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

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- 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 re-exposure to same antigen

RECOGNITION

HOW TO RECOGNIZE ANTIGEN?

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ANTIGEN PROCESSING- WHY IS IT NECESSARY?

Because it is needed to process to be recognized

YB

YY Y YYY

Y

B

Proliferation and antibody production

Cross-linking of surface membrane Ig

Y

B

Y

B Y

B

Y

B

Y

B Y

B

Y

B

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T cells do not recognise native antigens

Y

T

Y

T

production

No proliferationNo cytokine release

Cell surface

Antigens must be processed in orderto be recognised by T cells

Y

T

Solublenative Ag

Soluble peptidesof Ag

Cell surface peptides of Ag presented by cells that express MHC antigens

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Cell surfacepeptidesof Ag

T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

Cell surfacenative Ag

of Ag

ANTIGENPROCESSING

B2. Binding and internalisation via Ig induces expression

Fate of Antigens Internalised by B cells

1. Capture by antigenspecific Ig maximisesuptake of a single antigen

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B

Ig induces expressionof CD40

3. Antigen enters exogenous antigenprocessing pathway

4. Peptide fragments of antigen are loadedonto MHC molecules intracellularly.MHC/peptide complexes areexpressed at the cell surface

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

How to recognize Ag?

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a) MHC-I associate with peptides from endogenous Ags

b) MHC-II associate with peptides from exogenous Ags

MHC-I association

Most cells (target cells) can present Ag w/ MHC-I to TCs

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

MHC-II association

Only APCs can present Ag on MHC- II to THs

APCs are of 2 categories:

Professional APCs

Non-professional APCs

Association with MHC-II does

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allogeneic cells from transplants

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

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")

DROPBOX

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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.

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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

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 non-professional APC by certain cytokines such as IFN-.

Non-professional APCs include:

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Non-professional APCs include: Fibroblasts (skin) Thymic epithelial cells Thyroid epithelial cells Glial cells (brain) Pancreatic beta cells Vascular endothelial cells

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?

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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?

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

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3. Transport to cell surface for expression

Different cellular pathways for association of peptide with MHC-I &-II

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

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Peptide generation from proteolysis

Transport to ER

Peptide binding to MHC-I

Degradation in the proteasomeCytoplasmic cellular proteins, including non-self proteinsare degraded continuously by a multicatalytic protease of 28 subunits

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The components of the proteasome include MECL-1, LMP2, LMP7These 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

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

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a proteosome

Specific proteosomes generate peptides which can bind 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

Endogenous Ag processingpeptide binding to MHC-I

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peptide close to MHC assembly

Allows MHC-I to bind to peptides

MHC I-Ag exits ER to Golgi to plasma membrane

Endogenous Ag processingtransport 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

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ATP to help transport peptides (8-10 aas) to lumen of ER

Usually basic aas @ COOH end of peptide chain

Assembly and stabilization of MHC-I + Ag complex

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ENDOPLASMIC RETICULUM

Peptide antigens produced in the cytoplasm are physically separated from newly formed MHC class I

Newly synthesisedMHC class I molecules

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CYTOSOL

Peptides needaccess to the ER in

order to be loaded onto MHC class I molecules

PeptidePeptidePeptidePeptidePeptidePeptide

Maturation and loading of MHC class I

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Endoplasmic reticulum

Calnexin bindsto nascentclass I chainuntil 2-M binds

PeptidePeptidePeptidePeptidePeptide

B2-M binds and stabilises floppy MHC

Tapasin, calreticulin, TAP 1 & 2 form a complex with the floppy MHC

Cytoplasmic peptides are loaded onto the MHC molecule and the structure becomes compact

MHC-I Pathway

Viral protein is madeon cytoplasmic

ribosomes

Plasma membrane

Peptide passes

Peptide is presentedby MHC-I to CD8cytotoxic T cell

Globular viralprotein - intact

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Proteasome degrades protein topeptides

Peptide transporterprotein moves peptide into ER

MHC class I alpha and beta proteinsare made on the rER

Peptide associateswith MHC-I complex

Peptide with MHCgoes to Golgi body

Peptide passeswith MHC from Golgi

body to surface

Golgi body

rER

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

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Degraded peptide bind to MHC-II, returns to PM & recycling surface receptors

Exogenous Ags are typically phagocytized/ endocytized by M and APCs

Processing of Exogenous Ag:

in details

within ER, and chains of MHC-II combine with a protein the invariant

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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

In the endoplasmic reticulum

MHC class II maturation and invariant chain

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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

Removal of CLIP

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?

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

Competition between large number of peptides

HLA-DM assists in the removal of CLIP

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HLA-DM HLA-DR

HLA-DM: Crystallised without a peptide in the grooveIn space filling models the groove is very small

HLA-DM

Single pocket in grooveinsufficient to accommodatea peptide

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HLA-DR

Multiple pocketsin groove sufficient toaccommodate a peptide

HLA-DM catalyses the removal of CLIP

HLA-DM

Replaces CLIP with a peptide antigen using a catalytic mechanism (i.e. efficient at sub-stoichiometric levels)

Discovered using mutant cell lines that failed to

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MIIC compartment

cell lines that failed to present antigen

HLA-DO may also play a role in regulating DM

Sequence in cytoplasmic tail retains HLA-DM in endosomes

HLA-DMHLA-DR

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MHC-II Pathway

Globular protein

EndocytosisEndosome

Lysosome

Fusion of endosomeand exocytic vesicle

Immunodominant

Endosome fuses withplasma membrane

Peptide MHC-II complex is presentedto CD4 helper T cell

CD4 helper T cell

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Protein is processed topeptides in endosome

or lysosome

Lysosome

Endoplasmic reticulum

Class II MHCSynthesis

3 chains: , and Ii

Golgibody

Exocytic vesicle fuseswith endosome

releasing Ii from dimer

Ii

Immunodominantpeptide binds

to class II MHC

MHC-II Pathway

Globular protein

1a

b

2

6

7 e

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b

c

3

d.

4

Ii

5

Comparison of Ag-processing pathways

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Ag-processing pathways

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

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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.

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

Summary of Ag recognition

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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

EVASION of Ag presentation

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PeptidePeptide

Evasion of immunity by interference with endogenous antigen processing

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Endoplasmic reticulum

Peptide

Peptide

Peptide

Peptide

HSV protein blocks transportof viral peptides into ER

Sent to lysosomesfor degradation

Normally exported to the cell surface

Adenoviralproteinretains MHC

Evasion of immunity by interference withendogenous antigen processing

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Sent to lysosomes for degradation

retains MHCclass I in the ER

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

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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

Figure 6-1

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Figure 6-2

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Green: MHC class IIRed: Lysosomal protein

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