Anergy in Memory CD T Cells Is Induced by B Cells Sar - PDF document

AnergyinMemoryCD4TCellsIsInducedbyBCellsSaratK.Dalai,*SaiedMirshahidi,*AlexandreMorrot,FidelZavala,andScheherazadeSadegh-NasseriInductionoftoleranceinmemoryTcellshasprofoundimplicationsinthetreatmentofautoimmunediseasesandtransplantrejection.Previously,wereportedthatthepresentationoflowdensitiesofagonistpeptide/MHCclassIIcomplexesinducedanergyinmemoryCD4Tcells.Inthepresentstudy,weaddressthespecicinteractionofdifferenttypesofAPCswithmemoryCD4 *DepartmentofPathology,JohnsHopkinsSchoolofMedicineandDepartmentof differentdosesofcOVA.WedemonstratethatanergyisinducedinmemoryTcellsexvivowhenBcellspresentedtheAgatacertainrangeoflowpeptidedoses.Further,wevalidatedthisndinginvivobydemonstratingthattransferringrestingBcellspulsedwithlowdosesofpeptidetomicebearingmemoryCD4Tcellsinducedanergy.WeruledoutaroleforCD11corotherDCsintheinductionofanergyinmemoryTcells.ConsistentwiththeroleforrestingBcellsexpressinglowlevelsofB7-1intheinductionofanergy,weshowedthatanergyinthissystemisreg-ulatedbyCTLA-4.MaterialsandMethodsTCRTgmice(DO11.10)thatexpressTCRrecognizinganI-AstrictedcOVAonBALB/cbackgroundwereusedasthesourceofTcells.Non-TgfemaleBALB/cmiceandC57BL/6(B6)miceat5–6wkofage,TgfemaleDO11.10andOTIImice,andcell-decientmuMTmicewereallpurchasedfromTheJacksonLaboratory.Diphtheriatoxinreceptor(DTR)TgmiceonBALB/cbackgroundwerebredwithnon-TgBALB/cmice,andtheheterozygousoffspringforDTRwereusedforthestudy.AllmicewerehousedintheJohnsHopkinsUniversityanimalfacilities(Bal-timore,MD)undervirus-freeconditions.AllexperimentswereperformedinaccordancewithprotocolsapprovedbytheAnimalCareandUseCom-mitteeoftheJohnsHopkinsUniversitySchoolofMedicine.PeptidesandAbsThepeptidecOVA(ISQAVHAAHAEINEAGR)wassynthesizedbyGlobal-PeptideServices.Thepeptidewas90%pureasanalyzedbyre-verse-phaseHPLC.FluorescentlylabeledAbstomouseCD4,CD25,CD44,CD45RB,CD62L,CD69,CD45R(B220),CD19,CD11c,IL-2,,anti-mouseCTLA-4,CD80(B7.1),andCD86(B7.2)werepur-chasedfromBDPharmingen;theAbtoclonotypicTCR(KJ1.26)specicforDO11.10CD4TcellswaspurchasedfromCaltagLaboratories;andanti-mouseCTLA-4Abwaspuriedfromtheculturesupernatantofhy-bridoma(UC10-4F10-11;AmericanTypeCultureCollection(ATCC)).AdoptivetransferandimmunizationsTgCD4Tcells(2.0cellspermouse),preparedfrompooledLNsandspleensofDO11.10mice,wereresuspendedin100lofsterilePBS(Invitrogen)andtransferredi.v.intoBALB/crecipients.Twodayslater,micewereimmunizeds.c.(atthebaseofthetail)with15nmolofcOVApeptideemulsiedinCFA.InductionofanergyincOVA-specicmemoryCD4cellsinvivobyadministrationoflowdosesofpeptideinIFAFiveweekspostimmunization,toallowforthegenerationofmemoryTcells(veriedbystainingformemoryTcellmarkers;datanotshown),micewereinjecteds.c.withincreasingconcentrationsofcOVAtide(0.005–50,000pmol)mixedwithIFA.Tendayslater,inguinalLNswereremovedandTcellswerechallengedinvitrowithcOVAtide.ProliferationwasmeasuredbyCFSEdilutionassay.Briey,LNcellswerelabeledwith1MCFSEinPBSfor80satroomtemperatureandwashedtwicewithRPMI1640containing10%FCS.Cellswerecul-turedwithorwithoutpeptideat37Cfor72h.Followingtheculture,Tcellswerestainedwithuorescentlylabeledanti-mouseCD4AbandKJ1.26anticlonotypicTCRAbandanalyzedbyowcytometry.Tcelldivisioninresponsetoinvitropeptidechallengewascalculatedasde-scribed(14)usingthefollowingformula:numberofevents()ateachcelldividedby2.Thepercentageoftotaldividingcellsnumberofcohortsundergoing1–6divisions/totalnumberofcohortsun-dergoing0–6divisions)IsolationofTcellsSpleensfrom5-wkpostimmunizedmicewereremovedandasingle-cellsuspensionwasmadeafterlysisofRBCsandusedasthesourceofTcells.ThemicesacricedforthispurposehadreceivedasingleimmunizationafteradoptivetransferofTcells.ByusinganR&DSystemsCD3enrich-mentcolumn,Tcellswereisolatedfromsplenocytes.TcellpuritywasIsolationofinvivopulsedAPCsForty-eighthoursafters.c.injectionofpeptideemulsiedinIFA,miceweresacricedandinguinalLNswereremoved.ToseparatewholeAPCs,LNcellsweredepletedofTcellsbyusingeitherDynal-Thy1.2beadsorMACS-Thy1.2microbeads(MiltenyiBiotec)(95%depletion).ToisolateBcells,aMACSB220microbeadwasused(95%purity).ToobtainAPCsdevoidofBcells,LNcellsweredepletedofTandBcellsbyusingacombinationofThy1.2andB220microbeads(MACS).MACSCD43microbeadswereusedtopurifyB2Bcells.ToobtainAPCsdevoidofB2Bcells,LNcellsweredepletedofTcellsbyThy1.2microbeadsfollowedbypositiveselectionwithCD4microbeads(15).TheseAPCsaretermedinvivopulsedAPCsinthisstudy.InductionofanergyinmemoryCD4TcellsexvivobyinvivopulsedAPCsToinduceanergyinmemoryCD4Tcells,enrichedTcells(1wereincubatedwithinvivopulsedAPCs(irradiated,2000rad)ataratioof1:2for48hina96-wellplate.Totestforanergy,splenocytesfromnormalmicewerepulsedinvitrowithcOVApeptide,irradiated,andadded(2perwell)totriplicateculturewellsandincubatedfurtherfor72hbeforeadding[H]thymidineorfor10hbeforeassayingforintracellularcytokinesynthesis.Todeterminethepercentageofcellsmak-ingIL-2orIFN-,cellswerestainedwithAbstoCD4,clonotypicTCR(KJ1.26),andIL-2orIFN-andanalyzedbyowcytometry.Duringdataanalysis,thequadrantsweredrawnbasedonthematchedisotypeAbcon-trols.ThepercentageofDO11.10cellsmakingIL-2orIFN-wascalcu-latedaftersubtractingthepercentageofDO11.10cellsstainedpositiveforthematchedisotypes.InductionofanergyinmemoryCD4TcellsexvivobyinvitropulsedBcells,DCs,oractivatedBcellsWefollowedthesameanergyprotocolasaboveexceptthatBcellsorDCswerepuriedfromthespleensofunimmunizedmicebyusingMACs-B220 FIGURE1.AdministrationofsuboptimaldosesofcOVAtideinIFAinducesanergyinspecicmemoryCD4Tcellsinvivo.Eightgroupsofmice(threemicepergroup)bearingmemoryCD4Tcellswerechallengedwithincreasingdoses(0.005–50,000pmol)ofcOVApeptideinIFA.Tendayslater,cellsfromdrainingLNswereharvestedandanergywasassayedbypeptiderechallengefor72hinvitro.Tcellpro-liferationwasmeasuredbyCFSEdilutionassayofAg-specicKJ1–26Tcells.ThedosesfortheinvitrochallengewithcOVApeptidearerepresentedasfollows:lledblackhistogram,0M;thingrayline,0.1M;blackline,1M;thickgrayline,10M.Datashownrep-resentoneofthreeindependentexperiments.ThelowerpanelthepercentageofAg-speciccells(DO11.10)dividedinresponsetoinvitrochallenge.3222BCELLSANERGIZEMEMORYCD4TCELLS forBcells(purity95%)orMACs-CD11cmicrobeadsforDCs(purity80%)andpulsedinvitrofor3hat37°CwiththecOVAbeforeincubatingwithmemoryTcells.ForactivatedBcells,puriedBcellswerestimulatedwithCpG(6g/ml)for24h(16)andwashedtwicebeforepulsingwithpeptide.NotethattheamountofpeptideforpulsingAPCsinvitrowascalculatedbasedonmolarpeptideconcentration,whereasthepeptidesgiventomicewereinmoleunits.InductionofanergyinmemoryTcellsinvivointheabsenceofBALB/cmiceexpressingDTRundertheCD11cpromoter(17)wereadop-tivelytransferredwithDO11.10TgTcells.Twodayslater,micewereimmunizeds.c.withcOVApeptideinCFA.After5wk,DCsweredepletedbymultipleinjectionsofdiphtheriatoxin(DT)(Sigma-Aldrich)duringtheinductionofanergyinvivointhesemice.Briey,micewereinjectedi.p.with100ngofDTpermouse12hbeforetheinjectionofpeptideemulsiedinIFA.Then,micewereinjectedwiththesamedoseofDTtwicemore,at10and36hafterAgchallenge.SamplingofspleenandLNtissuesduringthetreatmentinaparallelsetofmiceconrmedthedepletionofDCsinDT-injectedmiceasdetectedbyselectiononCD11candGFP(expressedundertheCD11cpromoter),indicating0.2%DCsintheLNsofthosemice.Onday3(60hafterAginjection),miceweresacricedandcellsharvestedfromthedrainingLNswerechallengedexvivowithcOVApeptidefor72h.InductionofanergyinAg-specicTcellswasdeterminedbyCFSEdilutionassay.ThepercentageofAg-specic(DO11.10)CD4Tcelldivisionsinresponsetoinvitropeptidechallengewascalculatedasdescribedabove.InductionofanergyinvivoinmemoryTcellsbyinvitropulsedBcellsBcellswerepuriedfromthespleensofunimmunizedmiceandpulsedinvitrowithincreasingamountsofcOVApeptide(0.00001–1000nM)for3hat37°C.Followingincubation,Bcells(notirradiated)werewashedtwicetoremoveexcesspeptideand10cells/mousewerei.v.transferredtoBALB/cmicebearingcOVA-specicmemoryCD4Tcells.Tendayslater,miceweresacriced,andcellsfromdrainingLNsandspleenswereharvested.InductionofanergyinAg-specicTcellswasdeterminedbytheCFSEdilutionassayafterinvitrochallengewiththecOVApeptidefor72h.ThepercentageofAg-specic(DO11.10)CD4Tcelldivisioninresponsetoinvitropeptidechallengewascalculatedasde-scribedabove.InductionofanergyinmemoryCD4TcellsinvitrobyinvivopulsedB2BcellsAllBcells,B2Bcells,orAPCsdevoidofB2BcellswereobtainedfromthedrainingLNsofmiceimmunized48hearlierwithpeptideinIFAandincubatedwithmemoryCD4Tcellsfor48h.InductionofanergywastestedbyrestimulatingTcellswithcOVApeptide-pulsedsplenocytesfor72h,followedby[H]thymidineincorporation.InductionofanergyinmemoryCD4TcellsinBcell-decientmuMTmiceBecausemuMTmiceareonB6background,onlyforthisexperimentB6micewereusedasrecipientsandOT-IITgCD4TcellsspecicforwereusedforadoptivetransferandthegenerationofmemoryTcells.RecipientmicewereimmunizedwithcOVACFAfortheinductionofmemoryTcellsadayafterTcelltransferasdescribedpreviously.Intheexperimentsshown,memorycellswereiso-lated4–6moafterimmunization.CD4Tcells(7toincludeOT-IITcells)werethenpuriedandtransferredtomuMTmicebeforetheinjectionofAgforanergyinduction.B6micebearingmemoryOT-IIcellswerecalledBcell-sufcientmice.Allgroupsofmicewereimmu-nizeds.c.withcOVAinIFA.Ninedayslater,cellsfromdrainingLNswereharvestedandassayedforanergybytheirabilitytoproliferate FIGURE2.BcellsloadedwithlowdosesofAginduceanergyinmemoryCD4Tcellsexvivo.,Anergywasinducedinvivo(asdescribedinMaterialsandMethods).MicebearingmemoryCD4TcellsweregivencOVAinIFAfortheinductionofanergyand48hlaterdrainingLNswereharvestedandtestedforanergyinvitrousing[H]thymidineincorporation.,Anergywasinducedexvivo.MemoryTcellsisolated(APC-depletedTcells)frommiceimmunized5wkorlongerwereincubatedwithAPCpopulations(wholeLNcellsdepletedofTcells()orenrichedforBcells())isolatedfromBALB/cmicegivencOVAinIFA48hbeforethecellharvest.IsolatedAPCs(depletedofTcellsbyThy1.2microbeads)werepulsedwithdifferentdosesofpeptideasshownonthe-axis.MemoryTcellsandpulsedAPCswereincubatedfor48h.Culturedcellswereassayedfortheinductionofanergyduringtherst48hofexvivoculturebytheadditionoffreshlypulsedsplenocytesat0,0.1,1.0,and10McOVAinvitroandcellproliferation()andIL-2()andIFN-production()weremeasured.Thedatashownrepresentoneofthreeindependentexperiments.BarsrepresentmeanSDoftriplicatecultures.0.005,5,or50pmolvs0.05or5000pmolofpeptidedoses(Student’stest).TherewerevemicepergroupandcellsfromtriplicatewellswerepooledbeforemeasuringIL-2andIFN-synthesis.CytokinesynthesiswasanalyzedonKJ1.26cellsgatedonCD4Tcells.TheJournalofImmunology andtomakeIL-2andIFN-usingthymidineincorporationandintracel-lularcytokineassays.Forthymidineincorporation,cellswerestimulatedwithpeptidefor72hfollowedbytheadditionof[H]thymidineandincubatedfor18hbeforeharvest.Forintracellularcytokinesynthesis,cellswerestimulatedwithpeptidefor5.5hinthepresenceofGolgiStop.OT-IIcellswerethenstainedwithAbstoCD4,V2,V5.1,V5.2,andIL-2or.Duringdataanalysis,thequadrantsweredrawnbasedonthematchedisotypeAbcontrols.ThepercentageofOT-IIcellsmakingIL-2orwascalculatedaftersubtractingthepercentageofOT-IIcellsstainedpositiveforthematchedisotypes.ExpressionofCTLA-4inmemoryCD4TcellsMemoryCD4TcellswereincubatedwithinvivopulsedAPCs(BcellsorAPCsdepletedofBcells)for48h.SurfaceexpressionofCTLA-4onTcellswasdeterminedbyowcytometry.ThelevelofCTLA-4wasexpressedasCTLA-4index:meanuorescenceintensityfractionofcellspositiveforCTLA-4.ReversalofanergyinmemoryCD4Tcellsbyanti-CTLA-4MemoryCD4TcellswereincubatedwithinvivopulsedBcellsfor48hinthepresenceof10g/mlanti-mouseCTLA-4Ab(UC10-4F10-11;ATCC).AnergyorreversalwastestedbyrestimulatingTcellswithpeptide-pulsedsplenocytesfor72h,followedby[dineincorporation.StatisticalanalysesLevelofsignicance(value)wasdeterminedbyusingunpairedStu-0.02wasconsideredsignicant.specicCD4Tcellsacquirememoryphenotype35dayspostprimingWehavepreviouslyshowntheinductionofanergyinmemoryCD4Tcellsinresponsetolowdoseantigenicchallengeintwoantigenicsystems(11).ToextendthosendingstotheDO11.10Tgsystem(cOVAspecic),DO11.10TgTcellsweretransferredintowild-typeBALB/cmice(18)and2dayslater,whichallowedthehomingoftransferredcellstothelymphoidorgans,recipientswereimmunizedwith15nmolofcOVAinCFA.Thirty-vedaysafterimmunization,thetimerequiredforthegenerationofmemoryTcells(11,19),splenocyteswerehar-vestedandDO11.10CD4Tcellswerestainedformemorymarkers.LevelsofCD25,CD44,CD45RB,CD62L,andCD69expres-sionwereanalyzedonCD4andKJ1.26doublepositivecellsandcomparedwiththelevelsonnaiveTcellsfromcontrolmicetreatedwithIFAorCFAalone.Byday35postimmunization,80%oftheCD4TcellsacquiredthememoryphenotypeasevidencedbyanincreasedlevelofCD44anddecreasedlevelsofCD45RBandCD62LexpressionontheircellsurfacesInvivoadministrationoflowdosesofpeptideinIFAinducesanergyincOVA-specicmemoryCD4TcellsMicepreviouslyimmunizedwithcOVAforthegenerationofmemoryTcellswereadministereddifferentamountsofpeptideinIFA.After10days,cellsfromdrainingLNswerelabeledwithCFSEandCD4TcellswerestimulatedinvitrobyfreshAPCspulsedwithincreasingdosesofpeptidefor72h.TodetermineAg-speciccellproliferation,CFSEdilutionanalysiswasdoneondoublepositivecellsbyowcytometry.WefoundthatAg-specicTcellsthathadencountered0.5–50pmolofpeptideinvivoremainedundivided,whereasTcellsfromthemiceimmunizedwithdosesbeloworabovethatrangedividedseveralcycles(Fig.1).ThestartingnumbersofCD4TcellswerecomparableinallofthegroupsofmiceimmunizedwithpeptideinIFA,indicatingthatTcellsarenotdeletedbutratherareanergized.TheseresultsextendtheapplicabilityofourpreviousndingstoanentirelynewantigenicsysteminwhichmemoryTcellscanbeanergizedbyarangeofsuboptimaldosesofAgs(11,12).ExvivoanergyassaytotestrolesfordifferentAPCsintheinductionofanergyinmemoryCD4TcellsDeterminingwhichAPCsuniquelyinteractwithmemoryTcellsinvivoisexperimentallychallenging.Tosimplifytheproblem,anexvivoassaywasdesignedtoevaluatethecontributionofdifferentAPCstotheinductionofanergyinCD4memoryTcells.WehypothesizedthatAPCsloadedinvivowithlowdosesofpeptidecouldanergizememoryTcellsexvivoinasimilarmannerastheydoinvivo.Totestthis,memoryTcellswereisolatedfrommice35dayspostimmunizationandcoincubatedwithAPCsfromthedrainingLNsofmicethathadbeenimmunizedwithincreasingdosesofpeptide(range0–5,000pmol)inIFA48hearlier.Cellswereculturedfor48handthentestedforanergyinductionuponstimulationwithsplenocytespulsedwith0,0.1,1,and10peptide.Fig.2isacontrolproliferationexperimentindicatingthatanergyisestablishedwithin48hinvivo.Fig.2,,depictresultsoftheexvivoassayindicatingtheproliferationofTcellsexposedtoAPCsfrommiceinjectedwithdifferentdosesofpep-tideinIFA.Theseexperimentsclearlyshowthatduringthe48-hexposuretoAPCs,memoryTcellsbecameanergicinresponseto FIGURE3.InvitropulsedBcellsinduceanergyinmemoryCD4cellsexvivo.EnrichedB220Bcells()orCD11cenrichedDCs(fromspleensofvenormalBALB/cmicewerepulsedwiththeindicateddosesofcOVApeptidefor3h,incubatedwithmemoryCD4cellsfor48h,andtestedforanergyasdescribedinthelegendofFig.2using[H]thymidineincorporation.Datashownrepresentoneoffourindependentexperiments.BarsrepresentmeanSDoftriplicatecultures.0.001,0.15,or1.5nMvs0.0015or15,000nMpeptide3224BCELLSANERGIZEMEMORYCD4TCELLS wholeAPCs(LNcellsfreeofTcells)loadedwithtolerogenicdosesofpeptide,whileAPCsloadedwithnontolerogenicdosesofpeptidedidnotpreventthesubsequentproliferationofTcells(Fig.).TheproliferationofmemoryTcellsencounteringtolerogenicdosesofAg,asaccessedby[H]thymidineincorporation,wassignicantlyreduced(0.005)comparedwiththeproliferationofTcellsencounteringnontolerogenicdosesofAg,althoughnotcompletelyblocked.Thisbackgroundproliferationwaslikelybe-causeofnonspecicthymidineuptakebybystanderlymphocytes.ThismightbeduetothedistributionoftheMHCIImolecules/MHCII-peptidecomplexesonAPCsthatmostlikelyfollowthePoissondistributionand,thus,thoseAPCswithhighernumbersofMHCII-peptidecomplexesinduceactivationinTcellsratherthanBcellsastheAPCsthatinduceanergyinmemoryTcellsNext,wetestedtheabilityofpuriedB220-enrichedBcells(tobecalledpuriedBcellsthroughoutthearticle)loadedwithpep-tideinvivotoinduceanergyinAg-specicmemoryTcellsexvivo(Fig.2,)bymeasuringIL-2andIFN-synthesis.WefoundthatBcellsexposedto5–50pmolofpeptideinvivoaner-gizedmemoryTcellsasassessedbytheirreducedabilitytopro-duceintracellularIL-2andIFN-.Encouragedbytheseresults,weinvestigatedwhetherBcellspulsedwithAginvitrocouldinduceanergyexvivo.PuriedsplenicBcellsfromunimmunizedmicewerepulsedinvitrowithdifferentamountsofpeptideandtestedfortheinductionofanergyinmemoryTcellsexvivo.WefoundthatsplenicBcellspulsedwith0.1–1nMpeptideinvitroin-ducedanergy(Fig.3).Notethatwehaveusedmole(mol)unitsforabsoluteamountsofpeptideinjectedpermouseandconcentrationsofpeptideusedtopulseAPCsexvivoareinmolar(M)units.DCsdonotinduceanergyinmemoryCD4TcellsexvivoWenextexaminedthecontributionofDCstotheinductionofanergy.PuriedDCsfromthesplenocytesofunimmunizedBALB/cmicewerepulsedwithdifferentconcentrationsofpeptideandincubatedwithmemoryTcells.BecausethelevelsofMHCIIexpressiononDCswerethreetimeshigherthanthoseonBcells,theconcentrationofpeptideusedforpulsingwasadjusted3-foldtokeeptheabsolutenumbersofpeptide/MHCcomplexesbetweenBandDCsthesame.Fig.3depictsonerep-resentativeexperimentoffourshowingthatDCsdidnotanergizememoryCD4Tcellsatanyofthepeptideconcentrationstested.DCdepletiondoesnotaffecttheinductionofanergyinvivoAlthoughwefoundthatBcellsbutnotDCsinduceanergy,be-causeofthebroadimmunologicalsignicanceofDCs,itispos-siblethatourinvitroconditionswerenotidealandthatDCsmightcontributetoanergyinductioninmemoryTcellsinvivo.Toad-dressthisissue,weusedDTRTgmiceasDO11.10TgTcellrecipientsandgeneratedmemoryTcellsbyOVApeptideinCFAimmunizationasdescribedabove.DTRTgmiceexpressafusionproteinofDTRandenhancedgreenuorescenceprotein(EGFP) FIGURE4.AbsenceofCD11cDCsdoesnotaffecttheinductionofanergyinmemoryCD4Tcellsinvivo.DTRTgmiceexpressingDTRundertheCD11cpromoter(BALB/cbackground)wereusedasthesourceofmemoryCD4Tcells.Miceweregivendailyi.p.injectionsofDTfor3daysstarting12hbeforeAgchallengeinIFA.Miceweresacriced60hafterAgchallenge,andcellsfromdrainingLNswereharvested.Inductionofanergywasmeasuredby[H]thymidineincorporation()aswellasbytheCFSEdilutionassay().Forthymidineincorporation,cellswerechallengedexvivowithcOVApeptidefor54handpulsedwith[H]thymidinefor18h.ForCFSEdilutionassay,cellswerelabeledwithCFSEandsimilarlychallengedexvivowithcOVApeptidefor72h.Ag-specicTcellproliferationwasmeasuredbyCFSEdilutionassayusingowcytometryonCD4doublepositivecells.DosesforinvitrochallengewithcOVApeptidearerepresentedasfollows:lledblackhistogram,0M;blackline,0.1grayline,1.Datashownrepresentoneofthreeindependentexperiments.Datainaresummarizedasthepercentageofdividingcellsandshownin.Therewerethreemicepergroup.BarsrepresentmeanSDoftriplicatecultures.0.001,5,or50pmolvs0.005or5000pmolofpeptidedosesTheJournalofImmunology underthecontroloftheCD11cpromoter(17).ThispromoterisconstitutivelyactiveinnearlyallconventionalDCsubsets.MicedonotpossessanativeDTRandtheircellsarethushighlyresis-tanttoDT.TransgenicDCsexpresssufcientamountsofDTR-EGFPandthusaredepletedfollowinganinjectionofDT.BecauseDCsarereplenishedfromprecursors,depletionpersistsonlyfor24haftertreatmentandnewDCsaregenerated48hafterDTinjection(17).Ofnote,anergyinoursystemwasoptimallydevel-opedduringtherst48hofAgencounterinvivo(11).ToavoidthepresenceofDCsduringanergyinduction,threeinjectionsofDTweregiven:once12hbefore,andtwiceat10h,and36hafterpeptideinjectioninIFA.ThepercentageofDCs,asjudgedbyGFPuorescence,didnotexceed0.15%ofthetotalleukocytesindrainingLNs,i.e.,85–90%ofDCsremaineddepleted24hafterthelastDTinjection.Fig.4showsresultsfromtwosetsofexperiments.InFig,4experimentsweredoneinthepresenceofCD11cDCs,i.e.,DTRTgmicewithoutDTinjection,andin,withmultipleDTinjec-tions.WeobservedthatTcellsfromthemiceinjectedwith5–50pmolofpeptidewereanergizedintheabsenceofDCs(Fig.4,)astestedby[H]thymidineuptake.SimilarresultswereobtainedinanothersetofexperimentsinFig.4byCSFEdilutionassay,suggestingthatthecontributionofDCstotheinductionofanergyinmemoryTcellsisnotessential.InvitropulsedBcellsinduceanergyinmemoryCD4TcellsinvivoToclarifyfurthertheroleofBcellsintheinductionofanergy,weinvestigatedwhethermemoryTcellscouldberenderedanergicbyinvitropulsedBcellsinvivo.Totestthis,wetransferred10cellspermouse(pulsedinvitrowithpeptideconcentrationsrang-ing0.00001–1000nMfor3h)intodifferentgroupsofmicebear-ingmemoryTcells.Tendayslater,weusedaCFSEdilutionassaytodeterminewhetheranergywasinducedinthosememoryTcells.WefoundthatAg-specicTcellsceasedtodividebeyond1–2divisionsingroupsofmicethatwereinjectedwithpuriedBcellsloadedwith0.001–0.01nMpeptide,whereasTcellsinmicethatreceivedBcellspulsedwithpeptidebeloworabovethosecon-centrationsproliferatedmultipletimes(Fig.5).B2BcellsaremajorcontributorsforinductionofanergyinmemoryCD4TcellsTodissectthepopulationofBcellsthatinduceanergyinmemoryTcells,wepuriedB2Bcells(conventionalBcellsresidinginlymphoidfollicles;Refs.21and22)fromdrainingLNsofmiceimmunizedwithpeptideinIFA48hearlier,andincubatedthemwithCD4Tcellsfortheinductionofanergyexvivo.Inaparallelexperiment,wealsousedallAPCsdepletedofB2Bcellsandtestedtheirabilitytoinduceanergy.Wefoundthatbothpu-riedBcellsandB2BcellsinducedanergyinmemoryTcellswhereasAPCsdevoidofB2Bcellsfailedtodoso,indicatingthatB2BcellsaretheprimaryBcellsthatinduceanergyinmemoryTcells(Fig.6).Bcell-decientmicedonotsupportmemoryTcellanergyToestablishthatonlyBcellsandnotanyotherAPCsinduceanergyinmemoryTcellsinvivo,weusedcell-decientmuMTmice(23).BecausemuMTmicedonotsupportefcientdevelop-mentofCD4memoryTcells(24),werstgeneratedmemoryTcellsusingOTIITgcellsspecicforOVA(25,26)bytransferringOTIITgcellstoB6recipientsandimmunizingthem.MemorycellswerethentransferredtomuMTmice.AllgroupsofmiceweregivendifferentdosesofpeptideinIFAasbeforeandassayedforanergy9dayslater.Wefoundthatwhile5or50pmol FIGURE5.InductionofanergyinmemoryCD4cellsinvivobyinvitropulsedBcells.SplenicBcellswerepulsedinvitrowithdosesofcOVA(asshownonthe-axis)for3h,transferredtomicebearingmemoryTcells,and10dayslaterthemiceweresacriced.TcellproliferationwasmeasuredindrainingLNsbytheCFSEdilutionassayafterinvitrochallengewithcOVApeptide.DosesforinvitrochallengewithcOVApeptidearerepresentedasfollows:lledblackhistogram,0mM;blackline,0.1M;grayline,1.Datashownrepresentoneoftwoindepen-dentexperiments.Therewerethreemicepergroup.LowerpanelrepresentsthepercentageofAg-speciccells(DO11.10)dividedinresponsetoinvitro3226BCELLSANERGIZEMEMORYCD4TCELLS ofpeptideinducedanergyinBcell-sufcient(B6wildtype)mice,memoryOT-IIcellsfromBcell-decientmuMTmicedidnotshowsignsofanergyasevidencedbyIFN-andIL-2production(Fig.7).ThisexperimentconrmsthatBcellsarethecriticalAPCsforinducinganergyinmemoryCD4Tcells.InductionofanergyinmemoryTcellsbyBcellsismediatedbyCTLA-4isapotentnegativeregulatorofTcellactivation.TotestwhetherCTLA-4wasmediatingBcell-inducedanergyinmemoryTcells,weincubatedmemoryCD4Tcellsfor48hwithAPCs(BcellsorAPCswithoutBcells)pulsedinvivoandmea-suredCTLA-4expressionontheTcellsurface(Fig.8,Wefoundadirectcorrelationbetweentheinductionofanergyandtheup-regulationofCTLA-4(Fig.8).TcellsinteractingwithBcellspulsedwithanergizingdosesofAgexpressedhigher(fold)levelsofCTLA-4comparedwithTcellsinteractingwithBcellspulsedwithnonanergizingdosesofpeptide.APCsdevoidofBcellspulsedwithanydoseofAgdidnotinduceanergyandthelevelsofCTLA-4remainedunchanged.InonefurtherstudyoftheroleofCTLA-4,weincludedananti-mouseCTLA-4blockingAbduringexvivoanergyassayandfoundthatthepresenceofCTLA-4AbpreventedtheinductionofanergybyBcells(Fig.8InductionofTcelltoleranceiscriticalinprotectionagainstself-destructiveimmuneresponses.BecausememoryTcellsarekeyplayersinautoimmunepathogenesis(27–29),itisimportanttosilencethemtopreventautoimmunity.AlthoughtoleranceiswelldocumentedinnaiveTcells,itispoorlyunderstoodinmemoryTcells.OurearlystudiesshowedthatlowavidityengagementoftheTCRbyagonistpeptide-MHCIIcomplexesinducedanergyinmemoryCD4Tcells(10–12).AnergyinducedinthosestudieswaslonglastingandmetthedenitionofanergyofferedbySchwartzandcolleaguesas“astateofTcellunresponsivenessaccompaniedbyalackofresponsetoproliferationsignalsinclud-ingIL-2production,anditsreversibilitybyexogenousIL-2(30–32).”Inthepresentstudy,wehavetakenastepfurtherinunder-standinghowanergyisinducedandaskedwhichAPCstriggeranergyinmemoryCD4Tcells.TheabilitytomanipulatememoryTcellresponseshashighpotentialforprovidingnewtherapeuticavenues.Toaccomplishthis,anovelexvivoapproachwasdesignedinwhichmemoryTcellswereexposedtodifferentpuriedAPCpopulationspulsedwithdifferentdosesofantigenicpeptide.Wefoundthatresting,butnotactivated,BcellsaretheprimaryAPCsthatinduceanergyinmemoryCD4Tcells.Ourexperimentsalsostronglysuggested FIGURE6.B2BcellsloadedwithlowdosesofAginduceanergyinmemoryCD4Tcellsexvivo.DrainingLNcellswereharvestedfromthemicethathadbeenimmunized48hearlierwithcOVApeptideemulsiedinIFA.AllBcells()werepuriedbyB220microbeads,B2Bcells()werepuriedbyCD43microbeads,andAPCsdevoidofB2Bcells()wereobtainedbydepletingTcellsfromLNcellsbyThy1.2microbeadsfollowedbypositiveselectionwithCD43microbeads.AllthreedifferenttypesofAPCswereincubatedwithmemoryTcellsfor48h.InductionofanergywastestedbyrestimulatingtheTcellswithcOVApeptide-pulsedsplenocytesat0,0.1,1,and10Mfollowedby[H]thymidineincorporation.Thedatashownrepresentoneofthreeindependentexperiments.BarsrepresentmeanSDoftriplicatecultures.0.005,5,or50pmolvs0.05or5000pmolofpeptidedoses(Student’sTheJournalofImmunology thatCD11cDCsdidnotplayasignicantroleintheinductionofanergyinmemoryTcells.Thiswasconrmedinbothexvivoandinvivosettings.SeveralreportsindicatethatrestingBcellsmaybethepredominantAPCsthatinducetolerance.Thetolerogenicna-tureofrestingBcellsmaybeduetothelowabundanceofco-stimulatorymoleculessuchasB7,whichishighlyexpressedinDCsandactivatedBcells(33–35).AseeminglycontrarystudyreportsthatrestingBcellsfailedtoinduceanergyinmemoryTcellsspecicforHYAg(36).Failuretoinduceanergyinthatstudymightbebecauseofoneormoreofthefollowingrea-sons:1)theliganddensitypresentedbyBcellsisnotregulated;2)AgpresentationbyMHC-ItoCD8TcellsmightbedifferentfromthepresentationbyMHC-IItoCD4Tcells;and3)theactivationstatusofCD8TcellsinresponsetolowdensityofligandislikelydifferentfromthatofCD4Tcells.OurstudyrevealsthedualrelationshipofBcellswithmemoryTcells:Bcellsweretolerogenicwhenpresentinglimitednumbersofpeptide/MHCcomplexesandstimulatorywhenpresentinghighernumbersofcomplexes.ThelatterisconsistentwiththereportthatallAPCs,includingrestingBcells,canactivatemem-oryTcells,whichmaybeduetoalessstringentrequirementofmemoryTcellsforcostimulatorymolecules(34,37).Thedatapresentedhereprovidestrongevidenceinfavoroftheformer,inthatBcellsinduceanergyinmemoryCD4Tcells.Inthisstudy,memoryCD4TcellswereanergizedbothexvivobyBcellsthathadcapturedpeptideinvivo,andinvivobyBcellsthatwereloadedwithpeptideinvitro.Dendriticcellsarepotentinitiatorsofimmuneresponses,al-thoughtheyarealsoreportedtobeinvolvedintheinductionoftoleranceinvivo(38–40).WhentheavailabilityofpeptideAgislimited,aswasthecasehere,matureDCswithhighMHCclassIIexpressionontheirsurfacesmightbethelikelyAPCstocapturelowlevelsofAgandrenderTcellsanergic.Nevertheless,weclearlydemonstratethatDCswerenotresponsibleforinducinganergyinmemoryCD4Tcellsbyusingseveralseparateexperimentaldesigns.Inone,APCsdepletedofBcellsorenrichedfor FIGURE7.Bcell-decientmicedonotsupportmemoryTcellanergy.GroupsofB6andBcell-decientmuMTmicebearingmemoryOT-IITcells(18hafteradoptivetransferofpuriedCD4TcellscontainingmemoryTcells)werechallengedwithincreasingdoses(0.05–5,000pmol)ofcOVApeptideinIFA.Ninedayslater,cellsfromdrainingLNswereharvestedandtestedforanergyinvitro.TcellresponsesweremeasuredbyintracellularandIL-2production(middlepanels)and[H]thymidineincorporation(lowerpanels).ThequadrantsforIL-2-orIFN--producingcells()weredrawnbasedonthematchedisotypecontrols.Therewerethreemicepergroupandwereassayedindividuallywithsimilarresults.Thedatashownrepresentoneoftwoindependentexperiments.BarsrepresentmeanSDoftriplicatecultures.0.001or5pmolvs0.05or5000pmolofpeptidedoses(Student’s3228BCELLSANERGIZEMEMORYCD4TCELLS DCsfailedtoinduceanergyinourexvivoanergyassay.Intheother,anergywasinducedbyAPCsintheabsenceofDCsinvivo.Moreover,Bcellsthatwereloadedwithpeptideinvitrosuccess-fullyinducedanergyinmemoryCD4Tcellswhentransferredinvivo.Finally,Bcell-decientmice,whilepresumablysufcientinallotherAPCtypes,failedtoinduceanergyinCD4memoryTcells.Therefore,wefeelconvincedthatBcellsarethecriticalAPCstoinduceanergyinmemoryCD4Tcells.AroleforCTLA-4inthenegativeregulationofTcellactivationiswelldocumented(41–44).Interestingly,wefoundthatBcellspresentingtolerogenicdosesofpeptideinducedhigherlevelsofCTLA-4expressiononTcells.Upontheadditionofanti-CTLA-4Abtotheculture,anergywasprevented,stronglysuggestingthatanergyinducedbyBcellsismediatedbyCTLA-4.TheexpressionoflowlevelsofB7.1onBcells,whichdoesnotchangeatdifferentdosesofantigenicstimulation(datanotshown),isinagreementwiththenotionthatBcellsaretheAPCsinvolvedintheinductionofanergyinasystemthatisregulatedbyCTLA-4.BecauseCTLA-4/B7.1avidityishigherthanCD28/B7.1interaction,itiscertainlyfeasiblethatunderlimitingB7.1expressiontheinhibitoryeffectsofCTLA-4willbedominant(45).Inagreementwiththisnotion,CpG-stimulatedBcellsfailedtoinduceanergyinmemoryTcellsinallconcentrationsofthepeptide(0–10,000nM)tested,whereasthecontrolBcellspulsedwith0.1–1.0nMpeptideweresuccessfulininducinganergy(datanotshown).Recentreportshaveindicatedthatprogrammeddeath(PD)-1/PD-L1,andnotCTLA-4/B7,interactionsdictatethestateofex-haustiongeneratedinCD8Tcellsinchronicviralinfections(46,47).Basedonourdata,wesuggestthatanergymightberegulateddifferentlyinCD4vsCD8Tcells.Indeed,itisreportedthatanergyinCD8TcellswasinducedintheabsenceofCTLA-4/B7interaction(48).Thus,ourstudyopensnewavenuesforinvesti-gatingdifferencesinmolecularmechanismsunderlyingCD4vsCD8anergy.BecauseoftheinvolvementofCTLA-4intheinductionofan-ergyinmemoryCD4TcellsandinlightofexpressionofCTLA-4andCD25onregulatoryTcells(Tregs),onepossibleexplanationforourobservationsmightbethatthehyporespon-sivenessshownhereisinducedbyTcellsthathaveTregfunction.Interestingly,arecentreportdocumentedthatAgpresentationbyBcellscausedanincreaseinFoxP3expressioninTregcells(49).Despitethosendings,inoursystem,Tregsdonotseemtobetheinduceroftheobservedhyporesponsiveness.WehadpreviouslyreportedthatinductionofanergyisnotduetoregulatoryTcells(11).Inthoseexperiments,cellsfrominvivoanergizedgroupsweremixedwiththenonanergizedrespondinggroupsatdifferentratiostondoutwhetherpotentialTregspresentintheanergizedgroupswouldrendertherespondingcellsanergicexvivo.Theresultsindicatednoreductioninresponseinthecell-mixedculturewells.Additionally,amicroarraygenechipassaycomparingin FIGURE8.InductionofanergyinmemoryTcellsbyBcellsisCTLA-4mediated.Fourgroupsofmicewereimmunizedwithincreasingdoses(0.05–5000pmol)ofcOVApeptideinIFA.After48h,miceweresacriced;BcellsandAPCsdevoidofBcellswerepuriedfromthedrainingLNsandincubatedwithmemoryCD4Tcellsfor48h.ExvivoinductionofanergybyBcells()orAPCsdevoidofBcells()wastestedbyrestimulatingTcellswithcOVApeptide-pulsedsplenocytes(atconcentrationsshown)for72hfollowedby[H]thymidineincorporation.CTLA-4expressiononAg-specic(CD4)Tcellswasdeterminedbyow-cytometry().ReversalofanergybyBcellswastestedsimilarly,exceptthatanti-mouseCTLA-4AbwasaddedtothecultureinthebeginningofthecoincubationofBcellsandmemoryCD4Tcells().Therewerevemicepergroup.Datashowninrepresentoneoftwoindependentexperiments;thoseinhavebeenrepeatedfourtimes.Barsrepresentmeanoftriplicatecultures.0.001,5,or50pmolvs0.05or5000pmolofpeptidedoses(Student’sTheJournalofImmunology vivoanergizedcellstononanergizedcellsdemonstratednosignif-icantincreaseofFoxP3inanergizedTcells.Inthesameexperi-ment,thelevelsofexpressionofCD25,amarkerofTregs,re-mainedlowerintheanergizedcellscomparedwiththenonanergizedcells(datanotshown).WendthesedatatoargueagainstaroleforTregsintheinductionofanergyinoursystem.OnemightarguethattheinductionofanergyinmemoryTcellsmightnotbebenecialtothemaintenanceofahealthyimmuneresponse.WeproposethatareducedlevelofAg/MHCexpressiononthesurfaceofBcellsmightbeamechanismthathasevolvedtosignalmemoryTcellsofthe“endofaninfection”sothatcellswouldstopproliferatingandsecretinginammatorycytokines(50).Undoubtedly,excesscytokinesecretionisharmfulandnotneededoncetheinfectionisterminated.Followingtheterminationofinfection,Agloadisgraduallydiminishedwithinammation.BcellsbearingspecichighafnityAgreceptors,suchasB2Bcells,atacertainthresholdofAgloadwouldpreferentiallycaptureAgandpresentittomemoryCD4Tcells.Ourstudy,therefore,suggestsanewroleforBcellsastheAPCsofchoicewhenAgfallstononthreateninglowlevels(51).Thus,itislogicaltohy-pothesizethatBcellssignalmemoryTcellstoundergoanergy.Incontrast,atthebeginningofaninfection,whentheAgloadmightalsobelow,theinductionofanergyinmemoryTcellswouldnotbedesirable.AfeatureofanergyasdescribedinthisarticleisitsreversibilityuponanencounterwithIL-2andAg,aconditionthatismetduringtheonsetofaninfectionwhenasurgeofinamma-toryresponsesfromtheinnateimmunesystemcoincideswiththereleaseofmultiplecytokinesincludingIL-2(52).InsupportofthishypothesisareourpreviousreportsshowingthatanencounterwithlowdensitiesofAginthepresenceofIL-2doesnotleadtotheinductionofanergyandthatanergizedcells,whenincubatedwithIL-2andAg,arenolongeranergized(10–12).ThepointsdescribedabovesuggestthattheinductionofanergyinmemoryTcellsmightbeanevolutionarilybenecialmecha-nism.Abetterunderstandingofthisphenomenoncouldhelpinrevealingtheunderlyingmechanismsforviralandtumorsurveil-lance.ManyvirusesandseveraltumorsareknowntodecreasetheexpressionofcellsurfaceMHCclassII(53).Also,sometumor-associatedpeptidesbindpoorlytotheMHCmolecules(54).ThereducedsurfaceexpressionofMHCand/orlowafnitypeptide/MHCcomplexesleadstothepresentationoflowdensitiesofspe-cicpeptide/MHC,whichwouldinduceanergytotheviralortu-mor-derivedAgs.Thus,ourndingssuggestnewstrategiesforovercomingviralinfectionsandtreatingtumors.Ratherthanre-peatedimmunizations,itmightbemoreproductivetofocusonthereversalofanergy.Inthecaseofautoimmunity,self-reactivememoryTcellsmightbekeptundercheckfromeffectorfunctionbyencounteringlowdensitiesofpeptide-MHCpresentedbyBcells,whichcouldbeanimportantprocessinthepreventionoforgan-specicautoimmunediseases.Thus,memoryTcellsinau-toimmuneconditionscouldbetargetedspecicallyforinductionofanergybytransferringsyngeneicBcellspulsedexvivowiththespecicpeptides.ControversialobservationshavebeenreportedontherolesthatBcellsmightplayinautoimmunediseasemodels(55–57).TheidenticationoffollicularBcells(B2Bcells)asthecriticalAPCsforinductionofanergyinmemoryCD4TcellsleadsthewaytofurtherstudiesontheuniquecharacteristicsofthemembraneofmemoryCD4Tcellsand/ormoleculesexpressedonthesurfaceofmemoryTcellsandBcellsthatcontributespe-cicallytotheinductionofTcellanergy.WeespeciallythankDrs.DavidScott,RonGermain,andRonSchwartzforinsightfuldiscussions,AbdelHamad,ChungDang,andKedarNarayanfordiscussionandcriticalreadingofthemanuscript,andStanislavKho-ruzhenkoforFACSdataanalysisandpreparationofhighresolutionTheauthorshavenonancialconictofinterest.1.Moulton,V.R.,andD.L.Farber.2006.Committedtomemory:lineagechoicesforactivatedTcells.TrendsImmunol.27:261–267.2.Seder,R.A.,andD.L.Sacks.2004.Memorymaynotneedreminding.Nat.Med.10:1045–1047.3.Hataye,J.,J.J.Moon,A.Khoruts,C.Reilly,andM.K.Jenkins.2006.NaiveandmemoryCD4Tcellsurvivalcontrolledbyclonalabundance.114–116.4.McKinstry,K.K.,S.Golech,W.H.Lee,G.Huston,N.P.Weng,andS.L.Swain.2007.RapiddefaulttransitionofCD4Tcelleffectorstofunctionalmemorycells.J.Exp.Med.204:2199–2211.5.Chandok,M.R.,F.I.Okoye,M.P.Ndejembi,andD.L.Farber.2007.Abio-chemicalsignatureforrapidrecallofmemoryCD4Tcells.J.Immunol.3689–3698.6.Watson,A.R.,andW.T.Lee.2004.Differencesinsignalingmoleculeorgani-zationbetweennaiveandmemoryCD4Tlymphocytes.J.Immunol.33–41.7.Steinman,R.M.,D.Hawiger,andM.C.Nussenzweig.2003.Tolerogenicden-driticcells.Annu.Rev.Immunol.21:685–711.8.Blair,D.A.,andL.Lefrancois.2007.IncreasedcompetitionforantigenduringprimingnegativelyimpactsthegenerationofmemoryCD4Tcells.Proc.Natl.Acad.Sci.USA104:15045–15050.9.Patke,D.S.,andD.L.Farber.2005.ModulationofmemoryCD4Tcellfunctionandsurvivalpotentialbyalteringthestrengthoftherecallstimulus.J.Immunol.174:5433–5443.10.Korb,L.C.,S.Mirshahidi,K.Ramyar,A.A.SadighiAkha,andS.Sadegh-Nasseri.1999.InductionofTcellanergybylownumbersofagonistJ.Immunol.162:6401–6409.11.Mirshahidi,S.,C.T.Huang,andS.Sadegh-Nasseri.2001.AnergyinperipheralmemoryCD4TcellsinducedbylowavidityengagementofTcellreceptor.J.Exp.Med.194:719–731.12.Mirshahidi,S.,L.C.Ferris,andS.Sadegh-Nasseri.2004.ThemagnitudeofTCRengagementisacriticalpredictoro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