Immune Evasion by Murine Melanoma Mediated Through CC Chemokine

The Journal of Experimental Medicine

(301) 496-5370;email:[email protected] Center Dr.,Bethesda,MD20892-1908. Phone:(301)496-8724;Fax: The onlineversionofthisarticleincludes supplementalmaterial. cell deathmightenhancetheeffectivenessofcancertherapy. associated signalingpathwaysthatinterferewithFas-mediated in theFasdeathpathway(5).Thus,antagonismoftumor- hibitory protein(FLIP),whichblockstheactionofcaspase-8 tumors, selves inordertokillattackingimmunecells(4).Some avoid engagementbyFasLorbegintoexpressthem- death. Malignantcellsmaydown-regulatesurfaceFas(3) to have evolvedseveralstrategiestoescapeFas-mediatedcell mechanism bywhichimmunecellskilltumors,tumor (FasL)-expressing NKcellsandCTLsconstitutesamajor cancers toescapeimmune-mediatedcelldeath(2). mechanisms havebeendescribedwhich,intheory,enable progression ofmelanomaandothercancers,many avoid immunesurveillanceislikelytobecentralthe increasing inincidence(1).Theabilityoftumorcellsto Malignant melanomaisapotentiallyfatalskincancerthat Introduction

Address correspondencetoSamT.Hwang, Bldg.10/Rm.12N246,10 Because engagementofFasontumorcellsbyligand

including melanoma,up-regulateFLICE-likein-

1337 S. SiannaCastillo, e od:metastasis•chemokine receptor •cancercellsignaling Key words: through activationofPI3K/Akt),therebyprovidingameansfortumorprogression. CCL27 allowsmelanomacellstoescapehostimmuneantitumorkillingmechanisms(possibly CCR10-expressing melanomacells.WeproposethatCCR10engagementbylocallyproduced cutaneous injectionofneutralizingantibodiestoendogenousCCL27blockedgrowth kinase (PI3K)–dependentprotectionfromapoptosisinducedbyFascross-linking.Invivo, cell deathinducedbymelanomaantigen-specificcytotoxicTcells,andphosphatidylinositol-3- tumors. Invitro,exposureoftumorcellstoCCL27ledrapidactivationAkt,resistance overexpressed bothluciferaseandCCR10resistedhostimmuneresponsesreadilyformed melanoma, cellsrenderedmoreimmunogenicviaoverexpressionofluciferase,B16that whose ligand(CCL27)isconstitutivelyproducedbykeratinocytes.ComparedwithB16murine Human melanomacellsfrequentlyexpressCCchemokinereceptor(CCR)10,a 5 4 1 Nicholas P. Restifo, Abstract Takashi Murakami, CC ChemokineReceptor-10 Immune Evasion MelanomaMediatedthrough by Murine Department ofDermatology,Department Hospital, University ofZürich CH-8091Zürich, Switzerland National InstituteofNeurologicalDisorders andStroke, Bethesda, MD20892 Dermatology, National InstitutesofHealth(NIH), Bethesda, MD20892 http://www.jem.org/cgi/doi/10.1084/jem.20030593 The JournalofExperimental Medicine 2 Surgery, and 3 Phillip A. Dennis, 1 2 Adela R. Cardones, 3 Brenda A. Klaunberg, Cancer Therapeutics Branches, CCR, NationalCancerInstitute(NCI), (13). CCR10hastwoknown chemokineligands:CCL27 on endothelialcellsandcytokine-stimulatedmelano of skin-homingmemoryTcells,butitcanalsobe CXCR4 (8).CCR10(12,13)isexpressedbyapopulation mokine receptorCCR10inconjunctionwithCCR7and metastasis (viaCCchemokinereceptor[CCR]7)(10,11). CXC chemokinereceptor[CXCR]4)(8,9)andnodal receptors playdistinctrolesindistantorganmetastasis(via a limitedrepertoireofchemokinereceptors(7,8).These inflammatory cells,andproliferation(7),tumorcellsexpress volved inneoangiogenesis,theattractionandretention of produce avarietyofchemokineligandsthatmaybein- homologous chemokineligands(6).Althoughcancercells spanning proteinsthatbindprimarilytofourgroupsof of afamilyGprotein–coupled,seven-transmembrane– clearance mechanisms.Chemokinereceptorsarecomprised tors inthecontextoftumorescapefromimmunecell CXC chemokinereceptor;IHC,immunohistochemistry; Akt; PI3K,phosphatidylinositol-3-kinase. Abbreviations usedinthispaper: Interestingly, manymelanomacelllinesexpresstheche- In thispaper,weexploretheroleofchemokinerecep- •Vlm 9,Nme ,Nvme ,20 1337–1347 November3,2003 •Volume198,Number 9, 3 andSam T. Hwang 1 Steven E. Finkelstein, 5 FrankO. Nestle, CCR,CCchemokinereceptor;CXCR, 1 4

p-Akt, phospho- detected cytes The Journal of Experimental Medicine fly ( CCR10-transduced tumorlinesweretransducedagainwithfire- ously withG418-basedselection(9,10).ThepLNCX2-and cells weretransducedwithcDNAforCCR10asdescribedprevi- roviral vector(CLONTECHLaboratories).B16/F1melanoma stitute, Glasgow,Scotland)wassubclonedintothepLNCX2ret- CCR10 cDNA(12)(agiftfromDr.GeraldGraham,BeatsonIn- for 2hat37 and PD98059werepurchasedfromSigma-Aldrich. inactivated FCSandsupplements(10).Wortmannin,LY294002, cells (19)weregrowninDME(GIBCOBRL)with10%heat- Animal UseandCareCommittee.SyngeneicB16/F1melanoma mal experimentswereconductedwiththeapprovalofNCI [18]). Allani- ment ofcharacteristiclymphoproliferativedisease Laboratories andusedat8wkofage(i.e.,beforethedevelop- mice ontheC57BL/6backgroundwerepurchasedfromJackson through theexpressionofspecificchemokinereceptors. enable melanomacellstoevadethehostimmuneresponse lectively, ourresultssuggestanovelmechanismthatmay static characteristicsofCCR10-positivetumorcells.Col- the effectsofCCR10expressionongrowthandmeta- of thesecellsintoearskin.Usingthismodel,weassessed generate effectiveanti–tumorresponsesafterimplantation cells lessaggressive,whichallowedtherecipientmiceto served thattransductionwithluciferaserenderedtumor and aggressivelyformtumorsinsyngeneicmice,weob- lineated. CCL27’s roleinmelanomapathogenesishasnotbeende- to playaroleinTcellhominginflamedskin(16,17). skin byepidermalkeratinocytes(14),andithasbeenshown kines becauseitisselectivelyandconstitutivelyproducedin (14) andCCL28(15).CCL27isuniqueamongchemo- Materials andMethods toxin (Calbiochem)beforeaddition ofCCL27.Cellswerelysed (LY294002, wortmannin,PD98059; Sigma-Aldrich),andpertussis construct. from threeormoreseparatetransductionswitheachretroviral served usingCCR10-andpLNCX2-transducedcellsderived CCR10-B10). Similarinvivophenotypes(seeResults)wereob- was comparableinallcelllines(pLNCX2-,CXCR4-,and 10% FCS,G418,andpuromycin(9).Luciferaseactivitypercell transduced line,pLNCX2-B16,weremaintainedinDMEwith duced cellline,CXCR4-B16,andthecontrolvector-luciferase– full/jem.20030593/DC1). ThehumanCXCR4-luciferase–trans- (Fig. S1,AandB,availableathttp://www.jem.org/cgi/content/ and showedspecificcalciumfluxwiththeadditionofCCL27 expressed a42-kDproteinbandbyWesternblottingforCCR10 hereby referredtoasCCR10-B16.TheCCR10-B16cellline mycin. Forconvenience,theCCR10-luciferaseB16celllineis (8–12 wkold)wereusedinallexperiments.6-wk-oldFasL rum-starved CCR10-B16cells(5 ralogics. Fordetectionofphospho-Akt (p-Akt)atserine473,se- CCR10 (CI0127)polyclonalantibodies, respectively,fromCap- formed usinggoatanti–humanCCR10(CI0126)andanti–mouse chemistry (IHC)andWesternblottingforCCR10wereper- Whereas B16cellsarenormallypoorlyimmunogenic Immunohistochemistry andWesternBlotting. Retroviral TransductionofB16/F1MelanomaCells. Animals, Reagents,andCellLines. Photinus pyralis

C withorwithoutvariouskinase inhibitors

) luciferasecDNAandmaintainedwithpuro-

1338

CCR10 andMelanoma ImmuneEvasion

Female C57BL/6mice(9)

/well) werepreincubated

Immunohisto-

Mouse

gld

CAC-3 jections, 10 jected intothelefthindfootpadsofC57BL/6mice.Forearin- ml anti-FLAGM2mAb(Sigma-Aldrich)for16hplus1 Results werenormalizedtoexpressionofGAPDH:forward,5 CCL28 (mouse),forward,5 CCL27 (mouse),forward,5 performed asdescribed(9). package system(Xenogen).Invitroluciferasequantificationwas (Xenogen). ImageswereanalyzedwiththeLivingImagesoftware for 1–3mininapitch-blackchamberwithcooledCCDcamera otium) (2mg/mouse)viatheintraperitonealrouteandimaged ACCCACTCCTCCACCTTTGA-3 footpad injections,cells(4 trypsinization andwashedtwiceinPBSbeforeinjection.Fors.c. B16 cellsinexponentialgrowthphasewereharvestedby beled withcalcein-AM(Molecular Probes)at1 was usedat1 FCS asdescribed(21).VAD-fmk(CLONTECHLaboratories) man IL-2inthepresenceof1 with 10%heatinactivatedFCS) with30IU/mlrecombinanthu- incubation inculturemedium (CM) (consistingofRPMI1640 mouse gp100(mgp100)–specificpeptide(22)wereactivatedby for IHCofformalin-fixedtumorsectionsasdescribed(20). anti–p-Akt antibody(9277;CellSignalingTechnology)wasused anti-total Aktantibodies(CellSignalingTechnology).Arabbit and analyzedbyWesternblottingusinganti–p-Akt(Ser473) analysis software(TreeStar).Forinductionofapoptosis,IFN- sciences) (seeFig.5A)forflowcytometricanalysisusingFloJo stained withPE-conjugatedanti–mouseFasmAb(BDBio- firm Fasexpression,cellswereharvestedandwashed,then (50 ng/ml;Peprotech)for12–16hinDME/0.5%FCS.Tocon- with similarresults. ameters. Experimentswereperformedtwotofourtimeseach by measurementofthetwomaximumperpendiculartumordi- immediately abovethecartilage.Tumorgrowthwasmonitored neous spaceunderthecentraldorsalsurfaceofleftear mixture ofketamineandxylazine,injectedwith human FLAG-taggedFasL(Apotech)incombinationwith1 CCR10-B16cellswereexposedto10ng/mlrecombinant treated CAGGAAATGAGCTTGACAA-3 nonparametric Student’s tometry withFloJosoftware.Pvalueswerebasedontwo-sided, structions (BDBiosciences).Analysiswasperformedbyflowcy- ture platesforannexinVstainingaccordingtomanufacturer’sin- 16 h,attached(anddetached)cellswerecollectedfromtissuecul- After exposureofB16cellstoapoptosis-inducingconditionsfor were stainedwithannexinVforbaselineassessmentofapoptosis. get cellsweretreatedovernight withIFN- used onday5–10afterthestart of culture(23).CCR10-B16tar- chemokine (whenindicated)at37 that expressedTcellreceptorsspecificforaH-2D CATACT-3 CCR10-B16 cellsweretreatedwithrecombinantmurineIFN- pairs: humanCCR10,forward,5 formed asdescribedpreviously(10)usingthefollowingprimer GCT-3

–treated CCR10-B16cellsthathadnotbeenexposedtoFasL

For invitroCTLassays,Tcells(PMEL)fromtransgenicmice S.c. InoculationofTransducedCellLines. Quantitative RT-PCR. Apoptosis andCytotoxicityAssays. In vivoLuciferaseImaging.

and

and reverse,5

cellsin

reverse, 5 and M asaninhibitorofFas-mediatedcelldeath.IFN-

reverse, 5 20 -CCATGGGAAGTATGGCTTCTG-3 t tests(unlessotherwisespecified). -CACGACAGCCTGGAGGTGA-3 l PBSwereinjectedintothesubcuta- -CCTGGACATCGGCCTTGT-3 10 Quantitative RT-PCRwasper- Mice wereanesthetizedwitha -CTGCTGAGGAGGATTGTC- M humangp100 5 -CAGGGCTCACACTCATG- cellsin -GGGGATGAAGAGGACG- ) andthenmultipliedby10 C inthepresenceof0.5% To increaseFasexpression, andreverse,5 CCR10- andpLNCX2- 20 (asabove)andla- 25–33 M finalconcen- l PBS)werein- d -luciferin (Bi- peptideand b –restricted, -CATAC- g/ml g/ 7 – . - . ; ; The Journal of Experimental Medicine Results transduced B16/F1cellswith the full-lengthcDNA-encod- transduced in amurinemodelofmelanoma growthandmetastasis,we express CCR10insitu. both primaryandmetastatic human melanomasfrequently pared withskinfromhealthyvolunteers(Fig.1C).Thus, http://www.jem.org/cgi/content/full/jem.20030593/DC1. mRNA expressioninvariousorgans.Figs.S1–S6areavailableat footpad tumor.Fig.S6indicatesmouseCCL27andCCL26 (Fig. S4A).Fig.S5showsphospho-AktstainingofpLNCX2-B16 of pLNCX2-B16tumorinFasL-deficientmicebyluciferaseassay Fig. S4showsthefate expression inpLNCX2-B16footpadtumor. Fig. S3showsFax vivo luciferaseimagingoftumorprogression. (Fig. S1A)andcalciumfluxassayB).Fig.S2indicatesin sion oftransducedCCR10inB16cellsbyWesternblotanalysis pore). Specificlysis lease wasmeasuredusingaCytoFluor2350platereader(Milli- observed metastatic melanomatissuebyquantitativeRT-PCRand pression. Wealsoanalyzedthreesamplesofcutaneous sion, andthreetumorswithoutapparentCCR10ex- tive sample),threetumorswith10–50%CCR10expres- 100% expressionofCCR10(seeFig.1Bforarepresenta- CCR10 antibodyandobservedseventumorswithnearly cutaneous melanomasfrom13patientswithanti–human melanoma hasnotbeeninvestigated.Westainedprimary lines (8),proteinexpressionofCCR10inprimaryhuman mRNA expressionhasbeenreportedinmelanomacell spontaneous]) (1.5 tration, washed,andaddedtoround-bottommicrotiterplates the averageoftriplicateassays. target cellsalone.Thepercentageofcytotoxicitywascalculatedas ton X-100,whereasspontaneouslysiswasobtainedbyincubating CCR10-B16 Cells. 2.5 hat37 Enhanced RegionalLNMetastasis andLocalGrowthof Human MelanomasExpressCCR10. Online SupplementalMaterial. 10 4 /well) withspecificratiosofeffectorPMELcellsfor C. Thesupernatantswererecovered,andcalceinre- 800-fold increasesinCCR10mRNAcom- 100.Maximallysiswasachievedwith0.1%Tri- ([experimental To characterizetheroleof CCR10 1339 Fig. S1showsfunctionalexpres- Murakami etal. spontaneous]/[maximal Whereas CCR10 some pLNCX2-B16–injected miceexhibitedsmall( 13 of15pLNCX2-B16–injectedears(Fig.2C).However, pLNCX2-B16 cellsinthefootpad,tumorsdidnotform in lage. Incontrasttothegrowthpatternobservedwith pLNCX2-B16 cellsintotheskinsuperficialtoearcarti- a separatesetofexperiments,weinjectedCCR10-B16and with E).13of15(87%)mice injectedintheearskinwith 2 E)grewprogressivelyinthe earskin(Fig.2,Ccompared cells (notdepicted)andCCR10-transduced B16cells(Fig. (Fig. 2D).Interestingly,parental (untransfected)B16/F1 mm) residual,darkmacules at theinitialinoculationsites B16–injected miceweremoderatelyincreasedinsize(P not toCXCL12(Fig.S1B).FootpadtumorsofCCR10- cells underwentacalciumfluxinresponsetoCCL27but nized byaspecificanti-CCR10antibody.CCR10-B16 (CCR10-B16) expresseda42-kDproteinthatwasrecog- (Fig. S1A,lanes1and2),CCR10-transducedB16cells the vectoralone(pLNCX2-B16)orwithCXCR4cDNA and WesternblottinginlysatesofB16cellstransducedwith CCR10 wasnotdetectablebyRT-PCR(notdepicted) chemokine receptorstested,includingCCR10.Whereas cells shownegligiblelevelsofmRNAexpressionforseveral ing murineCCR10(12).Asnotedpreviously(10),B16 B16–injected mice(P injected miceversus21relativelightunitsforpLNCX2- relative lightunitsinthepoplitealLNfromCCR10-B16– quantification revealedameanluciferaseactivityof13988 these cellsthatwehadreportedpreviously(10).Luciferase metastasized tothedrainingLN(Fig.2B),aphenotypeof tastases (Fig.2B).Bycontrast,pLNCX2-B16cellsrarely CCR10-B16–injected miceshowedfrequentgrossme- mice(Fig.2A).Strikingly,thepoplitealLNfrom injected 0.003) comparedwiththetumorsfrompLNCX2-B16– pads ofmice. metastasis tothedrainingLNafterinjectionintofoot- trol cells,CCR10-B16cellsdisplayastrikingincrease in tage ingrowthoftheprimarytumorcomparedwithcon- test). Thus,inadditiontodemonstratingamoderateadvan- CCR10-B16 CellTumorProgressionintheEarSkin. are shownaboveindividualsampleresults. for CCR10mRNAexpression.Themeansofthesamples tastases ( indicates positivestaining.(C)Cutaneousmelanomame- nification is CCR10 antibodies(B)orspecies-matchedcontrol(A).Mag- cutaneous melanomawasprobedwithanti–human express CCR10proteinandmRNA.(AB)Primary Figure 1.

3) andnormalhumanskin(

Human primaryandmetastaticmelanomas

400. Theredcolor(amino-9-ethycarbazole)

0.003,

7, Mann-Whitney n

2) wereanalyzed In 1 The Journal of Experimental Medicine CCR10-B16 cellsdevelopedtumorsthatwere tion (Fig.S2,availableat http://www.jem.org/cgi/content/ nium ofpLNCX2-B16–injected mice20dafterinocula- detected inthecervicalregion, abdominalcavity,orcra- mice wasreadilydetected,whereas nolightsignalcouldbe mor andipsilateralcervicalLN fromCCR10-B16–injected ence ofmetastases.Lightemanating fromtheprimarytu- both typesoftumorcellswasalsousedtodetectthepres- tastasis (8). consistent withapostulatedroleforCXCR4innodalme- tastasis in8of10miceinjectedwiththesecells,whichwas form tumorsinears,wedidobserveenhancednodalme- CXCR4-B16 cellsdidnotdisplayanenhancedabilityto Fisher’s exacttest;Fig.2FcomparedwithG).Although diameter (P out of15)pLNCX2-B16–injectedmice(P gross cervicalLNmetastasiswasobservedinonly20% (3 animalsdevelopedgrossLNmetastases,whereas injected largement (Fig.2F).14of15(93%)CCR10-B16– mice developedpronouncedipsilateralcervicalLNen- oped tumors(unpublisheddata). into mouseearsandfoundthatonly1of10micedevel- in tumorformation,weinjectedCXCR4-B16cells(9) another chemokinereceptor,CXCR4,hadsimilareffects injected mice,Fisher’sexacttest).Todeterminewhether In vivoimagingbasedonthepresenceofluciferasein After inoculationofCCR10-B16cellsintoearskin, 0.001 comparedwithpLNCX2-B16– 1340 CCR10 andMelanoma ImmuneEvasion 4 mmin 0.001, lenge withthesamecells. The expressionofCCR10, tive hostresponsethatresists tumor formationuponrechal- pLNCX2-B16 cellsintothe earofmiceleadstoaneffec- ive mice(Fig.3E,right column). Thus,injectionof to thosearisingfrompLNCX2-B16 cellsinjectedintona- metastasize toregionaldrainingLN. contrast topLNCX2-B16cells)growintheearskinand was associatedwiththecapacityofCCR10-B16cells(in full/jem.20030593/DC1). Thus,expressionofCCR10 sponses. vaccinated miceresultedintumors similarinsize(P column). Interestingly,injectionofCCR10-B16cellsinto mors inthefootpadsofnonimmunized,naivemice(left The samelotofpLNCX2-B16cells,however,formedtu- oculation inrechallengedmice(Fig.3E,middlecolumn). growth ofpLNCX2-B16cellswasobserved17dafterin- pLNCX2-B16 cellsinthefootpads.Noevidence of with pLNCX2-B16cellswererechallenged30dlater mice thatdidnotdevelopeartumorsafterinitialchallenge mice wereprotectedfromrechallengewithtumorcells, (Fig. 3B).TodetermineifpLNCX2-B16cell–injected were composedofhighlyatypicalcellsthatfilledthedermis in sharpcontrasttoCCR10-B16cell–injectedsites,which cellsandraretumor(Fig.3,A,C,D) inflammatory scopically, theseinjectedsitescontainedpredominantly B16–injected skinsitesdidnotshowoverttumors.Micro- CCR10 ProtectsB16CellsFromHostImmuneRe- When injectedintoearskin, in mm. Scale barinB–Eiscalibrated animals areshowninFandG). mouse(G;representative injected but notinapLNCX2-B16– CCR10-B16–injected mouse(F), large cervicalLNmetastasisina tumor injectionsitesrevealsa cervical regionipsilateraltothe inoculation. Dissectionofthe B16 cells(E)areshown20dafter at analogoussiteswithCCR10- arrow (D) macule indicatedbythewhite cells (C)showingaresidualtumor of theearwithpLNCX2-B16 from miceinjectedinthedermis pads. (C–E)Representativeears popliteal LNfrominjectedfoot- n pads werecompared(P killed andtumorsizesinthefoot- mice. (A)After21d,animalswere were injectedintothefootpadsof CCR10- orpLNCX2-B16cells pLNCX2-B16 tumorcells. LN metastasisofCCR10-and Figure 2.

5 pergroup).(B)Draining 5 and frommiceinjected 90% ofpLNCX2- Growth andregional

0.003, 0.3) The Journal of Experimental Medicine expression ofFas ( middle column)orCCR10-B16cells( 4 A).Becauseinterferon(IFN)- cells invitroexpressedlowlevels ofsurfaceFasprotein(Fig. late theFasdeathpathway.RestingCCR10-B16melanoma prompted ustodetermineifCCR10activationcouldregu- http://www.jem.org/cgi/content/full/jem.20030593/DC1) pLNCX2-B16 tumorcellsinvivo(Fig.S3,available at 3) andourobservationofclearFasproteinexpressionby pLNCX2-B16 cells)toresistimmuneclearance(Figs.2and The abilityoftheCCR10-B16cells(incontrastto form progressivetumors. however, allowsB16cellstoescapethishostresponseand rechallenged inthehindfootpadwitheitherpLNCX2-B16cells( had beeninjectedpreviouslywithpLNCX2-B16cellsintheearwere ( hematoxylin andeosin.(AB)Lowpower( mice wasremoved18dafterinoculation,formalinfixed,andstainedwith skin frompLNCX2-B16(A,C,andD)CCR10-B16–injected(B) Figure 3. age/sex-matched naivemice( mice. Asacontrol,pLNCX2-B16cellswereinjectedintofootpadsof Note notumorgrowthwasobservedinthepLNCX2-B16–rechallenged 0.001, CCR10versuspLNCX2-B16injectedintoimmunizedmice. CCR10-B16 cellswithIFN- to increaseFasexpressionby melanomacells,wetreated form orasproducedbycytotoxic Tcells [24–26]) isknown control versusCCR10-B16–rechallengedgroup. 200), and(D)highpower( CCR10 MediatesResistancetoFas-mediatedApoptosis. Antitumor responsesinpLNCX2-B16–injectedmice.Ear 92% expression; Fig.4A). n 1341 400) views.(E)Tumor-freemicethat

5 mice,leftcolumn).P , whichresultedinuniform n Murakami etal. (eitherinrecombinant

7 mice,rightcolumn).P 25), (C)mediumpower

0.3, naive n

5, The difference intheabilityofpLNCX2-B16 cellstoform tastases wereobservedin jem.20030593/DC1). Moreover, largecervicalLNme- S4 A,availableathttp://www.jem.org/cgi/content/full/ both strains(Fig.4D,quantified byluciferaseassayin Fig. WT mice,whereasCCR10-B16 cellsformedtumorsin progressive tumorsintheFasL-deficient( region ofFasL(27).Strikingly,pLNCX2-B16cellsformed gous inactivatingpointmutationintheCOOH-terminal from theH-2D vitro–activated CD8 specific CTLs,weexposedCCR10-B16(target)cellstoin B16 cellsfromcytotoxicityinducedbymelanomaantigen- tected fromapoptosisbyexposuretoCCL27(Fig.4B). presence ofmultimerizedFasL,thesecellswerenotpro- B). WhilepLNCX2-B16cellsunderwentapoptosisinthe apoptosis, demonstratingthespecificityofassay(Fig.4 pase inhibitor(VAD-fmk)effectivelyblockedFas-mediated antibody alonedidnotinduceapoptosis,andageneralcas- CCR7) couldnotreducecelldeath(Fig.4B).Anti-FLAG apoptosis, whereasCCL21(achemokinethatactivates CCR10-B16 cellsweremoreresistanttoFas-mediated (not depicted).InthepresenceofCCL27,however, treatment ofmelanomacellswiththedrugcamptothecin nexin Vstainingwasnearlyidenticaltothatachievedby linking (Fig.4B,farleftcolumn).Themagnitudeofan- cells readilyunderwentapoptoticcelldeathafterFascross- nexin Vstaining.IntheabsenceofCCL27,CCR10-B16 from FasL-triggeredapoptosis,whichwasmeasuredbyan- determine ifCCR10engagementcouldprotectB16cells multimerized FasLinthepresenceorabsenceofCCL27to (PMEL-1), whichexpressedtheV CCR10-B16 cellkillingbyPMEL-1Tcells anti-FasL antibodies(notdepicted)botheffectivelyreduced caspase inhibitor,VAD-fmk(Fig.4C),andtheadditionof cells, evenatloweffector:target(E:T)ratios(Fig.4C).The mice andC57BL/6( pLNCX2-B16 cellsintotheearskinofWTC57BL/6 for thedestructionofpLNCX2-B16tumors,weinjected Cells. and byCTLsspecificforabonafidetumorantigen. mediated cellapoptosisinducedbyrecombinantFasligand the surfaceofmelanomacellsconferredresistancetoFas- T cells(notdepicted).Thus,engagementofCCR10on RT-PCR, CCR10expressionwaslargelyabsentinPMEL B16 cellsviaeffectsonTcells.Moreover,byquantitative that CCL27wasnotreducingapoptosisintheCCR10- killing ofpLNCX2-B16cellsbyPMELTcells,suggesting the presenceofVAD-fmk.CCL27,however,didnotaffect killing oftargetcellsthatwascomparabletoin The additionofCCL27alsoresultedinmarkedlydecreased #9 Tcell(22).PMEL-1CD8 highly efficientkillingofIFN- stimulation (23).PMEL-1Tcells(PMEL)demonstrated icating B16tumorsinvivoafteradoptivetransferandre- To determineifCCR10ligationcouldprotectCCR10- Fas andFasLintheHostResponsetopLNCX2-B16Tumor We thenexposedIFN- To determineifFas–FasLinteractionswerecritical b restricted,murinegp100–specificclone Tcellsfromatransgenicmouse gld Fasl –treated CCR10-B16cellsto but notWTmice(Fig.S4B). gld Tcellsarecapableoferad- ; gld)thathadahomozy- –stimulated CCR10-B16 2V 13 Tcell(TCR) gld ) micebutnot 80%.

The Journal of Experimental Medicine tor-mediated protectionfrom apoptosis. sized thatAktactivationcontributed tochemokinerecep- stresses includingadministration ofFas(30),wehypothe- tivation caninhibitapoptosis causedbyavarietyofcellular dylinositol-3-kinase (PI3K)/Akt pathway.BecauseAktac- activate MAPkinasefamily members andthephosphati- a complexcascadeofintracellularsignalingevents(29)that ment withappropriateligands,chemokinereceptorstrigger CCR10-mediated ProtectionfromApoptosis. cells fromformingtumorsinearskin. the hostantitumorresponsethatpreventspLNCX2-B16 B16 tumorcellsinvivo,andFasLappearstobecriticalfor and (28; notdepicted)readilyformedskintumorsinbothWT B16/F1 cells(Fig.4E)andRMAmurinelymphoma tumors inWTversus Involvement ofAktandPhosphatidylinositol-3-kinasein gld mice.Thus,FasantigenisinducedinpLNCX2- gld 1342 micewasspecificsinceparental CCR10 andMelanoma ImmuneEvasion After engage- exposed tomultimerizedFasL intheabsenceorpresenceof CCR10-B16 cells,IFN- PI3K/Akt signalinginCCL27-mediated protectionof ear (Fig.5B).Finally,todemonstrate thesignificanceof compared withresidualpLNCX2-B16 cellsfoundinthe of CCR10-B16cellsshowed increasedlevelsofactiveAkt activation state-specificAktantibodies.Tumorscomprised of miceinjectedwithCCR10-orpLNCX2-B16cells cells byCCL27invivo,westainedtissuesectionsfromears PD98059 (Fig.5A).TodetectactivationofCCR10-B16 LY294002), andbyPTX,butnottheMEKinhibitor, hibited byselectivePI3Kinhibitors(wortmanninand tion ofthiscriticalserineresiduewasnearlycompletelyin- for thecellsurvivalactivityofAkt(Fig.5A).Phosphoryla- phosphorylation atserine473,asiteknowntobenecessary CCR10-B16 cells,weobservedastrongincreaseinAkt Within 5minafteradministrationofCCL27to –treated CCR10-B16cellswere 10 parental dicated. mice (14-dtumorassay)asin- and gender-matchedWTC57BL/6 the earskinof condition). (C)IFN- 0.0016 versusFasL( B16 (100 toxicity. (D)pLNCX2-,CCR10- E:T ratiosandassessedforcyto- PMEL Tcellsattheindicated incubated withantigen-stimulated CCR10-B16 cells,asinB,were IFN- B16 cellswereeithertreatedwith exposure toCCL27.(A)CCR10- mediated CCR10-B16 cellsfromFas- Figure 4. right-most conditions. were notusedunderthe itive cells.pLNCX2-B16cells as percentageofannexinV–pos- FloJo softwareandarepresented B16 cellswerequantifiedusing V–FITC. DatafromPI-negative propidium iodide(PI)andannexin then collectedandstainedwith after IFN- which weresimilarlyup-regulated of Fas-stainingbeforetreatment, lines showedequivalentlowlevels antibodies. Notethatbothtumor multimerized nant FLAG-taggedFasLand cells wereexposedtorecombi- pLNCX2- antibody. (B)IFN- antibody. Iso:isotypecontrol staining withanti-CD95(Fas) 3 cells/ear)wereinjectedinto orwithPBSbefore B16/F1 cells(100 apoptotic celldeathvia treatment.Cellswere 10 and CCR10-B16 with anti-FLAG gld Protection of 3 perear),(E) miceorage- n –treated –treated * P 3 per three The Journal of Experimental Medicine (14, 16).Thus,wepostulated thatskin-derivedCCL27 produced attheproteinlevel byepidermalkeratinocytes CCR10-B16 TumorsinEarSkin. and G CCR10-B16 cellsfromFasL-induced apoptosisinaPI3K- row, righthistogram).Therefore, CCL27protects tom apoptosis eveninthepresenceofCCL27(Fig.5C,bot- rendered CCR10-B16cellssusceptibletoFas-mediated 5 C,middlerow,leftdotplot).TreatmentwithPTXalso LY294002 alonedidnotinducesignificantcelldeath(Fig. CCL27 (Fig.5C,middlerow,rightdotplot).However, LY294002, however,abrogatedprotectionconferredby row, rightdotplot).PretreatingCCR10-B16cellswith nexin Vstainingwascompletelyblocked(Fig.5C,top conjunction withmultimerizedFasL,theincreaseinan- top row,middledotplot).WhenCCL27wasadded in of multimerizedFasLaloneincreasedapoptosis(Fig.5C, CCL27 and/oraPI3Kinhibitor.Asbefore,administration (1 Figure 5. ( CCL27 (1 with thePI3Kinhibitor,LY294002(10 CCR10-B16 andresidualpLNCX2tumorsintheearswereprobedforp-AktexpressionbyIHC.(C)cellsthathadbeenpre ng/ml, PTX),andPD98059(20 -FLAG). Anti-CCL27 NeutralizingmAb PreventFormationof g/ml) i protein–dependentmanner. for theindicatedtimeinpresenceofcellsignalinginhibitors(wortmannin(200nM,WM),LY294002(10 One ofthreeexperimentswithsimilarresults. Involvement ofPI3KandAktinCCR10-mediatedprotectionfromapoptosis.(A)CCR10-B16cellswereexposedtoPBSorCCL27 g/ml) for16hat37 1343 C. Asanegativecontrol,cellswereexposedtotheanti-FLAGepitopeantibodywithoutpriorexposureFasL M, PD),lysed,andanalyzedbyWesternblotforp-AkttotalAkt.(B)14dafterinoculationintothedermis, Murakami etal. M), PTX(200ng/ml),orDMSOalonewereexposedtomultimerizedFasLinthepresenceandabsenceof CCL27 isconstitutively jected withthecontrolantibody developedtumors(P CCR10-B16 tumors,whereas sixoutofanimalsin- six animalsinjectedwithanti-CCL27 mAbdevelopedgross 6). Intwoindependentexperiments, atotalofzeroout anti-CCL27 mAbonCCR10-B16 cellswasspecific(Fig. tumor progression,demonstratingthattheeffectof agents undersimilarconditionsshowednodifferences in parental B16/F1cellsthatweretreatedwiththesamere- vented CCR10-B16tumorformation(Fig.6).However, data). Theanti-CCL27mAbtreatmenteffectivelypre- in vitroataconcentrationof100 hibited downstreamp-AktactivationinCCR10-B16cells influx ofTcellsintoskininvivo(16)andcompletelyin- anti-CCL27 mAbhasbeenshownbyotherstoinhibitthe mAb (16)orwithisotype-matchedcontrolantibodies.This B16 cellsintoearskineitherwithanti-CCL27neutralizing responses. Totestthishypothesis,weinjectedCCR10- protected CCR10-B16cellsintheskinfromhostimmune 0.0022, Fisher’sexacttest). M, LY),pertussistoxin(200 g/ml (unpublished treated for18h

The Journal of Experimental Medicine Discussion for abonafidemelanomatumor antigen,gp100(22,33, dent celldeath(Fig.4)mediated byCTLthatarespecific thermore, CCR10-B16cells wereresistanttoFas-depen- sponses directedatendogenous melanomaantigens.Fur- data), suggestingthatearimmunization alsoleadstore- pad injectionofparentalB16/F1 tumorcells(unpublished tially protectedfromtumorformationresultingfoot- that pLNCX2-B16–immunizedmice(Fig.3)arealsopar- cells isnotsolelydirectedatluciferase.Wehaveobserved sponse thatdevelopsafterearinjectionofpLNCX2-B16 rected atthisneo-antigen.However,theimmunere- of thehostresponsetopLNCX2-B16cellswasdi- cells lackedexpressionofluciferase,itispossiblethatpart B16 cellsarepoorlyimmunogenic(32).Becausethese in earskin,whichwasnotsurprisingsinceunmanipulated lacked CCR10expressionalsoformedprogressivetumors B16 tumorcellsfromFas-triggeredapoptosisinvitro. D andFig.S4)(2)CCR10-mediatedsignalingprotects for theeradicationofpLNCX2-B16cellsinear(Fig.4 Our resultsclearlysuggestedthat(1)Fas–FasLisrequired killer cellsandtotheefficacyofsomeantitumordrugs(31). the efficacyofantitumorresponsesbyCTLsandnatural ated celldeathbecausetheFaspathwaycanbecriticalto footpads, weexploredtheeffectofCCR10onFas-medi- ing FasexpressioninpLNCX2-B16cellsinjectedintothe responsible forthedestructionofthesecells.Afterobserv- in theskin,wesuspectedthatahostimmuneresponsewas matory reactionsurroundingresidualpLNCX2-B16cells mors afterearskininjection.Becauseoftheintenseinflam- draining regionalLN. ion, and(3)theymetastasizedwithhighfrequencyto Fas-mediated apoptosisinvitroaPI3K-dependentfash- ciferase-expressing B16cellscouldnot,(2)theyresisted ing cells:(1)theyformedtumorsatskinsiteswherelu- cDNA andfoundthreeremarkablefeaturesoftheresult- CCR10, wetransducedB16melanomacellswithCCR10 rine (B16)melanoma.Toexplorethepossiblefunctionof in primarycutaneoushumanmelanomasbutnotmu- Nontransduced parentalB16/F1melanomacellsthat Unlike controlB16cells,CCR10-B16cellsformedtu- Herein, wereportthatCCR10isfrequentlyexpressed 1344 CCR10 andMelanoma ImmuneEvasion that chemokine-mediatedup-regulation ofPI3K/Aktmay apoptosis invitro.Although these dataraisethepossibility critical forchemokinereceptor–mediated protectionfrom CCL27, suggestingthatthePI3K/Akt signalingpathwayis Fas-mediated celldeath(Fig. 5C)inthepresenceof LY294002 reestablishedthe susceptibility ofthesecellsto and B).Ofnote,exposureofCCR10-B16cellsto easily detectableinCCR10-B16tumorsvivo(Fig.5, A B16 cellsafterinvitroexposuretoCCL27andwasalso Fas-mediated apoptosis(37).AktwasactivatedinCCR10- Akt regulatescellularsurvival,motility,andprotectsagainst PI3K (29),wefocusedonthePI3K/Aktpathwaybecause Stat proteins,MAPkinases,focaladhesionand of multipleintracellularsignalingpathwaysinvolvingJak- fective antimelanomaresponses(36). noma tumorantigenintotheLNcanleadtoclinicallyef- sponses (35),anddirectinjectionofDCsloadedwithmela- organs maybeafactorinenhancedhostanti-tumorre- that earlymigrationofcancercellstosecondarylymphoid B16 cellsintoearskin.Interestingly,othershaveshown LN involvementwasobservedafterinjectionofCXCR4- footpad injection(9),whereasinthecurrentwork,cervical pLNCX2-B16 cellstometastasizethepoplitealLNafter differenceintheabilityofCXCR4-B16cellsand no gional LNmaybethereasonwepreviouslyobserved ferential site-specificaccumulationoftumorcellsinre- shortly afterfootpadinjections(unpublisheddata).Thisdif- whereas neithercelllineaccumulatedinthepoplitealLN cervical LNwithin5minofinjectionintoearskin, of bothpLNCX2-andCCR10-B16cellsinthedraining in vivoluciferaseimagingwehaveobservedthepresence cal responseagainstpLNCX2-B16cells(Fig.3).Indeed,by draining cervicalLNthatresultedinapotentimmunologi- mitted rapidmovementoftumorcellsfromtheskinto footpad), webelievethatanatomicalfactorsintheearper- pLNCX2-B16 cellsinjectedintheear(asopposedto endogenous targets. immune responsesdirectedatendogenousaswellnon- 34). Thus,CCR10activationprotectstumorcellsfrom Although chemokinereceptorligationleadstoactivation To explainthepotentimmuneresponseagainst with similarresults. 20 was coinjectedwithtumorcellsinavolumeof R&D Systems)or100 rat anti–mouseCCL27IgG(16)(clone68623; buffer containingeither100 of injection,thecellsweresuspendedininjection mice inan18-dtumorformationassay.Atthetime or B16/F1cellswereinjectedintotheearskinof formation withanti-CCL27treatment.CCR10-B16 Figure 6. l. Oneoftwoindependentexperiments Prevention ofCCR10-B16celltumor g/ml ofratIgG,which g/ml ofneutralizing The Journal of Experimental Medicine survival. Based onourresultsshowing protectionof chemokines maybeamechanism thatpromotescancercell mor-associated chemokinereceptors bylocallyproduced ability toaffectorgan-selective metastasis.Ligationoftu- play criticalrolesintumor progression apartfromtheir and LN(8). CCL27) arerelativelylowintheskincomparedwithlung in earskin,perhapsbecauselevelsofCXCL12(versus skin. Interestingly,CXCR4-B16cellsdidnotformtumors arises intheenvironsofskinandfrequentlymetastasizes to expression couldbeadvantageoustomelanoma,which CCL27 intheskinmaypotentiallyexplainwhyCCR10 brane aftersynthesisbykeratinocytes.Thepresence of dermis becauseofitsabilitytocrossthebasementmem- CCL27 mayhaveeffectsontumorcellsimplantedinto dermal cells)isalsopresentinthedermis(16).Thus, dermis (14),CCL27protein(presumablycomingfromepi- mune evasionbytumorcells. vivo demonstrationofchemokinereceptor–mediatedim- types ofcells(41,43,44),thepresentstudyisfirstin implicated chemokinereceptorsinthesurvivalofother effects inbrainmicroglia(42).Whereaspriorstudieshave vitro (41),andengagementofCX3CR1hasantiapoptotic from bothFas-andcycloheximide-inducedapoptosisin ple, activationofCCR9partiallyprotectslymphoidcells inhibit apoptosismaynotbeuniquetoCCR10.Forexam- metastatic potential. ing (40),andthispropertymaycontributetoenhanced ported toenhancecellmigrationviauniquenuclearsignal- injected intoskin(39).Alternatively,CCL27hasbeenre- the LNashasbeenshownforotherchemokinesthatwere the skinmayactuallyenterlymphaticsfortransportto However,CCL27from content/full/jem.20030593/DC1). skin (Fig.S6,availableathttp://www.jem.org/cgi/ CCL28, wasalsofoundatlowlevelsintheLNand possibility. TheonlyotherknownligandforCCR10, melanoma cellstothelymphaticsviaCCR10isanunlikely high levelsofCCR10ligand(8),directattractionthe Because neitherlymphaticsnorLNarelikelytoexpress B16 cellsthatarriveintheLNaftercutaneousinjection. lated toenhancedsurvivalofasmallnumberCCR10- mechanism underlyingthisresultisunclear,itmaybere- metastasis wasintriguingbutunexpected.Althoughthe PI3K/Akt (38). growth factorreceptor,thatareupstreamactivatorsof regulators ofcancergrowth,includingtheepidermal IHC haslimitations,thismayreflecttheactionofother tivation ofAkt.AlthoughprecisequantificationpAktby jem.20030593/DC1), suggestingCCR10-independentac- S5, availableathttp://www.jem.org/cgi/content/full/ footpad tumorsarisingfrompLNCX2-B16cells(Fig. Substantial stainingforactiveAktwasalsoobservedin tional studieswillbenecessarytovalidatethisproposal. be animportantfactorfortumorprogressioninvivo,addi- Our resultsdemonstratethat chemokinereceptorsmay Although CCL27isconstitutivelysynthesizedintheepi- The abilityofchemokinereceptor–mediatedsignalingto Our observationthatCCR10stronglyenhancedLN 1345 Murakami etal. 0 Wiley,H.,E.B. Gonzalez,W.Maki,M.Wu,andS.T. 10. clinical setting. may enhanceantitumorimmunityandbeusefulina therapy directedatspecificchemokinereceptorpathways bodies toCCL27(Fig.6).Thesefindingsdemonstratethat was markedlyreducedbytreatmentwithneutralizinganti- tumor response.Indeed,CCR10-B16formation make CCR10-B16cellsmoresusceptibletothehostanti- that inhibitionoftheCCL27-CCR10pathwayshould CCR10-B16 fromFas-triggeredapoptosis,wepredicted Accepted: 12September2003 Revised: 8August2003 Submitted: 11April2003 search Foundation. their invaluablehelpwithinvivoimaging. ments, andthemembersofMouseImagingFacility,NIH,for stitute ofAllergyandInfectiousDiseases)formanyhelpfulcom- (NCI), ScottAbramsandPhilipM.Murphy(NationalIn- melanoma specimens.WealsowishtothankDrs.MarkC.Udey CCR10 cDNAandtoDr.JohnWunderlich(NCI)forproviding stitute ResearchGroup,Glasgow,Scotland)forhisgenerousgiftof We wishtoextendourgratitudeDr.GerryGraham(BeatsonIn- References .Murakami,T.,W.Maki, A.R.Cardones,H.Fang,A.T.Kyi, 9. 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