Cytotoxic T Cell Functions Shomyseh (Shomi) Sanjabi, PhD [email protected] Micro 204, 11/05/18, CL220
CytotoxicTCellFunctions
Shomyseh(Shomi)Sanjabi,[email protected]
Micro204,11/05/18,CL220
RecommendedReading:Textbook:• Janeway’s 9th Edition:Chapters9and11.
Reviews:• Kaech andCui.TranscriptionalcontrolofeffectorandmemoryCD8+Tcell
differentiation(2012)NatRevImmunology12:749.• Farberetal.HumanmemoryTcells:generation,compartmentalization
andhomeostasis.(2014)NatRevImmunology14:24.• Bantug etal.ThespectrumofTcellmetabolisminhealthanddisease.
(2018)NatRevImmunology18:19.
Primaryliterature:• Arakietal.mTOR regulatesmemoryCD8T-celldifferentiation.(2009)
Nature 460:108.• Limetal.Neutrophiltrailsguideinfluenza-specificCD8+Tcellsinthe
airways.(2015)Science 349:aaa4352.• Schenkel etal.Tcellmemory.ResidentmemoryCD8Tcellstrigger
protectiveinnateandadaptiveimmuneresponses.(2014)Science 346:98.
TheCourseofaTypicalAcuteInfectionThatisClearedbyanAdaptiveImmuneResponse
Ultimategoal:1) clearpathogenwithminimaldamagetohost2) formimmunologicalmemory
BothInnateandAdaptiveCellsareInvolvedinCytotoxicity
Dranoff (2004)NatureReviewsCancer
•NaturalKillercells•γδ-TcR+Tcells•CD4+αβ-TcR+Tcells•CD8+αβ-TcR+Tcells
• AntibodiescanPREVENT infection
• CTLcanonlykillthecellAFTER itisinfected
LayeredImmuneResponsetoMinimizeCostandProvideSufficientProtection
IwasakiandMedzhitov NatureImmunology2015
• Viralinfectionsü Acutelyticviruses(polio,vaccinia,influenza)ü Persistentnon-lyticviruses(CMV,HSV,EBV,HIV)thatneedcontrolFOREVER
• Intracellularbacteria¶sitesü Listeria,Mycobacteria,Toxoplasma
§ IFNγ ¯ophageactivationimportant§ CTLimportantforclearance
• Tumors• Immunopathology
• Metaboliccost
Haringetal.2006Immunity
StagesofCD8TCellActivation,Differentiation,andMemoryFormation
SLECs(KLRG1hi) MPECs(IL-7R⍺hi)
I.TCellActivation
SpecializedDCsforCD8TCellPriming
Caminschi et al. 2012, Front. in Immunology
Cross-presentingability
ThreeSignalsareRequiredtoActivateNaïveTCells
1) TCRactivation:(Foreign-peptide:selfMHCcomplex&T-cellreceptor)
à Activation
2)Co-stimulation:(B7.1/B7.2&CD28)à Survivalandproliferation(IL-2)à Primingofmitochondriaformemory
3)Inflammation:(cytokines&correspondingreceptor)
à Differentiationà Inflammatoryenvironmentcan
affectCD8TcelldifferentiationintoSLECs(i.e.IL-2,IL-12,IFNγ)vs.MPECs(i.e.IL-10,IL-21,TGF-β)
ModelsforGeneratingEffectorandMemoryCellHeterogeneity
Kaech andCui2012NatRevImmunol
Cumulativehistoryofsignals.
Overallstrengthofthesignals.
Faterisesfromfirstcelldivision.
Kaech andCui2012NatRevImmunol;Changetal.2014NatImmunol
SignalsContributingtotheDiversityofMemoryCells
KimandSuresh,2013FrontiersinImmunol
OrchestrationofCD8TCellDifferentiationbythePI3K/Akt Pathway
WhydoweneedagradedCTLresponse?
AGradedViewofTCellMetabolicStatesinHealthandDisease
Bantug etal.2018NatRevImmunol
AlteringtheDifferentiationofCD8+TCellsCanInfluenceTheirFunction
Sukumar etal.2013JCI123:4479Raoetal.2012Immunity 36:374
Sade-Feldmanetal.2018Cell
ThreeSignalsareRequiredtoActivateNaïveTCells
1) TCRactivation:(Foreign-peptide:selfMHCcomplex&T-cellreceptor)
à Activation
2)Co-stimulation:(B7.1/B7.2&CD28)à Survivalandproliferation(IL-2)à Primingofmitochondria
3)Inflammation:(cytokines&correspondingreceptor)
à Differentiationà Inflammatoryenvironmentcan
affectCD8TcellsdifferentiationintoSLECs(i.e.IL-2,IL-12,IFNγ)vs.MPECs(i.e.IL-10,IL-21,TGF-β)
CD28Co-stimulationInducesIL-2andIL-2Rα(CD25)ExpressiononActivatedTCells
Note:IL-2atprimingalsoprogramssecondaryexpansionofmemorycells.
ProlongedInterleukin-2R⍺ ExpressiononVirus-SpecificCD8+ TCellsFavorsTerminal-Effector
DifferentiationInVivo
Kalia etal.2010Immunity
MitochondrialPrimingbyCD28
KleinGeltink etal.Cell2017
ActivatedTcellsExpressCD69toInhibitTheirEgressfromLymphNode
• TcellsthatdidnotseetheircognateantigenexittheLNbyfollowingsphingosine 1-phosphate(S1P)gradientviaS1PR1.
• ActivatedTcellsexpressCD69,whichinternalizesS1PR1,thusTcellscannotrespondtoS1PgradientandremaininLN.
• AfterTcellproliferationinLN,CD69expressiondecreasesandS1PR1reappears.
IfCo-stimulationisWeakonDCs,CD4TCellsCan“Help”CD8TCellPriming
1)CD4TcellshelpfurtheractivateAPC:• B7expressedbytheDCsfirstactivates
theCD4TcellstoexpressIL-2andCD40ligand.
• CD40ligandbindsCD40ontheDC,deliveringanadditionalsignalthatincreasestheexpressionofB7and4-1BBLbytheDC.
• Thisprovidesadditionalco-stimulationtothenaiveCD8Tcell.
2)TheIL-2producedbyactivatedCD4TcellsalsoactstopromoteeffectorCD8T-celldifferentiation.
• ActivatedDCsthatproduceIL-12andIL-18canstimulatenaïveCD8TcellstoproduceIFNγ.
• CD8producedIFNγ activatesmacrophagesforthedestructionofintracellularbacteriaandcanpromoteantiviralresponsesinothercells.
BystanderCD8TCellActivationviaIL-12andIL-18
ActivationofTCellsChangestheExpressionofSeveralCell-surfaceMolecules
LNhoming
BindingtoP- andE-selectins onendotheliumat
sitesofinflammation
ControlofLNEgress
Increasessensitivitytostimulationby
lowconcentrationofpeptide:MHC
complex.
Tcellarrestoninflamedvascular
endotheliumhoming
Increasesavidityofinteraction
withpotentialtargetcells
TechnicalSideNotes:
• Peptide:MHC Tetramers• Antigen-specificTCRTransgenics• AdoptivetransferofTcells• CFSElabelingtomonitorproliferation• MonitoringTcellpriminginvivo.
IdentificationofT-cellReceptorSpecificityUsingPeptide:MHC Tetramers
• Peptide:MHC tetramersareusedtoidentifypopulationsofantigen-specificTcells.
• Multimers ofthepeptide:MHC complexincreasetheavidityoftheinteraction withspecificTCR.
• Thestreptavidinmoietyislabeledwithafluorochrome toallowdetectionofthoseTcellscapableofbindingthepeptide:MHC tetramer.
Biotin
Peptide
GatedonCD3+cells:
AntigenSpecificTCRTransgenicMiceonCongenicBackground
TcellclonewithknownTCR
specificityandMHCrestriction
GeneraterearrangedαandβchaincDNA
constructs
Injectintofertilizedmouseovumtocreate
transgenicmice
BreedTCRtransgenicmicetocongenicand/orRAG-/-background
100%congenicallymarkedTcellswithknownspecificity.
CreatespecificgeneKOTcellswithknownAg
specificity.
AdoptiveTransferofCongenically MarkedCells
• CD45.1andCD45.2areallelicvariantsofCD45,whichisanabundantcell-surfacereceptor.
• Thesetwoallelescanbedistinguishedbyspecificantibodies(CD45.1orCD45.2).
• HeterozygousmiceareCD45.1.2,whichisusefulforco-adoptivetransfers.
FlowCytometric AssayforCellProliferationBasedonCFSEDilution
1)Incubatecellswithafluorescentdyesuchascarboxyfluoresceinsuccinimidyl ester(CFSE).
2)Dyebecomescovalentlycoupledtolysineresiduesoncellularproteins.
3)Eachtimethecelldivides,eachdaughtercellinheritsone-halfoftheCFSE-labeledproteins.
• Underoptimalconditions,thisassayiscapableofdetectingupto7–8celldivisions,afterwhichCFSEfluorescencecannolongerbemeasured.
• Bromodeoxyuridine (BrdU)orki67(antigeninnucleiofdividingcells)canbeusedtoidentifydividingcells.
II.Proliferation,Migration,CTLActivity
Proliferation,Differentiation,andEffectorFunction
• IL-2signalingenhancesclonalexpansionandcontributestothedifferentiationoftheTcellstoeffectorcellstatus(andmemoryprogramming).
• EffectorTcellsleavetheLNandmigratetositeofinfection.• AnyencounterthateffectorTcellshavewithspecificantigen,triggerstheir
effectoractionswithouttheneedforco-stimulation.• Thus,aCTLscankillanyvirus-infectedtargetcell,includingthosethatdonot
expressco-stimulatorymolecules.
HowdoprimedCTLsknowwheretogotoexerttheireffectorfunction?
AntigenSamplingandImprintingoftheTcellsforMigrationBacktoSiteofInfection
IslamandLusterNatureMedicine2012
MigratoryDCsfromvarioustissuescan“imprint”Tcellstoexpresstraffickingreceptorsthatenabletissue-specifichoming.
ShinandIwasakiImmunologicalReviews 2013
RestrictedEffectorTCellMigrationIntoSomeTissues
CD8(+)Tlymphocytemobilizationtovirus-infectedtissuerequiresCD4(+)T-cellhelp.(Nakanishietal.2009Nature)
Neutrophiltrailsguideinfluenza-specificCD8+Tcellsintheairways.(Limetal.2015Science)
InteractionofCTLswithTheirInfectedTargets
• Immunologicalsynapseorthesupramolecular activationcomplex(SMAC)isclusteringofT-cellreceptorsandtheirassociatedco-receptorsatthesiteofcell–cellcontact.
• CentralSMAC(cSMAC)containsmostofthesignalingproteinsforT-cellactivation.
• Peripheral(pSMAC)istheLFA-1andthecytoskeletalproteintalin,whichconnectsLFA-1totheactincytoskeleton.
FormationofImmunologicalSynapse
T-cellreceptorcontrolsthedeliveryofeffectorsignalsinthreeways:
1) itinducestightbindingofeffectorcellstotheirtargetcellstocreateanarrowspaceinwhicheffectormoleculescanbeconcentrated;
2) itfocusesdeliveryofeffectormoleculesatthesiteofcontactbyinducingareorientationofthesecretoryapparatusoftheeffectorcell;
3) ittriggersthesynthesisand/orreleaseoftheeffectormolecules.
EffectorT-cellactivityisthushighlyselectiveforappropriatetargetcells,eventhougheffectormoleculesthemselvesarenotantigen-specific.
DirectedReleaseofEffectorMolecules
CTLsrelease:• Perforin – Aidsindeliveringcontentsofgranules
intothecytoplasmoftargetcell• Granzymes – Serineproteases, whichactivate
apoptosisonceinthecytoplasmofthetargetcell• Granulysin (notinmice) – Hasantimicrobial
actionsandcaninduceapoptosis• EffectorcytokinestoincreaseMHC-I;activate
macrophagesandinduceNOproduction; activateNKcellsandneutrophils;andenhanceanti-viralactivity.
CTLscarry:• Fasligand(CD178) – membrane-boundeffector
molecule,andwhenitbindstoFas(CD95)onatargetcell,itactivatesapoptosisintheFas-bearingcell.
DifferentMechanismsofKillingbyCTLs
ExtrinsicPathwayofApoptosis(FasL/Fas)
CTL
Targetcell
IntrinsicPathwayofApoptosisisMediatedbytheReleaseofCytochromecfromMitochondria
• Pro-apoptoticexecutionersbindtomitochondrialmembranesandcandirectlycausecytochromecrelease.(Formporesinthemembranes?)
• Anti-apoptoticprotectorsbindtothemitochondrialmembranetoblockthereleaseofcytochromec.(Directblockingthefunctionofthepro-apoptoticfamilymembers?)
• Sentinelseitherblocktheactivityoftheanti-apoptoticproteinsorstimulatetheactivityoftheexecutionerpro-apoptoticproteins.
IntrinsicPathwayofApoptosisisRegulatedbytheBcl-2FamilyofProteins
TargetedInductionofApoptosisbyCTLs
TechnicalSideNotes:
• Methodstomeasureapoptosis– TUNEL– Annexin V– Ab detectionofactiveCaspase 3/7
• MeasuringCTLactivityinvitro• MeasuringCTLactivityinvivo
DetectionofapoptoticcellswithTUNELStain
TUNEL(TdT- dependentdUTP–biotinnickendlabeling)
• The3’endsoftheDNAfragmentsgeneratedinapoptoticcellsarelabeledwithbiotin-coupleduridine byusingtheenzymeterminaldeoxynucleotidyl transferase (TdT).
• Thebiotinlabelisthendetectedwithenzyme-taggedstreptavidin,whichbindstobiotin.
• Whenthecolorlesssubstrateoftheenzymeisaddedtoatissuesectionorcellculture,itproducesacoloredprecipitateonlyincellsthathaveundergoneapoptosis.
DetectionofapoptoticcellswithAnnexin V
• Whencellsundergoapoptosis,theenzymeresponsibleformaintainingphosphatidylserine (PS)polarity,calledflippase,isnolongeractive.
• Asaresult,PS’spolarheadgroupsbecomeexposedontheextracellularfaceoftheplasmamembrane.
• FluorescentlylabeledAnnexin Vcanbindtightlytotheexposedpolarhead.• Annexin Vstainingisoftencombinedwithaviabilitydyesuchaspropidium
iodide(PI)or7-aminoactinomycinD(7-AAD).
DetectionofApoptoticCellsbyIntracellularStainingforActiveCaspases
• Whencellsareundergoingapoptosis,pro-caspase 3iscleavedintotwosubunitsthatdimerize toformtheactiveenzyme.
• Fluorescentlylabeledantibodiescanbeusedtodetecttheactiveformofcaspase 3 infixedandpermeabilizedcells.
InVitroCTLActivityasMeasuredbyChromiumReleaseFromLabeledTargetCells
• Livecellswilltakeup,butdonotspontaneouslyrelease,radioactivelylabeledsodiumchromate,Na251CrO4.
• Whenlabeledcellsarekilled,theradioactivechromateisreleasedanditcanbemeasuredinthesupernatant.
InVivoCTLActivityasMeasuredbyKillingofCFSELabeledTargetCells
• TargetcellsareincubatedwithMHC-Irestrictedantigenicpeptide.
• ThesecellsareincubatedwithalowconcentrationofCFSE.
• AcontrolpopulationofcellsthatisnotgiventheantigenicpeptideisincubatedwithahighconcentrationofCFSE.
• Thetwocellpopulationsaremixed1:1andinjectedintoexperimentalanimals.
• Fourhourslater,spleencellsareanalyzedbyFACS.
• Specifictarget-celllysis iscalculatedfromtheratioofthetwoCFSE-labeledcellpopulations.
III.ContractionandMemoryPrecursorFormation
Commonγc CytokinesPlayanImportantRoleinEffectortoMemoryTransition
Schluns &Lefrancois 2003NatRevImmunol
CD25
CD122
CD127
Formitogenic andanti-apoptoticsignals:• NaïvecellsdependonIL-7• EffectorcellsdependonIL-2• MemorycellsdependonIL-7andIL-15
MemoryTCellsArisefromEffectorTCellsThatMaintainorRe-expressIL-7Rα
IL-7Rαhi CellsMayCompeteMoreEffectivelyfortheSurvivalSignalsDeliveredbyIL-7
TakadaandJameson2009NatRevImmunol
IV.MemoryCellSubsetsandCompartmentalization
Jandus etal.2017,MethodsinMolecularBiology1514:1
Mouse/HumanCD8MemoryTCellSubsets
CompartmentalizationofMemory TCellSubsetsinSpaceandTime
Farberetal.2014NatRevImmunol
TRMcellsmayoutnumbertherecirculatingTcellsthatmigratethroughthebody.
TRMCellsareDerivedFromEffectorTCells
Beura andMasopust Cell 2014
TRMsResideinTissuesattheSiteofOriginalInfectionandProvideProtectionAgainstReinfection
CD8TRMsTriggerProtectiveInnateandAdaptiveImmuneResponses
CarboneandGebhardt Science 2014
“Tissue-residentmemoryTcellsmaybetheimmunecellsofchoiceindesigningstrategiestostoppathogensbeforethey
establishameaningfulinfection.”
NK DC VCAM-1
MemoryCD8+ T&Bcells
recruitment
Bystanderprotection
“Alarmingfunction”
Localviralinfections
Schenkel etal.2014Science
HallmarkofImmunologicalMemory
• Vaccinationartificiallyinducesprotectiveimmunitythoughgenerationofimmunologicalmemory.
• Manyconsidervaccinationtobethemostoutstandingaccomplishmentofimmunologyinthefieldofmedicine.
Larger#ofprecursors
Physicallocationofmemorycells
CD4TCellsareRequiredfortheDevelopmentofFunctionalCD8MemoryTCells
• MHC-II-/- micefailtodevelopCD4Tcells.• ExpansionofeffectorCD8TcellsisnotalteredinMHC-II-/- mice.• Uponre-challengeAg-specificmemorycellsfailtoexpandinMHC-II-/- hosts.
HowdoCD4TcellshelpdevelopmentoffunctionalCD8memoryTcells?
IL-10ProducedbyCD4+ Tregs PromotestheMaturationofMemoryCD8+ TCells
Laidlawetal.2015NatImmunol; Laidlaw2016NatRevImmunol
Duringtheresolutionphase,Treg cell-derivedIL-10actstosuppressthematurationstateofDCsandlimittheirproductionofpro-inflammatorycytokines,whichallowforthecontinuedmaturationofeffectorCD8Tcellsintofunctionalmemorycells.
CD4TCellsPromotetheMaintenanceofCD8
MemoryCells
• WTMiceareimmunizedwithLCMV.• Memorycellsaretransferredinto
eitherWTorMHC-II-/- mice.• MemoryCD8TcellsdeclineinMHC-
II-/- mice.• Thishasimplicationsforconditions
suchasHIV/AIDSwhereCD4Tcellnumbersarediminished.
WhatwillittaketomakeaCTL-basedvaccineforHIV?