501732

Antigen Processing and Presentation

Presented to you by: Hercolanium Under the supervision of: Prof. M. F.

Introduction
A century ago the composite word antigen referred to that which

generates antibodies. This tautology developed before the concept of a receptor, before the definition of macromolecular proteins, and before it was known that antibodies actually bind to antigens. The antigenantibody tautology remains central to our concepts of immunity and to the tautology of self and non-self .
At a molecular level, an antigen can be defined today as any molecule

recognized by (i.e., binding specifically to) the antigen-binding domain of an antigen receptor (antibody or T-cell receptor TCR).

 The antigen bound by a particular antigen receptor is sometimes called

its cognate antigen. The concept of a cognate antigen is useful when identifying the molecular rules governing antigenantigen receptor interactions, for predicting antigens from sequence and structural information, for designing subunit and genetic vaccines, and for understanding what the immunologist means by such terms as self and

nonself.
In this lecture we shall focus on the antigens regarding their processing

and presentation by specialized cells in order for the immune system to be activated or sensitized so that the body can counter the attack.
In order for us to elaborate antigen processing and presentation we

should focalize on the antigen recognition process later on.

concepts
Endogenous Ags: antigens synthesized within cells, including self and non-self
protein----class MHC molecules.

Exogenous Ags: antigens comes from outside the cells, including self and non-self
protein----class MHC molecules.

Antigen processing: the conversion of native proteins to peptides which can

combine with MHC molecules.

Antigen presentation: the course of formation and display of peptide-MHC

complexes on the surface of APCs and the course of peptide-MHC complexes
recognition by T cells.

Ag capturing----Endocytosis (internalization)
Phagocytosis, Pinocytosis, Receptor-mediated endocytosis

Antigens and Antigen Presenting Cells (APCs)

Before we can explain the mechanisms of antigen processing, presentation and recognition by

the immune system we should focus on the antigen itself.

Antigens could be peptides, proteins, nucleic acids, polysaccharides, lipids, or small chemicals.

That does not evoke any problem if the cell acting is a B cell because a humoral immune

response will take place ; nonetheless when T cells are acting this will be a problem given that
most of them recognize peptide antigens which will elicit cell-mediated immune response.
T cells are specific for amino acid sequences of peptides while B cells recognize conformational

determinants of antigens, even proteins, in their native tertiary (folded) configuration. The

antigen receptors of T cells recognize very few residues even within a single peptide, and
different T cells can distinguish peptides that differ even at single amino acid residues.

 Antigen Presenting Cells (APCs/accessory cell) are cells that display foreign antigen complexes

with major histocompatibility complex (MHC) on their surfaces. T-cells may recognize these complexes using their T-cell receptors (TCRs). These cells process antigens and present them to

T-cells.
T cells cannot recognise, and therefore react to, 'free' antigen. T cells can only 'see' antigen that

has been processed and presented by cells via an MHC molecule. 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 (i.e., activate a T cell that
has not been exposed to antigen, naive T cell). 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.

 T cells recognize and respond to foreign peptide

antigens only when the antigens are attached to the

surfaces of APCs, whereas B cells and secreted

antibodies bind soluble antigens in body fluids as well as exposed cell surface antigens. This is because T cells can recognize only peptides bound to and displayed by MHC molecules, and MHC molecules are integral

membrane proteins expressed on APCs.

T cells from anyone individual recognize foreign

peptide antigens only when these peptides are bound to and displayed by the MHC molecules of that

individual. This feature of antigen recognition by T

cells, called self MHC restriction, can be demonstrated in experimental situations in which T lymphocytes from one individual are mixed with APCs from another individual.
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 Dendritic cells are the most effective APCs for activating

naive CD4+ and CD8+ T cells, and therefore for initiating T cell responses. Macrophages present antigens to differentiated (effector) CD4+T cells in the effector

phase of cell-mediated immunity, and B lymphocytes

present antigens to helper T cells during humoral immune responses. Dendritic cells, macrophages, and B lymphocytes express class II MHC molecules and

costimulators, and are, therefore, capable of activating

CD4+ T lymphocytes. For this reason, these three cell types have been called professional APCs; however, this term is sometimes used to refer only to dendritic cells because this is the only cell type whose principal function is to capture and present antigens, and the only APC capable of initiating T cell responses.

PHAGOCYTES Mononuclear cells

Macrophage

Press

Dendritic cells

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Types of Dendritic Cells

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Fibroblast
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:
o Fibroblasts (skin) o Thymic epithelial cells o Thyroid epithelial cells o Glial cells (brain) o Pancreatic beta cells o Vascular endothelial cells

Glial (Neuroglial) Cells

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Major Histocompatibility Complex (MHC)

Two groups of MHC genes: Structurally and functionally distinct
1. 2.

Class I Class II

recognition by CD8+ T cells recognition by CD4+ T cells

HLA molecules are responsible for the compatibility of the tissues of genetically different

individuals and for the rejection of transplant

MHC genes are codominantly expressed in each individual Monozygotic twins have the same histocompatibility molecules on their cells MHC genes are the most polymorphic genes present in the genome! (Up to 250 alleles identified for some loci) MHC expression
Class I On all nucleated cells (no MHC on red blood cells, weak expression on cells in brain)

Class II Found on antigen presenting cells

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MHC class I molecule

1. Heavy chain 1, 2 domain: polymorphic sites 3 domain: binding of CD8 2. -2 microglobulin 3. Peptide

Ig Domain

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MHC class II molecule

1. chain
1: polymorphic sites 2: binding of CD4

2. chain
1: polymorphic sites 2: binding of CD4

3. Peptide

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Different MHC molecules have different groove structure.

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Peptides in MHC Grooves

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Elaboration of MHC restriction

Because alloreactive T cells distinguish between self and nonself MHC
molecules, it is tempting to think that MHC restriction mediates self/nonself discrimination. However, it is clear that allospecific antibodies also distinguish self from nonself MHC molecules. MHC restriction may function in two opposing ways.

The agents that raise

endosomal and lysosomal pH, or directly inhibit

First, antigen processing increases the complexity of pathogen antigens by exposing epitopes not available on the surface of pathogens. MHC molecules are merely the mechanism for presenting processed epitopes to T cells. MHC polymorphism further increases the size of the species antigenic universe.

endosomal proteases,
block class II-restricted but not class I-restricted antigen presentation,

whereas inhibitors of
ubiquitination or proteasomes selectively block class I-restricted antigen presentation.

Second, the requirement that T cells do not respond unless activated by

co-stimulation from the APC is enforced by anchoring MHC molecules on the APC. This second mechanism thus minimizes autoimmunity. In this view, the experimental observations of genetic MHC restriction and

alloreactivity are by-products of the polymorphism of MHC molecules.

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Peptide binding by MHC class I and II molecules. Class I molecules are usually closed at both ends. The peptide termini must interact with terminal sockets. Peptides that are too long must be cleaved (arrows) prior to entry into the binding site. The clefts of class II molecules are open at the ends, permitting the binding of long peptides.

MHC restriction carries out two critical functions. First, by presenting processed peptides derived from within proteins and pathogens, MHC molecules sample a broader antigenic landscape than antibodies, whose epitopes are surface oriented. Second, nave T cells respond to cognate epitopes only when presented by an activated APC (B) but not when the APC is resting (A). Experimentally observed MHC restriction results when an activated APC cannot present the proper MHC/peptide pair (C).

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T cells do not recognize native antigens

Cross-linking of surface membrane Ig

YYY Y Y Y Y Y
Proliferation and antibody production

B B B B BB B B

Y Y Y Y Y Y
T
No proliferation No cytokine release
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Antigens must be processed in order to be recognised by T cells

Soluble native Ag Cell surface native Ag

Soluble peptides of Ag

APC

No T cell response

No T cell response

No T cell response

Y
No T cell response

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

ANTIGEN PROCESSING

T cell response
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Provision of additional stimuli to the T cell beyond those initiated by recognition of peptide-MHC complexes by the T cell antigen receptor

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The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used

EXTRACELLULAR OR ENDOSOMAL REPLICATION Vesicular Compartment

Contiguous with extracellular fluid Exogenous processing (Streptococcal, tumor antigens)

INTRACELLULAR REPLICATION Cytosolic compartment

Endogenous processing (Viral, tumor antigens )

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1. Antigens Foreign proteins or self-proteins 2. are transported into the roughwithin the cytosol are broken down into fragments that are endoplasmic reticulum by TAP antigens by the action of(Transporter proteasomes which appears as a cylinder composed of a stacked array Associated with Antigen Presentation). Interestingly, of two inner and two outer rings, each ring being composed of seven subunits. Three of the the TAP1 and TAP2 genes are next to the genes seven subunits are the catalytic sites for proteolysis. A larger, 1500-kD proteasome is likely to be encoding LMP-2 and LMP-7 in the MHC, and the most important for generating class I-binding peptides and is composed of the 700-kD structure synthesis of the TAP protein is also stimulated by plus several additional subunits that regulate proteolytic activity. Two catalytic subunits present IFN-y. in many 1500-kD proteasomes, called LMP-2 and LMP-7, are encoded by genes in the MHC, 3. Antigens combine with MHC and are particularly important for generating class I-binding peptides. Cytokine IFN-y treatment class I molecules. 3 2 increases production of LMP-2 and LMP-7. MHC class I 4. The MHC class I/antigen Protein
complex is transported to the Golgi apparatus, packaged into a vesicle, and transported to the plasma membrane.
molecule 1 Protein fragments (antigens) Membrane Lumen 5 Rough endoplasmic reticulum Golgi apparatus

5. Foreign antigens combined

with MHC class I molecules stimulate cell destruction.

4
Foreign antigen

6. Self-antigens combined with

MHC class I molecules do not stimulate cell destruction.

Self-antigen 6 31

Antigen processing for MHC class I. Two chief pathways for antigen process intersect within the cytosol. Most endogenous antigens are synthesized on cytosolic ribosomes, processed by proteasomes, and enter the ER through the TAP (Transporter associated protein)transporter. A minor set of antigens are processed within the ER from proteins secreted into the ER. Professional antigen-presenting cells transfer endocytosed antigens into the cytosol for processing.

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TAP (Transporter Associated with Antigen Presentation)

Transport associated protein -TAP is responsible
for the peptide transport from cytoplasm to ER. Proteins are degraded to peptide in proteasome. The peptides are picked up by TAP proteins and transported from the cytosol into the RER where they assemble with the transmembrane polypeptide and beta-2 microglobulin. this trimolecular complex then moves through the Golgi apparatus and is inserted in the plasma membrane

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The functions of class II MHC-associated invariant chains and HLA-OM. Class II molecules with
bound invariant chain, or CLIP, are transported into vesicles, where the CLIP is removed by the action of OM. Antigenic peptides generated in the vesicles are then able to bind to the class II molecules.

Another class II-like protein, called HLA-DO, may regulate the OM-catalyzed removal of CLIP. CIIV.
class II vesicle.

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2. The 3.The antigen vesicle is broken containing down the into processed fragments antigen to fuses with vesicles produced by the Golgi apparatus that 1. The unprocessed extracellular antigen is ingested form contain processed MHC antigens. classby IIInternalized molecules. A proteins class II-rich are subset of late endosomes that plays an important role in endocytosis and is within a vesicle. degraded antigen enzymatically presentation. inIn late macrophages endosomes and and human B cells, it is called the MHC class II compartment, or

lysosomes MIIC. to (In generate some mouse peptides B cells, that are a similar able to organelle containing class II molecules has been identified and bindnamed to the peptide-binding the class II vesicle.) clefts The of class MIIC IIhas MHC a characteristic multilamellar appearance by electron microscopy. containing
MHC class molecules. Importantly, The degradation it contains all of the protein components antigens required in for peptide-class II II association, including the enzymes molecules Vesicle

vesicles that is degrade an active protein process antigens, mediated the by class proteases II molecules, the Ii (or invariant chain-derived peptides), and a that molecule have acidic called pH optima. human leukocyte antigen DM (HLA-DM). Within the 2 MIIC, the Ii dissociates from class II MHC molecules by the combined action ofproteolytic enzymes and the HLA-DM molecule, and antigenic
Vesicle blocks access to the peptide-binding cleft of a class II MHC molecule, it must containing be removed before complexes processed 4. The MHC class II/antigen complex of peptide and class II molecules can form. The same proteolytic enzymes, such as cathepsin S, that generate antigen

peptides are then able to bind to the available peptide-binding clefts of the class II molecules. Because the Ii

is transported to the plasma Unprocessed peptides from internalized proteins also act on the Ii, degrading it and leaving only a 24-amino acid remnant membrane. 4
antigen

called class II-associated invariant chain peptide (CLIP).The processed antigen and the MHC class II
5. The displayed MHC class II/antigen molecule combine. complex can stimulate immune cells.
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MHC class II molecule Processed antigen 36

Two pathways for loading antigens onto class II MHC molecules. Autophagy and endocytosis transfer cytosolic and external antigens, respectively, into the endosomes. Nascent class II molecules are chaperoned to the endosomes from the Golgi by the invariant chain. The DM molecules catalyze the exchange of antigenic peptides for invariant chain. Mature class II molecules recycling from the cell surface can acquire peptides in a DM-independent manner. Antigens binding initially as polypeptides are trimmed into oligopeptides in the endosomes and at the surface.

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B lymphocyte as APC
Unlike DC and macrophages, B cells are not phagocytic or macropinocytic, and do not express multiple types of antigen receptors. Their capacity to endocytose antigen is restricted almost exclusively to those captured through their cell surface receptor, the BCR. While this makes B cells less efficient than other APC at presenting most antigens, it also makes them the most focused. The BCR consists of an mIg, which provides the antigen recognition component to the receptor, noncovalently associated to an Ig:Ig heterodimer. This dimer provides the signaling module to the receptor, as it contains an immunoreceptor tyrosine-based activation (ITAM) motif required for signal transduction that upon antigen engagement

triggers internalization of the receptorantigen complex and initiates a signaling cascade leading to B-cell
activation. Ig-mediated endocytosis allows B cells to concentrate in their endosomal compartments minute amounts of antigen due to the high specificity of their mIg molecules. It is easy to imagine how this process operates on soluble antigens, but less so for antigens associated with cellular membranes. However, it has

recently been demonstrated that B cells can indeed endocytose antigens ripped from cell surfaces, so
BCR-mediated endocytosis can account for presentation of even cell-associated antigens without the need to invoke a phagocytic mechanism for antigen capture

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Like all lymphocytes, B-lymphocytes circulate back and forth between the blood and the lymphoid system of the body. B-lymphocytes are able to capture and present

peptide epitopes from exogenous antigens to effector T4lymphocytes. The MHC-II molecules bind peptide epitopes from exogenous antigens and place them on the surface of the B-lymphocytes. Here the MHC-II/peptide complexes can be recognized by complementary shaped T-cell receptors (TCRs) and CD4 molecules on an effector T4lymphocytes. This interaction eventually triggers the effector T4-

lymphocyte to produce and secrete various cytokines that

enable that B-lymphocyte to proliferate and differentiate into antibody-secreting plasma cells.

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Activated B cells as APC

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B Lymphocyte

T Lymphocyte

B Lymphocyte
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T Cell Surveillance for Foreign Antigens

The class I and class II pathways of antigen presentation sample available proteins for display to T cells. Most of these proteins are self proteins. Foreign proteins are relatively rare; these may be derived from infectious microbes, other foreign antigens that are introduced into the body, and tumors.

T cells survey all the displayed peptides for the presence of these rare foreign peptides and respond to the
foreign antigens.

Self peptides do not stimulate T cell responses, either because T cells with receptors for these peptides were deleted during their maturation in the thymus or the cells have been rendered inactive by recognition of the self antigen.

MHC molecules sample both the extracellular space and the cytosol of nucleated cells, and this is important because microbes may reside in both locations.

Even though peptides derived from foreign (e.g., microbial) antigens may not be abundant, these foreign antigens are recognized by the immune system because of the exquisite sensitivity of T cells.

In addition, infectious microbes stimulate the expression of costimulators on APCs that enhance T cell responses, thus ensuring that T cells will be activated when microbes are present.

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Nature of T Cell Responses

The presentation of vesicular versus cytosolic proteins by the class II or class

I MHC pathways, respectively, determines which subsets of T cells will

respond to antigens found in these two pools of proteins.
The unique specificity of T cells for cell-bound antigen is essential for the

functions of T lymphocytes, which are largely mediated by interactions requiring direct cell-cell contact and by cytokines that act at short distances.

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Immunogenicity of Protein Antigens

MHC molecules determine the immunogenicity of protein antigens in two related ways:
The epitopes of complex proteins that elicit the strongest T cell responses are the peptides that are

generated by proteolysis in APCs and bind most avidly to MHC molecules. If an individual is immunized

with a multideterminant protein antigen, in many instances the majority of the responding T cells are
specific for one or a few linear amino acid sequences of the antigen. These are called the immunodominant epitopes or determinants. The proteases involved in antigen processing produce a variety of peptides from natural proteins, and only some of these peptides possess the characteristics that enable them to bind to the MHC molecules present in each individual.

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 The expression of particular class II MHC alleles in an individual determines the

ability of that individual to respond to particular antigens. The phenomenon of genetically controlled immune responsiveness. We now know that the immune response (lr) genes that control antibody responses are the class II MHC structural genes. They influence immune responsiveness because various allelic class II MHC molecules differ in their ability to bind different antigenic peptides and therefore to stimulate specific helper T cells.

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Presentation Of Lipid Antigens By CD1 Molecules

Unconventional T Cells

Some of these T cells do not follow the rule of MHC-peptide recognition.

An exception to the rule that T cells can see only peptides is the recognition of

lipid and glycolipid antigens by a numerically rare population of T cells called NK-T cells.
These lymphocytes have many unusual properties, including the expression of

markers that are characteristic of both T cells and NK cells, and the limited diversity of their antigen receptors. NK-T cells recognize lipids and glycolipids

displayed by the class I-like "non-classical" MHC molecule called CD I. There are
several CD1 proteins expressed in humans and mice.
Although their intracellular traffic pathways differ in subtle ways, all the CDI

molecules bind and display lipids by a unique pathway.

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1.

Newly synthesized CD1 molecules pick up cellular lipids and carry these to the cell surface.

2.

From here, the CD1-lipid complexes are endocytosed into endosomes or

lysosomes, where lipids that have been ingested from the external environment are captured and the new CD1-lipid complexes are returned to the cell surface.

3.

Thus, CD1 molecules acquired endocytosed lipid antigens during recycling

and present these antigens without apparent processing. The NK-T cells that recognize the lipid antigens may play a role in defense against microbes, especially mycobacteria (which are rich in lipid components).

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T cell bacteria

TCR

CD1

internalization
Transportation to cell surface

CD1 re-expression

Degradation

phagocytosis

Vesicle maturation/ Fusion

CD1 ?? GPI bacterial glycolipid

CD1

ENDOSOMES/MIC TGN

Glycosylation
CD1 HC ??

Golgi

ER

+
b2m calnexin

+
endogenous glycosylphosphatidyl inositol (GPI)
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 T cells
A minor T cell population in the peripheral blood and lymphoid organs of human expresses an alternative TCR made up of and chains. The high number of gamma/delta-expressing T cells also found in the epithelial lining layer (skin and guts) suggests that they form a first line of defense against invading pathogens. It is thought that they may form part of the early innate immune response to pathogens. Unlike T cells, T-cell antigen receptors composed of polypeptide chains (TCRs) can directly recognize antigens in the form of intact proteins or non-peptide compounds. About 5% of peripheral blood T cells bear TCRs, most of which recognize non-peptide phosphorylated antigens.

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Unconventional T Cells
Some of these T cells do not follow the rule of MHC-peptide recognition.

NKT cells recognize CD1 presented Ag

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Intracellular trafficking of CD1a and CD1b

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References:
- Abbas A., A. Lichtman & S. Pillai, Cellular and

MolecularImmunology, Sixth edition, Saunders- Elsevier,

2007. Rich R. R., Fleisher T. A., Shearer W. T., Schroeder Jr. H. W., Frew A. J., and Weyand C. M., Clinical Immunology: Principles and Practice, third edition, 2008 Websites

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