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I. Antigen Presentation: MHC II Presentation MHC I Presentation C. Additional Considerations

Antigen Presentation, Cytotoxicity and Anti-Viral Immunity Dr. Julia Rempel Section of Hepatology 789-3825 jdrempel@ms.umanitoba.ca 804D JBRC. OVERVIEW. I. Antigen Presentation: MHC II Presentation MHC I Presentation C. Additional Considerations II. Cytotoxicity:

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I. Antigen Presentation: MHC II Presentation MHC I Presentation C. Additional Considerations

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  1. Antigen Presentation, Cytotoxicityand Anti-Viral Immunity Dr. Julia RempelSection of Hepatology 789-3825jdrempel@ms.umanitoba.ca 804D JBRC

  2. OVERVIEW • I. Antigen Presentation: • MHC II Presentation • MHC I Presentation • C. Additional Considerations • II. Cytotoxicity: • A. Perforin/granzymes driven (CD8, NK cells) • B. Fas/FasL driven (CD4/CD8, NK cells) • C. TRAIL/IFN-γ • D. Apoptosis and necrosis • III. Pathogen Regulation of Antigen Presentation: • A. MHC II Presentation • B. MHC I Presentation

  3. How the APC receives the Ag information determines who the APC can give the information to Two ways to receive information:

  4. A. MHC CLASS II ANTIGEN PROCESSING • The Molecule • The Antigen • Antigen Processing • Antigen Presentation

  5. The Molecule • • MHC class II molecules are processed within the endoplasmic reticulum (ER). • • Challenges: • stabilizing complex • getting from the ER to the surface without the wrong peptide. Stabilizing: • Calnexin is an ER protein that binds and retains in the ER members of the Ig super family as they are being assembled: MHC class I, TCR, Ig. • Chaperon protein/peptide the invariant chain (Ii). • Ii and calnexin are critical to the exact folding of the MHC class II proteins. during the assembly phase.

  6. The Molecule Getting from the ER to the surface without the wrong peptide: • Within the ER, the MHC class II  dimers bind to Ii to form the nonameric complex (Ii)3. • The (Ii)3 complex can be exported out of the ER in an endosome. • The Ii is reduced by acidification/proteases - cathepsin S (over a 2 - 4 h period) to a truncated intermediate and then to CLIP (class II - associated invariant-chain peptide). Then MHC class II and peptide are transported to the surface.

  7. The Antigen Location: always ‘outside’ the cell Receptors Fc compliment scavenger mannose/fucose Macropinosomes (2.0 mm) ruffled surface membrane efficient for pinocytosis - on dendritic cells - - upregulated in epithelial cell and macrophages by growth factors and phorbol estors. Exogenous antigen is taken up by Clathrin coated pits (0.1 mm) Challenge: get the antigen outside the cell to the MHC class II molecule. Other possibilities

  8. Antigen Processing Formation of Phagosome/endosome upon internalization into the cell; triggers signaling events and remodeling of endosome i.e. change in expression of different endosomal markers • Early endosomes: neutral pH, endosomal proteases inactive. Endosome move toward the center of the cell: pH drops Interact with lysosomes: pH dependent proteases activated Degrade antigen into peptide fragments. • Results in mature phagolysosomes - these continue to interact with lysosomes/enzymes. • Enzymes include: Acid Proteases - Cystein proteases cathepsins B, D,S and L. • Endosome containing MHC class II + CLIP interacts with phagolysosomes.

  9. Antigen Presentation • Generally the exchange between CLIP and peptide is low, • except in the presence of the HLA-DM. • HLA-DM: • - gene in MHC class II region • - not expressed on the cell surface • - does not require peptide for stabilization. • - interaction with MHC class II molecule releases the CLIP and allows peptides to bind • the MHC class II molecule. • - stabilizes the empty MHC class II during • this transaction. • also appears to peptide edit: aid in the removal of weakly bound peptides. • IFN- upregulates HLA-DM • ADVANTAGE? CD4

  10. If the MHC class II molecules does not bind a peptide, it is quickly degraded.

  11. B. MHC CLASS I ANTIGEN PRESENTATION • The Molecule • The Antigen • Antigen Processing • Antigen Presentation

  12. MHC class I molecule is very unstable. • • Challenges: • stabilizing complex • getting from the ER to the surface without the wrong peptide. • Stabilizing: • When brought to ER to be processed: Calnexin + MHC class I  chain • In ER: Bind 2M and chaperone proteins • • Chaperone proteins: CalreticulinErp57 a protein disulfide isomerase - may break and remake the disulfide bond in the MHC class I  chain during peptide loading. Tapasin TAP-associated protein - link TAP1/2 heterodimer with MHC class I and allows for the transportation of peptide. The Molecule

  13. The Antigen - Viruses infecting the cell take over the cell’s biosynthetic mechanisms and make their own proteins. - Self antigens Location of Antigen: cytosol Challenge: get the antigen from the cytosol to the MHC class I in the ER

  14. Antigen Processing The Proteosome 7 subunits x 4 rings = 28 subunits Generally, the subunits are constituitively expressed. IFN inducible subunits: LMP2, LMP7 and MECL-1. When expressed, replace constituitively expressed units. May alter specificity: decrease cleavage following acidic residues and greater cleavage following hydrophobic and basic residues. Regulation: 1. IFN inducible units: Greater MHC class I groove binding and TAP transport of peptides. 2. Proteasome inhibitors inhibit MHC class I presentation. 3. Ubiguitin tagging of a protein enhances the MHC class I presentation of protein peptides.

  15. HYDROPHILIC TRANSMEMBRANE DOMAIN LUMEN OF ER ATP-BINDING CASSETTE (ABC) DOMAIN CYTOSOL TAP1 TAP2 Transport associated with Antigen Processing-1 and 2 (TAP1 and TAP2). Members of the ATP-binding cassette (ABC) family. ABC proteins are involved in ATP-dependent transport of many molecules: ions, sugars, amino acids, peptides. ATP hydrolysis Transport peptide from proteosome into the ER TAP1 and TAP2 genes located close to LMP2 and LMP7. Mutations in them impair MHC class I presentation. Interferons positively regulate these genes.

  16. Antigen Presentation MHC class I molecule correctly folded and peptide is inserted is stable enough to transport to the cell surface. Can interact with CD8 T cells. CD8

  17. C. Additional Considerations 1. Recycling of MHC Class I and Peptides. b. Recycling of MHC class I molecules. a. Retrograde translocation of peptides: transport of unbound peptides to the cytosol ATP-dependent transport complex (not TAP). c. ER-associated protein degradation: Misfolded proteins transported into cytosol, degraded by proteasome. Sec61 translocon channel, membrane proteins. In cytosol, protein are digested by the proteasome and (déjà vu) transported back into the ER.

  18. 2. Peptide loss: • pathogen would escape detection • another peptide could float in the groove • - if pathogen, healthy cell gets killed by CTL • - if self-peptide, autoimmunity. MHC class II and MHC class I molecules are generally produced in excess of pathogen peptide availability.

  19. 3. MHC Associations with Disease Chronic Disease Most of the D/DR associations involve autoimmune disorders (IDDM, rheumatoid arthritis, lupus, IBD, possibly MS) Tissue damage mediated by cellular or humoral immune response. Infectious Disease Mostly Class I genes, especially HLA B Thought to be the basis for differences in response to infectious agents, vaccines and allografts

  20. II. Cytotoxicity: A. Perforin/granzymes driven (CD8, NK cells) 1. Identifying target and contact (from Intro) - non-specific interactions through adhesion molecules. - formation of synapse/junction: “kiss of death” focal point - CD8 T cells - TCR:MHCpeptide polarization of cells due to cytoskeleton rearrangement nucleus is pushed toward back of cell golgi apparatus and granules pulled toward synapse granules released into synapse • after the strike, disengage and reorientation to another target cell • allows for killing of single cell without damage to surrounding cells.

  21. CTL Tumor cell http://www.biology.ucsc.edu/classes/bio111/Lecture%2011(Chapter%2014).pdf

  22. 2. Entry of granzymes A. Perforin: Forms polymers – pores in target cell membrane (ie C9). Mechanisms? 1. High concentrations: Diameter large enough to allow granzymes (~32 kDa) inside cell. Passive transfer of granzymes into cell. Perforin pores in erythrocyte membrane (EM) http://www.biology.ucsc.edu/classes/bio111/ Lecture%2011(Chapter%2014).pdf

  23. 2. Low perforin concentrations (sublytic): Granzyme/proteoglycan complexes - ~300 kDa Granzymes - cell entry perforin independent receptor-mediated endocytosis (mannose-6-phosphate) Perforin – release Granzymes from endosome, Granzymes accumulates in nucleus ( - Complement can not replace perforin. - Inhibited by: endosmolytic agents and adenovirus Brefeldin A, vesicular trafficking

  24. Two hypothetical but complementary models of perforin-granzyme synergy. Passive diffusion Facilitated access Browne, 1999

  25. 3. Granzyme activation and activity In T cell and NK cell granules: Granulysin Granzyme-A, B, C, D, E, F, G, H, K, and M Serine proteases with roles in caspase-dependent and -independent apoptosis What protects the CTL or NK cell: 1. Proteinase Inhibitor 9 - Serpin family protease inhibitor, binds Granzymes 2. Calreticulin – Ca++ binding, co-localizes with perforin, impairs perforin activity

  26. Granzyme B (from Intro): Granzyme B cleaves pro-enzyme caspase 3 to active caspase 3 cleaves target proteins after aspartate residues (Asp-ase activity). caspases: proapoptotic cysteine proteases constitutively expressed as proenzymes Granzyme B Caspase-3 degrades inhibitor of CAD (ICAD), bound to caspase-3-activated deoxyribonuclease (CAD) CAD is aendonuclease, degrades double stranded DNA (200-bp), responsible for DNA fragmentation that results in chromatin condensation. pro- Caspase 3 ICAD CAD

  27. In addition: 1. Granzyme B activates pro-caspase 8 to 2. Caspase 8 disrupts mitochondrial membrane, release of pro-apoptotic molecules: Cytochrome C endonucleases AIF (apoptosis inducing factor) loss of Δψ, increase in ROS. 1. 2. 3. 4. 5. 3. Cytochrome C dependent formation of apoptosome [APAF-1(Apoptosis Protease- activating Factor 1); CARD (Caspase- recruitment domains), pro-caspase 9] 6. 4. caspase 9 and 8 can cleave pro-caspase-3 to 5. caspase 3 cleaves ICAD releasing CAD translocates to nucleus and cleaves DNA 6. caspase 3 activating caspase 6, degrading lamin http://mcb.berkeley.edu/courses/mcb150/Lect20/Lect20.pdf

  28. Caspase activated apoptosis is inhibited by Bcl-2 proteins: Bcl-x proteins inhibit (Bcl-2) activate (Bax) For apoptosis to occur, cascade must 1) be activated; 2) not inhibited by anti-apopt. proteins

  29. Granzyme A: most abundant; serine protease perforin, ROS (reactive oxygen species) dependent caspase independent; not regulated by Bcl-2 not oligonucleosomal DNA fragmentation; single stranded DNA nicks inhibited by Antioxidants and Free Radical Scavengers the SET complex (270-420 kDa, in ER): APE (DNA repair enzyme), HMG-2 (DNA bending), pp32 (inhibits PP2A – viral replication and oncogenesis), NM23-H1 (DNAse, tumor metastases suppressor), SET (inhibits NM23-H1).

  30. Granzyme A induced apoptosis 1. direct, rapid release of ROS, loss of mitochondrial transmembrane potential Δψ note: no release of proapoptotic molecules, no disruption of membrane 1. 2. translocation of SET complex from ER to nucleus. ROS dependent. 2. 5. 3. cleaves APE, HMG-2 and SET (frees NM23-H1, nicks DNA). 3. 4. cleaves histones and core (DNA coiled) 5. degradation of nuclear lamina (Granzyme B and caspase 6) 4. Superoxide Scavengers Martinvalet, 2005

  31. The synopsis of CTL mediated apoptosis Cytotoxicity, extremely low peptide concentrations 10-12 to 10-15 M. IFNg production and IL-2 induced prolif, peptide concentrations >10-9 M. S Valitutti, JEM, 1996

  32. B. Fas:FasL aka CD95/Apo-1:CD95L (CD4, CD8) This pathway does not require antigen recognition on target cells. T cell and NK cell induced cytotoxicity FasL – on T cell membrane Fas – target cell membrane (any cell; tumors; macrophages) Quenching of T cell activity: Fas – on T cell membrane FasL – on other cell (any cell; tumors; macrophages) • Contribute to autoimmunity • Fas-mediated cytotoxicity is involved in tissue damage in cell-mediated autoimmune diseases; • defective Fas function impairs ability to shut off immune response • MRL/lpr mice, model of systemic lupus erythematosus • defective expression of Fas

  33. FAS induced apoptosis 1. FasL is a trimeric molecule. 2. FasL binding – trimerization of Fas, which binds dead domains. 3. Recruitment and activation of caspase 8. 4. The rest is history. 1. 2. 3. 4. Regulated through anti-apoptotic mechanisms (Granzyme B).

  34. C. TNF-α, LT- α (TNF- - FAS ligand) /TRAIL produced by T cells, NK cells, NKT cells potential for by-stander act; why? • Tumor Necrosis Factor family of ligands and receptors • Ligand binding to Receptor (Death Domains) eg. TNFR-I, TRAIL-R • Regulation more difficult. • NF-k B negative inhibitor of TNF-α induced apoptosis • in vitro TNF-α + inhibition NF-kB increases apoptosis.

  35. TRAIL [TNF-related apoptosis-inducing ligand] – cell bound • Hepatocytes: in hepatic flares, enhance TRAIL and TRAIL-R expression. • Cancer therapy: selective induction of apoptosis in many tumor cells IFN-γ regulation of TRAIL-induced apoptosis: Intraocular tumors: IFN-γ -/- mice – no apoptotic tumor cells WT mice – apoptotic tumor cells tumor cells express TRAIL-R IFN-γ increases CD4 T cell TRAIL expression enhances tumors susceptibility to apoptosis; inhibits NF-kB anti-TRAIL Ab block CD4 T cell induced apoptosis Balance of TRAIL and TRAIL-R may control TRAIL-mediated apoptosis.

  36. D. Overview of Apoptosis and Neurosis 1. Apoptosis Granzyme B Regulation

  37. What do apoptotic cells look like? http://www.sghms.ac.uk/depts/immunology/ %7Edash/apoptosis/

  38. 2. Comparison of Apoptosis and Necrosis Both mechanisms - normal and necessary. not enough vs too much Necrosis Apoptosis Chromatin clumping Swollen organelles Flocculent mitochondria Mild convolution Chromatin compaction and segregation Condensation of cytoplasm Nuclear fragmentation Blebbing Apoptotic bodies Phagocytosis Disintegration Release of intracellular contents Apoptotic body Phagocytic cells Inflammation

  39. Experimentally, how could you tell if your cells were undergoing apoptosis or necrosis? If your cells were undergoing apoptosis or necrosis, what can this tell you about what was happening in the culture? In cytotoxicity assays – apoptosis or neurosis?

  40. III. Pathogen Regulation of Antigen Presentation Probably every step is attacked by some pathogen. Specific pathogen proteins can interfere with MHC pathways.

  41. A. MHC class II interference: • Mycobacteria - inhibit acidification of endosomes that they live in or fusion with lysosomes. • Leishmania - endosomes acidify normally and fuse with lysosomes and even MHC class II molecules. But cells present parasite antigens poorly; may retain MHC class II in endosome. • Human herpesvirus 6 -lower antigen presentation in infected cells appears linked to impaired antigen capture. Hepatitis C virus - Genetic susceptibility associated with MHC class II Chronic HCV results in impaired T cell responses. HCV proteins did not alter MHC class II expression, but inhibited the ability of dendritic cells to stimulate CD4 T cells (IL-12 production).

  42. B. MHC class I interference: • SOME THINGS TO CONSIDER: • Balance between NK and CTL killing: • Why not take out 2m as oppose to all the mechanisms here? • Reduced MHC class I could target the cell for NK lysis. • Involved in viral replication. Yewdell and Bennink

  43. Breaking up is hard to do (Carpenters): Blocking peptide generation Epstein-Barr virus(EBV) EBV nuclear antigen (EBNA)-1 expressed in all latently infected B cells A 238-residue domain consisting of Gly-Ala repeats Resistant to proteasomal digestion. Consequence? How could you test this?

  44. Stop! in the name of love (or Herpesviridae) (Supremes): Blocking TAP-mediated peptide transport into the ER Herpes simplex virus(HSV) ICP47, small protein not required for replication Interferes with peptide binding to TAP, impairs peptide transportation into ER. Binds to TAP1-TAP2 10-1000x that of high affinity peptides. (cytosolic side) Very stable. Not inhibit ATP binding TAP; inhibit ATP hydrolysis May modify TAP confirm change HCMV US6, type I membrane anchor, binds to TAP in complex with MHC class I and chaperone proteins (lumenal side). Peptides can bind; but conformation change in TAP, inhibits ATP binding to TAP preventing peptide transport into ER. Advantages of targeting TAP 1. It is only involved in Ag processing; so deregulating its function is not going to alter cellular or viral replication. 2. ~90% of peptides to be presented are transported into ER via TAP. 3. Bottleneck

  45. You ain’t going nowhere (Bob Dylan): Impairing MHC class I transcription HIV TAT protein impairs MHC class I transcription: interacts with promoter

  46. Never say Good-bye (Bon Jovi): Retaining MHC class I in ER Adenovirus E19, type 1 membrane anchor, binds directly to TAP (TAP: tapasin interaction OK) interferes with MHC class I molecules interaction with tapasin, retaining them in the ER. Has dilysine motif, acts as a ER retrieval motif, retains proteins in ER HCMV US3 has a retention signal, binds to MHC class I/peptide molecules, the transport signal on MHC class I is ignored, MHC class I is retained in the ER. Transient binding. US10 slows down MHC transport out of ER, not block. HIV Vpu is not required for viral replication; but enhances viral replication. Appears to, by unknown mechanism, down regulates MHC class I expression on cell surface possibly by retaining MHC class I in ER. ADVANTAGE: Can work independent of MHC class I polymorphism. Does not appear to interfere with 2m binding to MHC class I  chain. How could you test this? Co-precipitation

  47. Return to Sender (Elvis Presley): Shipping MHC class I molecules back to cytosolER-associated protein degradation HCMV US2 and US11 type I integral membrane glycoproteins No effect on protein folding. appear to bind MHC class I molecules via cytosolic tail, ubiquitinates them, transport them out of ER into cytosol through the Sec61 ER channel, deglycosylates them and drag them to the proteasome to be digested. Autoimmune disease: 90.7% adult SLE patients were seropositive. How could you test this? Ubiquitin: 76 residue protein, attaches to lysines, forms polyubiquitin chains

  48. Another one bites the dust (Queen): Diverting MHC class I molecules to lysosomes MCMV gp48 directs MHC class I molecules to endosomes which have enzymes required for degradation, as opposed to endosome for transportation to cell surface. How could you test this? Lysosomal and proteasomal inhibitors Round and round we go (Trooper): Internalization of cell surface MHC class I molecules HIV Nef appears to act as a bridge between MHC class I molecules and Ap-1 and Ap-2 complexes: these associate with clahtrin-coated pits. How could you test this? Would have to differentiate between molecules coming from the ER and internalized from surface .

  49. Bloody Sunday (U2): hemorrhagic viruses • Upregulation of MHC class I • Hanta virus Bunyaviridae - there is an upregulation of MHC • class I and co-stimulatory molecules CD80, CD86 • Raftery et. al. 2002 J Virol 76:10724 • Denge virus Flaviviridae - pathogenesis appears to be • CD8 T cell driven. Severity of disease associated with HLA-A. • Loke et. al. 2001 J Infect Dis 184:1369 Down regulation of MHC class I Ebola virus Filoviridae Inhibition of MHC class I, IFN-, and IFN-. Pathogenesis thought to be cytokine (TNF-) driven.

  50. Vossen, et al, Immunogenetics, 2002, 54:527

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