Fundamentals of Immunology - Study Guide

Fundamentals of
Immunology
David Schidlow, M.D., Ph.D.

Professor, Department of Internal Medicine
Annenberg Dean & Senior Vice-President for
Academic Affairs
DREXEL UNIVERSITY | COLLEGE OF MEDICINE | 2015
BIOC 372x Study Guide

This study guide covers Part One: Antibodies and Innate
Immunity.
Lecture L01- Introducing the Metaphors

Lecture L02a- Surveying the Cells & Organs of the Immune System
Lecture L02b- Surveying the Cells & Organs of the Immune System
Lecture L03-Innate Immunity
Lecture L04- Antigens & Antibodies
Lecture L05-Organization & Expression of Immunoglobulin Genes
Lecture L06- Development of B Cells
Lecture L07- Complement
Glossary
Image Attribution
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Lecture L01
Introducing the Metaphor
Infectious disease is one of the few genuine adventures left in the

world. The dragons are all dead and the lance grows rusty in the
chimney corner....About the only sporting proposition that remains
unimpaired by the relentless domestication of a once free living
human species is the war against those ferocious little fellow
creatures, which lurk in the dark corners and stalk us in the
bodies of rats, mice, and all kinds of domestic animals, which fly
and crawl with the insects and waylay us in our food and drink
and even in our love. Hans Zinsser, 1935
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Metaphor is the lifeblood (ha!) of good scientific prose. Matt Ridley, 2003
I.
II.
Welcome
Staying Healthy
A. Context
1. All organisms (including plants and fungi) have defense mechanisms.
These are clearly derived from common ancestral forms, currently
classified as innate.
2. Vertebrates have an additional particularly effective defense - acquired or
adaptive immunity involving antibody production.
3. Insects, the group multicellular animals with the greatest number of
species and probably the highest overall biomass, also have a form of
immunity that allows for a flexible response.
4. The defenses are energetically expensive. (Figure 1.1)
Figure 1.1: ATP
5. These defenses represent a serious threat to your own body, and you
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control them to make sure that they don't wind up attacking the wrong
cells, which certainly does happen.
B. In Praise of Engineers - How We Stay Healthy
1. clean water
2. proper sewage disposal
3. mosquito (and other insect) discouraging buildings
4. communications and transportation infrastructure - allows delivery of
preventive health care and distribution of food.
5. vaccination- OK the engineers didn't give us this one, but without the
communications and transportation infrastructure, its hard to deliver
vaccines.
C. Disease Burden
1. Economic costs of being sick and having to tend to sick children.
2. Rates of infections disease and general ill health correlated with lowered
IQ. (Figure 1.2)
Figure 1.2: Happy Baby
a) Correlation is about 67%, which suggests that this is not the only
factor, but it does provide a possible explanation for the Flynn
effect.
b) Diarrheal diseases rob infant of nutrition at a period of critical brain
growth. 87% of the nutritional energy in newborns goes to the
brain.
c) Cerebral malaria can damage the brain directly.
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III.
Pathogens and Immunity

A. Types, with a few examples
1. Viruses: rhinovirus, flu, small pox, Ebola, polio (Figure 1.3)
Figure 1.3: Viruses
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2. Bacteria: Mycobacterium tuberculosis (TB), E. coli, anthrax, bubonic

plague, strep, Clostridium difficile (Figures 1.4-1.7)
Figure 1.4: Spirochetes
Figure 1.5: Bacteria Structures
Close up
view
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Figure 1.6: Gram Positive
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Figure 1.7: Gram Negative
LPS
Outer membrane
Porin
Peptidoglycan
Plasma membrane
Membrane proteins
CYTOPLASM
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3. Fungi: Candida albicans (yeast), athletes foot, ringworm, Cryptococcus

(Figure 1.8-1.11)
Figure 1.8:
Candida under
Microscope
Figure 1.9:
Candida Tropicalis
Figure 1.10:
Fungal organisms in
human tissue.
4. Unicellular eukaryotes: malaria, trypanosomes, amoebae, Giardia,

Chagas (Figure 1.11-1.12)
Figure 1.11: Malaria
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Figure 1.12: Protozoans
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5. Parasitic worms (Platyhelminthes and Nematoda): flukes, tapeworms,

hookworms, heartworms (Figure 1.13-1.15)
Figure 1.13: Schistoma

Larvae
Figure 1.14: Trichinella
Figure 1.15: Hookworm
B. Recognition Two general strategies to identify and neutralize the threat.

1. Innate recognition: pattern of molecules characteristic of general category
of pathogen. It does not require previous exposure to a pathogen.
2. Adaptive recognition: identifies molecules (usually specific proteins) found
only in a specific strain of pathogen (like mug shots, fingerprints, DNA
fingerprinting, facial recognition, text analysis.) Parallels: both are highly
specific, require previous exposure and are more recent innovations.
IV.
Words of Advice
A. Wash your hands
1. Hands are a big source of contamination.
a) Fecal-oral you pick up a bacterium and transfer it to your own
mouth or someone elses food - "employees must wash their
hands"
b) colds and flu viruses - picked up by hands and transferred to eyes
and nose
c) Think about what you touch.
d) Rub your eyes with the backs of your fingers.
2. Hand washing effectively prevents this.
a) Soap and water
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b) Gel alcohol
3. Doctors are a particularly lethal source of infections.
a) Ignaz Semmelweis and the prevention of puerperal fever
b) Hospital germs are much more likely to have multidrug resistance.
B. Think before you have sex.
C. If you're sick, stay in bed.
1. You'll keep your illness to yourself.
2. You'll force the virus into evolving strains that make everyone less sick.
V.
Metaphors if Power Politics

A. Policing Functions
1. Expensive-Malnutrition is associated with chronic infection.
2. Necessary
3. Deployed frugally
B. National Defense and the Defended Body
1. Variety of agents with complex interconnected controls and
communication.
2. Different levels of defense and hostile engagement.
a) All-out war: TH1 (destruction) - all-out war. If this response
consumes a lot of energy and destroys many of the bodys own
cells, so be it. The alternative is death and you risk all.
b) Cold war: TH2 (containment) diplomatic sanctions and trade
embargoes. This is the kind of response we make to chronic
infections and to many helminth (worm) parasites.
c) Non-Hostile or normal relations: Treg (peaceful coexistence): So
you want some kind of signaling process that tells you to leave
alone harmless bacteria.
3. Misdirected Defenses
a) Allergy - immune response to non-pathogens
b) Inflammatory tissue damage collateral damage during attack on
pathogen
c) Autoimmunity- harm by friendly fire when the immune system
attacks your own tissues.
d) Against transplanted tissues
VI.
Innate versus Adaptive Immune Response

A. Innate and Adaptive are parts if a whole
1. Innate evolved first
2. Adaptive later, in the vertebrates only
3. The two interact, cooperate and exchange information.
B. Innate Characteristics ready to go
1. Phagocytes (neutrophils and macrophages) and NK cells
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2. Defensive proteins complement, lysozyme

3. Barriers skin- mucus
4. Pattern recognition molecules sense general characteristics of
pathogens
C. Adaptive Characteristics
1. Requires more time
2. Requires gene rearrangement
Table 1.1: Innate Versus Adaptive

Innate
Adaptive
Fast (minutes) Always there
Recognizes
patterns
Slower-weeks requires gene Recognizes

initially, 3 or
rearrangement specific
more days
proteins
subsequently
Phagocytes,
NK cells,
proteins &
barriers
B and TH and
Tc cells
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D. Interactions in Action
1. TH cells (adaptive) are at the heart of the immune response.
2. Antigen presenting cells (innate) provide them with information.
3. TH cells in turn chemically stimulate innate cells, such as macrophages.
E. B-cells
1. TH cells will also stimulate B cells (adaptive) to develop and produce
antibodies.
2. Referred to as humoral immunity.
3. When stimulated they divide (clonal expansion).
4. After maturing, they secrete antibodies.
F. TC cells
1. Stimulated sick cells, which present antigen (on MHC I)
2. Gets OK from TH cells
3. Begins attacking sick self cells
VII.
The More you Know: Optional Resources and Fun stuff (You dont get tested on
this!)
A. We cover T cells in the second session of this course. However, I cant discuss
B cells without mentioning T cells, so heres a table (see next page) to help you
sort out some T cell traits:
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Table 1.2 Responding to Foreign Antigen (Inevitably simplified)

Responding Cell
TH (Helper)
Tc (Cytotoxic)
Response
Coordinates immune
response
Attacks and kills cell
Binds antigen with
T-cell receptor
T-cell receptor
Co-receptor
CD 4
CD 8
Antigen
presented/displayed on
Cells presenting/displaying
Class II MHC
Class I MHC
Sentinel dendritic,
macrophages, B cells
All nucleated cells except

sperm
Source of antigen
phagocytosis
synthesized in cell
Antigen hydrolyzed in
phagolysosome
proteosome
Response
Coordinates immune
response
B. Also, if youd like to follow up on some the issues raised in lecture, heres some
sources:
1. From a previous student on the death of a doctor who caught Nipah virus
from a corpse (There werent proper handwashing station at the hospital.
This is a horribly wasteful and tragic way to die.)
2. http://wwwnc.cdc.gov/eid/article/19/2/12-0971_article.htm
3. Importance of sanitation:
http://abcnews.go.com/Health/GlobalHealth/story?id=2805299&page=1
4. Disinfecting patients prevents staph:
http://www.msnbc.msn.com/id/34733342/ns/health-infectious_diseases/
5. Low national average IQs linked with infectious diseases:
www.physorg.com/news197179291.html and reported in The Economist,
July 3, 2010, pages 75-76
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Lecture L02a
Surveying the Cells & Organs of the Immune
System, part a
I can assure you that peace will not be built on poor nutrition
and human suffering. - Norman Borlag, 11/19/01 (from talk at
Rice University)
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I.
Orientation to Terminology
A. Analogies- At the end of your lecture outline, you can find a table that summarizes the
various cells and attempt to draw a parallel between their function and the function of
some element of military or policing defense.
B. Primary Classification Distinctions
1. Primary versus secondary organs:
a. Primary organs are where cells divide, decide on a developmental fate and, if
part of the plan, rearrange genes.
b. Secondary organs are site of co-ordination of information about pathogens and
the subsequent activation of cells.
2. Innate versus adaptive cells: innate cells dont rearrange genes, adaptive ones do.
3. myeloid versus lymphoid cells: two general categories define by an early branching
decision early in development. All adaptive cells are lymphoid, but some lymphoid
cells (NK cells) are innate. Most of the cells in the lymph are lymphoid, but some of
the cells in the blood plasma are lymphoid as well.
C. Cluster of Differentiation: The Term from Hell
1. Immune cells differ in their surface markers, which are characteristic proteins
extending from the plasma membrane.
2. Different cell surface properties cause a cell to sort differently during a process called
flow cytometry.
3. Scientists have a collection of different monoclonal antibodies that attach to and
identify these proteins.
4. Proteins are identified by a number preceded by CD, for cluster of differentiation.
5. Thus the names CD8 or CD25 simply indicate the relative order in which they were
identified.
II.
The Source of It All Hematopoiesis
A. Hematopoietic Stem Cell (Figure 2A.1)

1. This is a pluripotent stem cell that can give rise to any type of blood cell.
2.
It can divide to make more of itself - self-renew or it can begin to differentiate by

making a series of choices that narrows its options.
3. HSCs first form in the yolk sac membrane in the early embryo, migrate to the liver
and spleen and most settle in the bone marrow before birth.
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4. As few as 100 or so HSCs are enough to completely regenerate the whole

hematopoietic system.
5. Isolate lin- stem cells from various types of lin+ cells.
Figure 2A.1: Hematopoietic Stem Cell
B. Signaling Differentiation (Figure 2A.2)

1. Bmi-1 (transcription factor) keeps the HSCs undifferentiated and continuing to divide
and renew.
2. GATA-2 (transcription factor) triggers differentiation from the HSC into the general
path of division and development into a specialized cell.
3. The first decision or branch point, is whether the cell will go myeloid or lymphoid:
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a. CMP- If it turns into a common myeloid-erythroid progenitor, it may develop into a

huge number of types including different types of white blood cells, red blood
cells and platelet-producing megakaryocytes.
b. LMP- If it turns into a common lymphoid progenitor, it expresses the transcription
factor Ikaros, and can become the adaptive T and B cells, and also the innate NK
and dendritic cells. The notch family of transcription factors decides the T- or Bcell choice.
c. Dendritic cells (these are not nerve cells) can arise from either myeloid or
lymphoid lineages.
Figure 2A.2: Signaling
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C. Historical Baggage
1. red blood cells, or erythrocytes. (Figure 2A.3)
2. platelets, or cell fragments from the megakaryocytes. (Figure 2A.4)
3. white blood cells, or leukocytes, which include cells from both the myeloid and
lymphoid lineages.
Figure 2A.3: Erythrocyte
Figure 2A.4: Platelet
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III.
Myeloid Cells
A. Immune Involvement a Secondary Function

1. red blood cells only extrude nuclei in mammals
2. megakaryocytes pinch off platelets and cell fragments without nuclei.
B. Granulocytes: These cells all have specific granules that compartmentalize potentially
dangerous molecules.
1. neutrophils the infantry of the system, in the most modern techie sense.
a. strongly phagocytic first responders to infection and population expands if they
infection does not rapidly clear. (Figure 2A.5)
b. typically live for only a day (in some ways, these guy resemble Kamikaze pilots)
and remains accumulate in an infected region as pus. (Figure 2A.6)
c. granules stain with both acidic and basic stains (different granules with different
functions).
d. nucleus multilobed (sometimes called a polymorphonuclear leukocytes)
Figure 2A.5: Neutrophil
Figure 2A.6: Neutrophil
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2. basophils
a.
b.
c.
d.
granules with histamines stain with methylene blue, a basic stain

lobed nucleus (Figure 2A.7)
not phagocytic
respond to worms
Figure 2A.7: Basophil
3. mast cells similar to basophils, only they associate with tissues instead of circulating.
a.
b.
c.
d.
also basic granules with histamines (Figure 2A.8)

non-lobed nucleus (Figure 2A.9)
released as undifferentiated cells, maturing in their tissues.
have other immune regulatory functions
Figure 2A.8: Mast Cell
Figure 2A.9: Mast Cell
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4. eosinophils
a. granules stain with eosin red, an acidic stain, have hydrolytic enzymes (Figure
2A.10)
b. bilobed nucleus (Figure 2A.11)
c. phagocytic, though less important
d. target worms
Figure 2A.10: Eosinophil
Figure 2A.11: Eosinophil
C. Myeloid Antigen-Presenting Cells, called mononuclear because the nuclei are unlobed
and look like proper single nuclei. These cells are a little like cavalry or scouts: They
both patrol and report back and may kill bad guys.
1. monocytes
a. circulate in blood for about 8 hours (Figure 2A.12)
b. enlarge and give rise to - macrophages (Figure 2A.13)
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Figure 2A.12: Monocyte
Figure 2A.13: Monocyte
2. macrophages
a. migrate into tissues by amoeboid motion, enlarge five to 10 fold (Figure 2A.14)
b. phagocytize pathogens and debris from dead cells. Antibodies attached to
pathogens make this easier. (Figure 2A.15)
c. present antigen derived from phagocytosis to TH cells.
d. subtypes guard specific tissues, becoming a fixed part of the structure.
2A.14: Macrophage
2A.15: Macrophage
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3. sentinel dendritic cells

a. NOT related to nerve cells just have a lot of extensions
b. phagocytize pathogens and debris, but also use receptor mediated endocytosis
and pinocytosis (Figures 2A.16-17)
c. hang out in the peripheral tissues and only migrate to secondary lymphoid
organs if they sense something suspicious
d. present antigen to TH cells- most effective cell for initiating the immune response
e. Some types develop from the lymphoid lineage
Figure 2A.16: Sentinel Dendritic
Figure 2A.17: Follicular Dendritic
IV.
Lymphocytes make up 20 to 40% of the circulating leukocytes in the blood and
99% of the cells of the lymph.
A. B Cells (The equivalent of 007s M)
1. The name derives from their origin. They mature in the
a. bursa of Fabricius in birds (which is the dorsal wall of the cloaca) (Figure
2A.18)
b. bone marrow of most mammals
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Figure 2A.18: Bursa of Fabricius
2. B-cell surface markers

a. Membrane-bound antibody - arms of the Y stick out from the cell
b. Class II MHC molecules - allow B cells to present antigen to T cells after
activation
3. B-cell lineage (Figure 2A.19)
a. small lymphocytes - nave, unexposed to antigen. Only 6 ! in diameter and
indistinguishable from nave T cells. Enlarge during differentiation.
b. plasma cell - loses surface antibody and secretes soluble antibody, and then dies
in a couple of weeks by apoptosis. Lots of RER. (Figure 2A.20)
c. memory cell - held in reserve if the infectious agent should reappear.
Figure 2A.19: B-Cell
Figure 2A.20: Plasma Cell
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B. T Cells
1. The name derives from the fact that they mature in the thymus.
2. T-cell surface markers include T-cell receptor
a. antigen receptors, which differ in structure and function from embedded antibody
b. binds antigen bound to a MHC molecule of a presenting cell, infected cell, cancer
cell (altered-self)
3. T-cell lineage (Figures 2A.21-2A.23)
a. small lymphocytes - nave, unexposed to antigen - visually indistinguishable from
B version, but with different surface receptors. Also enlarge as during
differentiation.
b. TC (cytotoxic - attack altered-self cells) - receive antigen presented with class I
MHC, have CD8 in membrane- the sappers of the immune response, in the
sense of blowing things up or possibly the FBI in the sense that they check for
misbehaving members of the bodys citizenry.
Figure 2A.21: T Cell and Platelet
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Figure 2A.22: T Cell Interaction
c.
TH (helper) the officers of the immune response. They direct immune

response by signaling other cells via cytokines. - receive antigen presented
with class II MHC, have CD4 in membrane. The officers of the immune
response
i. TH1- direct all-out response against intracellular pathogens
ii. TH2- directs restrained containment response against chronic diseases,
including many worms.
iii. TH17 directs response against extracellular pathogens, especially fungi
iv. Treg down-regulated immune response- the diplomatic corps.
d. memory cells - held in reserve if the infectious agent should reappear.
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Figure 2A.23: T Cell Lineage
C. NK (Figure 2A.24-2A.25), or Natural Killer cells (the Seals or Green Berets of the
immune response) no T cell receptors, no CD4, no CD 8, but they do have:
1. MHC I receptors. They do not recognize antigen, but rather trigger an attack if a cell
doesnt have MHC I.
2. Antibody receptors (CD 16) can also recognize antibodies bound to altered cells,
which triggers NK attack.
3. large, with lots of granules containing enzymes and other molecules used to kill
aberrant cells, and apoptosis triggers extending from the plasma membrane.
4. released from the bone marrow ready to kill
Figure 2A.24: NK Cell
Figure 2A.25: NK Cell
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The More You Know: Optional Resources and Fun Stuff

(You dont get tested in this!)
Primary Organs of the Immune System
A. Youtube:
This last for 90 minutes, but it's a different approach to much of the material in the first
two lectures.UCTV Dr. Anthony de Franco.
http://www.youtube.com/watch?v=mFNxXfwlP3A
B. Leukocytes and Analogies tables. See next pages 32-33
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Table 2A.1 Leukocytes
Leukocytes
function
characteristics
Activates inOamrnation, Coordinates
:macroph.ages, and B cells immune response
name
lype
T" (helper)
lymphocyte
Surface expression
CD4
Receptors for class II
MHC olus anti!!en
"'
T
(regulatory)
lymphocyte
Tc
lymphocyte
or cytotoxic
T cell
:Suppresses immune
generally and
<>f speeific lymphocytes
Destroy altered-self cells
:Recognize antigen
present antigen to T
.cell
Plasma cell
:Secre-tes antibodies
Natural
Killer (NK)
cell (null)
Q.
"'
.5
neutrophil
Phago.cyiOSis. p311 of
inOammatory respotlse.
Move mto infected
tissues
basophil
hnponano in allergy
maso cell
Inflammatory response,
especially oo allergies
eosioophil
defense against parasites
!b
il
"
:2
"e>.
.\I
..e
CD4 and CD25 when

naive
Kills v iral infected
CD8
Receptors for class I
and malignanl
cells
lymphocyte
:2
allergy
and autoimmune
diseases
Develops into
plasma cell
Mature B cell.
lives I - 2 weeks
Kills viral infected
and malig.llant
cells. Can
recognize alteredself on first
exposure
picks up both
acidic and basic
dyes; hydrolytic
granules
picks up basis
dyes, e.g_
methYlene blue
Mature cells in
skin, mucosal,
digestive oraco and
other firso defense
Circulating
antigen, TH
MHC, complement
cymkine
receptors I and 2
No surface antibodies
CDI 6 - binds Fe
(stem) of antibodies.
MHC receptor-when
activated, inhibits
killing
Fe and complement
sig;n.als
TH cytokine
sig;llals
An.tlbodies
and downregulated
MHC I
antibodies
Perforin
and
g.ranzymes
chemokines
receptors
Fe re.cepoors fO< lgE
cbcmokines
pros10glandins
and
leukwienes
chemokines
adti-helminlh
agents
Fe re.cepoors for lgE
Sentinel
Process and present
dendritic cell antigen to TH cells
Fe re.cepoors for lgE
Class II MHC, B7,

toll-hke receptors
Class II MIIC, oolllike receptors
mac:ropha3"
.. ,J
follicular
dendritic
cells
PhagocyiOSis of
mierootganisms and
debris, presect antigen
after activation
feed antibody-anoigct
complexes oo 0 cells
Move through
tissues.
differentiated from
monocytes
found in secondary
lymphoid organs
!!
!!
membrane bound
antibodies, cla..;;s I I
action still
mysterioll.;;
tissues.
Macrophage precursors
.r"
MHC plus antigen
Mechanism of Cytokines:
IL- 10,
TGFJl
An.tigen
Perforin
presented on
and
ci"-<S I MHC
g.ranzymes
Pick up acidic
dyes. e.g.. eosin
mooocytc
presented on
ci"-<S II MHC
releases
Varie-ty of
cytokines
enzymes in
Covered with p.tn.

extensions. potent
anti!!<n oresettters
Circulate in blood
fO< 8 hours, then
differentiated into
macrophage
""
:2
!:.
Responds to
An.tigen
Interferon
fromTH
receptors to hold
antibody -<111li3"n
complexes
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Table 2A.2 Analogies
Analogies
element
hyp01hatrunu.<
T" (helper)
Jvmohocv1e
T
Jymphoc:y1e
Tc lympbocyle or
cytotoxic T cell
Narurat Killer (NK)
cell (null)
B Jympbocy1e
antibodies
neutrophil
basophil eosinophi l
ma..(jtcell
Sentin.e l den.dritic
cell
macrophage
Follicular dendritic
cells
Complemen.t
I oroteins
Megakaryocy1e:
(platele1s and
fibrinooenl
Assesses overall states
function
characteristics
Decides how muc.:h energy lhe
comoarison
Activates inOamrnation,
bodv snends on im munitv

Coordinates immune respon.!:;e
Federal govemmen1 - Congress, executive branch,

nennanent
Military officers rnajors and generals
Prevents allergy and autoimmune
Diplo1natic corps
and B cells
Suppresses immune
response, generally and of
snecific Jvmohocv1es
DestMy alter<d-setf celts
diseases
Recognizes signan ues on MHC I
Sappers, Seabees, military engineers
Can r<cogni2e alter<d-self on fi rst Navy S<!ats, Groen ber<H, etc.

expo.o:;ure
Recognize antigen
Develops into plasma cell
can nresem
to T cell
Recognize specific
Tie up pathogens, direc1 immune
pathogenic signarures
cells 10 palhogeos
Phagocytosis, pan of
Move in.to infecuxl tissues
inflammatory response.
lmponan.t defett.(ie again...o;t
May produce allergic response
parasites
Process an.d present an.tigen. Covered with p.m.
I potenl antigen. presenters;
toT" cells
Phagocytosis, present
Move through tissues,
anti!!en after activation
differentiated from mon.ocvtes
Presen.t antigen-an.tibody
Found in lymph nodes, improve
comnlexes to B cells
luftiniiV
Anach to pathogens and
Kill pathogens and summon
debris
immune cells
Recognize mechanical
Plug holes in vessels, summon.
damage
immune cells
James Bond's M
the guy who invents
Smart bombs with homin.g devices and signaling

unil(i
infan.try
Policing function where you want to keep the bad
contained without
lhe c.ilizens.
scouts
cavalry
M'..o; cadre oftechies, workin.g to improve defenses
Lan.d mines
City's Public Works departmen.t
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Lecture L02b
Surveying the Cells & Organs of the Immune
System, part b
I can assure you that peace will not be built on poor nutrition
and human suffering. - Norman Borlag, 11/19/01 (from talk at
Rice University)
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I.
Primary Organs of the Immune System
A. Bone Marrow (Figure 2B.1)

1. Location of HSCs, myeloid cell production, and initial division of lymphoid cells.
2. NK cells rise from here and B cells divide and rearrange their genes here.
3. T cells undergo initial commitment here, but then leave for the thymus to finish
rearranging genes and determining their specific roles.
4. Marrow of femur, humerus, hip bones and sternum are major sites.
Figure 2B.1: Bone Marrow
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B. Thymus (Figure 2B.2)

Figure 2B.2: Thymus
Figure 2B.3: Thymus Cross Section
1. located above the heart, below the thyroid and behind the upper part of the sternum.
a. cortex ( general word meaning outer layer) - Immature T cells (thymocytes) start
here.
b. medulla (general word meaning interior) Final Quality check.
2. Site of T-cell maturation (details of the process later) (Figure 2B.3)
a. Cells rearrange genes for TCR (the T-cell receptor) in cortex.
b. Cells are checked for the ability to recognize antigen on MHC with the correct
affinity (positive selection) in cortex.
c. Cells that survive selection travel through the medulla and undergo selection to
remove self-reactive cells (negative selection).
d. Cells that survive enter the circulation.
e. Cells that do not (over 95%) undergo apoptosis.
II.
Secondary Organs of the Immune System an interconnected surveillance
system, where the immune cells gather and exchange information.
A. Circulation among the organs: lymph makes a one-way trip, while blood makes a round
trip.
1. Blood moves immune cells throughout the body (along with erythrocytes) (Figure
2B.4)
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Figure 2B.4: Blood Movement
a. The lining of vessels (endothelium) responds to infections with inflammation and

this directs neutrophils and other immune cells to the infected site.
b. Proteins in blood plasma include antibodies, clotting proteins and complement
proteins that attack foreign cells.
c. Blood filtered by spleen, which recycles aged erythrocyte and picks of antigen
and other detritus.
2. Lymph also provides transport of immune cells, primarily lymphocytes, but no
erythrocytes (Figure 2B.5)
b. Drains interstitial fluid from tissues, picking up antigens and white cells (Figure
2B.6)
c. Lymph (fluid) filtered through lymph nodes, where antigen is trapped and acted
on.
d. Vessels join into larger ones that empty into the thoracic duct, which in turn
empties into left subclavian vein and then enters heart.
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Figure 2B.5: Return
Figure 2B.6: Lymphatic System
B. Lymph nodes - trap antigen and provides sites for the lymphocytes to interact with
antigen.
1. Basic structure (Figure 2B.7-2B.8)
a.
b.
c.
d.
e.
cortex receives incoming lymph (afferent)

follicles embedded in cortex receive and hold B cells
paracortex (immediately inside) hold T cells
mature B cells leave through this
exit out the efferent vessels
Figure 2B.7: Lymph Node
Figure 2B.8: Node Cross Section
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2.
Cell interactions
a.
B cells activated by antigen migrate to the paracortex to alert T cells.

Some get instruction to go forth and make antibodies.
b.
Secondary follicle develops after antigen exposure. Has active germinal

center where B cells develop in response to signal from follicular dendritic
cells, TH cells and macrophages.
c.
B cells that have spent time in a secondary follicle learn to make more
effective antibodies.
C. Spleen (Figure 2B.9)

1.
in abdomen, next to pancreas
2.
filters blood, not lymph (Figure 2B.10)
3.
red pulp with macrophages that recycle old red blood cells
4.
white pulp (PALS) has T cells
5.
marginal zone with B cells in follicles - system works like the lymph nodes:
6.
Removing the spleen can increase a person's risk for bacterial infections, but
there does seem to be some redundancy in the system as a whole.
Figure 2B.9: spleen
Figure 2B.10: Spleen Cross Section
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D. Mucosal-Associated Lymphoid Tissue MALT (Figure 2B.11)

Also gut associated GALT, and bronchial (lung epithelia) BALT, nasal
NALT
1.
The mucosa of the digestive, respiratory, and urogenital systems represents

the major site of entry of most pathogens.
2.
The epithelia of these systems contain defensive lymphoid tissues.
3.
Organized structures present include tonsils, appendix, and Peyr's patches in

the intestine (Figure 2B.12)
4.
Epithelial cells of the mucosa deliver antigen samples from the lumen,
delivering them via M cells
5.
M cells are large epithelial cells, each with a number of smaller immune cells
residing in the basolateral pocket it makes.
6.
Antigen crosses the plasma membrane to these. The B cell then migrates to
inductive sites.
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Figure 2B.11: GALT
Figure 2B.12: MALT
Skin- the largest organ of the body, not technically a Secondary Lymphoid Organ (Figure 2B.13)
1. Important in innate defenses
a. epithelial cells (keratinocytes) of the outer layer secrete cytokines
b. Also die, leaving behind keratin intermediate filament as a protective barrier.
2. Important in adaptive defenses
a. Keratinocytes can express class II MHC and present antigen.
b. Langerhans (dendritic) cell phagocytize antigen and carry it to lymph nodes.
Also carry class II MHC and activate TH cells.
c. Intraepidermal (a form of intraepithelial) lymphocyte, or IELs, many with
specialized T cell receptors) - activated or memory cells.
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Figure 2B.13: Skin
III.
Final Issues:
A. Apoptosis (Figure 2B.14)

One aspect of the immune system that makes it so energetically expensive is that it
produces huge numbers of cells and then gets rid of the great majority of cells before they
are even used.
1. Analogous to imploding a building.
a.
b.
c.
d.
Cell shrinks
Chromatin condenses
Membrane blebs
Cell fragments into intact pieces, easily phagocytized
2. Necrosis analogous to blowing up a building (Figure 2B.15)

a.
b.
c.
d.
Organelles swell and break down

Cell disintegrates
Contents released where they can cause tissue damage and inflammation
Much harder to clean up after
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Figure 2B.14: Apoptosis
Figure 2B.15: Apoptosis and Necrosis

Comparison
B. Evolution
Ancestral chordates, which gave rise to the vertebrate members of the phylum Chordata,
do not have an adaptive immune system.
1. The first fish to evolve were jawless and we have only a few remaining examples of
this type, among them the lamprey eel. These eels have B cells, GALT and some
thymic tissue with T cells at the tips of their gills.
2. Other fish have immune tissue around the gut, as well as spleens and defined thymic
tissue. (Figure 2B.16)
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3. Amphibians, reptiles, birds and mammals all have bone marrow, but their B cells
mature in a variety of places.
4. So, while its true that reptiles, bird and mammals have B cells and T cell along with
their innate defenses, there is a lot of variety in what gets made where and when.
5. Happily rodents and humans have reasonably similar immune systems, making mice
and rats good lab models for the study of the immune response.
Figure 2B.16: Evolutionary Immunology
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Lecture L03
Innate Immunity
Nothing in biology makes sense except in the light of evolution.

- Theodosius Dobzhansky
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Table 3.1 Immune Response Summary
Summary of Distribution of Immune Responses

organism
plants
sponges
arthropods
fish
reptiles
birds
mammals
innate defenses (PRR,

attack peptides, and
more)
yes
yes
yes
yes
yes
yes
yes
phagocytosis
transplant
(graft) rejection
no
yes
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
adaptive
defenses (B and
T cells)
no
no
no
yes
yes
yes
yes
I. Basic Considerations- An innate defense is one that you can produce prior to
exposure by a specific pathogen. Doesnt require changes to DNA/genes -- all
organisms have a form of innate immunity.
A. Ubiquity
1. Insects have amoeboid cells patrolling their body cavities and can make antibacterial or anti-fungal peptides when challenged by specific pathogens. (Figure 3.1)
2. Plants have an even wider array of pathogen sensors than do mammals. (Figure
3.2)
3. Stem chordates, like the sea squirts (tunicates) and Amphioxus (lancelets), have
innate, but not adaptive, defenses. (Figures 3.3-3.4)
a. Have spinal cord that runs down back, primitive members of our phylum.
b. Adaptive immunity only found in vertebrates fish, mammals, reptiles, birds,
amphibians.
Figure 3.1: Insect
Figure 3.2: Plant
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Figure 3.3: Lancelet
Figure 3.4: Tunicate
B. Anatomic Barriers.
1. Skin (tertiary immune organ) Few pathogens can penetrate intact skin, relying
instead on bites and abrasions. Water-borne schistoma parasites, causing
schistomiasis, or bilharzia, are an important example. (Figure 3.5)
a. Epidermis - thin layer of dead cells (keratin) over a thinner layer of live
regenerative cells over a basement membrane. Oil glands/hair follicles secrete
sebum, antibacterial peptides, and psoriacin to kill bad bacteria (like E. coli), help
promote good bacteria.
b. Dermis deeper thicker layer with blood supply, connective tissue and
mesenchymal tissue important in developmental signaling.
Figure 3.5: Skin
Figure 3.6: Epithelia
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2. Mucosa more vulnerable, typically single layer of epithelial cells over basement
membrane over connective tissue. (Figure 3.6
a. Secretions: tears, mucus, lysozyme - can tear up bacterial membranes.
b. Cilia (lungs and reproductive organs) sweep out pathogens and debris trapped in
mucus
3. Additional GI defenses:
a. stomach acid (vultures) (Figure 3.7)
b. enzymes
c. competing flora (good bacteria)
Figure 3.7: Vulture
II. Inflammation
A. Fever
1. Hypothalamus controls the bodys temperature set point.
2. Fever provides overt sign of immune up-regulation.
3. Higher body temperature associated with better survival rate, even in reptiles.
Reptiles are cold-blooded; a healthy reptile will want body temp. close to or a little
lower than a humans for optimal enzyme functionality. However, if a reptile is sick,
the reptile will go to hottest place available until it feels better. If you keep the reptile
cold and dont let it get warmer, it has a lower survival rate.
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4. Fever induction used with mixed success to treat cancer at the turn of the 20th
century (Coley toxins).
5. Inflammation is a primary response that involves marshalling the whole immune
system (especially the innate part at first) to fight a new pathogen threat. If you take
an antipyretic, you are downregulating this inflammatory response and mitigating its
effectiveness. Fever goes hand in hand with the inflammatory response, which is
why Coley toxins are effective stimulants of strong immune response.
Figure 3.8: Inflamed Finger
Figure 3.9: Inflammation
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B. Local Response: calls in phagocytic cells to the site of infection or injury.

1. Recognized in Roman times (heat, swelling, pain, reddening and loss of function).
TA Note: Sprained ankle wont get infected but definitely will get inflamed. Any type of
injury, even internal, triggers local inflammatory response. Involves swelling (tissue
leaks), redness (blood flow increased here), heating up, and pain. This helps by
preventing you from doing something to exacerbate the injury and allows the body part
to heal. (Figures 3.8-3.9)
2. Symptoms produced by vascular changes capillaries become more permeable so
that plasma (edema) and cells (extravasation) enter infected tissue.
3. Leukocyte extravasation is regulated by the changes in surface CAM molecules.
C. Signals and Receptors:
1. White cells and damaged tissues release chemokines, a type of cytokine, also
generally up-regulate inflammation, attracting cells to the site of the infection. (Figure
3.10)
2. Activation of cells by pattern receptors also activates inflammatory signals.
3. Neutrophils leave the blood stream and respond by changing the conformation of
their integrins, which makes them stick to the blood vessels near the problem.
(Figure 3.11)
a. Rolling
TA note: Eventually causes enough internal changes for the next step,
activation, to occur. Neutrophils sticking and releasing, bouncing on
endothelial surface.
b. Activation
c. Arrest
d. Transendothelial migration
TA Note: On its way to the tissue to instigate inflammatory response.
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Figure 3:10: Extravasation
Figure 3.11: Neutrophil
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4. The cytokines produced may also regulate the response so that it is most effective
for the particular type of pathogen that initiated it.
5. Activated macrophages and dendritic cells then travel to the TH cells and present
antigen, specifically activating the adaptive response.
6. The TH cells then coordinate an adaptive attack on the infections.
III. Innate Targeting of Pathogens
A. Reviewing the Bad Guys (Figures 3.12-3.17)
TA Note: How the innate immune system recognizes potentially dangerous pathogens.
Mainly characteristic cell surface proteins, types of genetic material.
Figure 3.12: Flu Single-stranded RNA
Figure 3.13: Bacterial Structures
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Figure 3.14: Candida (Yeast)
Figure 3.15: Giardia
Figure 3.16: Trypanosome
Figure 3.17: Tapeworm
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Table 3.2 Pathogen Recognition System

Pathogen
Examples
Factoids
Pattern recognition alerted by:
Viruses
flu, small
pox, HIV,
polio, Ebola,
rhinovirus,
hepatitis,
measles
Strep, staph,
TB, anthrax,
leprosy,
bubonic
plague,
pertussis,
diphtheria
Candida
(thrush)
athletes foot,
Cryptococcus
, ringworm
Can only
reproduce
inside cells
Foreign nucleic acids (double stranded RNA,

single stranded DNA, foreign methylation
patterns), reduced antigen presentation by
infected cells
Reproduce
intracellularly or
extracellularly,
depending on
type
Characteristic surface carbohydrates

(peptidoglycan, mannose repeats), flagellar
proteins (flagellin), lipids (lipotechoic acid)
characteristic DNA methylation patterns.
Bacteria cant evolve version of flagella that
cant be recognized anytime soon.
Eukaryotic,
unicellular,
multicellular or
multinucleate
Cell wall: zymosan ( 1-3 glucan) and

chitin (cellulose with N-acetyl glucosamine
instead of just glucose)
Protozoa
malaria,
Chagas,
sleeping
sickness,
amebic
dysentery,
leishmaniasis
Unicellular
eukaryotes
Characteristic cell surface proteins (profilin)

and lipids (glycosylated phosphatidyl inositols
- GPI)
Worms
(helminth
parasites)
pin worms,
hook worms,
heartworms,
schistosomia
sis, flukes,
tapeworms
Primarily
members of
Platyhelminthes
(flatworms) and
Nematoda
(roundworms)
Characteristic cell surface proteins
Bacteria
Fungi
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B. PAMPs (Pathogen Associated Molecular Patterns) Recognized by PRRs pattern

recognition receptors.
1. proteins - So far, we have identified very few proteins that we recognize innately by
their overall structure. The ones weve found are:
a. Flagellin (figure) bacteria (Figure 3.18)
b. profilin surface protein of protozoan (toxoplasmosis)
2. carbohydrates and glycopeptides
a. zymosan component of fungal cell walls a vague term because it has not
been exactly characterized.
b. peptidoglycan the cell wall component of both gram positive and gram negative
bacteria, although gram positive have a much thicker wall. (Figure 3.19)
3. lipids, especially attach to signature carbohydrates or peptides. (Figure 3.20)
4. nucleic acids
a. DNA with specific methylation patterns - recall restriction endonucleases.
b. wrong strandedness single stranded DNA and double stranded RNA are
usually signs of a potential threat.
c. Mice can specifically recognize the 23s component of bacterial ribosomes
Figure 3.18: Flagellin
Figure 3.19: Peptidoglycan
Figure 3.20: Lipids
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C. Categories of PRRs: Specific receptors bind to characteristic pathogen molecules.

1. Extracellular
a. Lysozyme (mucus and tears) against peptidoglycans (Figure 3.21)
b. Psoriacin (skin) against E. coli. Can be induced by sunlight (UVB) via vitamin
D.
c. AMPS defensive peptides that that typically kill by disrupting bacterial
membranes.
d. Mannose-binding lectin (plasma) activates complement.
e. C-reactive protein (CRP) (plasma) also recognizes microbes and damaged selfcells. (Figure 3.22)
f.
Lipopolysaccharide binding protein (LBP) specifically recognizes gram negative

bacteria.
TA Note: Gram negative bacteria have relatively thin peptidoglycan walls, also
have second membrane on outside often covered with lipopolysaccharide -setting off a HIGHLY inflammatory response can kill.
Figure 3.21: Lysozyme
Figure 3.22: CRP
2. cytoplasmic - NOD proteins (Nucleotide-binding oligomerization domains)

3. Membrane Bound the Toll-like receptors (TLRs) (Figure 3.23)
a. Work singly or in pairs. Singles extend onto the endoplasmic reticulum lumen,
pairs from the plasma membrane.
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b. Binding of a molecule characteristic of a pathogen on the outside of the

membrane triggers a signal on the opposite side.
c. The internal signal activates NF-B, the major internal inflammation regulator.
d. NF-B turns on the production of cytokines, alerting a variety of other immune
cells.
e. Identify the category of pathogen and set of inflammatory and adaptive response
TA Note: Internal TLRs (including 3, 7, 8, 9) largely involved in recognition of bacterial/viral
DNA. External (including 1, 2, 4, 5, 6) largely involved in recognition of characteristic cell surface
components. Usually paired TLRs on outside and unclear if endomembrane TLRs operate
singly or paired. For example, macrophages can use external TLRs to recognize pathogen cell
surface molecules during phagocytosis, and after it internalizes the pathogens it can use
internal TLRs to recognize internal pathogenic molecules such as their DNA or RNA.
Table 3.3 TLR
TLR
1
1&2
2&6
4&4
5 (&5?)
6
10 (Nonfunctional in
mice)
11 (mice only)
Recognizes
Triacyl lipopeptides
Peptidoglycan component
and triacylated lipopeptides
lipotechoic acid
zymosan (-glucan cell wall
component)
mucin
lipopeptides
Pathogen
mycobacteria
Gram+ bacteria
lipopolysaccharide (exterior
membrane)
F-protein
Flagellin
Diacyl lipopeptides
zymosan
unknown
Bacteria, Ni
RSV virus
plasma
Flagellated bacteria
Mycobacteria
Yeasts and fungi
allergens
plasma
plasma
Profilin
plasma
endomembrane
endomembrane
mycobacteria
yeasts and fungi
trypanosomes
Gram+ bacteria and
mycoplasma
12 (mice only)
profilin
13 mice only
bacterial 23sRNA
Eukaryotic parasites
(also recognizes
bacteria)
Eukaryotic parasites
(especially
trypanosomes)
prokaryotes
3
7
Double-stranded RNA
Single-stranded viral RNA
viruses
viruses (HIV)
Membrane
plasma
plasma
plasma
plasma
plasma
endomembrane
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8
9
Single-stranded viral RNA

DNA with unmethylated CpG
signatures
viruses
virus and bacteria
endomembrane
endomembrane
f. Mechanism for endomembrane TLRs

g. Negative regulation- you have to be able to turn these off or you get excess
inflammation and maybe and autoimmune disease.
h. Interact with: MD2 at surface for binding LPS (and also certain viral and cancer
proteins), MyD88 to initiate internal signal sequence.
Figure 3:23 TLR Location
IV. Cell Types and Function

A. Phagocytosis Macrophages and Neutrophils capture pathogens and kill them with a
complex toxic brew. (Figures 3.24-3.26)
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Figure 3.24: Macrophage
Figure 3.25: Neutrophil Anthrax
1. Capture pathogen in phagosome, promoted by PAMP, complement or antibody on

surface of pathogen. - activates membrane pump which
2. Triggers respiratory burst (O2 uptake) by NADPH phagosome oxidase (phox
enzymes) complex. This is not mitochondrial respiration, but rather the direct uptake
of oxygen by enzymes that use it to create toxic reactive oxygen species (ROS).
(Figure 3.27-3.28)
a. superoxide radicals (O2._)
b. hydrogen peroxide (H2O2)
c. HOCL (hypochlorous acid or bleach)
d. Can also produce reactive nitrogen species (RNS) including nitric oxide.
3. Superoxide radicals also generate reactive nitrogen species (RNS) including NO,
also toxic (Figure 3.29)
4. Oxidation is coupled to K+ transport, rendering the interior hypertonic. K+ enters to
compensate for the negative charges, resulting in a rise in pH (8.5). Once the pH
reaches this, further neutralization is done by transporting H+.
5. Change in K+ and tonicity dispersed protein granules. These release
a. hydrolytic enzymes
b. peptides that poke holes in the bacterial plasma membrane (BPI or defensin)
(Figure 3.30)
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6. Microfilaments package the exterior of the vacuole, preventing swelling (which would
normally occur after influx of K+ and subsequent increase in osmotic pressure) and
maintaining the concentration of toxic compounds inside.
Figure 3:26: Phagocytosis
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Figure 3.27: NADPH
Figure 3.28: Phox Complex
Figure 3.29: RNS, ROS
Figure 3.30: Defensin
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B. Cells
1. Neutrophils - the infantry in the modern sense, and the first on-site defenders. Along
with macrophages, most important phagocytes.
2. Macrophages - the cavalry of the outfit. Phagocytosis causes them to secrete IL-1,
IL-6 and TNF, all inflammation activators. Always on the lookout for pathogens,
and after phagocytizing, they relay info about the pathogen to the Th cells (unlike
neutrophils).
3. Dendritic cells - the scouts and patrols. Most important initial trigger of the adaptive
response. They phagocytize primarily in order to sample pathogens and activate
inflammation on the adaptive response. Specifically designed to activate Th cells and
therefore phagocytes, secrete a lot of cytokines and coordinate with Th cells. (Figure
3.31)
4. NK Cells - function by pattern recognition making them part of innate defenses.
Principally attack rogue-self by inducing apoptosis. Innate, but lymphoid cell that
recognizes self-cells acting out -- often because they have been commandeered by
pathogens. Also helps activate macrophages, which go on to activate Th cells.
Figure 3.31 Dendritic Cell
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V. Summary
Table 3. 4 Innate and Adaptive Immunity Compared
Characteristic
speed
Innate
response within minutes
recognizes
molecules characteristic of nonself usu. pattern recognition
diversity
hundreds of types, soluble and

membrane bound including TLRs
gene
rearrangement
needed
may attack self
memory
no
Cells
myeloid cells, especially

phagocytes, NK cells
(lymphocytes)
all living organisms (in various
forms) including bacteria
found in
no
no
Adaptive
first response: 2 weeks
second response: 3 days
(much greater specificity)
most proteins, many
carbohydrates and a few
lipids
VAST number of types,
soluble and membrane
bound, including every
possible antibody.
yes
yes
yes, prior exposure speeds
response.
B cells, T cells (instructed by
myeloid antigen-presenting
cells)
vertebrates only
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Table 3. 5 Innate Examples

secretions
across skin
secretion
across mucus
membranes
plasma,
interstitial
fluid
cell
membranes
cell cytoplasm
(lumen of
endomembrane
system)
Sebum
Mucus
complement
(MBL), LBP
TLRs
NOD2,
defensins,
hydrolytic
enzymes
antimicrobial
peptides
(psoriacin and
cathelicidin)
lysozyme
(hydrolytic
enzyme
attacking
peptidoglycan)
defensins
C-reactive
protein (bind
microbial
surfaces) also
used as a
blood test for
heart attacks.
pH adjustment
(slightly acid)
but nothing like
the stomach
acids (slight to
strong)
ROS/ RNS
pH adjustment
(basic)
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Lecture L04
Antigens & Antibodies
All models are wrong, but some are useful statistician George
Box, 1978
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I.
Context
A. A Riff on Models
1. Examples of Models
a. Physical aircraft carriers, the Mississippi river, antibodies, T-cell receptors,
MHC molecules and Toll-like receptors.
b. Computer important in epidemiology
c. Maps, house plans, circuit diagrams, flow charts showing signaling pathways
d. Model organisms: bacteria, Dictyostelium, yeast, C. elegans, Drosophila,
Arabidopsis, zebrafish, mice (Figures 4.1-4.8)
e. We try to use the simplest organism we can that still is similar enough to
us to imitate the complex processes were investigating.
Figure 4.1: Dictyostelium
Figure 4.3: DNA
Figure 4.4: Arabidopsis
Figure 4.2: Yeast
Figure 4.5: Drosophila
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Figure 4.6: C. elegans
Figure 4.7: Mouse
Figure 4.8: Zebrafish
2. A Good Model a. preserves the essential logical relationships or information pertinent to the
problem.
b. removes any details superfluous to the problem.
c. presents problem at a comprehensible scale.
d. allows you to manipulate, play and make mistakes at low cost.
e. may be quite different from the real thing.
B. Transferring Information
1. typical pathway (Mousetrap model)
a. Cell A secretes small protein (signal)
b. The small protein diffuses to the surface of Cell B, where it binds a largish protein
embedded in cell Bs membrane, extending into the cytosol (receptor).
c. Binding involves weak interactions
d. Upon binding, the receptor shifts shape, and transmits the change to its cytosolic
region (transduction).
e. The change at the inside sets off a cascade of changes: activates enzymes,
brings different molecules together, changes binding properties, etc.
TA Note: Ultimately the goal is often to influence the proteins being

churned out of cells. Transcription factors are often at the end of the
cascade, resulting in changes in the transcription pattern of DNA,
upregulating some genes and downregulating others.
f.
Cell B responds (subroutine in TTSP when a minor trigger leads to dropping a

whole piano.)
2. Variations
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a. signal may be not be protein.

b. signal may not come from one of your own cells (pathogen or the environment)
c. It may take more than one signal (repeats or two different ones at the same
time).
d. Paths may branch, inhibit other paths, and turn themselves off.
e. The response may involve a physiological change, a change in gene expression
or an overall increase in cell division.
II. The Immunoglobulin Superfamily, pipe cleaner models
A. What makes a protein a family member?
1. The molecule has at least one immunoglobulin domain. The domain is the
compact lump, packed together and stabilized.

2. In this domain, the peptide fan-folds into a compact lump. (Figures 4.9-4.10)
Figure 4.9: Peptide
Figure 4.10: Folding
3. Hydrogen bonds hold these switchbacks into pleated sheets

4. Disulfide linkages further stabilize the domain. They form by covalent joining of two
cysteine R groups. (Figure 4.11)
5. Blue regions are hydrophilic amino acid side chains and yellow regions are
hydrophobic, causing the peptide to fold into pleated sheets to avoid
unfavorable interactions.
6. You can refer to the whole domain as a bread and butter sandwich, because the
hydrophobic amino acid side chains wind up at the interior of the structure (butter)
the hydrophilic at the exterior (bread) and the disulfide bond function like a toothpick
in nailing everything together. (Figure 4.12)
7. Often represented by a structure looking like a capital C with the ends joined by
disulfide link. (Figure 4.13)
8. Most of these proteins extend from the plasma membrane, nailed there by
membrane-spanning regions. Antibodies are a rare exception.
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Figure 4.11: Disulfide Linkages
Figure 4.12: Sandwich
Figure 4.13: Domain Representation
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B. Tell me a story.
1. 650 million years ago, the oceans froze solid to a depth of a mile. Liquid water
remained on land around hot springs, and life also clung to the thermal vents in the
depths of the oceans.
2. At this time, organisms were small and simple in structure.
3. 600 million years ago, the earth warmed and melted.
4. Life multiplied, spread and evolved, using these molecules to construct complex
structures.
5. Animals used immunoglobulins to tag nerve cells and serve as signal receptors
during development. Cells of primitive organisms, such as sponges, initially
used immunoglobulin cell recognition molecules to reform after trauma.

Immunoglobulin molecules were thus initially employed in recognizing self-cell
molecules spatially and functionally.
6. Eventually animals began using immunoglobulins to recognize not-self, which is how
they came to be involved in immune responses.
III. The Structure of Immunoglobulin Receptors (BCR) and Antibodies.
Basically an Ig receptor (or B-cell receptor) is an antibody with a membrane-spanning and
cytosolic domain at the end (C-terminal). Thus the antibody is soluble and secreted from
the cell and the receptor version is stuck in the cell membrane with the business end facing
outside the cell.
A. Terminology
1. In the 1960s, chemists classified proteins as fibrous (silk, collagen) versus globular
proteins (most proteins, actually). Globular basically meant soluble in plasma.
2. immunoglobulins: the protein fraction in the plasma involved in fighting disease.
3. gamma (high mobility) fraction
4. Scientist purified this fraction and used it to study the structure of the antibody.
TA Note: Putting the blood plasma into the gel electrophoresis will give you 5
peaks (the first is much larger than others).
The peaks are albumins (small blood proteins to help carry things around and
keep osmotic pressure under control), alpha 1, alpha 2, beta, gamma (heavy
one). Majority of gamma globulins were identified as antibodies, conferring
the vast majority of immune protection among the plasma proteins.
B. Analytical History Porter and Edelman
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1. Gerald Edelman - treated antibodies with mercaptoethanol (Figure 4.14)

a. This treatment reduces the disulfide bond, thus breaking the covalent bond that
stabilizes the antibody.
b. The antibodies separated into two peptides.
c. We now know these are the intact light and heavy chains. You can this separate
them and study each in isolation. Heavy chains separated by gel electrophoresis,
about twice as massive.
2. Rodney Porter - cleaved antibodies with brief exposure to proteolytic enzymes
(Figures 4.15-4.16)
a. This treatment breaks up the peptide bonds between amino acids, targeting the
most accessible bonds first.
b. Very brief treatment with pepsin: cleaves preferentially at hinge between arms
and stem, separating the Fc (stem) section) from the top half, called Fab(ab')2.
c. Mix Fab(ab')2 with their antigen and they will precipitate. This has both heavy
chain parts and light chains -- still held together by disulfide bonds. Fab 2
fragments are the entire top half and retain both antigen binding sites. This
lets them bind an antigen on each arm and connect together to form a
chain, which readily precipitates out. This is only possible because
Fab2(TWO) fragments have TWO arms, and Fab fragments just have 1,
allowing several to bind a single antigen without forming a chain.
d. Brief treatment with papain produces FAB fragments, which are isolated arms.
e. These can bind antigen, but will not precipitate because they cannot cross-link
one antigen to two fragments.
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Figure 4.14: Edelman
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Figure 4.15: Porter - Pepsin
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Figure 4.16: Porter - Papain
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C. Form and Function (foam board model) (Figure 4.17)
1. Two light (L) chains (~25,000 MW; MW 50,000 total for both ), identical to each
other, composed of 2 immunoglobulin domains, variable and constant. Chains =
peptide. The bottom left and right blue and pink regions compose light chains.
The top right and left dark blue and pink regions leading all the way down into
the Fc stem are heavy chain polypeptides. These 4 polypeptides are
associated at the quaternary level in antibodies.. Sequins indicate disulfide
bonds.
2. Two heavy (H) chains (~50,000 MW; MW 100,000 for both), identical to each other,
composed of 4 or 5 immunoglobulin domains, one variable and 3 or 4 constant.
3. Overall molecular weight: ~150,000
4. The amino (NH2) end of the heavy chain joins to the light to form the Y arm.
5. The other ends (carboxyl or COOH) of the heavy chains join together to form the Y
base or stem.
6. Both L-H and H-H linkages involve weak interactions and covalent disulfide bonds.
7. The amino ends of both L and H peptides (the part found at the tips of the Y arms)
vary greatly from one antibody to the other. (Figure 4.18)
8. This (the loops at the ends of the arms) is the region that interacts with the antigen
9. An oligosaccharide (small carbohydrate) attaches to the second immunoglobulin
domain from the end, pushing open the Fc stem. This is the white fluffy part
between the Fc stem on the antibody diagram; it tells the immune system how
to treat what antibody is attached to.
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Figure 4.17: Foam Board Model
Figure 4.18: CDR
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D. Details of the Structure of the Light Chains

Two basic parts or domains (Figure 4.19)
Figure 4.19: Light Chain
1. Constant Region (CL)

a. Typical Ig domain: pleated stabilized by disulfide linkages, hydrophobic side
chains to the interior, hydrophilic to the exterior.
b. Forms part that connect to hinge/bend to base of Y
c. Two versions: for kappa and for lambda, differing in the constant region.
d. In humans, each lambda () gene has five different versions of the constant
region.
e. Either kappa () or lambda () can be in any immunoglobulin class and all
versions have very similar overall structures.
2. Variable Region (VL)
a. Most of variable domain is Ig domain and is actually pretty constant.
b. 3 loops that stick out at the end comprise hypervariable region composed of
three non-contiguous amino acid sequences (15 to 20% of the domain).
c. The rest of the domain is the framework region that basically holds the loops in
place.
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IV. Immunoglobulin Classes (Figure 4.20)

Figure 4.20: Antibody Ribbon Model
There are 5 classes of antibodies, which differ in function and in the exact amino acid
sequence and conformation of the stem part of the Y. In all cases, the basic unit has
two heavy and two light chains and the light can be either or .
A. Categorization by Heavy Chain Structure
1. flexible hinge or rigid bend number of constant C Ig domains 3 versus 4
(Figure 4.21)
Figure 4.21: Comparison
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2. oligosaccharide small carbohydrate added to second domain from the C terminalvaries, depending on exact type.
3. J chain compound antibodies that can cross epithelia
4. subclasses (different version of related Ig types)
Table 4. 1 Antibody Classes
Ab
Forms
comple
xes
J chain
Subclasses
Timing
Membranespanning Ig
receptor?
Role
Flexible
hinge or
rigid
bend?
rigid
yes
yes
no
yes, nave
and memory
general
hinge
no
no
no
on nave
cells, rarely
soluble or
memory
aids nave B
cell activation?
hinge
no
no
memory
cells
specific
responses to
acute infections
hinge
yes
yes
memory
cells
crosses
epithelia,
protects
boundaries
bend
no
no
first class
produced
in
maturing
B cells.
Produced
as Ig
receptor
on mature
but nave
B cells
after class
switching
in
activated
B cells
after class
switching
in
activated
B cells
after class
switching
in
activated
B cells
memory
cells
TH2 response:
allergies,
pollutants,
chronic
infections
B. Travelling Down the Heavy Chain

1. Variable Region
a. also composed of pleated sheets, with switchbacks
b. also "bread and butter sandwich" structure
c. 3 loops that stick out at the end show variation, framework region much less
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d. Hypervariable region (coupled with corresponding hypervariable region on the

light chain) composes the complementarity-determining regions (CDRs).
e. Thus each CDR is composed of 6 loops at the tip of the Y, 3 H and 3 L.
2. First Constant Domain
3. Hinge-Bend Region
a. M and E ( and heavy chains) rigid bend at C2 between C1 and two constant
stem domains, C3 and C4. C2 replaced by hinge in G, A and D.
b. G, A and D (, and heavy chains) has a longish sequence rich in proline and
secured with disulfide linkages that makes this region especially bendable. This
connects C1 and C2, occupying the same place in the antibody as C2 in the and
heavy chains.
4. Next Constant Domain (C2 of , , and and C3 of and )
a.
b.
c.
d.
also "bread and butter sandwich" structure

site of oligosaccharide attachment (added after protein synthesis)
opens these domains to the aqueous environment
interacts with complement (more later)
5. Carboxy-terminal Constant Domain (C3 of , , and and C4 of and )

a. Crucial function in determining whether or not the antibody is membrane-bound
or secreted.
b. Secreted version ends with short hydrophilic sequence.
c. Membrane-bound version ends with hydrophobic and then hydrophilic sequence.
d. Membrane bound version are expressed first in nave cells, secreted versions
after maturation to plasma cells, and membrane-bound versions in memory cells.
C. Specific Types
1. IgM - heavy chain rigid bend (Figure 4.22)
a. rigid bend 4 constant domains
b. Function- general purpose - First class expressed in plasma.
c. Monomeric form (actually 2H +2L) expressed as a membrane-bound antibody on
the nave B cell.
d. Secreted form occurs as pentamer, looking like 5 IgG's stuck together, stems in,
10 antigen-binding sites out.
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e. Held together by an additional peptide, the J chain. The J chain binds to a

secretory component, a peptide that allows structure to be secreted into mucus,
etc. (Figure 4.23)
f.
Very good at binding large complex structures and activating complement (to kill
foreign cells).
Figure 4.22: IgM Pentamer
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Figure 4.23: J Chain
2. IgD - heavy chain flexible hinge (Figure 4.24)

a. Function- aids recognition by nave B cell.
b. Primarily found (with IgM) as a membrane-bound receptor in nave B cells. While
M class antibodies also function in plasma, D class rarely does.
c. Rarely found in plasma (0.2% of total serum immunoglobulins)
d. superficially resembles IgG (different amino acid sequence).
Figure 4.24: IgD
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3. IgG - heavy chain (Figure 4.25)

a. flexible hinge, 3 constant domains
b. standard secreted antibody defending against bacterial and viral pathogens
c. comes in four versions, numbered 1 to 4, varying in biological specificity:
IgG1 activates complement, Fc receptors bind tightly
IgG2 weakly activates complement, Fc receptors bind weakly
IgG3- strongly activates complement and binds tightly to Fc (lots disulfides)
IgG4 does not activate complement, binds weakly to Fc.
Figure 4.25: IgG Subclasses
Table 4.2 G-class Antibodies

Clas
s
complement
activation
phagocyte
activator
function
hinge
length (#
disulfides)
2
strong
very strong
weak
no
inflammatory: TH1 response to

serious threats
only mildly inflammatory; may
cooperate with A and E antibodies
during TH2 responses
11
very strong
very strong
no
strong
highly inflammatory: TH1 response

to intracellular pathogens.
intermediate response (possibly
mop-up)
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4. IgA - heavy chain flexible hinge (Figure 4.26)

a. Function- primarily epithelial patrol.
b. Also occurs in serum as a monomer.
c. The secreted form occurs as dimer, looking like 2 IgG's stuck together, stems in,
4 antigen-binding sites out, and may occur as trimer or even tetramers
d. Two subclasses (1 and 2)
e. Also held together by an additional peptide, the J chain (binds secretory peptide)
which is identical to the one in IgM
f.
Secreted into mucus, tears, saliva, and breast milk- up to 15 grams per day!
g. Plasma cells that secrete this tend to home in on various epithelial linings.
h. Unfortunately these same pathogens often produce proteases that specifically
target the vulnerable hinge regions of this antibody.
5. IgE - heavy chain, rigid bend (Figure 4.27)
a.
b.
c.
d.
e.
Function defense again worm parasites.

Monomer superficially resembles IgM (somewhat different amino acid sequence)
rare, but potent
involved in allergic response
binds to FC (stem) receptors on mast cells and basophils, which causes them to
trigger the allergic reaction.
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Figure 4.26: IgA
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Figure 4.27: IgE
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V. Immunoglobulins in Action
A. Antigen Binding (Figure 4.28)
1. Antigen bound to the 6 loops at the tips of the Y arms by the same weak interactions
that produce enzyme-substrate interactions.
2. As with enzyme-substrate interaction, the binding can involve induced fit, distortion in
both structure of anybody and antigen.
3. usually proteins
a. B-cell epitopes are found at the surface of a protein often parts of the protein that
stick out.
b. Tertiary structure (shape) is important. Denatured proteins wont work.
c. Quaternary structure (association between two separate peptides) can be
important. An antibody may recognize the junction of two different proteins.
d. B-cell epitopes can therefore be formed by sequential or non-sequential
sequences of amino acids.
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Figure 4.28: Antigen Bonding Model
B. Prompting Immunogenicity in B cells: (Adaptive response)

1. differs from self (foreign)
2. big enough to crosslink two receptors
3. arrives with danger signal (activates PRR of the B cell) vaccine adjuvant, e.g. alum
which activate NOD receptors.
4. nutritional status and age
C. Manipulating Immunogenic Responses (Figures 4.29-4.31)
1. haptens are not inherently immunogenic
2. Can trigger an immune response is given after incorporation into large protein BSA,
making antibodies to:
a. BSA
b. Junction of BSA and hapten
c. Haptens - such as steroids hormones, drugs, pollutants or poisons.
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Figure 4.29: Hapten
Figure 4.30: Hapten & BSA
Figure 4.31: Hapten & BSA with Antibody
D. Biological Activity Antibody Signaling property of the Fc, or stem, region of the
antibody.
1. Allergic responses eosinophils, basophils and mast cells have receptors (FCRs) for
IgE. Binding triggers degranulation by these cells.
2. Signal for Phagocytosis: opsonization by macrophages and neutrophils.
a. Macrophages and neutrophils have cell surface receptors (FCRs) for IgG FCs.
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b. A bunch of FCs sticking out of a bacterium or cluster of viruses will bind to a

number of FCRs on the surface of a phagocytic cell.
c. The binding triggers a transmembrane response that leads to the particle being
phagocytized
3. Signal to Plasma Proteins: Activation of Complement (landmines in the plasma)
a. Activated by IgM and IgG when they are bound to a cell surface.
b. This sets of a cascade of activation, leading to the attack version.
c. Also opsonizes pathogen, improving phagocytosis by neutrophils and
macrophages.
4. Antibody Dependent Cell Mediated Cytotoxicity (ADCC)
a. When you are infected by a virus, your cells will display foreign antigen.
b. Antibodies against this antigen will bind to the surface of your cells.
c. This complex activates NK (natural killer) cells, which trigger apoptosis
5. Signal to Epithelia - Transcytosis - transfer of antibody across epithelia.
a. Mostly IgA, although IgM is transported in small amounts
b. Involves secretion into mucus, tears, and breast milk.
c. Also, IgG is transported across the placenta to the fetus of mice and humans
E. Yet More Terms It is possible to make antibodies to antibodies.
1. Isotype - constant region determinants. IgG3 is a different isotype from IgG4,
although they may recognize the same epitope.
2. Idiotype - refers to differences arising from variable domains. IgG and IgM that
recognize the same antigenic epitope are the same idiotype, but different isotypes.
F. B-cell Receptor (Figure 4.32)
1. If the antibody is stuck in the membrane, sticking out, its a receptor.
2. B-cells recognize foreign antigen when two neighboring receptors bind to it and
cross-link. (Figure 4.33)
3. The signal is transduced by the associated heterodimer of Ig/Ig, both of which
have long cytoplasmic tails. (Figure 4.34)
4. Nave B cells have M or D class receptors.
5. Memory B cells can have receptors of any class.
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Figure 4.32: Ig Receptor
Figure 4.33: Neighboring Receptors
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Figure 4.34: Ig/Ig
VI. Monoclonal Antibodies

A. Very Big Deal
Its incredibly powerful to be able to make a large amount of pure, defined antibodies.
You can target cells and proteins, and there are therapies and assays (ELISA) based on
this asset. While this lecture concentrates on the use of monoclonal antibodies to
identify, locate and quantify substances, these antibodies function in many of the cutting
edge therapeutics, especially for cancer.
1. Definition - Monoclonal antibodies are fractions of antibodies with an identical
defined specificity (CDR region) for a particular antigen.
2. The cells are clone from a single cell, a B-lineage cell, all of whose descendants
chuck out identical antibody. Ordinarily, you respond to an infection or other immune
challenge by making a number of cell lines, each producing antigen to a different
epitope.
3. Once you have a cell line producing a pure fraction of antibody to a particular protein
you can:
a. Use the antibodies to measure the presence and concentration of that protein or
antigen.
b. Label the antibodies with something fluorescent and localize the protein in the
cell.
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c. Label the antibodies with something radioactive toxic and localize the protein in
the body. Particularly helpful in tracing metastatic cancer cells.
d. Use the antibodies to specifically shut down signaling pathways leading to cell
division in cancers.
B. Hybridomas
The trick is to get a single cell line that will endlessly crank out a pure stream of
antibodies for you.
1. Challenge an organism with the antigen to which you want to make the antibodies.
2. Isolate an activated plasma (B) cell producing an antibody to one of the antigen
epitopes. Sadly, this will only live a few weeks on its own
3. Fuse the normal B cell with a myeloma cell. Myeloma cells live indefinitely. The
fusion is done by mixing the cells with polyethylene glycol.
4. The cells fuse randomly. There is no guarantee that one myeloma cell will fuse with
one normal B cell.
a. Myeloma plus B cell desired result
b. B plus B dies out
c. Myeloma plus myeloma (or unfused myeloma) lives forever. You must get rid
of these!
5. There are mutant lines of myelomas that lack the ability to make a component
necessary for growth. The typical tool is a myeloma missing HGPRT and thymidine
kinase, used to salvage nucleotides. These are OK as long as they can use the
regular de novo synthesis pathway.
Table 4.3 Plasma and Myeloma Cells Contracted
Plasma (B) Cell
Myeloma (B cancer) cell
Makes desired antibody
Selected line makes no antibody
Can synthesize nucleotides by

de novo pathway
Can synthesize nucleotides by de novo

pathway
Can synthesize nucleotides by

salvage pathway
Can NOT synthesize nucleotides

salvage pathway
Divides for only a couple of

weeks
Divides indefinitely
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6. Grow the mixture of fused cells on HAT medium. This has

a. aminopterin, which blocks de novo synthesis of nucleotides.
b. Hypoxanthine and thymidine, which supplies the salvage pathway for nucleotide
synthesis.
7. The B cells making the desired antibody have both nucleotide pathways intact
(although the de novo will not work in HAT with aminopterin), and any myeloma cells
that fuse with them will be able to make nucleotides and grow (hybrid cells).
Myelomas that dont fuse with B cells lack the ability to salvage and die out.
8. So, after fusing, the B cell brings in the ability to make a particular antibody along
with the salvage enzymes and the myeloma confers immortality.
9. Once you have a line going you have to check and make sure it's making the desired
antibody.
10. Once you've got the right antibody, you may need to prod the line into secreting the
antibody more effectively.
You have to go through this process every time you want a new antibody against a different
protein, but once you've done it, the cells will divide and you can grow large cultures and
share or sell them or share or cell the antibodies.

A. YouTube:
Rube Goldberg at its finest: This Too Shall Pass:
http://www.youtube.com/watch?v=qybUFnY7Y8w
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Lecture L05
Organization & Expression of Immunoglobulin
Genes
Although we are always taught about astonishing specificity in

biology, many mechanisms are only as specific as they need to
be. - Gerhart, John, and Marc Kirschner, 1997
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I. Uniqueness of the System

A. The Problem 1. The number of possible antibodies that an individual can produce is vast.
a. Vast is a good term here. Technically there can't be an infinite number.
b. Estimates range from 108 to 1011, but frankly no one really knows.
2. Any one antibody-producing plasma cell synthesizes antibodies with one and only
one CDR (complementarity determining region or antigen binding region).
3. Any one antibody-producing plasma cell can make more than one class of
antibodies, each with the same CDR.
B. The Solution
1. Mixing instructions for chains (heavy and light peptides).
2. Mixing instructions for domains within chains: Dryer and Bennet (in 1965).
3. Evidence for gene rearrangement, Tonegawa and Hozumi (1976)
C. THIS IS A VERY BIG DEAL.
1. Other Systems with Changes to the Germline DNA.
a. Chromosome Diminution. This is a loss that accompanies the decision to
become a somatic as opposed to a germ cell, and does not seem to be
involved in the regulation of genes involving differentiated state.
b. Gene amplification. There are a number of systems in which cells make extra
copies of particular genes (rRNA for example).
c. McClintock's jumping genes. She thought they were important in
developmental regulation, but they turned out just to be examples of DNA
parasitism
d. Neuron development. In mice, at least, as neurons in certain regions of the
brain develop, they relax their controls on transposable elements. Such
elements then move around randomly in the nuclei.
2. What Specifically Makes the Adaptive Immune System Unique
a. Differentiation of B (and T) cells involves clipping DNA out of specific
regions of the immunoglobulin genes.
b. The clipping is not precise within those regions.
c. The clipping takes place at different parts of the regions in different cells.
d. The clipping does not take place in regions other than those for immune
proteins.
e. The clipping results in different cells (and their progeny) ultimately having
different versions of the immunoglobulin genes.
f. These site-specific DNA rearrangements are unique to the vertebrate
adaptive immune system.
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II. Gene to RNA to Protein

A. Orientation: Im going to show the human version. Many texts show versions from
the mouse, which differ from the human. (Figure 5.1)
1. There are two different genes for the light chain, and one for the heavy, all on
different chromosomes.
2. Since we are diploid, there is a pair of each chromosome and therefore each of
3.
4.
5.
6.
the following arrangements may occur twice in the genome.

Many of the elements of these genes are repeated variations of
interchangeable parts.
When DNA elements occurs as tandem repeats, individual often vary in the
number of these repeats.
In this lecture, I refer to the Ig genes as genes and the subparts of the genes
are gene regions.
Do not confuse the terms "region" with "exon."
Figure 5.1: Human Version IgG Genes
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B. Lambda () Gene Expression (Figure 5.2)

1. Gene family includes a series of leader variable regions followed by a series of JC regions.
2. One leader-variable and one J-C pair come together at random.

3. This activates the promoter of the selected leader only, enabling transcription
from the beginning of that leader only.
4. RNA polymerase transcribes the VL and JC into a primary transcript. (Figure 5.3)
Figure 5.2: Lambda () Gene Expression
Figure 5.3: Primary Transcription of Lambda () Gene
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5. Message processing removes the introns from between the V-L and the J-C,
adds a poly A tail and 5 cap (not shown).
6. The ribosome attaches to the massage, begins translating, attaches to the
RER, and pushes the nascent peptide into the ER lumen.
7. Enzymes clip off the leader, leaving a light chain with a variable domain and a
constant domain.
C. Kappa () Gene Expression (Figure 5.4)

Figure 5.4: Kappa () Gene Expression
1. Gene family in humans includes a series of about 40 !! !(leadervariable)
regions, 5 functional !! (joining) regions, and 1 C constant region.

2. Gene rearrangement places 1 VL next to 1 J gene region, again activating only the
promoter of the selected VL.
3. RNA polymerase transcribes a message precursor with one VL, the selected J,
the constant region and any remaining Js and introns between them.
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4. During processing, introns and extra Js get clipped out, leaving a message with
the same structure as that of the lambda.
5. Translation proceeds as above, producing a light chain with the same overall
structure
D. Heavy Gene Expression (Figure 5.5-5.9)

Figure 5.5: Series of Constant Regions
The order seen below (in blue constant regions) is: , , 3, 1, 1, 2, 4, , and 2
1. As with the kappa light, family begins with sequence of about 40 V-Ls.
2. Next is a series of about 20 short D (diversity) segments, each coding
for 3 amino acids.
3. In humans, 5 or 6 J regions follow.
4. Finally there is a series of constant regions, 1, 1, 4 different s, 1
!,and 2 s. The order is , , 3, 1, 1, 2, 4, !, and 2.
5. First a D region joins with a J, cutting out all the extra downstream Ds
and upstream Js between them, but leaving any downstream Js.
Figure 5.6: D Regions Join with a J
6. Then one of the VLs joins with a D, removing all the extra downstream
VLs and up-stream Ds.
Figure 5.7: VL Joins with D
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7. The initial primary transcript starts with this LVDJ regions, continues
though any remaining Js and introns, and then copies through the and
constant regions and stops.
Figure 5.8: Initial Heavy Primary Transcript
8. The transcript now undergoes alternative message splicing to include

either the or the constant exons, but not both.
Figure 5.9: Results of Alternative Message Splicing
9. Translation proceeds as with the other messages.
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III. Rearrangement in Developmental Context in the bone marrow.

A. Variable Region Rearrangements (Figure 5.10)
Figure 5.10: B Cell Development
1. First, the developing B cell rearranges a heavy chain gene. If you get a
2.
3.
4.
5.
functioning gene, fine, you express it and shut down the other heavy
chain gene.
If not you try the other heavy chain gene, if that doesnt work, you
proceed to the light gene. If not, the cell undergoes apoptosis.
First you rearrange one kappa gene and then, if that does work, the
other, again turning off the unused genes.
If neither works, you proceed to the lambda, first one then the other.
Only if all four genes prove to be duds does the cell apoptose.
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B. Comments on the Final Peptides

1. Whether you start with a lambda or kappa gene, you wind up coding for peptides
that superficially look the same.
2. The peptides for these genes NEVER have membrane-spanning regions.

3. Once you have decided on a heavy chain gene the RNA from this gene can
be alternatively spliced into four different peptides.
(M class) with M1 and M2
(D class) with M1 and M2
(M class) soluble
(D class) soluble
a. Initially, a developing cell only makes the version with membrane-spanning

exons.
b. Upon maturity, the cell now makes both and peptides in the same cell.
c. Once stimulated to produce antibody, the cell makes primarily peptides, but
without the membrane spanning region. Thus they will make soluble M-class
antibodies.
d. A cell rarely makes peptides without the membrane-spanning regions.
IV. Mechanism Details: Breaking and Joining (Figure 5.11)

Figure 5.11: Recombination Signal Structure Overview
A. Signal Structure
1. Recombination signal sequences (RSSs) flank the V, D, and J segments
a. 3' end of V
b. both sides of the D
c. 5' end of the J
2. Each RSS has (Figure 5.12)
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Figure 5.12: Recombination Signal Structure Detail
a. 7 nucleotide palindrome (reads the same forward and back). This

region will participate in the ligation of adjacent V(D)J segments.
b. spacer, seems to be important in lining up the adjacent V(D)J regions
so that they join properly:
i. 12 base (1 turn of helix - V) or 23 base (2 turns of helix - J)
ii. 23 base (2 turns of helix - V) or 12 base (1 turn of helix - J)
iii. heavy chain, V and J, both are two turn, D is one turn
c. 9 nucleotides rich in AT aid in attachment to the multienzyme
complex that performs the whole complex function. (Figure 5.13)
Figure 5.13: Recombination Signal Structure Nucleotide Sequence
3. Signal sequences with one-turn spacers can only join with those with
two-turn spacers, so this protects against misjoining.
4. The enzyme complex responsible for joining is called the V(D)J
recombinase.
B. Mechanism of Rearrangement
1. The genes to be arranged moved to and specific location in the nucleus and
open up, detaching from the nucleosomes.
2. First the processing complex grabs one of the RSS signals, than it grabs
the complement.
3. One-turn signal juxtaposes to two-turn signal.
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4. One strand of the DNA between the coding and signal sequence cleaves.
5. The 5'OH end of the cut strand attacks the opposite side of the uncut strand of the
same DNA molecule.
6. This produces a hairpin loop at the downstream end of the V and the upstream
end of the J (light chains).
a. The nucleotides within the loop were originally part of the palindromic
sequences.
b. The one- and two-turn sequences, along with the AT region signal end in a
flush cut with a 5 phosphorylated end.
7. Enzymes clip the hairpin loop. Now the gene regions end in a double strand of
DNA with an open end, the clip site used to join the V(D)J and add variability at
the junction.
a. A few nucleotides get trimmed off.
b. The ends of two of the single strands are ligated.
c. Nucleotides are added to fill in the unmatched singled stranded regions (Pnucleotide addition), matching the ends generated by the cut.
d. For heavy chains only, up to 15 additional nucleotides can be added at random
in the junction
8. Repair and ligation of the coding joint, with release and digestion of the signal
sequences (which are retained if there an inversional joining.)
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9. At a later stage of development, the parts of the genes coding for the
hypervariable region can mutate, introducing further variation (much more later).
C. Commitment to Producing a Single Functional CDR (Antigen Binding Region)

1. Because the joining process between VL and JL and VH, DH, and JH regions
2.
3.
4.
5.
6.
7.
contains so many random elements, about 2/3 of the time, any one junction will
produce a frame shift.
If any one junction in any part of the gene gets out of phase, this produces a nonproductive rearrangement.
If the message remains in phase, this usually produces a productive
rearrangement and the message for a working peptide. N and P addition can also
throw in random stop codons, even if the message as a whole remains in frame.
Recall that these cells are diploid and that these rearrangements will take place
on both homologous chromosomes.
However you must get both a good heavy and a good light of some kind or the
cell dies by apoptosis. Only about 8% make if through this.
Allelic Exclusion - you only get one good productively rearranged gene.
a. The heavy rearranges first. Once there's a good heavy chain, this shuts off
rearrangement of the other heavy gene.
b. The light rearranges next. First it tries a , and if one works, the cell shuts
down rearrangement.
c. If the doesn't work, it tries a . Once one of them works, the cell shuts
down rearrangement.
d. If nothing works, the cell apoptoses.
Once a cell has a working heavy gene and a working light, then it shuts off its
recombination enzyme genes.
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D. Dangers and Mechanisms (Figure 5.14)

Figure 5.14: Dangers and Mechanisms
V. Reviewing the Generation of Antibody Diversity.

A. Mixing and Matching Germ Line Genes
1. Recall that both mice and humans have multiple VH, DH, JH, V, D, JH, and J
regions on homologous chromosomes.
2. You can put any heavy chain with any light.

3. So just from recombinatorial options in the germ line, there's a lot of potential
variability.
B. Fooling Around at the Junction (Figure 5.15)
Figure 5.15: Coding Junctions
1. Coding junctions do not always join precisely.

2. P-nucleotide addition. Generates a palindrome when it matches the
nucleotides opened at the clipping of the hairpin loop.
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3. N-nucleotides- those optionally inserted in the middle of this.

4. The region of the protein coded by the junction winds up in the third CDR loop of
the variable region.
C. Somatic Hypermutation (Figure 5.16-5.17)

Figure 5.16: Somatic Hypermutation Light Chain
Figure 5.17: Somatic Hypermutation Heavy Chain
1. The naive B cell leaves the bone marrow able to make one and only one kind of
CDR (although it can add this to the different classes of antibody heavy C
chains).
2. However, it can still change or mutate the region coding for the hypervariable
loops.
3. This occurs later in the process of antibody stimulation, maturation, and selection
outside the bone marrow.
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VI. Class Switching

A. Where and When
1. Class switching typically occurs in the lymph nodes after exposure to antigen.
2. Class switching takes place after the system has been producing antibodies for
a week or more.
3. Thus the first antibodies produced in response to an infection are Class M and you
dont start to see G (or other classes) until later in the infection.
B. How
1. Class switching involves further rearrangements to the DNA, but these are
brought about by a separate set of enzymes. (Figure 5.18)
2. TH cells synapse with the B cells and signal the specific switch.
3. Recombination sites are called switch regions, and are designated by 2-3
kb sequences of DNA with multiple copies of short consensus sequences.
4. Before switching, the cell expresses the and constant regions.
5. Switching involves removing these and whatever other constant gene region(s)
stand(s) between the VDJ recombined region and the constant to be
expressed.
These may be removed in sequence as a cell class-switches down its options.
Figure 5.18: Heavy Chain Constant Regions
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VII. Synthesis and Secretion

A. A Farewell Look at the Complexity of the Heavy Chain Gene (Figure 5.19)
Figure 5.19: Substructure of the 5 and Heavy Chain Gene
1. Our first look showed the different gene regions, but not the internal structure of
the constant region or most of the signals.
2. If we enlarge a portion of the rearranged gene showing just one upstream VL and
the downstream region into the first gamma exon, we can show more detail.
3. At a first pass you can see that the turquoise lines indicate splice signals, which
will allow introns to be processed from the message.
4. Focus on the part of the gene that codes for the variable domain, which is flanked
by an unused VL and an unused J, both of which still have the complete RSSs.
5. Locate the functional parts of the instructions for the variable domain:
a. The activated promoter
b. The VDJ in use
c. The rearrangement joints with the location of the N-nucleotide addition
6. Now focus on the constant chain instructions
7. The C and C domains are coded for by separate exons for each constant
domain with intervening introns.
a. The sequence is preceded by a switch signal.
b. Thus there are four C exons and three C, the D class having
the hinge region and therefore greater flexibility.
c. Both complex regions have two more exons (M1 and 2) downstream
from the constant exons.
d. Both C and C have a polyA splicing option in both the last Ig exon
and the second membrane spanning exon.
e. This explains the four possible options (M or D antibody, M or D Ig
receptor) that a message form this region could specify.
8. Beware of confusing terms! Class M refers to the whole antibody, IgM,
whether soluble or membrane bound. This class has the constant
region. If it is membrane-bound then the has M 1 and 2 at the end.
9. This drawing does not show most of the rest of the constant instruction,
but you can see the switch site and first exon for the 3 gene region.
10. All other gene regions have the two membrane-spanning exons,
expressed only in memory cells.
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B. Processing in the GERL (Golgi-ER-Lysosome)

1. L and H transcripts exit the nucleus separately, attach to ribosomes, and
2.
3.
4.
5.
6.
7.
begin peptide synthesis.

The initial signal sequence (leader) causes the ribosome to attach to the RER
and the insert the L and H peptide separately into the lumen.
Only if the message for the H chain ends on a membrane-spanning region,
the peptide will remain anchored in the membrane.
In the RER lumen, enzymes clip off the leader and being to add oligosaccharides
to the peptides.
Assembly and processing occurs in the primarily in the lumen of the RER and the
product is then sent to the Golgi.
First the heavy chains are put together, then the Ls are added (for the G class
first one heavy and one light associate).
After assembly, enzymes oxidize the disulfide bonds, nail the structure into
position, and adjust the oligosaccharide into the version characteristic of the
antibody.

Resource:
VIBE- Virtual Interdisciplinary Biology Education
http://bcs.whfreeman.com/immunology6e/content/cat_070/Stanford%20VIBE/index.html
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Lecture 6 (L06)
Development of B Cells
Most of what we once thought we knew about global health has

been proved wrong by the relentless advances of HIV/AIDS,
tuberculosis and malaria..There can be no more urgent cause
facing us today. In Africa, the enemy is already among us. In
Asia, the enemy is at the gates.
Richard G. A. Feachem, Houston Chronicle, 1/21/03.
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I.
B-Cell Maturation (Figure 6.1)
Figure 6.1: B-Cell Maturation
A. Initial Steps (Bone Marrow)

1. Stem cell commits to lymphoid line.
2. Lymphoid progenitor commits to progenitor B cell, or pro-B cell, first developing
signaling ability using Ig/Ig transmembrane proteins with ITAMs (Figure 6.2)
Figure 6.2: Pro-B Cell
3. Pro-B cells begin gene rearrangement and differentiate into pre-B cells upon
stimulation by the stromal cells
B. Differentiation and Gene Rearrangement (bone marrow), review
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1. Pro-B cells bring DH and JH together

2. Then they add leader plus VH to DHJH, and do the P and N nucleotide additions.
3. If this produces a non-productive (frame shifted) gene rearrangement, then they try
the other allele.
4. If the rearrangement is productive, then the heavy chain is put into the membrane
with a surrogate light chain, composed of the products of two genes that can function
without rearrangement (Figure 6.3)
Figure 6.3: Pre-B Cell
5. The immature receptor associates with the Ig/Ig transmembrane signal. This
signals allelic exclusion and initiates the light chain gene rearrangement.
6. Once there is a productive H chain gene, the cell is a pre-B cell. If there is no
productive rearrangement, the cell apoptosis.
7. The pre-B cell then undergoes rearrangements of first one , then the other, and one
, then the other, stopping as soon as there is a productive light chain arrangement
and ultimately apoptosing if there is not.
8. Once you have two productive rearrangements, you have an immature B cell, one
that has a determined antigenic specificity (CDR) and uses the CH region to
produce membrane-bound antibody. (Figure 6.4)
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Figure 6.4: Immature B Cell
9. Undergoes negative selection (below).

10. Shortly thereafter, the cell also begins to express membrane-bound CH, and
become a mature (but nave) B cell, expressing IgM and IgD on the cell surface.
11. At this point the cells are released into the plasma and head to the peripheral
(secondary) lymphoid organs: lymph nodes, spleen and mucosa.
C. Removing Self-Reactive B Cells.
1. 90% of the B cells produced by the above process never make it to the plasma. At
least some of the negative selection occurs as cell lines expressing antibodies to
self-antigen are eliminated.
2. The elimination is signaled by the crosslinking of IgM.
3. Artificially crosslinking IgM will lead to apoptosis of developing B cells.
4. However, B cells can sometimes get a second chance. If cross-linking occurs, the
cells may arrest and reactivate their RAG enzymes, and try rearranging again.
5. If they have a chain involved in the CDR for a self-antigen, they may try to replace
it with a .
II. B-Cell Activation and Proliferation - To survive, circulating B cell must encounter antigen
that can bind to its receptors or they will undergo apoptosis within a few weeks.
A. Initiating Antigen Exposure
1. Thymus-dependent activation: In most cases, when antigen cross-links a B cells Ig
receptors, this sets off a signal (signal 1) and the B cell seeks out a TH cell.
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2. Thymus independent activation; there are a few antigen (TI antigens) that can
prompt B-cell development independent of TH cell co-stimulus. These antigen also
simultaneously activate toll-like receptors.
a. Type 1 antigen - lipopolysaccharide such as those found in the outer bacterial
cell walls of gram negative bacteria, which also activates TLR4. (Figure 6.5)
b. Type 2 antigen - repetitive polymeric proteins, such as bacterial flagellin, can
cross link the membrane-bound immunoglobulins and kick off proliferation if the
simultaneously activate TLR5. (Figure 6.6)
Figure 6.5: Type 1 Antigen
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Figure 6.6: Type 2 Antigen
3. However, TI activation does not induce class switching (you mostly just make IgM)
and does not produce memory cells. For that you need TH cells.
B. Activating Signals- Generating signal 1.
1. Review Ig receptor.
a. mIgM or mIgD molecule
b. Ig/Igheterodimers
c. immunoreceptor tyrosine-based activation motif, or ITAM extend into the
cytoplasm
2. When an antigen cross-links one antibody with the next outside the cell, it brings
together the cytosolic Ig/Ig domains, activating the ITAMs. (Figure 6.7)
3. This causes the complex to change conformation and activates src-like kinases.
These are enzymes that add phosphate to molecules and they add them to the
ITAMs.
4. Once the ITAMs have phosphates, another kinase, syk, docks and triggers several
different enzymes cascades.
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Figure 6.7: Activation
5. These lead to the up-regulation of transcription factors, the inflammatory transcription

factor NF-B being involved in this.
6. The cells begin to divide and secrete antibodies.
7. As antibodies build up, they bind to CD-22. the Fc or antibody stem receptor, which
provides brakes on the system
C. Role of TH cells
1. However, the BCR does not signal effectively without contact with a TH cell, nor do
the cells divide rapidly without additional stimulus from TH cell cytokines.
2. When B cells bind antigen, they bring some it inside and hydrolyze it.
3. Some of the hydrolyzed peptide winds up attached to class II MHC molecules, the
genes for which are upregulated along with the one for B7.
4. Thus the B cell can present some of the antigen to a TH cell and also contact the T
cell using B7 to CD28.
a. Because of its ability to gather up the antigen using the BCR, a B cell is very
effective at presenting antigen, and can stimulate a TH cell at concentrations 100
to 10,000 times lower than those necessary for a macrophage or dendritic cell.
b. Of course the antigen received is different from the antigen presented. The
presented antigen is a peptide derived from the overall molecule.
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5. The cells attach, forming a conjugate or immune synapse.

6. This causes the TH cell to produce CD40L, which is a juxtacrine factor that turns
around and signals the B cell through CD40 receptor. (Figure 6.8)
Figure 6.8: Immune Synapse
7. The contact reorganizes the interior of the TH cell so that cytokines are released
toward the B cell.
8. The B cells begin producing receptors for the cytokines.
9. Cytokine signaling activates the B cells and they begin proliferating and
differentiating.
III. Primary Versus Secondary Response
A. The Primary Response
1. nave lymphocytes
2. 4 to 7 day lag time
3. produces antibody secreting plasma cells and memory cells
4. initial antibodies mostly IgM; IgG toward the end
B. The Secondary Response: The Sadder, but Wiser, Immune System
1. Produced primarily by memory cells
2. 1 to 3 day lag time
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a. The number of memory cells specific for the antigen increases.

b. These memory cells are more easily activated.
c. They have already been through affinity maturation, so they're better at binding
antigen
3. more antibody secreted, and over a longer time
4. much higher proportion of IgG and other isotypes
IV. B-Cell Maturation in Anatomical and Histological Context
A. Lymph Nodes
1. Lymph drains from tissues and passes through these.
2. Antigen enters. It can be
a. "free" - particle from pathogen, or the whole bacteria or viruses themselves
b. proteins or other antigens from the pathogens complexed with antibodies
c. carried in by presenting cell (dendritic or macrophages) that have picked it up
elsewhere
3. Free and antibody-bound antigen in the plasma is likely to be picked up by
a. interdigitating dendritic cells (Figure 6.9)
b. macrophages (Figure 6.10)
c. follicular dendritic cells (Figure 6.11)
Figure 6.9: Interdigitating

Dendritic Cell
Figure 6.11: Follicular

Dendritic Cell
4. Nave lymphocytes from the bone marrow enter via the lymph.
5. Activation begins in the paracortex, the layer between the outer cortex and the inner
medulla, where there is a high concentration of T cells, macrophages, and dendritic
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cells. (Figure 6.12)

6. First the macrophages and the dendritic cells activate the TH cells.
7. Nave B cells contact the TH cells, presenting any antigen they have internalized via
the class II MHC, and forming a conjugate (immune synapse).
8. The B cell begins to divide, producing a clonal cluster (focus) at the boundary
between the paracortex and cortex.
9. A few activated B and TH cells migrate together from one of these foci to a primary
follicle in the cortex.
10. The follicle becomes a secondary follicle, one with a germinal center where B, TH,
and follicular dendritic cells interact.
Figure 6.12: Lymph Node
11. A reminder about follicular dendritic cells: these are NOT regular dendritic cells.
They capture antigen-antibody complexes in beaded structures (iccosomes) and
present them to the B cells.
B. Germinal Centers. These are the sites of affinity maturation (somatic hypermutation
CDR selection), the processes that refine the ability of a B cell's CDR to bind antigen
effectively. (Figure 6.13)
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Figure 6.13: Germinal Center
1. Activated B cells (centroblasts) proliferate and move to one edge or the follicle,
forming a dark zone. At this stage the centroblasts:
a. enlarge and begun to divide rapidly
b. begin somatic hypermutation- mutating the regions in the heavy and light chain
genes that code for the variable loops. (Figure 6.14)
c. stop displaying the membrane Ig (recycles the original via membrane turnover)
Figure 6.14: Somatic Hypermutation CDR Selection
2. The centroblasts differentiate into centrocytes which

a.
b.
c.
d.
e.
stop dividing
begin expressing membrane Ig
move into light zone
contact follicular dendritic cells
undergo selection B cells during which more effective receptors will survive and
multiply at a greater rate.
3. What happens in the follicles is a highly unusual form of accelerated natural

selection. It works like evolution in general, except that the time frame is days and
not centuries.
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a. random mutation (dark zone)

b. excess reproduction (dark zone)
c. selection (light zone)
4. The centrocytes leave the germinal center as plasma cells, lose their surface
antibody again, and begin secreting antibodies.
5. Most centrocytes do not contact an antigen that fits with their surface receptors,
however, and these die by apoptosis, and are recycled by macrophages.
6. Purpose - The environment of the light zone selects for those centrocytes that
express the most effective antibodies. Cells with effective antibodies live, those
without may return to the dark zone for more mutation, or they may die by apoptosis.
a. The maturing B cell that enters the germinal center and begins dividing does so if
it can bind (with its surface antibody) to some degree an antigen currently
arriving in the lymph node.
b. It differentiates into a centroblast that undergoes random mutational events to the
very region of the gene responsible for determining the effectiveness of this
antigen binding (the CDR). (Figure 6.15)
c. The mutated centrocyte now displays the new antibody at its surface. (Figure
6.16)
d. As with most random mutations, most of the progeny centroblast cells produced
by this will bind antigen less effectively than the original cell.
e. A few however, will bind the antigen more effectively. . Cells with improved
receptors divide more rapidly than cells with less effective receptors.
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Figure 6.15: CDR
Figure 6.16: Antibody
7. Signals
a. Follicular dendritic cells play an important role in the selection process. If the
centrocyte can bind to one of the little beads with antigen-antibody complex, then
it gets a signal necessary (but not sufficient) for its survival.
b. However, the beads are essentially a scarce resource, and the centrocytes have
to compete for them.
c. Thus the more effectively the centrocyte surface antigen binds to the antibody
displayed by the follicular dendritic cell, the more likely it is to live.
d. In addition, the centrocytes have to receive signals from the TH cells, especially
the contact of CDC40L to the CDC40R. This doesn't work either if the B
centrocyte cell does not display processed antigen back to the TH cell using its
class II MHC.
C. Class Switching directed by TH cells
1. The next decision the future plasma cell must make is exactly what class of antibody
to send out with the refined CDR region produced by affinity maturation. (Figure
6.17)
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Figure 6.17: Heavy Chain Gene
2. Cytokine signals from the TH cells will determine this (more later).
D. To Remember or to Act: The Final Decision.
1. Centrocyte now decides whether to become a plasmablast and generate a plasma
cell or become a memory cell and wait for a subsequent exposure to antigen.
2. Recall that plasma cells do not express membrane-bound antibody. This means that
the sequence of differentiation in the lymph node involves:
a. dividing mature B cells with surface antigen
b. dividing centroblasts with no surface antigen (undergoing hypermutation)
c. non-dividing centrocytes expressing surface antibody and undergoing selection
d. dividing plasma cells not expressing surface antibody secreting soluble (humoral)
antibody
3. The final differentiation to a plasma cell involves the switch that generates the
splicing enzymes that do not add the membrane-spanning exons to the heavy
chain message.
4. Also transcription and translation levels generally rise as the cell begins cranking out
antibody, as does the proportion of RER.
5. Memory cells set aside from this process may resemble nave B cells, but they have
undergone class switching and make a variety of heavy chains. (Figure 6.18)
6. The receptors of memory cells may therefore also be membrane-bound versions of
IgG, IgA, and IgE, and the regions for these genes all also have a region coding for a
membrane-spanning portion of the antibody that is not spliced into the message for
the secreted form.
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Figure 6.18: Class Switching -- Heavy Chains
V. Regulation
A. B-Cell Differentiation
1. B-Cell Specific Activator Protein (BSAP) functions as master regulator.
2. Present ONLY in members of the B cell lineage.
3. Present in ALL members of the B cell lineage EXCEPT mature plasma cells, which
are done differentiating.
4. Binds to a variety of B-cell gene promoter regions, including those like the surrogate
L chains and class switching regions that are involved in developmental decision
making.
5. High levels tend to maintain a cell as a memory cell, low levels tend to promote
formation of plasma cells.
B. Overall Immune Effector Response - Tolerance
1. You would like NOT to make antibodies against your own proteins, and therefore
tolerate them.
2. On the other hand, you do NOT want to develop tolerance for foreign antigens,
especially those associated with pathogenic infection.
3. Constant monitoring of your antigens by Treg cells suppresses immune responses to
your own proteins and to those of benign commensal bacteria and fungi.
4. Moreover, you need to apply brakes once an infection is under control.
5. If you introduce foreign antibodies to an antigen, this will tie up the antigen and
prevent it from promoting an immune response on the part of the host:
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References and Links:
The Gates Foundation
http://www.gatesfoundation.org/default.htm
VIBE
http://bcs.whfreeman.com/immunology6e/content/cat_070/Stanford%20VIBE/index.html
Follicular Dendritic
http://home.comcast.net/~aszakal/
Table 6.1 B-lineage Development Summary
Name
Location
Pro B cell
Bone marrow
Pre B cell
Bone marrow
Immature B
cell
Bone marrow
Mature,
nave B cell
Circulates:
plasma,
secondary and
tertiary lymphoid
tissue
Peripheral
lymphoid tissue
(lymph nodes as
example) at
paracortex
Activated B
cell - 1
Surface
receptor
molecule
Ig/Ig coreceptor
Heavy chain ()
plus surrogate
light chain and
co-receptor
mIgM and coreceptor
mIgM and mIgD

and co-receptor
mIgM and mIgD

and co-receptor
Other surface
signaling
molecules
C-kit, CD45R
CD25 ( chain
of IL-2
receptor)
No CD 25
Activity
Rearranging heavy
chain gene
Rearranging light chain
gene
Undergoing selection
against selfrecognition, changing
RNA splicing
Trolling for antigen:
binding and activation
necessary for next
stage
Begins clonal
expansion
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Table 6.1 B-lineage Development Summary Continued
Name
Location
Activated B
cell - 2
Cortex, primary
follicle, which then
become
secondary, with
germinal center
secondary follicle,
periphery (dark
zone) of the
germinal center
Cortex, now
secondary follicle,
periphery, light
zone of germinal
center
Cortex, now
secondary follicle,
periphery, light
zone
Lymph node
centroblast
centrocyte 1
centrocyte 2
plasmablast
plasma cell
Circulation, site of
infection
Surface
receptor
molecule
mIgM and mIgD
and co-receptor
Other surface
signaling
molecules
Up-regulate
MHCII, CD
40R, various
cytokine
receptors
No surface Ig
receptor
altered mIgM
and mIgD and
co-receptor
CD40R (signal
from CD40L
necessary, too)
altered mIgM
and mIgD and
co-receptor
CD40R (signal
from CD40L
necessary, too)
none
none
Activity
Associate with T cells
Divide rapidly, mutate

Ig V regions in
sequences coding for
the CDR loops
Stop division, compete
for antigen displayed
on follicular dendritic
cells.
Decide whether or not
to class switch and
whether or not to be
memory
Switch to splicing out
membrane-spanning
exons, up-regulation of
RER and Ig synthesis
Secretes antibody
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Lecture 7 (L07)
Complement
Science is a social process. It happens on a time scale longer

than a human life. If I die, someone takes my place. You die;
someone takes your place. What's important is to get it done. Alfred Lothar Wegener, shortly before his death at age 50. He
died on an expedition to the North Pole to gather information to
support his hypothesis of continental drift.
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I.
Overview and Terminology of the Complement System A. What Is It? the landmines of the circulatory system.
1. A system of over 30 proteins, most soluble in the serum, but some part of the surface
of cells.
2. Organized in a cascade system reminiscent of the clotting cascade, only MUCH
more complicated.
3. Most produced by the liver (along with serum albumins and fibrinogen).
4. Some produced by monocytes, macrophages, and epithelial tissues.
5. Released in an inactive form.
6. Proteolytic cleavage activates them.
B. How Does It Work?
1. Membrane Lysis
a. Kills cellular invaders and those virus that use components of the plasma
membrane.
b. Produces active MAC, the membrane attack complex that punches holes in the
membranes.
c. Three different pathways can activate the production of active MAC (model)
2. Opsonization
a. promotes phagocytosis by macrophages and neutrophils
b. coats foreign cells, free antigen, and antibody-antigen complexes with proteins
that promote phagocytosis.
3. Immune Complex Clearing
a. coats antibody-antigen complexes
b. attracts phagocytes to them
4. Activating Other Immune Responses
a. Cleavage fragments produced during activation function as paracrine factors.
b. These factors activate B cells.
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c. Promote inflammatory response

C. The Name Game
1. Most complement factors are designated by numbers
a. examples C1 - C9
b. Unfortunately, they are numbered in the order of discovery, not action.
2. Sub factors
a. The cleavage fragments are designated with letters. For example, C4 is cleaved
into C4a and C4b.
b. The peptides that make up the active protein may also be designated with letters.
3. Other names a. factor D
b. homologous restriction factor
4. Active factors - Activation frequently involves this association of more than one factor
to form an activated protein, with the complex indicated by overlining.
.
II.
Complement Activation: The Big MAC Attack

A. Convergence of the Pathways
1. You can trigger the pathway with both innate and adaptive recognition.
2. The last stages are the same, so the end results are identical.
B. Sequence of Events
1. The cleavage of C5 to C5a and C5b.
2. C5a serves as an inflammatory signal.
3. C5b attaches to C6, begins formation of the attack complex. If unattached, 5b is
unstable.
4. C5b6 complex attaches to C7.
5. C5b67 complex shifts into amphiphilic form and attaches to membrane.
6. Complex attracts C8, and C5b678 inserts into membrane
7. Complex gathers a ring of C9 peptides around it
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a.
b.
c.
d.
shallow cylinder with large channel down the middle.

resembles perforins used by TC cells to kill rogue-self
multiple copies: 10-17 bind to complex, forming the cylindrical ring
produces tube with 70 to 100A pore - note exact size will depend on the number
of C9s in the pore wall.
8. Big MAC Attack: The C9 complex pokes holes in membranes.

III.
Three Roads to the Big MAC (Figure 7.1)

A. Classical (Figure 7.2)
1. C1
a. complex of proteins that resembles a bouquet of flowers or an amusement park
ride frame with struts.
i.
6 Cq units with the tails in a bunch and the heads sticking out. Each is made
up of three peptides, A, B and C
ii. 2 Cr, hooked end to end.

iii. 2 Cs, hooked on the ends of these
b. Cr2s2 complex
i.
When bound to Cq complex, it assumes a figure 8 form in the middle of the

spreading stems.
ii. Activation of C1 frees of this complex, which now wraps around the outside of
the stems, exposing the catalytic activity of the Cr and Cs peptides.
c. Antigen Binding to C1q (This is what frees the Cr2s2 complex)
i.
Any one of the 6 heads can bind to the FC (stem) part of an antibody.
ii. If any two of the heads bind, then the Cr2s2 complex is freed.
iii. The oligosaccharide and the domain it is attached to constitute the principal
agent of activation.
d. Binding to IgM, figure 7-4, page 171 and figure 4-17e page 97
i.
Circulating IgM is pentameric (Figure 7.3), so the C1 ought to be able to

attach at least two heads to one antibody.
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Figure 7.1: Preview of Three Pathways
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Figure 7.2: Classical Pathway Diagram
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Figure 7.3: Pentameric IgM
ii. However, when the IgM is circulating, it's usually in the planar conformation
and the heads can't get into the middle well enough to bind.
iii. When the IgM binds antigen, it flips into the staple conformation, bending the
arms away from the FC region, and making it more accessible. (Figure 7.4)
iv. At that point, even one IgM can activate the complement cascade.
v. IgM in someone with an infection, even its early stages, will activate the
cascade rapidly and effectively.
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Figure 7.4: IgM Planar to Staple
e. Binding to IgG (Figure 7.5)
Figure 7.5: IgG Subclasses
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i.
IgG is monomeric, so it's going to take two them to activate the C1.
ii. Usually this doesn't happen unless the IgGs are stuck on the surface of a
pathogen,
iii. This also prevents random activation of C1 by serum IgG.
iv. Recall that it is a variant of IgG3 that works best here, followed by IgG1.
v. IgG2 barely activates and IgG4 doesnt work at all.
2. Initiating the Cascade
a. Either IgM or IgG binds to the surface of a pathogen or viral cluster.
b. C1 binds to the antibody and changes conformation
c. Cr2s2 complex activates
d. C1r now functions as a serine protease, cleaving C1s.
3. C1s triggers production of C3 convertase
a. This is the enzyme that activates C3.
b. C3 convertase is the active complex C4b2a, identical to the one used by the
classical pathway
c. C1s (a serine protease) cleaves both C2 and C4.
d. C1s removes the C4a fragment from the subunit of C4, which diffuses away.
e. The remaining large component, C4b can now bind to the target surface near C1.
f.
C2 attaches to C4b, and is also cleaved by C1s, the smaller fragment (C2b - go
figure) diffusing away.
g. The resulting active complex, C4b2a, can now act on C3.

4. Activation of C3
a. C3 convertase clips a fragment (C3a) from the chain.
b. The remaining fragment C3b binds back to the C4b2a convertase complex.
c. The total package C4b2a3b is a C5 convertase.
d. Some activated C3b functions as an opsonin, coating immune complexes.
5. The C5 convertase (C4b2a3b) activates C5
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a. binds to C5 via C3b

b. clips off C5a fragment, which diffuses away
6. C5 initiates formation of the MAC
B. Alternative - a different way to generate C5b (Figure 7.6)
Figure 7.6: Alternative Pathway Diagram
1. Component of the innate immune system

2. Initiated by a variety of compounds characteristic of pathogenic surfaces: (Figure
7.7)
a.
b.
c.
d.
e.
LPS
Teichoic acid (bacterial)
Zymosan (fungi)
Some tumor compounds
Trypanosome (unicellular eukaryote) markers
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Figure 7.7: Surface Triggers
3. Activation of C3 (Figure 7.8)

a. C3 spontaneously hydrolyzes at very slow rate to C3a and C3b.
b. C3b ultimately binds to surface antigens (see above), which stabilizes it.
c. C3b otherwise decays and is even inactivated on host cells by sialic acid.
Figure 7.8: C3 Activation
4. B
a. C3b binds to B, bringing it to the surface.
b. Binding exposes substrate for D, an active serum proteolytic enzyme.
5. D (sometimes called Fd, for factor D)
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a. D cleaves B to Ba (diffuses away) and Bb, generating the active C3bBb which is
also a C3 convertase.
b. Properdin (sometimes called Factor F) binds C3bBb (C3 convertase) to the
pathogenic surface, stabilizing it.
7. C3 convertase: C3bBb autocatalyzes more conversion of C3 to C3b in a positive
feedback loop. The C3b proteins alone can cover the pathogen.
8. C5 Convertase: C3b then joins the C3bBb to form the C5 convertase, C3bBbC3b.
This complex is analogous to C4b2a3b.
C. Lectin - also an innate pathway. The Classical Pathway evolved from this.
1. Lectin is a general term for proteins that bind to specific carbohydrates.
2. MBL - mannose-binding lectin - (Figure 7.9) recognizes carbohydrate on surface of
microorganisms
Figure 7.9: MBL
3. MBL resembles C1q in that it occurs in clusters of 3, 6 or 18 heads.

4. Bound MBL activates a serum protease, MASP, analogous to C1r and s. (Figure
7.10)
5. After that, this resembles the classical pathway, with the cleavage of C4 and C2 to
produce a C3 convertase, the cleavage of C3 by this enzyme, and the production of
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a C5 convertase made up of C4b2aC3b. (Figure 7.11)

Table 7.1 Pathways
Lectin Pathway
Classical Pathway
Initiated by mannose and other

carbohydrates
Changes conformation of MBL
Initiated by antibodies, using the

domain with the oligosaccharide
Changes conformation of C1q
Activates MASP
Activates C1rC1s
Figure 7.10: Bound MBL
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Figure 7.11: Shared Pathway
IV. Endgame and Consequences of Complement Activation - or "What Have You Done
for Me Lately?"
A. Cell Lysis
1. membrane-coated viruses:
2. Gram negative (Figure 7.12) bacteria (the ones with the thin walls sandwiched
between membranes- gram positive, the ones with the thick walls, are hard to
puncture: (Figure 7.13)
a. are usually vulnerable to complement
b. smooth, (with a lipopolysaccharide capsule) are resistant (Figure 7.14)
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c. Other gram negatives (a gonorrhea strain) have compounds in the membrane the
prevent insertion of the complex.
3. eukaryotic parasites
4. erythrocytes (red blood cells) are especially vulnerable. (Figure 7.15)
5. cancer cells
Figure 7.12: Gram Negative
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Figure 7.13: Gram Positive
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Figure 7.14: LPS
Figure 7.15: Red Blood Cell
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B. Inflammation
1. C3a triggers degranulation of mast and basophil cells, with resulting permeability
changes in the endothelium (Figures 7.16-7.17)
2. C5a- summons monocytes and neutrophils (Figures 7.18-7.19)
C. Coating
1. Opsonization- Neutrophils monocytes and macrophages have receptors for
complement proteins (in addition to Fc receptors, TLRs etc.) Binding by these
receptors enhances phagocytosis.
2. Viral Neutralization- Even if the complement does not poke holes in a viral
envelope, simply being coated and glued to other viral particles makes it much
harder for them to attach to and infect a subsequent cell
3. Immune Complex Clearing- works with red blood cells
4.
Figure 7.16: Mast Cell
Figure 7.17: Basophil Cell
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Figure 7.18: Neutrophil
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My staff and I would like to acknowledge with deepest gratitude the animations produced by
Scott Barnum of the University of Alabama at Birmingham (UABRF).
You may order your own copy here:
Complement Activation and Biological Functions by Scott Barnum, Scott Barnum and UABRF,
http://www.microbio.uab.edu/faculty/barnum/complement/index.html
Unless you have already had some sort of immunology course, the chances are youve never
even heard of complement. Its rarely covered in introductory biology texts or even mentioned
in sources that non-specialists are likely to read, such as the New York Times Science Section
or Scientific American. If youre teaching a course and looking for more supplements or just
want to look at a different versions of the events, you can try the following:
Videos
Complement Proteins: mokhtarr
http://www.youtube.com/watch?v=xcYSGRid50I&feature=youtube_gdata_player
The Classical Pathway of Complement Activation: Carlos Jimenez
http://www.youtube.com/watch?v=gNvHLStz-VA&feature=related
Complement Cascade: Doxacurium
http://www.youtube.com/watch?v=y2ep6j5kHUc&feature=related
Alternative pathway of Complement Activation: Carlos Jimenez
http://www.youtube.com/watch?v=qga3Wn76d9w
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Glossary
Adaptive cells those that rearrange their genes
ADCC- antibody dependent cell-mediated cytotoxicity. A process where white blood cells
recognize the stems of antibodies attached to a cell and then attack it.
Allele- A version of the gene. There are two alleles for the enzyme that produces color in four
oclock flowers, one that codes for an enzyme used to make red pigment and a different DNA
sequence that does not produce a functional enzyme, leaving the flower white.
ALT associated lymphoid tissue. MALT (mucosal), GALT (gut), BALT (bronchial), NALT
(nasal)
Antibody- a soluble immunoglobulin
Antigen- a molecule that can bind to an antibody, B cell receptor or T cell receptor
APC antigen presenting cell. Cells that present antigen on MHC II to TH cells
Apoptosis programmed cell death
ATP- adenosine triphosphate, directly supplies energy to many biological reactions
BSA bovine serum albumin. A smallish soluble protein isolated from cows blood.
CAM cell adhesion molecule. Any one of a number of different molecules that help stick cells
together.
CD cluster of differentiation. Refers to the isolation of cells by flow cytometry. Depending on
exactly what proteins extend from a cells surface, which in turn influences how the cell moves
during the separation process.
CDR- complementarity determining region- the recognition side on the tips of the antibody arms
Chitin cell wall material of fungi, also an important component of insect exoskeletons.
Chordate member of the phylum Chordata. Includes vertebrates and invertebrates with a
dorsal nerve cord, gill slits, notochord and muscles in blocks.
CLP common lymphoid progenitor. Gives rise to lymphoid cells, including NK, T cells, B cells,
and more.
CMP common myeloid progenitor. Stem cell that can give rise to any myeloid cell type
(including red blood cells and platelets.
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Coley toxin inflammatory material isolated from bacteria used in cancer chemotherapy
around 1900.
Complement- a system of proteins that helps identify pathogens and debris for destruction and
phagocytosis (the landmines of the plasma.)
CTL Cytotoxic T cell. Activated TC cell, ready to kill rogue-self cells
Downstream- the end of the DNA or RNA with the free 3 carbon of the (deoxy) ribose. Nucleic
acid synthesis and translation proceeds 5 to 3.
Epitope the specific portion of a molecule that binds to an adaptive receptor. For example, a
viral protein is an antigen whose different epitopes bind to different antibody idiotypes.
Exon- the part of a gene that codes for a sequence of RNA that will wind up in a message and
get translated (expressed.) A gene or gene region may have one to many exons.
Gene region a sequence of DNA coding for a specific part of the Ig or T-cell receptor.
Granulocytes Cells with copious granular inclusions and that do not present antigen.
Includes neutrophils, basophils, and eosinophils (which have oddly-shaped nuclei) and mast
cells (which do not).
Hapten- a molecule that could potentially bind a CDR, but by itself is not large enough to kick
off an immune response.
HSC- hematopoietic stem cell. Can divide and regenerate of develop into any type of blood cell.
Found in bone marrow.
Humoral response Immune defense found in the plasma, the word humor derived from the
ancient Greek medical theory of body fluids. It really just refers to antibodies. Stupid term. If
people stop using it, maybe it will go away.
Hybridoma- a cell or cell line derived for the fusion of a blood cell cancer (myeloma) and a
normal, antibody-producing plasma (B lineage) cell.
Idiotype a category of antibodies that all have the same recognition region
Innate cells those that do not rearrange genes.
Introns- that DNA sequences of the gene that code for RNA sequences that get clipped out
during processing.
Isotype- a category of antibodies of the same class
Lymphoid cells white blood cell types (innate and adaptive) found in the lymph and (and
blood and immune organs as well).
MAC- membrane attack complex- terminal complement pathway produces this, which punches
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holes in plasma membranes.

MASP- mannose associated protein
MBL- mannose binding lectin
MHC- Major Histocompatibility Complex. Includes the genes and the proteins they code for.
These include the proteins (groups I and II) that hold small peptides so that T cells can
recognize them. They also include a variety of other proteins, including enzymes important in
immune recognition and promotion. The human versions are names HLA molecules for human
lymphocyte antigen.
Monoclonal- refers to a cell line of (theoretically) identical cells derived from the division of a
single cell.
Myeloid cells innate white blood cells rarely found in the lymph.
Necrosis- cell death from disease or injury
NK cell- Natural Killer cell. Kills rogue-self cells, recognizing them by innate mechanisms. Does
not require TH activation.
N-nucleotide addition- During gene rearrangement, when enzymes add nucleotides at random
in the palindromic regions of the joint.
NOD nucleotide oligomer detectors. Soluble pattern recognition receptors found in the
cytoplasm of cells. Despite the name, they often recognize cell wall materials.
Nucleic acid RNA or DNA
Peptidoglycan mesh-like macromolecules that compose the basic structure of the bacterial
cell wall.
Phagocytosis when a cell engulf large particles
Pinocytosis - when a cell gathers fluid in a vesicle and engulf the vesicle.
P-nucleotide addition- During gene rearrangement, when enzymes fill in the missing
nucleotides at the joint by copying the palindromic nucleotides on the other strand.
Receptor-mediated endocytosis when a cell binds material at its surface using a proteins
receptor and then internalized the complex into a vesicle that enters the cell.
RSS recombination signal sequence. The sequence of 28 or 40 nucleotides that the
upstream or downstream end of a gene region providing the signals for gene rearrangement.
Simple sugar single sugar unit, includes glucose, mannose and galactose. May be modified
into sugar units as sialic acid (NANA) or N-acetyl glucosamine.
TC cell- cells that recognize rogue self-cells by antigen they present on MHC I. They develop
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into CTLs after instructions from TH cells.

TCR T-cell receptor. Found extending from the surface of both TC and TH cells. Recognizes
antigen, coded for by rearranged genes.
TH cell- thyroid helper cell. Coordinates immune responses. TH 1 cells promote a serious
response; TH 2 promotes a containment response, and Treg tolerance. There are additional
types as well.
TLR Toll-like receptors. Pattern recognition receptors that recognize molecules characteristic
of pathogens. Found embedded in plasma membrane and endomembranes of many white
blood cells.
Transcription factor a protein that either up- or down- regulates the copying of RNA
(transcription) from DNA. They often have domains that attach to specific sequences of DNA
nucleotides. Some attach to other proteins that attach to the DNA. Or both.
Upstream- the end of the DNA or RNA with the free 5 carbon of the (deoxy) ribose. Nucleic
acid synthesis and translation proceeds 5 to 3.
Zymosan cell wall material of fungi
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Image Attributions
Figure 1.1 Credit: Alma Novotny
Figure 1.2 Shutterstock. ID
#50995822. Credit: Lichtmeister.

#14214415 Credit: Sebastian Kaulitzki
Figure 2A.1 Shutterstock. ID
#125581520 Credit: Alila Medical Media

#96427406. Credit: Alila Medical Media
Figure 2A.2 Credit: Alma Novotny

#61626418 Credit: BioMedical

#152927564 Credit: dream designs

#166921538 Credit: Designua

#163942121 Credit: Oscar Moncho

Figure 2A.5 Credit: Yan Jun (Daisy) Chung


Figure 1.8 Wikipedia. Credit: Y.
Tambe (http://en.wikipedia.org/wiki/Candid
a_%28fungus%29)
#1464383 Credit: Andre Nantel

Figure 2A.8 Wikipedia. Credit: Kauczuk
(http://en.wikipedia.org/wiki/Mast_cell)

#149456 Credit: Tom Mc Nemar
Figure 2A.10 Wikipedia. Credit: BruceBlas

(http://en.wikipedia.org/wiki/Eosinophil_gran
ulocyte)

#158258786 Credit: xrender
Figure 2A.11 Credit: Yan Jun (Daisy)

Chung

#53576431 Credit: Zuzanae
Figure 2A.12
Wikipedia. (http://en.wikipedia.org/wiki/Mon
ocyte)

#28610878 Credit: Jubal Harshaw
#72915091 Credit: D. Kucharski K.
Kucharska
Figure 2A.13 Wikipedia. Credit: BruceBlas

(http://en.wikipedia.org/wiki/Monocyte)
Figure 2A.14 Wikipedia.
(http://en.wikipedia.org/wiki/Macrophage)
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Figure 2B.10 Shutterstock. ID

#149425529 Credit: Jose Luis Calvo
Figure 2A.16 Wikipedia. Credit: NIH

(http://en.wikipedia.org/wiki/Dendritic_cell)
Figure 2B.11 Credit: Ayna Troshkina
Figure 2A.18 Credit: Ayna Troshkina

#79599691 Credit: Alex Luengo

Figure 2A.21 Wikipedia. Credit: NIH
(http://en.wikipedia.org/wiki/T_cell)
Figure 2B.14 Wikipedia.

(http://en.wikipedia.org/wiki/Apoptosis)
Figure 2B.16 Credit: Alma Novotny
Figure 2A.22 Wikipedia.

(http://en.wikipedia.org/wiki/Antigenpresenting_cell)
Figure 3.1 Credit: Edward Novotny

Figure 2A.23 Credit: Alma Novotny

Figure 2A.24 Credit: Yan Jun (Daisy)
Chung
(http://en.wikipedia.org/wiki/Bone_marrow)

#130068695 Credit: Ethan Daniels
Figure 2B.2 Credit: unknown

#79599691 Credit: Alex Luengo

(http://en.wikipedia.org/wiki/Thymus)




#72294895 Credit: CHEN WS
Figure 3.10 Credit: Yan Jun (Daisy) Chung



#172654067 Credit: Juan Gaertner

#150389330 Credit: O2creationz

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#1464383 Credit: Andre Nantel
Figure 3.30 Credit: Wikipedia.

(http://en.wikipedia.org/wiki/Defensin)

#53576431 Credit: Zuzanae
Figure 3.31 Wikipedia.

Figure 3.16 Shutterstock. ID #53576431

Credit: Zuzanae
Figure 4.1 Credit:

http://dictybase.org/Multimedia/LarryBlanton
/dev.html
Figure 3.17 Shutterstock. ID #116330686

Credit: Juan Gaertner
Figure 3.18 Credit: NIH.
(http://www.nih.gov/catalyst/back/95.01/sem
inar.figures.html)
Figure 3.19: Unknown
#101371408 Credit: Leonid Andronov
#155177696 Credit: molekuul.be
Figure 4.2: Unknown

#14703085 Credit: Studiotouch
Figure 4.4 Credit: Janet Braam
#57341071 Credit: kontur-vid
Figure 4.6 Credit: Wei Wei Zhong
Figure 4.7 Credit: Shutterstock. ID
#57341071 Credit: kontur-vid
Figure 4.8 Credit: Daniel Wagner

(http://en.wikipedia.org/wiki/White_blood_ce
ll)

#119402488 Credit: Knorre

(http://en.wikipedia.org/wiki/Nicotinamide_a
denine_dinucleotide_phosphate)
Credit: Wikimedia.
(http://commons.wikimedia.org/wiki/File:Maj
or_cellular_sources_of_Reactive_Oxygen_
Species_in_living_cells.jpg)

#111785756 Credit: petarg
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Figure 6.2 Credit: Marie Treuse Valovska



(http://en.wikipedia.org/wiki/Macrophage)
(http://en.wikipedia.org/wiki/Follicular_dendri
tic_cell_sarcoma)

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Figure 7.10 Credit: Ayna Troshkina



Figure 7.14 Credit: Credit: Edward Novotny
#152927564 Credit: dream designs
Figure 7.9 Credit: wiki or shutter

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BIOC 372x Study Guide

This study guide covers Part Two: T-cells and Signaling.
Lecture L08- MHC

Lecture L09a- Antigen Processing and Presentation, part 1
Lecture L09b- Antigen Processing and Presentation, part 2
Lecture L10- T-cell Receptors
Lecture L11-T-Cell Development
Lecture L12- Cytokines and Signaling
Lecture L13-Inflammation
Lecture L14- Cellular Immunity
Glossary
Attributions
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2
Lecture 8 (L08)
The Major Histocompatibility Complex
Theres only one person in the whole world like you. .. Theres
never been anybody exactly like you before, and there will never
be anybody exactly like you in the future. Youre the only one.
Fred Rogers, 1928 - 2003
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I.
Basic Characteristic: used to hold antigen for T cells, determine transplantation

success.
Figure 8.1 - Structure of the MHC Locus
A. Genes: Human HLA complex on chromosome 6, organized into 3 regions

(Figure 8.1):
1. Class I MHC
a. Codes for a three-domain protein
b. The main peptide ( ) associates with a second ( ), which is
coded for on an entirely different chromosome (#15)
c. Displays antigen (small peptide) to Tc (cytotoxic) cells.
d. Everyone has a block of 3 of these genes from Mom and 3 from
Dad (A, B & C below) (Figure 8.2)
e. The genes are wildly polymorphic: each one of these 6 could be
any one of as many as 100 different alleles
f. Expressed by almost all nucleated cells, but not found on red
blood cells, sperm or nerve cells
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Figure 8.2 - MHC Regions
2. Class II MHC
a. Each gene codes for 2 separate peptides ( and ) which
function together.
b. present antigen (small peptide at the end between and to TH
(helper) cells
c. Everyone has 3 of these genes from Mom and 3 from Dad.
d.
block of genes on the centromere end (DP, DQ and DR)
e. wildly polymorphic: each one of these 6 could be any one of as

many as 100 different alleles.
f. expressed by antigen presenting cells (macrophages, dendritic
cells, B cells)
3. Class III MHC
a. Completely different - generally not membrane components.
b. code for secreted proteins involved in the innate responses:
complement proteins, cytokines, and heat shock proteins.
c. included because the genes just happen to lie between I and II
genes
B. Haplotypes
1. Both Class I and Class II genes are tightly linked, with a crossover rate
of about 0.5%. This means that any one individual tends to pass on the
collection of 3 class I and II MHC from Mom as a unit and the collection
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of 3 class I and II MHC genes from Dad as a unit.

2. This unit or block is called a haplotype. l
3. Lets look at a marriage in which one partner has two different blocks of
these genes, haplotype A from her Mom and haplotype D from her Dad
and marries someone with a completely different block, M from Mom and
P from his Papa. (Figure 8.3)
Figure 8.3 - Haplotype Possibilites from Parents
4. These blocks can combine to produce AM, AP, DM and DP, illustrated
here as four possible genotypes in the children.
5. This is why, when people discuss the possibility of tissue transplants
from a sibling, they say you have a one-in-four chance of a match.
C. Recombinant Haplotypes
1. Crossing over can occur during meiosis of the germ cells of either Mom
or Dad.
2. Rarely, if the crossover happens between the I and II blocks, this
produces a recombinant haplotype. (Figure 8.4)
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Figure 8.4 - Recombinant Haplotypes
3. If a sperm with one of these joins and fertilizes an egg, the resulting
child is highly unlike to EVER have a sib match that would require the
exact same crossover event, the same choice of one of the two
crossover results and a union with the exact same egg haplotype.
D. Inbred Mice
1. If you inbreed mice with a series of brother-sistermatings, you can
produce different strains in which each mouse is histocompatible with
the others.
2. This is because each mouse of a given strain will have the same
haplotype on each homologous chromosome.
3. Different strains will have different haplotypes
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4. If you then take two-different inbred strains and cross them you get an
F1 that: (Figure 8.5):
a. can accept grafts from any other F1 (same types).
b. can accept graft from either parental strain (doesnt graft on any
nonself.)
c. cannot donate to either parent (will provide other parent's
antigen).
Figure 8.5 - F1 Generation from Inbred Strains
E. Practice
1. Lets look at those mice again.
a. How many different MHC I molecules can left hand (yellow) strain
make?
b. How many different MHC I molecules can the right hand (peach)
strain make? MHC II?
c. How many different MHC I molecules can F1 progeny make?
MHC II?
2. Heres the maternal and paternal haplotypes of an individual from an
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isolated village in remote area. (Figure 8.6)

a. The MHC I A haplotypes are the same form Mom and Dad,
although MHC I B and I C are different.
b. The MHC II DP s are different but the MHC II DQs and DPs
are identical.
Figure 8.6 - MHC Haploytypes
3. How does this influence variability of receptors?

a. How many different MHC I molecules can this person make?
b. How many different MHC II molecules can this person make?
c. This person leaves the village for the city and married someone
completely unrelated who shares not alleles for either MHC. How
many different MHC I molecules will their child make? MHC II?
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Figure 8.7 - MHC Shapes
II.
MHC Protein Structure - Both kinds are membrane-bound glycoproteins with

common structural elements, including a peptide-binding cleft (Figure 8.7)
A. Class I MHC
1. chain
a. 3 major domains, 1, 2, 3 (amino to carboxyl)
b. peptide binding site between 1 and 2
c. 3 connected to
d. transmembrane segment with cytoplasmic tail
2. microglobulin chain
a. gene actually located on a different chromosome, #15
b. peptide associates with 1 by weak linkages
c. necessary for membrane expression of whole molecule
d. This does not have a membrane-spanning region, but rather
attached by weak bonds to the domain of the class I MHC
immediately exterior to the membrane.
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3. Homologies
a. 3 domain and the chain resemble each other.
b. Both resemble the constant domains of immunoglobulins.
c. Note the bread and butter sandwich structure.
d. Gene transcript requires processing (no alternative splicing
forms), but the DNA is not changed.
4. Interactions
a. 1 and 2 form a platform structure with 8 antiparallel strands
connecting two helical regions.
b. The space between the helices forms a deep groove, the
peptide-binding cleft
c. Long enough to hold a peptide of 8 to 10 amino acids
d. The platform region also interacts with the microglobulin, which
kind of supports one side of the structure.
e. The peptide is necessary for the proper folding of the peptide
and its placement into the cell membrane.
B. Class II MHC
1. chain
a. two major external domains (1 and 2)
b. transmembrane domain with cytoplasmic tail
2. chain
a. two major external domains (1 and 2)
b. transmembrane domain with cytoplasmic tail
3. Homologies
a. 2 and 2 resemble the class I MHC 3 domain and chain
and immunoglobulin constant regions
b. 1 and 1 resemble the class I MHC 2 domain and 1domains
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respectively.
c. MHCs are also in the immunoglobulin superfamily.
d. Gene transcript requires processing (no alternative splicing
forms), but the DNA is not changed.
4. Interaction
a. The and dimers are joined by weak interactions
b. The antigen binding cleft is formed by the 1 and 1 interaction.
III.
Specifics of Peptide Binding

Class I and II molecules both bind peptide hydrolysis products of proteins, the peptides
being inserted into their respective cleft regions (Figure 8.8)
A. Common Elements
1. The peptides produced by hydrolysis may come from any part of the
molecule.
2. Peptides are extended in the cleft, and do not have their native
secondary or tertiary structure.
3. MHC molecules can bind a variety of peptides, although each MHC has
some (broad or promiscuous) specificity.
Figure 8.8 - Cleft Shapes of MHC
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B. Class I MHC
1. Sides of the cleft defined by x helices
2. Bottom of the cleft define by sheets
3. Ends of the cleft also defined.
4. Peptides bind best with 9 amino acids.
a. 8 or 10 can fit, because of bending.
b. Peptide bows outward slightly,
c. Bowing helps display middle of the peptide from out of the groove
of the cleft
5. Peptides held on the ends by their anchor residues, which interact with
specific side chains of the amino acids of the class I MHC.
a. Carboxy (COOH) terminal anchor (amino acid #9 of the peptide)
is typically hydrophobic.
b. Amino acid #8 and even #7 may be involved in anchoring.
c. Amino terminal anchor is #2
d. Middle amino acids may vary, although exactly what they are will
be important to the T cell receptor they will ultimately interact with.
C. Class II MHC
1. Sides of the cleft defined by helices
2. Bottom of the cleft defined by sheets
3. Ends of the cleft are undefined: the peptide can stick out like a long
hotdog in a short bun
4. Peptides bound have 13 to 18 residues, but only 13 of them fit the cleft
5. Peptides do not bow outward, but rather lie flat in the cleft.
6. Peptides therefore interact with the class II MHC molecule at a series of
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places in the cleft region.

7. Different MHCs will have different binding specificities based on these
interactions
8. The MHC amino acids involved in the interaction show the most of the
polymorphism of the molecule.
IV.
Genetic Expression
A. Polymorphism and Individual Expression
1. There are an enormous number of different possible alleles coding for
different versions of all the class I and II MHC classic genes and a fair
amount of variety in the non-classic ones as well.
a. For HLA I in humans, we have identified 60 versions of A, 110
versions of B, and 40 versions of C.
b. This underestimates the diversity, because we usually test
whoever is handy, and that means disproportionately from people
of European descent.
2. Any one individual, however, will express only those alleles present in his
genotypes
3. This means that any one individual will express 6 (3 maternal 3 paternal)
class I MHC alleles, 6 (3 maternal 3 paternal) class II MHC alleles.
4. The 12 classical alleles come from this incredible assortment of
possibilities, meaning that any two unrelated individuals are very unlikely
to share them.
5. Although it does happen
B. Cellular Expression
1. Class I MHCs are expressed by most cells, but to varying degrees:
a. Very high levels on lymphocytes - critical in presenting
b. Low on fibroblasts, muscle cells, and liver cells
c. None on nerve cells, red blood cells or sperm
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Table 8.1 - Locations of MHC I and II in Cell Types
neither MHC I nor

II
MHC I but not II
MHC I and II
MHC II, but not I
red blood cells
most cells,
including
neutrophils and T
cells, kidneys and
muscle
professional
antigen presenters:
B cells, sentinel
dendritic cells and
macrophages
no cells
sperm cells
nerve cells (with
interesting
exceptions)
2. Class I MHCs present antigen from normal turnover of interior proteins.

a. They will bind and present peptides from cytochromes , histones,
ribosomal proteins, and indeed any cell protein, cytoplasmic,
nuclear, or mitochondrial.
b. These proteins will be recognized as self-proteins early in
development.
3. Class I MHCs present antigen from turnover of abnormal interior proteins
as well.
a. Viral proteins degraded into peptides will also get bound to some
of the class I MHC and presented.
b. These will be recognized as non-self and trigger an immune
response, resulting in attack by TC cells.
4. Class II MHCs are not expressed by most cells.
a. Expressed by antigen presenting cells, B cells, macrophages,
and dendritic cells.
b. Expressed upon induction by thymic epithelial cells and a few
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other types responsive to certain cytokines.

c. Present exogenous antigen to TH cells, more in next lecture.
C. Expressing the complete genotype

Figure 8.9 - Possible MHC Types
1. Considering the class I MHC, the chances are that all 6 alleles, three
from Mom and 3 from Dad, will be different. (Figure 8.9)
a. This will code for 6 different surface binding molecules, each with
a different specificity for peptide binding.
b. What about the microglobulin? This is coded for by a different
gene on a different chromosome (15), and may well have
different alleles as well.
2. Consider class II MHC, multiple figure (Figure 8.9)
a. You have three from Mom and three from Dad.
b. You have three from Mom and three from Dad.
c. However, any DR from either parent may combine with any DR
, resulting in 4 different DR combinations.
d. This is also true for DP and DQ, so there are 12 different MHC II
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combinations, assuming no inbreeding.

e. This broadens the possibilities for effective presentation to TH
cells.
D. Comparing MHC Molecules and Lymphocyte Receptors

1. All cells in any one individual have the same genetic possibilities for
making either kind of MHC.
2. The genes for MHCs do not rearrange, nor do they generate different
splicing isoforms.
3. The extreme specificity of the response to the antigen presented is a
property of the T-cell receptor, not the MHC presenter.
4. Description of the complex figure show different combinations of
proteins:
V.
But Wait! Theres More! Non-classical Alleles

A. Non-Classical MHC Genes (more detailed map at the end of the handout)
1. Interspersed in the Class II MHC genes are genes that code for
elements of the system that processes antigen for Class I MHC
presentation (go figure.)
2. Interspersed in the Class I MHC genes are those for "non-classical"
membrane proteins involved in a variety of specialized receptor,
transport, or presentation functions.
3. Most have the same general three-domain shape as MHC I, associate
with microglobulin, and are typically named HLA something, for
Human Leukocyte Antigen.
B. CD1 and Lipid Presentation
1. T cells also recognize glycolipids produced by hydrolysis of bacteria.
2. Presenting cells add these lipids to special MHC molecules, loading them
in the phagolysosome.
3. The binding part looks a lot like a class I MHC and also uses the same
microglobulin.
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4. The lipid-binding MHCs come in 5 versions, CD1A-E, all coded for on

Chromosome 1. (Figure 8.10)
Figure 8.10 - Lipid-binding MHCs
C. Compare and contrast the Class I and II and CD1 MHCs

Table 8.2 - Compare and Contrast MHC I, MHC II, and CD1 MHCs
MHC I
MHC II
CD(A-E)
displays endogenous peptide
presents exogenous peptide
displays exogenous lipid
to TC cells
to TH cells
to TH cells
on most nucleated cells
on presenting cells
on presenting cells
1 peptide, 3 domains
2 peptides, 4 domains total
1 peptide, 3 domains
chromosome 6
chromosome 6
chromosome 1
plus globulin (c-some 5)
plus globulin (c-some 5)
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closed cleft between 1 and 2
open cleft between 1 and
deep cleft between 1 and 2
loaded in RER
loaded in phagolysosome
loaded in phagolysosome
VI.
MHC and Disease

A. MHC and Immunity
1. Determinant Selection: While MHC binding is promiscuous, it is not
universal. Some MHCs bind some antigens better than others, and
some antigens do not seem to get displayed properly.
a. The perils of inbreeding: Cheetahs are at risk of extinction in part
because they are so inbred. (Figure 8.11)
Figure 8.11 - Cheetah
b. Disease as an evolutionary selective force: Populations

constantly endure a sweep of infection that kills off a substantial
fraction of its members. Those remaining will usually have a
statistically significant difference in the distribution of MHC (and
other) alleles.
2. HIV elite controllers there are people who have contracted HIV, but
maintain low levels of the virus and stay healthy for decades.
a. Studies comparing their DNA to those of susceptible people
showed a number of common snips in the DNA of chromosome
6.
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b. Upon analysis, the relevant region was in the gene coding for
MHC I, HLA-B, specifically the part determining the binding
groove. Variant #27 is associated with slow progression. (Figure
8.12)
c. Thus elite controllers are particularly effective at binding and
displaying HIV-derived peptides and thus very effective at
activating Tc cells.
d. The Tc cells, in turn, keep the viral levels so low that the TH cells
remain protected and thus able to continue coordinating the
immune response.
e. However, the bad news is that #27 is also associated with
increased risk of ankylosing spondylitis, an autoimmune disease
which results in fusion of the spinal cord vertebrae.
Figure 8.12 - MHC variations
B. MHC and Susceptibility

1. Possession of a particular allele increases your relative risk, sometimes
very substantially.
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2. For example, a variant of DR2, that if you have it you are 130 times
more likely to exhibit narcolepsy as a typical member of the general
public. Narcolepsy is now classified as an autoimmune disorder.
3. On the other hand, possession of a particular allele does not predictably
doom you to a certain disease; environment and dumb luck also play a
role.
4. An exception is hereditary hemochromatosis, which results from having
two copies of a mutant variant of HLA- HFe.
5. This codes for a surface protein on the cells of the digestive tract, and
when it's there, it interferes with negative regulation of iron uptake.
Some Extras:
Video: Rusty, the narcoleptic dog, falls asleep every time he get excited.
http://www.youtube.com/watch?v=jTj3a2nHw8k
Narcolepsy as an autoimmune disorder:
http://www.nature.com/news/narcolepsy-confirmed-as-autoimmune-disease-1.14413
Hereditary hemochromatosis: http://emedicine.medscape.com/article/1878061-overview
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Lecture 09a (L09a)

Antigen Processing and Presentation, part 1
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Lecture 9
Antigen Processing and Presentation (Part 1)
I.
T Cell Antigen Recognition

A. Common Elements of TC and TH Cell Recognition
1. T cells only recognize peptide antigens not native proteins.
2. These peptides must be attached to some kind of MHC molecule extending from the
surface of a cell.
3. The T-cell receptors recognize the combination of self-MHC and antigen.
B. Specific T cells
1. TC Cells (Figure 9A.1)
a. Cells have CD8 assisting the receptor.
b. Cells bind to Class I MHC plus antigen.
c. Antigen displayed is endogenous - arises from proteins produced by the cell.
d. Most cells display antigen on MHC I.
e. TC cells respond by attacking the cells displaying abnormal antigens.
f. Thus the cells displaying foreign antigen are target cells.
Figure 9A.1: TC Cell With Class I MHC
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2. Specific to TH Cells (Figure 9A.2)

a. TH cells have CD4 assisting the receptor.
b. TH cells bind to Class II MHC plus antigen.
c. Antigen presented is exogenous - arises from proteins hydrolyzed after
phagocytosis or endocytosis.
d. B cells, macrophages or sentinel dendritic cells do most of the presenting, and
are called professional antigen presenting cells.
e. TH cells respond by producing cytokines and other signals that stimulate a
variety of immune responses.
Figure 9A.2: TH Cell With Class II MHC
C. Professional Antigen Presenting Cells (APCs)- the most important source of antigens
for TH cells.
1. sentinel (but not follicular) dendritic cells
a. most effective
b. constitutively express class II MHC and costimulatory (B7) molecules
c. can activate nave TH cells
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2. macrophages
a. activated by phagocytosis
b. express class II MHC and costimulatory (B7) molecules after activation.
3. B cells
a. constitutively express class II MHC
b. express costimulatory (B7) molecules after activation (receptor cross-linked by
antigen)
c. most effective at activating TH cells with small amounts of antigen
4. Table summarizes roles. Sentinel dendritic cells are constitutively active and B7
is a co-stimulatory molecule important in activating T cells.
Table 9.1: Professional Antigen Presenting Cells (APCs)
II. Self-MHC restriction

Table 9.2: Self-MHC restriction: Which of these can the immune system recognize?
Self MHC/
self-antigen
Foreign MHC/
foreign antigen
Self MHC/
foreign
antigen
Foreign MHC/
self-antigen
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Figure 9A.3: TH Cell
Figure 9A.4: Guinea Pig
A. TH cells and class II MHC, Rosenthal and Shevach (Figure 9A.3)

1. Used guinea pigs from two different highly inbred strains. (Figure 9A.4)
2. Added viral antigen to the macrophages from one strain (strain 2).
3. Gave the macrophages time to process and then isolated them. These are the
antigen-pulsed macrophages.
4. Exposed TH cells to these macrophages.
5. If the TH cells were from the same strain (2), they were stimulated to divide and
differentiate.
6. If the TH cells were from a different strain (13) then they would not respond.
Table 9.3: Test results of the Rosenthal and Shevach Experiment
TH cells from strain 2
Macrophages from strain 2
respond
TH cells from strain 13
no response
TH cells from 2/13 hybrid
respond
7. If the TH cells were from an F1 strain from the cross of 2 and 13, then they would
respond. Such cells will share a haplotype with either strain.
8. This showed that TH cells are class II MHC restricted - meaning they will only
respond to antigen presented on class II MHC molecules whose genes they
themselves possess.
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Figure 9A.5: TC Cell
Figure 9A.6: Mouse
B. TC cells and class I MHC, Zinkernagel and Doherty, used mice. (Figures 9A.5-9A.6)
1. Immunized mice with LCM (lymphocytic choriomeningitis) virus.
2. Extracted cells from the spleen, which included TC cells with receptors specific to
virus antigen.
3. They then mixed these cells with LCM virus-infected cells and checked to see if the
TC cells could kill the infected cells. (Figure 9A.7)
4. If the infected cells shared a haplotype with the TC cells, then the TC cells attacked
the infected cells.
5. If the infected cells did NOT share a haplotype, then the TC cells ignored them,
even through they could recognize the viral antigen perfectly well.
Table 9.4: Test results of the Zinkernagel and Doherty Experiment
TC cells from strain A
LCM-infected cells (strain A)
respond
TC cells from strain B
no response
TC cells from AB hybrid
respond
6. This showed that TC cells are class I MHC restricted: If the viral antigen was not
displayed on a self-class I MHC, then the TC cells could not respond to it.
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C. T-cell Antigens, Townsend et al.

1. The TC cell recognized internal matrix and nucleocapsid proteins better than
envelope proteins.
2. Moreover they were recognizing short peptide sequences, not the proteins as a
whole.
3. Synthetic peptides with these sequences worked just as well to target flu-infected
cells.
Figure 9A.7: LCM Infected Cell With TC Cell
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Lecture 09b (L09b)

Antigen Processing and Presentation, part 2
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Lecture 9
Antigen Processing and Presentation (Part 2)
I.
Cytosolic Pathway: Class I Processing and Presentation
Figure 9B.1: Proteasome
A.
Figure 9B.2: Presentation Pathways
Peptide Generation by Proteasomes HHMI (Skip to 40s)

https://www.youtube.com/watch?v=4DMqnfrzpKg
1. Protein turnover is a normal feature of cell metabolism.
2. Proteins are targeted for destruction by complexing with another small
protein, ubiquitin.
3. Complexing with ubiquitin sends a protein to the interior of a proteasome
(Figure9B.1).
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4. LMP2 and LMP7, (coded for by genes embedded in the class II region of
the MHC complex(Figure 9B.3)) two additional protein subunits, attach to the
proteasome and promote the degradation of proteins into peptides of the
correct size (9 amino acids) and composition (carboxyl terminal is
hydrophobic or basic).
B.
Transport to the RER

Figure 9B.3: MHC Genes
Figure 9B.4: TAP Proteins

and Transport
1. The peptides so generated bind to TAP 1 and 2 proteins ( also coded for by
genes embedded in the class II region of the MHC complex) embedded in
the RER,
2. TAP proteins (transporters associated with antigen processing) have the
following structure:
a. heterodimer
b. cytosolic binding site for peptides
c. ATP hydrolysis function near the binding site (variant of ATP-binding
cassette proteins)
d. hydrophobic anchoring regions with three switchbacks per peptide
3. The genes for the TAPs and the LMP2 and 7 all map within the class II (not
a typo) region and are polymorphic.
4. The TAP 1 and 2s bind to the peptides, hydrolyze ATP, and drive them into
the lumen of the RER (Figure9B.4).
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Figure 9B.5: MHC I chain
C.
Figure 9B.6: MHC I Complex with Associated Proteins
Assembly with and Display by Class I MHC

1. Recall that the class I MHC peptides are synthesized on the RER, and that
the chain is inserted into the membrane, and the microglobulin chain
sent through to the lumen (Figure9B.5).
2. Calnexin, a molecular chaperon, associates with the chain, promoting its
proper folding (Figure 9B.6).
3. The microglobulin chain can then associate, releasing the calnexin
(Figure9B.6).
4. The complex now associates with the chaperone calreticulin and with
tapasin, which brings the TAP transporter over to the complex.
Figure 9B.7: Stabilized complex
Figure 9B.8: MHC I with Peptide Bound
5. ERp57 then binds over the whole distal end of the complex (Figure 9B.7).
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6. When the TAP protein hands off the peptide to the cleft, the antigenic
peptide stabilizes the MHCI molecule and allows it to exit the RER to the
Golgi.
7. The antigenic peptide displace the ERp57 and the chaperones dissociate
from the class I MHC - peptide complex (Figure 9B.8).
8. After sorting in the Golgi, the vesicle with the class I MHC - peptide complex
is released by exocytosis and the complex becomes part of the plasma
membrane extending to the exterior (Figure 9B.9).
Figure 9B.9: MHC I Structure
II.
Exogenous Pathway: Class II Processing and Presentation (Figure 9B.10)

A.
Internalizing Antigen
1. Phagocytosis - engulfment of whole particles with pathogens - macrophages
(Figure 9B.11).
2. Endocytosis - internalizing membrane carrying whatever the membrane is
bound to - B cells internalize antigen as they recycle patches of membrane
containing M and D antibodies (Figure 9B.12).
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Figure 9B.10: Exogenous Pathway
Figure 9B.11: Receptor-Mediated

Endocytosis
B.
Figure 9B.12: Endosomes
Hydrolysis in Vesicles
1. Goes through a series of three vesicle types, each one more acidic and
hydrolytic (Figure 9B.12).
a. early endosome, pH 6.0 - 6.5
b. late endosomes of endolysosomes, pH 5.0 to 6.0
c. lysosomes (or phagolysosomes), pH 4.5 - 5.0
2. Lysosomes contain a variety of hydrolytic enzymes that will not only chop
up foreign proteins, but it will also remove any carbohydrate and lipids
attached to them and hydrolyze them.
3. The net result is a collection of peptides, most of which are 13 to 18 amino
acids long, and an array of miscellaneous digestive products.
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Figure 9B.13: Exogenous Pathway, a Different View
C.
Joining Up with the Class II MHC (Figure 9B.13)

1. Class II MHCs are synthesized by the RER, just like class I.
2. However, vesicles with class II MHC are targeted by the Golgi differently
from those with class I MHC, thus encountering peptides that not only come
from a different source, but which have also been processed somewhat
differently.
3. Three pairs of a complete class II MHC heterodimer associate with a
trimer of invariant chains (Ii).
4. The invariant chain plays an important role in the sorting process:
a. blocks the cleft, preventing premature binding of the wrong (cytosolic)
antigen.
b. acts as a chaperone, to help fold the peptides into proper
conformation
c. helps them exit the RER.
d. tags them for recognition by the Golgi in the sorting to fusion with
endocytic vesicles.
5. Class II MHC-invariant complexes leave the Golgi and fuse with the early
endosomes.
6. As the antigen and complexes move from the early to the late endosomes to
the lysosomes, the invariant chains get degraded, leaving behind a
fragment, called the CLIP, which still blocks the cleft.
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7. At the end of hydrolysis, a non-classical MHC (HLA-DM) removes the CLIP,

exposing the class II MHC to the antigenic peptides.
8. The binding of antigen stabilizes the class II MHC complex, allowing it to be
transported to the surface and displayed (Figure 9B.14)
Figure 9B.14: MHC II Structure
III.
Figure 9B.15: Comparison of Endogenous and

Exogenous Pathways
Making Sense of Variations

A. Cross presentation (Figure 9B.17)
1. APCs pick up exogenous antigen and put it on MHCI to activate TC cells.
2. Endosomes with peptides digested from a pathogen travel to RER and fuse.
3. Antigen in the lumen can now attach to MHCI
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Figure 9B.16: Comparison of MHC Structures
Figure 9B.17: Cross Presentation vs. Exogenous Pathway
B. Non-Peptides
1. T cells can also recognize bacterial lipid derivatives.
These are presented by a group of non-classical MHCs, the CD1 family of 5
genes (A through E), discussed previously.
2. The overall structure of the CD1 molecules resembles that of MHC I. Both
are peptides made up of 3 domains, binding antigen between the 1 and a2,
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and associating with microglobulin.

3. However, the lipid antigens that fit on these molecules must generated by
hydrolysis in an endosome (phagolysosome) and then presented to TH cells.
In this respect the CD1s function more like a class II MHC.
4. However the pathway is quite strange. The CD1 heads to the cell surface,
backs up into the lysosomes, picks up the antigen and the heads back to the
surface.
5. T cells seem to receive these rather differently as well. They may use a
somewhat different receptor, with the receptor, and no CD4 or CD 8. T
cells with receptors have been shown to bind these as well, using CD4
6. NK cells may also recognize members of the CD1 family displaying
autologous (non-bacterial) antigen, and participate in recycling damaged
cells identified by these signals.
7. iNKT cells plus CD4 also recognize lipids on CD1
C. Compare and Contrast (Figure 9B.16)
1. Compare and contrast the structure and function of Class I and Class II MHC
molecules with respect to:
a. structure (Indicate homologies between domains in the two classes and
distribution of membrane-spanning regions.)
b. location and structure of antigen binding site
c. size of antigen peptide bound and conformation in the binding site
d. intracellular location of synthesis of the MHC and where in the cell the
antigen is loaded onto the MHC
e. which cells synthesize either class
f. what type of T cell that they present/display to, the co-receptor they
associate with and interaction with the T cell that they promote
Follow up: The chains of class I and class II MHC molecules are similar in that they BOTH
______.
A. are composed of two domains
B. form the complete structure of the binding site
C. are anchored to the cell by membrane-spanning domains
D. are unimportant in the recognition process leading to transplant rejection
E. are coded for by genes rearranged in the bone marrow
2. Compare and contrast the processing and presentation of exogenous and
endogenous antigen with respect to:
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a.
b.
c.
d.
biological source of the antigen

class of MHC molecule that will present it
class of T cell that will respond to the antigen-MHC complex
sequence of events bringing the antigen from wherever it starts out to the
MHC molecule.
e. The accessory proteins used in processing antigen or chaperoning the
complex
f. process leading to display on the cell surface
Follow up: Processing and presentation of antigen for presentation on class I MHC differs
from the corresponding process involving class II MHC in that only in the processing for class
I _______.
A. can the resulting complex display lipids as well as proteins
B. does the MHC enter the Golgi body with the antigen in the binding cleft
C. will the resulting complex be recognized by both and TCRs
D. is the complex displayed after a vesicle fuses with the plasma membrane
E. requires that a protein be hydrolyzed into a peptide
3. Compare and contrast antigen presentation on the non-classical MHC
presenting molecule, CD1 with cross presentation on MHCI. Include:
a.
b.
c.
d.
e.
A comparison of the structure of CD1 and MHC1

Where the molecules are synthesized and ultimately displayed.
The source and chemical nature of the antigen.
Where the antigen is loaded onto the MHC and subsequently displayed.
The T cells binding to the antigen and co-receptors involved.
Follow up: Presentation of antigen on CD1 differs from cross presentation on MHC I in that
only the CD1 ______.
A.
B.
C.
D.
E.
binds the antigen at a binding site comprised of the junction of two distinct peptides
binds peptides of 11 to 12 amino acids
presents to TH cells
displays an antigen derived from exogenous sources
is a membrane-bound protein composed of three domains
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Table 9.5: Summary of MHC properties
Class II MHC
Class I MHC
Function
external antigen presentation
self antigen display
Cells
presenting/displaying
Sentinel dendritic,
macrophages, B cells
All nucleated cells except

sperm
Molecule structure
and peptides, two domains

each
peptide with 3 domains,

associated microglobulin
with 1 domain.
Source of antigen
phagocytosis
synthesized in cell
Antigen hydrolyzed in
phagolysosome
proteasome
Typical length of peptide
13 to 18 amino acids
8 to10 amino acids
Antigen loaded onto MHC

in
phagolysosome
rough ER
Antigen binding site
open-ended cleft between

and peptides
closed slot at junction of 1

and 2 peptide domains
antigen conformation
lies flat
bows outward
Responding cell
TH (Helper)
TC (Cytotoxic)
TCR binding to antigen
TCR
TCR
Co-receptor
CD 4
CD 8
Response
Coordinates immune response
References:
Heath, William R., and Francis R. Carbone (2005). Coupling and cross-presentation. Nature
434, 27 28, March 03
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235490/?tool=pmcentrez (Walker et al)
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Lecture 10 (L10)
T-cell Receptors
We should never have sought either solace or moral instruction

in Nature. Stephen Jay Gould
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I.
Early Work A. Learning (or Not) from the Immunoglobulin Receptor

Figure 10.1 - TCR vs Ig Receptor
TCR
Ig Receptor
B. Cloning the T-cell receptor gene

1. Isolated mRNA from membrane bound polysomes (chains of actively transcribing
ribosomes, bound to ER membrane receptors), on the assumption that the TCR was
inserted into the cells membrane by the standard mechanism. This eliminated 97%
of the cytoplasmic mRNA. (Figure 10.2)
Figure 10.2 - B and T cells were pelleted, to isolate mRNA (top fraction)
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2. Copied this fraction into 32P labeled cDNA

3. Used subtraction hybridization to get rid of mRNA that wasnt coding for the
receptor. That is, they isolated membrane-bound polysomes from B cells. This
fraction should have mRNA for proteins common to both types of lymphocytes,
but not those for the TCR. They could use the B-cell mRNA to hybridize and
remove any messages the two cells had in common. (Figure 10.3)
4. 10 different cDNA clones remained, presumably of messages unique to T cells,
and including the messages for the TCR. You would expect messages for the
and peptides, the , , , and peptides of the CD3, and the CD4 and
CD8.
Figure 10.3 - Workflow for isolating T cell specific mRNA
5. Checked to see which of these sequences bound to genes in T cells that had been
rearranged. The sequences would bind only to DNA that has been rearranged, and
the cDNA would not be able to recognize unarranged gene from macrophages or B
cells. (Figure 10.4)
6. Looked at 6 different T-cell lineages, each with a somewhat different version of the
gene. Presumably the receptor mRNA would be different in different lines of cells.
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Figure 10.4 - Assay to identify which cDNA coded for the TCR: Each cDNA (denoted Clone 1, 2,
etc.) was used to probe a liver cell, B cell, and 6 different T cell lines.
Clone 2 in this image is the TCR transcript, because: 1) it hybridized to a different-length gene for each T
cell (because they rearranged differently), and 2) liver and B cells have same non-rearranged gene.
II. T-Cell Receptors: Structure and Roles

Figure 10.5 - TCR Structures
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A. versus receptors (Figure 10.5)

1. The receptor as a heterodimer, made up of either or peptides.
2. A T cell can make one kind or the other. Moreover theres no such thing as a
hybrid ( or ) receptor.
3. Most T cells make receptors.
B. Common Elements
1. The structure of both types resembles that of a truncated immunoglobulin.
2. Each chain has a conserved region including a constant bread and butter
sandwich region typical of the immunoglobulin superfamily.
3. Each chain has a variable bread-and-butter sandwich region at the amino
terminus with three hypervariable regions in each chain.
4. The transmembrane chains (also conserved and ending at the carboxyl terminal)
are unusual in that they have positively charged (hydrophilic) residues that attract
other (negatively charged) R groups on the transmembrane signal transducer,
CD3.
5. The membrane signaling complex is the same for both receptors.
C. Functional Differences.
1. T cells must undergo selection to produce a highly specific recognition
molecule, each cell having only one kind and with a huge variety of different
possible kinds of these receptors produced by different cell lineages.
2. T-cell receptors also undergo selection to produce a specific receptor, but from
a defined repertory of receptor types evolved in response to pathogens most often
encountered by that organism.
3. T cells have receptors that target are relatively limited and defined range of
antigen. For example, most human T cells recognize lipid antigen characteristic
of tuberculosis bacteria and parasites (malaria and leishmaniasis).
4. T cells function very differently from T cells:
a. can react with antigen directly, that is, not presented on MHC
b. can also recognize lipid antigens presented on non-classical MHC
c. can bind to non-classical MHC (T22), which does not bind antigen and seems
to up-regulate their activity.
d. typically has neither CD4 nor CD8.
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e. can both signal and attack (attack resembles that of TC and NK cells, inducing
apoptosis)
f. can phagocytize and present antigen (Brandes, et al, 2005).
5. T cells are important in patrolling mucosa and epithelia and in wound healing.
6. Disruptions in T- cell regulation are associated with autoimmune disorders.
7. From now on, unless otherwise specified, when I talk about a T cell receptor, I
mean an .
Table 10.1: Differences between the and TCRs
receptor
structure
two peptides, + charged anchors,

147 bend
two peptides, + charged anchors

111 bend
diversity
TH and TC : highly
limited (quasi innate)
requires MHC
yes (MHC I, II and CD1)
no, but may use CD1 or T22
requires
co-receptors
CD4 or CD8
not usually, but may have Fc and

Toll-like receptors
phagocytosis
and antigen
presentation
no
yes
location
mucosa
III. T-Cell Receptor Genes

Figure 10.6 - TCR Gene Locations
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A. Gene Location (Humans) (Figure 10.6)

1. The gene family is located on chromosome 14, long arm
2. The gene family is located on chromosome 7, long arm
3. The gene family is located on chromosome 7, short arm
4. The gene family is also located on chromosome 14, very strangely in the middle
of the gene family, between the V and J genes.
B. Gene Regions (Humans)
1. Compare with Ig genes: the set-up resembles that of the immunoglobulin genes,
with the and equivalent to the light and the and equivalent to the heavy.
2. Numbers of Gene Regions: Please note that this table refers to humans. Use it as
an approximation.
Table 10.2: Genes and Numbers of Gene Regions
Gene\ Gene region

gene
V
~40
D
-
J
~50
C
1
gene
~40
2 (functional)
6 and 7
gene
~5
(functional)
~3 (or more)
~5
~3
~4
gene
C. Mechanisms of Gene Rearrangements: very much like those that go on in

immunoglobulin genes.
Figure 10.7 - TCR Joining Region Structure
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1. The Recombination Signal Sequences are the same (Figure 10.7)

a. Have a heptamer (7 base) palindrome next to the region to recombine
(downstream on V, upstream on J and on both sides of the D.
b. Next to the palindrome is either a one or a two- turn (12 or 23 base)
sequence.
c. Next to the turn sequence is the AT-rich nonamer, or nine base sequence.
2. The RAG1 and RAG2 enzymes are the same one used in B cells.
3. A one-turn sequence must join to a two-turn sequence.
4. The joint between two regions will have:
a. a variable number of palindromic bases
b. bases added through P-nucleotide addition
c. bases added trough N-nucleotide addition (this includes both and and
and genes.
d. none of the bases from either the turn sequences or the AAT rich nonamers
D. Gene Expression1. similar to the process in Ig genes
2. All messages have membrane-spanning exons.
3. All four possible peptides extend into the lumen of the ER, anchored into the
membrane.
4. Vesicles with receptor fuse with the plasma membrane.
5. More details in animation in the next clip.
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IV. Gene Rearrangement in Action

Figure 10.8 - TCR Genes
A. Alpha Rearrangement- (Figure 10.9)

1. removes the gene.
2. leaves variable number of VL regions in the gene.
3. only activates the promoter of the VL joined to the J
4. leaves variable numbers of Js
5. leaves intron preceding the C
Figure 10.9 - Alpha Rearrangement
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B. Beta Rearrangement (Figure 10.10)

Figure 10.10 - Beta Chain Gene

2. only activates the promoter of the VL joined to the D (or the J if the D gets left out)
5. In this you use the first C1, will leave the J2s and C2 as well.
6. (Figure 10.11a) shows a possible rearrangement and (Figure 10.11b) shows the
results.
Figure 10.11a - Example 1 Beta Rearrangement: Joined L-V-D1 to J2
Figure 10.11b - Example 1 Beta Rearrangement: Removed RSS
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7. (Figure 10.12a) shows a different rearrangement, with (Figure 10.12b) showing

results below (note absence of D and presence of remaining downstream
sequences.).
Figure 10.12a - Example 2 Beta Rearrangement: Joined L-Vn to Jn (removed D)
Figure 10.12b - Example 2 Beta Rearrangement: Removed RSS
C. Delta Rearrangement (Figure 10.13)

Figure 10.13 - Delta Chain Gene (within the alpha chain gene)
1. leaves both the whole gene and variable number of VL regions in the gene.
2. only activates the promoter of the VL joined to the D ( or J). (Figure 10.14)
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3. may add more than one D

Figure 10.14 - Delta Rearrangement (with more than one D region)

D. Gamma Rearrangement
Before rearrangement (Figure 10.15)
Figure 10.15 - Gamma Gene
After rearrangement (Figure 10.16)

Figure 10.16 - Gamma Gene after Rearrangement

2. only activates the promoter of the VL joined to the J
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E. Subsequent Expression: Common Elements:
1. The region is made up of 4 exons, each corresponding to a functional domain.
a. Constant Ig domain ( pleated sheet switchback)
b. Extracellular connecting sequence
c. Transmembrane region
d. Cytoplasmic anchoring tail
Figure 10.17 - Example of Gene Expression: Gamma Gene
2. Transcription begins at the promoter (activated by splicing) and transcribes through

whatever constant region it reaches first and then stops. (Figure 10.17)
3. The message precursor is capped and gets a poly A tail.
4. Processing removes any extra Js. (Figure 10.18)
Figure 10.18 - Example of Gene Expression: Gamma Gene Primary Transcript
5. Translation of the leader attaches the ribosome to the ER.

6. All four TCR peptides will insert into the lumen
a. The C terminal remains anchored to the ER membrane
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b. The leader is clipped off.

c. Enzymes in the Golgi oxidize the SH groups to form disulfide bonds
d. Vesicles with receptor fuses with the plasma membrane.
V. Diversity and its Constraints

Figure 10.19 - Expression of Genes
A. Sources
1. Combinatorial joining - The random joining of V and J segments is still a geometric
generator of diversity.
a. and have the following segments: multiple leaders plus Vs, multiple Js, and
single C (or constant).
b. has multiple leaders plus Vs, and two repeats of a single J, D and C.
c. multiple leaders plus Vs, (because it's in the middle of , it sometimes
exchanges Vs with it), multiple Ds and Js, and a single C
2. Alternative joining - sometime the chain leaves out the D segment altogether. The
can even add in more than one D segment.
3. Junctional flexibility - variation in exactly where the segments are joined.
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4. P and N nucleotide addition occurs in all chain rearrangements, whereas for

immunoglobulins, only the heavies do N.
B. Constraints
1. A functioning TCR must recognize an MHC, either I or II.
2. It is thought that the V regions accommodate this.
3. However, when it comes to recognizing antigen, the TCR must be prepared for
extreme diversity. This seems related specifically to one of the recognition regions,
CDR3.
4. On the other hand, one thing that the genes do not do is undergo affinity
maturation: no somatic mutation followed by selection.
VI. The TCR Receptor Complex: (Figure 10.20)
Figure 10.20 - TCR Receptor Complex
A. CD3, the receptor complex

1. complex of three dimers associated with the and chains (or and )
a. heterodimer
b. heterodimer
c. homodimer or (zeta-eta) heterodimer (only 10%)
2. and are splicing isoforms: coded for by the same gene.
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3. peptide structure
a. , , and chains are all part of the immunoglobulin superfamily.
b. and are not, having only 9 amino acids to the exterior and a long extended
cytoplasmic tail.
c. All CD3 peptides have a negatively charged aspartic acid in the transmembrane
regions which causes them to salt bridge with a positively charged R groups in
the transmembrane domain to the TCR.
d. All peptides have an ITAM (immunoreceptor tyrosine-based activation motif) at
the interior, the and cytoplasmic tail having three each.
e. The ITAMs function like those in the B cell receptor: they attract other tyrosine
kinases and do not autocatalyze the transfer of phosphates themselves.
B. CD 4 (Figure 10.21)
1. used by TH cells to bind class II MHC (Figure 10.22)
2. immunoglobulin superfamily protein
3. functions as monomer
4. 4 extracellular immunoglobulin domains
5. N-terminal domain binds to the base of MHC II in a hydrophobic pocket for by the
junction of the 2 and 2 domains.
6. transmembrane region
7. C-terminal cytoplasmic tail with 3 serines that can be phosphorylated
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Figure 10.21 - CD4
Figure 10.23: CD8
Figure 10.22 - MHCII
Figure 10.24: MHC I
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C. CD 8 (Figure 10.23)
1. used by TC cells to bind class I MHC (Figure 10.21)
2. immunoglobulin superfamily protein
3. functions as - heterodimer or - homodimer (link by disulfide)
4. extends out from the membrane as a stalk ending one Ig domain per peptide at the
N-terminal end.
5. double Ig domains wrap around the 3 and also touch 2 domains and the
macroglobulin.
6. transmembrane region
7. cytoplasmic tail with several amino acids that can be phosphorylated
VII. Alloreactivity: Once of Life's Great Mysteries

A. The Problem
1. Tissue transplants are rejected if the transplant has MHC molecules not found in the
host, which is almost always.
2. TH cells recognize foreign class II, and TC cells foreign class I.
3. The tissue is destroyed by TC cells.
4. But, given what we've studied up to now, they shouldn't be able to do that. The
TCRs seem to be recognizing the foreign MHC directly and then mounting an
immune attack.
B. Evidence and Speculation

1. 1 - 5% of all T cells are alloreactive - that is, they can bind non-self MHCs. This is
a lot more than can recognize any one native MHC-antigen complex.
2. There is some evidence that T cell recognize foreign MHC via a binding mechanism
somewhat different from that used to detect antigen attached to self MHC.
3. The problem may in part arise inability of the T-regulatory cells to recognize a
transplant and then protect it from the random attack of NK, TC or T cells.
4. Moreover, most authors ignore the possible role of a T cell initiating the
response. Which is odd, because we know they function in autoimmunity, do not
to require MHC for recognition, and both attack and signal other cells to attack. On
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the other hand, theyre supposed to recognize primarily lipids and we know little
about the selection process (if any) that precedes their release form the thymus.
Whats Where (Human):
Chromosome 1 - CD1 family of non-classical MHC molecules
Chromosome 2 - light chain immunoglobulin
Chromosome 6 - MHC complex
Chromosome 7 - and TCR chains
Chromosome 14 - heavy chain of immunoglobulin and TCR chains
Chromosome 15 - microglobulin of class I MHC
Chromosome 22 - light chain immunoglobulin
References:
Allison, Timothy J. and David N. Garboczi (2001) Review: Structure of receptors and their
recognition of non-peptide antigens.
Brandes et al, Science Express (sciencemag.org/cgi/content/abstract/1110267v1) on T
cells as antigen presenters.
Gamma-delta function:
http://www.nature.com/ni/journal/v13/n3/full/ni.2240.html?WT.ec_id=NI-201203
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Lecture 11 (L11)
T-Cell Development
For a successful technology, reality must take precedence over

public relations, for nature cannot be fooled. - Richard Feynman
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I.
Maturation in the Thymus A. Early Events

1. Hematopoiesis
2. Some stem cells differentiate into lymphoid precursors - ikaros, a Zinc
finger protein transcription factor. (Figure 11.1)
Figure 11.1 - Zinc Finger DNA Complex
3. Signals from surrounding cells to notch activate lymphoid precursors to

differentiate into T-cell precursor ( or T-cell progenitors or thymocytes)
(Figure 11.2)
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Figure 11.2 - Notch Signaling
B. Double Negative Transitions (division somewhat arbitrary) (Table 11.1, below)

1. DN 1 (first double negative) T-cell progenitors (thymocytes or pro-T
cells) analogous to that of a pro-B cell (progenitor B cell).
a. Cells migrate to thymus, no TCR (receptor)
b. neither CD4 nor CD8 (CD4- CD8- or double negative) NOR
initially CD25, which is one of three peptides of the IL-2 receptor.
Much more later, as this is fundamental in stimulating immune cell
proliferation and differentiation.
c. genes for TCR have not rearranged
d. express membrane-bound receptor, c-Kit (CD117), which
receives a paracrine factor call alternatively stem cell factor or
steel. The signal tends to keep a cell in an undifferentiated state.
e. membrane-bound CD44 (an adhesion molecule that come in
several forms. Some target to bone marrow, but this version
seems to target to thymus)
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2. DN2- The T-cell progenitors stop dividing and begin rearranging their
genes.
a. Cells remain in cortex just to the interior of the capsule (Figure
11.3)
Figure 11.3 - A Thymus
b. They begin reducing expression of c-Kit, reduce CD44

expression, express CD-25, allowing them to assemble the
complete IL-2 receptor.
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Figure 11.4 - T-Cell Receptor Auxillary Proteins
c. Turn on RAG genes, the same genes used to cut and paste Ig
genes.
d. Turn on CD3 co-receptors.
e. A subpopulation of cells rearranges the genes. Most frequent
during gestation and declines to about 5% in adulthood. These
cells exit the thymus and head to epithelia.
f. Most thymocytes begin to rearrange the gene (pro-T cell).
3. DN3
a. cells still in sub-capsular cortex
b.
c-kit or CD44 and CD 25 still present, but declining
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Figure 11.5 - Pre-T Cell Receptor
c. chains produced by productive rearrangement combine with

preT chains (analogous to surrogate light chains in B cells) and
CD3 signaling peptides (loosely analogous to the B-cell
dimer) to form a pre-T-cell receptor (pre-TCR of pre T cell).
(Figure 11.5)
4. DN4
a. Cell penetrate to cortex (which is still the outer layer).
b. gene undergoes allelic exclusion, allowing rearrangement of
the a gene, although this is at first held in check.
c. Induces expression of BOTH CD4 and CD8, leading to the
double positive state.
II.
Positive Double (DP) Events

A. Completing the Receptor
1. RAG-2 first rises
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2. Thymocytes begin to rearrange the gene and then express it.

3. RAG-2 declines
4. Expanded clones with the same gene have many cells, each of which
rearrange to a different gene, increasing diversity.
5. Cells making good TCR and CD3 have a functioning receptor and
survive
B. Displaying Co-Receptors
1. Cell no longer express any c-kit, CD44 or CD25, although they will
express the two other subunits of the IL-2 receptor.
2. Cells now express both CD 4 AND CD8 (double positive).
C. Beginning Selection the process demands that
1. Cells must decide which MHC to respond to, and thus whether they
should continue to make CD4 or CD 8.
a. Cells that can bind MHCI will keep the CD8 and lose the CD4.
b. Cells that kind bind MHCII will keep the CD4 and lose the CD8.
2. Cell must not recognize self-antigen.
a. Vast possible number of ab TCRs (1015 a number so large as to
be meaningless)
b. The receptors can theoretically attach to almost anything.
Table 11.1 - Double Negative to Double Positive Development: Characteristics
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III.
Selection: Fully 98% of all thymocytes that start out in the thymus die there.
A. Positive Selection: results in significantly more cell deaths than does
negative
1. Immature thymocytes in cortex of the thymus contact stromal cells
(epithelial, macrophages, and dendritic cells.)
2. Stromal cells express MHC, both class I (as will any cell) and class II.
3. Thymocyte TCR binding to MHC molecules, signals inhibition of
apoptosis.
4. Only developing T cells that can bind to and recognize self-MHC survive.
5. T cells become single-positive, expressing CD8 or CD4 depending on
which class of MHC they happen to recognize. (Figure 11.6)
Figure 11.6 - CD4 and CD8
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a. Those recognizing class I continue to express CD8.

b. Those recognizing class II continue to express CD4.
c. If you knock out the gene for MHC II, the mice will not produce
CD4+ cells.
d. If you knock out the gene for MHC I, the mice will not produce
CD8+ cells.
6. Thus cells with no affinity for MHC are deleted. Positive means that
there must be some affinity or the cells wont survive.
B. Negative Selection, against those cells that bind MHC plus self-antigen, which
results in self-tolerance.
1. Takes place in medulla
2. Stromal cells of the medulla produce a varied buffet of self-antigens,
including many proteins normally characteristic of differentiated tissues.
3. They do this by expressing an unusual transcription factor, AIRE (Figure
11.7)
Figure 11.7 - AIRE Transcription Factor
a. the Swiss army knife (Figure 11.8) of transcription factors,

having two plant Hodo Zn fingers, 4 nuclear receptor motifs, a
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SAND domain and N terminal similar to SP100.

Figure 11.8 - Swiss Army Knife
b. randomly up-regulates transcription in thymic epithelial and

dendritic cells, which present antigen. This exposes developing
thymocytes to many more of the bodys antigens than developing
B cells encounter in the bone marrow.
c. Mutations in the aire (autoimmune regulator) gene are inherited
in humans as a classical Mendelian autosomal recessive,
producing a type of multi-organ autoimmune disorder call
APECED.
4. The selection process removes:
a. cells with a very high affinity for MHC all by itself.
b. cells with affinity for MHC plus self-antigen.
5. The process leaves alive only those T cells that respond to altered-self:
MHCs with non-self-antigens.
6. Cell that survive all this exit into the circulation
C. Issues in Selection, the Goldilocks Principle
1. How then could not binding to self-MHC lead to apoptosis at the same
time that binding to it too avidly (whether or not it has antigen on it) will
also lead to apoptosis?
2. Something allows just those cells with binding specificity for self-MHC
displaying non-self antigen to survive the process. How?
3. It a cell can bind to MHC weakly, using the 1 and 2 loops of the
receptor, and if it encounters an antigen outside the thymus, that antigen
will be foreign and will cause the whole complex to bind tightly enough to
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kick off a response.

4. The strength of the binding (avidity) between the TCR complex and the
MHC-antigen complex of the presenting cell determines whether the T
cell will live or apoptose.
a. If binding between receptor and MHC too weak, the T cell dies. It
cant recognize the antigen on the MHC.
b. If binding between receptor and MHC is too strong, the T cell
dies. It will react whether there is an antigen present or not, or
will react to self-antigen plus MHC.
c. Only a medium binding strength permits survival, because this
means a cells is primed to bind an antigen it has yet to
encounter, presumable a foreign one.
IV.
CD4+ T H Cells: Types and Functions
Recall the TH activation is the necessary and central event for humoral response (production of
antibodies by B cells) and cell-mediated (TC ) responses.
A. Determination of Subclass (Figure 11.11)
1. TH 1
2. TH 2 (Figure 11.9)
3. Treg (Figure 11.10)
Figure 11.9 - TH2
4.
Figure 11.10 - Treg
TH 17 (TH 3) - extra-cellular bacteria, fungi and parasites

a. Reciprocally regulated with Treg. IL6 diverts Treg to highly
proinflammatory T17 IL 25 and retinoic acid negatively, diverts to
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Treg.
b. Involved in decision as to whether or not to produce an immune
response or ignore the antigen.
c. Gamma delta cells and some sentinel dendritic cells also secrete
IL-17.
d. Improper up-regulation of TH 17 also involved in autoimmune
diseases.
e. IL-17 (not just from TH cells), is involved in barrier integrity.
f. Production triggered by IL-23, versus IL-12 for TH 1
Figure 11.11 - T-Cell Development
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B. Overview of the Process (Figure 11.12)

1. APC presents an antigenic peptide on a class II MHC molecule.
Figure 11.12 - Antigen Presentation
2. TH cell binds this complex with a TCR - CD3 receptor, aided by

3. A cascade of signals, beginning at the cell surface and entering the
nucleus, pushes the TH cell into a cell division cycle (out of G0).
4. This signal process necessarily involves transcriptional activation (Figure
11.13)
a) This signal process necessarily involves transcriptional activation
b) early genes (1 - 2 hours) include interleukins and their receptors.
c) late genes - more than 2 days after stimulus - cell adhesion
molecules.
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Figure 11.13 - Signal Transduction Pathway
C. Superantigens - Not a Good Deal

1. Superantigens are viral or bacterial proteins that bind to ternary
TCR-antigen-MHC complex in such a way as to nail them together.
(Figure 11.14)
Figure 11.14 - Superantigen
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2. This excessively strong binding results in hyperstimulation of the TH cell.

3. Include exotoxins secreted by gram-positive bacteria, including those
that cause:
a) staphylococcal enterotoxin
b) toxic shock toxin
c) exfoliative dermatitis
4. Binding is non-specific for antigen, and so different populations of TH
cells are stimulated.
5. The overproduction of cytokines can kill you.
V.
T H Cell Activation Pathway - forming the immunological synapse. Specific

TCR-coupled Pathways
A. Signal binds to receptor.
1. TCR binds to MHC II plus antigen
a) heterodimer forming the ligand (MHC-antigen complex)
binding unit.
b) CD3 complex - and peptides
c) Homodimer of (zeta) chains
2. CD-4 assists binding, locking on to constant domain (carboxyl end) of
the exposed MHC II. (Figure 11.15)
Figure 11.15 - CD4 Binding
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3. TCR complexes cluster with one another.

B. Membrane signaling complex assembles.
1. TCR/CD3 initially excluded form lipid rafts, regions of the plasma
membrane rich in sphingomyelin, glycosphingolipids and cholesterol.
2. Lipid rafts have p56 Lck , associated with CD4 or CD8 co-receptors.
(Figure 11.16)
Figure 11.16 - Lipid Raft associated with CD4 and CD8
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3. Binding of antigen to TCR causes co-receptor to associate, drawing

CD3 complex into the lipid raft. (Figure 11.17)
Figure 11.17 - Formation of the Immunological Synapse
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4. Cell adhesion molecules associate (integrins bound to IgCAMs) - CD45

to CD 22 and reorganize into bulls-eye. (Figure 11.18)
Figure 11.18 - Mature Synapse: Bulls-Eye Lipid Raft Formation
C. Second messenger(s) generated. (Figure 11.19)

1. p56 Lck phosphorylates the ITAMs, providing a docking site for ZAP 70.
2. ZAP-70 (zeta associated protein) gets phosphorylated by p56Lck and
activated.
3. This kicks off a variety of pathways.
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Figure 11.19 - ZAP-70 Pathway
D. Proteins activity regulated by the addition (kinase) or removal (phosphatase)

of phosphates.
1. ZAP-70 phosphorylates a variety of molecules.
2. Recruits mediators to complex.
3. Activates phospholipase C.
4. Activates a G protein
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E. Signals amplified by enzymes cascades. (Figure 11.20)

1. DAG-IP3 via hydrolysis of membrane phospholipid by phospholipase C,
causes ER to release Ca2+.
2. Ca2+ release activates NFAT (T-cell specific transcription factor) via
pathways involving calmodulin and calneurin.
3. DAG activates the transcription factor NF-B.
4. Activation of Ras, the G protein.
Figure 11.20 - DAG-IP3 Pathway
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F. Transcription factors alter genes expression.

1. NFAT and NF-kB, which turn on cytokine genes, including IL-2:
up-regulates the innate inflammatory response as well
2. Transcription factors activated by MAP kinases turn on genes that
trigger cell division.
G. Process requires co-stimulation (Figure 11.21, next page)
1. Signal 1 - Binding of antigen to the CD3 complex is necessary, but not
sufficient,
2. Signal 2 A non-specific co signal is also required.
3. Signal 2 comes mostly from the B7 on the antigen presenting cell
binding to the CD28 on the T cell. CD28-B7 co-stimulation initiates a
primary response.
4. Within 48 hours the cell will leave G0, enlarge, and begin cell division,
begin transcribing the message for both IL2 and the chain of high
affinity version of its receptor and enter into a positive feedback loop.
5. However, binding of B7 to CTLA-4, a similar immunoglobulin superfamily
protein, will downregulate T-cell development. Two slightly different
forms of B7 are specific for each molecule,
6. Resting T-cells initially have no CTLA-4, but begin to produce it during
activation.
7. Clonal anergy: If the APC doesnt have B7 to bind to CD28, it turns it off.
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Figure 11.21 - Co-stimulatory interactions
References:
On Gleevec: www.msnbc.com/news/571486.asp
On TH 17; http://ndt.oxfordjournals.org/cgi/content/full/23/3/816
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Lecture 12 (L12)
Cytokines and Signaling
If all the matter in the universe except the nematodes were
swept away, our world would still be dimly recognizable, and if,
as disembodied spirits, we could then investigate it, we should
find its mountains, hills, vales, rivers, lakes and oceans
represented by a thin film of nematodes. The location of towns
would be decipherable, since for every massing of human beings
there would be a corresponding massing of certain nematodes.
Trees would still stand in ghostly rows representing our streets
and highways. The location of various plants and animals would
still be decipherable, and, had we sufficient knowledge, in many
cases even their species could be determined by their erstwhile
nematode parasites. - N. A. Cobb, 1914, Yearbook of the United
States Department of Agriculture, page 472.
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I.
Information Transfer
A. Cell to Cell Contact
1. TH cells are activated by APCs, using antigen presented on MHC II.
2. B cells, once activated by antigen, contact T cells (the immune
synapse) for information exchange.
3. Direct contact with infected or malignant cells activates TC cells.
4. Pathogens contacting macrophages trigger an inflammatory response
via toll-like receptors.
B. Ways to transfer information
1. juxtacrine- touching
2. paracrine- local
3. hormone- at a distance, via circulation
C. Paracrine Signaling: naming conventions
1. Cytokines (refers to cell to cell) or Interleukins (IL refers to white cell to
white cell)
a. secreted from one leukocyte and act on another, although they
often affect other cells as well.
b. numbered 1 through ?
c. Some cytokines (especially those identified early on before
scientists developed a theoretical framework to discuss their
function) have common names, for example:
i.
ii.
Interferon (IFN) Interferon is a type of interleukin, and

more generally, a type of cytokine
tumor necrosis factor (TNF) identified because it helped
in getting rid of malignant cells. Turns out to be a
powerful, general immune up-regulator
2. Chemokines
a. Signal movement of cells
b. Attach to seven-span receptors
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c. Activate G-proteins
II.
Cytokine Receptors and Ligands

Figure 12.1 - Cytokine Receptors
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A. Tumor Necrosis Factor Receptor (TNF is one ligand.)

Figure 12.2 - Tumor Necrosis Factor
Receptor
1. Receptor (TNFR or CD120) has four repeated domains of beta-pleated

sheets stabilized by cysteines. However these seem to be different from
Ig domains. (Figure 12.2)
2. Clusters in trimer upon binding TNFs. (Figure 12.3)
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Figure 12.3 - Tumor Necrosis Factor Receptor- Crystal Structure
3. Most TNFs are juxtacrine factors produced by monocytes and

macrophages (Figure 12.4)
4. Signal up-regulates immune responses and specifically results in
appetite suppression and, long term, cachexia (wasting).
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Figure 12.4 - TNF Receptor and Signaling Molecules
B. Immunoglobulin Superfamily
1. pleated sheets (bread and butter switch-backs) characteristic of
family (Figure 12.5)
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Figure 12.5 - Beta pleated sheets in the Immunoglobulin Superfamily
Figure 12.6 - Immunoglobulin Superfamily Crystal Structure
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2. Different version bind structurally different ligands

a. c-Kit (CD117) binds stem cell factor (also called c-kit ligand or
steel)
b. IL-1 general inflammatory up-regulator (Figure 12.7)
Figure 12.7 - IL-1 Structure
C. Hematopoietin (Class I) Includes the IL- receptor, one of our major

structural examples. (Figure 12.8)
1. largest class of immune-related signal systems. Dozens of specific
versions, including receptors for IL-4 and 5 (important in TH 2 response),
G-CSF (important in any defensive response) and receptors for peptide
hormones that are not even part of the immune response
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Figure 12.8 - Hematopoietin - Class 1 Receptor
2. function as heterodimers or trimmers. Generally no one part functions

optimally in any role without being bound to the others.
3. have four conserved cysteines at the N terminal domain (CCCC motif), a
conserved sequence of tryptophan-serine-whatever-tryptophan serine
(WSXWS motif), and a C terminal cytoplasmic signaling tail in both
dimers. If there is a third peptide, it aids ligand binding, and does not
have these features. (Figure 12.9)
Figure 12.9 - Hematopoietin - Crystal Structure
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4. Recruit cytosolic tyrosine kinases

D. Interferon (Class II)
1. includes receptor for IFNg, a major inflammatory signal. Also IFN and
, IL-12 and a dozen or so more. Well look at this signaling pathway in
detail. (Figure 12.10)
Figure 12.10 - Interferon - Class II Receptor
2. also have four conserved cysteines, distributed in N terminal domain

(CCCC motif) and cytoplasmic signal domain, but not the WSXWS
motif.(Figure 12.10,11)
Figure 12.11 - Interferon - Class II Receptor - Crystal Structure
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3. function as heterodimers (more rarely trimers)

4. Recruit cytosolic tyrosine kinases and signal via JAK-STAT pathway, a
very common sequence in all forms of cell signaling, not just immunity.
E. IL-17 Receptor Family (Figure 12.12)
1. Most recently identified, with many structural variations.
Figure 12.12 - IL-17 Receptor
2. Units have elements similar to TLRs as well as a fibronectin-like domain.

3. Variations have hetero and homodimers and trimers.
4. IL-17 paracrine factor is a small ovoid dimer with beta pleated sheets.
(Figure 12.13)
Figure 12.13 - IL-7 Crystal Structure
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5. Specifically identified as secreted by a class of TH cells, the TH 17,

which operate at the border of innate and adaptive immunity to trigger a
defensive response.
6. Works in boundary defense and against extracellular pathogens
F. Chemokine Receptor
1. 7-span receptor ((Figure 12.14)
Figure 12.14 - Chemokine Receptor
2. associate with G proteins

3. binds chemokines, for example IL-8, which are smallish (90 to 130
amino acids) proteins with alpha helix and beta pleated sheets stabilized
with conserved cysteines. (Figure 12.15)
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Figure 12.15 - Chemokine Receptor Crystal Structure
4. important in directing traffic during inflammation

5. Cross reception common: a given chemokine may bind more than one
receptor and a specific receptor may bind more than on chemokine
III.
Specific Examples of Signaling Pathways

A. IFN Signaling via the JAK-STAT Pathway. This is used as a TH 1 signal to both
B cells and macrophages, coordinating the innate and adaptive response to
acute infection. While it uses a similar signal transduction to the pathway
above, the receptor and specific internal pathway differs.
1. IFN is an interferon peptide that can signal
a. cells to resist viral infection
b. B cells to class switch to some of the g heavy chains
(complement-activating IgG3 and IgG1)
c. Up-regulation of phagocytes and activation of ability of
macrophages to present antigen
d. specific development of TH cells to the TH 1 response.
2. The receptor is composed of and subunits. JAK kinase bound to
the cytoplasmic part with kinase activity on the other side, and
subunits binding JAK 1 and 2 respectively
3. INF dimer binds outside the cell the and subunits, causing them
to associate.
4. The JAKs phosphorylate each others cytoplasmic receptor tyrosines,
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without recruiting an additional kinase. (Figure 12.16)

5. The receptor now provides a docking site for a STAT protein (potential
transcription factor).
6. JAKs then phosphorylate the STAT, activating it.
7. STATs leave the receptor and dimerize.
8. Dimer gets a serine phosphorylated (different cytoplasmic enzyme).
9. STAT enter nucleus and binds to promoters of interferon promoted
genes.
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Figure 12.16 - JAK-STAT Pathway
B. IL-2 : fundamental to adaptive signaling

Check out this animation showing stimulation pathway
http://www.rndsystems.com/Pathway.aspx?p=15457&r=15436
1. Structure:
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The complete activated receptor is a heterotrimer: (Figure 12.17)

a. subunit (CD 25) structure unrelated to other subunits, aids
binding, but doesnt bind IL-2 very well by itself.
b. subunit signal transduction, CCCC and WSXWS motifs. No
binding by itself.
c. subunit- signal transduction, CCCC and WSXWS motifs. No
binding by itself, but with b subunit can do some so-so binding.
Figure 12.17 - IL-2 Receptor and Subunits
2. Binding
a. Resting T cells (except for Treg) do not have the functioning
chain (CD 25)
b. Activation causes synthesis of the chain (one of the early
genes up-regulated by the immediate genes)
c. chain is constitutively expressed, but cannot bind and signal
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on its own.
d. and chains together can bind and signal, but the
intermediate affinity usually doesn't leads to a productive signal.
Found in NK cells and resting T cells.
e. All three together and the overall receptor has high affinity and
active signaling. Found in active TH , TC , and B cells.
3. JAK-STAT initiates activation of several internal signaling pathways
a. JAK (Janus- or Just Another Kinase) allows and chains to
phosphorylate each other. (Figure 12.18)
Figure 12.18 - IL-2 Receptor and JAK
b. STATs (Signal Transducer and Activation of Transcription) bind,

receive phosphates, activate and turn on genes. Several different
STATs get activated, which allows the signal to up-regulate a
variety of different immune genes. (Figure 12.19)
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Figure 12.19 - IL-2 Receptor and JAK-STAT
c. Be fruitful and multiply signals- IL-2 leads to clonal expansion of

lymphocytes by promoting cell division via the Ras pathway and
blocking apoptosis.
IV.
Cytokine Signaling by CD4+ T Cells: T H 1, T H 17, T H 2

A. TH subsets T cells with CD4 (TC and T set aside before decision to use
CD4)
1. TH 1 - cell mediated, activates for acute attack on active bacterial and
viral infections and cancers. Signals B cells to make opsonizing
(complement activating) IgG3 and IgG1 antibodies. May promote
excessive inflammation and tissue injury (scorched earth- sort of life
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Russia in WWII.).
2. TH 2 humoral defends body against entrenched enemies, especially
helminth parasites. Activates eosinophils and mast cells. Signals B cells
to make lots of IgM, non-opsonizing IgG and IgE. T cells secrete IL-4
and IL-5. Inappropriate response produces allergy.
3. TH 17- inflammatory, and seems to bridge adaptive and innate responses.
Secrete TNF, but not IFNg. Seriously hostile war against something not
inside your borders (sort of like US and England in WWII). However,
when it goes astray it CAN trigger autoimmune reactions
a. Cells secrete IL-17 as well as respond to it.
b. Mobilizes neutrophils and prompts secretion of antimicrobial
factors by epithelia.
c. The pathway triggered in the cells receiving IL-17 is not the
JAK-STAT, but rather a completely different pathway resembling
the one used by the innate system TLRs. Both activate the
transcription factor NF-B.
d. Interestingly T cells and NKT cells, which both respond to
lipids, protect epithelia, bridge the innate and adaptive systems,
secrete IL-17s.
4. Treg suppress immune responses
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B. Summary Table
Table 12.1 - TH (CD4 Positive) Cells Compared
THl-mediated Response
(cellular)
Acute infection (viral,
bacterial, and intracellular
pathogens) and inflammation
TH2-mediated
Response (humoral)
Chronic or parasitic
infection, allergy
Treg-mediated Response
(inhibitory)
Modulation of
inflammatory responses,
prevent allergy
TH17- mediated
(epithelial)
Acute infection
(extracellular bacteria
and fungal), opposes Treg
Moderately strong antibody

response, especially IgG I
and 3, which activates
complement and phagocytes
Very strong antibody

response, especially
IgE, also IgM and noncomplement activating
IgG4
Stimulates
development and
activity of mast cells
and eosinophils
Can attack B cells,

reducing antibody
production
Inflammatory and
humoral response (lgA)
while blocking CD-8
cells
Stimulates development and

activity ofTc cells and
macrophages (up-regulates
B7)
secrete IFN1, IL2,
andTNFa
Activated by macrophages
and dendritic cells. These
uses PAMPs to recognize
pathogens and then signal
with ILs
THl development promoted
by IL-3, GM-CSF, IL-12,18, IFNr from macrophages
Excess responseinflammatory damage to
nonnal tissue
(autoimmunity)
Activates transcription
factor: winged helix
Foxp3, leading to
production of inunune
suppressing Treg ceEls
Secrete TNFa, IL-6 and
TH2 cells secrete !Ls-3, Secrete IL-l 0 and
-4, -5, -6, -10, -13
IL-17
Activated by mast
Activated by resting
Activated by dendritic
cells with activated
cells, basophils, and
thymus epithelial cells
NK cells
and resting dendritic
bacterial PAMPS
cells. Blocked by TLR
(inflammatory) pathways
Promoted by IL-2:
TH2 development
Promoted by TGFJI with
promoted by IL-3,
express CD25 (IL-2
IL-6
GM-CSF, IL-4
receptor).promoted by
blocked by retinoic acid
TGFJI and retinoic acid,
blocked by IL-6
Excess responseExcess response Autoimmunity (although
allergy
survival of tumors or
often different from THI
infected cells
diseases)
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V.
Determining Development and Function of T H Subsets

A. Review Figure (Figure 12.20)
Figure 12.20 - T-Cell Differentiation
1. Notice that all of the defensive sets have up-regulated some form of
STAT transcription factor, although each has a different version.
2. This is a reminder that variation of the JAK-STAT pathway will have
different receptor-ligand triggers and different STAT targets.
3. T regulatory cells have a different role and different controls (Figure
12.21)
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Figure 12.21 - T-Reg Cells
a. CD4+ and CD25 + from the get-go, meaning that they can act
faster and to lower levels of IL-2 than the defensive TH cell types.
b. Also associated with class switch to non-inflammatory IgA, which
is produced copiously whether youre experiencing an infection
or not (Figure 12.22)
Figure 12.22 - IgA Dimers
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c. Down-regulating Treg function may prove an important

improvement in cancer therapy.
d. Up- regulating Treg cell production may help in treating allergies
and autoimmune responses
B. Summary of types of interactions
1. Pleiotropy - one cytokine may act on several different cell types. For
example IL-4 coordinates a TH 2 response by acting on B cells and mast
cells, promoting cell division in each type. IL-3 and GM-CSF both
promote development of TH 1 and TH 2 cells (Figure 12.23)
Figure 12.23 - Pleiotropy
2. Redundancy - more than one cytokine will produce the same effect. For
example, ILs -2, -4 and -5 each, by themselves, induces proliferation in
B cells. Different chemokine signals may cross-react with a variety of
receptors, and any one will up-regulate inflammation in a variety of cells.
(Figure 12.24)
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Figure 12.24 - Redundancy
3. Synergy - when combinations of stimulus have effects neither has by

itself. For example IL-4 and IL-5 together will cause B cells to class
switch and make IgE immunoglobulins. (Figure 12.25)
Figure 12.25 - Synergy
4. Antagonism - one cytokine blocks the effects of another. For example,

IFNg (a TH 1 promoter) blocks the ability of IL-4 (a TH 2 promoter) to
induce class switching to IgE. Retinoic acid and IL-6 mutually oppose
each other in the decision to go Treg or T17. (Figure 12.26)
Figure 12.26 - Antagonism

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5. Cascade induction - one cytokine stimulates other cells to produce a

different cytokine, which stimulates yet more cells, etc. We saw that
above with the macrophage IL-12 TH l IFN- activated
macrophage pathway of development. Ongoing macrophage TH cell
conversations are critical to determining the specific tweaking of the
immune response. (Figure 12.27)
Figure 12.27 - Cascade
VI.
Clinical Applications
A. Leprosy
1. Caused by Mycobacterium leprae, an intracellular parasite.
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2. Not particularly contagious; most people's macrophages can fend it off.

Rather benign really: can't migrate on its own, doesn't produce nasty
toxins.
3. In some people, however, it survives in within phagosomes of the
macrophages. Depending on how the immune system responds, the
disease can take one of two courses, or, for that matter, intermediate
versions.
4. Generally treatable by antibiotics (rifampicin) and (more recently)
thalidomide.
5. Tuberculoid form - more typical course, resulting from a TH 1 response
with delayed hypersensitivity.
a. The host's cell-mediated response leads to formation of
granulomas, walled-off regions of infected macrophages.
b. Most of the bacteria are destroyed by this process.
c. Skin and nerves are damaged slowly over a period of time.
d. Patients survive over the long term, but with some nerve damage
and possible deformity
6. Lepromatous form TH 2 response - this is the nasty version with the bad
reputation
a. humoral antibodies are released, often at excessive levels.
b. The bacteria continue to thrive, hiding in the macrophages.
c. Infection disseminates widely beneath the skin and throughout
the body; destroys bone cartilage and nerves, and can kill you.
Even in this form the progress of the disease is relatively slow.
d. Bacteria tend to prefer cooler temperatures, which is why
infected individuals tend to lose fingers, toes, ears and noses.
More evidence that fever is helpful in fighting disease.
B. Bacterial Septic Shock (as opposed to toxic shock, cause by superantigens)
1. Caused by overproduction of cytokines (general problem discussed
previously under superantigens).
2. Gram negative bacteria - recall these are the ones that have a second
membrane external to the plasma membrane and thin cell wall and
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usually a capsule (mostly acidic polysaccharides with some protein) as

well.
3. Shock generally refers to a dangerous drop in blood pressure. In the
case of bacterial septic shock, there is also high fever, diarrhea and
possible blood clotting in various organs. Also, the drop in blood
pressure stems primarily from a vasodilatation of peripheral blood
vessels (although dehydration doesn't help).
4. The endotoxins from the outer membrane and wall overstimulate the
macrophages via the toll-like receptors.
5. The macrophages release too much of the cytokines IL-1 and TNF-a.
Mechanism similar to toxic shock.
6. TNF- all by itself can induce toxic shock in mice.
7. Possible improvements in therapy may involve monoclonal antibodies to
these cytokines.
C. Chagas' Disease
1. Caused by Trypanosoma cruzi, a member of the trypanosomes which
cause a number of other nasty diseases, including African sleeping
sickness and related Leishmania donovani.
2. Unicellular eukaryotes - protozoans often using various insects as
vectors to transfer agents back and forth from people and cattle and
other domesticated mammals.
3. The group is generally good at changing surface antigen - one member
has over 100 genes coding for surface proteins.
4. T. cruzi is a particularly nasty version, living in the guts of insects that
live in adobe houses. The insects come out, suck blood from the
residents and void feces, which allow the trypanosomes to enter through
the break in the skin.
5. Once in the body they multiply and produce an unknown diffusible
substance that blocks the subunit of the IL-2 receptor.
6. The suppression of IL-2 signaling prevents the TH cells from multiplying
and signaling to the rest of the system.
7. The parasites then go on to destroy a variety of tissues, including heart
muscle, skeletal muscle and nerves. If they're lucky, people die of heart
failure. If they're unlucky, they die of peristaltic failure due to damage of
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the nerves coordinating the movement of gut muscles.

8. Treatment:
a. Hard to treat - drugs are dangerous and uncertain.
b. Heart transplants are not an option, as the immune suppressants
allow the parasites to kill the patient.
c. A disease of poverty, preventable by better housing.
References:
Gaffen, Sarah. (2009) Structure and signaling in the IL-17 receptor family. Nature Reviews
Immunology 9:556-567 (August)
Chagas' Disease:
Panisin, Claire (1998) The parasite of the poor. Vital Signs. Discover (December), page 38.
Hansen's Disease:
Norman, Robert A. (2002) Numbness of the Arm. Vital Signs. Discover (February), page 30.
http://www.oneworldhealth.org/diseases/chagas.php
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Lecture 13 (L13)
Inflammation
The fact that there are unsolved problems within the framework
of an existing theory does not of itself imply that the theory must
be thrown away, or replaced by another; unsolved problems are
the essence of science, the means by which theories are refined.
John Maddox in Nature, 1986, vol. 320. April 17, 1986.
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I.
What, Another Metaphor?!?

A. Full Red Alert
1. When the body perceives a threat, it directs defenses to meet that
threat. This means that resources used for growth, physical activity and
even higher mental function may be diverted, depending of the severity
of the threat. This can save your life.
2. However, if you are on extended alert or full lockdown) for a long period
of time, things dont get done that need to get done. This is comparable
to the dangers of chronic inflammation.
B. Roman Insights. The military surgeons of the Roman were the best in the world
of their time. Soldiers retired with a full pension at age 40 and the majority lived
to collect it, thanks in no small part to the care they received if they were
injured. The following are specifically attributed to Celsus and Galen:
1. Hallmarks of the inflammatory response (Figure 13.1):
a.
b.
c.
d.
e.
rubor (redness)
tumor (swelling)
calor (heat at tissue site)
dolor (pain)
functio laesa - loss of function
2. Cause by vascular changes

a.
b.
c.
d.
endothelium leaks
edema
positive feedback loop of cell signaling
further changes in cell adhesion molecules on both cells and
endothelium.
Figure 13.1 - Visible inflammation
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3. Medical Background
a. Long-time recognition that fever accompanies infection. Lower
fever, lower infection?
b. Bleeding lowers fever.
c. Giving blood is associated with lower heart disease (as are
statins, aspirin and fish oils, all of which are anti-inflammatory
and all of which tend to increase bleeding).
II.
Signals This sections reprises and extends material we introduced in Part 1

Lecture 3
A. Pattern Recognition
1. PAMPS (pattern-associated molecular proteins) - generally used to
describe a variety of molecules used to identify pathogens. The first
group identified was the toll-like receptors, but many more types exist.
(Figure 13.2)
2. DAMPS (death or damage-associated molecular proteins) - molecules
that are normal cell components but should be inside cytoplasmic or
nuclear proteins of membrane proteins belonging in the inner leaflet.
Figure 13.2 - Pattern Recognition Locations and Targets
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B. Response:
1. Activate complement, which in turn releases inflammatory signals
(anaphylatoxins)
2. Attracts neutrophils, macrophages and mast cells.
C. Inflammatory Chemokines
1. Smallish (90 to 130 amino acids), with conserved cysteines
2. Receptors are seven-span membrane proteins. Cross reception
common: a given chemokine may bind more than one receptor and a
specific receptor may bind more than on chemokine
3. Binding activates G-proteins (Figure 13.3a-b)
a. large (heterotrimeric with subunits) kicks off cytoskeletal
changes quick change in adhesiveness
b. Switches from active to inactive as it binds GTP and then
hydrolyzes it to GDP
4. G-proteins kick of several internal cascades:
a. hydrolyzes membrane phospholipids to IP3 and DAG
b. lets Ca2+ loose in the cytoplasm, polymerizing actin and
promoting movement
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c. kicks off Ras cascade that leads to activation of transcription

factors.
Figure 13.3a - G protein coupled
receptor structure
Figure 13.3b - GPCR mechanism
*Solely for your information - no need to memorize this
D. The High-endothelial Venules (Figure 13.4)

1. found at the end of capillaries in lymph nodes, Peyr's patches, and
tonsils (not spleen).
2. The endothelial lining here is composed of cells that do not look like
flattened paving stones, but are thicker.
3. Most lymph cells that extravasate attach to these specific cells.
4. The HEVs specifically attract lymphocytes and do NOT attract
neutrophils.
5. The HEV develops in response to foreign antigen exposure: you don't
see this in mice raised in a germ-free environment and you don't see
this in tissues that have the circulation to them blocked to that antigen
does not enter.
6. The unique morphology is associated with the induced expression of
specific selectins, mucin-like, and Ig CAMs, and they specifically allow
different classes of lymphocytes to home to different organs and regions
of the body. Examples in the next section.
Figure 13.4 - High-Endothelial Venules
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III.
Extravasation
A. Cell Adhesion Molecules- Selectins and Mucins
1. Selectin Family, representation and ribbon diagram of lectin domain.
(Figure 13.5)
a. membrane glycoprotein with lectin at amino terminus that binds
carbohydrates. Compare with complement lectin pathway
b. specific for sialic acid (mucins have a lot of these)
c. comes is L (Leukocyte), E and P (endothelium) versions
d. initiate initial sticking of leukocytes to the endothelial wall
Figure 13.5 - Selectin structure, with enlarged blue region at the right
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2. Mucin-Like Family (Figure 13.6)

a. protein part rich in serine and threonine (OH-containing R
groups)
b. LOTS of carbohydrate linked to these OHs.
c. Carbohydrates have lots of sialic acid, interact with selectins
d. The mucin-like versions of the endothelium interact with the
selectins on the leukocytes and vice-versa.
Figure 13.6 - Mucin structure
B. Cell Adhesion Molecules- Igs and Integrins

1. I-CAM - Immunoglobulin Super-Family (Figure 13.7)
a.
b.
c.
d.
endothelium has several versions

mucosa has another, which also has a mucin-like domain
bind to integrin on leukocytes
inflammation increases their expression
Figure 13.7 - Immunoglobulin superfamily receptor structure
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2. Integrin Family varied binding partners (Figure 13.8)

a. Heterodimer ( and chains)
b. Expressed in leukocytes
c. Different integrins bind to different immunoglobulin CAMs (cell
specificity)
d. Can also bind to fibronectin
e. Cytoplasmic portion can interact with cytoskeleton and signaling
proteins such as the tyrosine kinases, Fyn and Lck.
Figure 13.8 - Integrin binding to extracellular matrix (orange)
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C. Sequence (Figure 13.9)
Figure 13.9a - Neutrophil!
Figure 13.9b - Neutrophil Extravasation
Inner Life of the cell Narrated: http://multimedia.mcb.harvard.edu/innerHi.swf

1. rolling - attachment by low affinity P selectins (yellow) on the
endothelium to mucin-like molecules on the neutrophil (purple). Because
the connections are loose, they tend to break and the neutrophils kind of
roll along, attaching to one endothelial cell after the next as it is swept
along by the flow of blood.
2. activation the stick and release from the rolling response along the
endothelium triggers chemokine (IL-8) release by the endothelium. The
neutrophils activate G-protein cytoplasmic pathways via the 7-span
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receptor.
a. Video shows lipid raft. We looked at these in connection with T
cell activation, but they also form membrane compartment to
organize other interactions as well. Will return to this.
b. The mechanical pulling and tugging causes the endothelium to
release a chemokines.
3. arrest and adhesion - The G-proteins activate integrins, changing their
conformation, and increasing their affinity for Ig-related CAMs. This
nails down the neutrophils. Neutrophils cannot bind to non-inflamed
endothelium.
a. The chemokine binds to a 7-span chemokine receptor.
b. This receptor activates a G protein and sets off an internal
signaling cascade.
c. The integrins change conformation, enter the lipid rafts, deploy
and stick Ig tightly to the Ig CAMs of the endothelium
d. Some of the first proteins affected are those that associated with
the inner side of the plasma membrane and connect to the
cytoskeleton. (Figure 13.9c)
Figure 13.9c - Neutrophil Extravasation Interactions
4. transendothelial migration - the arrested neutrophil then finds the gap

between two adjacent endothelial cells and squeezes through it.
a. The cytoskeletal changes lead to the neutrophils changing shape
and moving by amoeboid motion.
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b. The neutrophil forms a leading wedge and ootches through a gap

between the endothelial cells (recall that inflammation makes the
endothelium somewhat leaky).
D. Lymphocyte Extravasation, Trafficking, and Homing (Figure 13.10)
Figure 13.10 - Lymphocyte extravasation and homing
While the processes that allow the lymphocyte to leave the blood vessel
(rolling,activation, arrest, and migration) are similar, the mechanisms that
control exactly where they lymphocyte will under go extravasation are more
complex and involve specific homing signals.
1. Nave lymphocytes
Lymphocytes migrate in and out of secondary lymph organs where they
contact presenting sentinel dendritic cells. Recall that the chance of any
one lymphocyte recognizing any one antigen are miniscule (1 in 105),
so the cells run repeated loops through secondary organs and in and out
of circulation, essentially kissing frog after frog and looking for the rare
prince.
a. T cells, for example are attracted by inflammatory signals and
cruise into sites of active infection, where they may be activated
by antigen presented by innate cells fighting the infection.
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b. T cells may also be activated by dendritic cells in the lymph

nodes in spleen. In that case the dendritic cells also
communicate the site of the infection by activating specific T-cell
surface receptors for cytokines and CAMs characteristic of
particular tissues. For example retinal/retinoic acid is produced
by the gut and only gut-homing T cells have receptors for it. For
skin, its vitamin D.
2. Effector and Memory Cells
a. Effector cells tend to head to the site of infection.
b. Memory cells tend to head to the tissues that initially had the
infection.
c. Cells are directed by a combination of cytokines and cell surface
receptors (above) but can also be retrained, which is necessary
if an infection spreads.
d. T cells, in particular, are programmed to home to a specific site
because they express a specific combination of cytokine and
CAM receptors.
IV.
Inflammatory Mediators
A. Clotting (Figure 13.11)
Figure 13.11 - Clotting
1. Clotting both up-regulates, and is up-regulated by, inflammation. This is

why chronic inflammation can lead to strokes and cardiac infarcts.
2. Initiated by platelets and RBCs contacting damages surfaces (collagen
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fibrils) and breaking open.

3. This releases and activates Hageman factor, with several consequences.
4. The clotting cascade ends with prothrombin activation, which clips
fibrinogen into fibrin and peptides.
5. The fibrin forms the clot.
6. The released peptides promote inflammation.
7. Hagemans factor also activates the fibrinolytic system, which dismantles
the clot using the enzyme plasmin.
8. Plasmin also produces inflammatory peptides and activates complement
B. Kinins
Figure 13.12 - Bradykinin
1. Specifically leads to pain, in addition to increased endothelial

permeability and smooth muscle contraction.
2. Cascade initiated by Hageman factor.
3. Also involve proteolysis.
4. Leads to clipping of kininogen to bradykinin. (ACE inhibitors) (Figure
13.12)
C. Lipid derivatives- these often depend of activations of specific enzymes.
1. Membrane phospholipids play a structural role but also serve as the raw
material for generating a lot of signaling compounds.
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2. Phospholipids have two fatty acids linked to glycerol and the glycerol
links up via a phosphate to a polar group (ethanoleamine or inositol for
example). (Figure 13.13a)
3. Depending on what you begin with and where you cut it, and how you
modify the results, you can get an array of different potent signaling
molecules.
4. Arachidonic acid- derived from one to the fatty acids. (Figure 13.13b)
5. Act on it with enzymes called cyclooxygenases (COX2 inhibitors) and
you can get
a. thromboxane (Figure 13.13d)
b. prostaglandins
6. Act on it with lipoxygenase and get a variety of leukotrienes. (Figure
13.13e)
7. In a separate origin pathway, phospholipids can provide the raw material
for Lyso-PAF, which is converted to PAF (Platelet Activating Factor,
Figure 13.13f)
Figure 13.13a - Phospholipid
Figure 13.13b - Arachidonic acid
Figure 13.13c - Phosphatidylinositol

4,5-bisphosphate
Figure 13.13d - Thromboxane
Figure 13.13e - Leukotriene
Figure 13.13f - Platelet activating factor
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V.
Clinical Considerations- All of this signaling and enzymatic activity provides a lot
of opportunity of diagnosis, monitoring and treating disease.
A. Disease
1. Systemic Acute-Phase Response defense from something like flu or
bacterial disease acts more globally- if your toe infection turns into
sepsis, for example.
a. Compromised by malnutrition or starvation
b. IL-6, TNF, IFN
c. Act on the hypothalamus to induce fever and CRP release, which
in turn causes the pituitary to release of ACTH which causes the
adrenals to produce steroids (cortisol etc.).
d. The liver responds with the release of acute-phase proteins (a
general term) - activates complement.
e. increased leukocyte production and activation, especially
neutrophils
2. Chronic Inflammatory Response IFN and TNFa, transcription factor
NF-B
a. persistent infection (gum disease), autoimmune response,
cancer, chronic injury.
b. old age, obesity, diet high in trans-fats, triglycerides (diet high in
sugar) wrong gut flora, diabetes, sleep disorders.
c. contributes to disease processes, including cancer and
cardiovascular diseases
d. Increased clotting
e. fibrosis - a type of scar tissue formed when chronic inflammation
leads to excess production of fibroblasts and collagen
f. granuloma (also called tubercle) - an attempt to wall off the
problem with macrophages and specialized TH cells.
B. Anti-inflammatories - Braking the System- Hey, that drug sound familiar!
1. Antibodies used to block leukocyte extravasation:
a. antibodies to integrins
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b. antibodies to CAMS
2. Corticosteroids
a. Pregnisone, cortisone, dihydrocortisone
b. High doses block adrenals and many immune functions
c. Side effects mood swings, edema, glaucoma, increased
susceptibility to infections
3. NSAIDs
a. Aspirin (acetylsalicylic acid), Advil (ibuprofen) Aleve (naproxin)
are OTC
b. prevent prostaglandin production from arachidonic acid.
Celebrex specifically blocks COX-2
c. NOT Tylenol/acetaminophen- acts on brain to raise pain
threshold and lower hypothalamic thermostat).
4. Cooling - Interferes with inflammatory process (recall that fever is often
a helpful part of the response to infection)
a. used to prevent excessive tissue damage following injury or
stroke- exercise raises levels of CRP and other inflammatory
indicators
b. used during surgery
c. used to cut down on symptoms of autoimmune disease multiple
sclerosis chilling via hands to allow athletic competition.
References:
Inner Life of the cell Narrated: http://multimedia.mcb.harvard.edu/innerHi.swf
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Lecture 14 (L14)
Cellular Immunity
The future is already here. It is just not evenly distributed. William Gibson
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I.
Division of Labor. While the primary agents of this form of cell-mediated (TH 1)
immunity are the TC and NK cells, other cells make important contributions to the
process.
A. Lymphoid cells developing in the thymus rearrange TCR genes
1. TH - signal to tailor and coordinate responses. (Figure 14.1)
Figure 14.1: TH Cell
Figure 14.2: TC Cell
2. TC attack and kill cells displaying specific antigens by disrupting

membrane and lysing cell. (Figure 14.2)
3. chain T cells - patrol epithelia, attack and kill pathogens and
rogue-self using same mechanisms as NK and TC cells, signal. Some
authors refer to these as primitive because their function seems less
specialized. (Figure 14.3)
Figure 14.3: Types of TCRs
Figure 14.4: NKT Cell Recognition
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4. NKT- innate recognition iNKTcells (type 1) respond to lipid antigens,

especially those characteristic of bacteria. Other NKT cells (type 2)
recognize a wider array of lipids and have more variation in the
receptor. (Figure 14.4)
a. Type 1 (iNKT) cells produce a semi-invariant ab TCR. They
always use the same a option, and use one of a limited number
of b options. There will still be some variation at the regions
where you put them together.
b. These cells develop in the thymus, are CD4+ (or sometime DN)
and bind to cells presenting lipids on CD1d. This is a typical TH
function.
c. has CD 16 (the Fc receptor a typical NK function)
d. requires TLR recognition of bacterial responds to activate
e. attacks bacterial cells with inflammatory cytokine signaling,
including IL-17, IFN
f. attacks tumors with specific lipids, using FAS ligand and
perforins (a typical NK and TC function.)
g. prevents autoimmunity (a Treg and gd function), does not form
memory cells, and seems to be quasi-innate
B. Lymphoid cells developing in the bone marrow do not rearrange TCR genes
1. B cells rearrange immunoglobulin genes, phagocytize, but do not
seem to be a primary killer of pathogens, and do not attack rogue self.
The antibodies that they produce are often important in directing attack
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by other cells. (Figure 14.5)

2. NK (natural killer) - do not rearrange any genes. They attack and kill
cells with suspicious surface displays by disrupting their membranes
and lysing the cells. (Figure 14.6)
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Figure 14.5: B Cell
Figure 14.6: Natural Killer Cell
C. Context
1. The variety of lymphoid cells (Figure 14.7)
Figure 14.7 - Lymphoid Cells in Order of Innate to Most Adapted
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2. Developmental Flow Chart (Figure 14.8)

Figure 14.8 - Immune Cell Development Flow Chart
D. Myeloid Cells
1. macrophages phagocytize, present antigen (Figure 14.9)
2. neutrophils phagocytize, do not present antigen (Figure 14.10)
3. eosinophils coordinate attack on eukaryotic parasites, do not present
antigen, weakly phagocytic (Figure 14.11)
Figure 14.9: Macrophages
Figure 14.10: Neutrophils
Figure 14.11: Eosinophils
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II. Attack of the T C cell (Figure 14.12)

A. Activation
1. The TC cell binds a foreign antigen on MHC I (TC cells that bind self-antigen have
been killed off during negative selection.)
2. CD45R on TC and TH attaches to CD22 (sometimes called CD45L) on target cells.
3. TH cell activates the cell, turning it into a CTL. (Figure 14.13)
4. Cross presentation speeds this process by making it easier for the Tc cells to
encounter the foreign antigen.
5. Activation leads to
a. Cell division
b. Up-regulation of cell adhesion molecules
c. Synthesis of the complete IL-2 receptor
Figure 14.12: Healthy Human T cell
Figure 14.13: Cytotoxic (Tc) T Cell
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Figure 14.14: Presentation Pathways
B. CTLs on the Attack

http://www.youtube.com/watch?v=WPaRKm2-YNY
http://www.youtube.com/watch?v=FWWMOb7z5GI
Figure 14.15: Normal Cell
Figure 14.16: Infected Cell Displaying Viral

Antigen
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1. CTL cell attached to target will up-regulate cell adhesion molecules, forming
conjugate
2. CTL attacks target membrane
a. cytoskeleton rearranges, directing Golgi and storage granules (perforins and
granzymes) to target cell
b. granule contents released by exocytosis (Ca2+ dependent)
c. Perforin monomers undergo conformational change and insert in the target
membrane (sequence homology with C9 of the complement system).
d. Granzymes (serine proteases) enter through pores and activate apoptosis.
(Figure 14.18)
Figure 14.17: Infected Cell Without Class 1

MHC
Figure 14.18: Apoptosis
3. Apoptosis can also be induced by Fas ligand binding to target Fas receptor on the
target cells
a. Ipr mice are mutant for Fas (receptor) production.
b. They laugh at Fas ligand.
c. Cells from mice that are both perforin knock-out AND Fas knock-out cannot be
killed by cytotoxic T cells.
d. Either pathway initiates apoptosis in target cells.
e. Ultimately, each sets off a pathway that activates a cascade of proteolytic
enzymes (caspases).
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Table 14.1: How Mutations Affect Ability to Induce Apoptosis
Infected, but
otherwise
normal, mice
Perforin mutant
Ipr mutant ,
does not make
FAS (the
receptor)
Double mutant
CTLs produce
perforin and FAS
ligand
CTLs produce FAS
ligand, but not
perforin
CTLs produce
perforin and FAS
ligand
All cells respond to

FAS ligand
Infected cells die

of apoptosis
All cells respond to

FAS ligand
Infected cells die

of apoptosis
Cells do not respond

to FAS ligand
Infected cells die

of apoptosis
CTLs produce FAS

ligand, but not
perforin
Cells do not respond

to FAS ligand
Infected cells
survive
4. CTL-dissociation
a. The integrin holding the cells together reverts to its less avid form after 5 to 10
minutes.
b. The CTL undocks and begins looking for another cell to hit.
c. The target cell has already received its death signal.
5. Target cell destruction
a. The target cell undergoes apoptosis.
b. The pieces are scavenged by the macrophages or neutrophils.
III. Natural Killer Cells yet another link between innate and immune systems
Figure 14.19: Natural Killer Cell
Figure 14.20: NK Cell
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A. Characteristics (Figure 14.19)

1. Large cells with lots of granular inclusions.
2. Lymphoid, sharing a common progenitor and surface markers with T cells,
including the use of signaling peptides similar to those in the complete CD3
complex.
3. Develop in the bone marrow and do not rearrange genes.
4. Also have TLRs and inflammatory cytokine receptors typical of those of
macrophages and neutrophils.
5. Do express the receptor of FC of IgG (CD 16) and the IL-2 receptor. This allows
them to recognize and attack antibody-coated cells during ADCC.
6. Have MHC I receptor, but it is inhibitory, and not used to recognize antigen, but
rather viral down-regulation.
7. Pre-programmed to attack cells with surface characteristics that are likely
indicators of trouble.
B. Mechanisms of Killing
http://www.youtube.com/watch?v=84MlWh1XN0Q
http://www.youtube.com/watch?v=NdxAIyn73sU
In this one, the NK cell is directed by an antibody on the surface of the cancer cell.
1. Granules containing perforins and granzymes directionally released at target cell.
2. Constitutively cytotoxic; do not require activation.
3. Do not require antigen or MHC recognition.
4. Adhesion to target triggers release.
C. Control of Killing
Because these cells run around in the ready, there are opposing controls that act to
either unleash the NKs or rein them in. (Figure 14.21)
1. Class I MHC receptor coupled to AR (inhibitory) receptor. Enough MHC and the
AR will inhibit killing no matter what the other signals.
2. However virus that down-regulate MHC I production may fool TC cells, but they will
activate NK cells.
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3. Stress receptor. There are a variety of stress indications displayed by sick cells,
and a variety of receptors used by NK cells to sense them. The interaction
promotes attack.
Figure 14.21: NK Cell Interactions
4. Cytomegalovirus not only lowers the cells MHC production, it attempts to fool the
NK cells by producing a fake MHC. The viral genome codes for a protein
resembling MHC I. This does not activate TC cells as it does not display foreign
antigen. However, it is recognized as MHC I by NK cells of susceptible mice. The
fake MHC binds to the same NK inhibitory receptor that real MHC binds to.
5. Some mice have evolved NK surface receptors that can distinguish fake viral MHC I
from the real thing. These mice have an NK receptor for the viral protein that
activates attack, instead of suppressing it. And these mice are quite resistant to
CMV.
Figure 14.22 NK Cell Interaction
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IV. Coordinated Killing - Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

A. How it Works
1. Targeting of cell harboring pathogens. (Figure 14.23)
a. The cells display abnormal antigen.
b. B-cells produce antibodies to this antigen
c. The antibodies attach so that their FC portion stick out from the cell.
2. Cells with Antibody (FC ) Receptors
a. NK
b. macrophages
c. neutrophils (Figure 14.24)
d. eosinophils
Figure 14.23: IgD Antibody
Figure 14.24: Ingestion by

Neutrophil
Figure 14.25: NK cell induced apoptosis
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Table 14.2: Cells participating in Antibody Dependent Cell Mediated Toxicity
cell
NK
macrophag
e
neutrophil
eosinophil
Antibody
recognized
IgG, IgM
Cells
attacked
rogue self
Result
Other results
apoptosis
IgG, IgM
pathogen
phagocytosis
IgG, IgM
IgE
pathogen
parasites
phagocytosis
surface attack
possible damage to
healthy tissues
antigen presentation
on MHC II
often break open
may break open or
release toxins
B. Graft Versus Host Cooperation from the immune system you would like to avoid.
1. Supply immuno-incompetent individual with competent lymphocytes from another
genetically different individual.
2. The Tc cells activate and begin to attack the host cells.
3. The B cell produce antibodies to the host cells surface antigen.
4. The patient experiences a cell-mediated attack from both NK and TC cells, with some
help from the macrophages, neutrophils and maybe even complement.
5. The cells under heaviest attack are GI epithelial (diarrhea), skin (which may slough
off in sheets), and spleen, (which may attack the red blood cells, resulting in
jaundice). The whole response can kill a person.
6. Spleen enlargement is generally used as a diagnostic indicator.
NK cells: http://www.youtube.com/watch?v=HNP1EAYLhOs&feature=fvwrel
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Glossary
Adaptive cells those that rearrange their genes.
ADCC- antibody dependent cell-mediated cytotoxicity. A process by which white blood
cells recognize the stems of antibodies attached to a cell and then attack it.
Allele- a version of the gene. There are two alleles for the enzyme that produces color in
four oclock flowers, one that codes for an enzyme used to make red pigment and a different
DNA sequence that does not produce a functional enzyme, leaving the flower white.
ALT associated lymphoid tissue. MALT (mucosal), GALT (gut), BALT (bronchial), NALT
(nasal)
Antibody- a soluble immunoglobulin.
Antigen- a molecule that can bind to an antibody, B cell receptor or T cell receptor.
APC antigen presenting cell. Cells that present antigen on MHC II to TH cells.
Apoptosis programmed cell death.
ATP- adenosine triphosphate, directly supplies energy to many biological reactions.
B7 an important juxtacrine signal from APCs to TH cells.
BSA bovine serum albumin. A smallish soluble protein isolated from cows blood.
Calnexin- first MHC I chaperone, displaced by beta macroglobulin.
Calreticulin- binds to tapasin- MHC I complex
CAM cell adhesion molecule. Any one of a number of different molecules that help stick
cells together.
Centromere- the region on a chromosome that attaches to the spindle during mitosis.
CD cluster of differentiation. Refers to the isolation of cells by flow cytometry depending
on exactly what proteins extend from a cells surface. This in turn influences how the cell
moves during the separation process. Recall that these are applied solely on the basis of the
order of discovery.
CD1 (A through E) - the peptides or genes that code for them that bind lipids to present
them to T cells.
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CD3- the peptides that associate with the TCR for signaling.
CD4- the co-receptor that helps TH cells bind to MHC II.
CD8- the co-receptor that helps TC cells bind to MHC I
CDR- complementarity determining region- the recognition side on the tips of the antibody
arms.
Chagas Disease- caused by a trypanosome (unicellular eukaryote) that is spread by an
insect vector. Changes surface coats and causes down-regulated IL-2.
Chordate member of the phylum Chordata. Includes vertebrates and invertebrates with a
dorsal nerve cord, gill slits, notochord and muscles in blocks.
Clip- portion remaining after hydrolysis of the invariant chain.
CLP common lymphoid progenitor. Gives rise to lymphoid cells: NK, T cells, B cells, and
more.
CMP common myeloid progenitor. Stem cell that can give rise to any myeloid cell type
(including red blood cells and platelets).
Coley toxin inflammatory material isolated from bacteria used in cancer chemotherapy
around 1900.
Complement- a system of proteins that helps identify pathogens and debris for destruction
and phagocytosis (the landmines of the plasma).
CTL Cytotoxic T cell. Activated TC cell, ready to kill rogue-self cells.
DAG- diacyl glycerol. Two fatty acids joined by ester condensation bonds to glycerol,
produced by clipping a phospholipid.
DAMPS (death or damage-associated molecular proteins) - molecules that are
normal cell components but should be inside the cell, not identifiable on the exterior.
Down-regulate- turn off cellular processes (often a result of negative feedback).
Downstream- the end of the DNA or RNA with the free 3 carbon of the (deoxy) ribose.
Nucleic acid synthesis and translation proceeds 5 to 3.
DN 1 through 4- stages of T cell development before cells express either CD4 or CD8.
Early genes- genes for signaling proteins and receptors activated within hours after T-cell
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activation.
Epitope the specific portion of a molecule that binds to an adaptive receptor. For
example, a viral protein is an antigen whose different epitopes bind to different antibody
idiotypes.
ERp57- final chaperone binding to MHC I prior to antigen binding.
Exon- the part of a gene that codes for a sequence of RNA that will wind up in a message
and get translated (expressed.) A gene or gene region may have one too many exons.
FAS/FAS ligand- A tripartite juxtacrine signal set, used by immune cells to induce
apoptosis in damaged cells.
G protein (trimeric) an important internal signal transducer. It responds to receptor
activation by exchanging GTP for GDP and triggering a signal cascade to the cells interior.
Gamma-delta T cells- those with the quasi-innate gamma-delta receptor, involved in
boundary defense.
Gene region a sequence of DNA coding for a specific part of the Ig or T-cell receptor.
Goldilocks Principle- a general principle that in many biological system the intermediate
value of a process or level or a signal is most effective.
Granulocytes Cells with copious granular inclusions and that do not present antigen.
Includes neutrophils, basophils, and eosinophils (which have oddly-shaped nuclei) and mast
cells (which do not).
Granuloma (also called tubercle) a nodule of macrophages and specialized TH
cells surrounding a walling off macrophages infected with intracellular bacteria.

Haplotype- a group of genes next to each other on a chromosome that tend to be inherited
as a block.
Hapten- a molecule that could potentially bind a CDR, but by itself is not large enough to
kick off an immune response.
HLA- human lymphocyte antigen, the MHC types of humans.
HLA-DM helps load MHC II by displacing clip.
HSC- hematopoietic stem cell. Can divide and regenerate of develop into any type of
blood cell. Found in bone marrow.
Humoral response Immune defense found in the plasma, the word humor derived from
the ancient Greek medical theory of body fluids; it really just refers to antibodies. Stupid term.
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If people stop using it, maybe it will go away.

Hybridoma- a cell or cell line derived for the fusion of a blood cell cancer (myeloma) and a
normal, antibody-producing plasma (B lineage) cell.
Idiotype a category of antibodies that all have the same recognition region
IL (-1, -2, -4, -5, etc.) interleukins- a general name for a heterogeneous collection of
paracrine signaling molecules.
Immediate genes- T cell activation gene for transcription factors, activated in minutes.
iNKT cells quasi innate T cells that bind to lipids.
Integrin- proteins that spans the plasma membrane, connecting to the cytoskeleton at the
interior and capable of binding a variety of extracellular proteins at the exterior.
Innate cells those that do not rearrange genes.
Introns- that DNA sequences of the gene that code for RNA sequences that get clipped out
during processing.
Invariant chain- MHC II chaperone.
IP2 or IP3- inositol with two or three phosphates. Produced by clipping inositol from a
phospholipid and adding one or two phosphates to it (It has one to begin with).
Isotype- a category of antibodies of the same class.
Janus kinase- two-faced receptor. The signaling domains phosphorylate each other
during signaling.
Kinase- and enzyme that adds phosphate to proteins or other molecules.
Late genes- gene for CAMS up-regulated by T cell stimulus. These take days to activate.
Leprosy- a relatively non-contagious bacterial disease. An ineffective immune response
results in the disfiguring loss of extremities.
Lipid raft- a patch of plasma membrane with a distinct lipid composition and a more rigid,
thicker structure. Helps to separate different functional membrane protein from each other.
LMP 2 and 7- proteins that determine the peptide length of peptide hydrolyzed in the
proteasome.
Lymphoid cells white blood cell types (innate and adaptive) found in the lymph and (and
blood and immune organs as well).
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MAC- membrane attack complex- terminal complement pathway produces this, which
punches holes in plasma membranes.
MASP- mannose associated protein. Associated with lectin pathway of complement.
MBL- mannose binding lectin. Associated with lectin pathway of complement.
MHC- Major Histocompatibility Complex. Includes the genes and the proteins they code
for. These include the proteins (groups I and II) that hold small peptides so that T cells can
recognize them. They also include a variety of other proteins, including enzymes important in
immune recognition and promotion. The human versions are named HLA molecules for
human lymphocyte antigen.
Monoclonal- refers to a cell line of (theoretically) identical cells derived from the division of
a single cell.
Mucin- CAM with proteins attached to extensive amounts of branched carbohydrate
chains, ending in sialic acid.
Myeloid cells innate white blood cells rarely found in the lymph.
Necrosis- cell death from disease or injury.
NFAT- inflammatory transcription factor activated by phosphatases in turn activated by
MAP kinases.
NFB- inflammatory transcription factor activated by phosphatases in turn activated by
MAP kinases.
NK cell- Natural Killer cell. Kills rogue-self cells, recognizing them by innate mechanisms.
Does not require TH activation.
N-nucleotide addition- During gene rearrangement, when enzymes add nucleotides at
random in the palindromic regions of the joint.
NOD nucleotide oligomer detectors. Soluble pattern recognition receptors found in the
cytoplasm of cells. Despite the name, they often recognize cell wall materials.
NSAID- non-steroidal anti-inflammatory.
Nucleic acid RNA or DNA.
PAMPS (pattern-associated molecular proteins) - generally used to describe a
variety of molecules characteristic of proteins- recognized by PRRs such as TLRs.

Peptidoglycan mesh-like macromolecules that compose the basic structure of the
bacterial cell wall.
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Perforins- monomer secreted by NK and Tc cells that assemble into pores that construct
holes in the plasma membranes of self-cells under immune attack. Resemble the pores
produced by complement C9.
Phagocytosis when a cell engulfs large particles.
Pinocytosis - when a cell gathers fluid in a vesicle and engulfs the vesicle.
Plasma cell- activated antigen-secreting B cell.
P-nucleotide addition- During gene rearrangement, when enzymes fill in the missing
nucleotides at the joint by copying the palindromic nucleotides on the other strand.
Proteasome- organelle responsible for hydrolyzing and recycling cytosolic proteins.
Receptor-mediated endocytosis when a cell binds material at its surface using a proteins
receptor and then internalized the complex into a vesicle that enters the cell.
RAG enzymes- those responsible for gene rearrangement (related to gene used during
meiosis for gene recombination).
RSS recombination signal sequence. The sequence of 28 or 40 nucleotides that the
upstream or downstream end of a gene region providing the signals for gene rearrangement.
Selectin- class of CAMS that have a lectin domain that bind to mucins carbohydrates.
Simple sugar single sugar unit, includes glucose, mannose and galactose. May be
modified into sugar units as sialic acid (NANA) or N-acetyl glucosamine.
STAT- transcription factor activated by phosphorylation and subsequent dimerization
(Signal Transduction Activator of Transcription). Typically activated by Janus kinases.
TAP 1 and 2- transport peptides into the ER using ATP.
Tapasin- bring TAP to MHC I.
TC cell- cells that recognize rogue self-cells by antigen they present on MHC I. They
develop into CTLs after instructions from TH cells.
TCR T-cell receptor. Found extending from the surface of both TC and TH cells.
Recognizes antigen, coded for by rearranged genes.
TH cell- thyroid helper cell. Coordinates immune responses. TH 1 cells promote a serious
response; TH 2 promotes a containment response, TH 17 promotes inflammation and boundary
defense, and Treg tolerance. There are additional types as well.
TLR Toll-like receptors. Pattern recognition receptors that recognize molecules
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characteristic of pathogens. Found embedded in plasma membrane and endomembranes of

many white blood cells.
Transcription factor a protein that either up- or down- regulates the copying of RNA
(transcription) from DNA. They often have domains that attach to specific sequences of DNA
nucleotides. Some attach to other proteins that attach to the DNA. Or both.
Up-regulate- turn on pathways (sometimes related to positive feedback).
Upstream- the end of the DNA or RNA with the free 5 carbon of the (deoxy) ribose.
Nucleic acid synthesis and translation proceeds 5 to 3.
Zymosan cell wall material of fungi.
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Fundamentals of Immunology - Study Guide

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Fundamentals of Immunology - Study Guide

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Fundamentals of

David Schidlow, M.D., Ph.D.

BIOC 372x Study Guide

Lecture L01- Introducing the Metaphors

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Infectious disease is one of the few genuine adventures left in the

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Figure 1.1: ATP

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Pathogens and Immunity

Figure 1.3: Viruses

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2. Bacteria: Mycobacterium tuberculosis (TB), E. coli, anthrax, bubonic

Figure 1.4: Spirochetes

Figure 1.5: Bacteria Structures

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Figure 1.6: Gram Positive

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Figure 1.7: Gram Negative

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3. Fungi: Candida albicans (yeast), athletes foot, ringworm, Cryptococcus

4. Unicellular eukaryotes: malaria, trypanosomes, amoebae, Giardia,

Figure 1.11: Malaria

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Figure 1.12: Protozoans

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5. Parasitic worms (Platyhelminthes and Nematoda): flukes, tapeworms,

Figure 1.13: Schistoma

Figure 1.14: Trichinella

Figure 1.15: Hookworm

B. Recognition Two general strategies to identify and neutralize the threat.

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Metaphors if Power Politics

Innate versus Adaptive Immune Response

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2. Defensive proteins complement, lysozyme

Table 1.1: Innate Versus Adaptive

Fast (minutes) Always there

Slower-weeks requires gene Recognizes

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Table 1.2 Responding to Foreign Antigen (Inevitably simplified)

Attacks and kills cell

Binds antigen with

All nucleated cells except

Attacks and kills cell

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The Source of It All Hematopoiesis

A. Hematopoietic Stem Cell (Figure 2A.1)

It can divide to make more of itself - self-renew or it can begin to differentiate by

4. As few as 100 or so HSCs are enough to completely regenerate the whole

Figure 2A.1: Hematopoietic Stem Cell

B. Signaling Differentiation (Figure 2A.2)

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a. CMP- If it turns into a common myeloid-erythroid progenitor, it may develop into a

Figure 2A.2: Signaling

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Figure 2A.3: Erythrocyte

Figure 2A.4: Platelet

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A. Immune Involvement a Secondary Function

Figure 2A.5: Neutrophil

Figure 2A.6: Neutrophil