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Steps in AI response

1. Recognition of the Ag by specific lymphocytes


2. Activation of these specific lymphocytes
3. Proliferation and differentiation into effector cells;
- The effector cells eliminate the Ag
- Return of homeostasis and development of memory cells
4.

Memory cells evoke a more rapid and long response on reexposure to same antigen

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RECOGNITION
HOW TO RECOGNIZE ANTIGEN?
ANTIGEN PROCESSING- WHY IS IT NECESSARY?
Because it is needed to process to be recognized

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Y
Y
Y
Y
Y
Y

Y
Cross-linking of surface
membrane Ig

YYY Y
Y Y Y Y

B B
B B BB
B B

Proliferation and antibody


production

No proliferation
No cytokine release

T cells do not recognise native antigens


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Antigens must be processed in order


to be recognised by T cells
T

Y
Soluble
native Ag
Cell surface
native Ag

Soluble
peptides
of Ag

Cell surface peptides of Ag


presented by cells that
express MHC antigens

Cell surface
peptides
of Ag

ANTIGEN
PROCESSING
No T cell
response

No T cell
response

No T cell
response

No T cell
response

T cell
response
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Fate of Antigens Internalised by B cells

1. Capture by antigen
specific Ig maximises
uptake of a single antigen

2. Binding and internalisation via


Ig induces expression
of CD40
3. Antigen enters exogenous antigen
processing pathway
4. Peptide fragments of antigen are loaded
onto MHC molecules intracellularly.
MHC/peptide complexes are
expressed at the cell surface

B
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How to recognize Ag?


Ag recognition by T cells REQUIRES presentation by MHC on a
cell membrane (MHC restriction)
Pathways for Ag presentation:
a) MHC-I associate with peptides from endogenous Ags
b) MHC-II associate with peptides from exogenous Ags

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MHC-I association

MHC-II association

Most cells (target cells) can


present Ag w/ MHC-I to TCs

Only APCs can present Ag on


MHC- II to THs

Nearly all nucleated cells


infected by microbes/virus, or
abnormal proteins produced by
cancer cells, aging cells, or by
allogeneic cells from transplants

APCs are of 2 categories:


Professional APCs
Non-professional APCs

Associate with MHC- I requires


replication of foreign entity (i.e.,
abnormal protein synthesis)
within the target cell

Association with MHC-II does


not require replication of entity
within target cells
Phagocytosis is important in
Ag-processing

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DROPBOX

Antigen Presenting Cells (APCs)

APCs fall into two categories: professional or non-professional.


Most cells in the body can present antigen to CD8+ T cells via
MHC class I molecules and, thus, act as "APCs"; however, the
term is often limited to those specialized cells that can prime T
cells. These cells, in general, express MHC class II as well as
MHC class I molecules, and can stimulate CD4+ ("helper")
cells as well as CD8+("cytotoxic") T cells, respectively.
To help distinguish between the two types of APCs, those that
express MHC class II molecules are often called professional
antigen-presenting cells.

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Professional APCs
There are 3 main types of professional antigen-presenting cells:
Dendritic cells (DCs), which have the broadest range of antigen
presentation, and are probably the most important APC. Activated DCs are
especially potent TH cell activators because, as part of their composition,
they express co-stimulatory molecules such as B7.
Macrophages, which are also CD4+ and are therefore also susceptible to
infection by HIV.
B-cells, which express (as B cell receptor) and secrete a specific antibody,
can internalize the antigen, which bind to its BCR and present it
incorporated to MHC II molecule, but are inefficient APC for most other
antigens.
Certain activated epithelial cells

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Non-professional APCs
A non-professional APC does not constitutively express the
Major Histocompatibility Complex class II (MHC class II)
proteins required for interaction with naive T cells;
These are expressed only upon stimulation of the nonprofessional APC by certain cytokines such as IFN-.
Non-professional APCs include:
Fibroblasts (skin)
Thymic epithelial cells
Thyroid epithelial cells
Glial cells (brain)
Pancreatic beta cells
Vascular endothelial cells
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Antigen Presenting Cells- some more details

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Experimental Basis for


Ag Processing and Presentation
Pulse macrophages with a protein antigen short time wash cells, fix
immediately mix with T cells and measure proliferation
PROLIFERATION OF T CELLS?
Pulse macrophages with same protein antigen short time wash cells, fix
after 4-5 hoursmix with T cells and measure proliferation
PROLIFERATION OF T CELLS?

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Ag Processing and Presentation


Ag processing and presentation take sometime because of .

1. Fragmentation of protein into peptides


2. Association of peptide with an MHC molecule
3. Transport to cell surface for expression
Different cellular pathways for association of peptide with MHC-I &-II

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Ag processing
*MHC I interacts w/ peptides from cytosolic degradation
*MHC II interacts w/ peptides from endocytic degradation

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Processing of endogenous Ag involves 3 activities:


Peptide generation from proteolysis
Transport to ER
Peptide binding to MHC-I

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Degradation in the proteasome


Cytoplasmic cellular proteins, including non-self proteins
are degraded continuously by a multicatalytic protease of 28 subunits

The components of the proteasome include MECL-1, LMP2, LMP7


These components are induced by IFN- and replace constitutive
components to confer proteolytic properties.
LMP2 & 7 encoded in the MHC
Proteasome cleaves proteins after hydrophobic and basic amino acids
and releases peptides into the cytoplasm
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Endogenous Ag processing in proteasome of cytosol


peptide generation
Proteins targeted for
lysis combine w/ a small
protein- ubiquitin;
Ubiquitin-protein
complex is degraded by
a proteosome
Specific proteosomes
generate peptides which
can bind to MHC-I

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Endogenous Ag processing
peptide binding to MHC-I
MHC I assembly occurs with the
aid of chaperone proteins to
promote folding (calnexin + MHC I
chain)
Tapasin + calreticulin brings TAP/
peptide close to MHC assembly
Allows MHC-I to bind to peptides
MHC I-Ag exits ER to Golgi to
plasma membrane

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Endogenous Ag processing
transport to ER
Peptides from proteolysis bind to
a transporter protein assoc w/
Ag processing (TAP)
TAP is a heterodimer which uses
ATP to help transport peptides
(8-10 aas) to lumen of ER
Usually basic aas @ COOH end
of peptide chain

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Assembly and stabilization of MHC-I + Ag complex

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Peptide antigens produced in the cytoplasm are


physically separated from newly formed MHC class I

ENDOPLASMIC RETICULUM
Newly synthesised
MHC class I molecules

CYTOSOL

Peptides need
access to the ER in
order to be loaded onto
MHC class I molecules
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Maturation and loading of MHC class I

Peptide
Peptide

Peptide

Endoplasmic reticulum

Calnexin binds
to nascent
class I chain
until 2-M binds

B2-M
binds and
stabilises
floppy
MHC

Tapasin, calreticulin, TAP Cytoplasmic peptides


1 & 2 form a complex with are loaded onto the
the floppy MHC
MHC molecule and the
structure becomes
compact
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MHC-I Pathway
Peptide is presented
by MHC-I to CD8
cytotoxic T cell
Plasma membrane

Viral protein is made


on cytoplasmic
ribosomes
Globular viral
protein - intact

Peptide passes
with MHC from Golgi
body to surface

rER
Peptide associates
with MHC-I complex

Proteasome
degrades
protein to
peptides
Peptide transporter
protein
moves
peptide into ER

Golgi body

Peptide with MHC


goes to Golgi body

MHC class I alpha


and beta proteins
are made on the rER

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MHC-II pathway

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Processing of exogenous Ag within endocytic vacuole of endocytic


pathway involves 2 activities:
Ag degradation: Early endosomes (pH 6-6.5); Late endosomes or
endolysosome (pH 5-6); Lysomes (pH 4.5 5); Ag is degraded into 13-18
aa polypeptides
Degraded peptide bind to MHC-II, returns to PM & recycling surface
receptors
Exogenous Ags are typically phagocytized/ endocytized by M and APCs

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Processing of Exogenous Ag:

in details

within ER, and chains of MHC-II combine with a protein the invariant
chain (IC, Ii, CD74)
the IC binds to MHC @peptide binding cleft + then exits the ER to Golgi
apparatus
as proteolytic activity continues, the IC is degraded to a small fragment (CLIP*)
another MHC II (HLA-DM (found in endosomes)) substitutes Ag for CLIP within
lysosome
MHC II Ag complex is transported to the PM
*CLIP = class II associated invariant chain peptide

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MHC class II maturation and invariant chain


In the endoplasmic reticulum

Need to prevent newly


synthesised, unfolded self
proteins from binding to
immature MHC

Invariant chain stabilises MHC class II by


non- covalently binding to the immature
MHC class II molecule and forming a
nonomeric complex
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Removal of CLIP

How can the peptide stably bind to a floppy binding site?


Competition between large number of peptides
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HLA-DM assists in the removal of CLIP

HLA-DM

HLA-DR

HLA-DM: Crystallised without a peptide in the groove


In space filling models the groove is very small
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HLA-DM
Single pocket in groove
insufficient to accommodate
a peptide

HLA-DR

Multiple pockets
in groove sufficient to
accommodate a peptide

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HLA-DM catalyses the removal of CLIP


HLA-DM
Replaces CLIP with a
peptide antigen using a
catalytic mechanism (i.e.
efficient at substoichiometric levels)
Discovered using mutant
cell lines that failed to
present antigen
HLA-DR

MIIC compartment

HLA-DM

HLA-DO may also play a


role in regulating DM

Sequence in cytoplasmic tail


retains HLA-DM in
endosomes
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MHC-II Pathway
Peptide MHC-II
complex is presented
to CD4 helper T cell

Endosome fuses with


plasma membrane
Immunodominant
peptide binds
to class II MHC
Golgi
body

Globular
protein

CD4 helper T cell

Endosome
Fusion of endosome
and exocytic vesicle

Endocytosis
Lysosome

Exocytic vesicle fuses


with endosome
releasing Ii from dimer

Protein is processed to
peptides in endosome
or lysosome

Class II MHC

Synthesis
3 chains: , and Ii
Ii
Endoplasmic reticulum

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MHC-II Pathway
7

Globular
protein

2
b
5
4
d.

Ii
c

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Comparison of
Ag-processing pathways

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Processing of Exogenous Ag:


the Endocytic pathway

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Points Concerning Ag Processing& Presentation


1. Location of pathogen
viruses in cytosol, MHC class I pathway, Tc response
extracellular bacteria, MHC class II pathway, Th2 response,
Ab formation
intracellular bacteria, MHC class II pathway, Th1 response
2. Peptides derived from both self and non-self proteins can
associate with MHC- I &- II molecules.
3. Chemical nature of MHC groove determines which
peptides it will bind.
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Summary of Ag recognition
T and B cells recognise Ag differently
Ag must be catabolised before T cells can recognise it
Ag processing generates antigenic peptides
Exogenous antigen processing takes place in lysosomes, uses invariant
chain and HLA-DM
Endogenous processing is non-lysosomal, uses proteasomes and peptide
transporters in antigen processing
The mechanism of Ag processing depends upon the compartment in which
the pathogen replicates
Endogenous and exogenous Ag processing both involve uptake,
degradation, complex formation and presentation
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EVASION of Ag presentation

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Evasion of immunity by interference with endogenous


antigen processing

Peptide

Peptide

Endoplasmic reticulum

Sent to lysosomes
for degradation

HSV protein blocks transport


of viral peptides into ER
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Evasion of immunity by interference with


endogenous antigen processing

Normally exported to the cell surface

Adenoviral
protein
retains MHC
class I in the ER

Sent to lysosomes for degradation

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FYI

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DC as APC
Dendritic cells = the only APC that can initiate Ag specific
response
Presumably from myeloid and lineage
Precursor DCs in blood; differentiate into immature DCs (iDC) iDC
recognized antigen with TLR and other receptors; activated
Pinocytosis/phagocytosis and cytokine production, now DC DCs can
no longer phagocytose; go to T-cell area of lymph nodes where they
present Ag to T cells
Langerhans cells are skin DC

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Figure 6-1

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Figure 6-2

Green: MHC class II


Red: Lysosomal protein

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