The earliest intrathymic precursors of CD thymic dendritic cells correspond to myeloidtype - PDF document

TheearliestintrathymicprecursorsofCD8thymicdendriticcellscorrespondtomyeloid-typedouble-negative1ccells,LaurenceArdouin,PearlineTeo,PeterSeeSandrineHenri,MiriamMerad,FlorentGinhouxBernardMalissen SupportingInformationavailableonline SeeaccompanyingCommentarybyKruegerIntroductionTcellsdevelopinthethymusthroughdiscretestagesdenedonthebasisoftheexpressionofCD4andCD8molecules.Immaturedouble-negative(DN)CD4Tcellsgiverisetodouble-positive(DP)CD4 Theseauthorscontributedequallytothiswork. Correspondence:Prof.BernardMalissen Frontline tothemedullaryregionofthethymus[1].Followingthistransitionknownaspositiveselection,SPcellsexpressingTCRwithahighafnityforpeptide-MHC(pMHC)complexespresentonmedullarycellsaredeleted,aprocessreferredtoasnegativeselection.Thepoolofthymicself-peptidesavailableforTCRselectionderivesfromproteinsexpressedbythymicepithelialcells(TECs)andbythymicDCs(tDCs).TheTECsfoundinthecortex(cTECs)areprimarilydevotedtopositiveselection[2].Incontrast,themedullaisthemajorsiteofnegativeselectionduetotheabilityofmedullaryTECs(mTECs)toectopicallyexpressself-proteinsthatarenormallyfoundoutsidethethymus[3].mTECsareshort-livedandupondeathcantransfertheirproteincontenttothedensenetworkoftDCspresentinthemedulla[4–6].TherespectivecontributionofmTECsandtDCsincentraltoleranceinductionandintheshapingoftheTregulatorycelllineageremainstobeestablished[7].ThreesubsetsofCD11cDCscoexistinmousethymus[8].OnesubsetexpressesCD45RandcorrespondstoplasmacytoidDCs(pDCs),whereasthetwoothertDCsubsetsareCD45RandcanbedistinguishedbasedonthedifferentialexpressionofCD8andCD172(Sirp).TheCD8lowCD172tDCsarisefromquasi-differentiatedbloodprecursorsthatcontinuouslyenterthethymus[9,10].Incontrast,theCD8highCD172tDCsdevelopintra-thymicallyandstandapartfromallotherDCsinthattheyarereportedtooriginatefromearlyT-cellprogenitors(ETPs)[10–12].BasedontheexpressionofCD25andCD44,DNcellscanbeorganizedaccordingtothefollowingdevelopmentalseries:DN1(CD44CD25DN2(CD44CD25DN3(CD44CD25DN4(CD44CD25)[13].DN1cellscanbesubdividedfurtherintoDN1a,b,c,dandesubsetsbasedontheexpressionofCD24andofthec-Kittyrosinekinasereceptor(alsocalledCD117)[13–15].DN1a(CD24CD117)cellsconstitutetheprecursorsofDN1b(CD24intCD117)cellsthatgiverisetotheT-celllineage.DN1aplusDN1bthymocytesaretermedETPs[16,17],anditistheseETPsthatarethoughttogenerateCD8highCD172tDCsinadditiontoTcells[10–12].Incontrast,theCD8lowCD172tDCsderivefrombonemarrow(BM)-derivedDCprogenitors.SuchBM-derivedDCprogenitorsarecomposedofthecommonmacrophage-DCprogenitors(MDP),thecommonDCprecursors(CDP)andofpre-DCsthatconstituteanintermediatestagebetweenCDPsandclassicalDCs[18,19].Therefore,CD8highCD172tDCswouldconstituteanexceptionamongauniverseofDCsthatoriginatefrommyeloid-restrictedprecursors.Contradictorydatahave,however,suggestedthatCD8highCD172tDCsalsoderivefrommyeloidprecursors[20],orfromprecursorsthatareunrelatedtotheT-celllineageandthatremaintobecharacterized[21].TofurtherexploretheoriginofCD8tDCsandcharacterizetheirearliestintrathymicprecursors,wetookadvan-tageofknockinmiceinwhichanenhancedgreenuorescentprotein(Langerin-EGFPmice)orahumandiphtheriareceptor(DTR)fusedtoanEGFP(Langerin-DTREGFPmice)wasplacedunderthecontrolofthegenecodingforlangerin(CD207)[22].LangerinisaC-typelectintheexpressionofwhichwasoriginallydescribedonepidermalLangerhanscells(LC)andlateridentiedonadditionalDCsubsets[23].UsingthehighlysensitiveLangerin-reportermice,wecharacterizedtheabundantCD207(EGFP)DCpopulationthatisfoundinthethymusandshowedthatitcorrespondsentirelytoCD8tDCs.Moreover,sinceLangerin-DTREGFPmiceareparticularlysuitabletoanalyzetheprecursor–productrelationshipthatexistsbetweenCD207[24,25],weusedthemtodemonstratethattheearliestintrathymicprecursorsofCD207tDCsarenotcontainedamongETPsbutcorrespondtoDN1c(CD24int)cells.DN1ccellshavenopotentialtogenerateTcellsandtheirdevelopmentalpotentialhasremainedunclear[14].ConsistentwiththeconclusionthattheDN1ccellsarethesourceoftheCD207tDCsandaredevelopmentallyseparatedfromtheT-celllineage,weshowedthatamutationintheinterferon-regulatoryfactor8(IRF-8)genepreventedthedevelopmentofbothDN1ccellsandCD207tDCswithoutaffectingtheETPsandtheT-celllineage.Finally,tocorroboratethemyeloidoriginoftheCD207tDCs,wealsoshowedthattheycanbegeneratedviaadoptivetransferofMDPs,CDPsorpre-DCsthatrepresentthedifferentstagesofthedevelopmentalseriesleadingtocanonicalDCs.tDCscorrespondtoCD8TocharacterizethephenotypeoftheCD207DCsthatarefoundinadultmousethymus[22],thymifromweredigestedwithcollagenaseandlight-densitycellswerepreparedusingOptiprepgradient.Afterexcludingeosinophils[26],CD11c[27]andCD11cNKcells(Fig.1),theremainingtDCswereanalyzedfortheexpressionofCD207(EGFP)andCD172permittingustodistinguishandCD207subsetsthatrepresented5.2and34.94.6%oftheCD45RtDCs,respectively(Fig.1).CD207wereCD8lowtohightDCswereCD8tolow(Fig.1).Therefore,incontrasttoCD8thathasacontinuousdensitydistributionontDCs,thebimodaldistributionofCD207(EGFP)permittedunambiguousdenitionofCD207tDCsubsets.CD207tDCswerepredominantlyfoundinthemedulla(datanotshown)andcorrespondedtothepreviouslydescribedCD8[9]andareherereferredtoasCD207DN1ccellscompriseCD207myeloid-typecellsTodeterminewhetherprecursorsoftheCD207CD8hightDCscanbedetectedamongtheDN1a-bETPsaspreviouslysuggested[10–12],wetookadvantageofthehighsensitivityaffordedbyLangerin-EGFPreportermiceoverstainingwithanti-CD207antibodies.Forthesakeofconsistency,thymitobeusedforsuchanalysisweredigestedwithcollagenaseandsubjectedtoisopycniccentrifugationonOptiprepsolutionasdescribedfortheisolationEur.J.Immunol.2011.:2165–2175Lucheetal. 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu oflight-densitytDCs.Thepelletofheavy-densitycellsthatformedaftercentrifugationcontainedthewholeconstellationofthymicTcellsincludingETPs(seebelow)and,basedonworkperformedonsplenicDCs[28],shouldentailtheearlieststagesofCD207CD8hightDCdevelopment.ToenrichforDNcells,heavy-densitycellpelletspreparedfromLangerin-EGFPmiceweredepletedofCD4Tcellsbycomplement-mediatedkilling.Aswillbedescribedbelow,theearliestintrathymicprecursorsoftheCD207CD8hightDCscorrespondtoDN1ccellsandan‘intermediate’develop-mentalstagelinksDN1ccellstotheterminallydifferentiatedCD207CD8hightDCsthatareconnedtothelight-densitycellfraction.Cellsbelongingtosuchintermediatestagestartexpres-singCD8(Fig.S1).Topreservethem,CD8antibodieswerethusomittedfromthecomplement-mediatedkillingstepaimedatenrichingDNcells,andresidualCD8SPTcellswereexcludedfromfurtheranalysisbystainingwithanti-CD3andanti-CD5antibodiesandgatingoutCD3CD5cells.Accordingly,onanoperationalbasisourDN1ccellscomprisebothbonadeCD4CD8DN1ccells[14]andCD4CD8lowtointermediatecellsthatlinkstheDN1ccellstotheterminallydifferentiatedCD207CD8hightDCs.Analysisofthecellsremainingintheheavy-densitycellfrac-tionattheendoftheenrichmentstepsshowedthattheirCD25–CD44prolecorrespondedtotheoneexpectedforDNcells[13]andthattheCD11ccellstheycontainedwereexclu-sivelyfoundintheDN1subset(Fig.2A).Consistentwithpreviouswork[14],theanalysisoftheDN1cellsforCD24andCD117expressionshowedthattheycomprisedDN1c(CD24DN1d(CD24)andDN1e(CD24)subsetsinadditiontotheDN1a-bsubsets(Fig.2B).CD11cDN1cellssegregatedintoDN1c(58.05.6%),DN1d(9.642.3%)andDN1e(25.15.3%)subsets(Fig.2B).Importantly,theDN1csubsetwasprimarilymadeofCD11ccellsandincludedalmostalltheCD207(EGFP)cellsfoundintheheavy-densitycellfraction(Fig.2CandD).FurthercharacterizationoftheDN1ccellsshowedthattheymarkedlydifferedfromtheDN1a-b,DN1dandDN1esubsetsinthattheyexpressedMHCIImoleculesandtheFms-liketyrosinekinasereceptor3(Flt3,alsocalledCD135;Fig.2C),areceptorrequiredforthedevelopmentofmostDCsubsetsandfortheirmaintenanceinlymphoidandnon-lymphoidtissues[19,29,30].DN1ccellswereCCR7(datanotshown)andexpressedlowlevelsoftheCX3CR1chemokinereceptor(Fig.2C).Consistentwithpreviousdata[21],DN1ccellsdidnotexpressdetectablelevelsoftheIL-7receptorchain(CD127)(datanotshown).DN1ccellswerecomposedofaminorCD207fractionandofamajorCD207fraction(Fig.2C).WhencomparedwithCD11cDN1ccells,CD11cDN1ccellsweresmaller,expressedlowerlevelsofMHCIImoleculesandonlyone-fourthexpressedCD8(Fig.2D).CD11cDN1ccellswere,whereasCD11cDN1ccellswereCX3CR1(seebelow).Therefore,DN1ccellsexpressedmarkersthatarenormallypresentonall(CD11candMHCIImolecules)oronsome(CD207,CD135,CD8,CX3CR1)DCsubsets.Incontrast,DN1a-bcellsthathavebeenthoughttoconstitutetheprecursorsofCD207tDCswerenegativeforallthosemarkers(Fig.2BandC).DN1ccellsaretheearliestintrathymicCD207tDCprecursorsTodeterminetheprecursor–productrelationshipthatexistsbetweenthedistinctpopulationsofCD11ccellsthatwerefoundintheheavy-andlight-densitycellfraction,wefollowedtheirkineticsofrestorationafterinjectionofDTintomice(Fig.3).AnalmostcompletedepletionoftheCD207DN1ccellswasmanifested16hafterDTinjection(Fig.3).At16and24hpost-DTinjection,thefewCD11ccellsthatwerelefthadaCD207phenotype(Fig.3C).DN1ccellsreappearedaroundday3andtheirpoolwasgraduallyrestoredoverthenext5days.TherstDN1ccellsthatreappearedweremainlyCD8(Fig.3C).SuchprotractedkineticsofCD8expressionascomparedwiththatofCD207suggeststhattheprogenyofDN1CcellsprogressesthroughCD11cstagesuptotheCD11ctDCsfoundintheheavy-densitycellfraction.Importantly,theanalysisofCD11c–CD117dotplotspriortoandaroundthetimeofreappearanceoftheCD207DN1ccellsshowedthattherewasnostreakoftransitionalcellsconnectingtheCD11cDN1a-bcellclusterwiththeCD11cDN1ccellcluster(Fig.3A).Therefore,thislastresultsuggeststhattheCD11cDN1ccellsconstitutethedirectsourceoftheandCD11cstagesfoundinthelight-densitycellfraction(Fig.3). Figure1.tDCscorrespondtoCD8tDCs.Single-cellsuspensionswerepreparedfromthymidigestedwithcollagenase-DNaseI,andlight-densitycellswereseparatedbycentrifugationoveranOptiprepgradient.Afterexcludingeosinophils(CD11cCD11b),pDCs)andNKcells(CD11c),theremainingCD11cinttohiDCswereanalyzedfortheexpressionofCD207(EGFP)versusCD172,CD8versusCD172andCD24versusCD172.Dataarerepresentativeofatleasttenindependentexperiments.Eur.J.Immunol.2011.:2165–2175HIGHLIGHTS 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu FurtheranalysisofDT-treatedshowedthattheCD207tDCsfoundinthelight-densitycellfractionreappearedwithdelayedkineticsrelativetothatofthecellsfoundintheheavy-densitycellfraction(Fig.4A),anobservationconsistentwiththeviewthatthelatterconstitutedthereservoiroftheformer.Tofurthersupportthatprecursor–-productrelationship,micewerecontinuouslyexposedto5-bromo-2deoxyuridine(BrdU)forupto12days,andthekineticsofBrdUincorporationoftheCD207tDCsfoundintheheavyandlightfractionswasdetermined(Fig.4B).‘Heavy’and‘light’CD207tDCshadahighturnoverresemblingthatoflymphoid-tissueresidentDCs[19]andofmigratoryDCspriortotheirmigrationtodrainingLNs[23].Congruentwiththeviewthatthe‘light’CD207tDCswererapidlygeneratedfromthe‘heavy’CD207tDCs,aone-daydelaywasobservedintheascendingpartoftheBrdUlabelingcurvecorrespondingtothe‘light’CD207tDCsascomparedwiththatofthe‘heavy’tDCs(Fig.4B).Altogether,thesedatademonstratetheexistenceofanintrathymicdevelopmentalseriesthatstartswithbonadeDN1ccellsandthatproceedsthroughCD207andCD207intermediatestagestotheCD207tDCsfoundinthelight-densitycellfraction.IRF-8isrequiredforDN1ccellandCD207tDCdevelopmentBXH2C57BL/6JC3H/HejmicelackbothCD8tissue-residentDCs[31,32]andCD8-typemigratoryDCs[30]duetoamutationinthegene[33].AnalysisofsuchmiceshouldrevealwhetherthemutationalsoaffectsthedevelopmentoftheCD207tDCsandoftheDN1ccellsthatweproposetobetheirimmediateintrathymicprecursors.ComparisonoftheDNcellsfoundintheheavy-densitycellfractionofwild-typeandofthymishowedthatDN1ccellsweredramaticallyreducedin Figure2.DN1ccellsexpressmarkersthatarenormallypresentonDCs.ThymocytesofLangerin-EGFPmiceweresubjectedtoOptiprepgradientseparation,andtheheavy-densitycellfractionwasisolatedanddepletedofDPandofCD4SPTcells.ResidualTcells,BcellsandpDCswereexcludedfromfurtheranalysisusingacocktailofanti-CD3,anti-CD5andanti-CD45Rantibodies.(A)TheremainingDNcellswereanalyzedfortheexpressionofCD44versusCD25.CD11ccellsconstituted0.5%oftheDNcellsandwereexclusivelyfoundamongtheDN1(CD44)subset.(B)DN1cellswerefurthersubdividedintoDN1a-b,DN1c,DN1dandDN1esubsetsusingCD24andCD117expression[14].CD11ccellsfellintheDN1c,DN1dandDN1egates.(C)ExpressionofCD11c,CD207(EGFP),CD135,CD8,MHCIIandCX3CR1(EGFP)amongthefourDN1subsetsdenedin(B).Percentagesofcellspositiveforthespeciedmarkerareindicated.(D)CharacterizationoftheDN1csubsetofLangerin-EGFPmice.CD11cDN1cellswereseparatedintoaCD207(EGFP)andaCD207(EGFP)fractionandanalyzedfortheexpressionofCD24versusCD117.GatedCD207andCD207DN1ccellswereanalyzedfortheexpressionofCD11c,CD207(EGFP),CD135,CD8,MHCIIandfortheirsize(FSC-A).ThesmallfractionofCD135DN1a-bcellscorrespondedtotheearliestthymusseedingprogenitors[16,17].TheDN1dandDN1esubsetscontainedsmallpercentagesofCD11cCD207cellsthatmightcorrespondtoCD172tDCprecursors.Percentagesofcellspositiveforthespeciedmarkerareindicated.ThemeanFSC-Avalueisindicated.Dataarerepresentativeofthreeindependentexperiments.Eur.J.Immunol.2011.:2165–2175Lucheetal. 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu Figure3.KineticsofreappearanceoftheCD11ccellsfoundintheheavy-densitycellfractionafterDTtreatment.(A)DNcellsfromtheheavy-densitycellfractionofLangerin-EGFPthymiwereanalyzedatvarioustimepointsafterthelastDTinjectionfortheexpressionofCD24versusCD117andCD11cversusCD117.GatescorrespondingtotheDN1a-b,DN1c,DN1dandDN1esubsetsareasspeciedinFig.2B.(B)GatedDN1cellswereanalyzedasin(A).(C)GatedCD11cDN1ccellswereanalyzedforCD207versusCD8expressionatvarioustimepointsafterDTinjection.Dataarerepresentativeofthreeindependentexperimentsandthepercentagesofcellsfoundineachgateareindicated. Figure4.KineticsofreconstitutionoftheCD207DCsfoundintheheavy-densityandlight-densitycellfractions.(A)KineticsofdisappearanceandreappearanceoftheCD207tDCsfoundintheheavy-densityandlight-densitycellfractionsofLangerin-DTREGFPthymiatvarioustimepointsafterthelastDTinjection.AbsolutenumbersofcellsweredeterminedusingFlowcountuorospheres(Coulter)andnormalizedtotheabsolutenumbersofcellspresentinthymiofLangerin-DTREGFPmicethatreceivednoDT.(B)BrdUwasadministeredcontinuouslyfor12daystoagroupoftwotothreeB6micetocomparetheBrdU-labelingkineticsoftheCD207tDCsfoundinthelight-densityandhigh-densityfractionsofanOptiprepgradient.Datain(A)arerepresentativeofatleastthreemicepertimepointandcorrespondtothreeindependentexperimentsanddatain(B)arerepresentativeofthreetofourindependentexperiments.ErrorbarscorrespondtotheSEM.Eur.J.Immunol.2011.:2165–2175HIGHLIGHTS 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu (Fig.5A).Incontrast,themutationhadnodemonstrableeffectontheotherDN1subsets,includingtheDN1a-bETPs.FurtheranalysisoftheCD45RtDCsfoundinthelight-densitycellfractionofthymishowedthattheylackedCD207tDCs(Fig.5B).Themutationhad,however,noeffectonthedevelopmentofthetDCs.Therefore,theanalysisofmicedemonstratesthedevelopmentalinterdependencethatexistsbetweenDN1candCD207tDCsandconverselyshowsthatDN1a-bETPsandTcellsdevelopindependentlyofDN1candCD207MDPs,CDPsandpre-DCsgiverisetoCD207thymicDCsTheCD8phenotypeoftheCD11cDN1ccells(Fig.2)resemblesthatofthepre-DCsthatarefoundintheBMandtheblood[34–36]andsuggeststhattheydirectlyderivefromthem.AnalysisofbonadeCD8DN1ccellsmiceshowedthattheyexpresslowlevelsofCX3CR1(EGFP)andloseitastheyprogresstotheCD8lowtointermediatestagethatlinkDN1ccellstotheCD207tDCs(Fig.6A).Interestingly,analysisoftheCD11ccellspresentintheBMandinperipheralbloodofmicefortheexpressionofCD24andCD117showedthepresenceofcellsresemblingbonadethymicDN1c(Fig.6B).ThymicCD11cDN1ccellsexpressedhigherlevelsofMHCIImoleculesascomparedwiththepre-DCsfoundintheBMandtheblood(Fig.6C),suggestingthattheyaremorematurethanpre-DCs.TodeterminewhethercanonicalDCprecursorscangiverisetotheCD207tDCs,MDPs,CDPsandpre-DCswerepuriedfromBMandinjectedintonon-irradiatedcongenicmice.Oneweekafteradoptivetransfer,theircapacitytodifferentiateintoCD8andCD8tDCswasmeasured.Donor-derivedDCsweredetectableinthethymusofmiceinjectedwithMDPs,CDPsandpre-DCsandsegregatedintobothCD8subsets(Fig.7A).Thesmallergenerativepotentialofpre-DCsascomparedwiththatofMDPsandCDPsislikelyduetotheirmorelimiteddivisionpotential[30].Asexpectedonthebasisofpreviousstudies[36],MDPs,CDPsandpre-DCswerealso Figure5.IRF-8isrequiredforbothDN1candCD207tDCdevelopment.(A)DNcellsfromtheheavy-densitycellfractionofwild-type(WT)micewereanalyzedfortheexpressionofCD24versusCD117.GatescorrespondingtotheDN1a-b,DN1c,DN1dandDN1esubsetsareasspeciedinFig.2B.Numbersindicatethepercentagesofcellswithinthespeciedgates.(B)tDCsfromthelight-densitycellfractionofwild-type(WT)andmicewereanalyzedfortheexpressionofCD24versusCD172.In(A),acocktailofantibodiesdirectedagainstCD3CD25,CD45R,NK11andCD11bpermittedtoexcludecellspositiveforthosemarkersandtofocuson‘lineagenegative’(Lin)cells.Datain(A)and(B)arerepresentativeoffourmicepergenotypeandcorrespondtotwoindependentexperiments.Numbersindicatethepercentagesofcellswithinthespeciedgates. Figure6.CellsresemblingthymicDN1ccellscanbefoundinthebonemarrowandtheblood.(A)LinDN1cellsofCX3CR1-EGFPmicewerepreparedasdescribedinFig.2.DN1andCD11cDN1cellswereanalyzedforCD24andCD117expression.ThepatternofCX3CR1(EGFP)versusCD8isshownforCD11cDN1c(CD24CD117int)cells.(B)CD11ccellsfrombonemarrow(BM)peripheralblood(PB)andthymusofCX3CR1-EGFPmicewereenrichedbyMACSseparation.AfterremovingcellsexpressingCD25,CD45R,NK1.1,CD3,CD19,Gr-1,CD115,CD172a,Sca-1orCD8,theremainingLinCX3CR1(EGFP)cellswereanalyzedfortheexpressionofCD24versusCD117.(C)CD24cellswerepreparedfrombonemarrow(BM)peripheralblood(PB)andthymusofCX3CR1-EGFPmiceasdenedin(B)andanalyzedforCD11cversusMHCIImolecules.GatehasbeensetupusingtheMHCIICD24lowCD117cellsshowninpanel(B).Resultsarerepresentativeoftwoindependentexperiments.Eur.J.Immunol.2011.:2165–2175Lucheetal. 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu capableofgivingrisetotheCD8andCD8DCsfoundinthespleen(Fig.7B).ThesedatatogetherwiththefactthatmicedeprivedofIrf8(Fig.5)orofBatf3[37]transcriptionfactorlackedbothsplenicandthymicCD8DCshighlightstheirrelateddifferentiationprogram.Therefore,theseresultsformallydemonstratethemyeloidoriginoftheCD8tDCsandcorro-borateourdatashowingthattheearliestsignsofintrathymicCD207expressionoccurredinDN1ccellsandcoincidedwiththeexpressionofmarkersrestrictedtothemyeloidlineage.TheCD8DCsfoundinthethymushavebeenthoughttohavealymphoidorigin[10–12].ConsideringthatCD207isnotexpressedoutsideoftheDClineage[22,38]andthatCD8hightDCscorrespondtoCD207tDCs(thisstudy),weusedCD207(EGFP)expressiontorevisittheoriginoftheCD207CD8hightDCs.ByvisualizingtheonsetofCD207(EGFP)expressioninthethymusofLangerin-EGFPmice,weshowedthattheintrathymicprecursorsoftheCD207CD8hightDCsdonotderivefromETPsbutunexpect-edlyoriginatefromDN1ccells.IncontrasttoETPs,DN1ccellsexpressedmarkersthatarenormallypresentonall(CD11candMHCIImolecules)oronsome(CD207,CD135,CD8,CX3CR1)DCsubsets.DN1cwerefoundtohavelimitedTlineagepotential[14]andtheirdevelopmentalpotentialremainedunclearpriortothepresentstudy.Inaddition,usingLangerin-DTREGFPmice,wedocumentedthatthedevelopmentalseriesleadingtoCD207CD8hightDCsstartswithCD11cCD207CD8MHCIIlowDN1ccellsproceedsthroughtheimmatureCD207CD8MHCIIintstageandendsupwiththeterminallydifferentiatedCD207CD8MHCIItDCs(Fig.8).TocorroboratethemyeloidoriginofCD207weshowedthatMDPs,CDPsandpre-DCsisolatedfromtheBMgaverisetoCD207tDCs.Inaddition,wedemonstratedthatamutationinthegenepreventedthedevelopmentofbothDN1ccellsandCD207tDCswithoutaffectingtheETPsandtheT-celllineage.Altogether,ourdatademonstratethattheCD207tDCshaveamyeloidoriginandarethuscongruentwithfatemappingstudiesvisualizingthehistoryexpressioninthethymus[21]andwithotherobservations[39,40]thatindirectlysuggestedalackofdevelopmentalrela-tionshipbetweentDCsandTcells.ConsistentwithourviewthatDN1ccellsareofmyeloidoriginanddevelopmentallyinsulatedfromtheT-celllineage,conditionalablationoftheNotchligandDelta-like4(Dll4)inthymicepitheliumaffectedDN1a-bcellsbutsparedDN1ccells[41].ThepreviousclaimthatCD8derivefromanintrathymicT-DCbi-potentprecursormightstem Figure7.ThymicandsplenicCD8DCsarisefromMDPs,CDPsandpre-DCs.MDPs,CDPsandpre-DCspuriedfromtheBMofCD45.2micewereadoptivelytransferredintounconditionedCD45.1congenichost.Thymus(A)andspleen(B)wereanalyzedaweekafteradoptivetransfer.CD11ccellswereenrichedusingmagneticbeadscoatedwithanti-CD11cantibodyandCD11cDCswereidentiedonaCD11cversusMHCIIdotplot.CD11cDCsweresubsequentlyanalyzedandseparatedintoCD8andCD8lowfractions.Percentagesofhost-derived(CD45.1anddonor-derived(CD45.2)cellsareshownfortheCD11candCD11cfractions.Resultsarerepresentativeofthreeindependentexperiments.Eur.J.Immunol.2011.:2165–2175HIGHLIGHTS 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu fromthefactthatCD11ccellsthatconstitutetheprecursorsoftheCD8tDCswerepresentamongthesortedCD117DNcellsthatwereusedinpreviousstudies[11].Inconclusion,usingknockinmiceinwhichanEGFPreporterorahumanDTRwasplacedunderthecontrolofthewehaveidentiedtheearliestintrathymicprecursorsofCD207tDCsandshowedthattheycorrespondtomyeloid-typeDN1ccellsandnottoETPs.TheseresultsareconsistentwithrecentfatemappingexperimentsthatshowedthattDCshavenolymphoidpast[42].Altogether,thoseresultssupporttheviewthatmyeloid-restrictedprogenitorsgeneratethewholeconstel-lationofDCspresentinthebodyincludingthethymus. MaterialsandmethodsMicewerehousedunderspecicpathogen-free(SPF)conditionsandusedbetween6and8weeksofage.micehavebeendescribed[22]andwerebackcrossedontoB6CD45.2miceforatleasttengenerations.AsinglecopyofissufcienttorenderCD207DCssusceptibletoDT.SincethealleleprovideshigherlevelsofEGFPuorescencethantheheterozygousmicewereusedtofacil-itatedepletionmonitoring.Hemizygousmice[43]wereusedtoallowtheexpressionofCX3CR1fromthewild-typeallele.BXH2C57BL/6JC3H/Hejmicethatcarryaspontaneousmutation(R924C)inthegene[33]werepurchasedfromTheJacksonLaboratories.AllexperimentsweredoneinaccordancewiththeFrenchandEuropeanguidelinesforanimalcare(DDSVdesBouches-du-RhoInvivodepletionofCD207ForsystemicinvivodepletionofCD207tDCs,micewereinjectedtwiceand15hapartwith1gdiphtheriatoxin(DT)(Calbiochem).MicewereanalyzedforthereappearanceoftDCsatdifferenttimepointsafterthelastDTinjection.CellpreparationDCswereisolatedfromlymphoidorgansaspreviouslydescribed[44].Briey,organswererstcutintosmallpiecesandincubatedfor20mininRPMI-1640mediumwith2%fetalcalfserumcontaining1mg/mLoftypeIIcollagenase(WorthingtonBiochemical)and0.15mg/mLofDNAseI(Sigma-Aldrich).Theresultingcellsuspensionwastreatedwith5mMEDTAfor5minatroomtemperaturetodisruptDC–T-cellconjugatesandlight-densitycellsthatincludeDCswereenrichedbyisopycniccentrifugationonanOptiprepsolution(1.32g/mL,Abcys).Thepelletofhigh-densitycellsthatformsafterOptiprepgradientcentrifugationcontainsimmaturetDCsamongothercells[28].ToenrichforimmaturetDCs,thepelletwasincubatedfor20minatroomtemperaturewithratIgMdirectedagainstmouseCD4(cloneRL172.4),followedbytheadditionofa1:10dilutionofLow-Toxrabbitcomplement(CedarlaneLaboratories).Aftera30-minincubationat37Cwithfrequentgentleagitation,viablecellswererecoveredthroughcentrifugationonaFicollgradient1.077g/mL,Pharmacia).Aspreviouslydemonstrated[15],theIgManti-CD4antibodyusedinourstudydoesnotdepleteDN1andDN2cells.MACSpuriÞcationBeadscoatedwithanti-CD11cantibody(MiltenyBiotec)wereusedtoisolateCD11c-expressingcellsfrombone-marrow,bloodandcollagenase-DNAseItreatedthymus.Asensitivemodeofseparation(Posselds)wasusedontheautoMACSProseparatortoensureahighyieldofbothCD11candCD11cDCs.TopurifyCD11c-expressingcellsfromperipheralblood,bloodsamples Figure8.AmodelofCD207tDCdevelopment.Commonmacrophage-DCprogenitors(MDP),commonDCprecursor(CDP)andclassicalDC-restrictedprecursors(pre-DCs)presentinthebonemarrow(BM)generateCD207tDCs.TheearlieststageoftheintrathymicdevelopmentalseriescorrespondstotheDN1c(CD11c)cellspreviouslyidentiedbyPorrittandcolleagues[14].TheDN1ccellsarefoundintheheavy-densitycellfraction(1.32g/mL)ofanOptiprepgradientandprogressthroughCD207andCD11cCD207intermediatestagestothematureCD207stagethatisfoundinthelight-densitycellfraction(1.32g/mL)ofanOptiprepgradient.Thedevelopmentalblockobservedinmiceexpressingamutationintheinterferon-regulatoryfactor8()geneisshown.Ourndingsdonotformallyruleoutthepossibilitythat,undersomeexperimentalconditions,earlyT-cellprecursorscangiverisetoCD207tDCs.Asrecentlystressed[42],itisimportanttodistinguishphysiologicalfatechoices–asdocumentedinthepresentstudy–fromcellfatesthatarepossibleexperimentally.Eur.J.Immunol.2011.:2165–2175Lucheetal. 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu werecollectedfromadultmicebydirectheartpuncture.ErythrocyteswerelysedwithaNHClsolutionpriortoincubationwithbeadscoatedwithanti-CD11cantibody.FlowcytometryPacicblue-conjugatedanti-CD8(53-6.7,BDBiosciences),anti-CD11b(M1/70,BDBiosciences)andanti-CD24(M1/69,eBioscience);uoresceinisothiocyanate-conjugatedanti-CD172(P84,BDBiosciences);peridinin–chlorophyll–proteincomplexindodicarbocyanine(Cy5.5)-conjugatedanti-CD11b(M1/70,BDBiosciences);phycoerythrin-conjugatedanti-CD4(RM4-5,BDBiosciences),anti-CD117(2B8,BDBiosciences)andanti-CD172(P84,BDBiosciences);phycoerythrin–indodicarbocyanine(Cy5)-conjugatedanti-CD24(M1/69,eBioscience),anti-Sca-1(D7,eBioscience);phycoerythrin–indodicarbocyanine(Cy5.5)-conju-gatedanti-CD11c(N418,eBioscience);phycoerythrin–indotricar-bocyanine(Cy7)-conjugatedanti-CD8(53-6.7,BDBiosciences),anti-CD11c(N418,eBioscience),anti-CD44(IM7,eBioscience);allophycocyanin-conjugatedanti-CD117(2B8,BDBiosciences);Alexa-700anti-MHCII(M5/114,eBioscience);allophycocyanin-H7-conjugatedanti-CD25(PC64,BDBiosciences),anti-CD45R(RA3-6B2,BDBiosciences)andanti-CD161c(PK136,BDBios-ciences);biotin-conjugatedanti-CD135(A2F10,eBioscience),anti-CD45.1(A20,BDBiosciences)andanti-CD45.2(104,eBioscience).Biotin-conjugatedantibodiesweredetectedusingstreptavidinconjugatedwithQuantum-Dot605(Invitrogen).Unlessstated,alineagecocktail(‘Lin’)consistingofAPC-Cy7-conjugatedanti-CD25,anti-CD45Randanti-CD161cwassystem-aticallyusedinallstainingmixestoexcludeCD117DN2cells,pDCsandNKcells,respectively.Detectionofintracellularlangerin(CD207)wasperformedonpermeabilizedcellsusingaBDCytox/Cytopermkit(BDBiosciences).LangerinwasdetectedwithanAlexa647-conjugatedanti-CD207antibody(929F3,Dendritics).Beforestaining,cellswerepre-incubated10minonicewiththe2.4G2antibodytoblockFcreceptors.Inallexperiment,SytoxBlue(Invitrogen)wasusedtoexcludedeadcellsfromtheanalysis.MultiparameterFACSacquisitionwasperformedonaLSRIISORPsystem(BDBiosciences).AnalysiswasperformedusingFACSDiva6.3(BDBiosciences)andFlowJo7(TreeStar)software.Doubletsweresystematicallyexcludedbasedonsidescatter(SSC)andforwardscatter(FSC)parameters.BrdUincorporationMicewereinjectedintraperitoneally(i.p.)with1.5mgBrdU(Sigma-Aldrich)toensureitsimmediateavailability,andtheirdrinkingwaterwassupplementedfortheindicatedtimewith0.8mg/mLofBrdUand2%glucoseandchangeddaily.TwelvedaysaftercontinuousBrdUlabeling,somemicereceivedBrdU-freedrinkingwaterforanadditional2–3weeks.TheamountofBrdUincorporationwasdeterminedusingtheBrdUlabelingFlowkit(BDBiosciences).AdoptivetransferofDCprecursorsForadoptivetransferexperiment,MDPs,CDPsandpre-DCswereisolatedasdescribed[30].Puriedprecursorcells(fromto4)wereinjectedintravenouslyandtheircontributiontosplenicandtDCswasanalyzedaweekafteradoptivetransfer.StatisticalanalysisAllresultsareexpressedasthemeanSEM.Statisticaltestswereperformedusingatwo-tailedStudent’s WethankMarieMalissen,MartinGuilliams,SamiraTamoutounour,MarcDalod,Claudegoire,ShoYamasaki,PhilippeNaquet,LionelPoulin,Anne-MarieSchmitt-VerhulstandLeeLesermanfordiscussionsandDanLittmanfortheCX3CR1-EGFPmice.WethankMarcBarad,PierreGrenotandAtikaZouineforassistancewithcellsorting.ThisworkwassupportedbyCNRS,INSERM,EuropeanCommunitiesFrameworkProgram7(MASTERSWITCHIntegratingProject;HEALTH-F2-2008-223404),INCA(Melan-Immproject),ARCandbypostdoctoralfellowshipfromCNRS(H.L.)andSYBILLACollaborativeProject(H.L.).Conictofinterest:Theauthorsdeclarenonancialorcommercialconictofinterest.Petrie,H.T.andZuniga-Pßucker,J.C.,Zonedout:functionalmappingofstromalsignalingmicroenvironmentsinthethymus.Annu.Rev.Immunol.:649–679.McCaughtry,T.M.,Baldwin,T.A.,Wilken,M.S.andHogquist,K.A.,Clonaldeletionofthymocytescanoccurinthecortexwithnoinvolvementofthemedulla.J.Exp.Med.:2575–2584.Klein,L.,Hinterberger,M.,Wirnsberger,G.andKyewski,B.,presentationinthethymusforpositiveselectionandcentraltoleranceNat.Rev.Immunol.:833–844.Gray,D.,Abramson,J.,Benoist,C.andMathis,D.,ProliferativearrestandrapidturnoverofthymicepithelialcellsexpressingAire.J.Exp.Med.:2521–2528.Koble,C.andKyewski,B.,Thethymicmedulla:auniquemicroenviron-mentforintercellularself-antigentransfer.J.Exp.Med.Gallegos,A.M.andBevan,M.J.,Centraltolerance:goodbutimperfect.Immunol.Rev.:290–296.Klein,L.,Hinterberger,M.,vonRohrscheidt,J.andAichinger,M.,Autonomousversusdendriticcell-dependentcontributionsofEur.J.Immunol.2011.:2165–2175HIGHLIGHTS 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu medullarythymicepithelialcellstocentraltolerance.TrendsImmunol.:188–193.Wu,L.andShortman,K.,Heterogeneityofthymicdendriticcells.Immunol.:304–312.Proietto,A.I.,vanDommelen,S.,Zhou,P.,Rizzitelli,A.,DÕAmico,A.,Steptoe,R.J.,Naik,S.H.etal.,DendriticcellsinthethymuscontributetoT-regulatorycellinduction.Proc.Natl.Acad.Sci.USALi,J.,Park,J.,Foss,D.andGoldschneider,I.,Thymus-homingperipheraldendriticcellsconstitutetwoofthethreemajorsubsetsofdendriticcellsinthesteady-statethymus.J.Exp.Med.:607–622.Ardavin,C.,Wu,L.,Li,C.L.andShortman,K.,ThymicdendriticcellsandTcellsdevelopsimultaneouslyinthethymusfromacommonprecursor:761–763.Corcoran,L.,Ferrero,I.,Vremec,D.,Lucas,K.,Waithman,J.,OÕKeeffe,M.,Wu,L.etal.,Thelymphoidpastofmouseplasmacytoidcellsandthymicdendriticcells.J.Immunol.:4926–4932.Ceredig,R.andRolink,T.,Apositivelookatdouble-negativethymocytes.Nat.Rev.Immunol.:888–897.Porritt,H.E.,Rumfelt,L.L.,Tabrizifard,S.,Schmitt,T.M.,Zuniga-Pßucker,J.C.andPetrie,H.T.,HeterogeneityamongDN1prothymocytesrevealsmultipleprogenitorswithdifferentcapacitiestogenerateTcellandnon-Tcelllineages.Immunity:735–745.Laurent,J.,Bosco,N.,Marche,P.N.andCeredig,R.,Newinsightsintotheproliferationanddifferentiationofearlymousethymocytes.Int.Immunol.:1069–1080.Sambandam,A.,Maillard,I.,Zediak,V.P.,Xu,L.,Gerstein,R.M.,Aster,J.C.,Pear,W.S.andBhandoola,A.,NotchsignalingcontrolsthegenerationanddifferentiationofearlyTlineageprogenitors.Immunol.:663–670.Tan,J.B.,Visan,I.,Yuan,J.S.andGuidos,C.J.,RequirementforNotch1signalsatsequentialearlystagesofintrathymicTcelldevelopment.Immunol.:671–679.Geissmann,F.,Manz,M.G.,Jung,S.,Sieweke,M.H.,Merad,M.andLey,K.,Developmentofmonocytes,macrophages,anddendriticcells.:656–661.Liu,K.,Waskow,C.,Liu,X.,Yao,K.,Hoh,J.andNussenzweig,M.,ofdendriticcellsinperipherallymphoidorgansofmice.Nat.Immunol.:578–583.Manz,M.G.,Traver,D.,Miyamoto,T.,Weissman,I.L.andAkashi,K.,Dendriticcellpotentialsofearlylymphoidandmyeloidprogenitors.:3333–3341.Schlenner,S.M.,Madan,V.,Busch,K.,Tietz,A.,Lauße,C.,Costa,C.,Blum,C.etal.,FatemappingrevealsseparateoriginsofTcellsandmyeloidlineagesinthethymus.ImmunityKissenpfennig,A.,Henri,S.,Dubois,B.,Laplace-Builhe,C.,Perrin,P.,Romani,N.,Tripp,C.H.etal.,DynamicsandfunctionofLangerhanscellsinvivodermaldendriticcellscolonizelymphnodeareasdistinctfromslowermigratingLangerhanscells.ImmunityHenri,S.,Poulin,L.F.,Tamoutounour,S.,Ardouin,L.,Guilliams,M.,deBovis,B.,Devilard,E.etal.,dermaldendriticcellscross-presentkeratinocyte-derivedantigensirrespectiveofthepresenceofLangerhanscells.J.Exp.Med.:189–206,Poulin,L.F.,Henri,S.,deBovis,B.,Devilard,E.,Kissenpfennig,A.andMalissen,B.,ThedermiscontainslangerindendriticcellsthatdevelopandfunctionindependentlyofepidermalLangerhanscells.J.Exp.Med.:3119–3131.Bursch,L.S.,Wang,L.,Igyarto,B.,Kissenpfennig,A.,Malissen,B.,Kaplan,D.H.andHogquist,K.A.,Identicationofanovelpopulationofdendriticcells.J.Exp.Med.:3147–3156.Aguado,E.,Richelme,S.,Nunez-Cruz,S.,Miazek,A.,Mura,A.-M.,Richelme,M.,Guo,X.-J.etal.,InductionofThelpertype2immunitybyapointmutationintheLATadaptor.:2036–2040.Ferrero,I.,Held,W.,Wilson,A.,Tacchini-Cottier,F.,Radtke,F.andMacDonald,H.R.,MouseCD11c()B220()Gr1()plasmacytoiddendri-ticcellsdevelopindependentlyoftheT-celllineage.Naik,S.H.,Metcalf,D.,vanNieuwenhuijze,A.,Wicks,I.,Wu,L.,OÕKeeffe,M.andShortman,K.,Intrasplenicsteady-statedendriticcellprecursorsthataredistinctfrommonocytes.Nat.Immunol.Karsunky,H.,Merad,M.,Cozzio,A.,Weissman,I.L.andManz,M.G.,ligandregulatesdendriticcelldevelopmentfromFlt3lymphoidandmyeloid-committedprogenitorstoFlt3dendriticcellsinvivo.J.Exp.Med.:305–313.Ginhoux,F.,Liu,K.,Helft,J.,Bogunovic,M.,Greter,M.,Hashimoto,D.,Price,J.etal.,TheoriginanddevelopmentofnonlymphoidtissueCD103J.Exp.Med.:3115–3130.Suzuki,S.,Honma,K.,Matsuyama,T.,Suzuki,K.,Toriyama,K.,1Akitoyo,I.,Yamamoto,K.etal.,Criticalrolesofinterferonregulatoryfactor4inCD11bhighCD8alpha-dendriticcelldevelopment.Proc.Natl.Acad.Sci.USA:8981–8986.Tamura,T.,Tailor,P.,Yamaoka,K.,Kong,H.J.,Tsujimura,H.,OÕShea,J.J.,Singh,H.andOzato,K.,IFNregulatoryfactor-4and-8governdendriticcellsubsetdevelopmentandtheirfunctionaldiversity.J.Immunol.:2573–2581.Turcotte,K.,Gauthier,S.,Tuite,A.,Mullick,A.,Malo,D.andGros,P.,mutationintheIcsbp1genecausessusceptibilitytoinfectionandachronicmyeloidleukemia-likesyndromeinBXH-2mice.J.Exp.Med.:881–890.Naik,S.H.,Sathe,P.,Park,H.Y.,Metcalf,D.,Proietto,A.I.,Dakic,A.,Carotta,S.etal.,Developmentofplasmacytoidandconventionaldendriticcellsubtypesfromsingleprecursorcellsderivedinvitroandinvivo.Nat.Immunol.:1217–1226.Onai,N.,Obata-Onai,A.,Schmid,M.A.,Ohteki,T.,Jarrossay,D.andManz,M.G.,IdenticationofclonogeniccommonFlt3plasmacytoidandconventionaldendriticcellprogenitorsinmousebonemarrow.Nat.Immunol.:1207–1216.Liu,K.,Victora,G.D.,Schwickert,T.A.,Guermonprez,P.,Meredith,M.M.,Yao,K.,Chu,F.F.etal.,Invivoanalysisofdendriticcelldevelopmentand:392–397.Atibalentja,D.F.,Murphy,K.M.andUnanue,E.R.,redundancybetweenthymicCD8alphaandSirpalphaconventionaldendriticcellsinpresentationofblood-derivedlysozymebyMHCclassIIproteins.J.Immunol.:1421–1431.Valladeau,J.,Ravel,O.,Dezutter-Dambuyant,C.,Moore,K.,Kleijmeer,M.,Liu,Y.,Duvert-Frances,V.etal.,Langerin,anovelC-typelectinspecictoLangerhanscells,isanendocyticreceptorthatinducestheformationofBirbeckgranules.Immunity:71–81.Rodewald,H.R.,Brocker,T.andHaller,C.,Developmentaldissociationofthymicdendriticcellandthymocytelineagesrevealedingrowthfactorreceptormutantmice.Proc.Natl.Acad.Sci.USAEur.J.Immunol.2011.:2165–2175Lucheetal. 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu Radtke,F.,Ferrero,I.,Wilson,A.,Lees,R.,Aguet,M.andMacDonald,H.R.,Notch1deciencydissociatestheintrathymicdevelopmentofdendriticcellsandTcells.J.Exp.Med.:1085–1094.Hozumi,K.,Mailhos,C.,Negishi,N.,Hirano,K.,Yahata,T.,Ando,K.,Zuklys,S.etal.,Delta-like4isindispensableinthymicenvironmentspecicforTcelldevelopment.J.Exp.Med.:2507–2513.Schlenner,S.M.andRodewald,H.R.,EarlyTcelldevelopmentandthepitfallsofpotential.TrendsImmunol.:303–310.Jung,S.,Aliberti,J.,Graemmel,P.,Sunshine,M.J.,Kreutzberg,G.W.,Sher,A.andLittman,D.R.,AnalysisoffractalkinereceptorCX(3)CR1functionbytargeteddeletionandgreenuorescentproteinreportergeneMol.Cell.Biol.:4106–4114.Vremec,D.,Pooley,J.,Hochrein,H.,Wu,L.andShortman,K.,CD4andCD8expressionbydendriticcellsubtypesinmousethymusandspleen.J.Immunol.:2978–2986.Abbreviations:commonDCprecursorsdoublenegativedoublepositivediphtheriatoxinT-cellprogenitorsinterferon-regulatoryfactor8macrophage-DCprogenitorsmedullaryTECsplasmacytoidDCsorCD8singlepositivedendriticcellsthymicepithelialcellsFullcorrespondence:Prof.BernardMalissen,Centred’ImmunologiedeMarseille-Luminy,UniversitedelaMee,Marseille,FranceFax:e-mail:bernardm@ciml.univ-mrs.frSeeaccompanyingCommentary:http//dx.doi.org/10.1002/eji.201141850Received:5/5/2011Revised:20/5/2011Accepted:26/5/2011Acceptedarticleonline:1/6/2011Eur.J.Immunol.2011.:2165–2175HIGHLIGHTS 2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheimwww.eji-journal.eu