Major Histocompatibility Complex&

Antigen Processing and Presentation

Sreeraj EBPS051318PlantScience

MHC molecules

• Major Histocompatibility Complex

– Cluster of genes found in all mammals

– Its products play role in discriminating self/non-self

– Participant in both humoral and cell-mediated immunity

• Act as antigen presenting structures

• Polymorphic (genetically diverse) glycoproteins

• Cross over rate is low (0.5 %)

• Alleles are co-dominantly expressed

• Promiscous binding to peptides

• In Humans - Chromosome 6 & referred to as HLA complex

• In Mice - Chromosome 17 & referred to as H-2 complex

MHC-encoded -chain of 43kDa

Structure of MHC class I molecules

3 domain & 2m have structural & amino acid sequence homology with Ig C domains Ig GENE SUPERFAMILY

2-microglobulin, 12kDa, non-MHC encoded, non-transmembrane, non covalently bound to -chain

3 Highly Conserved Among MHC I Molecules Interacts

with CD8+ TCyt cell

Peptide antigen in a groove formedfrom a pair of -helicies on a floor of anti-parallel strands

-chain anchored to the cell membrane

MHC Class I

• The α3 segment of the MHC I serves as a binding site for CD8.

• The β2-microglobulin interacts with the α3 non-covalently.

• Class I MHC is found in almost all nucleated cell.

• Presents endogenous antigen.

Structure of MHC class I molecules

Properties of the inner faces of the helices and floor of the cleft determine which peptides bind to the MHC molecule

Chains Structures

• The α chains forms a platform of 8-stranded anti-parallel β pleated sheet supporting two parallel strands of α-helix.

• The formed cleft can bind peptides of 8 -10 amino acids in a flexible extended conformation.

Structure of MHC class II molecules

• MHC-encoded, -chain of 34kDa

• A -chain of 29kDa

• and chains anchored to the cell membrane

• NO -2 microglobulin

• Peptide antigen in a groove formed from a pair of -helicies on a floor of anti-parallel strands

• Found in APCs,presents exogenous antigen.

• 2 & 2 domains have structural & amino acid sequence homology with Ig C domains Ig GENE SUPERFAMILY

MHC class I

MHC class II

Cleft geometry

Peptide is held in the cleft by non-covalent forces

MHC class I accommodatepeptides of 8-10 amino acids

Cleft geometry

MHC class II accommodatepeptides of >13 amino acids

2-M

-chain

Peptide

-chain

-chainPeptide

Anchor residues and T cell antigen receptorcontact residues

Cell surface

MHC class ISliced between-helicies to revealpeptide

T cell antigen receptor contactresidue side-chains point up

MHC anchor residueside-chains point down

Anchor residue: The peptide residue that anchor the antigenic peptide into the MHC groove.

YI

MHC molecule

YI

MHC molecule

Complementary anchor residues & pockets provide the broad specificity of a particular type of MHC molecule for peptides

MHC molecules can bind peptides of different length

P S

ASI

K

S

P SA IK S

Peptide sequence between anchors can varyNumber of amino acids between anchors can vary

Archedpeptide

A flexible binding site?

A binding site that is flexible at an early, intracellular stage of maturation Formed by folding the MHC molecules around the peptide.

Floppy Compact

Allows a single type of MHC molecule to • bind many different peptides• bind peptides with high affinity• form stable complexes at the cell surface• Export only molecules that have captured a

peptide to the cell surface

Peptides can be eluted from MHC molecules

Purify stable MHC-peptide complexes

Fractionate and microsequencepeptides

Acid elute peptides

Eluted peptides from MHC molecules have different sequences but contain motifs

Peptides bound to a particular type of MHC class I molecule have conserved patterns of amino acids

P E IYS F H I

A V TYK Q T L

P S AYS I K I

R T RYT Q L VN C

Tethering amino acids need not be identical but must be relatedY & F are aromaticV, L & I are hydrophobic

Side chains of anchor residues bind into POCKETS in the MHC molecule

S I I FN E K L

A P G YN P A L

R G Y YV Q Q L

Different types of MHC molecule bind peptides with different patterns of conserved amino acids

A common sequence in a peptide antigen that binds to an MHC molecule is called a MOTIF

Amino acids common to many peptides tether the peptide to structural features of the MHC moleculeANCHOR RESIDUES

Genetic map of MHC

Human Leukocyte Antigen (HLA)

• In humans, there are three MHC Class I α-chain genes, called:

HLA -A, HLA-B, and HLA-C

• There are also three pairs of MHC Class II α- and β-chain genes, called :HLA-DR, HLA-DP, and HLA-DQ.

The genes of the MHC locus

Other genes in the MHC

MHC Class 1b genes

Encoding MHC class I-like proteins that associate with -2 microglobulin:

HLA-G binds to CD94, an NK-cell receptor. Inhibits NK attack of foetus/ tumoursHLA-E binds conserved leader peptides from HLA-A, B, C. Interacts with CD94HLA-F function unknown

MHC Class II genesEncoding several antigen processing genes:HLA-DM and , proteasome components LMP-2 & 7, peptide transportersTAP-1 & 2, HLA-DO and DOMany pseudogenes

MHC Class III genesEncoding complement proteins C4A and C4B, C2 and FACTOR BTUMOUR NECROSIS FACTORS AND

Immunologically irrelevant genesGenes encoding 21-hydroxylase, RNA Helicase, Caesin kinaseHeat shock protein 70, Sialidase

B C ADP DQ DR

1

Polygeny

B C ADP DQ DR

1Variant allelespolymorphism

Genes in the MHC are tightly LINKED and usually inherited in a unit called an MHC HAPLOTYPE

B C ADP DQ DR

1Additional set of variant alleles on second chromosome

MHC molecules are CODOMINANTLY expressedTwo of each of the six types of MHC molecule are expressed

Diversity of MHC molecules in the individual

HAPLOTYPE 1

HAPLOTYPE 2

Populations need to express variantsof each type of MHC molecule

• Populations of microorganisms reproduce faster than humans

• Mutations that change MHC-binding antigens or MHC molecules can only be introduced to populations after reproduction

• The ability of microorganisms to mutate in order to evade MHC molecules will always outpace counter evasion measures that involve mutations in the MHC

• The number of types of MHC molecules are limited

To counteract the superior flexibility of pathogens:

Human populations possess many variants of each type of MHC molecule

Variant MHC may not protect every individual from every pathogen.However, the existence of a large number of variants means that the population is prevented from extinction

Limited diversity in HLA gene polymorphism due to breeding bottle-neck in recent past leaves cheetahs exceptionally susceptible to viral infections. (6th Ed. P. 206)

• Expression is increased by cytokines such as IFN, IFN, IFN and TNF

• Transcription factors like CIITA (Transactivator), RFX (Transactivator) increase MHC gene expression

• Some viruses (CMV, HBV, Ad12) decrease MHC expression

• Reduction of MHC may allow for immune system evasion

MHC Expression

HLA vs. Transplantation

• HLA matching between the donor and recipient - a predictor of graft survival in the kidney allograft patient

• HLA mis-matches may cause chronic pathologies in the allograft

• It is clear that a negative HLA-A2 recipient receiving an allograft expressing HLA-A2 will have a much higher risk of humoral rejection of the vascular endothelium.

IMMUNE GRAFT REJECTION

TcRTcRTcR

Molecular basis of transplant rejection

MHC A MHC B MHC C

Normal peptiderecognition

Indirect peptiderecognition

Direct peptiderecognition

Differential distribution of MHC molecules

Cell activation affects the level of MHC expression.

The pattern of expression reflects the function of MHC molecules:

• Class I is involved in the regulation of anti-viral immune responses

• Class II involved in regulation of the cells of the immune system

Anucleate erythrocytes can not support virus replication -hence no MHC class I.

Some pathogens exploit this -e.g. Plasmodium species.

Tissue MHC class I MHC class II

T cells +++ +/-

B cells +++ +++Macrophages +++ ++Other APC +++ +++

Thymus epithelium + +++

Neutrophils +++ -Hepatocytes + -Kidney + -Brain + -Erythrocytes - -

Superantigens Bacterial superantigensact by binding to both the MHC protein and the TCR at positions outside the normal binding site

superantigens can interact with large numbers of cells, stimulating massive T-cell activation, cytokine release and systemic inflammation

Conventional Antigen

αC βC

CHO CHO

CHOCHO

βVαV

α2 β2

β1α1CHO CHO

CHO

αC βC

CHO CHO

CHOCHO

βVαV

α2 β2

β1α1CHO CHO

CHO

MHC Class II

T cell receptor

AntigenSuper

antigen

T lymphocyte

Antigen presenting cell

Superantigen

Antigen Processing and Presentation

MHC-restricted antigen recognition by T cells

• Any T cell can recognize an antigen on an APC only if that antigen is displayed by MHC molecules

– Antigen receptors of T cells have dual specificities:

1. for peptide antigen (responsible for specificity of immune response) and

2. for MHC molecules (responsible for MHC restriction)

– During maturation in the thymus, T cells whose antigen receptors see MHC are selected to survive and mature; therefore, mature T cells are “MHC-restricted”

Antigen processing

• Conversion of native antigen (large globular protein) into peptides capable of binding to MHC molecules

• Occurs in cellular compartments where MHC molecules are synthesized and assembled (ER)

– Determines how antigen in different cellular compartments generates peptides that are displayed by class I or class II MHC molecules

What Does the αβ T Cell Receptor (TCR) Recognize?

1. Only fragments of proteins (peptides) associated with MHC molecules on surface of cells

• Helper T cells CD4+(Th) recognize peptide associated with MHC class II molecules

• Cytotoxic T cells CD8+ (Tcyt) recognize peptide associated with MHC class I molecules.

Antigen-presenting cells

Dendritic cells

Protein antigen in cytosol (cytoplasm) --> class I MHC pathway Protein antigen in vesicles --> class II MHC pathway

Pathways of antigen processing

Class I MHC moleculesEndogenous Antigens: The Cytosolic Pathway

• Cytotoxic T lymphocytes need to kill cells containing cytoplasmic microbes, and tumor cells (which contain tumor antigens in the cytoplasm)

• Cytosolic proteins are processed into peptides that are presented in association with class I molecules

• Most cytosolic peptides are derived from endogenously synthesized (e.g. viral, tumor) proteins

• All nucleated cells (which are capable of being infected by viruses or transformed) express class I

The class I MHC pathway of processingof endogenous cytosolic protein antigens

Cytoplasmic peptides are actively transported into the ER;class I MHC molecules are available to bind peptides in the ER

Cytosolic proteolytic system for degradation of intracellularproteins.

• Proteins to be degraded are often covalently linked to a small protein called ubiquitin.

• A ubiquinating enzyme complex links several ubiquitin molecules to a lysine-amino group near the amino terminus of the protein.

• Degradation of ubiquitin-protein complexes occurs within the central channel of proteasomes, Proteasomes are large cylindrical particles whose subunits catalyze cleavage of peptide bonds.

• In the cytosol,association of LMP2, LMP7, and LMP10 with a proteasome changes its catalytic specificity to favor production of peptides that bind to class I MHC molecules. All three are induced by increased levels of the T-cell cytokine IFN-γ

• Peptides generated in the cytosol by the proteasome are translocated by TAP (transporter associated with antigen processing )into the RER by a process that requires the hydrolysis of ATP.

• TAP has the highest affinity for peptides containing 8–10 amino acids, which is the optimal peptide length for class I MHC binding.

• Within the RER membrane, a newly synthesized class I chain associates with calnexin until β2-microglobulin binds to the chain.

• The class I αchain/β2-microglobulin heterodimer then binds to calreticulinand the TAP-associated protein tapasin.

• When a peptide delivered by TAP is bound to the class I molecule, folding of MHC class I is complete and it is released from the RER and transported through the Golgi to the surface of the cell.

Assembly and stabilization of class I MHC molecules.

• Newly formed class I chains associate with calnexin, a molecular chaperone, in the RER membrane.

• Subsequent binding to β2-microglobulin releases calnexin and allows binding to the chaperonin calreticulin and to tapasin, which is associated with the peptide transporter TAP.

• This association promotes binding of an antigenic peptide, which stabilizes the class I molecule–peptide complex, allowing its release from the RER

Class I MHC Pathway

Viral protein is made

on cytoplasmic

ribosomes

Plasma membrane

Proteasome

degrades

protein to

peptides

Peptide transporter

protein moves

peptide into ER

MHC class I alpha

and beta proteins

are made on the rER

Peptide associates

with MHC-I complex

Peptide with MHC

goes to Golgi body

Peptide passes

with MHC from Golgi

body to surface

Peptide is presented

by MHC-I to CD8

cytotoxic T cell

Golgi body

rER

Globular viral

protein - intact

CD8 CTL

Tumour cell

Exogenous Antigens: The Endocytic Pathway

Loading of antigen to MHC class II

Generation of antigenic peptides in the endocyticprocessing pathway.

• Internalized exogenous antigen moves through several acidic compartments, in which it is degraded into peptides that ultimately associate with class II MHC molecules transported in vesicles from the Golgi complex.

Assembly of class II MHC molecules

• Within the rough endoplasmic reticulum, a newly synthesized class II MHC molecule binds an invariant chain.

• The bound invariant chain prevents premature binding of peptides to the class II molecule and helps to direct the complex to endocyticcompartments containing peptides derived from exogenous antigens.

• Digestion of the invariant chain leaves CLIP, a small fragment remaining in the binding groove of the class II MHC molecule.

• HLA-DM, a nonclassical MHC class II molecule expressed within endosomal compartments, mediates exchange of antigenic peptides for CLIP.

How class I- and class II-associated antigen presentationinfluence the nature of the host T cell response

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