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M261 ANTIGEN PRESENTATION/MHC CLASS I • • • • 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 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. MOLECULES OF LYMPHOCYTE RECOGNITION 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. T-CELL B-CELL Iga CD3 a b Igb TCR Surface Ig Complete molecule (conformational epitope) MHC b1 a1 b2 a2 Processed peptide (linear epitope) ANTIGEN PRESENTING CELL MOLECULES OF T LYMPHOCYTE RECOGNITION 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, noncovalently associated with b2microglobulin. MHC class II is composed of two polypeptides, referred to as a and b. 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 RELATIONSHIP BETWEEN PATHOGEN AND MHC COMPARTMENTS: A TWO-PRONGED APPROACH TO ANTIGEN PRESENTATION Text Figure 5.2 • • • 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. 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-g. FUNCTION OF MHC CLASS I MOLECULES • Provide genetic basis for T cell recognition of foreign molecules, e.g. virus infection. Nobel prize % 51Cr release 70 CBA/H (H2k) 60 C57Bl (H2b) 50 awarded 1996. CBA/HxC57Bl (H2k/b) 40 nu/nu 30 20 10 C3H (H2k) 0 Infected 51Cr Uninfected Zinkernagel and Doherty, Nature 248:701, 1974 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, MHCa (left panel), will not recognize the complex of peptide x with a different MHC allele, MHCb (center panel), or the complex of peptide y with MHCa (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 H2k with LCMV pep H2b with LCMV pep H2k with HSV pep GENETIC ORGANIZATION OF MHC IN HUMANS AND MICE • Human HLA (Chrom. 6) – Three class I genes, HLAA, 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 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. Upper case=H2 gene Lower case=H2 allele H2kxH2d 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 b-pleated sheets (bottom of groove) and ahelices (sides of groove). a1 and a2 of MHC class I and a1 and b1 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). HOW THE T CELL RECEPTOR “SEES” PEPTIDE-MHC View from bystander perspective View from TCR perspective 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 STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE • 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). Uninfected Infected CTL MHC CLASS I Anti-HLA Ab Infected cell Lyse cells MHC CLASS I Mild H+ HPLC HPLC, CTL, sequence peptides 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. Anchor residue= H2Kb H2Kd PEPTIDES BOUND TO THE GROOVE OF HUMAN MHC CLASS I Housekeeping function Immune function 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 b2microglobulin (b2m) 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 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-g. These Treatment Protein signaled for subunits direct the proteasome to degradation generate peptides that have -IFN-g carboxyl ends that fit MHC (e.g., uninfected cell) molecules well and are easily transported by TAP. IFN-g converts proteasome from +IFN-g housekeeper to specialized (e.g., infected cell) factory worker. Proteasome LMP2 Peptides Result Difficult to transport to ER and load in MHC class I Easy to transport to ER and load in MHC class I LMP7 THE ROLE OF PROTEOLYSIS IN MHC CLASS I PRESENTATION -IFN-g • Peptide profile (HPLC) of IFN-g -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-g. • 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-g HPLC Housekeeping function Immune function TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING (TAP) ER (glycosylation takes place) Shepherd, et. al., Cell, 74:577, 1993 RYWANATRSX Nature, 367:648, 1994. TAP- TAP-/ratTAP TAP X • Two proteins (TAP-1,2) that form a heterodimer spanning the ER membrane. • Facilitates the ATPdependent 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). Momburg, et. al., ROLE OF TAPASIN IN THE MHC CLASS I PATHWAY • Components of the MHC class I-TAP complex affinity purified by antiTAP-1 column. • Tapasin transfection restores MHC class I surface expression and CTL lysis. Ortmann, et. al., Science 277:1306, 1997 CUSTOM PEPTIDES GENERATED FOR MHC CLASS I BY ERAAP TAP-deficient fibroblasts No NH2-terminal amino acids Seven NH2-terminal amino acids Serwold et. al. Nature, 419:480-483, 2002 SIGNIFICANCE OF ERAP ERAP (endoplasmic reticulum-associated aminopeptidase 1) an IFN-g inducible ER protease that trims peptides to fit MHC class I MECHANISMS OF IMMUNE EVASION (MHC CLASS I) gp34 (mCMV) Nef (HIV) gp48 (mCMV) gp40 (mCMV) E19 (adenovirus) US3 (hCMV) E19 (adenovirus) US2, US11 (hCMV) Vpu (HIV) EBNA1 (EBV) US6 (hCMV) ICP47 (HSV) MECHANISMS OF IMMUNE EVASION (MHC CLASS I) -HSV • 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. +HSV X Hill, et. al., Nature 375:411, 1995 MECHANISMS OF IMMUNE EVASION (MHC CLASS I) Tumor cell Reduce MHC class I expression Loss of tumor antigen expression Secrete anti-inflammatory cytokines Defective death receptor signaling Lack of costimulation T cell Dendritic cell HOW DO NAÏVE CD8 T CELLS BECOME ACTIVATED WHEN THE INFECTED CELL IS NOT A PROFESSIONAL APC?