1Cytotoxic T Lymphocytes(CTLs) and NK CellsAfter activation, naïve T cells differentiateinto effector and memory T cellsAfter activation, T cells remain in lymph nodes for 5-6 daysEffector T cellsCell Function Released Effector Molecules Membrane Effector Molecules Th1 Cytokines to activate CTLs and macrophages IL-2, IFN TNF , GM-CSF TNF Th2 Activate B cells IL-4, IL-10, CD40L CTL Kill Target cells Perforin, Granzymes Fas Ligand CTLs do not require costimulation to kill infected targetsChromium Release AssayExperiments demonstrating CTL killing of target cells are usually done with achromium (51Cr) release assay, which measures target cell lysis in 4-8 hours.Infect mouse with virus to generate CTLs specific for virus infected targets. Wait until mouse has generated immune response. Use Spleen as source of CTLs. Incubate with syngeneic targetsinfected with virus that are radioactively labeled with51Cr that will be released when cell is lysed. Chromium attaches toproteins in the cytoplasmof target cells2Chromium Release CTL Assay•Target cells mixed with effector cells at various ratios.•Measure release of 51Cr into media.•Express as percentage lysis relative to non-specific effector cell. Perforin/Granzyme BPathwayDirectional release ofgranules (in red). How do CTL kill target cells?CTL Killing Perforin• Contained in CTL granules• Pore forming protein.• Pokes holes in target cell membrane.• Homologous to C9.Perforin forms polymers thatpoke holes in membranes to allowenzymes inside cell.CTL KillingCTL Killing Granzymes• A family of proteases which are involved ininduction of apoptosis in target cells after entrythrough perforin pores.• Granzyme = granule enzyme• Granzyme B is most active granzyme.3CTL Killing Perforin/Granzyme pathwayAntigen specific killing of infected target cells allows for reduced bystander killing of uninfected cells. CTL Killing Selective (antigen specific) killing happens in minutes.CTLs can be serial killers (repeatedly killing).Why don't CTLS kill themselves?Cathepsin B-- a proteasewhich cleaves perforin!!!controlAnti-cathepsinBWildtype Perforin KO VirusVirus51Cr labeled targetsHarvest splenocytesHarvest splenocytesPerforin deficient mice can still killvirally-infected target cells. How?Specific killing ofvirally infected targetsSpecific killing ofvirally infected targetsFas/Fas Ligand PathwayCTLs express Fas ligand interact with Fasexpressed on the target cell surface.Fas Induced Apoptosis Cascade of CaspasesProteases that cut at C-terminal side of anaspartate. Pro-enzyme form becomes active throughcleavage into subunits.Proteolytic cascade must be activated for eventualDNA fragmentation.4Fas Induced ApoptosisBcl-2 Proteins Control ApoptosisPro-apoptosis and anti-apoptosis proteinsBcl-2 proteins can inhibit or activate apoptosis.Proteolytic cascade must be activated and notinhibited by anti-apototic bcl-2 proteins.(Target cell has to want to commit suicide).CTLPathways ofCytotoxicitySummaryNaïve CD8 T cells are activated in secondary lymphoidorgans and differentiate into CTL effector cells.CTLs can kill targets independently of costimulation.Once a CTL encounters a target cell it releases cytotoxicgranules containing perforin and granzymes.Perforin forms pores in the membrane of the target cellallowing granzymes to enter the cell.Granzymes induce apoptosis in the target cell by cleavingcaspases.CTL also express FasL and can kill targets via Fas expressedon target cells.But CTLS are not enough…..Viruses are tricky!Class I MHC inhibition by viruses•Virus Protein Effect on class I•Adenovirus E3-k19 Retain in ER•HSV-1,2 ICP47 Blocks TAP•EBV EBNA1 Block peptides•CMV US2 ER to cytosol•CMV US3 Retain in ER•CMV US6 Blocks TAPNK Cells Detect "Missing Self"Protection Against VirusesN on-infec te danti-c la ss IC MV i n fect e danti-c la ss IcIgCMV infection down-regulated MHC class I on human fibroblastsImmune evasion mechanism of viruses to decrease Class I MHC. NK cells preferentially kill cells that have lost expression of MHC class I.5NK Cells Detect "Missing Self"Immune surveillance for TumorsTumor cells often have decreased expression of Class I MHC to escape T cell recognition.NK cells kill tumor cells.NK cellsDistinct lineage of lymphocytes.Do not rearrange α,β,γ or δ TCR.CD3-,CD56+ in humans.CD3-,NKR-P1+ (NK1.1) in rodentsEffector functions include cell-mediatedcytotoxicity & cytokine secretion.CD3+Ste m Ce llT / NK Prog e nitorPre-T Pre-TMature TThymusCD34++CD34+CD16+, CD56+Mature NKPre-NKCD4+ or CD8+CD3+CD3+CD3+TCRRe arrange mentCD3-CD4+, CD8+NK and T cell DevelopmentBone MarrowStromaIL-2, IL-7, stem cell factor (SCF)CD3-, CD4-, CD8-Thymus not requiredfor development. Normal NK cells inscid mice and micewithout RAG1 orRAG2NK Cells - Distribution~5-20% peripheral blood lymphocytes~5% lymphocytes in spleenRare in uninfected lymph nodes>90% of lymphocytes in placentaNK Cells - Effector FunctionsCell mediated-cytotoxicity– Perforin granzyme pathway– Secreted or membrane TNF-αAntibody-dependent cellular cytotoxicity(ADCC)Cytokine secretion– Early γ-interferon production– Secretion of TNF-α, LT-α, GM-CSF, IL-5, M-CSF, IL-3, IL-10, IL-13.Antibody-dependent cellularcytotoxicity (ADCC)Cells that performADCC must haveFC receptors to bind Ig moleculesand trigger killing of target cell.6Cytokine secretion of NK cellsRole for IFN-γVirusesVaricella zoster virus & CMV are life-threatening in humans lacking NK cells.BacteriaNK cells protect against intracellular bacteriawhich tend to infect macrophages. (e.g.Listeria Toxoplasma, Leishmania)Natural Killer (NK) CellsPart of Early Immune ResponseNK are lymphocytes without traditional antigen receptorsHow do they get activated?• NK cells express both activating and inhibitory receptors• Inhibitory receptors recognize MHC class I (self) on target cells• Activating receptors recognize ligands upregulated on infectedcells or tumor cells+–NK cell Activating ReceptorsHave ITAMsIntracellular signal oflymphocyte receptorsthrough ITAMs. Immunoreceptor Tyrosine basedActivating motifs (ITAMs). NK cell Activating ReceptorsHave ITAMsSTIMULATORYRECEPTOR(FcR, NKR-P1,NKG2D)YXXLLigandPPhosphorylation of substratesImmunoreceptortyrosine-basedActivation motif(ITAM)YxxL x 2e.g. SYKProteinTyrosineKinaseYXXLDAP12+--Class I MHC Specific NK Inhibitory ReceptorshumanmouseIg SFC-type lectin-related family=