Antigen Recognition by T-lymphocytes

1. Antigen Recognition by T-lymphocytes T-cell receptor structure/specificity MHC structure and function Antigen processing and presentation Human MHCs (HLA s� human leukocyte antigens)

2. T-lymphocyte epitope-specific receptor Similarities with the Fab portion of a BCR/antibody Two identical length chains (? and �) One antigen-binding site/receptor (one V? and one V�) C? and C� Beta sheets, intersheet bonds (non-covalent & disulfide) Domains HV and FR amino acid sequences

3. Diversity generated by somatic recombination(virtually identical to process for BCR) V? uses two gene fragments (V,J) on chromosome 14 (where C? is located) V� uses three gene fragments (V,D,J) on chromosome 7 (where C� is located) Many nucleotide sequences for each fragment Junctional diversity (P and N nucleotides � addition/deletion) Only membrane-spanning versions (all with MCs) � no secreted equivalents Initiated by RAG-1 and RAG-2 genes

4. T-cell receptor signaling TCR and �flanking� CD3 (similar to BCR and flanking Ig? and Ig�) Involved in initiating intracellular signaling cascade

5. Some clinically relevant monoclonal antibodies Antibodies for a T-cell surface molecule that can suppress the response Prevent transplantation rejection CD3 � involved in signaling T-cell to respond Anti-CD3 antibodies bind to CD3 and interfere with (prevent) the signal initiation Essentially suppresses all T-cells Not as desirable as suppression using Mab specific for CDRs of the antigen receptor of only those T-cells that specific for the �foreign� (non-self) MHCs on donor tissues (to block ability to attach to antigen)

6. TCR + MHC-presented peptide MHC presentation selects type of responding T-cell

7. The MHC molecules Class I ? chain forms the peptide-binding �groove� �-2 microglobulin provides stability Class II ? and � chains form peptide-binding �groove� TCR binds to peptide CD binds to MHC

8. The MHC molecules (in humans) Major HCs Involved in antigen presentation and also most important in determining possibility of transplant rejection (histocompatability) 6 MHC gene loci on chromosome 6 (3 for Class I and 3 for Class II) Two homologous chromosomes 6 12 total gene loci Large number of different alleles available for each locus Can be homogeneous or heterogeneous for each locus Set of 12 MHCs is your �tissue type� (for transplantation purposes) Each MHC can bind/present many similar peptides/groove Set of 12 different MHCs can present millions/trillions of different peptides (this is usually good enough to take care of �everything�)

9. Peptide characteristics Size is different for Class I and Class II grooves Class I � peptides are usually 8-10 amino acids in length Class II � wider range; 10/13-25/30 amino acids in length

10. Peptide characteristics HLA is name for MHCs in humans (human leukocyte antigens) MHC Class I � HLA-A, HLA-B, HLA-C MHC Class II � HLA-DP, HLA-DQ, HLA-R All peptides require certain amino acids occupying �anchor� amino acid positions (regardless of size) These anchor amino acids form non-covalent bonds with groove amino acids during �presentation� Other positions can be any amino acid

11. Antigen processing and presentation(all details are NOT shown in the diagram below) Produces peptides suitable for presentation by Class I and Class II MHC that presents selects for T-cell that responds (T-helper, T-cytotoxic) Virus and tumor peptides -> Class I presentation -> T-cytotoxic cells �extracellular� antigens -> Class II presentation -> T-helper cells

12. Location of antigen degradation is important Peptides generated in the cytoplasm (cytosol) will be presented by MHC Class I Peptides generated in membrane-bound vesicles will be presented by MHC Class II

13. MHC Class I antigen processing and presentation Peptides in cytoplasm viral proteins Tumor proteins (phenotypic expression of activated oncogenes) Self-proteins (damaged, no longer needed, excess)

14. Protein degradation into MHC Class I peptides Proteosome � complex of subunits/enzymes in the cytoplasm Degrades proteins into peptides Variety of sizes (many too large for MHC Class I groove) INF? (produced by NK cells during response to virus-infected cells and tumor cells) Modifies proteosome (now called an �immuno�proteosome) Favors production of peptides suitable for Class I groove (8-10 amino acids in length) Peptides are moved into ER through TAP (Transporter associated with antigen processing) TAP favors transport of peptides suitable for Class I Non-functional TAP No MHC Class I presentation No stimulation of T-cytotoxic cells �bare� lymphocyte syndrome

15. MHC Class I in endoplasmic reticulum ? chain initially stabilized by calnexin �-2 microglobulin attaches and provides stability (calnexin no longer needed) Several other molecules become associated with MHC MHC still not fully configured, but now ready to receive peptide into groove Positioned near TAP (where peptides are entering the ER) If any peptide �fits� (size and anchor amino acids) MHC-peptide moves into golgi, then by vesicle to surface to present peptide

16. MHC Class I in endoplasmic reticulum If peptide is not a perfect fit (but almost good enough) Some �trimming� of peptide may be done by ERAP (endoplasmic reticulum aminopeptidase) Immunoproteosomes, TAP, ERAP � all favor production of MHC Class I peptides

17. MHC Class II antigen processing and presentation Any type of antigen/biomolecule can be �processed� Bacteria, viruses, carbohydrate, protein, lipid, nucleic acid Peptides generated in membrane-bound vesicles (phagolysosomes) B-lymphocytes Phagocytic cells (e.g., macrophages) Tuberculosis bacteria prevent formation of phagolysosome

18. MHC Class II antigen processing and presentation Peptides produced are of various sizes (MHC Class II can bind wider range of peptide sizes, no need for any further �sizing�) MHC Class II also initially in endoplasmic reticulum Don�t want any peptides in ER to bind to MHC Class II groove Temporarily fill in Class II groove with an �invariant� chain Is a signal for MHC Class II to leave ER in a membrane-bound vesicle Still needs to bind to a peptide (has not happened in ER)

19. MHC Class II antigen processing and presentation Enzymes in vesicle are activated and cleave off most of the invariant chain Smaller CLIP remains in the groove (will be easier to remove than larger invariant chain) CLIP � class II invariant chain peptide MHC vesicle fuses with phagolysosome (which contains peptide fragments) HLA-DM removes CLIP and inserts peptide into groove MHC-peptide leaves fused vesicle and now moves to surface

21. Distribution of MHC Class I and Class II Related to cell �needs� Class I - all nucleated cells (excludes red blood cells) - can be infected with viruses and become tumor cells Class II Cells that may need �help� will be able to present MHC Class II B-lymphocytes Macrophages Cells in thymus involved in T-cell development INF? (secreted by NK cells and activated T-helper cells) Upregulates (increases) MHC Class I expression MHC Class II expressed on some cells that do not normally express it

22. TCR must also �fit� with MHC Established during �positive� selection in thymus

23. �Cross-presentation� by Dendritic Cells Usual endocytosis of bacteria, soluble antigens Class II presentation as expected (peptides in membrane-bound vesicles) Infection by viruses Class I presentation as expected (peptides in cytoplasm) DC involved in �priming� T-cells in lymph node Need to present peptides using MHC Class II and Class I Class II using �normal� endocytosis into membrane-bound vesicles Class I presentation Endocytosis of viruses (not an infection) Peptides moved into cytoplasm (Class I presentation) Viral peptides also transferred to other DC

24. DC-SIGN receptor on Dendritic Cells Dendritic Cell-specific intracellular adhesion molecule-3 grabbing non-integrin Binds to mannose-like carbohydrates (bacteria) glycoproteins (viral envelopes and some tumor cell membrane fragments) Often involved in designing vaccines that initiate T-cell priming Virus vaccines Cancer vaccines

25. Human MHCs Called HLAs (Human Leukocyte Antigens) Gene loci all on chromosome 6 Major HLAs (most important for transplant rejection) Class I � HLA-A, HLA-B, HLA-C Class II � HLA-DP, HLA-DQ, HLA-DR Also all other molecules involved in antigen processing and presentation TAP, LMP (proteosome)

26. Human MHCs Your set of MHCs (your tissue type) Two homologous chromosome 6 (one from mother, one from father) 6 MHC gene loci/chromosome (3 for Class I, 3 for class II) 12 total MHC gene loci in any one person Very large number of possible alleles for each gene locus Most persons are completely heterozygous for each locus Set of 12 MHC is �unique� except for: Identical twins Some siblings if inherit same chromosomes from mother and father Random chance of tissue type match US caucasian population ~ 1:20,000

27. HLAs in transplant rejection Positive selection � shape of TCR will �fit� shape of MHC Often called MHC �restriction� (i.e., TCR can only fit with set of MHCs in that person�s body) Some T-cells survive positive selection Still could not respond to peptides presented by that person�s own MHC Some can respond to peptides presented by another person�s MHCs (allogeneic MHCs) T-cytotoxic cells responding to �foreign� MHC-presented peptides Destroy presenting cells Transplant rejection

28. Why so many different HLAs in human population? Immune system has evolved over eons Fewer different HLAs would make transplantation much less complicated Immune system not designed to deal with transplants (a very recent phenomenon) Is designed to deal with pathogenic �threats� Diversity is probably influenced by Continuous appearance of new pathogens Mutations that affect peptide-binding grooves (where most diversity among HLAs occur) Mutated HLAs that contribute to survival from pathogens are retained HLA heterozygosity may influence survival from HIV infection Red line � completely heterozygous for all HLA Class I loci Yellow line � homozygous for one of the HLA Class I loci Blue line � homozygous for two or three HLA Class I loci