1. Pete Sieling Phone: 825-6964 email: psieling@mednet.ucla.edu psieling@mednet.ucla.edu Office: 52-127 CHS Reading for Wednesday (4-20) and Friday (4-22) lectures: Chapter 5 (pp169-198), Janeway et al. 6th edition. M261 ANTIGEN PRESENTATION/MHC CLASS II

2. 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.

3.   CD4 T-CELL TCR  CD4 CD3 9 aa peptide 15 aa peptide CD8 T-CELL CD8   TCR  CD3   ANTIGEN PRESENTING CELL MHC CLASS II 11 11 22 22 MOLECULES OF T LYMPHOCYTE RECOGNITION CLASS I MHC 2m2m 22 11 33 ANTIGEN PRESENTING CELL

4. WHAT DISTINGUISHES MHC CLASS I FROM MHC CLASS II ANTIGEN PRESENTATION? MHC class IMHC class II Expression:all nucleated cellsprofessional APCs Source of Ag:endogenousexogenous T cell co-receptor:CD8CD4 MHC protein:single chain (heavy)two chains (  and  ) Salient characteristic:peptide transporterpeptide loading comp.

5. DENDRITIC CELLS INITIATE T CELL RESPONSES BY PRESENTING ANTIGENS TO NAÏVE T CELLS Text Figure 8.14

6. TWO SIGNAL HYPOTHESIS OF NAÏVE T CELL ACTIVATION (e.g. mature dendritic cell)

7. TWO SIGNALS ARE REQUIRED FOR A NAÏVE T CELL TO BECOME ACTIVATED, OTHERWISE ANERGY (UNRESPONSIVENESS) IS INDUCED

8. CONSERVATION OF STRUCTURE IN MHC MOLECULES AND PEPTIDE-BINDING GROOVE The  1 and  2 portions of MHC class I and  1 and  1 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).

9. 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 I-A k Human HLA-DR3 Mouse H2K b

10. STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE MHC class I + peptide MHC class II + peptide

12. MHC CLASS II ANTIGEN PROCESSING MHC class II molecules present antigens taken up by the cell through endocytosis. MHC polypeptides (  and  ) 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. Text Figure 5.10 pH 7 pH 5

13. 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.

14. 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. Nakagawa et. al., Immunity 10:207, 1999 Cathepsin S seems to be especially important in B cells and dendritic cells, less so for macrophages. Nakagawa et. al., Science 280:450, 1998 I-A b IiCLIP

15. ROLE OF HLA-DM IN THE MHC CLASS II PATHWAY HLA-DM facilitates the removal of CLIP and binding of antigenic peptide into the MHC class II binding groove. HLA-DM activity is pH- dependent. pH Fluorescence measurement MHC class II 11 11 22 22 Anti-MHC in microtiter plate 11 11 22 22 HLA-DM or control protein Protein concentration Sloan, et. al., Nature 375:802, 1995 Biotinylated peptide Streptavidin fluorescent tag

16. lysosome MHC class II 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. Text Figure 5.9 MHC class II Late endosome Lysosomes ER Golgi Plasma membrane Early endosome 0 2 4 6 8 10 12 05101520 Fraction number Amount (arbitrary units) Tulp et. al., Nature 369:120, 1994 particle

17. 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. Sturgill-Koszycki, et. al., Science 263:678, 1994 Mycobacteria Control Leishmania Control pH

18. HOW DO NAÏVE CD8 T CELLS BECOME ACTIVATED WHEN THE INFECTED CELL IS NOT A PROFESSIONAL APC?

19. BONE MARROW CHIMERAS Used to determine the function of certain immune cells of one background against the non- immune 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.

20. 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? Polio-OVA Hematopoietic cell (donor) gPVR gPVR-expressing cell (recipient) Peptides Sigal, et. al., Nature 398:77-80, 1999.

21. CONTROL OF CROSS PRESENTATION BY THE MHC CLASS I CYTOPLASMIC DOMAIN Lizee, et. al., Nature Immunol 4:1065-1073, 2003. Transfected fibroblasts/CTL Transgenic mice

22. ER-PHAGOSOME FUSION CREATES A CROSS PRESENTATION COMPARTMENT Guermonprez, et. al., Nature 425:397-402, 2003 OVA TAP2 MHC class I

23. CROSS PRESENTATION PATHWAY Houde, et. al., Nature 425:402-406, 2003.

24. 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

25. HLA AND DISEASE ASSOCIATION Some diseases are associated with specific MHC alleles, though the role that MHC plays in the disease isn’t clear.

26. CD4 T-CELL   TCR  MHC CLASS II CD4 CD3 CD8 T-CELL ANTIGEN PRESENTING CELL   TCR  MHC CLASS I CD8 CD3 T-CELL (ALL PHENOTYPES)   TCR  CD1 CD3 MOLECULES OF T-LYMPHOCYTE RECOGNITION 9 aa peptide 2m2m 2m2m 15 aa peptide lipid

27. DIFFERENT CD1 MOLECULES TRAFFIC TO DISTINCT INTRACELLULAR LOCATIONS IN HUMAN DC Sugita, et al Traffic 2000 CD1 LAMPCD1+LAMP CD1a CD1b CD1c Sugita, et al. Immunity 2000

28. MYCOBACTERIAL ANTIGENS THAT ACTIVATE CD1-RESTRICTED T CELLS Mannosyl-  1- phosphoisoprenoid Glucose monomycolate Mycolic acid Phosphatidylinositol mannoside CD1b antigensCD1c antigenCD1a antigen Mycobactin

29. EXAMPLES OF MHC II EPITOPES THAT REQUIRE PROCESSING IN DISTINCT SUBCELLULAR COMPARTMENTS Antigen: Early Endosome: MIIC: HEL (Zhong et al. 1997) 46-61 -  35-45  - 116-129  - S. pyogenes M5 (Delvig et al. 1998) 17-31  - 308-319 -  Influenza Virus (Pinet et al. 1998) HA H3 307-318  - M1 18-29 - 

30. CD4+ T-CELL 1 TRANSLATIONAL THERAPIES TARGETING MHC ANTIGEN PRESENTATION PATHWAYS EARLY ENDOSOME TCR CD4 LATE ENDOSOME/ LYSOSOME/M II C GOLGI Ag CD4+ T-CELL 2 TCR ? MHC II ? ? M II C Classical Pathway Alternative Pathway

31. TRANSLATIONAL THERAPIES TARGETING MHC ANTIGEN PRESENTATION PATHWAYS Leader LAMP-1Ag Immunize with DNA Measure immunological functions Challenge with microbial pathogen and measure survival CD4 IFN-  UnimmunizedImmunized 0 20 40 60 80 100 120 0510152025 Time (weeks) % survival Immunized Unimmunized

32. CD8+ T-CELL CD4+ T-CELL PROTEOSOME MHC CLASS I PATHWAY MHC CLASS II PATHWAY pH<5 ENDOSOME TAP MHC CLASS I TCR CD8 CD4 M II C ER MHC CLASS II GOLGI Ag