1. M261 ANTIGEN PRESENTATION/MHC CLASS I 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.

2. ANTIGEN PRESENTATION MHC CLASS I How do T cells “see” antigen and what does MHC have to do with it? MHC class I antigen processing and presentation pathway. Mechanisms of immune evasion.

3. MOLECULES OF LYMPHOCYTE RECOGNITION   T-CELL TCR CD3 ANTIGEN PRESENTING CELL MHC 11 11 22 22 B-CELL Surface Ig Ig  Ig  Although B and T cell receptor genes are rearranged similarly to generate a high degree of specificity for antigen, the receptors “see” antigen in entirely different ways. Processed peptide (linear epitope) Complete molecule (conformational epitope)

4.   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 Major histocompatibility complex (MHC); human=Human Leukocyte Antigen (HLA); mouse=H-2 Gorer and Snell identified a genetic basis for graft rejection and Snell named it histocompatibility 2 (H-2). Nobel prize awarded to Snell. Highly polymorphic genes organized in a complex on chromosome 6 (human) and 17 (mouse). Glycoproteins expressed on the surface of cells. MHC class I is composed of one polypeptide, non- covalently associated with  2microglobulin. MHC class II is composed of two polypeptides, referred to as  and . MOLECULES OF T LYMPHOCYTE RECOGNITION CLASS I MHC 2m2m 22 11 33 ANTIGEN PRESENTING CELL

5. RELATIONSHIP BETWEEN PATHOGEN AND MHC COMPARTMENTS: A TWO-PRONGED APPROACH TO ANTIGEN PRESENTATION Two places for pathogens to be found within eukaryotic cells, cytosol/endogenous and vesicles/exogenous. Two pathways for antigen presentation for peptides (MHC class I and II). One place for antigens to end up in order for the TCR to recognize foreign antigen-on the surface of the cell in the context of MHC. Text Figure 5.2

6. TISSUE DISTRIBUTION OF MHC EXPRESSION SUPPORTS ANTIGEN PRESENTATION PATHWAYS The expression of MHC molecules differs between tissues. MHC class I molecules are expressed on all nucleated cells, although they are most highly expressed in hematopoietic cells. MHC class II molecules are normally expressed only by a subset of hematopoietic cells and by thymic stromal cells, although they may be expressed by other cell types on exposure to the inflammatory cytokine interferon- .

7. FUNCTION OF MHC CLASS I MOLECULES Provide genetic basis for T cell recognition of foreign molecules, e.g. virus infection. Nobel prize awarded 1996. C57Bl (H2 b ) nu/nu CBA/H (H2 k ) CBA/HxC57Bl (H2 k/b ) 51 Cr 0 10 20 30 40 50 60 70 InfectedUninfected % 51 Cr release C3H (H2 k ) Zinkernagel and Doherty, Nature 248:701, 1974

8. FUNCTION OF MHC CLASS I MOLECULES T cell receptor recognition requires correct MHC as well as correct peptide. The antigen-specific T-cell receptor (TCR) recognizes a complex of antigenic peptide and MHC. One consequence of this is that a T cell specific for peptide x and a particular MHC allele, MHC a (left panel), will not recognize the complex of peptide x with a different MHC allele, MHC b (center panel), or the complex of peptide y with MHC a (right panel). The co-recognition of peptide and MHC molecule is known as MHC restriction because the MHC molecule is said to restrict the ability of the T cell to recognize antigen. This restriction may either result from direct contact between MHC molecule and T-cell receptor or be an indirect effect of MHC polymorphism on the peptides that bind or on their bound conformation. Text Figure 5.17 H2 k with LCMV pep H2 k with HSV pep H2 b with LCMV pep

9. GENETIC ORGANIZATION OF MHC IN HUMANS AND MICE Human HLA (Chrom. 6) –Three class I genes, HLA- A, HLA-B, HLA-C. –Three pairs of class II genes, HLA-DR, HLA-DP, HLA-DQ. Mouse H-2 (Chrom. 17) –Three class I genes, H2-K, H2-L, H2-D. –Two pairs of class II genes, IE and IA. Text Figure 5.11

10. IMMUNOLOGICAL DIVERSITY GENERATED BY MHC LOCUS Expression of MHC gene products are co-dominant, meaning that each gene encoding these proteins on the parental chromosome of the diploid cell, is expressed. Polymorphism and polygeny contribute to the diversity of MHC molecules expressed by any individual. H2 k xH2 d Upper case=H2 gene Lower case=H2 allele

11. STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE Crystal structure (solved in late 1980’s) revealed a binding groove formed by anti-parallel  -pleated sheets (bottom of groove) and  - helices (sides of groove).  1 and  2 of MHC class I and  1 and  1 of MHC class II form “mirror images of each other to create the peptide binding groove. Amino acids along the edges of the groove interact (through hydrogen bonds and ionic attractions) with the amino acids of the peptide to stabilize peptide binding (Figure).

12. HOW THE T CELL RECEPTOR “SEES” PEPTIDE-MHC View from TCR perspectiveView from bystander perspective

13. STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE Allelic variation occurs at specific sites along the MHC molecules. These sites correspond to amino acids that line the antigen binding groove. Similar variability is seen in MHC class II. Text Figure 5.16

14. STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE MHC CLASS I Anti-HLA Ab The peptides that bind to the MHC groove were identified by lysing virus-infected cells with detergent, (in some cases) affinity purifying the MHC molecules, isolating peptides with HPLC, and identifying the active peptides using CTL assays (Rotzschke, et. al., Nature 348:252, 1990). UninfectedInfected HPLC CTL Mild H + Lyse cells HPLC, CTL, sequence peptides MHC CLASS I Infected cell

15. PEPTIDES BOUND TO THE GROOVE OF MOUSE MHC CLASS I MHC class I molecules bind 8-10 amino acid peptides whereas MHC class II bind 12 amino acid or longer peptides. Peptides that bind to a particular MHC protein share a motif. H2K b H2K d Anchor residue=

16. PEPTIDES BOUND TO THE GROOVE OF HUMAN MHC CLASS I

17. Immune function Housekeeping function

18. MHC CLASS I ANTIGEN PROCESSING AND PRESENTATION PATHWAY Peptides presented by MHC class I come from cytosolic proteins and peptides presented by MHC class II come from endocytosed proteins. This is an oversimplification. Many of the components of MHC class I processing are encoded in the MHC locus. Cytosolic proteins are degraded in the cytoplasm by a complex called the proteasome, often after the protein is ubiquitinylated. Peptides are transported into the ER via the transporter associated with antigen processing (TAP), a heterodimeric, ATP-dependent transporter. In the meantime, MHC class I heavy chain is being translated in the ER and folded into the proper conformation. The MHC protein is stabilized by a protein, calnexin, a resident ER protein that binds to glycoproteins. MHC class I then binds to  2microglobulin (  2m) and calreticulin, a chaperone, that trafficks MHC class I to tapasin which stabilizes a TAP-tapasin-MHC class I complex. Peptides are loaded into the binding groove, stabilizing MHC class I, which then moves to the cell surface. Peptide deficient MHC class I is unstable and is rapidly degraded whether or not it reaches the cell surface. Text Figure 5.6

19. THE ROLE OF PROTEOLYSIS IN MHC CLASS I PRESENTATION The proteasome is a complex of polypeptides that sequester proteins signaled for proteolysis. It degrades all proteins tagged with ubiquitin, regardless of whether they will end up in the ER. Two subunits, LMP2 and LMP7, are encoded in the MHC and can be up-regulated by IFN- . These subunits direct the proteasome to generate peptides that have carboxyl ends that fit MHC molecules well and are easily transported by TAP. IFN-  converts proteasome from housekeeper to specialized factory worker. LMP2 LMP7 Proteasome Protein signaled for degradation Peptides Easy to transport to ER and load in MHC class I Difficult to transport to ER and load in MHC class I Result -IFN-  Treatment (e.g., uninfected cell) +IFN-  (e.g., infected cell)

20. THE ROLE OF PROTEOLYSIS IN MHC CLASS I PRESENTATION Peptide profile (HPLC) of IFN-  -treated cells differs from untreated cells. When peptides are sequenced, it becomes apparent that some proteolytic products are favored in the cells treated with IFN- . The favored products are those that will transport easily into ER and load on MHC class I. Boes, et. al., J.Exp.Med. 179:901, 1994. Centifugation, column purification -IFN-  +IFN-  HPLC

21. Immune function Housekeeping function

22. TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING (TAP) Two proteins (TAP-1,2) that form a heterodimer spanning the ER membrane. Facilitates the ATP- dependent transport of peptides across the ER membrane. Selectivity in transporting peptides. Bias towards transporting peptides that bind well to MHC class I (amino acids with hydrophobic or bulky side chains). Shepherd, et. al., Cell, 74:577, 1993 ER (glycosylation takes place) TAP X RYWANATRSX TAP- TAP-/ratTAP Momburg, et. al., Nature, 367:648, 1994.

23. ROLE OF TAPASIN IN THE MHC CLASS I PATHWAY Components of the MHC class I-TAP complex affinity purified by anti- TAP-1 column. Ortmann, et. al., Science 277:1306, 1997 Tapasin transfection restores MHC class I surface expression and CTL lysis.

25. CUSTOM PEPTIDES GENERATED FOR MHC CLASS I BY ERAAP TAP-deficient fibroblasts No NH 2 -terminal amino acidsSeven NH 2 -terminal amino acids Serwold et. al. Nature, 419:480-483, 2002

26. SIGNIFICANCE OF ERAP ERAP (endoplasmic reticulum-associated aminopeptidase 1) an IFN-  inducible ER protease that trims peptides to fit MHC class I

28. MECHANISMS OF IMMUNE EVASION (MHC CLASS I) EBNA1 (EBV) US2, US11 (hCMV) US6 (hCMV) ICP47 (HSV) E19 (adenovirus) US3 (hCMV) Vpu (HIV) gp40 (mCMV) gp48 (mCMV) Nef (HIV) gp34 (mCMV)

29. MECHANISMS OF IMMUNE EVASION (MHC CLASS I) Herpes simplex virus encodes for a protein, ICP47, that prevents TAP from transporting peptides into the lumen of the endoplasmic reticulum. Prevention of peptide transport results in an inability to load peptide into nascent MHC class I proteins. X Hill, et. al., Nature 375:411, 1995 -HSV +HSV

30. MECHANISMS OF IMMUNE EVASION (MHC CLASS I) Tumor cell T cell Reduce MHC class I expression Loss of tumor antigen expression Secrete anti-inflammatory cytokines Defective death receptor signaling Lack of costimulation Dendritic cell

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