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M261 ANTIGEN PRESENTATION/MHC CLASS II • • • • Pete Sieling Phone: 825-6964 email: [email protected] Office: 52-127 CHS • Reading for Wednesday (4-20) and Friday (4-22) lectures: Chapter 5 (pp169-198), Janeway et al. 6th edition. ANTIGEN PRESENTATION MHC CLASS II • Importance of dendritic cells to T cell activation. • Compare and contrast MHC I and MHC II peptide binding. • MHC class II antigen processing and presentation pathway. • Mechanisms of immune evasion. • Cross-presentation. • CD1 antigen presentation pathway. • Translational therapies targeting MHC antigen presentation pathways. MOLECULES OF T LYMPHOCYTE RECOGNITION CD8 T-CELL CD4 T-CELL a b TCR ab CD4 b1 MHC CLASS II b CD3 b2 a1 a CD8 15 aa peptide a2 ANTIGEN PRESENTING CELL a b TCR ab a2 MHC CLASS I CD3 a3 a1 9 aa peptide b 2m ANTIGEN PRESENTING CELL WHAT DISTINGUISHES MHC CLASS I FROM MHC CLASS II ANTIGEN PRESENTATION? Expression: Source of Ag: T cell co-receptor: MHC protein: Salient characteristic: MHC class I MHC class II all nucleated cells endogenous CD8 single chain (heavy) peptide transporter professional APCs exogenous CD4 two chains (a and b) peptide loading comp. DENDRITIC CELLS INITIATE T CELL RESPONSES BY PRESENTING ANTIGENS TO NAÏVE T CELLS Text Figure 8.14 TWO SIGNAL HYPOTHESIS OF NAÏVE T CELL ACTIVATION (e.g. mature dendritic cell) TWO SIGNALS ARE REQUIRED FOR A NAÏVE T CELL TO BECOME ACTIVATED, OTHERWISE ANERGY (UNRESPONSIVENESS) IS INDUCED CONSERVATION OF STRUCTURE IN MHC MOLECULES AND PEPTIDE-BINDING GROOVE • • The a1 and a2 portions of MHC class I and a1 and b1 of MHC class II form mirror images of each other to create the peptide binding groove. Despite modest sequence homology, MHC class I and II have evolved to create similar, though not identical binding grooves from a single chain (MHC I) or two chains (MHC II). STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE • • MHC class I molecules bind 8-10 amino acid peptides whereas MHC class II bind 12 or longer peptides. MHC class II more promiscuous about length of peptide bound. Mouse H2Kb Mouse I-Ak Human HLA-DR3 STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE MHC class I + peptide MHC class II + peptide MHC CLASS II ANTIGEN PROCESSING • • • • • MHC class II molecules present antigens taken up by the cell through endocytosis. MHC polypeptides (a and b) are synthesized on ER and are chaperoned to a specialized antigen loading compartment by invariant chain (Ii); invariant chain serves two purposes, it functions as a chaperone and occupies the peptide binding groove to stabilize MHC class II and prevent other peptides (self) from binding until MHC arrives in the loading compartment. Invariant chain is cleaved by acid proteases until it leaves only the peptide-binding portion (CLIP) in MHC. Endocytosed antigens are also cleaved by acid proteases; low pH requirement for proteases to become activated. HLA-DM catalyzes the removal of CLIP and binding of antigenic peptide. pH 7 Text Figure 5.10 pH 5 pH 5 pH 5 THE ROLE OF PROTEOLYSIS IN MHC CLASS II PRESENTATION • • • Proteolysis for MHC class II antigen presentation occurs in endocytic vesicles by proteases that are active at low pH. Cysteine proteases such as the cathepsins degrade proteins into short peptides for MHC class II presentation. Invariant chain is also degraded by cathepsins into CLIP. THE ROLE OF CATHEPSINS IN MHC CLASS II ANTIGEN PRESENTATION • Cathepsin L is especially important in thymic epithelial cells, which present antigen for positive selection of thymocytes. • Cathepsin S seems to be especially important in B cells and dendritic cells, less so for macrophages. Nakagawa et. al., Immunity 10:207, 1999 I-Ab Ii CLIP Nakagawa et. al., Science 280:450, 1998 ROLE OF HLA-DM IN THE MHC CLASS II PATHWAY Sloan, et. al., Nature 375:802, 1995 • HLA-DM facilitates the removal of CLIP and binding of antigenic peptide into the MHC class II binding groove. • HLA-DM activity is pHdependent. Biotinylated peptide b1 a1 b2 a2 MHC class II b1 Protein concentration Streptavidin fluorescent tag a1 b2 a2 HLA-DM or control protein Anti-MHC in microtiter plate Fluorescence measurement pH MHC CLASS II PROTEINS ARE LOADED WITH PEPTIDE IN A SPECIALIZED ENDOCYTIC COMPARTMENT (MIIC) • MHC class II proteins are enriched in multilamellar vesicles called MIICs (MHC class II compartments) that are distinct from other intracellular vesicles. MHC class II Late endosome Tulp et. al., Nature 369:120, 1994 Early endosome Amount (arbitrary units) 12 Lysosomes Plasma membrane lysosome 10 MHC class II 8 particle 6 4 Golgi 2 ER 0 0 5 10 15 20 Fraction number Text Figure 5.9 MECHANISMS OF IMMUNE EVASION (MHC CLASS II) • Mycobacteria prevent acidification of endosomes. • Inhibition of acidification will prevent proteases from being activated. • Without proteases, mycobacterial proteins won’t be processed or loaded into MHC class II. Mycobacteria Control Leishmania Control Sturgill-Koszycki, et. al., Science 263:678, 1994 pH HOW DO NAÏVE CD8 T CELLS BECOME ACTIVATED WHEN THE INFECTED CELL IS NOT A PROFESSIONAL APC? BONE MARROW CHIMERAS • Used to determine the function of certain immune cells of one background against the nonimmune cells of another background. Irradiate recipient mouse (kills hematopoietic cells from which immune cells are derived) and inject bone marrow cells from donor mouse. In this way, you populate a mouse of one genetic background with the immune system from a mouse of another genetic background. CROSS PRESENTATION • CTL immunity to virus-infected non-hematopoietic cells requires presentation of exogenous antigen. Question addressed: Do non-hematopoietic cells infected with virus activate CTLs to kill infected cells or do hematopoietic cells take up exogenous peptides from environment, present them to T cells? gPVR gPVR-expressing cell (recipient) Polio-OVA Peptides Hematopoietic cell (donor) Sigal, et. al., Nature 398:77-80, 1999. CONTROL OF CROSS PRESENTATION BY THE MHC CLASS I CYTOPLASMIC DOMAIN Transfected fibroblasts/CTL Transgenic mice Lizee, et. al., Nature Immunol 4:1065-1073, 2003. ER-PHAGOSOME FUSION CREATES A CROSS PRESENTATION COMPARTMENT OVA Guermonprez, et. al., Nature 425:397-402, 2003 TAP2 MHC class I CROSS PRESENTATION PATHWAY Houde, et. al., Nature 425:402-406, 2003. THE POWER OF MHC TETRAMERS • MHC heavy chains are engineered with linker to create tetramer. • Peptide is added in solution or engineered to covalently bind to MHC binding groove. • HLA-A2 tetramers are labeled with a fluorescent tag that allows one to use flow cytometry to determine the frequency of antigen-reactive T cells; previously this was evaluated by limiting dilution analysis. Altman, et. al., Science 274:94, 1996 HLA AND DISEASE ASSOCIATION • Some diseases are associated with specific MHC alleles, though the role that MHC plays in the disease isn’t clear. MOLECULES OF T-LYMPHOCYTE RECOGNITION CD4 T-CELL a b CD4 CD3 a b TCR ab CD8 CD3 a b MHC CLASS I b 2m ANTIGEN PRESENTING CELL CD3 TCR ab TCR ab 9 aa peptide 15 aa peptide MHC CLASS II T-CELL (ALL PHENOTYPES) CD8 T-CELL lipid CD1 b 2m DIFFERENT CD1 MOLECULES TRAFFIC TO DISTINCT INTRACELLULAR LOCATIONS IN HUMAN DC CD1 LAMP CD1+LAMP CD1a CD1b CD1c Sugita, et al. Immunity 2000 Sugita, et al Traffic 2000 MYCOBACTERIAL ANTIGENS THAT ACTIVATE CD1-RESTRICTED T CELLS Mycobactin CD1a antigen Mycolic acid Phosphatidylinositol mannoside Glucose monomycolate Mannosyl-b1phosphoisoprenoid CD1b antigens CD1c antigen EXAMPLES OF MHC II EPITOPES THAT REQUIRE PROCESSING IN DISTINCT SUBCELLULAR COMPARTMENTS Antigen: Early Endosome: MIIC: 46-61 - 35-45 - 116-129 - 17-31 - 308-319 - - HEL (Zhong et al. 1997) S. pyogenes M5 (Delvig et al. 1998) Influenza Virus (Pinet et al. 1998) HA H3 307-318 TRANSLATIONAL THERAPIES TARGETING MHC ANTIGEN PRESENTATION PATHWAYS CD4+ T-CELL 1 TCR CD4 LATE ENDOSOME/ LYSOSOME/M II C Ag Classical Pathway Alternative Pathway MHC II ? EARLY ENDOSOME ? ? GOLGI M II C TCR CD4+ T-CELL 2 TRANSLATIONAL THERAPIES TARGETING MHC ANTIGEN PRESENTATION PATHWAYS Leader Ag LAMP-1 Immunize with DNA Measure immunological functions Challenge with microbial pathogen and measure survival % survival 120 100 IFN-g IFN-g 80 CD4 Unimmunized 60 Immunized 40 Unimmunized 20 CD4 Immunized 0 0 5 10 15 Time (weeks) 20 25 CD8+ T-CELL TCR MHC CLASS I PATHWAY CD8 Ag PROTEOSOME CD4+ T-CELL TAP TCR ER MHC CLASS I M II C CD4 MHC CLASS II pH<5 GOLGI Ag MHC CLASS II PATHWAY ENDOSOME