2020研究生讲座肿瘤微环境简介课件

肿瘤微环境简介苏州大学免疫学系居颂光...肿瘤微环境简介苏州大学免疫学系居颂光...1？...？...2......3SidP.KerkarandNicholasP.Restifo.

CancerRes2012;72:3125-3130.肿瘤微环境如果仅仅关注肿瘤细胞，那我们在盲人摸象...SidP.KerkarandNicholasP.4肿瘤研究≠肿瘤细胞研究一个世纪的观念：肿瘤是局部组织/器官细胞异常生长形成的疾病肿瘤是个系统性疾病肿瘤是一种组织（间质、血管、微环境），不是一堆肿瘤细胞免疫功能失控，肿瘤患者的免疫系统不是简单的低下：

保护肿瘤生长的免疫细胞：功能增强抑制肿瘤细胞的免疫细胞：功能低下

...肿瘤研究≠肿瘤细胞研究...5肿瘤的免疫编辑免疫成分在肿瘤发生过程中的作用...肿瘤的免疫编辑免疫成分在肿瘤发生过程中的作用...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肿瘤细胞...肿瘤细胞...13肿瘤抗原OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...肿瘤抗原OliveraJ.Finn,Ph.D.NEn14肿瘤抗原的加工提呈OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...肿瘤抗原的加工提呈OliveraJ.Finn,Ph.D15肿瘤干细胞...肿瘤干细胞...16KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFNƔIL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun17NEnglJMed2006;355:1253-1261....NEnglJMed2006;355:125318CancerstemcellNatureReviewsCancerdoi:10.1038/nrc1232TumorHeterogeneityandCancerStemCells...CancerstemcellNatureReviews19肿瘤微环境中的免疫稳态失衡...肿瘤微环境中的免疫稳态失衡...20Nature.2005,5:263...Nature.2005,5:263...21......22......23......24......25......26肿瘤患者的免疫状态是有别于正常人的免疫稳态的。...肿瘤患者的免疫状态是有别于正常人的免疫稳态27肿瘤微环境中的免疫细胞...肿瘤微环境中的免疫细胞...28TH17cellsintumourimmunityandImmunotherapy.NATuREREVIEWS|Immunology.doi:10.1038/nri2742DifferentiationofhelperTcellsubsets...TH17cellsintumourimmunity29......30TH17cellsandantitumourimmunity...TH17cellsandantitumourimmu31......32肿瘤微环境中的抑制性成分——Treg细胞...肿瘤微环境中的抑制性成分——Treg细胞...33Treg细胞定义1995由Sakaguchi等发现了机体重要的负性免疫调节成分——调节性CD4+CD25+T细胞（regulatoryTcell,Treg）。随后的研究根据细胞内标记分子Foxp3和细胞膜分子CD127表达与否，将经典的Treg细胞定义为CD4+CD25+Foxp3+CD127low/negT细胞。通过细胞间接触依赖机制发挥作用或分泌IL-10和TGF-β等细胞因子，Treg细胞抑制CD4+25-T细胞、CD8+T细胞和树突状细胞等其他免疫细胞的活性和功能。...Treg细胞定义...34大量的研究报道表明:CD4+CD25+Treg细胞是抑制机体抗肿瘤免疫应答的关键成分之一，例如：Woo等发现在非小细胞肺癌和卵巢癌患者的肿瘤浸润淋巴细胞中Treg的含量明显增多；在胃肠道肿瘤患者中CD4+CD25-T细胞明显减少而Treg细胞数量增多，而且Treg细胞数量的增高与较差的预后和较低的生存率高度相关；Curiel等报道，在卵巢癌中Treg细胞能抑制T细胞介导的特异性抗肿瘤免疫应答，并且有利于肿瘤的生长；因此研究者希望通过减少Treg细胞的数量或降低其活性来开辟治疗肿瘤免疫治疗的新途径。例如，以Foxp3为靶点清除荷瘤小鼠体内的Treg细胞后肿瘤会消退。...大量的研究报道表明:CD4+CD25+Treg细胞是抑制机体35Treg细胞的作用机制...Treg细胞的作用机制...36非小细胞性肺癌（NSCLC）与Treg新亚群...非小细胞性肺癌（NSCLC）与Treg新亚群...37Fig1...Fig1...38Fig.2...Fig.2...39Fig3...Fig3...40Fig4...Fig4...41CD8+T细胞在肿瘤免疫中的作用肿瘤微环境中的“正性”免疫成分...CD8+T细胞在肿瘤免疫中的作用肿瘤微环境中的“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肿瘤微环境中的免疫分子...肿瘤微环境中的免疫分子...59肿瘤微环境中的ImmunecheckpointsNATUREREVIEWS|CANCER2012

doi:10.1038/nrc3239...肿瘤微环境中的ImmunecheckpointsNATUR60Multipleco-stimulatoryandinhibitoryinteractionsregulateTcellresponses...Multipleco-stimulatoryandin61Immunecheckpointsregulatedifferentcomponentsintheevolutionofanimmuneresponse...Immunecheckpointsregulatedi62Twogeneralmechanismsofexpressionofimmune-checkpointligandsontumourcells...Twogeneralmechanismsofexpr63Implicationsoftheadaptiveimmuneresistancemechanismforcombinatorialimmunotherapyofcancer....Implicationsoftheadaptivei64......65Clinicalresponsesandimmune-mediatedtoxicitiesonantibodyblockadeoftheCTLA4‑mediatedimmunecheckpoint....Clinicalresponsesandimmune-66BlockadeofPD-1orCTLA-4SignalinginTumorImmunotherapy.10.1056/nejme1205943...BlockadeofPD-1orCTLA-4Sig67肿瘤微环境中的免疫成分正与负的相对性...肿瘤微环境中的免疫成分正与负的相对性...68SGN-40CP870,893CHIR-12.12BlymphomaSolidtumorMultiplemyelomaThreeCD40mAbsinpreclinicaltrials...SGN-40CP870,89369CD40signalingregulatesangiogenesis.Givingblood:anewroleforCD40intumorigenesisBergmannS,PandolfiPP.JExpMed.2006Oct30;203(11):2409-12.Epub2006Oct16...CD40signalingregulatesangio70GrowthfactorsTumorvasculatureDisseminatingtumorcellsInvasionCellcyclecontrolProliferatingtumorcellsAngiogenesisSignaltransductionDouble-edgedweapon→single-edgedfortumor???...GrowthfactorsTumorvasculature71KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFNƔIL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun72谢谢！...谢谢！...73肿瘤微环境简介苏州大学免疫学系居颂光...肿瘤微环境简介苏州大学免疫学系居颂光...74？...？...75......76SidP.KerkarandNicholasP.Restifo.

CancerRes2012;72:3125-3130.肿瘤微环境如果仅仅关注肿瘤细胞，那我们在盲人摸象...SidP.KerkarandNicholasP.77肿瘤研究≠肿瘤细胞研究一个世纪的观念：肿瘤是局部组织/器官细胞异常生长形成的疾病肿瘤是个系统性疾病肿瘤是一种组织（间质、血管、微环境），不是一堆肿瘤细胞免疫功能失控，肿瘤患者的免疫系统不是简单的低下：

保护肿瘤生长的免疫细胞：功能增强抑制肿瘤细胞的免疫细胞：功能低下

...肿瘤研究≠肿瘤细胞研究...78肿瘤的免疫编辑免疫成分在肿瘤发生过程中的作用...肿瘤的免疫编辑免疫成分在肿瘤发生过程中的作用...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肿瘤细胞...肿瘤细胞...86肿瘤抗原OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...肿瘤抗原OliveraJ.Finn,Ph.D.NEn87肿瘤抗原的加工提呈OliveraJ.Finn,Ph.D.NEnglJMed2008;358:2704-15...肿瘤抗原的加工提呈OliveraJ.Finn,Ph.D88肿瘤干细胞...肿瘤干细胞...89KnutsonK,etal.CancerImmunolImmunother2005,54:721–728APCTHTregCytoxicTcellsOthersIFNƔIL-2IL-12GranzymePerforinAbsothersTumormicroenvironment:...KnutsonK,etal.CancerImmun90NEnglJMed2006;355:1253-1261....NEnglJMed2006;355:125391CancerstemcellNatureReviewsCancerdoi:10.1038/nrc1232TumorHeterogeneityandCancerStemCells...CancerstemcellNatureReviews92肿瘤微环境中的免疫稳态失衡...肿瘤微环境中的免疫稳态失衡...93Nature.2005,5:263...Nature.2005,5:263...94......95......96......97......98......99肿瘤患者的免疫状态是有别于正常人的免疫稳态的。...肿瘤患者的免疫状态是有别于正常人的免疫稳态100肿瘤微环境中的免疫细胞...肿瘤微环境中的免疫细胞...101TH17cellsintumourimmunityandImmunotherapy.NATuREREVIEWS|Immunology.doi:10.1038/nri2742DifferentiationofhelperTcellsubsets...TH17cellsintumourimmunity102......103TH17cellsandantitumourimmunity...TH17cellsandantitumourimmu104......105肿瘤微环境中的抑制性成分——Treg细胞...肿瘤微环境中的抑制性成分——Treg细胞...106Treg细胞定义1995由Sakaguchi等发现了机体重要的负性免疫调节成分——调节性CD4+CD25+T细胞（regulatoryTcell,Treg）。随后的研究根据细胞内标记分子Foxp3和细胞膜分子CD127表达与否，将经典的Treg细胞定义为CD4+CD25+Foxp3+CD127low/negT细胞。通过细胞间接触依赖机制发挥作用或分泌IL-10和TGF-β等细胞因子，Treg细胞抑制CD4+25-T细胞、CD8+T细胞和树突状细胞等其他免疫细胞的活性和功能。...Treg细胞定义...107大量的研究报道表明:CD4+CD25+Treg细胞是抑制机体抗肿瘤免疫应答的关键成分之一，例如：Woo等发现在非小细胞肺癌和卵巢癌患者的肿瘤浸润淋巴细胞中Treg的含量明显增多；在胃肠道肿瘤患者中CD4+CD25-T细胞明显减少而Treg细胞数量增多，而且Treg细胞数量的增高与较差的预后和较低的生存率高度相关；Curiel等报道，在卵巢癌中Treg细胞能抑制T细胞介导的特异性抗肿瘤免疫应答，并且有利于肿瘤的生长；因此研究者希望通过减少Treg细胞的数量或降低其活性来开辟治疗肿瘤免疫治疗的新途径。例如，以Foxp3为靶点清除荷瘤小鼠体内的Treg细胞后肿瘤会消退。...大量的研究报道表明:CD4+CD25+Treg细胞是抑制机体108Treg细胞的作用机制...Treg细胞的作用机制...109非小细胞性肺癌（NSCLC）与Treg新亚群...非小细胞性肺癌（NSCLC）与Treg新亚群...110Fig1...Fig1...111Fig.2...Fig.2...112Fig3...Fig3...113Fig4...Fig4...114CD8+T细胞在肿瘤免疫中的作用肿瘤微环境中的“正性”免疫成分...CD8+T细胞在肿瘤免疫中的作用肿瘤微环境中的“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