CytotoxicTCellFunctions

Shomyseh(Shomi)Sanjabi,PhDShomyseh.Sanjabi@gladstone.ucsf.edu

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&parasitesü Listeria,Mycobacteria,Toxoplasma

§ IFNγ &macrophageactivationimportant§ 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?