Micro 204 Cytotoxic T Lymphocytes (CTL) Lewis Lanier Lewis.Lanier@ucsf.edu
Lymphocyte-mediated Cytotoxicity CD8 + αβ-tcr + T cells CD4 + αβ-tcr + T cells γδ-tcr + T cells Natural Killer cells
CD8 + αβ-tcr + T cells Recognition of antigen Generation. priming via dendritic cells Effector functions..killing and cytokines Cytolytic process Effector molecules - perforin & granzymes Avoiding auto-destruction Maintenance & memory
MHC class I cross-priming by dendritic cells
CTL and Immunity to Intracellular Infections Viral infections -Acute lytic viruses (polio, vaccinia, influenza) -Persistent non-lytic viruses (CMV, HSV, EBV,HIV) need control FOREVER Intracellular bacteria & parasites Listeria, Mycobacteria, Toxoplasma IFNγ & macrophage activation important CTL important for clearance
Antibodies can PREVENT infection CTL can only kill the cell AFTER it is infected
Priming a CTL DC eats apoptotic cells, delivers peptides to class I pathway (still merky how this happens) DC migrates to lymph node and presents antigen to naïve T cell Clonal expansion of CD8 + T cells in lymph node CD8 + T cells exit lymph node and traffic to site of infection
Priming a CTL CTL precursors - low frequency, no lytic granules, non-dividing Naïve T cell and APC - interactions: TcR----- MHC class I (α1 & α2)- peptide CD8---- MHC class I (α3) LFA-1 --ICAM (adhesion & signaling) CD28 --CD80 & CD86 or other co-stimulation (4-1BB) Results in: Synthesis of granzymes & perforin Cytokine production (IFNγ, TNF, Fas ligand, some IL-2) Massive Proliferation Requires transcription & protein synthesis (6 hrs minimum)
Different dendritic cells prime helper T cells Cell 162:1322, 2015 Immunity 43554 2015
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
CTL - Target Interactions Adhesion Low affinity adhesion TcR recognition - shift to high affinity LFA-1 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
CTL forms synapse with target cell Red = lck Green = cathepsin
Measuring CTL activation by degranulation CD107 (LAMP-1) Uhrberg Leukemia 2005
Editorial Comment Detecting degranulation of a CTL DOES NOT prove that the target cells is dead If you want to claim that you are detecting cell KILLING then you need to show that the target cells is dead! CD107a+ Target cell death!
Lytic Mechanisms Granule Exocytosis - predominant pathway (FAST KILLING- minutes) granzymes & perforin Cell surface TNF-family effector molecules (SLOW KILLING - hours) Membrane TNF, lymphotoxin, Fas ligand, Trail Secretion of soluble toxic cytokines (SLOW KILLING - hours) TNF & Interferon-g
Granule exocytosis model
Perforin Protein with homology to C9 Expressed only in CTL & NK, not naïve T cells Ca ++ dependent assembly to form pore in target membrane..(binds phosphotidylcholine) Stored in granules (low Ca ++, bound to calreticulin) Purified perforin from granules lyses RBC, but not nucleated cells
Granzymes Granzyme B - Aspase activates caspases Stored in granules & released Grmb-/- mice show CTL defects Granzyme A - Lys/Arg-ase role? Stored in granules & released Grma-/- mice minimal effect on CTL activity
Granzyme A (green) at CTL synapse with target cell
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)
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 IgEcoated tumor RBL- transfect perforin & granzme - kill IgE-coated tumor
Cytotoxicity via TNF family
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
Direct anti-microbial function Human CTL granules contain granulysin member of defensin family Together with perforin is directly lytic for certain bacteria and viruses, including Mycobacteria tb
Expansion & contraction of T cells in an immune response
Primary CD8 + cell response does not require CD4 + T cell help - maintenance of memory CD8 + T cells does Sun & Bevan
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 Naïve CD8 + T cells require class I in host to survive memory CTL don t require class I to live
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
Naive Effector Memory Effector and Memory CTL differ in: Quality (proliferation, cytokines, cytotoxicity, turnover, phenotype) Location David Masopust
Memory CD8 + T cells are delineated into T CM and T EM 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
Generating 1, 2, & 3 CD8 + T cell responses Heterogeneous boost David Masopust
David Masopust Boosting results in quicker responses & more memory VSV-infected mice
Boosting results in quicker responses due to higher frequency of antigen-specific T cells - VSV-immunized mice David Masopust Days post infection
Individual T cells yield phenotypically distinct progenies Recipients of 500 to 700 naïve, barcode-labeled OT-I T cells were infected with LM-OVA C Gerlach et al. Science 2013;340:635-639
Chronic infections Chronic infections result in CD8 + T cell exhaustion Senescence Poor cytokine production CD8 + T cell exhaustion is major barrier to clearance of persistent pathogens or tumors Can we restore CD8 + T cell responses against chronic infections?
Nature, 2006 PD-1 inhibitory receptor induced on activated CD8 + T cells If unable to control the infection, PD-1 causes CD8 + T cell exhaustion Blocking PD-1 re-activates CTL - allows viral clearance
David Masopust
CTL cause immunopathology Too vigorous CTL response is bad CTL response to hepatitis and influenza in humans CTL response to LCMV in mice