Lectures 5 & 6 Antigen Recognition by T Lymphocytes

Lecture objectives Big questions? How TCR recognizes antigens? Why many autoimmune diseases are associated with particular types of MHC genotypes? Why MHC molecules are the major antigens responsible for transplantation rejections? Structure of TCRs CDRs Differences of recognized antigen between TCR and immunoglobulins (Ig) TCR germ line configuration and rearrangements TCR specificity Clonality of T cells Differences between TCR a and b chains Names of human MHC I and II genes. How antigens are processed for presentation on MHC I and II? MHC class I and II molecules sample and present antigens of different origins. How? MHC polymorphism How many different MHCs a person can express? Why? Structures of MHC I and II HLA typing

Core content

A big picture: How do T cells recognize antigens? MHC molecule TCR

Similarity between TCR and Ig Both: Bind antigen Have variable region Constant region Each binding site is a heterodimer (composed of 2 different chains) TCRs act only as receptors Igs act as receptors and effector molecules (soluble antigen-binding molecules)

Figure 3-2

TCR genes undergo DNA rearrangement in thymus *No Ds in Va gene; DJ first then VDJ in b gene rearrangement Un-rearranged rearranged expression rearranged Un-rearranged

If you can not make antigen receptors as in the case of SCID patients, Figure 3-4 you can not make T and B cells, and are susceptible even to opportunistic pathogens (e.g. C. albicans)

TCR complex Figure 3-6 CD3 chains transmit signals

Figure 3-7

Figure 3-8 part 1 of 2

Figure 3-8 part 2 of 2 gd T cells recognize a limited set of antigens: Host MHC class 1b: T10/22, MICA/B Nonproetin alkylamines Bacterial products: Mycobacterial Hsp, superantigens (SEA) Host heat shock proteins Transformed or stressed host cells

Antigen processing is required to present antigen peptides to TCR Antigen processing is required to present antigen peptides to TCR. TCRs bind short antigen peptides but not whole antigen proteins Figure 3-9

The structures of CD4 and CD8 Figure 3-10

MHC class I molecules present antigens to CD8+ T cells, and MHC class II molecules present antigens to CD4+ T cells: Figure 3-12

Figure 3-13 part 1 of 2

Figure 3-13 part 2 of 2

Figure 3-14

The peptide-binding groove of MHC molecules Figure 3-15

Figure 3-16

Two different types of antigens: Extracellular for MHC II and intracellular for MHC I They are generated in different compartments

Transport of Cytosolic Peptides into ER Role of transporter associated with antigen processing, TAP Figure 3-17 Bare lymphocyte syndrome: No TAP expression  no MHC I expression  no CD8 T cells  defective cytotoxic activity against virus-infected cells chronic respiratory infection TAP=TAP1+TAP2 Proteosome: a barrel shaped protein complex composed of 28 subunits 8 or more AA-long polypeptides are transported MHC I cannot leave ER without loaded peptides

Assembly of antigen peptide/MHC class I complex Assembly of antigen peptide/MHC class I complex. Molecular chaperons (calnexin, calreticulin and tapasin) aid the folding of MHC I and loading of peptides Figure 3-18 In the absence of infection, self peptides are presented on MHC, but do not activate T cells. A HSV protein inhibits TAP function, and an Adeno virus protein inhibits MHC I expression.

Figure 3-20 Assembly of antigen peptide/MHC class II complex -Extracellular microorganisms are taken up by macrophages via phagocytosis, and by B cells by cell surface Ig-mediated endocytosis -MHC II molecules bind peptides in the fused vesicles, not in ER -Invariant chain, CLIP and HLA-DM guide the peptide loading -After losing CLIP, MHC II must bind peptides or gets degraded. -Certain pathogens (e.g. mycobacteria), when engulfed, prevent the fusion of phagosomes and lysosomes, and persist in phagosomes.

Figure 3-21

TCR recognition of antigens induce T cell activation, functional maturation, and killing/activation of target cells Figure 3-11

Different cell distribution of MHC class I and II Figure 3-22 All cells express MHC I for comprehensive surveillance by CD8 T cells Only some cells express high levels of MHC II and MHC I These are B cells, macrophages, dendritic cells and thymic epithelial cells. B cells, macrophages and dendritic cells are called professional antigen- presenting cells (APC). IFN-g increases the expression of MHC II in APC and induces the expression in non-APC cells at sites of infection

Figure 3-23

Figure 3-24 part 1 of 2 CD8/NK NK Remains intracellular NK MHC CLASS I molecules form ligands to activate CD8+ cells and inhibit NK cells

Polymorphism: presence of multiple alternative forms (alleles) of a gene. Help peptide loading With antigen peptides activate CD4+ T cells Polymorphism allows the population can handle a variety of pathogens.

Major Histocompatibility Complex (=HLA locus) MHC molecules in human is also called HLA (human leukocyte antigen) Class I and II locus. HLA-DR alpha chain is monomorphic HLA-DRB1 is most polymorphic in MHC II genes HLA-DRB1 is always present in any individual HLA-DRB3/4/5 is present in some but not all people. A heterozygote person expresses two haplotypes. A person can express 3-6 class I and 3-8 class II isoforms. 2198 possible class II isoforms in the human population. 440 MHC I isoforms in the human population. [MHC isoforms]  [presentable antigen peptides]  [TCR repertoire]

Figure 3-25

Figure 3-27

Genetics of MHC gene expression: both alleles are expressed (co-dominant) In any mating, four possible combinations of haplotypes can be found in the offspring; thus siblings are also likely to differ in the MHC allele they express. Halplotype: The particular combination of MHC alleles found on a given chromosom 6.

Figure 3-26

Figure 3-28

Each MHC isoform binds a characteristic set of peptides -Anchor residues in peptides are important for binding to MHC -Not all residues are important Figure 3-29 Degenerate binding allows each MHC molecule handles many peptides.

T cell receptor recognition of antigens is MHC-restricted Figure 3-30

Figure 3-31 Being heterozygous for MHC is advantageous in presenting antigens

Figure 3-32 Pathogens can select MHC polymorphism in human population A scenario: Selection 2 as the result of epidemic of a new pathogen Selection 1 as the result of multiple successive infections

MHC Heterozygocity makes AIDS+ people more resistant to HIV Complete Heterozygocity Homozygocity

HLA typing: how? Microcytotoxicity assay for detection of HLA antigens Anti-HLA serum, or monoclonal antibody, is mixed with live lymphocytes. Specific Ig binds to the polymorphic protein moiety of the HLA molecule expressed on the cell surface. Exogenous complement is added to the well which will result in lysis of cells to which antibody has been bound. Cell death is determined by ethidium bromide vital stain exclusion. Flow cytometry or ELISA Monoclonal antibodies to different MHC alleles have been generated. Using panels of these antibodies, HLA typing before transplantation is possible. RFLP: Restriction Fragment Length Polymorphism Digestion of genomic DNA with certain restriction enzymes followed by hybridization with radio-labeled MHC gene probes gives MHC isotype-specific digestion patterns. PCR: Polymerase Chain Reaction PCR using MHC gene-specific primers