for cross-matching to detect pre- tive interest in the possibility of using animal organs
sensitized recipients is now taken very seriously.Bone (see below) or mechanical substitutes, while some are
marrow grafts, however, require a high degree of com- even trying to prevent the disease in the first place!
patibility and the greater accuracy of DNA typing
methods can be most helpful in this respect.
Because of the many thousands of different HLA
phenotypes possible (figure 17.9), it is usual to work Graft rejection can be held at bay by the use of agents
with a large pool of potential recipients on a continen- which nonspecifically interfere with the induction or
tal basis (Eurotransplant),so that when graft material expression of the immune response (figure 17.10).
becomes available the best possible match can be Because these agents are nonspecific, patients on
made. The position will be improved when the pool of immunosuppressive therapy tend to be susceptible
Figure 17.10. Immunosuppressive agents used to control graft re- understood manner. Leflunomide effectively blocks T-dependent
jection. The new name for FK506 is tacrolimus. Mycophenolic acid, and -independent antibody synthesis in viva by inhibiting dihy-
a purine analog produced by metabolism of mycophenolate mofetil, droorotatedehydrogenase,an enzymerequired for de novo synthesis
is a powerful new immunosuppressant undergoing early trials of uridine 5'-PO4. Simultaneous treatment with agents acting at
which inhibitsproliferation but also suppressesexpression of CD25, sequential stages in development of the rejection response would
-71, -154 (CD40L) and CD28. Another potent drug is deoxysper- be expected to lead to strong synergy and this is clearly seen with
gualin which interferes with lymphocyte function in an, as yet, ill- cyclosporinAand rapamycin.
to infections; they are also more prone to develop which has a preferential effect on T-cell-mediated
lymphoreticular cancers, particularly those of viral reactions. It is broken down in the body first to 6-
etiology. mercaptopurineand then converted to the active agent,
the ribotide. Because of the similarity in shape, this
competes with inosinic acid for enzymes concerned
Targeting Iymphoid populations
with the synthesisof guanylic and adenylic acids; it also
Anti-CD3 monoclonals are in widespread use as anti- inhibitsthe synthesisof 5-phosphoribosylamine,a pre-
T-cell reagents to reverse acute graft rejection. The cursor of inosinic acid, by a feedback mechanism. The
potential therapeutic benefits though have been net result is inhibition of nucleic acid synthesis.Anoth-
constrained by their immunogenicity and their er drug, methotrexate, through its action as a folic acid
propensity to activate a severe cytokine release syn- antagonist also inhibits synthesis of nucleic acid. The
drome involving complex 'flu-like' symptoms. These N-mustard derivative cyclophosphamide probably
problems can be circumvented by 'humanizing' the attacks DNA by alkylation and cross-linking, so pre-
antibody (see p. 123) and by mutating position 297 to venting correct duplication during cell division. These
prevent glycosylation and consequently binding to Fc agents appear to exert their damaging effects on cells
receptors and to complement. An IgM monoclonal to during mitosis and, for this reason, are most powerful
conserved regions on the TCR now available seems to when administered after presentation of antigen at a
provoke fewer untoward reactions than conventional time when the antigen-sensitivecellsare dividing.
anti-CD3. An exciting group of fungal metabolites is having a
The It-2 receptor a chain (CD25),expressed by acti- dramatic effect in human transplantation and in the
vated but not resting T-cells, represents another ex- therapy of immunological disorders through its ability
ploitable target. A humanized version of a murine to target T-cells. Cyclosporin A (CsA), a neutral hy-
monoclonal anti-IL-2Ra, dubbed daclizumab (a name drophobic cyclical peptide containing 11amino acids
seemingly derived from a Harry Potter story), had which is extremely insoluble, selectively blocks the
a longer half-life and of course reduced immuno- transcription of IL-2 in activated T-cells. Resting cells
genicity. When used to supplement standard base- which carry the vital memory for immunity to micro-
line immunosuppression consisting of cyclosporin bial infections are spared and there is little toxicity for
plus corticosteroids, acute rejection episodes at 6 dividing cells in gut and bone marrow. Some studies
months in recipients of cadaveric kidney grafts oc- also point to an 'exquisite' sensitivity of dendritic
curred in only 28% as compared with 47% in the place- APCs to the drug. Another T-cell-specificimmunosup-
bo controls. The results were sufficiently encouraging pressive drug, FK506, isolated from a species of Strep-
for the FDA to provide market clearance for the use tomyces, also blocks cytokine production. The latest
of daclizumab in the prevention of acute kidney addition to the stable is rapamycin, a product of
transplant rejection. Following the relative ease of the fungus Streptomyces hygroscopicus, which is a
engraftment of allogeneic bone marrow in patients macrolide like FK506, but in contrast acts to block sig-
with a deficiency of the 'adhesin' molecule LFA-1 (see nals induced by combination of IL-2 with its receptor.
p. 306), attention is now turning to the use of anti-LFA- We now have greater insight into the mode of action
1as an immunosuppressant for such grafts. of these drugs (figure 17.11).Both CsAand FK506 com-
plex with different specific binding proteins termed
immunophilins (cyclophilin and FK-binding protein
Immunosuppressive drugs
respectively), which for obscure reasons have pep-
The development of an immunological response tidyl-prolyl isomerase activity; these complexes then
requires the active proliferation of a relatively small interact with and inhibit the calcium- and calmodulin-
number of antigen-sensitive lymphocytes to give dependent phosphatase, calcineurin A, which is re-
a population of sensitized cells large enough to be sponsible for producing the transcription factors for
effective. Many of the immunosuppressive drugs IL-2, apoptosis and exocytosis in activated T-cells.
now employed were first used in cancer chemothera- Transcriptionof IL-10 is spared suggestingthat switch-
py because of their toxicity to dividing cells. Aside ing from Thl to Th2 responses may be a consequence.
from the complications of blanket immunosuppres- Although rapamycin also binds to the FK-binding pro-
sion mentioned above, these antimitotic drugs are es- tein, the complex has a quite differentbiological activi-
pecially toxic for cells of the bone marrow and small ty in that it blocks the IL-2-induced activation of the
intestine and must therefore be used with great care. p70 S6 kinase which phosphorylates ribosomal S6
A commonly used drug in this field is azathioprine prior to cell proliferation.
Figure 17.11. The mode of action of cyclosporin, FK506 and ra- chains to form a patch which can bind calcineurin, rather like
pamycin. The complexes of CsA with cyclophilinand of FK506 with double-sided tape. Aminodextran derivatized with approximately
FKBP12 (one of a family of FK506-bindingproteins) bind to and in- 10 molecules of CsA agglutinated T-cells and, although it cannot
activate the phosphatase calcineurin responsible for activating nu- penetrate the lymphocytes, it inhibited phorbol ester-induced IL-2
clear factor of activated T-cells (NFAT)(and possibly OAP and Oct-l; synthesis, suggesting a reaction with cyclophilin on the cell mem-
cf. p. 168),whichis a transcription factor for IL-2 synthesis. Transfec- brane rather than in the cytoplasm. The rapamycin-FKBP12
tion with calcineurin decreases the inhibitory powers of CsA and complex blocks the activation of p70 S6 kinase by transduced IL-2
FK506. On binding to cyclophilin, CsA undergoes an awesome con- signals, thereby inhibiting cell growth.
formational change enabling it to exteriorize hydrophobic side-
Cyclosporin now has a proven place as first-line doses below those causing renal fibrosis due to stimu-
therapy in the prophylaxis and treatment of transplant lation of TGFP production by several cell types. There
rejection. Figure 17.12 gives an example of its use in is also some cause for concern that cyclosporin may
kidney transplantation, but it has also been evaluated make patients susceptible to EB virus-induced lym-
in a wide range of disorders where T-cell-mediated phomas since the drug inhibits T-cells which control
hypersensitivity reactions are suspected. Indeed, the EB virus transformationof B-cells in vifro;however, the
benefits of cyclosporin in diseases such as idiopathic latest results suggest that the incidence of lymphoma
nephrotic syndrome, type 1 insulin-dependent dia- is relatively low in comparison with that reported
betes, Beh~et's syndrome, active Crohn's disease, for allografted patients on conventional immuno-
aplastic anemia, severe corticosteroid-dependent suppressive therapy. Although not itself carcinogenic,
asthma and psoriasis have been interpreted to suggest cyclosporin can promote tumor progression, probably
or confirm a pathogenic role for the immune system. through its effect on TGFP.
However, effects not only on Langerhans' dendritic FK506 is greatly superior to CsA on a molar basis in
cells but also on the proliferation of normal and trans- vifrobut is not substantially more effective when used
formed keratinocytes in vifromay contribute to the fa- in kidney grafting; however, its tropism for liver could
vorable outcome in psoriasis. A rapid onset of benefit be exploited.Because they act at different stages in the
and relapse when treatment is stopped are common activation of the T-cell, CsA and rapamycin show a
features of cyclosporin therapy. most impressive degree of synergy which allows the
There are, of course, side-effects. It has to be used at two drugs to be used at considerably lower dose levels
tension by growth-associated protein-43, leading to
the propositions that FK506 could be clinically em-
ployed in stroke patients and in the treatment of nerve
degeneration. A close association of FKBP12 with the
surface receptor for transforming growth factor-p
(TGFP) has also been revealed. With good fortune
these drugs will take us down some very unexpected
and intriguing pathways.
Figure 17.12. Actuarial survival of primary cadaveric kidney If the disadvantages of blanket immunosuppression
grafts in 877 patients treated at the Oxford Transplant Centre with are to be avoided, we must aim at knocking out only
triple therapy. Cyclosporin: l0 mg/kg/day orally given as a single
dose (first dose given before surgery) and reduced according to the reactivity of the host to the antigens of the graft,
whole-blood trough levels (200400ng/ml in first 3 months and leaving the remainder of the immunological appara-
then maintained at 100-200ng/ml). Azathioprine: 100mg/day tus intact -in other words, the induction of antigen-
orally; reduced if leukocyte count < 5000. Prednisolone: 20 mg/day specific tolerance.
given in divided doses orally and reduced to a maintenance dose of
10mg/day by 6 months. The initial dose is reduced to 15mg/day in It turns out that bone marrow represents a privi-
patients of less than 60 kg body weight. In patients with stable renal leged source of tolerogenic alloantigens, and the
function at 1 year, prednisolone is discontinued over a period of production of stable lymphohematopoietic mixed
some months. (Data kindly provided by Professor Peter J. Morris.) chimerism by bone marrow engraftment is proving to
be a potent means of inducing robust specific trans-
plantation tolerance to solid organs across major MHC
with correspondingly less likelihood of side-effects mismatches. However, successful allogeneic bone
(figure 17.10).Another possible synergistic partner for marrow transplantation in immunocompetent adults
cyclosporin is fludarabine which, unlike cyclosporin, normally requires cytoablative treatment of recipients
blocks signaling by STAT-1, an intracellular intermedi- with irradiation or cytotoxic drugs and this has tended
ate activated by interferons and important for cell- to restrict its use to malignant conditions. A most en-
mediated immunity. Combination therapies may also couraging recent study has now shown the feasibility
include azodicarbonamide which blocks the calcium of inducing long-lasting tolerance not only to bone
flux associated with CD3 activation and the drugs marrow cells but also to fully MHC-mismatched skin
mycophenolic acid and leflunomide which limit the grafts in naive recipients receiving high-dose bone
availability of DNA precursors. marrow transplantation and costimulatory blockade
Steroids such as prednisolone intervene at many by single injections of monoclonal anti-CD154
points in the immune response, affecting lymphocyte (CD40L) plus a CTLA-4-Ig fusion protein (figure
recirculation and the generation of cytotoxic effector 17.13).A persistent hematopoietic macrochimerism is
cells, for example; in addition, their outstanding anti- achieved with a significant proportion of donor-type
inflammatory potency rests on features such as inhibi- lymphocytes in the thymus indicating intrathymic
tion of neutrophil adherence to vascular endothelium deletion of donor-reactive T-cells.
in an inflammatory area and suppression of mono- While this protocol permits long-term engraftment
cyte/macrophage functions such as microbicidal ac- of bone marrow and solid organs, it seems that direct
tivity and response to cytokines. Corticosteroids form blockade with just anti-CD154 and CTLA-4-Ig is suffi-
complexes with intracellular receptors which then cient to induce tolerance to solid organ grafts. Stimula-
bind to regulatory genes and block transcription of tion of alloreactiveT-cellsby the graft in the presence of
TNF, IFNy, IL-1, -2, -3, -6 and MHC class 11, i.e. they costimulatory blockade leads to apoptosis, a process
block expression of cytokines from both lymphocytes promoted by rapamycin which improves the tolerant
and macrophages, whereas cyclosporin has its main state. Bcl-XL(cf.figure 11.5,p. 203) prevents both T-cell
action on the former. apoptosis and tolerance induction by this treatment re-
In parentheses, the immunophilins may be involved vealing the importance of apoptotic T-cell deletion for
in other cellular functions, such as the regulation of the the establishment of antigen-specific unresponsive-
N-methyl-D-aspartatesubtype of neural glutamate re- ness. In a further twist to the tale, the apoptotic T-cells
ceptors and the augmentation of neuronal process ex- 'reach from beyond the grave' by producing IL-10, so
Figure 17.13. Induction of tolerance and macrochimerismby fully
allogeneic bone marrow transplantation plus costimulatory
blockade. B6 mice received bone marrow cells from the fully allo-
geneic BIO.A strain with injections of anti-CD154 (CD40L) and the Figure 17.14. Induction of allograft tolerance by nondepleting
CTLA-4-Ig fusion protein which blocks B7-CD28 interactions anti-CD4 plus anti-CDS. Tolerance to skin grafts from donors with
(CTLAis a T-cell receptor for B7 which downregulates T-cell activa- multiple minor transplantation antigen mismatches was achieved
tion; cf. pp. 165 and 170).Eight mice showing long-term persistence by concurrent injection of IgG2a monoclonal antibodies to CD4 and
of multilineage donor cells (macrochimerism)were fully tolerant to CD8 which do not induce cell depletion (green arrow). The mainte-
BIO.A skin grafts. Five mice with transient chimerism showed mod- nance of tolerance depends upon the continued presence of antigen
erate prolongation of skin graft survival relative to unrelated 3rd which enables the unresponsive cells to interact with newly arising
party grafts. (Data taken from Wekerle T. et al. (2000)NatureMedicine immunocompetent cells on the surface of the same antigen-present-
6,464, with permission.) ing cells and render them unresponsive through an infectious toler-
ance mechanism (cf.figures 12.9 and 20.24).Whether the mechanism
involves 'infectious anergy', direct suppression, 'immune devia-
that their phagocytosis along with antigen leads to the tion' of potentially aggressive Thl cells by suppressor Th2, down-
presentation of the antigen in a tolerogenic form which regulation of the antigen-presenting cell, or all four in various
maintains tolerance through the production of im- combinations, is unresolved. Loss of tolerance on depletion of CD4
munoregulatory cells. but not CD8 cells (red arrows) shows that active tolerance is main-
tained by the CD4 subset. (Figure synthesized from data kindly
Despite the role of the mature dendritic cell as the
provided by Dr S.P. Cobbold and Professor H. Waldmann.)
champion stimulator of resting T-cells, the dendritic
cell precursors may present antigen in the absence of B7
costimulators and, by mechanisms echoing those de- alloreactive T-cells by administration of tolerogenic
scribed above in the costimulatory blockade experi- peptides represents quite a challenge, and the strategy
ments, would appear to have a powerful potential of using costimulatory blockade with the antigens
for tolerance induction. This concept is of particular being provided by the graft itself looks to be a more
relevance to the specific unresponsiveness generated promising route.
by grafts of liver, which being a hematopoietic organ,
continually exports large numbers of these immature
I S XENOGRAFTING A PRACTICAL
dendritic cells.
PROPOSITION?
Nondepleting anti-CD4 and -8 monoclonals, by de-
priving T-cells of fully activating signals, can render Because the supply of donor human organs for trans-
them anergic when they engage antigen through their plantation lags seriously behind the demand, a wide-
specific receptors. These anergic cells can induce unre- spread interest in the feasibilityof using animal organs
sponsiveness in newly recruited T-cells ('infectious is emerging. Pigs are more favored than primates as
tolerance', p. 233) and so establish specific and indefi- donors both on the grounds of ethical acceptability
nite acceptance of mouse skin grafts across class I or and the hazards of zoonoses. The first hurdle to be
multiple minor transplantation antigen barriers (fig- overcome is hyperacute rejection due to xenoreactive
ure 17.14). It should be noted that skin allografts natural antibodies in the host. Humans lack a-1,3-
provide the most difficult challenge for tolerance in- galactosyl transferase and galactose cc-1,3-galactose
duction, and transplants of organs such as the heart, and therefore develop antibodiesto this epitope which
which are less fastidiousthan skin, require less aggres- is present on many common bacteria and expressed
sive immunotherapy. abundantly on the xenogeneic vascular endothelium.
Given the wide variety of different peptide epitopes The natural antibodies bind to the endothelium and
presented by the graft MHC, full-frontal attack on the activate complement in the absence of regulators of the
Figure 17.15. Strategies for avoiding complement-mediated hy- pig cells with a-1,2-fucosyl transferase converted the terminal sug-
peracute rejection of a xenograft caused by reaction of natural anti- ars into the blood group H and rendered the cells resistant to lysis by
galactose antibodies with Gala-1,3-Gal on the surface of the pig graft the antigalactose. Other strategies involve transfection with genes
cells. Heart xenografts from transgenic pigs expressing the human encoding an a-galactosidase or intracellular recombinant scFv react-
complement regulatory proteins decay accelerating factor (DAF) or ing with a-1,3-galactosyltransferase.
CD59 functioned for prolonged periods in baboons. Transfectionof
human complement system, such as decay accelerat- gans, but the most exciting development has come
ing factor, CD59 and MCP (cf. figure 15.2),precipitat- from cloning 'Dolly' the sheep. This has revealed the
ing the hyperacute rejection phenomenon. Novel feasibility of taking the nucleus from a differentiated
genetic engineering strategies for the solution of this adult cell and reprograming it into an embryonic state
problem are outlined in figure 17.15. responsive to early growth factors,which can be select-
The next crisis is acute vascular rejection occurring ed to determine its future destiny as an islet, brain or
within 6 days as antibodiesproduced by Th2 cells react liver cell or what have you (figure 17.16).
with the xenoantigens on donor epithelium. In con-
trast with the response to allografts, IL-12 and IFNyac-
CLINICAL EXPERIENCE I N GRAFTING
tually inhibit acute vascular rejection and, over the
long term, IFNymay protect the graftby promoting the
formation of NO-which prevents constriction of blood
vessels. Brequinar sodium (figure 17.10), an inhibitor Cornea1 grafts survive without the need for immuno-
of pyrimidine biosynthesis and suppressor of both B- suppression. Because they are avascular they tend not
and T-cell-mediated responses, has been evaluated for to sensitize the recipient. This privileged protection is
efficacy, but induction of tolerance would clearly be boosted by the local production of immunosuppres-
more desirable although tricky. sive factors such as TGFP, IL-lRa,limited expression of
Even when the immunological problems are over- MHC and the strategic presence of FasL which can in-
come, it remains to be seen whether the xenograft will duce apoptosis in infiltrating lymphocytes. Nonethe-
be compatible with human life over a prolonged peri- less, they do become cloudy if the individual has been
od, latent pig viruses being an obvious worry. presensitized. Grafts of cartilage are successful in the
same way but an additional factor is the protection af-
forded the chondrocytes by the matrix. With bone and
TISSUE ENGINEERING
artery it doesn't really matter if the grafts die because
The ideal would be to create the tissue for a graft en- they can still provide a framework for host cells to
tirely from cells of the recipient, i.e. an autograft, colonize.
which would eliminate the need for immunosuppres-
sion. Science fiction? Probably not. We will steadily
accumulate the knowledge concerning the various
growth factors required to guide relatively undifferen- Thousands of kidneys have been transplanted
tiated stem cells into the desired mature form under and with improvement in patient management there
culture conditionsin vitro. In many instances, it is prov- is a high survival rate. In the long term (5 years
ing possible to isolate stem cells from various adult or- or more), the desirability of reasonable matching at
Figure 17.16. Anticipated production of
autologous grafts by tissue engineering.
Undifferentiated cells are obtained directly
from the patient either as stem cells (if
available) or by transplanting the nuclei of
mature cells into enucleated embryonic
cells. They are cultured in a biodegradable
matrix with appropriate growth factors to
provide a tissue populated with
differentiated cells which can function as
an autologousgraft.
suppressant such as TGFP, which can be produced in by the donor-type intrahepatic hematopoietic stem
high concentrations within the milieu of a cardiac graft cells and immature dendritic cells (seeabove) and pos-
without invoking systemic side-effects. sibly also by the liver parenchyma itself, known to pro-
duce copious amounts of soluble MHC class I.
Work is in progress on the transfer of isolated hepa-
tocytes attached to collagen-coated microcarriers in-
Survival rates for orthotopic liver grafts are not quite jected i.p. for the correction of isolated deficiencies
as high as those achieved with heart transplants (figure such as albumin synthesis. This attractive approach
17.18). The hepatotrophic capacity of FK506 is an has much wider application as a general vehicle for
added bonus which makes it the preferred drug for gene therapy.
liver transplantation. Rejection crises are dealt with by
high-dose steroids and, if this proves ineffective, anti-
lymphocyte globulin. The use of a totally synthetic
colloidal hydroxyethyl starch solution, containing lac- Patients with certain immunodeficiencydisorders and
tobionate as a substitute for chloride, allows livers to aplastic anemia are obvious candidates for treatment
be preserved for 24 hours or more and has revolution- with bone marrow stem cells with their potential to
ized the logistics of liver transplantation. To improve differentiate into all the formed elements of the blood;
the prognosis of patients with primary hepatic or bile so, too, are acute leukemia patients treated radically
duct malignancies, which were considered to be inop- with intensive chemotherapy and possibly whole-
erable, transplantation of organ clusters with liver as body irradiation in attempts to eradicate the neoplastic
the central organ has been designed, e.g. liver and pan- cells, as will be discussed in the next chapter.
creas, or liver, pancreas, stomach and small bowel or Bone marrow contains not only hematopoietic but
even colon. Nonetheless, the outcome is not very also mesenchymal stem cells which can give rise to car-
favorable in that up to three-quarters of the patients tilage, tendons and bone; after expansion in culture by
transplanted for hepatic cancer have recurrence of a factor of 5-10 times, they provide an excellent treat-
their tumor within 1year. For the future we must look ment for children with osteogenesis imperfecta, a
forward to the creation of autologous liver from adult genetic disorder in which the osteoblasts produce de-
cells when tissue engineering techniques have been fective type I collagen with resulting osteopenia and
developed sufficiently. severe bony deformities. Good results are being ob-
Experience with liver grafting between pigs re- tained with stem cell transplantation in utero for inher-
vealed an unexpected finding.Many of the animals re- ited blood disorders using populations from paternal
tained the grafted organs in a healthy state for many bone marrow enriched for the stem cell marker, CD34.
months without any form of immunosuppression and From the practical standpoint, it has been recognized
enjoyed a state of unresponsivenessto grafts of skin or that cord blood contains sufficient hematopoietic stem
kidney from the same donor. True tolerance is induced cells for bone marrow replacement, but what is even
more convenient is to enhance the number of CD34- sclerosis, the outcome is poor. Patients are treated with
positive progenitor cells in relatively small volumes of cyclosporin and prednisolone, but recently thalido-
peripheral blood by recombinant stem cell factor, IL- mide has been found to be very helpful because of its
1p, IL-3, IL-6 and erythropoietinin the presence of stro- anti-TNF effect. The pathogenesis is not straightfor-
mal cells or fibronectin monolayers ex vivo prior to ward and chronic disease could arise by a curious
transplantation. It is encouraging also that both mechanism involving the sneaking through of autore-
colony-stimulating factors G-CSF and GM-CSF great- active T-cells, which fail to be deleted in the thymus
ly accelerate the return of myeloid cells after lethal possibly because of thymic damage due to pre- or post-
doses of myelotoxic agents preceding autologousbone transplant therapy, e.g. associated viral infection, irra-
marrow transplantation; there are fewer infections, diation or even cyclosporin itself. It may be significant
less use of i.v. antibodies and earlier discharge from that cyclosporin inhibits the programed cell death of
hospital. They also accelerate the engraftment of immature thymocytes which occurs on activation by
allogeneic bone marrow without exacerbating g.v.h. anti-CD3,and it is known that an autologous 'g.v.h.' re-
disease. action supervenes on termination of prolonged cy-
closporin administration to young irradiated rats
which had received syngeneicbone marrow.
Graft-vs-host disease is a major problem in
bone marrow grafting
G.v.h. disease resulting from the recognition of recipi-
ent antigens by allogeneic T-cells in the bone marrow It is to be expected that improvement in techniques of
inoculum represents a serious, sometimes fatal, com- control of the rejection process will encourage trans-
plication, and the incidence of g.v.h. disease is reduced plantation in several other areas-not in cases of en-
if T-cells in the grafted marrow are first depleted with a docrine disorders such as Hashimoto's disease (cf. p.
cytotoxic cocktail of anti-T-cell monoclonals. Unex- 397) where exogenous replacement therapy is conve-
pectedly, complete purging of the T-cells leads to an nient, but, for example, in diabetes where the number
alarmingly high incidence of graft failure, apparently of transplants recorded is rising rapidly and the cur-
due to the loss of relatively rare facilitating cells rent success rate is around 4O0/0,although major prob-
expressing TCR, CD3, CD8 and a previously unknown lems still are inadequate output of insulin by the
33 kDa cell surface glycoprotein, FCp33. How these grafted islets and an adequate supply of tissue. Con-
and other facilitating cells, such as CD8+ dendritic siderable advances have been made in very substan-
cells, work is, to put it mildly, unknown. tially expanding the number of islet cells which can be
It is fondly hoped that successful engraftment and derived from pancreatic duct precursors, while mon-
avoidance of g.v.h. reactions will be achieved in the key islets may not prove to be a bad bet in that they
clinic -in the not too distant future(?)-by strategies have low surface MHC and are nonvascularized, so
such as those involving allogeneic marrow transplan- avoiding the presentation of the dangerous Gal.Ga1
tation under costimulatory blockade without cytoab- epitope (cf. p. 361) which is enriched on vascular en-
lative treatment of graft or recipient, as described dothelium in some marked species.In vivo transfection
earlier (figure 17.13).Until then, successful results are of hepatocytes with an adenoviral vector encoding the
more likely with highly compatible donors, particular- homeoprotein PDX-1, which is a glucose-dependent
ly if fatal g.v.h. reactions are to be avoided, and here transactivator of the insulin gene, induced them to
siblings offer the best chance of finding a matched make insulin in diabetic mice -very impressive, lots of
donor (figure 17.9). Undoubtedly, non-HLA minor potential, but early days yet. The 5-year survival rate of
transplantation antigens are important and are more 47% for lung transplants is improving but is still less
difficult to match. Acute g.v.h. disease occurring than satisfactory. One also looks forward to the day
within the first 100 days following infusion of allo- when the successful transplantation of skin for lethal
geneicmarrow primarily affectsthe skin, liver and gas- burns becomes more commonplace.
trointestinal tract. Antibodies to TNF or IL-1R block Reports are coming in of an experimentalforay into
mortality. Current therapy uses cyclosporinwith pred- the graftingof neural tissues.Mutant mice with degen-
nisolone, but inclusion of methotrexate in this regimen erate cerebellar Purkinje cells, which mimic the human
is said to improve efficacy. Chronic g.v.h. disease (i.e. condition cerebellar ataxia, can be restored by engraft-
later than 100 days) has a relatively good prognosis if ment of donor cerebellar cells at the appropriate sites;
limited to skin and liver, but if multiple organs are these express insulin-like growth factor-l (IGF-l),mi-
involved, clinically resembling progressive systemic grate to form a layer in lieu of the missing cells, induce
sprouting in host neurons and become synaptically in-
tegrated. Clinical trials with transplantation of human
ASSOCIATION OF HLA TYPE WITH DISEASE
embryonic dopamine neurons to reverse the neurolog-
ical deficit in Parkinson's disease have been severely
hampered by the excessive death of the grafted cells.
The hypothesis that this was due to oxidative stressled An impressive body of data is accumulating which
to a study showing that grafted neurons from trans- links specificHLAs with particular disease statesin the
genic mice overexpressing Cu/Zn superoxide dismu- human (table 17.1) and even more striking relation-
tase (cf. p. 6) had a greatly increased survival rate. ships may be uncovered as the complexityof the HLA-
Another candidate is Huntington's chorea which has D region is unraveled. The relationships are influenced
been tracked down to a mutation producing a protein by linkage disequilibrium, a state where closely
with an expanded polymorphic glutamine repeat, linked genes on a chromosome tend to remain associ-
toxic to spiny striatal neurons. Embryonic striatal allo- ated rather than undergo genetic randomization in a
grafts, producing the nontoxic wild-type protein, sur- given population, so that the frequency of a pair of alle-
vive for a prolonged period,become integrated into the les occurring together is greater than the product of the
host brain and induce functional alleviation of motor individual gene frequencies (figure 17.19a).This could
deficits, all in the absence of immunosuppression. To result from natural selection favoring a particular hap-
date, the need to use embryonic tissue has been a bug- lotype or from insufficient time elapsing since the first
bear, but the recent identification of neurogenic precur- appearance of closely located alleles to allow them to
sor cells from the adult human hippocampus may give
a glimpse of the Promised Land.
Cryopreservation of sperm is a successfulstrategy
Table 17.1. Association of HLA with disease. (Data mainly from
in the management of adult cancer sufferers to protect Ryder et al., see legend to figure 17.19, and Thorsby E. (1995) The
the sperm from mutagenic cancer treatment. This is Immunologist 3,51.)
not available to prepubertal boys, but an alternativefor
them is cryopreservationof their spermatogonialstem
cells for reintroduction post-treatment, since the Ser-
toli cells which support differentiation into mature
spermatozoa will function normally. As knowledge
from the human genome sequence accumulates, there
is a potential for identifying and correcting genetic
defects in the spermatogonia before their reintrodu-
ction, but ethical committees fight shy of this sort of
'Frankenstein' tinkering. More acceptably, in cases of
male infertility due to dysfunctional Sertoli cells, it
should be possible to develop mature spermatids by
culture of the spermatogoniawith Sertoli cells derived
from a normal individual.
Work proceeds apace to produce a successful pros-
thetic vascular graft which would remain patent as a
blood conduit, prevent thrombosis of the blood in low
flow or adverse states and control anastomotic cellular
hyperplasia with overgrowth of smooth muscle cells
and matrix production. This has proved difficult to
achieve, but there are encouraging attempts to engi-
neer a composite vascular graft in which microvessel
endothelial cells present in a small sample of subcuta-
neous abdominal wall fat, obtained by the relatively
simple cosmetic liposuction technique, grow as an
antithrombotic endothelial layer to line the surface of
an expanded polytetrafluoroethylene tube. Expect
development in this field, particularly for the replace-
ment of arteries. *DR specificities relate to 'old nomenclature', figure 17.9.
DQBl"0602. However, in those instances where two
susceptibilitygenes encoding a and P chains synergize
and are in the tram configuration (i.e.on different chro-
mosomes), linkage disequilibrium is less likely to be
the explanation.
Ethnic studies may help by making availablerecom-
bination events which alter haplotypes and permit the
identification of susceptibility determinants outside
their normal context. Thus, the DQa (DQA1*0201)
linked to DR7 on the Caucasian haplotype has a
neutral effect on diabetes susceptibility but, in the
black population, DR7 is associated with a high-
risk DQa (DQAl"0301) and now becomes a suscepti-
bility haplotype.
Table 17.2. Shared sequences on the DRPl a-helix in different haplotypesconfer susceptibilityto rheumatoidarthritis.(Data from Bell J.I. &
McMichael A.J. (1993)In Lachmann P.J., Peters D.K., Rosen F.S. & Walport M.J. (eds) Clinical Aspects of immunology, p. 748. Blackwell Scientific
Publications, Oxford.)
Last, it is worthy of note that the relationship of
MHC to disease resistance and vaccine efficacyin farm
animals is beginning to preoccupy veterinary scien-
tists; it is known, for instance, that susceptibility to
Marek's disease in White Leghorn chickens is associat-
ed with distinctMHC haplotypes.
REPRODUCTIVE I M M U N O L O G Y
Figure 17.21. Immunological contraception. (a) Target hormones. this should avoid the problem of inducing antibodiescross-reacting
(b) Purely diagrammatic representation of human chorionic go- with LH. (d) Structure of P-hCG showing some of the residues al-
nadotropin (hCG) showing epitopes specific for hCG (green) and tered (magenta, yellow and cyan) to produce epitope-loss mutants,
structures similar if not identical to luteinizinghormone (LH) (dark fulfilling the criteria in (c). (Reproduced from Jackson A.M. et al.
red). (c) P-hCG mutant in which the hCG-specificepitopeshave been (1996) Journal of Reproductive Immunology 31, 21, with permission
preserved but epitopes shared with LH lost.As a candidate vaccine from Elsevier Science, Ireland Ltd.)
Most attention has focused on a human vaccine exciting as an immunogen. The Indian vaccine com-
based on human chorionic gonadotropin (hCG) bines P-hCG with ovine a chain. This evokes much
which is made by the preimplantationblastocyst and is better antibody responses, and in Phase 2 clinical trials
essential for the establishment of early pregnancy (fig- only one pregnancy was observed in 1224cycles when
ure 17.21a);it is also the antigen assayed in the urine neutralizing antibodieswere above a certain level. De-
tests for pregnancy which cause so much pleasure or spite strong cross-reactionswith LH there was said to
consternationas the case may be. The a chain of hCG is be no disturbance of ovulation or loss of libido. As
common to follicle-stimulating hormone (FSH), thy- contraceptive cover while antibodies were rising,
roid-stimulating hormone (TSH) and LH while the P- cell-mediated hypersensitivity was induced by local
subunit shows around 80% homology with LH (figure intrauterine implantation of purified extracts from
17.21b).Clearly immunizationwith whole hCG would Neem, an ancient Indian tree. As antibody levels
elicit some exceedingly unwelcome reactions and two fall, fertility is regained. Long-term maintenance of
vaccines try to avoid them wholly or in part. Both cou- adequate antibody titers should be possible with
ple the antigen to tetanus or diphtheria toxoid carriers. biodegradable microspheres or a recombinant Salmo-
The WHO vaccine uses the 37-amino acid C-terminal nella construct incorporating the PhCG gene.
peptide (CTP)which is wholly specificfor hCG but not
Gm releafon Is an Immunologlml readon allc-MHC plus self-peptides plus a small number which
It showsspecificity, thesecond set responseisbrisk, it is directly recognize the allo-MHC molecule itself (the
mediated by lymphocytes and antibodies s p e a k for the direct pathway); later rejection inaeasingly involves
graft are formed. allogeneic peptides presented by self-MHC (the indirect
pathway).
~camoldnonrplonhmon~ Acute late rejection of organ grafts from 11 days on-
In each vertebrate speciesthem is a majorhistcampati- wards is caused by Ig and C bindiig to graft vessels.
bility complex (MHC) which is responsible for provoking Insidious and late rejection may be due to immune
themost intense graft reactions. complex deposition or breakthrough against
Parental MHCantigensarecodominantlysrpressedon immunosuppression.
cell surfaces.
Pmwlln011o t g m f t m ~ ~ o n
o f h r ~ d M mImmpHbllHy: This can be m i n i m i i by ems-mtching donor and
Class U MHC molecules provoke a mixed lymphocyte graft for AB0 and MHC tissue types. Individual MHC
reaction of proliferation and blast transformation when antigens are typed by the PCR using discriminating
genetically dissiilar lymphocytesinteract primer pairs.
Class U differences are largely responsible for the reac- Rejection can be blocked by agents producinggeneral
tionof tolerated grafted lymphocytesagainst hostantigen immunosuppression such as antimitotic drugs (e.g. am-
(graft-vs-host (g.v.h.) reaction). thioprine), anti-inflammatory steroids and antilymph*
Siblings have a 1:4 chance of identity with mspect to cyte monoclonals. Cyclosporin A, Fl<SO6and rapamycin
MHC. represent exciting groups of T-cellspecific drugs; com-
plexes of cyclosporin and R(506,with their cellular lig-
Medum~otgmR~on ands, blockcalcineurin,a phosphatasewhichactivatesthe
Preformed antibodies cause hyperacute rejection with- IL-2 tansaiption factor NFAT, while rapamycin com-
inminutes. plexes with the FK-binding pratein to block kinases in-
CMllymphocytesplayamajorroleintheacuteearlyre- volved in cell proliferation.
jection of first set nsponses. Antigen-specific depression through tolerance induc-
The sbength of allograft rejection is d w to the surpris- tioncan beachiwed withinjectionof allogeneicbonemar-
ingly large number of allospecific prervsor cells. These row with costimulatory blockade by anti-CM54 (CWOL)
derive mainly from the variety of T-cells which recognize plusa CI'LA-Plg fusionprotein. Dendriticcell precursors
(continuedp. 372)
can also induce tolerance through antigen presentation in with myasthenia gravis, DR17 with Sjogren's syndrome,
the absence of B7 costimulators. DR4 with rheumatoid arthritis, DQ2 and 8 with insulin-
dependent diabetesmellitus and DR2, DQ6 with multiple
Tissue engineering sclerosis.
A distinct goal is to create autograftsfrom stem cells or The associationmay be related to an abiity to b i d par-
embryonically reprogramed adult nuclei differentiating ticular antigenicpeptides or to cross-react with certain in-
in culture under the influence of specified growth factors. fectiousagents.
Mellor A.L. & Munn D.H. (2000)Immunology at the maternal-fetal Thorsby E. (1995) HLA-associated disease susceptibility The Immu-
interface:lessons for T-cell tolerance and suppression.Annual Re- nologisf 3/51.
view ofImmunoEogy 18,367. Transplantation (2001) Current Opinion in Immunolo~.(Critical re-
Morris P.J. (ed.) (2000) Kidney Transplantation:Principles and Practice, views of high quality appearing annually.)
5th edn. W.B.Saunders Company, Philadelphia. Vince G.S. & Johson P.M. (1996) Reproductive immunology:con-
Murphy W.J. & Blazar B.R. (1999) New strategies for preventing ception, contraception, and the consequences. TheImmunologist 4,
graft-versus-host disease. Current Opinion in Immunology 11, 172.
509. Wilson S.E. & Aston R. (1994) Overview: recent developments in
Pazmanty L., Mandelboim O., Vales-Gomes M. et al. (1996) Protec- macrolide imunosuppressants. Expert Opinion on Therapeutic
tion from natural killer cell-mediated lysis by HLA-G expression Patents 4, 1445. (Academics should be aware of the enormous
on target cells. Science 274,792. amount of data in the patent literature.)
Tumor immunology
inhDduction, 374 Diirent lymphoid malignanciesshow maturationarrest at
Chanpwon~ewlloceoftumorceils, 374 characteristic stages in differentiation, 382
Vimlly controlledantigens, 374 lrnmunohistologlcaldiagnosis of lymphoid neoplosios, 382
Expression of normally silent genes, 376 Plasma cell dyscrasias, 383
Mutant antigens, 377 lrnrnunodeficiency secondoiy to lymphoprolitemtive
Changes in carbohydratestructure, 378 disorders, 386
Chongss on the surface of cyclingcells, 378 Appmachesto m c e r Immunothempy, 386
Moleculesrelatedtometastaticpotential, 378 Antigen-independsntcytokine therapy, 386
Spontaneousimmune respmestotumors, 379 Exploitationof cell-mediated immune responses, 387
lmmunesurveillanceagainststrongly immunogenictumors, 379 Ttwopywiihrnonoclonalantibodies, 391
A mle for Innate imrnuniQ?, 3380 Immunodiagmkotsolidhlmon, 392
Unregulated developmentgives riseto lymphopmltlemtive Circulatingand cellular turnor markers, 392
disorders, 380 Tumor imaging inWJ,393
Deregulationof pmtooncogenes is a charactensticfeotureof many Detectionof mimmetostases in bone marrow, 393
Iymphocyticturnors, 38 1 Summory, 393
Chromosome translocuiionsore m r n o n in lymphopmliiemtive FwMer readfng, 395
disorders, 381
Figure M18.1.1. The specificity of immunity induced by with much lower numbers of hunor cells, a greater degree of
tumors. (a) A chemically induced tumor MCA-l can induce crossprotection between tumors may be observed, which has
resistance to an implant of itself but not to a tumor produced in a beenaxribed toa 44 kDa oncofetalantigen,possiblyanirnmature
syngeneicmousebythesamecaminogen.Thuseachhmorhasan version of a laminin receptor protein. @) Tumors produced by
individual antigen, now thought to be a processed mutant a given oncogenic virus immunize against tumors produced in
endogenousprotein complexed with a heat-shock protein. More syngeneic mice by the same but not other viruses. Thus hunors
recent data suggest that, if immunized animals are challenged produced by anoncogenicvi~sshareacommonantigen.
nism. Cytotoxic T-cells specific for tumor cells, ob-
tained from mixed cultures of peripheral blood cells
The dysregulated uncontrolled cell division of the with turnor, can be used to establish the identity of the
cancer cell creates a milieu in which the products of antigen employing the strategy described in figure
normally silent genes may be expressed. Sometimes 18.2, By something of a tour deforce a gene encoding a
these encode differentiation antigens normally asso- melanoma antigen, MAGE-1, was identified. It be-
ciated with an earlier fetal stage. Thus tumors derived longs to a family of 12genes, six of which are expressed
from the same cell type are often found to express such in a significant proportion of melanomas as well as
oncofetal antigens which are also present on embry-
onic cells. Examples would be a-fetoproteinin hepatic
carcinoma and carcinoembryonic antigen (CEA) in
cancer of the intestine. Certain monoclonal antibodies
also react with tumors of neural crest origin and fetal
melanocytes. Another monoclonal antibody defines
the SSEA-1 antigen found on a variety of human
tumors and early mouse embryos but absent from
adult cells with the exception of human granulocytes
and monocytes.
But the exciting quantum leap forward stems from
the original observation that cytosolicviral nucleopro-
tein could provide a target for Tc cells by appearing on
the cell surface as a processed peptide associated with
MHC class I (cf. p. 94). This established the general
principle that the intracellular proteins which are not
destined to be positioned in the surface plasma mem-
brane can still signal their presence to T-cells in the
outer world by the processed peptide-MHC mecha- Figure 18.1. Tumor-associatedsurface changes.
Figure 18.3. The role of heat-shock proteins (hsps) in tumor im- as stimulatory danger signals to dendritic antigen-presentingcells
munogenicity. (1) Stress factors upregulate hsps which can form and penetrate the cytoplasm, where (4)they can enter the MHC class
complexes with processed tumor antigen and increase surface pre- I processing pathway by so-called cross-priming. ( 5 ) CD8 resting
sentation of antigenic peptide by MHC class I. (2) They can also lead T-cells become activated and (6) kill the tumor cells. (Based on
to necrosis and release of hsp-peptide complexes, which (3)can act Wells A.D. & MalkowskyM. (2000)Immunology Today 21,129.)
individual Tc cells not reacting with other tumors or the colon, breast, lung and pancreas, making them a
normal cells and it would be surprising if this were not novel target for immunological attack since normal
a further example of turnor-specific mutant peptide adult tissues and benign epithelia1 tumors express
antigenicity. zero or very low levels. In like vein, the highly sialy-
lated cell surface glycoprotein endosialin (FEE) is
present in the newly generated vasculature of a signif-
icant proportion of malignant tumors but not in blood
The chaotic internal control of metabolism within neo- vessels of normal tissues.
plastic cells often leads to the presentation of abnormal
surface carbohydrate structures. Sometimes one sees
blocked synthesis, e.g. deletion of blood group A. In
other cases there may be enhanced synthesis of struc- Changes in surfacecarbohydrates can have a dramatic
tures absent in progenitor cells: thus some gastro- effect on malignancy. For example, colonic cancers
intestinal cancers express the Lewis Lea antigen in expressing sialyl Lexhave a poor prognosis and higher
individuals who are Le(am,b-)and others produce propensity to metastasize.Lung cancer patients whose
extended chainsbearing dimeric Leaor Le(a,b). tumors showed deletion of blood group A had a much
Abnormal much synthesis can have immunological worse prognosis than those with continuous A; the
consequences. Consider the mucins of pancreatic and finding that patients expressing H / L ~ Y / L ~ ~had
also
breast tissue. These consist of a polypeptide core of a poorer prognosis than antigen-negative subjects is
20-amino acid tandem repeats with truly abundant consistent with this observation.
0-linked carbohydrate chains.Amonoclonal antibody The role of CD44 (HERMES/Pgp-1) in cell traf-
SM-3 directed to the core polypeptide reacts poorly ficking, based on its interaction with vascular
with normal tissue where the epitope is masked by endothelium, has afforded it some prominence in
glycosylation, but well with breast and pancreatic the facilitation of metastatic spread. CD44 occurs in
carcinomas possessing shorter and fewer 0-linked several isoforms with a varying number of exons be-
chains. Tc cells specific for tumor mucins are not MHC tween the transmembrane and common N-terminus.
restricted and the slightly heretical suggestion has Normal epithelium expresses the CD44H isoform with
been made that the T-cell receptors (TCRs)are binding hyaluran-binding domains, but lacking the interven-
multivalently to closely spaced SM-3 epitopes on un- ing vl-v10 exons; expression of certain of these exons
processed mucins; alternatively, and closer to the party on tumors is indicative of a growth advantage, since
line, recognition is by y6 cells. they are present with higher frequency on more ad-
vanced cancers. Stable transfection of a nonmetastatic
turnor with a CD44 cDNA clone encompassing exons
v6 and v7 induced the ability to form metastatic
In some instances it is possible that the changes which tumors -a most striking effect. Further, injection of a
occur in the carbohydrate moiety of tumor surface monoclonal anti-CD44 v6 prevented the formation of
membrane glycoproteins are a natural consequence lymph node metastases. Exons v6 and v10 have now
of cell division. For example, Thomas found that the been shown to bind blood group H and chondroitin 4-
density of surface sugar determinants cross-reacting sulfate, respectively, and the latest hypothesis is that
with blood group H fell as murine mastocytoma cells these carbohydrates can bind to CD44H on endothe-
moved into the G1 phase of the division cycle, while, lium and thence homotypically to each other so gen-
reciprocally, group B determinants increased. Surface erating a metastatic nidus.
components binding the lectin, wheat-germ aggluti- Changes have quite frequently been observed in the
nin are poorly represented on resting T- and B-cells expression of class I MHC molecules. For example,
but, within 24 hours of stimulation by lymphocyte oncogenictransformation of cells infected with adeno-
polyclonal activators and before DNA synthesis virus 12 is associated with highly reduced class I as
begins, high concentrations of lectin-binding sites a consequence of very low levels of TAP-1 and
appear on the surface. -2 mRNA. Mutation frequently leads to diminished or
Unusual events may be observed in active cells as- absent class I expression linked in most cases to in-
sociated with tumor growth and spread as distinct creased metastatic potential, presumably reflecting
from the tumors themselves. For example, the 95 kDa decreased vulnerability to T-cells but not NK cells. In
surface glycoprotein, F19, is expressed in the reactive breast cancer, for example, around 60% of metastatic
stromal fibroblasts in more than 90% of carcinomas of tumors lack class I.
SPONTANEOUS I M M U N E RESPONSES TO
TUMORS
Waldenskom's macroglobulinernia
This disorder is produced by the unregulated prolif-
eration of cells of an intermediate appearance called
Figure 18.8. Myeloma paraprotein demonstrated by gel elec-
trophoresis of serum. Lane 1, normal; lane 2, y-paraprotein; lane 3, lymphoplasmacytoid cells which secrete a monoclon-
near P-paraprotein;lane 4, fibrinogenbandin the yregion of a plasma a1IgM, the Waldenstrom macroglobulin (figure 18.71).
sample; lane 5, normal serum; lane 6, immunoglobulin deficiency Remarkably, many of the monoclonal proteins have
(low y); lane 7, nephrotic syndrome (raised a,-macroglobulin, autoantibody activity, anti-DNA, anti-IgG (rheuma-
low albumin and Igs); lane 8, hemolysed sample (raised hemoglo-
bin/haptoglobin in a, region);lane 9, polyclonal increase in Igs (e.g. toid factor), and so on. It has been suggested that, like
infection, autoimmune disease);lane 10, normal serum. (Gel kindly the CLL cells, they are of the B-1 lineage which secrete
provided by Mr A. Heys.) 'natural' antibodies (cf. p. 236). Since the IgM is se-
creted in f arge amounts and is confinedto the intravas- Table 18.2, Potential tumor antigens for immunotherapy. (Repro-
cular compartment,there is a marked rise in serum vis- duced with permission from Fong L. & Engleman E.G. (2000) Den-
dritic cells in cancer irnmunotherapy. Annual Review of Immunology
cosity, the consequences of which can be temporarily 18,245.)
mitigated by vigorous plasmapheresis. The disease
runs a fairly benign course and the prognosis is quite
good, although the appearance of lymphoplasmacy-
toid tumor cells in the blood is an ominous sign.
APPROACHESTOCANCER
IMMUNOTHERAPY
since not only is it unreasonableto expect the immune
Although immune surveillance seems to operate only system to cope with a large tumor mass, but consider-
against strongly immunogenic tumors, the exciting able amounts of antigen released by shedding would
new information on the antigenicity of mutant and tend to prevent the generation of any significant
previously silent proteins, and the changes in carbohy- response in some cases due to the stimulation of T-
drate structures, should be of tremendous encourage- suppressors. This leaves the small secondary deposits
ment to any red-blooded investigator with an eye as the proper target for immunotherapy.
to develop new immunotherapies for cancer which
exploit the already impressive range of antigens cur-
rently available (table18.2).
On one point all are agreed: if immunotherapy is to Sporadic successes have greeted the use of compo-
succeed, it is essential that the tumor load should first nents of the immune system for antigen-independent
be reduced by surgery, irradiation or chemotherapy, therapy.
gates the capacity of the malignant clone to divide.
Interleukin treatment
Along these lines, GM-CSFhas been shown to enhance
On activation by IL-2 or IL-12, NK cells are capable of the differentiation, decrease the self-renewal capacity
killing a variety of fresh tumor cells in vitro and, on the and suppress the leukemogenicity of murine myeloid
basis of studies on mice with mammary glands carry- leukemias. Recombinant human products are now
ing the HER-2/neu oncogene, it would not be unrea- undergoing trials.
sonable to conduct a trial of systematic IL-12 treatment It is over 100years sincethe physician Coley gave his
in cancer patients with minimum residual disease name to the mixture of microbial products termed
in an attempt to prevent recurrence and to inhibit in- Coley's toxin. This concoction certainly livens up the
cipient metastases. innate immune system and does produce remission in
a minority of patients. The suggestion has been made
that these beneficial effects are due to the release
Interferon therapy
of TNF since the vascular endothelium of tumors
In trials using IFNa and IFNP, a 10-15% objective re- is unduly susceptible to damage by this cytokine
sponse rate was seen in patients with renal carcinoma, and hemorrhagic necrosis is readily induced. It is
melanoma and myeloma, an approximate 20% re- questionable whether the critical levels of TNF are
sponse rate among patients with Kaposi's sarcoma, reached in the human since these would be very
about 40% positive responders in patients with vari- toxic, although one study involving perfusion of
ous lymphomas and a remarkable response rate of an isolated limb with TNF, IFNy and melphalan pro-
80-90% among patients with hairy cell leukemia and voked lesions in the tumor endothelium without
mycosis fungoides. affecting the normal vasculature. Opinion is coming
With regard to the mechanisms of the antitumor round to the view that the Coley phenomenon may be
effects, in certain tumors IFNs may serve primarily as linked more to boosting a pre-existing weak antitumor
antiproliferative agents; in others, the activation of NK immmity
cells and macrophages may be important, while aug-
menting the expression of class I MHC molecules may
make the tumors more susceptible to control by im-
mune effector mechanisms.In some circumstancesthe
antiviral effect could be contributory. The current dogma is that T-cells rather than antibod-
For diseases like renal cell cancer and hairy cell ies are capable of savaging solid tumors, particularly
leukemia, IFNs have induced responses in a signifi- those expressing processed intracellular antigens on
cantly higher proportion of patients than have conven- their surface, and, since the majority are MHC class
tional therapies. However, in the wider setting, most I1 negative, it looks as though we are aiming at essen-
investigators consider that their role will be in com- tially CD8 cytotoxic T-cell responses, although CD4 T-
bination therapy, e.g. with active immunotherapy or cells can be involved in protective reactions against
with various chemotherapeutic agents where syner- tumor-associated vasculature.
gistic action has been observed in murine tumor sys-
tems. IFNa and P synergize with IFNy and the latter
Virally induced k m o r s
synergizes with TNF. IFNa acts as a radiation sensitiz-
er and its ability to increase the expression of estrogen Based on the not unreasonable belief that certain forms
receptors on cultured breast cancer cells suggests the of cancer (e.g. lymphoma) are caused by oncogenic
possibility of combining IFN with anti-estrogens in viruses, attempts are being made to isolate the virus
this disease. and prepare a suitable vaccine from it. In fact, large-
scale protection of chickens against the development
of Marek's disease lymphoma has been successfully
Colony-stimu katingfactors
achieved by vaccination with another herpes virus na-
Normal cell development proceeds from an immature tive to turkeys. In human Burkitt's lymphoma,work is
stem cell with the capacity for unlimited self-renewal, in progress to develop a vaccine to exploit the ability of
through committed progenitors, to the final lineage- Tc cells to target EBV-related antigenson the cells of all
specific differentiated cells with little or no potential Burkittfstumors. It may be an advantage to treat the
for self-renewal.Therapy aimed at inducing tumor cell patient at the same time with cytokines to upregulate
differentiation is founded on the idea that the induc- the expression of ICAM-1, LFA-3 and possibly of the
tion of cell maturation decreases and possibly abro- virus itself.
irradiated melanoma cells together with BCG which,
Immunization with whole tumor cells
by generating a plethora of inflammatory cytokines,
This has the advantage that we do not necessarilyhave increases the efficiency of presentation of tumor anti-
to know the identity of the antigen concerned. The dis- gens derived from necrotic cells. In a large-scalestudy
advantage is that the majority of tumors are weakly of over 1500 patients, 26% of vaccinees were alive at 5
immunogenic, and do not present antigen effectively years compared with only 6% of those treated with the
and so cannot overcome the barrier to activation of best available conventional therapy. It would be excit-
resting T-cells. Remember, the surface MHC-peptide ing to suppose that in the future we might expose a
complex on its own is not enough; costimulation with tumor surgically and then transfect it in situ by firing
molecules such as B7.1 and 87.2 and possibly certain gold particles (cf. p. 302) bearing appropriate gene
cytokines is required to push the GO T-cell into active constructssuch as B7, IFNy (to upregulate MHC class I
proliferation and differentiation. Once we get to and 11),GM-CSF, IL-2, and so on (figure 18.9).There is a
this stage, however, the activated T-cell no longer real risk of inducing autoimmune responses to cryptic
requires the accessory costimulation to react with its epitopes (cf. p. 407) shared with other normal tissues
target, for which it has a greatly increased avidity which the prudent investigatorwill not overlook.
due to upregulation of accessory binding molecules
such as CD2 and LFA-l (cf. p. 165; figure 18.9).
Therapy with subunit vaccines
The system works! Vaccination with B7-transfected
murine melanoma generated CD8+cytolytic effectors The variety of potential protein targets so far identified
which protected against subsequent tumor challenge; (table 18.2)has spawned a considerable investment in
in other words, transfection enabled the melanoma clinical therapeutic trials using peptides as vaccines.
cells to present their own antigensefficiently, while the Because of the pioneering work in characterizing
untransfected cells were vulnerable targets for the melanoma-specific antigens, this tumor has been the
cytotoxic T-cells so produced. A further telling focus of numerous studies which exploit to the full the
observation was that an irradiated nonimmunogenic academic background to modern immunology. En-
melanoma line which had been transfected with a couraging results in terms of clinical benefit, linked to
retroviralvector carryingthe GM-CSF gene stimulated the generation of cytolytic T-cells (CTLs), have been
potent and specific antitumor immunity, almost cer- obtained following vaccination with peptides com-
tainly by enhancing the differentiation and activation plexed with heat-shock proteins or modified at class I
of host antigen-presenting cells. A less sophisticated anchor residues to improve MHC binding. The inclu-
but more convenient approach ultimately utilizing sion of accessory factors, such as IL-2 or GM-CSF, and
similar mechanisms involves the administration of the CTLA-4 blockade can be crucial for success. Poten-
factors which facilitate engraftment, defense against has been shown to render them cytotoxic for cells
viral infection and the graft-vs-leukemia action at a expressing the surface antigen.
time when the recipient patient will have a low tumor
burden. With time, as graft-vs-host disease develops,
the dividing aggressor donor T-cells can be switched
off by administration of ganciclovir through the
T h e strategies
mechanism explained in the legend to figure 18.13.
An alternative which avoids g.v.h. disease alto- Antibodies reacting with antigens on the surface of
gether is to inject the purged bone marrow together tumor cells can protect the host by complement-
with an allogeneic cytotoxic T-cell clone specific for a mediated opsonization and lysis (modified by host
leukemia-associated peptide presented by the MHC complement regulatory proteins) and through recruit-
allele of the prospective recipient patient. This usually ment of macrophage and NK ADCC function by
works because the residues on the MHC helices which engagement of FcyRIII receptors, although for
contact the T-cell receptor are relatively conserved (un- macrophages this is partially countered by inhibitory
like those within the groove), so that the allo-T-cells FcyRII signals. These FcR-bearing cells serve not only
can recognize the MHC-peptide complex from the as cytotoxic effectors but also as multivalent surfaces
leukemia. Potential targets are cyclin-D1 and mdm- which hyper-cross-link antibody-coated target cells so
2 which are overexpressed in tumor cells and, in providing, in many cases, a transmembrane signal
leukemic cells in particular, the transcription factors which leads to apoptosis or premature exit from the
WT-1 and GATAl and the differentiation antigens cell cycle. This effect appears to sensitize neoplastic
myeloperoxidase and CD68, which are expressed ex- cells to irradiation and DNA-damaging chemothera-
clusively in hematopoietic cells and are likely to have py and holds out the exciting prospect of novel syner-
established tolerance in the patient but not in the gistic treatments whose efficacy may be enhanced by
allogeneic CTL donor (who will have been exposed the increased immunogenicity of the dying cells.
to different processed peptides) (cf. p. 353). A rather Immunologists have long been bemused by the idea
masterful development would be to transfect the of eliminating tumor cells by specific antibody linked
recipient with the genes encoding the T-cell receptor to a killer molecule and there is a truly impressive
of the allo-CTL clone. Again, looking to the future, array of ingenious initiatives. It is axiomatic that
transfection of T-cells in vitro with a humanized multimeric fragments bind much more avidly than
scFv-Fcy-transmembrane segment-CD3c construct monomeric fragments due principally to the lower
off-rates (cf. p. 87), and that constructs in the 60-120 27 to 87months. Likewise, radionuclide conjugates of a
kDa range are optimal for targeting solid tumors -too humanized anti-CD33 score very highly in myeloid
large and penetration is difficult, too small and kidney leukemia and can root out large tumor burdens. Trials
secretion is excessivelyfast. Monovalent fragments in- of 90~-anti-M~C-l in ovarian cancer provide further
clude Fv, scFv selected by antigen from phage libraries encouragement for the intrepid immunotherapists.
(cf. p. 124) and VH domains based on the large CDR
loops of the camels and llamas. For polymers we have
Attack on the turnor blood supply
bivalent and bispecific (thinkabout the difference)dia-
bodies (cf. p. 125),trivalent and trispecific triabodies, For solid tumors, the focus is upon two main targets.
even tetrabodies, and Fabs have been linked into The first would be minimal residual micrometastases
dimers or trimers. in the bone marrow which occur in one-third to one-
Radioimmunotherapy, using isotopes such as 90Y, half of patients with epithelia1 cancer after curative
"'In or 1311linked to antibody, delivers doses of radia- radical treatment of the primary lesion. The second
tion to tumor tissue which would be impossibly toxic would be the reactive tissue evoked by the malignant
with external beam sources. Results are even better process, such as stromal fibroblasts expressing the F19
when used in synergy with chemotherapy. Immuno- glycoproteinand newly formed blood vessels.
toxins represent another class of magic bullet in which Tumors generally cannot grow beyond the size of
the toxin of plant or bacterial origin is attached to an 1mm in diameter without the support of blood vessels
antibody or growth factor; after binding to the cell sur- and, because the tumor is new tissue, its blood vessels
face, the toxin is internalized and kills the cell by will also have to be new. These vessels form through
catalytic inhibition of protein synthesis. Along the the process known as angiogenesis, the sprouting
same lines, internalizing a photosensitizer renders the of new blood vessels from existing ones, but they are
cells vulnerable to photodynamic therapy. Delivery biochemically and structurally different from normal
of anticancer drugs by attachment to antibodies would resting blood vessels and so provide differential
seem to be an obvious goal, but a more sophisticated targets for therapeutic monoclonal antibodies, even
strategyinvolves the grand sounding antibody-direct- though the cancer cells themselvesin a solid tumor are
ed enzyme-prodrug therapy (ADEPT).After injection less vulnerable to antibodies directed to specific anti-
of the prodrug, each antibody-enzyme conjugate at- gens on their surface. Thus, receptors for VEGF and
tached to its cell target produces a large number of Eph, oncofetal fibronectin, matrix metalloproteases
small toxic molecules which can diffuse into the tumor MMP-2 and MMP-9 and the pericyte markers
mass and also give a bystander effect on adjacent aminopeptidase A and the NG2 proteoglycan are all
tumor cells even if they express only low levels of anti- highly and selectivelyexpressed in vasculature under-
gen. Aminopeptidase which acts on 2-L-pyroglutamyl going angiogenesis.
methotrexate to liberate methotrexate is one of the A noteworthy maneuver, which is unexpectedly
conjugates used. successful, is to identify peptides which home specifi-
cally to the endothelial cells of certain tumors by inject-
ing peptide phage libraries in vivo. One of the panel of
Clinical results
peptide motifs which has emerged from this probing
Useful responses to unmodified monoclonal anti- strategy includes RGD in the cyclic peptide CDCRGD-
bodies have been chalked up. These include anti-Her- CFC, a selective binder of the and ava,-integrins
2 (an epidermal growth factor receptor also known as known to be upregulated in angiogenic tumor en-
erbB2 and neu) for metastatic breast cancer, anti-CD52 dothelial cells. For therapeutic exploitation, these
(Campath-1H) for a range of hematological tumors peptides can be linked to appropriate drugs, such as
and 17-1Afor colorectal cancer. doxorubicin, or a pro-apoptotic peptide. Overall, there
1311-labeled anti-CD20 kills B-cell lymphomas by are undoubtedly a substantial number of targets for
ADCC and by signaling-induced apoptosis, acting the 'magic bulletsf.
in synergy with DNA damage due to y-radiation,
with perhaps a contribution from enhanced tumor im-
IMMUNODIAGNOSIS OF SOLID TUMORS
munogenicity. Therapy with this agent has been spec-
tacularly successful;in one long-term trial, injection of
the radiolabeled anti-CD20 into lymphoma patients
prepared by myeloablation with stem cell rescue gave Analysis of blood for the oncofetal antigens, a -
major responses in 86% with remissions lasting from fetoprotein in hepatoma and carcinoembryonic anti-
gen in &mors of the colon, has provided valuablediag- Table 18.3. Detection of bone marrow micrometastasesby stain-
ing for epithelial cytokeratin in colorectal cancer patients. (From
nostic informaeon, but enthusiasm has been slightly data of Schlimok G. et al. (1990)Journafof Clinical Oncology 8,831.)
curtailed by the knowledge that there is a high inci-
dence of so-called 'false positives'. Reappearance of
these proteins after surgical removal of the primary is
strongly indicative of fresh tumor growth. A hefty in-
crease in the ratio of free to bound prostate-specific
antigen (PSA) in the blood may signal cancer of the
prostate. The GM1 monosialoganglioside has been
demonstrated in the blood of 96%of patients with pan-
creatic carcinoma and 64% of patients with colorectal
carcinomas, as against 2% in normal subjects.
Identification of the cell type by monoclonal anti-
bodies is of value for the diagnosis and treatment of an
increasing number of tumors, including the lympho-
proliferative disorders as discussed earlier (seep. 382).
The same principles which govern the localization Because of the difficulty in detecting individual tumor
of monoclonal antibodies for tumor therapy apply cells in distant organs, the diagnosis of early dissemi-
equally to imaging. Maximizing the binding to tumor nated cancer has not been possible, and attempts to
relative to normal tissue and surrounding fluids is the identify earlier stages and to monitor the immuno-
name of the game. For example, the use of a bifunc- therapy of early disease have been hindered. A major
tional antibodywhich targets the tumor and an isotope advance was made when micrometastases were
chelator can be followed 24-120 hours later with the demonstrated by immunocytochemistry in the bone
chelate-containing radionuclide which allows clear- marrow of patients with colorectal cancer and were
ance of uncombined antibody. related to more widespread disease (table 18.3) and a
The Thomson-Friedenreich (T) antigen (GalP1-3- high relapse rate. The method involves scanning
GalNAca-0-Ser), expressed in the mucins of various pelvic crestbone marrow aspirates taken at surgery for
types of epithelial cancer, has proved to be a highly epithelial cells by staining for cytokeratin (cf. figure
successful target for antibody imaging. So has the F19 18.7f)and proliferation markers such as the Ki67 nu-
glycoprotein associated with proliferating fibroblasts clear antigen and receptors for transferrin and epider-
in the stroma of many carcinomas, as presumably the mal growth factor. Detection of micrometastases in the
many antigens associated with tumor angiogenesis marrow of patients with small cell lung carcinoma also
will prove to be. predicted early relapse.
(continuedp. 394)
The v6 and v10 exons of CD44 are intimately involved significant in Kaposis sarcoma and various lymphomas;
with metastatic potential. Loss of blood group A determi- they may be used in synergy with other therapies.
nants leads to a poor prognosis. GM-CSF enhances proliferation and decreases leukemo-
genicity of murine myeloid leukemias.
Immune response to tumors Cancer vaccines based on oncogenic viral proteins can
T-cells generally mount effective surveillance against be expected.
tumors associatedwith oncogenicviruses or UV induction Weakly immunogenic tumors provoke effective anti-
which are strongly immunogenic. cancer responses if given with an adjuvant, such as BCG,
More weakly immunogenic tumors are not controlled or if transfected with costimulatory molecules, such as B7
by T-cell surveillance, although sometimes low-grade re- and cytokines IFNy, IL-2, -4 and -7. CD8 CTLs are favored
sponses are evoked. for the attack on solid tumors.
NK cells probably play a role in containing tumor A variety of potential protein targets have been
growth and metastases. They can attack MHC class I- identified and intense effort is being expended in the
negative tumor cellsbecause the class I molecule imparts a investigation of peptides as subunit vaccines. Their
negative inactivation signal to NK cells. The A-NK subset, immunogenicity can be enhanced by complexing with
which expresses high levels of adhesion molecules, is heat-shock proteins and by accessory factors such as GM-
more cytolytic for fresh tumor cells. CSF, CTLA-4blockade and anti-CD40stimulation.
Tumors utilize a variety of mechanisms to escape host Naked DNA and self-replicating RNA vaccines are
immune responses. strong candidates.
Powerful immunogens have been created by pulsing
Unregulated development gives rise to dendritic antigen-presenting cells with peptides from
lymphoproliferativedisorders melanoma antigens and framework regions of CLL Ig.
Deregulation of the c-myc protooncogene is a character- A graft-vs-leukemia effect is achieved by allogeneic
istic feature of many B-cell tumors. CTLs or by allogeneic bone marrow transplantation with
Chromosome translocations are common. measures to limit graft-vs-host disease.
Lymphoid malignancies show maturation arrest at Monoclonalantibodiesconjugated to toxins or radionu-
characteristicstages in differentiation. clides can target tumor cells or antigens on new blood ves-
The surface markers of leukemias and lymphomas sels or the reactive stromal fibroblasts associated with
identified by monoclonal antibodies are important aids in malignancy. Encouraging, even impressive, therapeutic
diagnosis. Most non-Hodgkins lymphomas are of B-cell results have been obtained with antibodies to CD20 in B-
origin, are associated with EBV and express a monoclonal cell lymphoma,CD33in myeloid leukemia,anti-MUC-1in
surface Ig. ovarian cancer and c-erbB2 overexpressed on breast can-
Multiple myeloma represents a malignant proliferation cers. Bifunctional antibodies can bring effectors such as
of a single clone of plasma cells producing a single M NK and Tc close to the tumor target.
band on electrophoresis. 10-20% have widespread
amyloid deposits containing the variable region of the lmmunodiagnosisof solid tumors
myeloma light chain. Many circulating tumor markers are diagnostic, e.g. a-
Waldenstromsmacroglobulinemia is produced by un- fetoprotein in hepatic carcinoma and carcinoembryonic
regulated proliferation of a clone producing monoclonal antigen in colorectalcarcinoma.
IgM causing a marked rise in serum viscosity. Monoclonal antibody to tumor surfaces can provide a
Malignant lymphoid cells produce secondary im- basis for imaging. Certain tumor mucins, the F19 glyco-
munodeficiency by suppressing differentiation of the protein on reactive stromal fibroblasts and VEGF on new
correspondingnormal lineage. blood vessels around the tumor are good targets.
Detection of micrometastases in bone marrow by im-
Approaches to cancer immunotherapy munocytochemistry provides valuable information on
Immunotherapy is only likely to work after a tumor prognosis and the efficacy of new therapies.
mass has been debulked.
Innate immune mechanisms can be harnessed. Sys-
temic IL-12 may be effective against minimal residual dis- See the accompanying website (www.roitt.com)
ease. IFNy and p are very effective in the T-cell disorders, for multiple choice questions.
hairy cell leukemia and mycosis fungoides, less so but still
detection of residual marrow disease at clinical remission predicts
F U R T H E R READING metastatic relapse in small cell lung cancer. Cancer Research 50,
Begent R.H.J. et al. (1996)Clinical evidence of efficient tumor target- 6545.
ing based on single-chain Fv antibody selected from a combinato- Mannel D., Murray C., Risau W. & Clauss M. (1996)Tumor necrosis:
rial library.NatureMedicine 2,979. factors and principles. Immunology Today 17,254.
Blachere N.E. et al. (1997)Heat shock protein-peptide complexes,re- Morton D.L. & Barth A. (1996) Vaccine therapy for malignant
constituted invitro, elicit peptide-specific cytotoxicT-lymphocyte melanoma. CA:A Cancer Journalfor Clinicians46,225.
responses and tumor immunity. (Strategy initiated by P.K. Srivas- Ruoslahti E. & Rajotte D. (2000)An address system in the vasculature
tava.) Journal of Experimental Medicine 186,1315. of normal tissues and tumors. Annual Review of Immunology 18,
Cancer (2001) Current Opinion in Immunology 13(5). (Critical 813.
overviews of the whole field appearing annually, which are well- Stein H. & Mason D.Y. (1985) Immunological analysis of tissue sec-
worth reading.) tions indiagnosis of lymphoma. 1nA.V.Hoffbrand (ed.) Recent Ad-
Castellino F. et al. (2000)Receptor-mediated uptake of antigen/heat vances in Haematology, Vol. 4, p. 127. Churchill Livingstone,
shock protein complexes (by antigen-presenting cells) results in Edinburgh.
MHC class I antigen presentation via two distinct processing Van Elsas A., Hurwitz A.A. & Allison J.P. (1999) Combination im-
pathways (TAP/proteasome and endosomal). Journal of Experi- munotherapy of B16 melanoma using anti-CTLA-4 and GM-CSF-
mental Medicine 191,1957. producing vaccines induces rejection of S.C.and metastatic tumors
Fong L. & Engleman E.G. (2000) Dendritic cells in cancer im- accompanied by autoimmune depigmentation. Journal of Experi-
munotherapy. Annual Review of Immunology 18,245. mental Medicine 190,355.
Forbes I.J.& Le0ngA.S.-Y. (1987)Essential Oncology of the Lympkocyte. Van Ginderachter J.A. et al. (2000) B7-1, IFN-y and anti-CTLA-4
Springer-Verlag,Berlin. co-operate to prevent T-cell tolerization during immunotherapy
Leonard R.C.F., Duncan L.W. & Hay F.G. (1990)Immunocytological against a murine T-lymphoma.Journal of Cancer 87,539.
Autoi mmune diseases
1 -Scope and etiology
Although Dacies studies on red cell autoantibodies in cer- thyroid autoantibodies and chronic inflammatory de-
tain forms of hemolytic anemia were amongst the earliest struction of the thyroid gland architecture (figure
to implicate autoimmunity in the pathogenesis of disease, M19.1.laandb).
a direct link to disorders affecting whole organs was not Having noted the fall in serum y-globulin which fol-
established until 1956 when three major papers on thyroid lowed removal of the goiter in Hashimotos thyroiditis
autoimmunity appeared. and the similarity of the histology (figure M19.1.l~)to
In an attempt to confirm Paul Ehrlichs concept of that of Rose and Witebskys rabbits, Roitt, Doniach and
horror autotoxicus-the bodys dread of making anti- Campbell tested thehypothesis that the plasma cells in the
bodies to self-Rose and Witebsky immunized rabbits gland might be making an autoantibody to a thyroid com-
with rabbit thyroid extract in complete Freunds adjuvant. ponent, so causing the tissue damage and chronic inflam-
To what one might hazard was Witebskys dismay and matory response. Sure enough, the sera of the first patients
Roses delight, this procedure resulted in the productionof tested had precipitating antibodies to an autoantigen in
(continuedp. 398)
Figure M19.1.2. Thyroid autoantibodies in the serum of a pa-
tient with Hashimotos disease demonstrated by precipitation
in agar. Test serum is incorporated in agar in the bottom of the
tube; the middle layer contains agar only, while the autoantigen is Figure M19.1.3. The long-acting thyroid stimulator in Graves
present in the top layer. As serum antibody and thyroid autoanti- disease. Injection of TSH causes a rapid release of l3II from the
gen diffuse towards each other, they form a zone of opaque pre- prelabeled animal thyroid in contrast to the prolonged release
cipitate in the middle layer. Saline and kidney extract controls which follows injection of serum from a thyrotoxic patient. (Based
are negative. (Based on Roitt I.M., Doniach D., Campbell P.N. & on Adams D.D. & Purves H.D. (1956)Abnormal responses in the
H U ~ S OR.V.
I I (1956) Autoantibodies in Hashimotos disease. assay of thyrotrophin. Proceedings of the University ofOtago Medical
Lancet ii, 820.) School 34,ll.)
normal thyroid extracts which was soon identified as thy- thyroid-stimulating hormone (TSH) produced a peak
roglobulin (figure M19.1.2). in serum radioactivity some 4 hours or so after injection
In far off New Zealand (depending on your geographi- of the test animal, serum from thyrotoxic patients had
cal location!),Adams and Purves, in seeking a circulating a prolonged stimulatory effect (figure M19.1.3). The
factor which might be responsible for the hyperthy- so-called long-acting thyroid stimulator (LATS) was ulti-
roidism of Graves thyrotoxicosis,injected patients serum mately shown to be an IgG mimicking TSH through its
into guinea-pigs whose thyroids had been prelabeled with reaction with the TSH receptor but differing in its time-
1311, and followed the release of radiolabeled material course of action, largely due to its longer half-life in the
from the gland with time. Whereas the natural pituitary circulation.
Moving towards the center of the spectrum are those kidney glomeruli,joints, serousmembranes and blood
disorders where the lesion tends to be localized to a vessels. In addition, the formed elements of the blood
single organ but the antibodies are nonorgan-specific. are often affected.A bizarre collection of autoantibod-
A typical example would be primary biliary cirrhosis ies is found, some of which react with the DNA and
where the small bile ductule is the main target of other nuclear constituentsof all cells in the body.
inflammatory cell infiltration but the serum anti- An attempt to fit the major diseases considered to
bodies present -mainly mitochondrial-are not be associated with autoimmunity into this spectrum is
liver-specific. shown in table 19.1.
At the other end of the spectrum are the 'nonorgan-
specific or systemic autoimmune diseases' broadly
belonging to the class of rheumatological disorders,
exemplified by systemic lupus erythematosus (SLE), At this stage in the discussion it may be of value to have
where both lesions and autoantibodies are not con- a more precise account of the major autoantibodiesde-
fined to any one organ. Pathologicalchanges are wide- tected in the different diseases to provide a framework
spread and are primarily lesions of connective tissue for reference. Table 19.2 documents a list of these anti-
with fibrinoid necrosis. They are seen in the skin (the bodies and the methods employed in their detection.
'lupus' butterfly rash on the face is characteristic), The notes accompanying the table amplify specific
Table 19.1. Spectrum of autoimmune diseases. in their serum. Conversely, thyroid antibodies have
been demonstrated in up to 50% of pernicious anemia
patients. It should be stressed that these are not cross-
reacting antibodies. The thyroid-specific antibodies
will not react with stomach and vice versa. When a
serum reacts with both organs it means that two popu-
lation~of antibodies are present, one with specificity
for thyroid and the other for stomach.
At the nonorgan-specific end of the spectrum, sys-
temic autoimmune disease such as SLE is clinically as-
sociated with rheumatoid arthritis and several other
disorders which are themselves uncommon: hemo-
lytic anemia, idiopathic leukopenia and thrombo-
cytopenic purpura, dermatomyositis and Sjogren's
syndrome. Antinuclear antibodies and antiglobulin
(rheumatoid)factors are a general feature.
Sjogren's syndrome occupies an interestingposition
(table 19.3);aside from the clinical and serological fea-
tures associated with systemic disease mentioned
above, characteristics of an organ-specific disorder are
evident. Antibodies reacting with salivary ducts are
demonstrable and there is an abnormally high inci-
dence of thyroid autoantibodies;histologically the af-
fected lacrimal and salivary glands reveal changes of a
similar nature to those seen in Hashimoto's disease,
namely a replacement of the glandular elements by
points, while some of the tests are illustrated in figures patchy lymphocytic and plasma cell granulomatous
19.1, 6.9, 6.10 and 7.3. As antigens are characterized tissue. Associations between diseases at the two ends
and become available in purified form, the convenient of the spectrum have been reported, but, as might be
ELISA is becoming a dominant technique. predicted from the serological data (table 19.3), they
are not common.
Patients with organ-specific disorders are slightly
more prone to develop cancer in the affected organ,
There is a tendency for more than one autoimmune whereas generalized lymphoreticular neoplasia
disorder to occur in the same individual and when this shows up with uncommon frequency in nonorgan-
happens the association is often between diseases specific disease.
within the same region of the autoimmune spectrum
(cf. table 19.1).Thus patients with autoimmune thy-
roiditis (Hashimoto's disease or primary myxedema)
have a much higher incidence of pernicious anemia Both spontaneous and induced animal models have
than would be expected in a random population given tremendous insights into the nature of human
matched for age and sex (10% as against 0.2%). autoimmune disease and, to assist our discussions, we
Conversely, both thyroiditis and Graves' disease are felt it would be helpful to list them (table19.4).
diagnosed in pernicious anemia patients with an unex-
pectedly high frequency. Other associations are seen
NATURE AND NURTURE
between Addison's disease and autoimmune thyroid
disease and occur in the rare cases of juveniles with
pernicious anemia and polyendocrinopathy which
includes Addison's disease, hypoparathyroidism, Autoimmune phenomena tend to aggregate in certain
diabetes and thyroiditis. families. For example, the first degree relatives (sibs,
There is an even greater overlap in serological find- parents and children) of patients with Hashimoto's
ings. Thirty per cent of patients with autoimmune thy- disease show a high incidence of thyroid autoantibod-
roid disease have concomitant parietal cell antibodies ies (figure19.2)and of overt and subclinicalthyroiditis.
Table 19.2. Autoantibodies in human disease.
DISEASE ANTIGEN DETECTION OF IMMUNOLOGICAL REACTIVITY I
Hashimotos thyroiditis Thyroglobuiin Precipitins; passive hemagglutination; ELISA
Primary myxederna Thyroid peroxidase: Cytoplasmic IFT on unfixed thyroid; passive hemagglutination; ELISA
Cell surface IFT on viable thyroid cells; C-mediated cyiotoxiciiy
Graves disease Cell surface TSH receptors Bioassay-stimulation of mouse thyroid in WO;blocking
combination TSH with receptors; stimulation adenyl
cyclase
Growth receptors Induction of cell division in thyroid fragments
Pernicious anemia Intrinsic factor Neutralization; blocking combination with vit-B,,; binding
to intrinsic factor-B,, by coprecipitation
Parietal cell H+-K+ ATPase IFT on unfixed gastric mucosa
Addisons disease Cytoplasm adrenal cells (1 7a-/21 -hydroxylase) IFT on unfixed adrenal cortex
Premature onset of menopause Cytoplasm steroid-producing cells IFT on adrenal and interstitial cells of ovary and testis
Male infertility (some), Spermatozoa Sperm agglutination in ejaculate
Insulin-dependentdiabetes3 Cytoplasm of islet cells IFT on unfixed human pancreas
Insulin, GAD and CA512 ELISA
Type B insulin resistance with acanthosis Insulin receptor Block hormone binding to receptor
nigricans
Goodpastures syndrome Giomerular and lung basement Linear staining by iFT of kidney biopsy with
membrane fluorescent anti-lgG
Radioimmunoassay with purified Ag; ELISA
Pemphigus vulgaris Desmosomes between prickle cells in IFT on skin
epidermis (cadherin)
Pemphigoid Basement membrane IFT on skin
Phacogenic uveitis Lens Passive hemagglutination
Sympathetic ophthalmia uvea (Delayed skin reaction to uveal extract)
Autoimmune hemolytic anemia5 Erythrocytes Coombs antiglobulin test
ldiopathic thrombocytopenic purpura Platelets Shortened platelet survival in vivo
Primary biliary cirrhosis Mitochondria (pyruvate dehyrogenase) IFT on mitochondria-richcells (e.g. distal tubules of kidney)
Active chronic hepatitis (HBV & HCV -ve) Smooth muscle/nuclear lamins/nuclei IFT (e.g. on gastric mucosa)
Kidney/liver microsomes (cyt P450) IFT (kidney)
Ulcerative colitis Colon lipopolysaccharide IFT; passive hemagglutination(cytotoxic action of
iymphocytes on colon cells)
Colon epithelia1 cell surface protein ADCC on colon cancer cell line
Ab data in this disease not universally accepted
Sjogrens syndrome SS-A(R0) SS-B(L0) IFT; gel precipitation; ELISA
Ducts/mitochondria/nuciei/thyroid IFT
IgG Antiglobulin (rheumatoid factor) tests
Rheumatoid arthritis7 IgG Antiglobulin test; latex agglutination; sheep red cell
agglutinationtest (SCAT; commercial product, RAHA
test) and ELISA; agalocto-glycoform
Collagen Passive hemagglutination
Discoid lupus erythematosus Nucleor/lgG IFT/antiglobulin test
Sclerodermas Nuciear/lgG/centromere IFT
Nuclear/lgG/Scl-70 IF1 countercurrent electrophoresis; ELISA
Dermatomyo~itis~ Nuclear/lgG/Jo-1 IFT; countercurrent electrophoresis; ELISA
Mixed connective tissue disease0 Extractable nuclear IFT; countercurrent electrophoresis; ELISA
Systemic lupus erythematosus DNA ELISA; IFT on Crithidia
snRNP (Sm & ribonucleoprotein) IFT; gel precipitation techniques; E L M
Nucleoprotein IFT
Array of other Ag including formed elements
of blood/lgG
Cardiolipin/p2-glycoproteinl Radioimmunoassay
Wegeners granulomatosis Neutrophil cytoplasm (ANCk myeloperoxidase/ IFT on alcohol fixed poiymorphs; ELISA
serine proteinose)*
IFT, immunofluorescent test (cf. figure 7.3); ELISA, enzyme-linked immunosorbent assay (cf.figure 6.10).
Table 19.3. Organ-specific and nonorgan-
specific serologicalinterrelationships in
human disease.
*Immunofluorescencetest.
tRheumatoid factor classical tests.
$Incidenceincreaseswith age and females>males.
Parallel studies have disclosed similar relationships twins is unequivocal. When thyrotoxicosisor insulin-
in the families of pernicious anemia patients, in that dependent diabetes mellitus (IDDM) occurs in twins,
gastric parietal cell antibodiesare prevalent in the rela- there is a far greater concordance rate (i.e. both twins
tives who are wont to develop achlorhydria and at- affected) in identical than in nonidentical twins. Sec-
rophic gastritis. Turning to SLE, disturbances of ond, we have already noted that lines of animals have
immunoglobulin synthesis and a susceptibility to de- been bred which spontaneouslydevelop autoimmune
velop 'connective tissue diseases' have been reported, disease (table 19.4).Inother words, the autoimmunity
but there are some conflicting accounts still not is genetically programed. There is an Obese line of
resolved. chickens with autoimmune thyroiditis, the Nonobese
These familial relationships could be ascribed to en- diabetic (NOD) mouse modeling human IDDM and
vironmental factors such as infective microorganisms, the New Zealand Black (NZB) strain succumbing
but there is powerful evidence that important genetic to autoimmune hemolytic anemia. The hybrid of
components must be involved. The data on NZB with another strain, the New Zealand White
normally unresponsive because of clonal deletion, have failed to reveal an abnormalityin the antigen. Re-
clonal anergy, T-suppression or inadequate autoanti- member the experiment in which neonatal thyroidec-
gen presentation. Immediately, one could conceive of tomized Obese strain chickens make autoantibodies if
an abnormal degree of responsiveness to self-antigens injected with thyroglobulin prepared from nonnal
as a result of relatively low intrathymicexpression of a chickens, suggestingthat the immunological response
particular molecule (cf. p. 405). Abnormalities in the rather than the antigen is abnormal.Nonetheless, there
signaling pathways affecting the thresholds for posi- may be defects in the iodine metabolism of the gland it-
tive and negative selection in the thymus would also self in this strain (figure 19.4), and recent work has
affect subsequent responsiveness to peripheral auto- shown that the severity of thyroiditis is ameliorated
antigens. So might defects in apoptotic cell death. It when the birds are put on a low iodine diet.
would be interesting to know whether the innate re- Modification can also be achieved through combi-
sistance of the NZB mouse to tolerization by a protein nation with a drug (figure 19.1213.3).The autoimmune
antigen such as bovine serum albumin can be nailed to hemolytic anemia associated with the administration
one of these causes or whether it is a consequence of of a-methyldopa might be attributable to modification
defects in regulatory cells (seebelow). of the red cell surface in such a way as to provide a car-
rier for stimulating B-cells which recognize the rhesus
antigen. This is normally regarded as a 'weak' antigen
AUTOIMMUNITY CAN ARISE THROUGH
and would be less likely to induce B-cell tolerance than
BYPASS OF T-HELPERS
the 'stronger' antigens present on the erythrocyte. Iso-
niazid may produce arthritis associated with nuclear
antibodies and, unlike most other cases of drug-
Allison and Weigle argued independently that, if au- induced autoimmunity, synthesis of these antibodies
toreactive T-cells are tolerized and thereby unable to is said to continue after cessation of drug therapy. A
collaborate with B-cells to generate autoantibodies high proportion of patients on continued treatment
(figures 19.12aand 19.15),provision of new carrier de- with procainamide develop nuclear antibodies and
terminants to which no self-tolerancehad been estab- 40% present with clinical signs of SLE. Myasthenia
lished would bypass this mechanism and lead to gravis and symptoms of pemphigus have been de-
autoantibody production (figures 19.12band 19.15). scribed in some patients on penicillamine.It is not clear
in every case whether the drug provides carrier help
through direct modification of the autoantigen or of
1 Modification of the autoantigen
some independent molecule concerned in associative
A new carrier could arise through some modifica- recognition.
tion to the molecule, for example by defects in synthe-
sis or by an abnormalityin lysosomalprocessing yield-
2 Cross-reactions with B-cell epitopes
ing a split product exposing some new groupings
(figure 19.12b.l).Aside from the possibility of post- Many examples are known in which potential human
translational modifications, such as subtle changes in autoantigenic B-cell epitopes are present on a micro-
glycosylation patterns as seen in the low galactosyla- bial exogenous cross-reacting antigen which provides
tion of the Fcy sugar chains in rheumatoid arthritis, the new carrier that provokes autoantibody formation
many studies on spontaneous autoimmune disease (figure 19.1213.2).The mechanism is spelt out in more
Figure 19.12. T-helper bypass through new carrier epitope ( ) eliminated from the body and with it the T-cell epi-
generates autoimmunity.For simplicity, processing for MHC asso- tope, the only way that the autoimmunity can be sus-
ciation has been omitted from the diagram, but is elaborated in fig-
ure 19.13. (a) The pivotal autoreactive T-helper is unresponsive
tained is for the activated B-cell to capture circulating
either through tolerance or inability to see a cryptic epitope. (b) Dif- autoantigen and, after processing, present it to the T-
ferent mechanisms providing a new carrier epitope. helper (figure 19.13~).This is not possible for cell-
associated antigens but their special link with T-cell
recognition puts them in a totally different ballpark.
In this case, if an infecting agent mimics an autoanti-
detail in figure 19.13a. Two low molecular weight en- gen by producing a cross-reacting T-cell epitope, the
velope proteins of Yersinia enterolytica share epitopes resulting T-cell autoimmunity could theoretically per-
with the extracellular domain of the human thyroid- sist even after elimination of the infection. The au-
stimulating hormone (TSH) receptor; in rheumatic toantigen will normally be presented to the resting
fever, antibodies produced to the Streptococcus also autoreactive T-cell as a cryptic epitope and by defini-
react with heart, and the sera of 50% of children with tion will be unable to provide an activating signal.The
the disease who develop Sydenham's chorea give cross-reactinginfectious agent will provide abundant
neuronal immunofluorescent staining which can be antigen on professional APCs which can prime the T-
absorbed out with streptococcal membranes. Colon cell and upregulate its adhesion molecules so that it
antibodies present in ulcerative colitis have been now has the avidity to bind to and be persistently acti-
found to cross-react with Escherichia coli 014. There is vated by the cryptic self-epitope presented on the tar-
also some evidence for the view that antigens common get tissue cell provided that it is associated with the
to Trypanosoma cruzi and cardiac muscle and peripher- appropriate MHC molecule (figure 19.13b).Remem-
al nervous system provoke some of the immunopatho- ber the transgenic cytotoxic T-cells (Tc) which could
logical lesions seen in Chagas' disease. only destroy the pancreatic p-cells bearing a viral
transgene when they were primed by a real viral infec-
tion (cf. figure 12.10).Recall also the tumor cells that
3 Molecular mimicy of T-cell epitopes
could only be recognized by primed not resting T-cells
The drawback with the Allison-Weigle model of cross- (cf. figure 18.9).Theoretically, the resting T-cell could
reaction of B-cell epitopes and the provision of a new T- also be primed in a nonantigen-specific manner by a
cell carrier is that, once the cross-reacting agent is microbial superantigen.
Figure 19.13. Mechanisms of microbial induction of autoimm- antigen activator could also fulfil the same function of responding
unity and epitope spread. (This is a complex mouthful but diges- to a cryptic epitope. (c) If the autoantigen is soluble or capable of
tion is recommended because these ideas are crucial. The more uptake and processing after capture by the activated autoreactive
faint-hearted may require an ice-pack and persistence, but follow- B-cell (1) (either from (a) or through nonspecific polyclonal activa-
ing the numbers should help.) (a) A microbial antigen bearing an tion), a new epitope can be presented on the B-cell class I1 which
epitope Y which cross-reacts with self and a foreign T-cell epitope now stimulates an autoreactive (anti-Z)T-helper (2) which can now
X is (1) processed by an antigen-presenting cell, (2) activates the sustain an autoimmune response entirely through autoantigen
T-helper which (3) recognizes the processed X after capture by an stimulation (3).It can also produce epitope spread within the same
anti-Y B-cell and (4) stimulates the B-cell to secrete anti-Y autoan- molecule through helping a B-cell which captures the autoantigen
tibody. (b) The activated anti-X T-helper, as distinct from the through a new epitope W (4). It can also permit epitope spread
resting cell, may recognize and be stimulated by a cross-reacting to another componentin an intermolecularcomplex such as nucleo-
cryptic T-cell epitope expressed by a tissue cell. This will maintain somal histoneDNA or idiotype-positive (Id+) anti-DNA-DNA
the autoimmune response even after elimination of the microbe, which is 'piggy-backed' into the B-cell (5)which presents processed
because of the persistence of the self-epitope. The tissue expressing antigen to the T-helper (6)in the cases cited, specific for histone or Id,
the epitope will also be a target for immunological attack. Note respectively.*Denotesactivation.
also that a T-helper primed nonspecifically by a polyclonal super-
Although we have ascribed the dominant role of rived (figure 5.22), then the mature T-cells leaving the
MHC allelesas risk factorsfor autoimmunediseases to thymus will have been selected with a strong bias to
their ability to present key antigenic epitopes to au- weak recognition of self-MHC peptides presented by
toreactive T-cells, they might also operate in a quite class 11. There must therefore be a major pool of self-
distinct way. We may recollectthat, during intrathymic reactive T-cells vulnerable to stimulation by exoge-
ontogeny, T-cells are positively selected by weak inter- nously derived cross-reacting epitopes which mimic
action with self-peptides complexed with MHC. Now these MHC peptides. Just so. The critical sequence
since around 5ooh of the class I1 peptides are MHC de- QKRAAVDTY of the rheumatoid arthritis susceptibil-
ity allele HLA-DRBl"0401(table 17.2)is closely similar cell epitope. When this is recognized by the B-cell re-
to the QKRAAYDQY of the dnaJ heat-shock protein of ceptor, the helper component will be 'piggy-backed'
E. coli, and this peptide presented by DQ causes prolif- into the B-cell, processed and presented as an epitope
eration of synovial T-cells from RA patients. In fact, a for recognition by T-cells (figure 19.13~).
By the same
large number of microbial peptide sequences with token, the autoimmune response can spread to other
varying degrees of homology with human proteins epitopes on the same molecule.
have been identified (table 19.5),although it should be
emphasized at this stage that they only provide clues
for further study. The mere existence of a homology is
no certainty that infection with that organism will nec- We have argued the evidence for internal regulated id-
essarily lead to autoimmunitybecause everything de- iotype networks involving self-reactivity at some
pends on several contingencies, including the manner length. This raises the possibility of involving autore-
in which the proteins are processed by the APCs, and active lymphocytes with responses to exogenous
we cannot predict, as yet, which peptides will be pre- agents through idiotype network connections, partic-
sented and in what concentration. ularly since some autoimmune diseases are character-
ized by major cross-reactiveidiotypes.
Thus, knowing that T-helperswith specificityfor the
4 'Piggy-back' T-cell epitopes and epitope spread
idiotype on a lymphocytereceptor can be instrumental
One membrane component may provide help for the in the stimulation of that cell, it is conceivable that an
immune response to another (associativerecognition). environmental agent such as a parasite or virus, which
In the context of autoirnmunity, a new helper determi- triggered antibody carrying a public idiotype (cross-
nant may arise through drug modification as men- reactive idiotype, CRI), which happened to be shared
tioned above, or through the insertion of viral antigen with the receptor of an autoreactive T- or B-cell, could
into the membrane of an infected cell (cf. figure provoke an autoimmune response (figure 19.14b).
19.1210.4).That this can promote a reaction to a pre-ex- Similarly, if it is correct that the germ-line idiotypes
isting cell component is clear from the studies in which on autoantibodies generate a whole range of anti-
infection of a tumor with influenza virus elicited resis- idiotypes which mediate the response to exogenous
tance to uninfected tumor cells. The appearanceof cold antigens, then, by the same token, it is conceivable
agglutinins often with blood group I specificity after that antibodies produced in response to an infection
Mycoplasma pneumoniae infection could have a similar may react with the corresponding idiotype on the
explanation. In a comparable fashion, T-cell help can autoreactive lymphocyte (figure 19.14a).For example,
be provided for a molecule such as DNA, which cannot a hybridoma from a myasthenia gravis patient secret-
itself form a T-cell epitope, by complexing with a T- ed an anti-Id to an acetylcholine receptor autoanti-
dependent carrier, in this example a histone, or an anti- body; this anti-Id was found to react with the bacterial
DNAidiotype to which T-cellswere sensitized.For this product 1,3-dextran. Finally, it is possible for Id net-
mechanism to work, the helper component must still work interactions to allow a viral infection to give rise
be physically attached to the fragment bearing the B- to autoantibodies reacting with the viral receptor
(figure 19.14~). Since viruses all bind to specific com-
plementary receptors on the cells they infect, this se-
Table 19.5. Molecular mimicry: homologies between microbes quence of events may have serious consequences; we
and body components as potential cross-reacting T-cell epitopes. note for example that P-adrenergic receptors are the
surface targets for certain reoviruses and that rabies
virus binds to the acetylcholine receptor.
The immune system balances precariously between effec- where the autoantibodies have widespread reactivity and
tive responses to environmental antigens and regulatory the lesionsresemble those of serum sickness relating to de-
controlof anarray of potentially suicidalresponses toself- position of circulating immune complexes.
molecules. There is a tendency for organ-specificdisorders such as
thyroiditis and pernicious anemia to overlap in given indi-
The scope of autoimmune diseases viduals, while overlap of rheumatological disorders is
Autoimmunity is associated with certain diseases greater thanexpected by chance.
which form a spectrum. At one pole, exemplified by There are a number of models of organ-specificand sys-
Hashmotos thyroiditis, the autoantibodies and the le- temic autoimmune diseases which occur spontaneously
sions are organ-specific with the organ acting as the target (e.g. nonobese diabetic mice or NZBxW hybrids with
forautoimmuneattack; attheotherpolearethenonorgan- SLE) or canbeinduced experimentally (e.g.thyroiditis by
specific 01 systemic autoimmune diseases, such as SLE, thyroglobulin in complete Freunds adjuvant (CFA) and
(continuedp. 420)
perhaps SLE by immunization with Id+ anti-DNA Autoimmunitycan arise through bypass of 1-helpers
monoclonal in CFA). Abnormal modification of the autoantigen, cross-
reaction with exogenous antigens or 'piggy-back' recogni-
Genetic and environmental influences tion of T-helper epitopes can provide new carrier determi-
Multifactorialgenetic factors increase predisposition to nants and epitope spread.
autoimmune disease: these include HLA tissue type, the T-helpers could also be bypassed by idiotype network
predisposition to aggressiveautoimmunity and the selec- interactionswith cross-reactionsbetween public idiotypes
tion ofpotential autoantigens. on autoantibodies and microbial antibodies or microbes
Females have a far higher incidence of autoimmunity themselves, or by antibodies formed to antiviral idiotypes
than males, perhaps due to hormonal influences. which behaved as internal images of the virus and reacted
Feedback control of lymphocytes through the cy- with thecell surfaceviral receptor.
tokine-hypothalamus-pituitary-adrenal loop may be Finally, B-cells and T-cells can be stimulated directly b
defective as shown for rheumatoid arthritis. polyclonal activatorssuch as EB virus or superantigens.
Twin studies indicate a strong environmentalinfluence
in many disorders; both microbial and nonmicrobial Autoimmunity con arise through bypass of regulatory
factors have been suspected. mechanisms
T-helper bypass alone may be insufficient to maintain
Autoreactivity comes naturally autoimmunity and it is generally considered that, in addi-
5 1 cells form a pool of mutually stimulating cells tion, a defect in cells which normally regulate autoimmu-
spontaneously producing 'natural antibodies' which nity is required.
interact idiotypically and frequently show multispecific This could occur through an inability of the central T-
autoreactivity. helper cell to be tolerized or to respond to or induce T-
The immune system appears to have a set of T-cells suppressors.
directed to a limited number of dominant autoantigens It could also arise through defects in antigen-specific,
which are tightly controlled. idiotype-specific and hsp and perhaps other nonspecific
T-suppressor systems.
Is autaimmunitydriven by antigen? Another possibility would be the derepression of classI1
In spontaneous models of diabetes and thyroiditis, re- genes giving rise to inappropriate cellular expression of
moval of antigen prevents autoimmunity. class II so breaking the 'silence' between cellular autoanti-
The development of high affinity mutated antibodies gen and autoreactiveT-inducer.
9 This would make the cell a target for activated T-cells
and immune responses to clusters of anatomically related
antigens strongly imply B-cell selection of autoantigen. but, without costimulators such as 87, perhaps only pm-
T-cell specificities in systemic autoimmunity are un- fessionalAPCscould prime the resting autoreactiveT-cell.
knownbut may be anti-idiotype. Cytokme imbalanceprovides the circumstancesfor this
Autoantigens are, for the most part, accessible to circu- to occur although the situation may be very complex.
lating lymphocytes which normally include autoreactive
T- and B-cells. Dominant autoantigens will induce toler- Autoimmune disorders are multifoctorial
ance but T-cells specific for peptides presented at low Given the polygenic predisposition, these changes
concentrations (cryptic epitopes) will be potentially could come about by some spontaneous internal dysregu-
autoreactive. lation related perhaps to aging, and/or through enviror-
mental factors, particularly microbes, which could act
The 1-helper is pivotal for control an uncomfortablylarge number of different ways.
It is assumed that the key to the system is the control
of autoreactive T-helper cells which are normally unres- eetheaccompanying website
ponsive because of clonal deletion, clonal anergy, T- )r multiple choice questions
suppression or inadequate autoantigen processing.
FURTHER READING
Suggestions for further reading are given.atthe end of Chapter 20.
CHAPTER20
Autoimmune diseases
2 -Pathogenesis, diagnosis and treatment
resulting in lower fecundity rates and recurrent fetal uptake by the gland is unaffected by the adminis-
loss. The effect seems to be mediated through reaction tration of thyroxine or tri-iodothyronine, whereas
of the autoantibodies with a complex of cardiolipin normally this would cause feedback inhibition and
and P2-glycoprotein1which inhibits triggering of the suppression of uptake; this forms the basis of an
coagulation cascade. The placental trophoblast is a important diagnostic test for thyrotoxicosis.
primary target of these antibodies since the villous There is reason to believe that enlargement of the
cytotrophoblast is one of the few cell types which ex- thyroid in this disorder is due to the action of antibod-
ternalizes phosphatidyl serine during development. ies which react with a 'growth' receptor and directly
stimulate cell division as distinct from metabolic
hyperactivity. In contrast, sera from patients with pri-
mary myxedema (atrophic thyroiditis) contain anti-
bodies capable of blocking the stimulation of growth
Thyroid
by TSH, thereby preventing the regeneration of folli-
Under certain circumstances antibodies to the surface cles which is a feature of the enlarged Hashimoto
of a cell may stimulate rather than destroy (cf. 'stimu- goiter. Graves' disease is often associated with
lating hypersensitivity'; Chapter 16).This would seem exophthalmos which might be due to cross-reaction of
to be the case in Graves' disease (thyrotoxicosis or antibodies to a 64kDa membrane protein present on
Basedow's disease) where a direct link with autoim- both thyroid and eye muscle.
munity came with the discoveryby Adams and Purves
of thyroid-stimulating activity in the serum of these
Muscle and nerve
patients (Milestone 19.1)' ultimately shown to be due
to the presence of antibodies to TSH receptors (TSH- The transient muscle weakness seen in a proportion of
Rs), which seem to act in the same manner as TSH babies born to mothers with myasthenia gravis calls
itself (cf. p. 345). Both operate through the adenyl to mind neonatal thrombocytopenia and hyperthy-
cyclase system as indicated by the potentiating effect roidism and would certainly be compatible with the
of theophylline, and both produce similar changes transplacental passage of an IgG capable of inhibiting
in ultrastructural morphology in the thyroid cell, neuromuscular transmission. Strong support for this
but it is one of Nature's 'passive transfer experiments' view is afforded by the consistent finding of antibodies
which links TSH-R antibodies most directly with to muscle acetylcholine receptors (ACh-Rs) in myas-
the pathogenesis of Graves' disease. When thyroid- thenics and the depletion of these receptors within the
stimulating antibodies (TSAbs) from a thyrotoxic motor endplates. In addition, myasthenic symptoms
mother cross the placenta, they cause the production can be induced in animals by injection of monoclonal
of neonatal hyperthyroidism (figure 20.1), which antibodies to ACh-R or by active immunization with
resolves after a few weeks as the maternal IgG is the purified receptors themselves. Nonetheless, the
catabolized. majority of babies with myasthenic mothers do not
There is a good correlation between the titer of TSAb display muscle disease and it may be that they are pro-
and the severity of hyperthyroidism. Because TSAbs tecting themselves through production of antibodies
act independently of the pituitary-thyroid axis, iodine directed to idiotypes on the maternal autoantibodies.
Many myasthenics develop thymomas bearing mole- tors are a rare exotic species found in patients with
cules which cross-react with ACh-R although struc- acanthosis nigricans (type B) and ataxia telangiectasia
turally not belonging to that gene family. It must be on associated with insulin resistance.
the cards that molecular mimicry based on compara-
ble peptide sequences may prime autoreactive T-cells
which then drive genuine anti-ACh-R responses. An
association with ACh-R polymorphism hints at some
Gut
contribution to risk from the autoantigen.
Neuromuscular defects can also be elicited in Some patients with autoimmune atrophic gastritis
mice injected with serum from patients with the Lam- diagnosedby achlorhydria and parietal cell antibodies
bert-Eaton syndrome containing antibodies to presy- (see above and table 19.2)just meander on year after
naptic calcium channels. Autoantibodies to sodium year without developing the vitamin B,, deficiency
channels which cross-react with Campylobacter bacilli which precipitates pernicious anemia. It is probable
have been identified in Guillain-Barre syndrome, a that autoallergic destruction is roughly balanced by
self-resolving peripheral polyneuritis. Rather more regeneration of mucosal cells, an explanation which
wayout is Rasmussen's encephalitis, a childhood dis- could account for the observation that high doses of
ease of relentless and intractable focal seizures with an steroidsmay restore gastric function in certain patients
inflammatory histopathology in the brain; these pa- with pernicious anemia. However, the balance would
tients have antibodies which act as agonists and kill be upset were the patient now to produce antibodies
kainic acid-responsive neurons through overstimula- to intrinsic factor in the lumen of the gastrointestinal
tion of type 3 glutamine receptors. Would one uncover tract; these would neutralize the small amount of in-
yet more phenomena of this kind in other neurological trinsic factor still available and the body would move
disorders if the search was widened and intensified? into negative balance for B,,. The symptoms of B,, de-
ficiency pernicious anemia and sometimes subacute
degeneration of the cord, would then appear some
Stomach
considerable time later as the liver stores became
The underlying histopathological lesion in pernicious exhausted (figure20.2).
anemia is an atrophic gastritis in which a chronic in- The normally acquired tolerance to dietary proteins
flammatory mononuclear invasion is associated with seems to break down in celiac disease where T-cell
degeneration of secretory glands and failure to pro- sensitivity to wheat gluten in the small intestine can be
duce gastric acid. The development of achlorhydria is demonstrated. Since gluten can bind strongly to the
almost certainly accelerated by the inhibitory action extracellular matrix protein, endomysium, one could
of antibodies to the gastric proton pump, an H+,K+- hypothesize that uptake of the complex by IgA B-
dependent ATPase located in the membranes of the cells specific for endomysium would 'piggy-back' the
secretory canaliculi, and possibly also the gastrin gluten into the B-cell for processing and presentation
receptors. on MHC class I1 to gluten-specific T-helpers (cf. figure
The idea that some cases of gastric ulcer may result 19.13). Stimulation of the B-cell would now follow
from the stimulation of acid secretion by activation with secretionof the IgAendomysialantibodieswhich
through antibodiesto histamine receptors is appealing are exclusive to patients with celiac disease. Together
and we still await the further work required to estab- with the increased expression of Fca receptors in the
lish its validity. lamina propria and evidence of complement and
eosinophil activation, it is conceivable that antibody-
mediated mechanisms could be pathogenic.
Other cellular receptors
Some patients with atopic allergy have serum block-
S kin
ing antibodiesto P-adrenergic receptors and these may
represent just one of many different types of factor An antibody pathogenesis for pemphigus vulgaris is
which could alter the baseline sensitivity of mast cells favored by the recognition of a 130kDa autoantigen on
and make the individual more at risk for the develop- stratified squamous epithelia1cells which is a member
ment of disease. The flip side of the coin is revealed of the cadherin family of Ca2+-dependentadhesion
in the cardiomyopathy of Chagas' disease where anti- molecules. Likewise, antibodies to desmoglein 1
bodies to these receptors act as agonists and increase probably mediate the blistering of the epidermis in
the heart rate. Antibodies which block insulin recep- pemphigus foliaceus.
Figure 20.2. Pathogenesisof pernicious
anemia. Patientswith long-standing
atrophic gastritishaving parietal cell but
no intrinsicfactor antibodiesdo not go into
negative B,, balance. Pernicious anemia
develops when intrinsic factor antibodies
become superimposed upon the atrophic
gastritis.(AfterDoniachD. & Roitt I.M.
(1964)Seminars in Hematology I, 313.)
these will bind through the histone (and presumably antibodies gave rise to the production of new anti-
cationic anti-DNA) to extracellular heparan sulfate bodies of similar idiotype and specificity-in biblical
where they can accumulate and damage end-organ terms, 'antibody begets antibody'. Suppose a major
targets such as the kidney glomerulus. public idiotype network is kicked into action by a
There is yet another 'piggy-backf pathway. The pos- microbial infection (cf. figure 19.14); for example, the
sible involvement of idiotypes was mooted in the last 16/6 idiotype on a human anti-DNA circulating as
chapter by reference to experiments in which immu- a natural autoantibody is also carried on a germ-line
nization of mice with human monoclonal antinuclear antibody to Klebsiella. A T-helper cell recognizing
Figure 20.7. Conceivable pathogenetic pathway leading to end- merulonephritis. The high incidence of lupus in Clq deficient indi-
organ damage in SLE. Nucleosomes derived from apoptotic cells viduals and the susceptibility of lupus patients to skin rashes on
can stimulate anti-DNA production by a 'piggy-back' mechanism exposure to UV in sunlight, which induces apoptosis in skin
in susceptible hosts. The resulting complexes bind to heparan sul- cells, are well known. SAP, serum amyloid precursor; APC, antigen-
fate in the glomerular basement membrane where they induce glo- presenting cell; GN, glomerulonephritis.
processed 16/6 Id could stimulate anti-DNA B-cells IgG autosensitization and immune
which had captured a complex of DNA with the complex formation
Id-positive natural autoantibody (cf. figure 19.13and
Autoantibodiesto the IgG Fc region (see figure 20.lOa),
p. 208).
knownas antiglobulins or rheumatoid factors, are the
hallmark of the disease, being demonstrable in virtu-
ally all patients with RA. The majority have IgM
antiglobulins which react in the classical latex and
Morphological evidencefor
sheep cell agglutination tests (table 19.2; note 7), and
immunological activity
both they and the 'seronegative' patients who fail to
The joint changes in RA are in essence produced by react in these tests can be shown to have elevated levels
the malign growth of the synovial cells as a pannus of IgG antiglobulins detectable by solid-phase im-
overlaying and destroying cartilage and bone (figure munoassay (cf.p. 429; figure 20.9).
20.8a-f). The synovial membrane which surrounds If, therefore, au tosensitization to IgG is an almost
and maintains the joint space becomes intensely cellu- universal feature of the disease, one might expect plas-
lar as a result of considerable immunological hyper- ma cells in the synovium to be synthesizingantiglobu-
reactivity, as evidenced by large numbers of T-cells, lins. In fact 10-20% bind fluoresceinated IgG, either in
mostly CD4, in various stages of activation, usually as- the form of heat-aggregated material (figure 20.8j) or
sociated with dendritic cells and macrophages (figure immune complexes (rheumatoidfactor is a low affinity
20.81); clumps of plasma cells are frequently observed antibody and good binding is only seen when multiva-
and sometimes even secondaryfollicleswith germinal lent IgG is used as antigen).We must take into account
centers are present as though the synovium had be- a strange and unique feature of IgG antiglobulins;
come an active lymph node (figure 20.8g-i). Indeed, it because they are both antigen and antibody at the
has been estimated that the synthesis of immunoglo- same time, they are capable of self-association (figure
bulins by the synovial tissue ranks with that of a stimu- 20.10b) and this hides the majority of free antiglobulin
lated lymph node. There is widespread expression of valencies. Cleverly realizing that destruction of the Fc
surface HLA-DR (class11);T- and B-cells, dendritic and regions by pepsin would liberate these hidden binding
synovial lining cells and macrophages are all positive, sites, Munthe and Natvig observed that a greater per-
indicative of some pretty lively action (figure 20.8k). centage of the plasma cells in the synovium displayed
The thesis is that this fiery immunological reactivity an anti-IgG specificity following treatment with this
provides an intense stimulus to the synovial lining enzyme.
cells which undergo a Dr Jekyll to Mr Hyde trans- IgG aggregates, presumably products of these plas-
formation into the invasive pannus which brings ma cells, can be regularly detected in the synovial
about joint erosion through the release of destructive tissues and in the joint fluid where they give rise to typ-
mediators. ical acute inflammatory reactions with fluid exudates.
Analysis shows them to consist almost exclusively era1 structure shown in figure 20.11a and the terminal
of immunoglobulins and complement, while a major galactose lies in a special 'lectin-like' pocket (figure
proportion of the IgG is present as self-associated 20.11b). Some chains end in N-acetylglucosamine
antiglobulin as shown by binding to an Fcy im- and lack the terminal galactose sugars. What is ex-
munosorbent after treatment with pepsin. traordinary is that the percentage of sugars com-
pletely lacking galactose in the IgG of both juvenile
and adult RA patients is nearly always higher than
Abnormal patterns of IgGglycosylation
in the controls and can be as high as 60%. This
The two C,2 domains in the Fc region are held apart abnormal glycosylation could have four possible
(cf. p. 48) by two asparagine-linkedsugars of the gen- consequences.
Figure 20.8. (Opposite)Rheumatoid arthritis (RA). (a) Hands of a sue stained simultaneouslyfor IgM (with fluorescein-labeledF(ab'),
patient with chronic RA showing classical swan-neck deformities. anti-p) and rheumatoid factor (with rhodamine-labeled aggregated
(b) Diagrammatic representation of a diarthrodial joint showing Fcy).Two of the four IgM-positiveplasma cells appear to be synthe-
bone and cartilagenous erosions beneath the synovial membrane- sizingrheumatoid factors. (k)Rheumatoid synoviumshowinglarge
derived pannus. (c) Proximal interphalangeal joint depicting numbers of cells stained by anti-HLA-DR (anti-class11).(1)Rhema-
marked bony erosion and marginal erosion of the cartilage. (d)Early toid synovium showing class 11-positive accessory cells (green) in
pannus of granulation tissue growing over the patella. (e)Histology intimate contact with CD4+T-cells (orange).(m) Large rheumatoid
of pannus showing clear erosion of bone and cartilage at the cellular nodules on the forearm. (n) Granulomatous appearance of the
margin. (f) Histology of the pannus stained for macrophage nonspe- rheumatoid nodule with central necrotic area surrounded by epithe-
cific esterase; note long, stained dendritic processes. (g) Chronic in- lioid cells, macrophages and scattered lymphocytes. Plasma cells
flammatory cells in the deeper layers of the synovium in RA. (h) A making rheumatoid factor are often demonstrable and the lesion
hypervillous synovium revealing well-formed secondary follicles probably represents a response to the formation of insoluble anti-
with germinal centers (relatively rare occurrence). (i)A high power IgG complexes. (Kindly given by (a) Dr D. Isenberg; (c), (d), (e),(g),
view of an area of diseased synovium showing collections of classi- (h) and (i) Dr L.E. Glynn; (f) Professor J. Edwards; (j) Professors
cal plasma cells. (j) Plasma cells isolated from a patient's synovial tis- P. Youinou and P. Lydyard; and (k) and (1)Professor G. Janossy.)
Figure 20.11. The Fc sugars and their role
in bridging the two C,2 domains of IgG.
(a)Typical structure of each N-linked
sugar. Some chains lack one (Gl)or both
terminal galactoses (agalacto-IgG;GO).
(b)Structure of the C,2 regions and the
association between the terminal galactose
on the 1,6 arm and the protein surface. The
1,3 arms, one of which must lack galactose,
bridge the two domains. GlcNAc, N-
acetylglucosamine;Man, mannose; Gal,
galactose; Fuc, fucose.
(p. 414). The plot deepens with the realization that or of the enteric tract with Yersinia, Salmonella, Shigella
QKRAA binds to a second E. coli heat-shock protein or Campylobacter.The synovial tissue in reactive arthri-
dnaK and that HLA-DR containing the QKRAA se- tis remarkably still retains antigenic descendants or
quence binds the selfanalogue of dnaK, namely hsp73, memorials of the initiating bacteria many years after
which targets selected proteins to lysosomes for pro- infectionwhich can drive local T-cells.All the microbes
cessing. What this all means remains to be resolved are either obligate or facultative intracellular bacteria
but note the involvement of the hsp family yet again. and so may escape the immune system by hiding
Suspicion of previous microbial encounters is en- inside cells, probably aided by high local production of
gendered by the discovery by PCR amplification of IL-4. However, we may be dealing with molecular
nucleotide sequences characteristic of Mycoplasma mimicry. Natural infection with Salmonella typhimu-
fermentans, Chlamydia and Epstein-Barr virus in a sub- rium generates CD8 cytotoxic T-cells which recognize
stantial proportion of synovial tissues removed from an immunodominant epitope of the GroEL molecule
RApatients.Further work on the intracellular location, presented by the class Ib Qa-l and cross-react with a
molecular status and possible expression of this ma- peptide from mouse hsp60, so permitting a reaction
terial is awaited with interest. with stressed macrophages. HLA-B27 individuals are
The antigenic history of reactive arthritis is more particularly at risk and the importance of the microbial
amenable to study since it is triggered by an infection component is emphasized by experiments on mice
either of the urogenital tract by Chlamydia trachomatis bearing the B27 transgene; if reared in a germ-free
environment, lesions are restricted to the skin, but in sponses can downregulate destructive Thl cells. Last,
the microbiological wilderness of the normal animal the Thl/Th2 polarization is not apparent in diseases
house, the skin, gut and joints are all affected. Why, such as myasthenia gravis, Graves' thyrotoxicosis,
as in RA, are the joints targeted and what does B27 Sjogren's syndrome and primary biliary cirrhosis.
do? Only one in 300 of the T-cells in the reactive arthri-
tis synovium is CD8 and therefore class I restricted. It
Autoimmune thyroiditis
could be that a cross-reactive B27 sequence functions
as a cryptic epitope perpetuating a gentle microbial The inflammatory infiltrate in autoimmune thyroiditis
stimulus with an amplifying autoimmune response. is usually essentially mononuclear in character (see
Two experimentally induced models of arthritis are figure Ml9.l .lc) and, although not an infallible guide,
heavily dependent on T-cells. Adjuvant arthritis re- this has been taken as an expression of T-cell-mediated
sulting from immunization of rats with complete hypersensitivity. Firm evidence for a direct participa-
Freund's adjuvant (CFA) only can be transferred to tion of T-lymphocytes has yet to be provided, although
naive recipients with a T-cell clone specific for the the demonstration of class I1 molecules on patients'
mycobacterialheat-shock protein hsp60. The collagen thyrocytes and the presence of antigen-specific Thl
arthritis model involves injection of type I1 collagen in cells in the thyroid would accord with an involvement
complete Freund's, but here a synergy between cell- of these cells.
mediated hypersensitivity and antibody seems to We must turn to the animal models for further
operate:sensitizationwith denatured collagenin CFA- evidence albeit indirect. Draconian stamping out of
induced T-cell-mediated immunity but no arthritis T-cells in the Obese strain chicken by neonatal thymec-
unless the mice were also given IgG from animals tomy, and repeated injection of anti-T-cell serum,
primed with native collagen (figure20.13). prevented the spontaneous development of atrophic
Spontaneousmodels of arthritis are hard to come by. autoimmune thyroiditis. It is also of interest to note
Up to 30% of MRL/lpr mice have arthritic lesions but that, at the target cell level, the threshold for induction
this incidence varies with the animal house. If animals of MHC class I1 on OS thyrocytes by IFNy is far lower
harbor Sendai virus, they are protected from arthritis; than that reported for normal thyroid cells, further
what is that telling us? To complete the circle, they also reinforcing the notion that a thyroid abnormality is a
have raised levels of agalacto-IgG. contributory factor to the susceptibility phenotype.
Before leaving the subject, one should not overlook The other model, in which thyroiditis is induced by
the curious behavior of fibroblasts isolated from thyroglobulin in complete Freund's adjuvant (see
synovial tissue in established rheumatoid arthritis. In figure M19.1.lb), can be transferred to naive histo-
culture, they secrete collagenase which would con- compatible recipients with CD4+T-cell clones specific
tribute to cartilage breakdown, but more ominously for peptides containing thyroxine established from im-
they proliferate spontaneously due to unrestrained munized animals. The cells infiltrate between the thy-
control of the cell cycle by cyclin-dependent kinases 4 roid follicles and probably kill the epithelia1 cells by a
and 6 and their respective D-cyclins. This implies that combination of locally released IFNy and TNF. We see
they would be refractory to conventional therapies now that there is considerable diversity in the auto-
and might be a cause of apparently intractable disease. immune response to the thyroid leading to tissue des-
truction, metabolic stimulation, growth promotion or
mitotic inhibition which in different combinations ac-
count for the variety of forms in which autoimmune
To make a fairly sweeping statement, inflammatory thyroid disease presents (figure20.15).
organ-specific diseases are generally linked to T-
helper-l (Thl) responses. Clones producing EAE or
Insulin-dependent diabetes mellitus (IDDM)
transferring diabetes from NOD mice produce IL-2
and y-interferon (IFNy), while in collagen arthritis IL- Just as in autoimmune thyroiditis, IDDM involves
12 can be substituted for the mycobacteria in the com- chronic inflammatory infiltration and destruction of
plete Freund's adjuvant. On the other hand, Th2 CD4s the specific tissue, in this case the insulin-producing
are responsible for the polyclonal activation in murine 0-cells of the pancreatic islets of Langerhans. The delay
lupus, the glomerulonephritis and necrotizing vas- in onset of disease achieved by early treatment with
culitis induced in Brown Norway rats by mercuric cyclosporin, at levels which have little effect on anti-
chloride, and the chronic autoimmunity generated body production, points an accusing finger at effector
during graft-vs-host disease. We will see that Th2 re- T-cells as the agents of destruction, since this drug tar-
young nondiabetic NOD mice inhibited this transfer,
but the same subset from older diabetic mice had lost
its regulatory power and had become pathogenic. This
change to a Thl-type response coincided with the
onset of diabetesand is consistent with the observation
that IL-12, which favors Thl responses, exacerbates
disease.Just as in the human disease, MHC class I1alle-
les hold a pivotal controllingposition and introduction
of a transgene, in which residues at position 56 or 57 of
the H-2Ap chain are altered, drastically inhibits the de-
velopment of diabetes. One of the non-MHC suscepti-
bility loci in NOD mapped to the IL-1R and Bcg genes
on chromosome 1 was associated with natural resis-
tance to infectionby intracellular parasites.As a result,
the NOD mouse is resistant to Mycobacterium avium,
but after recovery from infection the onset of diabetes
is prevented. That responses to hsp60 may be involved
Figure 20.15. Relationship of different autoallergic responses to is implied by reports that a 24-amino acid peptide from
the circular spectrum of autoimmunethyroid diseases. Responses
involving thyroglobulin and the thyroid peroxidase (microsomal)
this protein is the target of diabetogenic T-cells in both
surface microvillous antigen lead to tissue destruction, whereas patients and the NOD mouse, and treatment with this
autoantibodies to TSH (and other ?) receptors can stimulate or peptide or with a NOD peptide-specific Thl clone
block metabolic activity or thyroid cell division. 'Hashitoxicosis' downregulates spontaneous disease. Cohen has inter-
is the down-to-earth term used by our Scots colleagues to describe
a gland showing Hashimoto's thyroiditis and thyrotoxicosis
preted this as a result of dysregulationof the 'immuno-
simultaneously. (Courtesy of Professors D. Doniach and G.F. logical homunculus' (cf.p. 208) and notes that the level
Bottazzo.) of a particular idiotype associated with a TCR CDR3
consistently falls prior to the onset of diabetes, while
mice reared under germ-free conditions which might
inhibit the development of a natural idiotype network
are more susceptible to IDDM. Time will test the valid-
gets T-cell cytokine synthesis so specifically. In vitm T- ity of this hypothesis. Notwithstanding this evidence
cell responses to islet cell antigens including glutamic relating to hsp and idiotypes, in the final analysis, one
acid decarboxylase (GAD) directly reflect the risk of has to take into account the following: up to 50% of
progression to clinical IDDM. The strength of the risk the infiltratingT-cells isolated from pre-diabetic NOD
factors associated with certain HLA-DQ alleles also islets are insulin-specific and can transfer disease to
has a strong whiff of T-cell action although, quite young NOD mice; GAD-specific T-cells can also be
mysteriously, monocytes (and dendritic APCs?) con- recovered and are also diabetogenic; and tolerance to
stitutively expressing cyclooxygenase-2 (COX-2), the either insulin or GAD prevents the onset of disease.
inducible enzyme responsible for the synthesis of Presumably the latter can be accommodated by an
prostaglandin E2 and other prostanoids, were present organ-related bystander tolerance mechanism de-
in relatives (of IDDM patients) bearing these alleles scribed below (p. 445). Overall, the data seem to be
and in the patients themselves. consistent with the necessity for two pathogenic
To obtain further insight into the cellular siege pathways, one dependent on hsp and the other on an
and destruction of the islet p-cells, one has to turn to organ-specific response, operating either synergisti-
the Nonobese diabetic (NOD) mouse which sponta- cally or serially, to achieve the final destruction of the
neously develops diabetic disease closely resembling pancreatic p-cells.
human IDDM in its range of autoimmune responses GAD in the central and peripheral nervous system
and the association of islet breakdown with a chronic produces y-aminobutyric acid (GABA), a major in-
infiltration by T-cells and macrophages (figure 20.16). hibitory neurotransmitter, from glutamine. Autoanti-
T-cells infiltrating the islets in diabetic mice had a bodies to GAD are seen not only in early diabetes, but
Thl-type cytokine profile and could transfer disease also in Stiff man syndrome (sounds like a cue for a
to NOD recipients congenic for the severe com- Western) where the GABA-ergic pathways controlling
bined immunodeficiency ( S C D ) mutation. The motor neuron activity are defective. The antibodies
C D ~ / C D ~ ~ Rmemory
B I O subset of splenocytes from cannot be pathogenic because GAD is present on the
Figure 20.16. Destruction of pancreatic islet p-cells by infiltrating stained by rhodamine-conjugated antibodies and T-cells by fluores-
T-cells in the Nonobese diabetic (NOD) mouse. (a) Normal intact ceinated anti-CD3. (Data reproduced from Quartey-Papafio R.,
islet. (b) Early peri-islet infiltration. (c) Penetration of the islet by Lund T., Cooke A. et al. (1995)Journal of Immunology 154,5567; pho-
infiltrating T-cells. (d) Almost complete destruction of insulin- tographs kindly provided by Dr Jenny Phillips.)
producing cells with replacement by invading T-cells. Insulin
inner surface of the plasma membrane, but T-cells ably the T-cell incites a local inflammation affecting
could be. How the brain as distinct from the pancreatic the endothelial cells at the blood-brain barrier which
islet could be specifically targeted is a conundrum but opens the gate for antibody to penetrate the brain
30% of patients do develop IDDM. tissue.
How much of this is relevant to human disease?
First, the serologically determined Caucasian DR2
phenotype (DRBl"1501, DQAl"0102, DQBl"0602) is
The idea that MS could be an autoimmune disease strongly associated with susceptibility to MS. At least
has for long been predicated on the morphologi- 37% of activated T-cells responsive to IL-2/4 in cere-
cal resemblance to experimental autoimmune en- brospinal fluid were specific for myelin components,
cephalomyelitis (EAE),a demyelinating disease lead- compared with a figure of 5% for subjects with other
ing to motor paralysis (figure 20.17) produced by neurological disturbances.A Leu.Arg.Gly. amino acid
immunization with myelin, usually myelin basic pro- sequence motif found in around 40% of TCR VP5.2
tein (MBP) in complete Freund's. T-cell clones specific N(D)N rearrangements in T-cells from MS lesions was
for MBP belong to restricted TCR VP families. They present in a VP5.2 clone from an MS patient cytotoxic
will transfer disease but this can be exacerbated by towards targets containing the MBP 89-106 peptide
injection of a monoclonal antibody to Theiler 'S virus, and in encephalitogenic rat T-cells specific for MBP
a murine encephalomyelitis virus, cross-reactingwith peptide 87-99. One is greatly encouraged to continue
an epitope on myelin and oligodendrocytes. Presum- with attempts to induce tolerance.
Figure 20.17. Experimental autoimmune encephalomyelitis lesions in dorsal columns, in both left (large) and right (small)
(EAE), a demyelinating model for multiple sclerosis induced by columns, as well as on lower left.Also gray matter involved with on-
immunization with brain antigens in complete Freund's adjuvant going inflammation, in particular affectingleft dorsal horn. Normal
(CFA). (a)Early lesion of EAE in the rat at 9 days after immunization myelin is stained brown. (c) Chronic relapsing EAE in guinea-pig.
with rat spinal cord homogenate in CFA. The lesion in brain white Large demyelinated plaques in brain white matter (arrows)closely
matter, which is probably a few hours old, shows perivenous infil- similar to plaques of multiple sclerosis. (d) Acute EAE in cat with
tration of lymphocytes and monocytes (a pure mononuclear inflam- optic nerve involvement. (Legend and slides provided by Dr
mation) with cells invading the nervous parenchyma.Myelin is not B. Waksman; (b) originally from Dr Trotter, (c) from Drs Lassmann
stained. (b)Lumbar spinal cord of rat with chronic EAE after immu- and Wisniewski and (d) from Dr Patterson.)
nization with myelin proteolipid protein. Large demyelinating
Figure 20.19. Expression of heat-shock protein 60 in an early The majority of approaches to treatment, not un-
human arterioscleroticlesion.Frozen, unfixed, 4 pm thick sectionof naturally, involve manipulation of immunological
a fatty streak (=early lesion) of a human carotid artery stained in responses (figure 20.20). However, in many organ-
indirect immunofluorescence with a monoclonal antibody to heat-
shock protein 60 and a fluorescein-labeledsecond antibody.Astrong specific diseases, metabolic control is usually suffi-
reaction with endothelial cells as well as cells infiltratingthe intima, cient, e.g. thyroxine replacement in primary myxe-
including foam cells, is evident. (Original magnification X 400.) dema, insulin in juvenile diabetes, vitamin Bl, in
(Photographkindly provided by Professor G. Wick.) pernicious anemia, antithyroid drugs for Graves' dis-
ease, and so forth. Anticholinesterase drugs are com-
monly used for long-term therapy in myasthenia
dietary intake of antioxidants, and the observationthat gravis; thymectomy is of benefit in most cases and it
the induction of atherosclerotic fatty streaks by high is conceivable that the gland contains acetylcholine
cholesterol diets in rabbits can be prevented by the (ACh) receptors in a particularly imrnunogenic form
antioxidant probucol, despite its lack of influence on (?associated with HLA class I1expression).
plasma cholesterol levels. Yet another piece in the It is worth recording that maintenance therapy to
jigsaw: p*-glycoprotein-l, which figures prominently replace the loss of an organ-specific molecule, such as
in the antiphospholipid syndrome (cf. p. 421), is also insulin in IDDM, might have the effect of subduing
abundantly present in atherosclerotic lesions. Clarifi- metabolic activity and reducing expression of the tar-
cationisawaited. get antigen. Help should be on the way for patients
with burnt-out pancreatic p-cells. Xenografts of ge-
netically engineered fetal or neonatal pig islets (cf. fig-
DIAGNOSTIC VALUE OF
ure 17.15)are under study, and other good news is that
AUTOANTIBODY TESTS
stem cells contained within ductal structures of the
Serum autoantibodies frequently provide valuable adult pancreas can be differentiated in culture to pro-
diagnostic markers. The most useful routine test is vide a 10000-fold increase in the number of available
screening of the serum by immunofluorescence on a islets per organ, although any recurrence of immuno-
frozen section prepared from a composite block of un- logically mediated damage would need to be dealt
fixed human thyroid and stomach, and rat kidney and with. Perhaps transfection of the grafted cells with
liver. This is supplemented by agglutination tests for TGFp might provide an appropriately suppressive
rheumatoid factors and for thyroglobulin, thyroid microenvironment. A slightly cheeky but novel ap-
peroxidase and red cell antibodies and by ELISA for proach to the repair of damaged target organs is to
antibodies to intrinsic factor, DNA, IgG, extractable target a nonpathogenic specific T-cell clone expressing
nuclear antigens, and so on (see table 19.2).The salient a transgenic growth factor to the inflamed area. Thus,
information is summarized in table 20.1. ELISAs a nonaggressive Th2 clone, specific for the proteolipid
are taking over and tests with purified gene-cloned (PLP) brain antigen, delivered a platelet-derived
antigens arranged in minispot arrays will one day growth factor (PDGF-A) transgene to the inflamed
supplant the need for immunofluorescence which is brain of an animal with EAE; contact with antigen
time-consumingand more skilled. stimulated the clone and the secreted growth factor
The tests will also prove of value in screening for induced proliferation of the oligodendrocyte pro-
people at risk, e.g. relatives of patients with autoim- genitor cells involved in remyelination.This would be
mune diseases such as diabetes,thyroiditis patients for a highly customized therapy only suitable for the well-
Table 20.1. Autoimmunity tests and diagnosis.
ANTIBODY COMMENT
Pernicious anemia Stomach deip n oiagnosis 01 latent PA, in diierential dlagnosis of non-auta mmune
megaloblon c memo and in s u s w e a SJbOCute m b i n e d aegenerotion of me cord
Poncreas InsLlin Ab early in dswse GAD Ab standard test for IDDM. Two or more autoAb se8n
diabetes meliillls (IDDM) in 80% of new onset cnlldren or prediabmc relatives but not in conmis
Muscle When postive suggests ossociotedlhymomo (more likely fl HLA-BI 2). m n N e in >80%
ACh receptor
Autoimmune hemlylic Erylhrmyte (bmbs' test) Ditimdon from other forms of onemia
Primary Uliory cirrhosis Mtochmdriol Distinclmn from omer lams ot obstructivejound:ce where iesl rarely I v e
Remgnize suogroup wnhin cryptogeniccirfhosis reioted to PBC with +ve mitachondnal Ab
Active chronic hepatitis Smaoth muscle onn-nuc.eor Smoorh muscle ~b 0.3n g m from S E
and 2090 m lochondnol ~ y p eI c.os.co~in women wrm Ab 10 nLclei, smoom muscle. actin and osmlogn/coprotein
receptor (these Ab disappear on remlsion indicating reductan :n steroids)
rype 2 in giris and yomg wamen wdh anti-LKM-1 (cy P450)
Rheumataid arthritis Antiglobuiin,e.g. SCAT and Hign lser inaim ve ot bcd prognosis
latex fixation
+
Anfialobulin mised Prognosis ot rhwmotoid anhnhs
og$acta-lg
Perinuclear v sp for eady RA Dom non1resiaue citruiiine (post-nonslat onoi moolflcmon at orginine)
High titer antinuclear, DNA DNA anti& es present in active phase ~b to doLblestranded DNA CharadenBQ high
aninry campiement-fixing ~b give kmney damage. IOW omnrly CNS lesions
I Wegener's granuiomotasis Neulmphiicytoplasm Antiserlne protease cioseiy associatedwith disease; treatment urgent
heeled, but a cheaper gambit is to inject a plasmid jected to y-irradiation, which induces the synthesis
DNA coding for Fas ligand in liposomes directly into of the ~ 1 senescence
6 protein.
~ ~This is~a tumor
the organ under attack. This manipulation, carried out suppressor which blocks the stable association of
in an experimental autoimmune thyroiditis model, in- cyclin-dependentkinases 4 and 6 with their respective
duced persisting expression of FasL on thyroid folli- D-cyclins and inhibits their ability to mastermind the
cular cells and total abrogation of antithyroglobulin passage of cells into the G1 phase of the cell growth
cytotoxicT-lymphocytes. cycle. Injection of the RA fibroblasts with a recombi-
The reader may recall the spontaneousproliferation nant adenovirus encoding the gene halted
of RA synovial fibroblasts when placed in culture. their growth and reduced synovial cell hyperplasia in
These cells undergo irreversiblecell cycle arrest if sub- the adjuvant arthritis model. If focus on expression of
Figure 20.20. The treatment of
autoimmune disease. Current
conventional treatmentsare in dark orange;
some feasible approachesare given in
lighter orange boxes. (In the case of
a live graft, bottom right, the
immunosuppressivetherapy used may
protect the tissue from the autoimmune
damage which affected the organ being
replaced.)
this gene fosters a new therapeutic approach to RA, it In RA, steroidsare very effective, but the recogrution
might also find utility in other disorders such as ath- of a defective pituitary-adrenal feedback loop in these
erosclerosis, scleroderma and possibly late stage asth- patients has inspired a novel approach aiming to
ma. Conceivably, the benefit of adding methotrexate to restore normal corticosteroid levels by a depot of
anti-TNF therapy in RA (see below) might be partly methylprednisolone (Depomedrone)which delivers a
attributable to an effect on fibroblastproliferation. low daily dose-the earlier in the disease the better.
Based on the possibility that multiple sclerosis is This treatment accelerates the induction of remission
virally driven, patients have been treated with IFNP; and decreases the side-effects of second-line agents
relapse rates were reduced by a third in relapsing- such as gold salts. Selectinsand adhesion molecules on
remitting disease, but there was only a modest effect endothelial cells and leukocyte integrins appear to be
on progressive disease. Note, however, that IFNP downregulated and this would seriously impede
influences some T-cell functions in addition to its the influx of inflammatory cells into the joint. Anti-
effect on viral proliferation. inflammatory drugs such as salicylates, innumerable
synthetic prostaglandin inhibitors and metallopro-
teinase poisons are widely used. The so-called second-
line drugs, sulfasalazine, penicillarnine, gold salts
Patients with severe myasthenic symptoms respond and antimalarials such as chloroquine, all find an
well to high doses of steroids and the same is true for important place in therapy but their mode of action is
serious cases of other autoimmune disorders, such as unknown.
SLE and immune complex nephritis, where the drug Treatment with antibodies to adhesion molecules
helps to suppress the inflammatory lesions. such as CD44 effectivelyblocks experimental arthritis
but there are considerable practical and economic Transfection of synovial cells with the natural IL-1 re-
problems in adapting to human disease. Therapeutic ceptor antagonist IL-1Ra may turn out to be a useful
blocking of other mediators directly concerned in im- long-term strategy.
munological tissue damage will be feasible as cytokine
and complement antagonists become available. Neu-
tralizing TNF with a humanized monoclonal antibody
is most effective in the short term, so revealing In a sense, because it blocks cytokine secretion by T-
the pathogenetic role of this cytokine. Most signifi- cells, cyclosporin is an anti-inflammatory drug and,
cantly, synergistic administration with methotrexate since cytokines like IL-2 are also obligatory for lym-
does seem to offer more lasting benefit (figure 20.21). phocyte proliferation, cyclosporin is also an antimito-
tic drug. It is of proven efficacy in uveitis, early type
I diabetes, nephrotic syndrome and psoriasis and
of moderate efficacy in idiopathic thrombocytopenic
purpura, SLE, polymyositis, Crohn's disease, primary
biliary cirrhosis and myasthenia gravis. In a double-
blind randomized control trial, cyclosporin demon-
strated significant though not complete disease sup-
pression over 12 months in a group of previously
refractory rheumatoid arthritis (RA) patients. Unfor-
tunately, high toxic doses were used but the synergy
with rapamycin is a strong indicationfor a trial of com-
bined therapy. Leflunomide is a promising new agent
for treatment of RA. Its active metabolite inhibits de
novo rUMP synthesis leading to G1 arrest of cycling
lymphocytes.
While awaiting more selective therapy, convention-
al nonspecific antimitotic agents such as azathioprine,
cyclophosphamide and methotrexate, usually in com-
bination with steroids, have been used effectively in
SLE, RA, chronic active hepatitis and autoimmune he-
molytic anemia for example. High-dose i.v. cyclophos-
phamide plus adrenocorticotropic hormone (ACTH)
or total lymph node irradiation through its effect
on the peripheral immune system either slowed or
stopped the advance of disease in approximately two-
thirds of progressive multiple sclerosis (MS) patients
Figure 20.21. Synergy of anti-TNF and methotrexate in the treat- for 1-2 years, a strong indication that the disease is
ment of rheumatoid arthritis. Top panel: Duration of response mediated by immune mechanisms. This is further sup-
to therapy as defined by 20% Paulus criteria at three doses of
monoclonal chimeric anti-TNF (infliximab) with and without
ported by the unfortunate finding that IFNy exacer-
methotrexate (MTX) and placebo (Plbo) plus MTX. Results shown bates disease in the majority. Pulsing MS patients with
are the proportion (%) of patients responding at weeks 1,2,4,8,12,16 the antileukocyte humanized monoclonal Campath-
and 26. The Paulus response is achieved by 20% improvement in 1H (anti-CD52) produced a brutal and surprisingly
four out of six of the following:tender joint and swollen joint scores,
duration of morning stiffness, erythrocyte sedimentation rate and a
persistent reduction in T-cell numbers. Over a 2-year
two grade improvement in the patient's and observer's assessment period virtually no new lesions were detected,
of disease severity. Lower panel: Serial measurements (median val- although in half the patients there was progression
ues) of the tender joint count,before (day 0), during (weeks 1-14) and of pre-existing lesions. A startling 33% developed
after (weeks 14-26) treatment. Results are included only up to the
point at which 250% of patients remained in the trial (up to week 6
Graves' disease (wow!), although this disturbing
for the placebo plus MTX group). Arrows indicate the timing of infu- statistic was not a feature of many other disorders,
sions of infliximab at weeks 0, 2, 6, 10 and 14. Methotrexate was including RA, where Campath-1H has been used.
given weekly and virtually eradicated the production of antibodies It is true that the EAE animal model can be countered
to the human chimeric antibody. (Data kindly provided by Profes-
sors R.N. Maini, M. Feldmann et al., see Maini R.N. et al. (1998)re-
by some very diverse manipulations: the antidepres-
produced with permission from Lippincott, Williams & Wilkins, sant, roliprim, suppresses cytokine production, a non-
MD, USA, Arthritis and Rheumatism 41,1552.) hypocalcemic derivative of vitamin D3potently blocks
IL-12 secretion, type IV phosphodiesterase inhibitors for many strategies (figure 20.22). Injection of mono-
suppress Thl cells and blockade of non-N-methyl-D- clonal anti-MHC class I1 and anti-CD4 successfully
aspartate (NMDA) receptors ameliorates the neuro- fends off lupus in spontaneous mouse models, and it is
logical sequelae. The problem is that, as it is so difficult relevant to record the preliminary clinical observa-
to carry out clinical trials in MS, few if any of these tions that injection of immunoglobulins eluted from
approaches are likely to see the light of day. placentas, and shown to contain anti-allo-class11, sig-
nificantly ameliorates the symptoms of RA. Immu-
nization with a cyclic peptide from a polymorphic
region of the p chain of the Nonobese diabetic
(NOD) class I1 molecule protected a large proportion
Cellular manipulation
of the mice from disease. It is unclear whether the
It should one day be practical to correct any relevant mechanism involves a block on MHC antigen pre-
defects in stem cells or in thymus processing by gene sentation or relates to the MHC mimicry hypothesis
therapy, bone marrow or thymus grafting or perhaps, (cf.p. 413).
in the latter case, by thymic hormones. Many centers Some take the anti-IL-2receptor approach to deplete
are trying out autologous stem cell transplantation fob activated T-cells (figure20.22 (l)),but we would like to
lowing hemato-immunoablation by cytotoxic drugs refer back to our discussion of the long-lasting effect of
in severe cases of autoimmune disease. Around two- nondepleting anti-CD4 for the induction of tolerance
thirds of a series of difficult cases of SLE, scleroderma, (figure 20.22 (2)and (3)),particularly when reinforced
juvenile and adult RA and so on, stabilized or im- by repeated exposure to antigen (cf. p. 361). Antigen
proved. Transplant-related mortality risk at 2 years reinforcement of course is an obvious continuing fea-
was 8*6%, comparable to that seen with cancer ture in autoimmune disease, so that anti-CD4 should
patients. be ideal as a therapy in disorders where the natural
If defects in programed cell death in antigen- 'switch-off' tolerogenic signals are still accepted by the
activated T-cells contribute in any way to the develop- CD4 cells. Ongoing trials in RA still look promising.
ment of certain autoimmune diseases, bisindolyl- Excellent remissions have been recorded in patients
maleimide, which potentiates weak and moderate with Wegener's granulomatosis who were refractory
apoptotic signals, might be therapeutic. to normal treatment, following sequential injection of
Because T-cell signaling is so pivotal, it is the target anti-CD52 (Campath-1H)and nondepleting anti-CD4
monoclonals.
We can manage perfectly well in life without a com-
plete set of our TCR VP genes; after all, wild mice, and
presumably ourselves, delete large tracts of VD fami-
lies during thymic differentiation and it does not seem
to do them much harm. So, the argument runs, if the
autoimmune T-cell clones specific for the autoantigen
in a given disease happen to be restricted to mem-
bership of a particular VP family, we could delete all
members of that family in vivo with the appropriate an-
tiserum and yet not make irreparable holes in the
host's defenses (figure 20.22 (6)). In PL mice immu-
nized with the N-terminal peptide of myelin basic pro-
tein, anti-VD8 eliminated experimental autoallergic
encephalitis almost entirely, so the strategy can work.
Not so good with SJL mice which respond to peptide
89-101 with 50%of the T-cells using VD17 receptors; in
this case, anti-Vp17did not block disease. Clearly, until
we have a wider knowledge of the extent of VD restric-
tion for each antigen and also for each individual, we
must suspend judgment on the general feasibility of
the strategy.
Figure 20.22. Strategic options for therapy based on T-cell Now, if one takes the view that rheumatoid factor
targeting. immune complexes are major players in the patho-
genesis of the RA joint lesions, logic suggests the to compensation by idiotype-negative clones. On the
radical approach of B-cell ablation with monoclonal other hand, much more profound changes have been
anti-CD20 as used in the treatment of B-cell leukemia. achieved by treatment with monoclonal autoanti-
Results to date are provocatively encouraging. bodies (idiotypes) derived from the autoimmune
strain in question. Thus, prior administration without
adjuvant of two peptides taken from the CDRs of a
Manipulation of vegulatoy mediators
pathogenic murine l6 / 6 Id+ anti-DNA monoclonal
We can correct some spontaneous models of autoim- was reported to inhibit the induction of SLE by immu-
mune disease by injection of cytokines: IL-1 cures the nization with the intact monoclonal in CFA.
diabetes of NOD mice; TNF prevents the onset of SLE Curiously, intravenous injection of Ig pooled from
symptoms in NZBx W hybrids; and transforming many normal donors is of positive benefit in a number
growth factor-P1 (TGFP1) is known to protect against of autoimmune blood diseases, recurrent abortions
collagen arthritis and relapsing EAE. We have al- associated with cardiolipin antibodies, juvenile der-
ready reminded ourselves of the maintenance doses matomyositis and patients with autoantibodiesto pro-
of steroids to restore the defective adrenal feedback coagulant factor VIII. The latter has been studied in
control on leukocytes in RA. some detail and the inhibitory effects of F(abf), frac-
If we now take it as almost gospel that the Thl subset tions from the normal Ig pool suggest that we are deal-
is pathogenic in solid organ-specific disease, attempts ing with anti-idiotypic reactions; it is as though the
to switch the phenotype to Th2 should be beneficial. normal pool was re-establishing a properly controlled
On the assumption that IL-4 or IL-10 can effect this network. These are intriguing observations which
switch, then treatment of an autoimmune individual deserve serious consideration, although to some
with these cytokines should defuse the Thl cells, as extent their use is marred by expense.
was indeed observed in an EAE model. The exacerba-
tion of Thl-mediated disease by IL-12 raises the pos-
Vaccination with T-cell idiotypes
sibility that the highly avid dimer of one of the IL-2
receptor chains might be a potent inhibitor. However, It is possible to protect animals against the induction
with the exception of steroids in RA, there is quite a of experimental allergic encephalomyelitis by immu-
gap between therapy in experimental animals and its nization with an attenuated T-cell clone specific for
application to human disease. Our earlier suggestion myelin basic protein (MBP).This must be mediated by
of transfection of the target organ with a protective the induction of suppressor T-cells specific for the ef-
cytokine like TGFP (figure 20.22 (8)) may prove to fector cell receptor idiotype. Confirmation has come
be a good bet (visions of firing a biolistic gun at islets from experiments showing that the encephalitis can be
pretransplantation?). prevented if mice are first immunized with a synthetic
peptide from the VP chain of the encephalitogenic
clone; this procedure generates CD8 T-cells specific
Idiotype control with antibody
for the receptor peptide presented by class I MHC,
The powerful immunosuppressive action of anti- and which transfer protection against induction of
idiotype antibodieshas led to much rumination on the encephalitis.
feasibility of controlling autoantibody production by This gambit (figure 20.22 (5))has now been played
provoking appropriate interactions within the im- in human disease. A TCR peptide vaccine embodying
mune network. We have focused previously on the the VP5.2 sequence expressed in MS plaques and on T-
intimate network interactions between hormone cells specific for MBP was used to treat MS patients in
receptors, hormones and their respective antibodies a double-blind trial (10pg weekly for 4 weeks and
(cf.p. 209) and it might be that the autoimmune disor- then monthly for 10 months). Lack of response to vac-
ders involvingthese receptors are especially amenable cination was associated with increased response to
to idiotype control. There is a growing realization that, MBP and clinical progression, but successful vaccina-
in general, more fundamental suppression can be tion boosted the frequency of TCR peptide-specific T-
achieved by utilizing the internal elements of the idio- cells, reduced the frequency of MBP-specific cells and
type network rather than anti-idiotypereagents raised prevented clinical progression without side-effects.
in other species. Thus, xenogeneic anti-idiotypeshave The reactive cells were predominantly Th2-like and di-
only won transient and partial improvement in the rectly inhibitedMBP-specific Thl responses, primarily
spontaneous thyroiditis of the Buffalo rat and the au- through release of IL-10 and probably through an anti-
toimmune lupus of NZB X W mice, presumably due idiotypic regulatory network. Although many of the
T-cells in the lesions would not belong to the VP5.2 tive inserted in liposomes, can block EAE and an hsp60
family,they could be switched off by organ-related by- peptide can prevent the onset of diabetes in the
stander tolerance (see figure 20.22). NOD mouse (cf. p. 434).Awareness of the therapeutic
T-cell vaccination has been used to protect against benefit of injected insulin in the NOD model has
the spontaneous development of diabetes in NOD fostered a large-scale trial in the human disease and
mice and the production of arthritis following sensiti- considerable clinical improvement has been achieved
zation with type I1collagen. It has also proved possible in patients with exacerbating-remitting MS given
to switch off Freund adjuvant-induced arthritis with Copl, a random copolymer of alanine, glutamic acid,
an attenuated clone of T-cells generated in response to lysine and tyrosine meant to simulate MBP. Many
the 65kDa mycobacterial heat-shock protein. This ad- thousands of patients have been injected with Copl
juvant model has been looked at in depth (I. Cohen). subcutaneously every day, thereby reducing the risk
Perversely, the earliest T-cell responses preceding the of relapse from 1.5 per year at onset to less than 1 in
adjuvant-induced arthritis were antigen-specific sup- every 5 years without an increase in neurological dis-
pression and anti-idiotypicreactivity; responses to the ability. The copolymer competes with MBP and other
antigen itself emerged a few days before the appear- brain antigens for binding to the MHC and to the cog-
ance of clinical arthritis. T-cell vaccination accelerated nate T-cellreceptors and induces T-suppressorswhich,
the kinetics of the antigen response, abolished antigen- it may be said, are also generated when the antigen
specific suppression, activated anti-idiotypic T-cells is fed.
and inhibited arthritis. The extremely rapid appear- We have already noted that, because the mucosal
ance of anti-idiotypeand antigen-specificsuppressors surface of the gut is exposed to a horde of powerfully
so soon after immunizationwith the 65 kDa heat-shock immunogenic microorganisms, and since enterocytes
protein again strongly suggestsa pre-existing network are especiallyvulnerable to damage by IFNy and TNF,
linked to epitopes on this antigen as envisaged in the it has been important for the immune defenses of the
'immunological homunculus' concept (cf. p. 208). If gut to evolve mechanisms which deter Thl-type re-
malfunctioning of the network produces autoimmune sponses.This objectiveis attained by the stimulationof
disease, vaccination with T-cell receptor epitopes cells which release cytokines such as TGFP, IL-4 and
would represent a logical attempt to re-establish IL-10 and suppress the unwanted responses. Thus
natural control. feeding antigens should tolerize Thl cells and this has
proved to be a successful strategy for blocking EAE,
the collagen I1 arthritis model and the development of
Manipulation by antigen
diabetes in NOD mice. Accordingly, MS patients are
The object is to present the offending antigen in suffi- now being fed MBP and RA patients type I1 chicken
cient concentration and in the form which will turn off collagen.
an ongoing autoimmune response. Since T-cells have The tolerogen can also be delivered by inhalation of
been accorded such a pivotal role, it is natural to devise peptide aerosols (figure2O.23),and this could be a very
the strategy in terms of T-cell epitopes rather than attractive way of generating antigen-specific T-cell
whole antigen, obviously a far more practical proposi- suppression in many hypersensitivity states. Induc-
tion because this reduces the problem to dealing with tion of anergy or active suppression may contribute to
relatively short peptides. One strategy is to design different extents. Intranasal peptides have been used
high affinity peptide analogs that will bind obstinately successfully to block collagen-induced arthritis, EAE,
to the appropriate MHC molecule and antagonize the spontaneous diabetes (NOD) and a mouse model of
response to autoantigen (figure 20.22 (4)). S'ince we allergy to the house dust mite antigen Der PI. Sig-
express several different MHC molecules, this should nificantly, treatment can be effective even after induc-
not impair microbial defenses unduly. However, we tion of disease (figure 20.23), although in established
are now talking of patients not mice and this could human disease this may be more difficult to achieve
involve repeated very high doses of peptide, although, and might require supplementary therapy, such as
much in their favor, peptides are well defined anti-CD4, and preliminary reduction of primed T-cells
chemically and relatively cheap to produce. Antigen- with cyclosporin or steroids. There is no shortage of
specific suppression of T-cells (figure 20.22 (3))would strings to pull.
be advantageous in this respect, and giving the pep- Now this is really important. A single internal
tide under an umbrella of anti-CD4 or using partial epitope of MBP can inhibit disease induced by the mix-
agonists (cf.figure 9.8)could be feasible. Injection of an ture of epitopes or antigens contained within whole
MBP peptide, particularly as a palmitoylated deriva- myelin. In other words, a single epitope can induce
Figure 20.23. Influence of peptide inhalation on experimental duce tolerance to other autoantigenic epitopes on the same protein
autoimmune encephalomyelitis (EAE) induced with pig spinal (linkedsuppression)and to epitopeson differentantigenswithin the
cord in complete Freund's adjuvant. Aerosols of the peptides nervous tissue used for k h z a t i o n (bystander tolerance). PBS,
were inhaled (a) 8 days before and (b) 8 days after injection of the phosphate-buffered saline; the peptide was an acetylated N-termi-
encephalitogen. A single dose can give long-lived protection nal 11-merfrom myelin basic protein with lysine at position 4 substi-
which is extended indefinitely if the mice are thymectomized. tuted by alanine. (Data from Metzler B. &WraithD.C. (1996)Annals
Regulation is IL-l0 dependent and both Thl and Th2 can be of the Nao York Academy of Science 778,228, with permission of the
tolerized. Administration of a single peptide T-cell epitope can in- publishers.)
suppressionof the pathogenic T-cells specific for other genic Thl cell recognizing a separate epitope pro-
epitopes on the same or other molecules provided that cessed from the same or another molecule in the same
they are generated within the same organ or locality. organ (figure20.24).
We have referred to this already as organ-related by- Another potentially valuable approach for the fu-
stander tolerance, a phenomenon best understood in fare involves 'switching off' primed B-cellsby present-
terms of interactions on the same antigen-presenting ing hapten linked to a thymus-independentcarrier like
cell between the regulatory cell, be it Th2 or anergic, the copolymer of D-glutamicacid and D-lysine(D-GL)
recognizing the suppressor epitope and the patho- or IgG of the same species, particularly when given
with high cortisone doses. This has certainly worked
well in NZB X W hybrid mice where anti-DNA levels
have been reduced using nuc\eosides as the haptens:
we shall have to see whether human and mouse really
are that different.
Another way to manipulate antigen is to remove it.
For nucleosomal DNA this can be accomplished by in-
jection of DNase. Sure enough, treatment with DNase I
suppressed the manifestations of lupus nephritis in the
NZB X W model. And last, back to the Holy Grail busi-
ness, several groups are trying to evolve a strategy
based upon the 'magic bullet', the essence of which
is to fashion different types of cytotoxic weaponry by
coupling bacterial toxins or lots of radioactivity to the
antigen which selectively homes on to the lympho-
cytes bearing specific surface receptors. Something
good has got to come out of all this!
Plasinapheresis
Plasma exchange to lower the rate of immune complex
deposition in SLE provides only temporary benefit,
although it may be of value in life-threatening cases
of arteritis. Successful results have been obtained in
cy for antigen-reactive cells to divide as the negative to loss of autoantibody (the dashed Line represents the amount of
feedback effectof is lowered followingremovalof g.b.". antigen bound in the assay by normal serum)and restoration
of kidney function. (Courtesyof Dr C.M. Lockwood.)
plasma proteins.
(cont i m e d )
* -
These give rise to acute innammation in the joint space thyroid disease are direct targets for locally activated Th
and stimulatethe synoviallining cells to grow as a malign cells specific for thyroid peroxidase.
pannus which produces erosions in the underlying That autoimmunity can cause thyroiditis is further
cartilage and bone through the release of IL-1, IL-6, shown by the deliberate induction of disease in rodents
TNF, prostaglandin 5,collagenase, neutral proteinase through immunization with thyroid antigem in complete
and reactive oxygen intermediates. Freund's adjuvant.
The onset of IDDM is delayed by cyclosporin, HLA-
T-cell-mediatedhypersensilIvIty as a paihogenicfactor DQ risk factors are prominent, and T-cell proliferative
Suppression of disease by cyclosporin or anti-CD4 responses to pislet cell antigens reflect prognosis of
treatment is strong evidence for T-cell involvement. So is disease.
anHLA-wedrisk factor. Thl cells from diseased NOD mice, which mimic the
Thereisaprev~gviewthatorgan-specificinflamma- humandisorderinhistopathology and autoimmunity,can
tory lesions are caused by autoreactive pathogenic Thl produce typical pancreatic lesions in young mice of the
cells. same strain. Jntroductionof a transgene encoding changes
Activated T-cells are abundant in the rheumatoid atresidues560157intheH-ZBchaindramaticallyamelio-
synovium and their production of TNF and GM-CSF rates disease.
complements the immune complex stimulus for pannus Similarity to experimental allergic encephalomyelitis,
formation. a demyelinating disease induced by immunization with
RA patients have poor corticosteroid responses to trig- myelin in complete Freund's adjuvant, has made autoim-
gering of the pituitary-adrenal feedback loop and treat- munity the front-running hypothesis in MS. Approxi-
ment with low, virtually maintenance doses of steroids is mately one-third of the IL-2or -4 activatable T e l l s in the
beneficial. CSF of MS patients are specific for myelin and the DF
T h p y t e s expressing MHC class II in autoimmune phenotypeis a strong risk factor.
(continued p. 448)
Some systemic disorders with a vascular component of the use of anti-inflammatory and immunosuppressive
unknown pathogenesis drugs.
Immunologically mediated vascular lesions are of cen- A whole variety of potential immunological control
tral importance in Wegeners granulomatosis, temporal therapies are under intensive investigation.These include
arteritis. scleroderma and atherosclerosis. antibody and T-cell idiotype manipulations and attempts
to induce antigen-specificunresponsiveness particularly
Diagnosticvalue of outoontibody tests to T-cells using peptides administered parenterally, orally
A wide range of serum autoantibodies now provide or nasally
valuable diagnosticmarkers. Organ-related bystander tolerance means that single
Routine immunofluorescent screening is carried out on epitopes can induce suppression of pathogenic cells with-
compositesections of human thyroid and stomach and rat in an organ reacting to other epitopes on the same or other
kidney and liver, supplemented by agglutinationtests for antigens.
rheumatoid factors, thyroid and red cell antibodies, and Plasma exchange may be of value especially in combi
by radioimmunoassays for intrinsic factor and acetyl- nation with antimitoticdrugs.
choline receptor antibodies. The accompanying comparison of organ-specific and
Solid-phase ELISA tests are used for antibodies to nonorgan-specific autoimmune disorders (table 20.3)
DNA and other nuclear antigens and will increasingly gives an overall view of many of the points raised in the
displace fluorescence as purified autoantigens become last two chapters.
available.
CDla Non-MHC, class I-like Presentation of lipid & glycolipid Thymocyte,DC, LC, B(sub) Lipid & glycolipid antigens,
molecule antigens aP TCR
CDlb Non-MHC, class I-like Presentation of lipid & glycolipid Thymocyte, DC, LC, B(sub) Lipid & glycolipid antigens,
molecule antigens aP TCR
CDlc Non-MHC, class I-like Presentation of lipid & glycolipid Thymocyte, DC, LC, B(sub) Lipid & glycolipid antigens,
molecule antigens aP TCR
CDld Non-MHC, class I-like Presentation of lipid & glycolipid Leukocytes, epithelium Lipid & glycolipid antigens,
molecule antigens aP TCR
CDle Non-MHC, class I-like Presentation of lipid & glycolipid Unknown Lipid & glycolipid antigens,
molecule antigens ap TCR
CD2 LFA-2, T11 T cell adhesion to target cells or Thymocyte, T, NK CD58 (LFA-3), CD59, CD48,
APC. T cell activation. Regulation CD15
of cytolysis
CD36 TCR-associated molecule T cell activation Thymocyte,T
CD~E TCR-associated molecule T cell activation Thymocyte,T
CD3y TCR-associated molecule T cell activation Thymocyte,T -
CD4 T4, L3T4 CO-receptorfor MHC class 11- Thymocyte (sub),T(sub), MHC class 11, HIV gp120
restricted antigen-induced T cell M01 M@
activation
CD5 Modulates signaling through Thymocyte, T, B(sub)
TCR & BCR
CD6 T cell activation & thymocyte- Thymocyte,T, B(sub) CD166 (ALCAM)
stromal cell interaction
CD7 Regulation of cytokine SC, Thymocyte, T, NK Unknown
production & sensitivityto LPS-
induced shocksyndromes
CD8a CO-receptorfor MHC class I- Thymocyte (sub),T(sub) MHC class I
restricted antigen-induced T cell
activation
CD8p CO-receptorfor MHC class I- Thymocyte (sub), T(sub) MHC class I
restricted antigen-induced T cell
activation
CD9 Platelet activation & aggregation Pre-B, Plt, *B,*T,eosinophils,
basophils, endothelium,
epithelium
CD10 CALLA, NEP Neutral endopeptidase Unknown
(Continued on p. 452)
CD markers (continued)
(Continued)
453
CD markers (continued)
(Continuedon p. 454)
CD markers (continued)
(Continued)
CD markers (continued)
(Continuedon p. 456)
CD markers (continued)
(Contimed)
457
CD markers (continued)
(Continuedon p. 458)
CD markers (continued)
(Continued)
CD markers (continued)
(Continuedon p. 460)
CD markers (continued)
(Continued)
CD markers (continued)
(Continued on p. 462)
CD markers (continued)
Abbreviations: APC, antigen-presenting cell; BM, bone marrow; T, T-cell; B, B-cell; NK, natural killer cell; MO,monocyte; M@,macrophage; Gr,
granulocytes (neutrophils, basophils, eosinophils); Plt, platelet; LC, Langerhans' cell; DC, interdigitating dendritic cell; FDC, follicular dendrit-
ic cell; SC, stem cell; *, activated; (mat), mature; (sub), subset; EBV, Epstein-Barr virus; ECM, extracellular matrix; VWF, von Willebrand factor.
Further information on each CD antigen can be freely accessed through Protein Reviews on the Web (PROW: Shaw S., Turni L.A. and Katz K.S.
[editors], http://www.ncbi.nlm.nih.gov/prow). Another excellent reference source is The Leucocyte Antigen Facts book (Barclay A.N., Brown
M.H., Law S.K.A., McKnightA.J., TomlinsonM.G. and van der Merwe P.A. 2nd edn, 1997,Academic Press, London).
acquired immune response: Immunity mediated triggered by IgE or anaphylatoxin-mediated mast
by lymphocytes and characterized by antigen- cell degranulation, leading to anaphylacticshock due
specificity and memory. to vasodilatation and smooth muscle contraction.
acute phase proteins: Serum proteins, mostly pro- anergy: Potentially reversible specific immunological
duced in the liver, which rapidly change in concen- tolerance in which the lymphocyte becomes func-
tration (some increase, some decrease) during the tionally nonresponsive.
initiation of an inflammatory response. antibody-dependentcellular cytotoxicity (ADCC):A
adjuvant: Any substance which nonspecifically en- cytotoxic reaction in which an antibody-coated tar-
hances the immune response to antigen. get cell is directly killed by an Fc receptor-bearing
affinity (intrinsic affinity): The strength of binding leukocyte, e.g. NK cell, macrophage or neutrophil.
(affinity constant) between a receptor (e.g. one antigen:Any molecule capable of being recognized by
antigen-binding site on an antibody) and a ligand an antibody or T-cell receptor.
(e.g.epitope on an antigen). antigen-presenting cell (APC): A term most com-
allele: Variants of a polymorphic gene at a given monly used when referring to cells that present
genetic locus. processed antigenic peptide and MHC class I1
allelic exclusion: The phenomenon whereby, follow- molecules to the T-cell receptor on CD4+ T-cells,
ing successful rearrangement of one allele of an e.g. macrophages, dendritic cells, B-cells. Note, how-
antigen receptor gene, rearrangement of the other ever, that most types of cell are able to present anti-
parental allele is suppressed, thereby ensuring each genic peptides with MHC class I to CD8+T-cells, e.g.
lymphocyte expresses only a single specificity of as occurs with virally infected cells.
antigen receptor (although this does not occur for a antigenic determinant: A cluster of epitopes (see
chains in T-cells). epitope).
allergen:An antigen which causes allergy. apoptosis:A form of programmed cell death, charac-
allergy: IgE-mediated hypersensitivity, e.g. asthma, terized by endonucleasedigestion of DNA.
eczema, hayfever and food allergy. atopic allergy: IgE-mediated hypersensitivity i.e.
allogeneic: Refers to the genetic differences between asthma, eczema, hayfever and food allergy.
individuals of the same species. autologous:From the same individual.
allograft: Tissue or organ graft between allogeneic avidity (functional affinity):The binding strength be-
individuals. tween two molecules (e.g. antibody and antigen)
allotype: An allelic variant of an antigen which, be- taking into account the valency of the interaction.
cause it is not present in all individuals, may be im- Thus the avidity will always be equal to or greater
munogenic in members of the same species which than the intrinsic affinity (seeaffinity).
have a different version of the allele. P,-microglobulin:A 12kDa protein, not itself encoded
alternative pathway (of complement activation): within the MHC, but forming part of the structure of
Activation pathway involving complement compo- MHC class I-encoded molecules.
nents C3, Factor B, Factor D, and Properdin which, B-11B-2 cells:The two major subpopulations of B lym-
in the presence of a stabilizing activator surface such phocytes. B-1 cells bear high levels of surface IgM,
as microbial polysaccharide, generates the alterna- lower levels of surface IgD, are CD43+,CD23- and
tive pathway C3 convertase C3bBb. most express the cell surface antigen CD5; they are
anaphylatoxin:A substance (e.g. C3a, C4a or C5a) ca- self-renewing, and frequently secrete high levels of
pable of directly triggering mast cell degranulation. antibody which binds to a range of antigens ('poly-
anaphylaxis:An often fatal hypersensitivity reaction, specificity') with a relatively low affinity. The major-
ity of B cells, however, are B-2 which express low mentalization within lymphoid tissues, Thl / T h .
levels of surface IgM, higher levels of surface IgD, development,angiogenesis and wound healing.
do not express CD5, and are CD43-, CD23+;they are chemotaxis: Movement of cells up a concentration
directly generated from precursors in the bone gradient of chemotactic factors.
marrow, and secretehighly specific antibody. chimeric: Composite of genetically distinct individu-
basophil: A type of granulocyte found in the blood als, e.g. following an allogeneic bone marrow graft.
and resembling the tissue mast cell. class switching:The process by which a B-cell changes
BCG (bacille Calmette-Gu6rin): Attenuated the class but not specificity of a given antibody it
Mycobacterium tuberculosis used both as a specific produces, e.g. switching from an IgM to an IgG
vaccine for tuberculosis and as an adjuvant. antibody.
biolistics: The use of small particles, e.g. colloidal classical pathway (of complement activation):Activa-
gold, as a vehicle for carrying agents (drugs, nucleic tion pathway involving complement components
acid, etc.) into a cell. Following coating with the de- Cl, C2 and C4 which, following fixation of Clq, e.g.
sired agent(s),the particles are fired into the dermis by antigen-antibody complexes, produces the clas-
of the recipient using a helium-powered gun. sical pathway C3 convertase C4b2a.
bispecific antibody: An artificially produced hybrid clonal deletion:A process by which contact with anti-
antibody in which each of the two antigen-binding gen (e.g.self antigen)at an early stage of lymphocyte
arms is specific for a different antigenic epitope. differentiation leads to cell death by apoptosis.
Such antibodies, which can be produced either clonal selection: The selection and activation by anti-
by chemical cross-linkage or by recombinant DNA gen of a lymphocyte bearing a complementary re-
techniques, can be used to link together two differ- ceptor, which then proliferates to form an expanded
ent antigens or cells, e.g. a cytotoxic T-cell and a clone.
tumor cell. clone:Identical cells derived from a single progenitor.
bursa of Fabricius: A primary lymphoid organ in colony stimulating factors (CSF):Factors that permit
avian species, located at the cloacal-hind gut junc- the proliferation and differentiation of hematopoiet-
tion; it is the site of B-cell maturation. ic cells.
capping: An active process whereby cross-linking complement: A group of serum proteins, some of
of cell surface molecules (e.g. by antibody) leads which act in an enzymatic cascade, producing effec-
to aggregation and subsequent migration of the tor molecules involved in inflammation (C3a, C5a),
molecules to one pole of the cell. phagocytosis (C3b),and cell lysis (C5b-9).
carrier: Any molecule which when conjugated to a complementarity determining regions (CDR): The
non-immunogenic molecule (e.g. a hapten) makes hypervariable amino acid sequences within anti-
the latter immunogenic by providing epitopes for body and T-cell receptor variable regions which
helper T-cells which the hapten lacks. interact with complementary amino acids on the
CD antigen: Cluster of differentiation designation as- antigen or peptide-MHC complex.
signed to leukocyte cell surface molecules which ConA (concanavalinA):A T-cell mitogen.
are identified by a given group of monoclonal congenic:Animals which only differ at a single genetic
antibodies. locus.
CD3: A trimeric complex of y, 6 and E chains which to- conjugate:Covalently-linked complex of two or more
<<
gether with a homodimer or <q heterodimer acts molecules (e.g.fluoresceinconjugated to antibody).
as a signal transducing unit for the T-cell receptor. Coombs' test: Diagnostic test using anti-immuno-
CD4: Cell surface glycoprotein, usually on helper globulin to agglutinateantibody-coated erythrocytes.
T-cells, that recognizes MHC class I1 molecules on cortex:Outer (peripheral)layer of an organ.
antigen-presentingcells. C-reactive protein: An acute phase protein which is
CDS: Cell surface glycoprotein, usually on cytotoxic able to bind to the surface of microorganisms where
T-cells, that recognizes MHC class I molecules on it functions as a stimulator of the classical pathway
target cells. of complement activation, and as an opsonin for
cell-mediatedimmunity (CMI):Refers to T-cell medi- phagocytosis.
ated immune responses. cyclophosphamide: Cytotoxic drug used as an
chemokines: A family of structurally-related cy- immunosuppressive.
tokines which selectively induce chemotaxis and cyclosporin A: A T-cell specific immunosuppressive
activation of leukocytes. They also play important drug used to prevent graft rejection.
roles in lymphoid organ development,cell compart- cytokines:Low molecular weight proteins that stimu-
late or inhibit the differentiation, proliferation or erythropoiesis: Erythrocyte production.
function of immune cells. exotoxin:Pathogenic protein secreted by bacteria.
cytophilic:Binds to cells. exudate: The extravascular fluid (containing pro-
cytotoxic: Kills cells. teins and cellular debris)which accumulates during
cytotoxic T lymphocyte (Tc):T-cells (usually CD8') inflammation.
which kill target cells following recognition of Fab: Monovalent antigen-binding fragment obtained
foreign peptide-MHC molecules on the target cell following papain digestion of immunoglobulin.
membrane. Consists of an intact light chain and the N-terminal
delayed-type hypersensitivity (DTH):A hypersensi- V and CH1domains of the heavy chain.
I?
tivity reaction occurringwithin 48-72 hours and me- F(ab :), Bivalent antigen-binding fragment obtained
diated by cytokine release from sensitized T-cells. following pepsin digestion of immunoglobulin.
differentiation antigen: A cell surface molecule ex- Consists of both light chains and the N-terminal part
pressed at a particular stage of development or on of both heavy chains linked by disulfidebonds.
cells of a given lineage. Fas: A member of the TNF receptor gene family. En-
DiGeorge syndrome:Immunodeficiency caused by a gagement of Fas (CD95) on the surface of the cell
congenital failure in thymic development resulting by the Fas ligand (CD178)present on cytotoxic cells,
in a lack of mature functional T-cells. can trigger apoptosis in the Fas-bearing target cell.
diversity (D) gene segments: Found in the im- Fc: Crystallizable,non-antigenbinding fragment of an
munoglobulin heavy chain gene and T-cell receptor immunoglobulin molecule obtained following pa-
p and 6 gene loci between the V andJ gene segments. pain digestion.Consists of the C-terminal portion of
Encode part of the third hypervariable region in both heavy chains which is responsible for binding
these antigen receptor chains. to Fc receptors and Clq.
edema: Swelling caused by accumulation of fluid in Fc receptors: Cell surface receptors which bind the Fc
the tissues. portion of particular immunoglobulinclasses.
effector cells: Cells which carry out an immune func- fibroblast: Connective tissue cell which produces
tion, e.g. cytokine release, cytotoxicity. collagen and plays an important part in wound
ELISA (enzyme-linked immunosorbent assay): Assay healing.
for detection or quantitation of an antibody or anti- fluorescein isothiocyanate (FITC): Green flu-
gen using a ligand (e.g. an anti-immunoglobulin) orescent dye used to 'tag' antibodies for use in
conjugated to an enzyme which changes the color of immunofluorescence.
a substrate. fluorescent antibody: An antibody conjugated to a
endocytosis:Cellular ingestion of macromolecules by fluorescent dye such as FITC.
invagination of plasma membrane to produce an follicular dendritic cell: MHC class 11-negative Fc
intracellular vesicle which encloses the ingested receptor-positive dendritic cells which bear im-
material. mune complexes on their surface and are probably
endogenous:From within. involved in the generation of antibody-secreting
endosomes: Intracellular smooth surfaced vesicles in cells and maintenance of B-cell memory in germinal
which endocytosed material passes on its way to the centres. (N.B. a different cell type to interdigitating
lysosomes. dendritic cells).
endotoxin: Pathogenic cell wall-associated lipopoly- framework regions: The relatively conserved amino
saccharides of Gram-negativebacteria. acid sequences which flank the hypervariable re-
eosinophil: A class of granulocyte, the granules of gions in immunoglobulin and T-cell receptor
which contain toxic cationic proteins. variable regions and maintain a common overall
epitope: That part of an antigen recognized by an structure for all V-region domains.
antigen receptor (see antigenic determinant). Freund's adjuvant: Complete Freund's adjuvant is an
Epstein-Barr virus (EBV): The virus responsible for emulsion of aqueous antigen in mineral oil that con-
infectious mononucleosis and Burkitt's lymphoma. tains heat-killed Mycobacteria. Incomplete Freund's
Used to immortalize human B-cells in vitro. adjuvant lacks the Mycobacteria.
equivalence:The ratio of antibody to antigen at which gammaglobulin: The serum proteins, mostly im-
immunoprecipitation of the reactants is virtually munoglobulins, which have the greatest mobility
complete. towards the cathode during electrophoresis.
erythema: The redness produced during inflamma- germ line: The arrangement of the genetic material as
tion due to erythrocytes entering tissue spaces. transmitted through the gametes.
germinal center: Discrete areas within lymph node causes increased vascular permeability and smooth
and spleen where B-cell maturation and memory muscle contraction.
development occur. HLA (human leukocyte antigen): The human major
giant cell:Large multinucleatecell derived from fused histocompatibility complex (MHC).
macrophages and often present in granulomas. humanized antibody: A genetically engineered
glomerulonephritis:Inflammation of renal glomeru- monoclonal antibody of non-human origin in which
lar capillary loops, often resulting from immune all but the antigen-binding CDR sequences have
complex deposition. been replaced with sequences derived from human
graft versus host (g.v.h.)reaction: Reaction occurring antibodies. This procedure is carried out to mini-
when T lymphocytes present in a graft recognize mize the immunogenicity of therapeutic mono-
and attack host cells. clonal antibodies.
granulocyte: Myeloid cells containing cytoplasmic humoral:Pertainingto extracellular fluid such as plas-
granules (i.e. neutrophils, eosinophils and ma and lymph. The term humoral immunity is used
basophils). to denote antibody-mediatedimmune responses.
granuloma: A tissue nodule containing proliferating hybridoma:Hybrid cell line obtained by fusing a lym-
lymphocytes, fibroblasts, and giant cells and phoid tumor cell with a lymphocyte which then has
epithelioid cells (both derived from activated both the immortality of the tumor cell and the effec-
macrophages), which forms due to inflammation in tor function (e.g.monoclonal antibody secretion) of
response to chronic infection or persistence of anti- the lymphocyte.
gen in the tissues. hypersensitivity: Excessive immune response which
granzymes:Serine esterases present in the granules of leads to undesirable consequences, e.g. tissue or
cytotoxic T lymphocytes and NK cells. They induce organ damage.
apoptosis in the target cell which they enter through hypervariable regions: Those amino acid sequences
perforin channels inserted into the target cell mem- within the immunoglobulin and T-cell receptor
brane by the cytotoxic lymphocyte. variable regions which show the greatest variability
gut-associated lymphoid tissue (GALT): Includes and contributemost to the antigen or peptide-MHC
Peyer's patches, appendix, and solitary lymphoid binding site.
nodules in the submucosa. idiotope: An epitope made up of amino acids within
H-2: The mouse major histocompatibility complex the variable region of an antibody or T-cell receptor
(MHC). which reacts with an anti-idiotope.
haplotype:The set of allelic variants present at a given idiotype: The complete set of idiotopes in the variable
genetic region. region of an antibody or T-cell receptor which react
hapten: A low molecular weight molecule that is with an anti-idiotypic serum.
recognized by preformed antibody but is not itself idiotype network: A regulatory network based on
immunogenic unless conjugated to a 'carrier' mole- interactions of idiotypes and anti-idiotypespresent
cule which provides epitopes recognized by helper on antibodies and T-cell receptors.
T-cells. immune complex: Complex of antibody bound to
helper T lymphocyte (Th):A subclass of T-cells which antigen which may also contain complement
provide help (in the form of cytokines and/or cog- components.
nate interactions)necessary for the expression of ef- immunoadsorption:Method for removal of antibody
fector functionby other cells in the immune system. or antigen by allowing it to bind to solid phase anti-
hemagglutinin: Any molecule which agglutinates gen or antibody.
erythrocytes. immunofluorescence: Technique for detection of cell
hematopoiesis: The production of erythrocytes and or tissue-associated antigens by the use of a fluores-
leukocytes. cently-tagged ligand (e.g. an anti-irnrnunoglobulin
high endothelial venule (HEV): Capillary venule conjugated to fluorescein isothiocyanate).
composed of specialized endothelial cells allowing immunogen: Any substance which elicits an immune
migration of lymphocytes into lymphoid organs. response. Whilst all immunogens are antigens, not
hinge region:Amino acids between the Fab and Fc re- all antigens are immunogens (seehapten).
gions of immunoglobulinwhich permit flexibility of immunoglobulin superfamily: Large family of pro-
the molecule. teins characterized by possession of 'immunoglobu-
histamine:Vasoactive amine present in basophil and lin-type' domains of approximately 110amino acids
mast cell granules which, following degranulation, folded into two P-pleated sheets. Members include
immunoglobulins, T-cell receptors and MHC joining (J) gene segments: Found in the immuno-
molecules. globulin and T-cell receptor gene loci and, upon
immunological synapse:A contact point between the gene rearrangement,encode part of the third hyper-
T-cell and antigen-presentingcell which is generat- variable region of the antigen receptors.
ed by reorganization and clustering of cell surface K (killer) cell: Large granular lymphocyte whch
molecules in lipid rafts. The synapse facilitates in- mediates antibody-dependent cellular cytotoxicity
teractions between TCR and MHC and between (ADCC), is Fc receptor positive, but does not re-
adhesion molecules, thereby potentiating the TCR- arrange or express either immunoglobulin or T-cell
mediated activation signal. receptor genes.
inflammation: The tissue response to trauma, charac- kinins: A family of polypeptides released during in-
terized by increased blood flow and entry of leuko- flammatory responses and which increase vascular
cytes into the tissues, resulting in swelling, redness, permeability and smooth muscle contraction.
elevated temperature and pain. KIRs: Killer cell Immunoglobulin-like Receptors
innate immunity: Immunity which is not intrinsically found on NK cells, some y6 and some ap T-cells.
affected by prior contact with antigen, i.e. all aspects KIRs recognize MHC class I molecules and, like the
of immunity not directly mediated by lymphocytes. C-type lectin receptors also found on these cells, can
interdigitating dendritic cell: MHC class 11-positive, either inhibit or activate the killer cells. If ITIM se-
Fc receptor-negative, antigen-presenting dendritic quences are present in their cytoplasmic domain
cell found in T-cell areas of lymph nodes and spleen. they are inhibitory.KIRs lacking ITIMs can associate
(N.B. a different cell type to follicular dendritic with ITAM-containingadaptor molecules, in which
cells). case they can activate the killer cell.
interferons (IFN): IFNa is derived from various knockout:The use of homologous genetic recombina-
leukocytes, IFNP from fibroblasts and IFNy from tion in embryonal stem cells to replace a functional
T lymphocytes. All three types induce an anti-viral gene with a defective copy of the gene. The animals
state in cells and IFNy acts as a cytokine in the regu- that are produced by this technique can be bred to
lation of immune responses. homozygosity, thus allowing the generation of a
interleukins (IL): Designation for some of the cy- null phenotype for that gene product.
tokines secreted by leukocytes. Kupffer cells: Fixed tissue macrophages lining the
internal image: An epitope on an anti-idiotypewhich blood sinuses in the liver.
binds in a way that structurally and functionally Langerhans' cell: Fc receptor and MHC class II-
mimics the antigen. positive antigen-presenting dendritic cell found
invariant chain: A polypeptide which binds MHC in the skin.
class I1 molecules in the endoplasmic reticulum, di- large granular lymphocyte (LGL):Large lymphocytes
rects them to the late endosomal compartment and which contain cytoplasmic granules and function
prevents premature associationwith self peptides. as natural killer (NK) and killer (K) cells. Activated
Ir (immune response) genes: The genes, including CDBf cytotoxic T lymphocytes (Tc) also assume an
those within the MHC, that together determine the LGL morphology.
overall level of immune response to a given antigen. lectins: A family of proteins, mostly of plant origin,
isotype: An antibody constant region structure pre- which bind specific sugars on glycoproteins and
sent in all normal individuals, i.e. antibody class or glycolipids. Some lectins are mitogenic (e.g. PHA,
subclass. ConA).
ITAM: Immunoreceptor Tyrosine-based Activation leukotrienes: Metabolic products of arachidonic
Motifs are consensus sequences for src-family tyro- acid which promote inflammatory processes (e.g.
sine kinases. These motifs are found in the cyto- chemotaxis, increased vascular permeability) and
plasmic domains of several signaling molecules are produced by a variety of cell types including
including the signal transduction units of lympho- mast cells, basophils and macrophages.
cyte antigen receptors and of Fc receptors. ligand: General term for a molecule recognized by a
ITIM: Immunoreceptor Tyrosine-based Inhibitory binding structure such as a receptor.
Motifs present in the cytoplasmicdomains of certain linkage disequilibrium:The occurrence of two alleles
cell surface molecules, e.g. Fcy RIIB, inhibitory NK being inherited together at a greater frequency than
cell receptors, and which mediate inhibitory signals. that expected from the product of their individual
J chain:Amolecule which forms part of the structure of frequencies.
pentameric IgM and dimeric IgA. lipid raft: Cholesterol- and glycosphingolipid-rich
membrane subdomain in which moleculesinvolved secondary immune response; faster, greater and
in cellular activation become concentrated. longer lasting than the primary immune response.
lipopolysaccharide (LPS): Endotoxin derived from memory cells: Clonally expanded T- and B-cells
Gram-negative bacterial cell walls which has in- produced during a primary immune response and
flammatory and mitogenic actions. which are 'primed' to mediate a secondary immune
lymph:The tissue fluid which drains into and through response to the original antigen.
the lymphatic system. MHC (major histocompatibility complex): A genetic
lymphadenopathy:Enlarged lymph nodes. region encoding molecules involved in antigen pre-
lymphokine: Cytokine produced by lymphocytes. sentation to T-cells. Class I MHC molecules are pre-
lymphokine-activated killer cells (LAK): Killer (K) sent on virtually all nucleated cells and are encoded
and natural killer (NK)cells activated in vitro by IL-2 mainly by the H-2K, D, and L loci in mice and by
to give enhanced killing of target cells. HLA-A, B, and C in man, whilst class I1 MHC mole-
Lymphotoxin (also called TNFP): A T-cell derived cules are expressed on antigen-presenting cells
cytokine which is cytotoxic for certain tumor cells (primarilymacrophages, B-cells and interdigitating
and also has immunoregulatoryfunctions. dendritic cells) and are encoded by H-2A and E in
lysosomes: Cytoplasmic granules containing hy- mice and HLA-DR, DQ, and DP in man. Allelic dif-
drolytic enzymes involved in the digestion of ferences can be associated with the most intense
phagocytosed material. graft rejection within a species.
lysozyme: Anti-bacterial enzyme present in phago- MHC restriction:The necessity that T-cells recognize
cytic cell granules, tears and saliva, which digests processed antigen only when presented by MHC
peptidoglycansin bacterial cell walls. molecules of the original haplotype associated with
macrophage: Large phagocytic cell, derived from the T-cell priming.
blood monocyte, which also functions as an antigen- minor histocompatibility antigens: Non-MHC-
presenting cell and can mediate ADCC. encoded cell surface processed peptides which, in
mannose binding protein: A member of the collectin association with MHC-encoded molecules, contri-
family of calcium-dependent lectins, and an acute bute to graft rejection, albeit not usually as severe as
phase protein. It functions as a stimulator of the clas- that due to MHC mismatch.
sical pathway of complement activation, and as an mitogen:A substance which non-specifically induces
opsonin for phagocytosis by binding to mannose, lymphocyte proliferation.
a sugar residue usually found in an exposed form mixed lymphocyte reaction (MLR):A T-cell prolifera-
only on the surfaceof microorganisms. tive response induced by cells expressing allogeneic
marginal zone: The outer area of the splenic periar- MHC.
teriolar lymphoid sheath (PALS) which is rich in monoclonal antibody: Homogeneous antibody de-
B cells, particularly those responding to thymus- rived from a single B-cell clone and therefore all
independent antigens. bearing identical antigen-bindingsites and isotype.
margination:Leukocyte adhesion to the endothelium monocyte: Mononuclear phagocyte found in blood
of blood vessels in the early phase of an acute inflam- and which is the precursor of the tissue macrophage.
matory reaction. mononuclear phagocyte system: A system compris-
mast cell: A tissue cell with abundant granules which ing blood monocytes and tissue macrophages.
resembles the blood basophil. Both these cell types mucosal-associated lymphoid tissue (MALT):Lym-
bear Fc receptors for IgE, which when crosslinkedby phoid tissue present in the surface mucosa of the res-
IgE and antigen cause degranulation and the release piratory, gastrointestinaland genitourinary tracts.
of a number of mediators including histamine and multiple myeloma: Plasma cell malignancy resulting
leukotrienes. in high levels of monoclonal immunoglobulin in
medulla:Inner (central)region of an organ. serum and of free light chains (Bence-Jones protein)
megakaryocyte:Abone marrow precursor of platelets. in urine.
membrane attack complex (MAC): Complex of com- murine:Pertaining to mice.
plement components C5b-C9 which inserts as a myeloma protein: Monoclonal antibody secreted by
pore into the membrane of target cells leading to cell myeloma cells.
lysis. negative selection: Deletion by apoptosis in the thy-
memory (immunological):A characteristic of the ac- mus of T-cells which recognize self peptides pre-
quired immune response of lymphocytes whereby a sented by self MHC molecules, thus preventing the
second encounter with a given antigen produces a development of autoimmune T-cells. Negative se-
lection of developing B-cells is also thought to occur associated molecular patterns (PAMPs). Amongst
if they encounter high levels of self antigen in the the large number of different PRRs are the mannose
bone marrow. receptor (CD206)and the macrophage scavenger re-
neutrophil: The major circulating phagocytic poly- ceptor (CD204).
morphonuclear granulocyte. Enters tissues early perforin: Molecule produced by cytotoxic T-cells and
in an inflammatory response and is also able to NK cells which, like complement component C9,
mediate antibody-dependent cellular cytotoxicity polymerizes to form a pore in the membrane of the
(ADCC). target cell leading to cell death.
NK (natural killer) cell: Large granular lymphocyte periarteriolar lymphoid sheath (PALS): The lym-
which does not rearrange nor express either im- phoid tissue which forms the white pulp of the
munoglobulin or T-cell receptor genes but is able to spleen.
recognize and destroy certain tumor and virally- Peyer's patches: Part of the gut associated lymphoid
infected cells in an MHC and antibody-independent tissue (GALT) and found as distinct lymphoid
manner. nodules mainly in the small intestine.
NK-T cell: NKl.lf lymphoid cells with a morphology PHA (phytohemagglutinin):Aplant lectin which acts
and granule content intermediate between T-cells as a T-cell mitogen.
and NK cells. They are potent producers of IL-4, may phage antibody library: A collection of cloned anti-
be CD4-8- or CD4'8-, and express low levels of ap body variable region gene sequences which can be
TCR with an invariant a chain and very restricted expressed as Fab or scFv fusion proteins with bacte-
p chain specificity. Many of these TCR recognize riophage coat proteins. These can be displayed on
antigens presented by the non-classical MHC-like the surface of the phages. The gene encoding a
molecule CD1. Their lectin-like NK1.l receptor may monoclonalrecombinant antibody is enclosed in the
recognize microbial carbohydrates. phage particle and can be selected from the library
nude mouse: Mouse which is T-cell deficient due to a by binding of the phage to specific antigen.
homozygous gene defect (nu/nu) resulting in the phagoeyte: Cells, including monocytes/macrophages
absence of a thymus (and also lack of body hair). and neutrophils, which are specialized for the
oncofetal antigen: Antigen whose expression is nor- engulfment of cellular and particulate matter.
mally restricted to the fetus but which may be ex- phagolysosome: Intracellular vacuole where killing
pressed during malignancy in adults. and digestion of phagocytosed material occurs
opsonin: Substance, e.g. antibody or C3b, which en- following the fusion of a phagosome with a
hances phagocytosis by promoting adhesion of the lysosome.
antigen to the phagocyte. phagosome:Intracellular vacuole produced following
opsonization: Coating of antigen with opsonin to en- invagination of the cell membrane around phagocy-
hance phagocytosis. tosed material.
PAF (platelet activating factor): An alkyl phospho- phorbol myristate acetate (PMA):A mitogenic phor-
lipid released by a variety of cell types including bol ester which directly stimulates protein kinase C
mast cells and basophils, which has immuno- and acts as a tumor promoter.
regulatory effects on lymphocytes and monocytes/ plaque forming cell (PFC):Antibody-secreting plas-
macrophages as well as causing platelet aggregation ma cell detected in vitro by its ability to produce a
and degranulation. 'plaque' of lysed antigen-sensitized erythrocytes in
paracortex: The part of an organ (e.g. lymph node) the presence of complement.
which lies between the cortex and the medulla. plasma cell: Terminally differentiated B lymphocyte
pathogen-associated molecular pattern (PAMP): which actively secretes large amounts of antibody.
Molecules such as lipopolysaccharide, peptidogly- pokeweed mitogen (PWM):Aplant lectinwhichis a T-
can, lipoteichoic acids and mannans, which are cell dependent B-cell mitogen.
widely expressed by microbial pathogens as repeti- polyclonal: Many different clones, or the product of
tive motifs but are not present on host tissues. They many different clones, e.g. polyclonal antiserum.
are therefore utilized by the pattern recognition re- poly-Ig receptor: A receptor molecule which specifi-
ceptors (PRRs)of the immune system to distinguish cally binds J-chain containing polymeric Ig, i.e.
pathogens from self antigens. dimeric secretory IgA and pentameric IgM, and
pattern recognitionreceptor (PRR):Receptors onpro- transports it across mucosal epithelium.
fessional antigen-presenting cells and phagocytes positive selection: The selection of those developing
which enable them to recognize pathogen- T-cells in the thymus which are able to recognize self
MHC molecules. This occurs by preventing apopto- rosette: Particles or cells bound to the surface of a
sis in these cells. lymphocyte (e.g. sheep erythrocytes around a
precipitin: Precipitate of antibody and multivalent human T-cell).
antigen due to the formation of high molecular scFv:Asingle chain molecule composed of the variable
weight complexes. regions of an antibody heavy and light chain joined
primary immune response: The relatively weak together by a flexible linker.
immune response which occurs upon the first SCID (severe combined immunodeficiency): Im-
encounter of naive lymphocytes with a given munodeficiencyaffectingboth Tand B lymphocytes.
antigen. secondary immune response: The qualitatively and
primary lymphoid organs: The sites at which im- quantitatively improved immune response which
munocompetent lymphocytes develop, i.e. bone occurs upon the second encounter of primed lym-
marrow and thymus in mammals. phocytes with a given antigen.
prime: The process of giving an initial sensitization to secretory component:Proteolytic cleavage product of
antigen. the poly-Ig receptor which remains associated with
prostaglandins:Acidic lipids derived from arachidon- dimeric IgA in sero-mucussecretions.
ic acid which are able to increase vascular perme- secretory IgA: Dimeric IgA found in sero-mucus
ability, mediate fever, and can both stimulate and secretions.
inhibit immunologicalresponses. somatic hypermutation: The enhanced rate of point
proteasome: Cytoplasmic proteolytic enzyme com- mutation in the immunoglobulin variable region
plex involved in antigen processing for association genes which occurs following antigenic stimulation
with MHC. and acts as a mechanism for increasing antibody
protein A: Stapkylococcus aureus cell wall protein diversityand affinity.
which binds to the Fc region of IgG. stem cell: Multipotential cell from which differ-
protein tyrosine kinases: Enzymes which are able to entiated cells derive.
phosphorylate proteins on tyrosines, and often act superantigen: An antigen which reacts with all the
in a cascade-like fashion in the signal transduction T-cells belonging to a particular T-cell receptor V
systems of cells. region family, and which therefore stimulates (or
prozone effect: The loss of immune precipitation or deletes) a much larger number of cells than does
agglutinationwhich occurs when antibody concen- conventional antigen.
tration is increased to an extent that the antibody is surface plasmon resonance: A technique based upon
in such excess that it is no longer able to effectively changes in the angle of reflected light which occur
cross-link the antigen. A similar phenomenon may upon ligand binding to an immobilized target
occur in antigen excess. molecule on a biosensor chip. This permits the
Qa antigens:'Non-classical' MHC class I molecules of observation of protein-protein interactions (suchas
mice. antibody binding to an antigen) in 'real-time', i.e.
recombination signal sequence (RSS): Conserved by continuous monitoring of the association and
heptamer (7-nuc1eotide)-nonamer (9-nucleotide) dissociationof the reversible reaction.
sequences, separated by a 12 or 23 base spacer, switch sequences: Highly conserved repetitive se-
which occur 3' of variable gene segments,5' and 3' of quences which mediate class switching in the im-
diversity gene segments, and 5' of joining gene seg- munoglobulin heavy chain gene locus.
ments, in both immunoglobulin and T cell receptor syngeneic: Genetically identical, e.g. a fully inbred
genes. They function as recognition sequences for strain of mice.
the recombinase enzymes that mediate the gene TAP: The Transporters associated with Antigen Pro-
rearrangement process involved in the generation cessing (TAP-1 and TAP-2) are molecules which
of lymphocyte antigen receptor diversity. carry antigenicpeptides from the cytoplasminto the
respiratory burst: The increased oxidative metabo- lumen of the endoplasrnic reticulum for incorpora-
lism which occurs in phagocytic cells following tion into MHC class I molecules.
activation. T-cell receptor (TCR):The heterodimeric antigen re-
reticuloendothelial system (RES):A rather old term ceptor of the T lymphocyte exists in two alternative
for the network of phagocytes and endothelial cells forms, consistingof a and p chains, or y and 6 chains.
throughout the body. The ap TCRrecognizespeptide fragments of protein
rheumatoid factor: IgM, IgG and IgA autoantibodies antigens presented by MHC molecules on cell sur-
to IgG, particularlythe Fc region. faces. The function of the y6 TCR is less clearly de-
fined but it can recognize native proteins on the cell toxoid: Chemically or physically modified toxin that is
surface. no longer harmful but retains immunogenicity.
T-dependent antigen: An antigen which requires tumor necrosis factor (TNF, also called TNFa):
helper T-cells in order to elicit an antibody Together with the related cytokine lymphotoxin
response. (TNFP),was originally named for its cytotoxic effect
T-independent antigen: An antigen which is able to on certain tumor cells, but also has immunoregula-
elicit an antibody response in the absence of T-cells. tory functions.
thymocyte: Developing T-cell in the thymus. variable (V) gene segments: Genes that rearrange to-
titer: Measure of the relative 'strength' (a combina- gether with D (diversity) and J (joining) gene seg-
tion of amount and avidity) of an antibody or anti- ments in order to encode the variable region amino
serum, usually given as the highest dilution which acid sequences of imrnunoglobulins and T-cell
is still operationally detectable in, for example, an receptors.
ELISA. vasoactive amines: Substances including histamine
tolerance:Specific immunological unresponsiveness. and 5-hydroxytryptaminewhich increase vascular
tolerogen:An antigen used to induce tolerance. Often permeability and smooth muscle contraction.
depends more on the circumstances of administra- xenogeneic:Genetic differences between species.
tion (e.g. route and concentration) than on any in- xenograft:A tissue or organ graft between individuals
herent property of the molecule. of different species.
This page intentionally left blank
Note: page numbers in italic refer to figures, AIDS 313-19 content 108
page numbers in bold refer to tables. CD4 T-cell depletion in 316 engineered 123-5,125,2834
clinical features 313-14 estimation 109-15
AB0 blood groups 333 control 319 evolution of generation of diversity 63-9
abzymes 122 diagnosis 317 excess 110,336
acanthosis nigricans 400,423 factors affecting progression 317-19 in graft rejection 354,355
acquired immune response 21-36 natural history 316-17 high affinity 86
and aging 218 AIDS-related complex 314 human anti-mouse 122
antigen specificity 30 alkaline phosphatase 114 immunoassay 113-15
evolution 2424 allelic exclusion 227,238 indirect test 131,133
genetic factors in 211-14,215 importance of 240 as inhibitor 126
lymphocyte activation 245,164-76 allergens, inhaled, clinical responses to maternally acquired 281,283
neuroendocrine control 214-17 325-7 monoclonal see monoclonal antibodies
ontogeny 22142,246-8 allergic bronchopulmonary aspergillosis natural 208,407
phylogeny 242-5,247-8 338 and neutralization of biological activity
primary 29 allergic rhinitis 326 126,258
production of effectors 177-99 allergy progesterone neutralizing 370
secondary 29 atopic see atopic allergy purification by affinitychromatography
specific 1,21 food 327 125-6
acquired immunodeficiencysyndrome see wheal and erythema 330 sequence diversity 194
AIDS allograft 350 specificity of antigen recognition 89-90
ACTH 215,276,441 fetus as 369-70,369 structure 190
activation-induced cell death (AICD) 201-3, response 3534 synthesis 190
215 tolerance to 360-1 in invertebrates 2434
acute early graft rejection 354 type I1hypersensitivity reactions to 335 thyroid stimulating 345
acute inflammatory response see see also grafts titer 108
inflammation, acute inflammatory allotypes 41-2 see also immunoglobulins
response alphaviral genome 291 antibody-dependent cellular cytotoxicity
acute late graft rejection 354 aluminum compounds as adjuvants 289, (ADCC) 32,51,70,332,333
acute lymphoblastic leukernia 382-3 300 in bacterial infections 260
acute phase proteins 16-17 ambient analyte principle 118 in parasitic infections 273
acute vascular rejection 362 amidopyrine 336 antibody-dependent cytotoxic
adaptor proteins 167,168 amino acid polymers 445-6 hypersensitivity 70,332-6,332
ADCC see antibody-dependent cellular amyloid 385 alloimmune reactions 333-5
cytotoxicity amyloid A protein 385 autoimmune reactions 335-6
Addison's disease 399,400 anaphylactic hypersensitivity 322-32 antibody-directed enzyme prodrug therapy
adeno-associated virus (AAV) 144 atopic allergy 325-32 (ADEPT) 122
adenosine deaminase deficiency 312 anaphylatoxin 12,283 antibody-secreting cells, enumeration
adherence 129 anaphylaxis 322 137-9,139
adjuvant arthritis 433 discovery 323 anti-CD3 358
adjuvants anchor positions 97,98 anti-CD4 361
depot effects 300 angiogenesis 392 anti-CD8 361
effects on lymphocytes 300-1 angiotensin 16 anti-idiotypes 61,207,294-5
macrophage activation 300 ankylosing spondylitis 368 antigen 24,27,80
in vaccines 299-302 antagonists of T-cell activation 169 antibody binding 37,109-12
adult respiratory distress syndrome 345 antibody 21-3,22,37-58 affinity of 85-9,86,87,88
affinity 85-9,86,87,88 in activation of phagocytic cells 23,24 bonus effects of multivalency 87,89,90
factors affecting 195 antibacterial effects 256,263 forces 85
maturation of 195 antigen binding 37,109-12 reversibility 84/85/87
affinity chromatography 125-6 affinity of 85-9,86,87,88,110-12 spatial complementarity 83,84
affinity constant 86,108-12 bonus effect of multivalency 87,89,90 avidity of antiserum for 87
agglutination reactions 112,113 forces 85 B-cell surface receptor 59-60
aging, effects on acquired immune response reversibility 84,85,87 binding site 47
218 spatial complementarity 83,84 and control of acquired immune response
agonists antiparasitic effects 272-3,273 195
P,-agonists 331 antiviral 269 determinant 82
T-cell activation 169 catalytic 122 dose effect 195
agranulocytosis, drug-induced 336 cellular basis of production 24-8 excess 110,336
antigen (continued) pathogenic effects of complexeswith basophils 5
identification of B-cell epitopes 82 425-31 BCG see bacille Calmette-GuPrin
identification and measurement 115-20 autografts 350,362,363 biolistics 302
immunoassay 118 autoimmune atrophic gastritis 423 biotin 114
interference within a mixture 200-1 autoimmune disease 33,396--420,421-49 bone marrow
mutant 377-8 animal models 399 antibody synthesisin 157-8
oncofetal 376 antigen-driven 409-10 B-cell differentiationin 234-6
presentation 301-2 and bypass of regulatory mechanisms detectionof micrometastases 393
by liposomes 301 415-19 grafting 360,361,364-5,390,391
processing 93-5,95,172-3,268 and bypass of T-helper cells 411-15 stem cells 223,231
purification by affinity chromatography environmentalfactors 406-7 bonus effect of multivalency 23,51,87,89,90
125-6 genetic factors 399405 Bordetella pertussis, vaccine 297
recognition multifactorial 419 bradykinin in acute inflammatoryresponse
generationof diversity for 63-9 organ-specificand nonorgan-specific 251
specificityof 89-90 3968,409 5-brornodeoxyuridine (BUDR) 129
slow release 302 overlap 399 Brucella spp. 262,431
T-cell surfacereceptor see T-cell receptors pathogenesis, humoral autoantibody Bruton's congenital a-y-globulinemia 308
thymus-dependent 171-3,172 4054,421-5 Bruton's tyrosine kinase (Btk)gene 308,325
thymus-independent 171 scope 396-9 buoyant density 129
transplantation 349,351 sex influences 4054,405 Burkitt's lymphoma
virally controlled 374 spectrum 396-8 associationwith malaria 277
antigen-presentingcells 300,407 treatment 438-46 c-my gene in 381
follicular dendritic cells as l60 autoimmunity genes 404 immunodiagnosis 383
interactionwith T-cells 164-5 autoinflammatorydisorders 306
interdigitatingdendritic cells as 15840 avidin 114,129 Cl inhibitor 308
macrophages as 158 avidity 412 Clq 21,23,47
antigen-specifictolerance 30,360 of a n t i s e m for antigen 87 Clr 22,23
antigenic drift 267-8,267 azathioprine 360 Cls 22,23
antigenic shift 267-8,267 after transplantation 360 C2 22
antigenic variation C3 10,13
in bacteria 255-6 B7 see CD80, CD86 cleavagemechanism 10,21,22
in parasites 276 B-cell-specific activator protein (BSAP) 235 C3 convertase 10,11,22
antiglobulins 427 B-ceUs/lymphocytes 24,50,56 C3nephritic factor 345
anti-inflammatory drugs in autoimmune activation 1 7 3 4 C3a 12,13,47
disease 440-1 antigen recognition by 104-5 C3b 12,13,15
antiphagocytes 254 B-l and B-2 subsets 236-7 control of levels 10-11,11
antiphospholipidsyndrome 421 developmentof specificity 23740 c m 11,13,15,22,254,268
antisensemolecules 141 differentiation 234-6 activation 10
antisem evolutionof lineages 244 C3bH 11
antibody content 108 gene rearrangements 239 C3i 10
antibody titer 108 irnmunoglobulin class switching in 191- 4 C4 22,47
avidity for antigen 87 primary deficiency 308-9 C4a 23
cross-reactivity 89,90 receptor 62 C=2,23,47,255
specificityfor 89,91 amplificationof diversity 64,65,68 C4b2a 21,22,23,255,268
a,-antitrypsin 16 role of cytokines in proliferation and C4bp 23
apoptosis 6,18,19,137,201-3 maturation 189,190 C5 12,241
appendix 154 surface receptors 59-60 C5a 12,15,47
Arthus reaction 337 tolerance 240-2 in acute inflammatoryresponse 13,251
intrapulmonary 337 bacille C a h e t t d u e r i n 7,286,287,296 C5b 12
in rheumatoid arthritis 339 Bacillus anthrax infection 262 C7 1
arylsulfatase B 19 bacteria 2 C8 12
Ascaris 276 antigenic variation 255-6 C9 12,19
association reaction 86 capsule 254,258,262 c-myc protooncogene, deregulationin B-cell
asthma 55,326-9 cell wall structure 254 tumors 381
occupational 326 challenge to complement system 254-5, C-reactive protein 16,116
ataxia telangiectasia 311 255 C region 40
atopic allergy 325-32,326,423 evasion of phagocytosis 254 calcineurin 140,141,167
cellular receptors 328 exotoxins 254 California hagfish, immune responses 244
clinical tests 330 Gram-negative 259,345 CALLA 383
etiology 329-30 Gram-positive 255 calmodulin 167
genetic factors 329 host response to 256-60 calnexin 93
therapy 330-2 intracellular 262-7 CAMP 14,345
atopic dermatitis 326,329 opsonization 256,258 Candida albicans 2,312
attenuation 286 pyogenic 4 phagocytosis of 8
by recombinant DNA technology 286-7 survival strategies 254-6 capsule 254,258,262
classical methods 286 bacterial infection carcinoembryonicantigen 376
autoanti-idiotypes 209 extracellular 254-62 cartilage grafts 362
autoantibodies 30,113 intracellular 262-7 caspases 19,202
in human disease 398-9,400,401 bacterial toxins 103 cathepsinG 4,9
tests and diagnosis 438,439 bactericidal/permeability(BPI)factor 9 CD markers 148
thyroid 398 bactericidins 242,313 CD1 78,102,148,159
autoantigen 404,410 bare lymphocytesyndrome 310 CD2 148
CD3 63,64,148 codominantexpression of MHC 76 multiple effects 181
in T-cell differentiation 63 Coley's toxin 387 network interactions 181,183
CD4 collagen arthritis 433 origin and functions 179
depletion of T-cells in AIDS 316 collectins 16,17,258 in pregnancy 370
in HIV infection 316 colloidal gold 133 in proliferation and maturation of B-cells
as marker of helper T-cells 184 colony stimulatingfactors see G-CSF; GM- 189,190
in T-cell differentiation 226,227 CSF; M-CSF structures 178
CD8 148,159 combinatorial library 123,124 cytolysin 18
as marker of cytotoxic T-cells 188 common acute lymphoblastic leukemia cytomegalovirus infection 268,270,314
in T-cell differentiation 226,227 antigen (CALLA) 383 cytotoxic T-cells 188
CD14 6,7,159 common variable immunodeficiency 308-9 adoptive transfer 29,284
CD19 148 complement 10 CD8 as marker 188
CD21, in B-cell differentiation 191 activation 10-12 control of 170-1
CD23 148 alternativepathway 11,15,22 generation 188-9
in germinalcenters 191 classicalpathway 10,21-3,22 in intracellular viral infection 32,271
CD25 148,205,358 diversionof 262 memory 285
CD28 148 in acute inflammatory response 13-15 Tcl clones 184
CD40 148,174,189,389 biologicalfunctions 12-13 Tc2 clones l84
CD40L 174,189,311 coat 259
CD44, in hunors 378 deficiencies 307-8 D gene segments 40,62
CD45 383 evasion of system by bacteria 254-5 and amplification of receptor diversity 65
CD45RA 132, 148,196 and immune complex formation 201 rearrangement 65/66! 67
CD45RO 132,l48,l96 in opsonization 258 daclizumbab 358
CD54 191 proteins of the system 23 defensins 4,8,9,14,260,284
CD77 191 receptors 258 dehydroepiandrosterone 216
CD80 148,205 complement control protein 23 delayed-typehypersensitivityreactions 244
CD86 148,205 complementaritydetermining regions deoxyuridinetriphosphate 137
CD95 148 (CDR) 40,45,64,123 Derpl 326
celiac disease 344,423 complete Freund's adjuvant 192,215,300, diabetesmellitus, insuh-dependent
cell cycle progression genes 381 341 HLA association 367
cell cycle suppressor gene products 381 concomitant immunity 272 pathogenesis 433-5
cell recognition molecules (CRMs) 245 confocal microscope 133,134 twin studies 401,407
cell shutdown 149 conglutinin 17,258 diapedesis 151,250,252
cell-mediated hypersensitivityreactions conjunctivitis 326 Dick reaction 261
341-5 contact dermatitis 341,3434,344 diet, effect on acquired immune response
cell-mediated immunity (CMI) 31,184 contraception, immunological 370 217
in intracellularbacterial infection 263 control mechanisms 200-20 differential splicing mechanisms 59,60
in intracellularviral infection 269-70 Coombs' test 334 DiGeorge syndrome 309-10
in parasitic infections 273,274 cornea1grafts 362 dinitrophenyl 37,85-6,89,171
T-cell effectorsin 185-9 corticosteroids,in treatment of type I diphtheria, effect of immunizationon
cellular techniques 12945 hypersensitivityreactions 331 notification of 284
centroblasts 190 countercurrentimmunoelectrophoresis 110 direct pathways of immunization 354
centrocytes 190 coxsackie virus 268 discoid lupus erythematosus 340,400
ceruloplasmin 16 CR1 258 disseminatedintravascularcoagulation
cetirizine 331,332 CR3 258 345
Chagas' disease 277,412,423 CR4 259 dissociation constant 87
Chediak-Higashi disease 306 CR5 259 disulfidebonds
cheesewashers' disease 338 Creutzfeldt-Jakob disease, new variant 277 interchain 45
chemokines 186,251 Crohn's disease 343,407 intrachain 45
C-C subfamily 187 cross-priming 96 DNP see dinitrophenyl
C-X-C subfamily 187 cross-reactingidiotypes 44,414 double-strandedDNA 425
receptors l78-9,18Ofl87 cross-reactivityof antisera 89 drugs
chemoluminescence 115 Cyptococcus neoformans 271 anaphylactic reaction 327
chemotaxis 10,186 CTLA-4 l65,168,l7O immunosuppressive 358-60
chlorpromazine, adverse effects 336 cutaneousbasophil hypersensitivity 342 type I1reactions to 335-6
cholera 262 cutaneous leukocyte antigen (CLA) 150,159 Duncan's syndrome 312-13
vaccine 297,299 cyclophosphamide 358
chondroitin sulfateA 17,19 cyclosporinA E-cadherin 156
choroid plexus, immune complex deposition in autoimmune disease 441 E-selectin 250,251
340 in prevention of graft rejection 358,359 echoviruses 268
chromosomalbreakage syndrome 311 cyclosporin G 363 Edelman, Gerald 38
chromosome translocations in cytochrome (cyt)b,, oxidase system, defects Ehrlich, Paul 28
lymphoproliferativedisorders 381-2 in 305 elastase 4
chronic granulomatous disease 305,306,343 cytochrome (cyt)b,,, 4,6,305 electrophoresis 115,116
class 11-associatedinvariant chain peptide cytokines 31/73 electrostaticforces in antigen-antibody
(CLIP) 95 as adjuvants 300 complexes 85,87
clonal anergy 233 and autoimmune disease 418-19 ELISA 111,112,114
and T-cell tolerance 233 in chronic inflammatory responses 185-7, ELISPOT technique 136,l37,l39,14Of183
clonal selection 26,28 186 embryonic stem cells, in development of
cloning 362,363 as intercellular messengers 177-81 transgenic animals 141,142
cluster of differentiation markers see CD in intracellularviral infection 187,271 endotoxin 6,7
markers length and range of action 177-8 enhancement 354
eosinophils 19,50,56 transmissible spongiform inheritance 76
i n a s t h a 326 encephalopathies 277 nomenclature 75,76
in parasitic infections 273,274 food allergy 327 see also major histocompatibility complex
eotaxin 251-2 formoterol 331 HAART see highly active antiretroviraldrug
epitope 81 fos 381 therapy
Bcell 81-2,81,119-20 functional affinity 87 Haemophilus influenzae infection 262,308
cross-reactionswith 411-12 fungi 2 Hageman factor 250
identification 82/83 immunity to 271 hagfish, immune response 244
in vaccine development 292 furrier's lung 338 HAMA 122,123
continuous/discontinuous 81 haplotype 75/76
cryptic 99,200,412 G-CSF 273 restriction 91
dominant clusters 82,99,200 source and function 179 segregation 76
helper 205 gene hapten 80,81
mapping 118-20 autoimmunity 404 physical linkagewith carrier 172
spatial complementaritywith paratope conversion 75 variation in structure 83
834 expression 134-5,136,284 haptoglobin 16
specific vaccines 291-2 silent, expression in tumors 376-7 Hashimoto's thyroiditis 335,365,400,403
suppressor 205 gene mapping, major histocompatibility goiter 422
T-cell 118-19,268 complex 73-5,74,75 organ specificity 397,401
link between suppressor and helper genetic engineering pathogenesis 422
205 antibodies 284 hay fever 55,326
molecular mimicry of 412-14,414 cells 1414 hCG 371
piggy-back 414 in vaccine development 286-7 heart block, congenital 424
in vaccine development 2934 genetic factors heart transplants 3634,364
Epstein-Barr virus 123,268,314,387,415 in acquired immune response 211-14 heat-shock proteins 377
equilibriumconstant 86 in atopic allergy 329 heavy chain disease 386
erythema nodosum leprosum 338 in autoimmune disease 399405 heavy chains 39,40
Escherichia coli infection 103,301,412 germinalcenter 152,190,191 variability 40
estrogen 216 globulines transporteurs 237 helminthic infection 19,273
exercise, effect on acquired immune glomerulonephritis hematoidin 5
response 218 in Goodpasture's syndrome 335 hematopoiesis 223
exotoxins 254,261 immune complex 33940,339 role of cytokines in l82
experimentalallergic encephalomyelitis passive transfer 425 hematopoietic stem cells 221-3
(EAE) 215,435,436 glucocorticoids hematopoietinreceptors 178,180
external mucosa, protection by secretory influence on immune responses 215,216 hemolytic anemia
immune system 25940,260 in regulation of inflammation 253 autoimmune 335,399
extracellular killing 18-19,18 glycos~lphosphatidylinositol(GM) 51 as drug reaction 336
extrinsic allergic alveolitis 338 GM-CSF 387 pathogenesis 421
adjuvant properties 300 hemolytic disease of newborn 334
Fab fragment 37,38,48,64,84,126 source and function 179 P-hemolytic streptococci 261
FACS see fluorescenceactivated cell sorting Gm groups 41-2 hemorrhagic shock syndrome 340
factor B 17 Goodpasture's syndrome 335,400,406 hepatitis B 268
factorD 10 pathogenesis 424 vaccination 287,297
familial Mediterraneanfever 307,385 plasmapheresis 446 herd immunity 284
farmer's lung 337 Gorer, Peter 71 hereditary angioedema 307
Fas and FasL 19,203,417 graft-versus-host reaction 352,365 herpes simplex virus 268,271
Fc fragment 38,47 grafts 335,349-73 herpes zoster virus 313
FcERIreceptor 159 clinical experience 362-6 herpesviruses 268,271
in anaphylactic hypersensitivity 324 genetic control of antigens 349,351 high-walled endothelium of post-capillary
FcyRI receptor 50,51,56 privileged sites 362 venules (HEV),lymphocytepassage
FcyRII receptor 50,51,159 rejection 33 across 150
FcyRIIA receptor 50,56 acute early 354 highly active antiretroviraldrug therapy
FcyRIIBreceptor 50,56 acute late 354 (HAART) 319
FcyRIII receptor 50,51 antigens involved 349-51 histaminase 19
FcyRn receptor 50,51 first and second set 350 histamine
FDC see follicular dendritic cells hyperacute 354,361 in acute inflammatory response 14,251
fetus immunologicalbasis 350 in anaphylaxis 322
as allograft 369-70,369 insidious late 354 histamine antagonists 331
liver, B-cell differentiation in 234-6 and MHC incompatibility 352 histocompatibility antigen 71
fibrin clot 250 mechanisms 352-5 HIV see human immunodeficiencyvirus
fibrinogen 16 prevention 355-61 HLA supertypes 293
fibronectin 16,312 role of humoral antibody 354 HLA system
fish oils 217 granulocyte-colonystimulating factor see diseaseassociations 366-9,404
FK-506 see tacrolimus G-CSF HLA-A 72/92
FKBP12 360 granulocyte-macrophage colony HLA-B 366
flow cytofluorimetry 133,135 stimulatingfactor see GM-CSF HLA-C 366
fluorescenceactivated cell sorting (FACS) granuloma 254 HLA-D, and tissue typing 355
130,131 granzymes 19 HLA-DP 366
fluorescenceantibody tests 115,132,402 Graves' disease 398,400,405,422 HLA-DQ 366
follicle center cell lymphoma 383 green fluorescentprotein 133 HLA-DR 366
follicular dendritic cells (FDC) 152,160,191, Guillain Barre syndrome 423 genes encoding 73
277 HLA-E 78
antigen presentation 160,389 H-2 system 101 HLA-F 78
HLA-G 78 see also antibody, antigen-binding IgM
HLE 78 immune surveillance 379 in bacteremia 54
polymorphism 355,356 immunization see vaccination biologicalproperties 49
and tissue typing 355,356 immunoassay expression by B-cells 59
see also major histocompatibility complex of antigens 113,114 monomeric 54
Hodgkinfsdisease 383 solid phase 114 physical properties 49
homing receptors 150,152 immunoblotting 117,317 structure 54
homograftsee allograft immunochemicaltechniques 108-28 isotypes 41
homologous recombination 142,143 immunoconglutinin 258 physical properties 49
horse globulins 281,283 immunodeficiency 33,305-21 structural variants 41-5
horseradish peroxidase 114 combined 3Ilf3l2-l3 subclasses 46-56
house dust mite 326 common variable 308-9 synthesis 192,193
human anti-mouse antibodies ( H A M ) 122 primary 308-11 immunohistochemis&y 130-4
human chorionic gonadotropin 371 recognition of 313 immunological escape mechanisms 379
human immunodeficiencyvirus (HIV) 104 secondary 313,386 immunological homunculus 208,294,434
characteristics 314-16,315 see also AIDS immunological synapse 169,170
infection of cells by 314-15 immunoelectrophoresis, countercurrent immunological tolerance
human T-cell leukemia virus-l 380 110,111 discovery of 230
humoral antibody 354 imrnunofixation 115,116 induction in T-cells 229-31
humoral immunity 25-8,184,272-3 immunofluorescencesandwich test 137-8 immunopathology 33
acquired 26 immunofluorescencetechniques 130-4 immunophilins 358
innate mechanisms 14-18 immunogen 80 immunoproteasome 93
see also antibody immunoglobulins immunosuppressivetherapy
Huntington's chorea 366 allotypes 41-2 after transplantation 357-60
hybridoma 120 amino acid sequence variability 37-9 in autoimmune disease 441-2
T-cell 130 basic structure 38 immunotherapy in cancer 386-92
hydrogen bonding, in antigen-antibody biological properties 49 incomplete Freundfsadjuvant 300
complexes 85,88 class switchingin B-cells 190,191-4 infection
hydrophobictransmembraneregions 59 classes 46-56 acute phase proteins in 16-17
hydroxylradicals 6 coated 259 adversarial strategiesduring 249-80
hypersensitivityreactions 32/33!322-48 domains 45-6 bacterial 254-67
innate 345 fold 245 barriers against 1,2
type I (anaphylactic) 322-32 genes 3941,42 parasitic 271-7
type I1 (antibody-dependentcytotoxic) rearrangementsduring B-cell protozoa1 2,273
332-6,332 differentiation 237-40 viral 207
type 111(immunecomplex-mediated) superfamily 245 cytokine responseto 32-3,32
33641 idiotypes 42-5,44 immunity to 267-71
type IV (cell-mediated/delayedtype) Ig-a 60 response of NK cells 271-2
341-5 Ig-P 60 infectious anergy 233,234,361
type V (stimulatory) 345 IgA 5 2 4 infectious tolerance 204,361
hypogamrnaglobulinemia, transient of biological properties 49 infertility,male 424
infancy 309 deficiency 308-9 inflammation 249-54
in human milk 52,281 acute inflammatoryresponse 13,23
iC3b 11 in the neonate 281 initiation 13,250
ICAM-1 see intercellular cell adhesion physical properties 49 mediators 13,249-50,252
molecule-l in protection of external mucosa 52-3 in type I11hypersensitivityreactions
ICAM-2see intercellular cell adhesion secretory 53 337-9
molecule-2 subclasses 56 chronic 2534
iccosomes 161 IgD 54-5 ongoing 250-2
IDC see interdigitatingdendritic cells biologicalproperties 49 regulation and resolution 189,252-3
IDDM see diabetesmellitus, insulin- physical properties 49 inflammatorybowel disease 343
dependent IgE 55 influenza
idiopathic thrombocytopenicpurpura 335, activation by parasites 275 epidemics, and antigenicdrift and shift
399,423 in anaphylaxis 322-5,324 l67
idiotypes 42-5,389 biologicalproperties 49 vaccine 290
in autoimmune disease 410 in inflammatory reactions 55 innate immunity 1-20,380
cascade 210 in parasitic infections 275 deficiencies of 305-8
cross-reacting 44 physical properties 49 iNOS 7
as epitope-specificvaccines 294 physiological role 55 inositol triphosphate see IP3
manipulation of the immune response structure 55,56,324 integrins
through 211 surface receptors 56 superfamily 150,151
networks 207-11 IgG 37,46-7,109 in thymus 245
private 43 in activation of classical complement intercellular adhesion molecule-l 159,165,
public 44 pathway 47 185,191,267
in regulation of immune responses antiglobulins 427 intercellular adhesion molecule-2 159
209-10 biologicalproperties 49 interchainamplification 68
in therapy of autoimmune disease 443 domains 46,48 interdigitatingdendritic cells (IDC),antigen
immune complex disease 368 glycosylationin rheumatoid arthritis presentationby 158-60,160
immune complexes 429-30 interferons 17-18,269
deposition 339 physical properties 49 IFNa
detection of formation 115 subclasses 55,56 in cancer therapy 387
hypersensitivityreactions mediated by synthesis 193 source and function 179
33641 univalent 5 2 4 IFNP, in cancer therapy 387
interferons (continued) leishmaniasis, evasion strategies of parasite traffic between lymphoid tissues 149-51,
IFNy 32 274,276 149,150
source and function 179 leprosy 266-7 transmigration 150-1,152
in viral infections 32 lepromatous 266 trapping 149
receptors 178 tuberculoid 266 ultrastructure 26
interledins leukemia inhibitoryfactor (LIP), source and see also specific cell types
IL-l function 179 lymphocytic choriomeningitisvirus (LCMV)
in acute inflammatoryresponse 250, leukemias 196
251 immunohistological diagnosis 382-3 lymphoid tissue
source and function 179 treatment 390-1 anatomy and organization 147-9,148,149
IL-2 see also lymphoproliferativedisorders H N i n 327
adjuvant properties 300 leukocyte adhesion deficiency 306 lymphocyte traffic between 149-51,149,
receptors 185 leukocytes 151
source and function 179 adherence 129 lymphomas, immunohistological diagnosis
and T-cell proliferation 185 binding to endothelialcells in 383
transcriptionon T-cell activation 167, inflammation 250 lymphoproliferativedisorders
l69 buoyant density 129 B-cell 313
IL-3, source and function 179 isolation of subpopulations 129-30 deregulationof c-myc protooncogene
IL-4 sedimentationrate 129 381
source and function 179 leukotrieneantagonists 331 chromosometranslocations 381-2
and T-cell proliferation 185 leukotrieneB,, in acute inflammatory immunodeficiencysecondaryto 386
IL-5, sourceand function 179 response 14,252 immunohistological diagnosis 382-3
IL-6 16 leukotrieneC,, in acute inflammatory maturation arrest and cellular phenotypes
source and function 179 response 14,251 382
and T-cell proliferation 185 leukotrieneD,, in acute inflammatory plasma cell dyscrasias 383-6
IL-7, source and function 179 response 14,251 X-linked 312-13
IL-8 LFA-1 see lymphocyte function associated lymphotoxin 19,73,103
in acute inflammatory response 251 molecule-l lysosomal-associatedmembrane proteins
source and function 179 LFA-3 see lymphocyte function associated (LAMPS) 95
IL-9, source and function 179 molecule3 lysozyme 1,4,81
IL-10 LHRH see luteinizinghormone releasing lytic granules 189
in regulation of inflammation 253 hormone
source and function 179 ligand binding assays 119 M-cells 162,162
XL-11, source and function 179 light chains 39'40 M-CSF, source and function 179
IL-12, sourceand function 179 limiting dilution analysis 137,138 M-protein (myeloma) 39,385
IL-13, sourceand function 179 limulin 16,243 M-protein (streptococcal) 261
origin and functions 179 linkage disequilibrium 366-7,367 macrophage activatingfactors 31,186-7
intraepitheliallymphocytes 155 lipid rafts 166 macrophage-colonystimulatingfactor see
invertebrates,microbial defense lipopolysaccharide 15,192,215,251 M-CSF
mechanisms 242-3 liposomes as antigen presenters 301,302 macrophage-derived chemokine (MDC) 159
Xr genes 213-14 Listeria monocytogenes 103,184,262,264-5, macrophages 2,4,50,56,92
Iscoms 301 266 activation 186-7,264
isohemagglutinins 54,333 liver by adjuvants 300
ITAM 51,60,63,70,324 fetal, B-cell differentiation in 234-5 in acute inflammatory response 13-14
ITIM 51/70 transplants 364 as antigen-presentingcells 158
LMP2 74 intracellular killing by 31,264
J gene segments 62 LMP7 74 MAdCAM-l 157
and amplification of receptor diversity LolPI-V 326 MAGE-1 376
65 long-acting thyroid stimulator 398 major basic protein 328
rearrangement 65,66,67 IoxP 142 major histo~ompatibilitycomplex (MHC)
JAK family 180,268 luteinizinghormone releasing hormone 31,70-8
Jarisch-Herxheimer reaction 338 370 Class I kolecules 32,70-3,72
Jenner,Edward 30-1,282 lymph nodes 149,153 antigen processing and presentation
Jerne's network hypothesis 207 B-cell areas 1514 93-5,95
Jones-Mote sensitivity 342 encapsulated 1514 classical 74
jun 381 T-cell areas 154 peptide complex 97,98,99
lymphocyte functionassociated molecule-l structure 72,77
K-cells 332,335 165,196 tissue distribution 77
Kaposi's sarcoma 314,387 lymphocytefunctionassociated molecule3 Class I1 molecules 31
kidney grafts 362-3 165,196 antigen processing and presentation
Kweim reaction 343 lymphocytes 95-6,96
activation 164-78 deficiency 310
lactic acid 1 adoptive transfer 29 invariant chain 95
Lactobacillus lactis 431 assessment of responsiveness 135-7,135, peptide complex 97-9,99
lactoferrin 8,9,16 136 structure 72
lactoperoxidase 1 effects of adjuvants on 300 tissue distribution 77
lac2 135 in graft rejection 352-3 Class I11molecules 73
Lambert-Eaton syndrome 400,423 homing receptors 150,152 discovery 71
lamina propria 155 intestinal 155-7 functions 77
Langerhans' cells 159,343 intraepithelial 155 gene maps 71,73-5,74,75
large granular lymphocytes 24 responsiveness 135-7 grooves 96-9
Lawrence's transfer factor 342 role in antibody production 24,25,26 inheritance 76
Lck 166 small 24,25,26,29 nomenclature 75-6
non-classicalor class I-related 74,77-8 multiple myeloma 3834 parasites
polymorphism 75,77 multiple sclerosis,pathogenesis 400,435 antigenic variation 276
and restriction of T-cell reactivity 91 multivalency,bonus effect of 23 evasive strategiesby 275-7
restriction of T-suppressor cells 203,204 muramyl dipeptide (MDP) 300 host responsesto 276-7
superantigen interaction 104 myasthenia gravis 335,400,405,411,422 immunity to infection 271-7
major histocompatibility locus 351 myb 381 immunopathology 277
malaria 273,276 Mycobacterium leprae 215,296 paratope 81,208
vaccine development 297-9,298 Mycobacterium tuberculosis 216,263,2656, spatial complementaritywith epitope
malnutrition,effect on acquired immune 286 834
response 217 Mycoplasma arthritidis mitogen (MAM) 103 paroxysmal nocturnal hemoglobinuria 307,
Mycoplasmafermentans 432 345
myeloma protein see M-protein (myeloma) partial agonists 169
mantle zone lymphoma 383 myeloperoxidase 4 passively acquired b u n i t y to infection
Mantoux reaction 341 myocardial infarction, acute 307 281-5
maple bark stripper's disease 338 myxedema, primary autoantibodies 399, Pasteur, Louis 282
Marek's disease 387 422 pathogen-associated molecular patterns
MASP 17,22 (PAMPs) 4,6,17,242,256
mast cells 5,13,50,56 N-CAM 245 pattern recognition receptors (PRRs) 4,6,
in acute inflammatory response 13,14 N-ramp gene 7 257
in anaphylactichypersensitivity 322-3 naked DNA therapy 290-1,296 Pax5 gene 235,236
inasthma 326 natural antibodies 208,407 pemphigus foliaceus 423
comparisonof types 323 natural killer (NK) cells 18,47,50,56,70,102 pemphigus vulgaris 400,423
triggering 14,325 cytotoxic activity 70,380 penicillin
MBP see major basic protein; myelin basic in intracellularviral infection 18,270-1 allergy 327
protein ontogeny 242 Jarisch-Herxheimer reaction 338
MCP see membrane cofactorprotein receptors 69-70 pentraxins 16
measles 296,313,343 natural suppressor cells 206 peptide infection marker 105
vaccination against 285,296 Neisseria gonorrhoeae infection 255,256,262, perforin 19,144
Medawar, Sir Peter 350 308 pernicious anemia 400,401,424
membrane attack complex 12,18,23,307 nematode infection 274,275 histopathology 423
membranecofactor protein (MCP) 268 neonate Peyer's patches 154,156,240,277
memory,acquired 28-30 alloimmunethrombocytopenia 335 M-cellsin 161
and vaccination 30-1,30 immune response in 242 phagocytosis 2,3,7,8
memory cells 27,154,195-7,297 maternallyacquired antibody 242,281, cells involved 2-10
B-cells 190 283 defects 305-6
cellular basis for generation 27 thymectomy in 224,416 evasion by bacteria 254,263
induction by DNA vaccine 292 thyrotoxicosis 422 frustrated 336
Metchnikoff, Elie 3 nephelometry in invertebrates 242-3
methotrexate 358 in assay of antigen 116-17,117 mechanisms 9
MHC see major histocompatibility complex in detection of antibodies 110 phagolysosome formation 8
mice nephrotic syndrome 340 phenacetin, adverse effects 336
designer 141 neural tissue grafts 365 phosphatidylinositol3-kinase 167
knocked in 143 neuroendocrinesystems, interactionwith phosphatidylinositolpathway 167
knockout 142,235 immune processes 214-17 phospholipase C 167,181
motheaten 313 neutrophils 2,4,5,8,15,50,56 phospholipase D 19
New Zealand (NZB) 401,416 evaluationof function 136 pigeon-fancier'S disease 337
NOD 401,407,408,409,434,435 Nezelof syndrome 309-10 plantibodies 125
Notch knockout 225 NEAT 167 plants, antibody gene expression in 284
NZB X W (hybrid) 401-3 NFKB 314 plaque techniquesin enumeration of
pure strain 142 nicotinamide adenine dinucleotide antibody-secreting cells 138,139
SCID 139,222-3,299 phosphate (NADPH) 6,114 plasma cells 24/25! 26,27
microbialvectors, in vaccine production Nijmegen breakage syndrome 311 plasmapheresis 446
287-8 Nippostrongtjlus brtasiliensis 273,276,407 platelet activatingfactor in acute
0,-microglobulin 70,72,94 nitric oxide 9,251 inflammatoryresponse 14,251,252
micrometastases 393 in macrophage leishmanicidal activity Pneumocystic carinii 308,314
mimotopes 120 274 poliomyelitis
minibodies 125 NK cells see natural killer cells notification 285
mitogen-activated protein kinases (MAPKs) NK-T cells 102 vaccine
167 NOD mice 401,407,408,409,434,435 killed 285
mixed lymphocyte reaction 352 non-Hodgkin's lymphoma 383 local IgA response 286
monoclonal antibodies 39,84,120-5 non-self, discriminationfrom self 30 poly-Ig receptor 52
as abzymes 122 Notch signalingpathway 222,226 polyarteritisnodosa 337
in cancer therapy 391-2 nurse cells 224 polyclonal BIT-cell activators 414-15
engineered 123-5,125,2834 polymerasechain reaction 355,356
human 122,123 Obese strain chickens 403,404,409,416 polymorphonuclearneutrophils 2,4,5,284
production of 121 Omenn's syndrome 310,312 polyvinylpyrrolidone 117
monocytes 5,50,56 oncofetal antigens 376 PorB protein 262
mononuclear phagocyte system 4,6 opsonins 256 Porter, Rodney 38
monophosphoryllipid A 300 opsonization 17,288 precipitin reaction 109
mouse mammary tumor viruses 1034 original antigenic sin 210,292 prednisolone,in prevention of graft rejection
mucosal-associatedlymphoid tissue 360
(MALT) 151,154-7,156,283 primary azurophil granule 4,5
multiple antigen peptide system 294 primary biliary cirrhosis 398
primary follicles 151 vaccine development 299 TCRl(yS) 62/63!102-3,271
primary immune response 29 SCID see severe combined TCR2 (aS) 62/63
prime and boost strategy 291 immunodeficiency development 228
private idiotypes 43 scleroderma 437 ternary complexwith MHC and antigenic
privileged immunological sites 158 second set rejection 350 peptide 99-100,101
procaspases 137 secondaryfollicles 152 T-cells 31,56
progesteroneneutralizing antibodies 370 secondaryimmune response 28,29 ap 227
progressive lymphoproliferativesyndrome secondaryspecific granule 4 activation, signals 165-6,168,169
313 Sedormid 336 anergy 233
prophylaxis 281-304 L-selectin 150 antigen recognition 90-3,104-5
prostaglandin E, self-association 427 defective repertoire 214
in acute inflammatoryresponse 251 self-tolerance differentiation, in thymus 223-5,226
in regulation of inflammation 253 discovery of 30 evolution 244
proteasome 93,94 induction in T-cells 229-31 evolutionof lineages 244
protein kinase 167 mechanisms of 242 extrathymic 233
protein tyrosine kinase (PTK),in T-cell semaphorins 222,268 y6 62-3,155,227
activation 166-7 serum amyloid P component 16 hybridomas 130
protein-calorie malnutrition, effect on severe combined immunodeficiency 139, interactionwith antigen-presentingcells
acquired immune response 217 144,205,222-3,311,312 164-5
protozoa1infection 2,273 signalrecognition particle 192 in intracellularbacterial infection 263,
psoriasis 344,436 singleradial immunodiffusion 115-16,116 265
psychoimmunology 216-17 Sjogren's syndrome 40,399,401 killer 92,93,183,188-9
PTK see protein tyrosine kinase SLC 150 memory 195-7,197
public idiotypes 44 SLE see systemic lupus erythematosus MHC restriction of reactivity 92
purine nucleoside phosphorylase 310 small cleaved cell lymphoma 383 ontogeny 225-9
small intestine 154 in parasitic infections 274
quinidine, adverse effects 336 smallpox, cell-mediated hypersensitivityin primary deficiency 309-11
282 primed 293
radiation chimeras 139 Snell, George 71 proliferation in responseto cytokines
radioallergosorbenttest (RAST) 113-14,330 sodium cromoglycate 327,330,331 184-5
radioimmunoassay 119 sodium dodecyl sulfate-polyacrylamide gel in regulation of acquired immune
RAG-l gene 40,65,66,227 electrophoresis (SDS-PAGE) 117 response 203-7
RAG-2 gene 40,65,66,227 SP-A 16 resting 165,293,388
RANTES 314,328 SP-D 16 surfacereceptors 60-3
rapamycin 358,359 Spl 314 in thymus 22,223-5,228-9
ras 167,180 sperm, cryopreservation 366 tolerance 229-34
Rasmussenfsencephalitis 423 spleen 154,155 T-helper cells 31,91,173
rate nephelometry 117 S taphylococcus aureus 1,308 and autoimmune disease 411-15
reactive arthritis 407,432 infection 261 in B-cell activation 174
reactive nitrogen intermediates 7,9 STATs 180,360 cytokine patterns 182
reactive oxyg& intermediates (ROI) 6-7,9, stem cell factor 222,389 in regulation of acquired immune
380 stem cells response 203
reactive stromal fibroblasts in carcinoma embryonic, in development of transgenic in T-cell regulation 203
378 animals 141,142 Thl /Th2 1814,182,328,344
receptor editing 66-8,67 hematopoietic 221-3 T-suppressor cells 203,204,214
receptor tyrosine kinase 245 maturation in thymus 224-5 tacrolimus 167,357,359
recombinant DNA technology see genetic Stiff man syndrome 434 TAP gene 93,144
engineering streptavidin 114 TdT 65,137
recombination signal sequences (RSS) 43, Streptococcus spp., infectionby 255 tears 1,260
65,67 Streptococcus mutans 261 temporal arteritis 437
Reed-Sternberg cell 383,384 Streptococcus pyogenes 261,308 tetanus toxoid 30
renal transplants 362-3 subacute sclerosingpanencephalitis 340 TGFP
reproductiveimmunology 369-71 suicide gene therapy 390 receptors 180
restriction elements 102 superantigens 1034,345,412 in regulation of inflammation 253
reticular dysgenesis 312 superoxideanion 6 source and function 179
reverse immunization 295 superoxidedismutase 6,366 and T-cell proliferation 185
reverse transcriptase 69,314 surface plasmon resonance 112 theophylline 332
rhesus blood groups 334-5 Sydenham's chorea 412 Thomson-Friedemeich antigen 393
rheumatic fever 261 systemic acquired resistance (SAR) 243 thrombocytopenic purpura, drug-induced
rheumatoid arthritis 400,401 systemic lupus erythematosus (SLE) 400, 336
adrenal feedback defect 406 401,406 thrombus 250
HLA association 368 genetic factors 405 thymectomy neonatal 224,244,416
pathogenesis 427-31,428,432 immune complex depositionin 340 thymic humoral factor 224
rheumatoid factors 113,385,427 as non-organ specific disorder 398 thymocytes
Ribi adjuvant 300 pathogenesis 425-7 double-negative 225
ricin 124 double-positive 226
T-cell receptors 31,60-3,61 thymopoietin 224
saliva 1,260 amplification of diversity 64,65,68 thymopontin 224
Salk vaccine 285 CD3 complex as integral part 63,64 a,-thymosin 224
salmeterol 331 classes 62 thymulin 224
Salmonella spp. infection 7,103,262,432 discovery 61 thymus 31,223
Scatchard plot 89,109 encoding 62-3 absence 224
schistosomiasis 272,275 genes 61,63,69 immunological function 225
induction of self-tolerancein 231 tumors vasoactive intestinal peptide (VIP) 251
T-cell differentiation in 223-5,226 changes on cell surface 374-8 VL)Jrecombination 40-1,312
T-cell selectionfor self-MHCrestriction in escape mechanisms 379 and amplification of receptor diversity 62,
228-9 immune response to 375,379-80 63,65,67,68
thymus- and activation-regulated immunodiaposis of solid tumors 392-3 vertebrates,evolution of immune response
chemokine (TARC) 159 imunotherapy 386-7 2434
thymus-dependent antigens 1713,172 in vivo imaging 393 Vibriocholevaeinfection 262
processing by B-cells 172-3 see also lymphoproliferativedisorders viral infection 207
thyroid autoantibodies 398 TUNEL method 137 combating 187
thyroid stimulatingantibodies 345 twin studies in autoimmune disease 401, cytokine response to 32-3/32
thyroiditis, autoimmune 397,433 407 immunity to 267-71
thyrotoxicosis 422 response of NK cells to 271-2
tingible bodies 152 ulcerative colitis 343,400 viral interference 268
tissue typing 357 urine 1 viral replication, inhibitionby interferons
TNF receptor-associatedperiodic syndrome 17-18
(TRAPS) 306 V genes 62,63 viruses 2
TNF-related activation-induced cytokine and amplification of receptor diversity antigenicdrift and shift 267-8,267
(TRANCE) 159 65 budding 270
TNF-related apoptosis-inducingligand rearrangement 65,66,67 inhibitionof replication by interferons
(TRAIL) 201-2 somatic mutation 68-9 17-18
tonsils 154 V region 40,42 oncogenic 374,375
toxic shock syndrome 103,261,345 vaccination 282 see also viral infection
toxins 15,103,258 and acquired memory 30-1 vitaminA deficiency 217
toxoid 289 DNA 290 vitamin B,, binding protein 4
Toxoplasma gondii 274 and herd immunity 284 vitamin C deficiency 217
transfection 141,388 history 282 vitamin D, regulatory role 217
transforminggrowth factor-psee TGFP mucosal 288 vitamin E deficiency 217
transfusionreactions 333 nasal 155,288 VLAsubfamily 151,185-6
transgenic animals 142,229,382 oral 287 von Behring, Emil 257
transmissible spongiform encephalopathies strategic considerations 284-5
277 T-cell 2934 Waldenstrom's macroglobulinemia 313,
transplantation see grafts vaccines 281-304 385-6
trauma, effect on acquired immune response acellular 290-1 Wegener 'S granulomatosis 400,436-7
218 adjuvants 299-302 Western blot analysis 117,118
Trichinella spiralis 273 controlled release 302 wheal and erythema (allergy) 330
triple therapy 363 current 296-7 Whipple's disease 407
Trypanosoma brucei 275 epitope-specific 291-6 Wiskott-Aldrich syndrome 311,379
Trypanosoma cruzi 272,292,412 experimental 297-9 Wiskott-Aldrich syndrome protein (WASP)
tuberculosis 265-6 factors for success 285 311
vaccination against 284,296,297,299 idiotypes as epitope-specific 294-5 worms 2,275
tum- variants 375 killed organisms as 285 Wright, Sir Almroth 257
tumor markers 392-3 live attenuated organisms 285-6 Wucheveria bancrofti 338
tumor necrosis factor (TNF) 73,124 peptide 293,294
in cancer therapy 387 subunit 288-91,388-90 X-linked immunodeficiencysyndromes 309
receptors 178 vaccinia virus, vaccine production 287 xenograft 361-2
TNFa 19 Van der Waals forces in antigen-antibody Xenopus, immune system 244
in acute inflammatory response 250, complexes 85/88 XL hyper-IgM syndrome 311
251 variolation 282
source and function 179 vascular addressins 150 Yersinia spp. 103,262,412
TNFP vascular cell adhesion molecule-l (VCAM-l)
anti-viral activity 187 151,185 ZAP-70 166
sourceand function 179 vascular endothelium growth factor 390 zinc deficiency 217