2020研究生講座腫瘤微環(huán)境簡介課件

腫瘤微環(huán)境簡介蘇州大學(xué)免疫學(xué)系居頌光...腫瘤微環(huán)境簡介蘇州大學(xué)免疫學(xué)系居頌光...1？...？...2......3SidP.KerkarandNicholasP.Restifo.

CancerRes2012;72:3125-3130.腫瘤微環(huán)境如果僅僅關(guān)注腫瘤細(xì)胞，那我們在盲人摸象...SidP.KerkarandNicholasP.4腫瘤研究≠腫瘤細(xì)胞研究一個世紀(jì)的觀念：腫瘤是局部組織/器官細(xì)胞異常生長形成的疾病腫瘤是個系統(tǒng)性疾病腫瘤是一種組織（間質(zhì)、血管、微環(huán)境），不是一堆腫瘤細(xì)胞免疫功能失控，腫瘤患者的免疫系統(tǒng)不是簡單的低下：

保護(hù)腫瘤生長的免疫細(xì)胞：功能增強抑制腫瘤細(xì)胞的免疫細(xì)胞：功能低下

...腫瘤研究≠腫瘤細(xì)胞研究...5腫瘤的免疫編輯免疫成分在腫瘤發(fā)生過程中的作用...腫瘤的免疫編輯免疫成分在腫瘤發(fā)生過程中的作用...6DunnG,etal.Natureimmunology.2002.3(11):991EliminationcorrespondstoimmunosurveillianceThehostimmunesystemandanytumorvariantthathassurvivedintheeliminationprocessenterintothedynamicequilibrium:*lymphocytesandIFN-γexertpotentselectionpressure.*Atumorbediscontainingmanygeneticallyunstableandrapidlymutatingtumorcells.Survivaltumorvariantsthathaveacquiredinsensitivitytoimmunologicdetectionand/oreliminationbegintoexpandinuncontrolmanner.DunnG,etal.Natureimmunology.2002.3(11):991...DunnG,etal.Natureimmunology7Solidtumorsreachacertainsize:Growinvasively,requireanenhancedbloodsupply.Inflammatorysignalsleadingtorecruitmentofcellsofinnateimmunesystem:macrophages,DCs,γδ-T,NKandNKT.ProduceIFN-γ...Solidtumorsreachacertains8TheinitialIFN-γstartsacascadeofimmuneresponse:1.Inductionofchemokines:CXCL10,CXCL9,CXCL11.Blockneovascularizationoftumor,recruitmacrophages,DCs,γδ-T,NKandotherimmunecells.2.Antiproliferationofdevelopingtumor.3.TheactivationofcytocidalactivityinmacrophagesandNKcellsenteringthetumor.DeadtumorcellsortumorcellsdebrisareingestedbyDCandaretraffickedtothedraininglymphnode....TheinitialIFN-γstartsacas91.TumorgrowthiskeptincheckbythecytocidalNKandmocrophages.2.CD4+TandCD8+Tcellsthatarespecificfortumorantigensaredevelopingindraininglymphnode....1.Tumorgrowthiskeptinchec10TumorspecificCD4+TandCD8+Tcellshometotumorsitealongachemokinegradient....TumorspecificCD4+TandCD8+T11DunnG,etal.Natureimmunology.2002.3(11):991EliminationcorrespondstoimmunosurveillianceThehostimmunesystemandanytumorvariantthathassurvivedintheeliminationprocessenterintothedynamicequilibrium:*lymphocytesandIFN-γexertpotentselectionpressure.*Atumorbediscontainingmanygeneticallyunstableandrapidlymutatingtumorcells.Survivaltumorvariantsthathaveacquiredinsensitivitytoimmunologicdetectionand/oreliminationbegintoexpandinuncontrolmanner....DunnG,etal.Natureimmunology12腫瘤細(xì)胞...腫瘤細(xì)胞...13腫瘤抗原OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...腫瘤抗原OliveraJ.Finn,Ph.D.NEn14腫瘤抗原的加工提呈OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...腫瘤抗原的加工提呈OliveraJ.Finn,Ph.D15腫瘤干細(xì)胞...腫瘤干細(xì)胞...16KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFN?IL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun17NEnglJMed2006;355:1253-1261....NEnglJMed2006;355:125318CancerstemcellNatureReviewsCancerdoi:10.1038/nrc1232TumorHeterogeneityandCancerStemCells...CancerstemcellNatureReviews19腫瘤微環(huán)境中的免疫穩(wěn)態(tài)失衡...腫瘤微環(huán)境中的免疫穩(wěn)態(tài)失衡...20Nature.2005,5:263...Nature.2005,5:263...21......22......23......24......25......26腫瘤患者的免疫狀態(tài)是有別于正常人的免疫穩(wěn)態(tài)的。...腫瘤患者的免疫狀態(tài)是有別于正常人的免疫穩(wěn)態(tài)27腫瘤微環(huán)境中的免疫細(xì)胞...腫瘤微環(huán)境中的免疫細(xì)胞...28TH17cellsintumourimmunityandImmunotherapy.NATuREREVIEWS|Immunology.doi:10.1038/nri2742DifferentiationofhelperTcellsubsets...TH17cellsintumourimmunity29......30TH17cellsandantitumourimmunity...TH17cellsandantitumourimmu31......32腫瘤微環(huán)境中的抑制性成分——Treg細(xì)胞...腫瘤微環(huán)境中的抑制性成分——Treg細(xì)胞...33Treg細(xì)胞定義1995由Sakaguchi等發(fā)現(xiàn)了機體重要的負(fù)性免疫調(diào)節(jié)成分——調(diào)節(jié)性CD4+CD25+T細(xì)胞（regulatoryTcell,Treg）。隨后的研究根據(jù)細(xì)胞內(nèi)標(biāo)記分子Foxp3和細(xì)胞膜分子CD127表達(dá)與否，將經(jīng)典的Treg細(xì)胞定義為CD4+CD25+Foxp3+CD127low/negT細(xì)胞。通過細(xì)胞間接觸依賴機制發(fā)揮作用或分泌IL-10和TGF-β等細(xì)胞因子，Treg細(xì)胞抑制CD4+25-T細(xì)胞、CD8+T細(xì)胞和樹突狀細(xì)胞等其他免疫細(xì)胞的活性和功能。...Treg細(xì)胞定義...34大量的研究報道表明:CD4+CD25+Treg細(xì)胞是抑制機體抗腫瘤免疫應(yīng)答的關(guān)鍵成分之一，例如：Woo等發(fā)現(xiàn)在非小細(xì)胞肺癌和卵巢癌患者的腫瘤浸潤淋巴細(xì)胞中Treg的含量明顯增多；在胃腸道腫瘤患者中CD4+CD25-T細(xì)胞明顯減少而Treg細(xì)胞數(shù)量增多，而且Treg細(xì)胞數(shù)量的增高與較差的預(yù)后和較低的生存率高度相關(guān)；Curiel等報道，在卵巢癌中Treg細(xì)胞能抑制T細(xì)胞介導(dǎo)的特異性抗腫瘤免疫應(yīng)答，并且有利于腫瘤的生長；因此研究者希望通過減少Treg細(xì)胞的數(shù)量或降低其活性來開辟治療腫瘤免疫治療的新途徑。例如，以Foxp3為靶點清除荷瘤小鼠體內(nèi)的Treg細(xì)胞后腫瘤會消退。...大量的研究報道表明:CD4+CD25+Treg細(xì)胞是抑制機體35Treg細(xì)胞的作用機制...Treg細(xì)胞的作用機制...36非小細(xì)胞性肺癌（NSCLC）與Treg新亞群...非小細(xì)胞性肺癌（NSCLC）與Treg新亞群...37Fig1...Fig1...38Fig.2...Fig.2...39Fig3...Fig3...40Fig4...Fig4...41CD8+T細(xì)胞在腫瘤免疫中的作用腫瘤微環(huán)境中的“正性”免疫成分...CD8+T細(xì)胞在腫瘤免疫中的作用腫瘤微環(huán)境中的“正性”免疫成42Gadd45β-/-miceorHet/WTmicewereinjectedintointradermallyofflankskinwith2.5X104B16cells/mouse.Tumorsizesweremeasuredevery2days.TumorinjectsiteGadd45bKOmicewerechallengedwithmelanomacells:B16

Freezedownthetissueoftumorsite....Gadd45β-/-miceorHet/WTmic43Fig4Gadd45β-/-miceorWTmicewereinjectedsubcutaneouslywith2.5X104B16cells/mouse,tumorsizesweremeasuredevery2days.Thesedataarefrom3separatedexperiments....Fig4Gadd45β-/-miceorWTm445.BothWTandGadd45bKOTcellsareabletoinfiltrateintotumorsites....5.BothWTandGadd45bKOTcel45Gadd45β-/-miceorWTmicewereinjectedintradermallyofflankskinwith5X104B16cells/site/20ulPBSSkinofinjectedsitesoroppositesiteswasresectedandfreezedownimmediatelySpleenCellsAdoptiveTransferandB16inoculationTumorinjectsiteChecktumorformationfrominjectedsites500RadWTKO500RadDonorHost...Gadd45β-/-miceorWTmicewe466.Gadd45βisimportantforlymphocytesintumorsurveillanceP<0.05*...6.Gadd45βisimportantforl47CD4+TCellsAdoptiveTransferandB16inoculation500RadWTCD4+TKOCD4+T500RadDonor

HostWTsplenocytesdepletedCD4+TWTsplenocytesdepleteCD4+T++...CD4+TCellsAdoptiveTransfer487.Gadd45bKOdoesnotaffectfunctionofCD4+TcellsintumorsurveillanceKO:TransferwithCD4+TcelldepletedspleencellsfromWTmice+CD4+TcellsfromGadd45bKOmicePBS:OnlytransferPBSWT:TransferwithCD4+TcelldepletedspleencellsfromWTmice+CD4+TcellsfromGadd45bHetorWTmice...7.Gadd45bKOdoesnotaffect49CD8+TCellsAdoptiveTransferandB16inoculation500RadWTCD8+TKOCD8+T500RadDonor

HostWTsplenocytesdepletedCD8+TWTsplenocytesdepleteCD8+T++...CD8+TCellsAdoptiveTransfer508.Gadd45bKOcausesfunctiondefectofCD8+TcellsintumorsurveillanceKO:TransferwithCD8+TcelldepletedspleencellsfromWTmice+CD8+TcellsfromGadd45bKOmicePBS:OnlytransferPBSWT:TransferwithCD8+TcelldepletedspleencellsfromWTmice+CD8+TcellsfromGadd45bHetorWTmiceP<0.05*...8.Gadd45bKOcausesfunction51Gadd45bisexpressedinCD8+TcellsduringtheiractivationFigure1Gadd45bmessageisinducedbyTCRsignalingandcostimulation.NaiveCD8+Tcellswerestimulatedwithplateboundanti-CD3(α-CD3)plusα-CD28andTh1conditionfor0h,1h,12h,24h,48hand96h.TheGadd45bmessagewasdetectedwithRT-PCR....Gadd45bisexpressedinCD8+T522.Gadd45βisimportantforthefunctionofCD8+Tcells.IL12/IL18α-CD3...2.Gadd45βisimportantforth53......54MAPkinaseprofileactivatedinCD8+Tcellsα-CD3(10μg/ml)IL-12(10ng/ml)+IL-18(30ng/ml)Figure3Gadd45βmediatesp38activationbyTCRsignalingorIL-12plusIL-18inCD8+Tcells.(a)EffectorCD8+Tcellswerestimulated(times,abovelanes)withplate-boundanti-CD3(α-CD3;10μg/ml)or(b)IL-12(10ng/ml)andIL-18(30ng/ml).Cellextractswereanalyzedbyimmunoblotwithanti-phospho-ERK(α-phospho-ERK),α-phospho-JNKandα-phospho-p38.p-,phosphorylated....MAPkinaseprofileactivatedi55Gadd45bKOcausesfunctiondefectoftumorinfiltratingCD8+TcellsFig5Gadd45βKOcausetumorinfiltratingCD8+Tcellfunctiondefectinvivo.Tumormasswasremovedatday13afterB16F0cellsinoculationanddigestedwithcollagenaseI.Cellswerestimulatedwithphorbol12-myristate13-acetate

(50ng/ml)andionomycin(500ng/ml)for6handincubatedforthelast3hwithBrefeldinA.CellsproducingIFN-γwasidentifiedwith

intracellularcytokinestaining....Gadd45bKOcausesfunctiondef56Type,Density,andLocationofImmuneCellsWithinHumanColorectalTumorsPredictClinicalOutcomeGalonJ,etal.29SEPTEMBER2006VOL313SCIENCE...Type,Density,andLocationof57Type,Density,andLocationofImmuneCellsWithinHumanColorectalTumorsPredictClinicalOutcomeGalonJ,etal.29SEPTEMBER2006VOL313SCIENCE(B)ComparisonofthemeanofimmunecelldensitiesintheCTandIMfrompatientswithtumorrecurrence(blackbars)orwithouttumorrecurrence(whitebars)....Type,Density,andLocationof58腫瘤微環(huán)境中的免疫分子...腫瘤微環(huán)境中的免疫分子...59腫瘤微環(huán)境中的ImmunecheckpointsNATUREREVIEWS|CANCER2012

doi:10.1038/nrc3239...腫瘤微環(huán)境中的ImmunecheckpointsNATUR60Multipleco-stimulatoryandinhibitoryinteractionsregulateTcellresponses...Multipleco-stimulatoryandin61Immunecheckpointsregulatedifferentcomponentsintheevolutionofanimmuneresponse...Immunecheckpointsregulatedi62Twogeneralmechanismsofexpressionofimmune-checkpointligandsontumourcells...Twogeneralmechanismsofexpr63Implicationsoftheadaptiveimmuneresistancemechanismforcombinatorialimmunotherapyofcancer....Implicationsoftheadaptivei64......65Clinicalresponsesandimmune-mediatedtoxicitiesonantibodyblockadeoftheCTLA4?mediatedimmunecheckpoint....Clinicalresponsesandimmune-66BlockadeofPD-1orCTLA-4SignalinginTumorImmunotherapy.10.1056/nejme1205943...BlockadeofPD-1orCTLA-4Sig67腫瘤微環(huán)境中的免疫成分正與負(fù)的相對性...腫瘤微環(huán)境中的免疫成分正與負(fù)的相對性...68SGN-40CP870,893CHIR-12.12BlymphomaSolidtumorMultiplemyelomaThreeCD40mAbsinpreclinicaltrials...SGN-40CP870,89369CD40signalingregulatesangiogenesis.Givingblood:anewroleforCD40intumorigenesisBergmannS,PandolfiPP.JExpMed.2006Oct30;203(11):2409-12.Epub2006Oct16...CD40signalingregulatesangio70GrowthfactorsTumorvasculatureDisseminatingtumorcellsInvasionCellcyclecontrolProliferatingtumorcellsAngiogenesisSignaltransductionDouble-edgedweapon→single-edgedfortumor???...GrowthfactorsTumorvasculature71KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFN?IL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun72謝謝！...謝謝！...73腫瘤微環(huán)境簡介蘇州大學(xué)免疫學(xué)系居頌光...腫瘤微環(huán)境簡介蘇州大學(xué)免疫學(xué)系居頌光...74？...？...75......76SidP.KerkarandNicholasP.Restifo.

CancerRes2012;72:3125-3130.腫瘤微環(huán)境如果僅僅關(guān)注腫瘤細(xì)胞，那我們在盲人摸象...SidP.KerkarandNicholasP.77腫瘤研究≠腫瘤細(xì)胞研究一個世紀(jì)的觀念：腫瘤是局部組織/器官細(xì)胞異常生長形成的疾病腫瘤是個系統(tǒng)性疾病腫瘤是一種組織（間質(zhì)、血管、微環(huán)境），不是一堆腫瘤細(xì)胞免疫功能失控，腫瘤患者的免疫系統(tǒng)不是簡單的低下：

保護(hù)腫瘤生長的免疫細(xì)胞：功能增強抑制腫瘤細(xì)胞的免疫細(xì)胞：功能低下

...腫瘤研究≠腫瘤細(xì)胞研究...78腫瘤的免疫編輯免疫成分在腫瘤發(fā)生過程中的作用...腫瘤的免疫編輯免疫成分在腫瘤發(fā)生過程中的作用...79DunnG,etal.Natureimmunology.2002.3(11):991EliminationcorrespondstoimmunosurveillianceThehostimmunesystemandanytumorvariantthathassurvivedintheeliminationprocessenterintothedynamicequilibrium:*lymphocytesandIFN-γexertpotentselectionpressure.*Atumorbediscontainingmanygeneticallyunstableandrapidlymutatingtumorcells.Survivaltumorvariantsthathaveacquiredinsensitivitytoimmunologicdetectionand/oreliminationbegintoexpandinuncontrolmanner.DunnG,etal.Natureimmunology.2002.3(11):991...DunnG,etal.Natureimmunology80Solidtumorsreachacertainsize:Growinvasively,requireanenhancedbloodsupply.Inflammatorysignalsleadingtorecruitmentofcellsofinnateimmunesystem:macrophages,DCs,γδ-T,NKandNKT.ProduceIFN-γ...Solidtumorsreachacertains81TheinitialIFN-γstartsacascadeofimmuneresponse:1.Inductionofchemokines:CXCL10,CXCL9,CXCL11.Blockneovascularizationoftumor,recruitmacrophages,DCs,γδ-T,NKandotherimmunecells.2.Antiproliferationofdevelopingtumor.3.TheactivationofcytocidalactivityinmacrophagesandNKcellsenteringthetumor.DeadtumorcellsortumorcellsdebrisareingestedbyDCandaretraffickedtothedraininglymphnode....TheinitialIFN-γstartsacas821.TumorgrowthiskeptincheckbythecytocidalNKandmocrophages.2.CD4+TandCD8+Tcellsthatarespecificfortumorantigensaredevelopingindraininglymphnode....1.Tumorgrowthiskeptinchec83TumorspecificCD4+TandCD8+Tcellshometotumorsitealongachemokinegradient....TumorspecificCD4+TandCD8+T84DunnG,etal.Natureimmunology.2002.3(11):991EliminationcorrespondstoimmunosurveillianceThehostimmunesystemandanytumorvariantthathassurvivedintheeliminationprocessenterintothedynamicequilibrium:*lymphocytesandIFN-γexertpotentselectionpressure.*Atumorbediscontainingmanygeneticallyunstableandrapidlymutatingtumorcells.Survivaltumorvariantsthathaveacquiredinsensitivitytoimmunologicdetectionand/oreliminationbegintoexpandinuncontrolmanner....DunnG,etal.Natureimmunology85腫瘤細(xì)胞...腫瘤細(xì)胞...86腫瘤抗原OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...腫瘤抗原OliveraJ.Finn,Ph.D.NEn87腫瘤抗原的加工提呈OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...腫瘤抗原的加工提呈OliveraJ.Finn,Ph.D88腫瘤干細(xì)胞...腫瘤干細(xì)胞...89KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFN?IL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun90NEnglJMed2006;355:1253-1261....NEnglJMed2006;355:125391CancerstemcellNatureReviewsCancerdoi:10.1038/nrc1232TumorHeterogeneityandCancerStemCells...CancerstemcellNatureReviews92腫瘤微環(huán)境中的免疫穩(wěn)態(tài)失衡...腫瘤微環(huán)境中的免疫穩(wěn)態(tài)失衡...93Nature.2005,5:263...Nature.2005,5:263...94......95......96......97......98......99腫瘤患者的免疫狀態(tài)是有別于正常人的免疫穩(wěn)態(tài)的。...腫瘤患者的免疫狀態(tài)是有別于正常人的免疫穩(wěn)態(tài)100腫瘤微環(huán)境中的免疫細(xì)胞...腫瘤微環(huán)境中的免疫細(xì)胞...101TH17cellsintumourimmunityandImmunotherapy.NATuREREVIEWS|Immunology.doi:10.1038/nri2742DifferentiationofhelperTcellsubsets...TH17cellsintumourimmunity102......103TH17cellsandantitumourimmunity...TH17cellsandantitumourimmu104......105腫瘤微環(huán)境中的抑制性成分——Treg細(xì)胞...腫瘤微環(huán)境中的抑制性成分——Treg細(xì)胞...106Treg細(xì)胞定義1995由Sakaguchi等發(fā)現(xiàn)了機體重要的負(fù)性免疫調(diào)節(jié)成分——調(diào)節(jié)性CD4+CD25+T細(xì)胞（regulatoryTcell,Treg）。隨后的研究根據(jù)細(xì)胞內(nèi)標(biāo)記分子Foxp3和細(xì)胞膜分子CD127表達(dá)與否，將經(jīng)典的Treg細(xì)胞定義為CD4+CD25+Foxp3+CD127low/negT細(xì)胞。通過細(xì)胞間接觸依賴機制發(fā)揮作用或分泌IL-10和TGF-β等細(xì)胞因子，Treg細(xì)胞抑制CD4+25-T細(xì)胞、CD8+T細(xì)胞和樹突狀細(xì)胞等其他免疫細(xì)胞的活性和功能。...Treg細(xì)胞定義...107大量的研究報道表明:CD4+CD25+Treg細(xì)胞是抑制機體抗腫瘤免疫應(yīng)答的關(guān)鍵成分之一，例如：Woo等發(fā)現(xiàn)在非小細(xì)胞肺癌和卵巢癌患者的腫瘤浸潤淋巴細(xì)胞中Treg的含量明顯增多；在胃腸道腫瘤患者中CD4+CD25-T細(xì)胞明顯減少而Treg細(xì)胞數(shù)量增多，而且Treg細(xì)胞數(shù)量的增高與較差的預(yù)后和較低的生存率高度相關(guān)；Curiel等報道，在卵巢癌中Treg細(xì)胞能抑制T細(xì)胞介導(dǎo)的特異性抗腫瘤免疫應(yīng)答，并且有利于腫瘤的生長；因此研究者希望通過減少Treg細(xì)胞的數(shù)量或降低其活性來開辟治療腫瘤免疫治療的新途徑。例如，以Foxp3為靶點清除荷瘤小鼠體內(nèi)的Treg細(xì)胞后腫瘤會消退。...大量的研究報道表明:CD4+CD25+Treg細(xì)胞是抑制機體108Treg細(xì)胞的作用機制...Treg細(xì)胞的作用機制...109非小細(xì)胞性肺癌（NSCLC）與Treg新亞群...非小細(xì)胞性肺癌（NSCLC）與Treg新亞群...110Fig1...Fig1...111Fig.2...Fig.2...112Fig3...Fig3...113Fig4...Fig4...114CD8+T細(xì)胞在腫瘤免疫中的作用腫瘤微環(huán)境中的“正性”免疫成分...CD8+T細(xì)胞在腫瘤免疫中的作用腫瘤微環(huán)境中的“正性”免疫成115Gadd45β-/-miceorHet/WTmicewereinjectedintointradermallyofflankskinwith2.5X104B16cells/mouse.Tumorsizesweremeasuredevery2days.TumorinjectsiteGadd45bKOmicewerechallengedwithmelanomacells:B16

Freezedownthetissueoftumorsite....Gadd45β-/-miceorHet/WTmic116Fig4Gadd45β-/-miceorWTmicewereinjectedsubcutaneouslywith2.5X104B16cells/mouse,tumorsizesweremeasuredevery2days.Thesedataarefrom3separatedexperiments....Fig4Gadd45β-/-miceorWTm1175.BothWTandGadd45bKOTcellsareabletoinfiltrateintotumorsites....5.BothWTandGadd45bKOTcel118Gadd45β-/-miceorWTmicewereinjectedintradermallyofflankskinwith5X104B16cells/site/20ulPBSSkinofinjectedsitesoroppositesiteswasresectedandfreezedownimmediatelySpleenCellsAdoptiveTransferandB16inoculationTumorinjectsiteChecktumorformationfrominjectedsites500RadWTKO500RadDonorHost...Gadd45β-/-miceorWTmicewe1196.Gadd45βisimportantforlymphocytesintumorsurveillanceP<0.05*...6.Gadd45βisimportantforl120CD4+TCellsAdoptiveTransferandB16inoculation500RadWTCD4+TKOCD4+T500RadDonor

HostWTsplenocytesdepletedCD4+TWTsplenocytesdepleteCD4+T++...CD4+TCellsAdoptiveTransfer1217.Gadd45bKOdoesnotaffectfunctionofCD4+TcellsintumorsurveillanceKO:TransferwithCD4+TcelldepletedspleencellsfromWTmice+CD4+TcellsfromGadd45bKOmicePBS:OnlytransferPBSWT:TransferwithCD4+TcelldepletedspleencellsfromWTmice+CD4+TcellsfromGadd45bHetorWTmice...7.Gadd45bKOdoesnotaffect122CD8+TCellsAdoptiveTransferandB16inoculation500RadWTCD8+TKOCD8+T500RadDonor

HostWTsplenocytesdepletedCD8+TWTsplenocytesdepleteCD8+T++...CD8+TCellsAdoptiveTransfer1238.Gadd45bKOcausesfunctiondefectofCD8+TcellsintumorsurveillanceKO:TransferwithCD8+TcelldepletedspleencellsfromWTmice+CD8+TcellsfromGadd45bKOmicePBS:OnlytransferPBSWT:TransferwithCD8+TcelldepletedspleencellsfromWTmice+CD8+TcellsfromGadd45bHetorWTmiceP<0.05*...8.Gadd45bKOcausesfunction124Gadd45bisexpressedinCD8+TcellsduringtheiractivationFigure1Gadd45bmessageisinducedbyTCRsignalingandcostimulation.NaiveCD8+Tcellswerestimulatedwithplateboundanti-CD3(α-CD3)plusα-CD28andTh1conditionfor0h,1h,12h,24h,48hand96h.TheGadd45bmessagewasdetectedwithRT-PCR....Gadd45bisexpressedinCD8+T1252.Gadd45βisimportantforthefunctionofCD8+Tcells.IL12/IL18α-CD3...2.Gadd45βisimportantforth126......127MAPkinaseprofileactivatedinCD8+Tcellsα-CD3(10μg/ml)IL-12(10ng/ml)+IL-18(30ng/ml)Figure3Gadd45βmediatesp38activationbyTCRsignalingorIL-12plusIL-18inCD8+Tcells.(a)EffectorCD8+Tcellswerestimulated(times,abovelanes)withplate-boundanti-CD3(α-CD3;10μg/ml)or(b)IL-12(10ng/ml)andIL-18(30ng/ml).Cellextractswereanalyzedbyimmunoblotwithanti-phospho-ERK(α-phospho-ERK),α-phospho-JNKandα-phospho-p38.p-,phosphorylated....MAPkinaseprofileactivatedi128Gadd45bKOcausesfunctiondefectoftumorinfiltratingCD8+TcellsFig5Gadd45βKOcausetumorinfiltratingCD8+Tcellfunctiondefectinvivo.Tumormasswasremovedatday13afterB16F0cellsinoculationanddigestedwithcollagenaseI.Cellswerestimulatedwithphorbol12-myristate13-acetate

(50ng/ml)andionomycin(500ng/ml)for6handincubatedforthelast3hwithBrefeldinA.CellsproducingIFN-γwasidentifiedwith

intracellularcytokinestaining....Gadd45bKOcausesfunctiondef129Type,Density,andLocationofImmuneCellsWithinHumanColorectalTumorsPredictClinicalOutcomeGalonJ,etal.29SEPTEMBER2006VOL313SCIENCE...Type,Density,an