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Micro 204 Cytotoxic T Lymphocytes CTL

CTL. Recognition of antigenGeneration?."priming" via dendritic cellsEffector functions..killing and cytokinesCytolytic processEffector molecules..perforin

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Micro 204 Cytotoxic T Lymphocytes CTL

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    1. Micro 204 Cytotoxic T Lymphocytes (CTL) Lewis Lanier Lewis.Lanier@ucsf.edu

    2. CTL Recognition of antigen Generation….”priming” via dendritic cells Effector functions..killing and cytokines Cytolytic process Effector molecules..perforin & granzymes Avoiding auto-destruction Maintenance & memory

    6. MHC class I “cross-priming” by dendritic cells

    7. CTL and Immunity to Intracellular Infections Viral infections Lytic viruses (polio, vaccinia, influenza) Antibodies important, CTL less important Persistent non-lytic viruses (CMV, HSV, EBV,HIV) CTL & Ab important Intracellular bacteria & parasites Listeria, Mycobacteria, Toxoplasma IFNg & Macrophage activation important, CTL important for clearance

    8. Priming a CTL DC eats apoptotic cells, delivers peptides to class I pathway (peptides transported by heat shock proteins? Or delivery of intact protein?) DC migrates to lymph node and presents antigen to naďve T cell Clonal expansion of CD8+ T cells in lymph node CD8 + T cells escape lymph node and traffic to site of infection

    9. Priming a CTL CTL precursors - low frequency, no lytic granules, non-dividing Naďve T cell and APC - interactions: TcR----- MHC class I (a1 & a2)- peptide CD8---- MHC class I (a3) LFA-1 --ICAM (adhesion & signaling) CD28 --CD80/CD86 or other costimulation (4-1BB) Results in: Synthesis of granzymes & perforin Cytokine production (INF-g, TNF, Fas L, some IL-2) Massive Proliferation Requires transcription & protein synthesis (6 hrs minimum)

    10. CD4+ T helper-dependent and independent CTL priming

    12. CTL Clonal Expansion Rapid proliferation of antigen-specific T cells In LCMV (mice) or EBV (human) infection - 70% of T cells can be antigen-specific! Response is antigen-specific, little bystander expansion (ELISPOT & tetramers vs. limiting dilution assays) Oligoclonal - a few specificities dominate the response

    14. CTL - Target Interactions Adhesion Low affinity adhesion TcR recognition - shift to high affinity adhesion Synapse formation & re-orientation of granules Lethal Hit Directional release of granule content Does not require RNA or protein synthesis Rapid (minutes) Release CTL hit & run…kill multiple targets

    16. CTL forms ‘synapse’ with target cell

    18. Lytic Mechanisms Granule Exocytosis - predominant pathway (FAST KILLING) granzymes & perforin Expression of cell surface TNF-family effector molecules (SLOW KILLING) Membrane TNF, lymphotoxin, Fas ligand, Trail Secretion of soluble toxic cytokines (SLOW KILLING) TNF & Interferon-g

    19. Granule exocytosis model

    20. Perforin Protein with homology to C9 Expressed in CTL & NK, not naďve T Ca++ dependent assembly to form pore in target membrane..(binds PC head groups) Stored in granules (low Ca++, bound to calreticulin) Purified perforin from granules lyses RBC, but not nucleated cells

    22. Granzymes Granzyme B - Aspase…activates caspases Stored in granules & released KO mice show CTL defects Granzyme A - Lys/Arg-ase…role? Stored in granules & released KO mice minimal effect on CTL activity

    23. Granzyme A (green) at CTL ‘synapse’ with target cell

    24. Granule Exocytosis Model Activation-induced re-orientation of granules to synapse Release of perforin & granzymes Perforin creates holes in membranes Granzymes B (aspase) cleaves pro-caspases Induces apoptosis in target cells Caspase activation - DNA fragmentation Mitochondrial damage (cytochrome C release)

    25. Proof that granzyme/perforin cause CTL activity - Henkart Rat basophil line (RBL) Has high affinity IgE receptor Has histamine containing granules Degranulates when IgE is cross linked Coat targets with IgE - trigger granular release RBL doesn’t kill IgE-coated RBC or tumor RBL - transfect perforin - kill IgE-coated RBC, not IgE-coated tumor RBL- transfect perforin & granzme - kill IgE-coated tumor

    26. Cytotoxicity via TNF family

    27. Why don’t released lytic granules kill the CTL? Surface cathepsin B protects CTL from self-destruction after degranulation Proteinase inhibitor 9 - serpin that inhibits granzyme B….expressed by CTL, dendritic cells, endothelial cells, some tumors

    29. Direct anti-microbial function Human CTL granules contain “granulysin” Together with perforin is directly lytic for certain bacteria and viruses, including Mycobacteria tb

    30. Expansion & contraction of T cells in an immune response

    31. Primary CD8+ cell response does not require CD4 + T cell help - maintenance of memory CD8 + T cells does

    32. CTL Maintenance After elimination of antigen, apoptosis of >90% of effector CTL (TNF-induced apoptosis?) Persistence of small subset of oligoclonal CD8 + T cell provides memory…allows rapid re-activation Naďve CD8 + T cells require class I in host to survive…memory CTL don’t require class I to live

    33. CTL Maintenance Maintenance of CTL memory requires IL-15 Adoptively transferred CTL effectors require exogenous IL-2 or antigen-specific CD4+ T cells to thrive E.g. adoptive transfer of CMV-specific autologous CTL in bone marrow transplant patient

    35. Memory CD8+ T cells are delineated into TCM and TEM subsets Central Memory CD62L+ & CCR7+ High proliferative potential IL-2 Non-cytolytic Secondary lymphoid tissue Effector Memory CD62L- & CCR7- Low proliferative potential Inflammatory cytokines Immediately cytolytic Non-lymphoid tissue

    36. Generating 1°, 2°, & 3° CD8+ T cell responses Heterogeneous boost

    37. Boosting results in quicker responses & more memory VSV-immunized mice

    38. Boosting results in quicker responses & more memory - VSV-immunized mice

    39. Prime-boost vaccination preferentially drives TEM generation: location

    40. Prime-boost vaccination preferentially drives TEM generation

    41. Chronic infections Chronic infections result in CD8+ T cell exhaustion Senescent Poor cytokine production CD8+ T cell exhaustion is major barrier to clearance Can we restore CD8+ T cell responses against chronic infections?

    42. PD-1 inhibitory receptor is preferentially expressed by CD8+ T cells specific for chronic infection PD-1 maintains CD8+ T cell exhaustion Blocking PD-1 “reactivates” CTL - allows viral clearance

    46. CTL-mediated immunopathology Too vigorous CTL response is bad CTL response to hepatitis CTL response to LCMV

    47. Selected Reading - CTL Persistence of Memory CD8 T Cells in MHC Class I-Deficient Mice, Science 286:1377-1381, 1999 Virus-specific CD8+ T cells in primary and secondary influenza pneumonia, Immunity 8:683-91, 1998 A non-cytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene, Cell 64:1175-1181, 1991 Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation, JEM 196:493-503, 2002 CD8+ but not CD8- dendritic cells cross-prime cytotoxic T cells in vivo. JEM 192:1685-96, 2000 Restoring function in exhausted CD8+ T cells during chronic viral infection by blocking PD-1. Nature 439:682-7, 2006

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