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Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells

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Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells
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Han, Shuhong
Wang, Yichen
Wang, Bei
Patel, Ekta
Okada, Starlyn
Yang, Li-Jun
Moreb, Jan S.
Chang, Lung-Ji
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Journal of Immune Based Therapies and Vaccines
BioMed Central
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English

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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund.

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ORIGINALRESEARCHOpenAccess Exvivo development,expansionand invivo analysisofanovellineageofdendriticcellsfrom hematopoieticstemcells ShuhongHan 1 ,YichenWang 2 ,BeiWang 1 ,EktaPatel 1 ,StarlynOkada 1 ,Li-JunYang 3 ,JanSMoreb 4 Lung-JiChang 1* Abstract Dendriticcells(DCs)playakeyroleininnateandadaptiveimmunitybuttheaccesstosufficientamountofDCs forbasicandtranslationalresearchhasbeenlimited. Weestablishedanovel exvivo systemtodevelopandexpandDCsfromhematopoieticstem/progenitorcells (HPCs).BothhumanandmouseHPCswereexpandedfirstinfeederculturesupplementedwithc-Kitligand(KL, stemcellfactor,steelfactororCD117ligand),Flt3ligand(fms-liketyrosinekinase3,Flt3L,FL),thrombopoietin (TPO),IL-3,IL-6,andbasicfibroblastgrowthfactor(bFGF),andtheninasecondfeedercultureectopicallyexpressingallabovegrowthfactorsplusGM-CSFandIL-15. Inthedualculturesystem,CD34 + HPCsdifferentiatedtowardDCprogenitors(DCPs),whichexpandedmorethan fiveordersofmagnitude.TheDCPsshowedmyeloidDCsurfacephenotypewithup-regulationoftranscriptionfactorsPU.1andId2,andDC-relatedfactorshomeostaticchemokineligand17(CCL17)andbeta-chemokinereceptor 6(CCR6).MultiplexELISAarrayandcDNAmicroarrayanalysesrevealedthattheDCPssharedsomefeaturesofIL-4 andIL-15DCsbutdisplayedapronouncedproinflammatoryphenotype.DCP-derivedDCsshowedantigen-uptake andimmuneactivationfunctionsanalogoustothatoftheperipheralblood-derivedDCs.Furthermore,bonemarrowHPC-derivedDCPvaccinesoftumor-bearingmicesuppressedtumorgrowth invivo ThisnovelapproachofgeneratingDCP-DCs,whicharedifferentfromknownIL-4andIL-15DCs,overcomesboth quantitativeandqualitativelimitationsinobtainingfunctionalautologousDCsfromasmallnumberofHPCswith greattranslationalpotential. Background Dendriticcells(DCs)initiateprimaryandmemory immuneresponsesaswellasactivateinnateimmunity andtherefore,playapivotalroleinimmunotherapy[1]. Accountingforonly0.02-0.2%ofthetotalwhiteblood cells,thenumberofDCsthatcanbeisolatedfromperipheralbloodislimited[2].WhenculturedwithsupplementofGM-CSFandIL-4,PBMCsorCD14-selected monocytesgenerateDCsatabout50%ofthestarting cellnumber.Furthermore,patientswithcanceror chronicinfectionsoftensufferfromacompromised immunesystemwithincreasedmyeloidsuppressorcells anddysfunctionalDCs[3-9]. Thedevelopmentaloriginandtissuedistributionof variouslineagesofhumanversusmouseDCsarestill notwelldefined[10-15].Transgenicmousestudieshave reportedseveraltranscriptionfactorsimplicatedinregulatingDCdifferentiation,whichincludezincfingerproteinIkaros,PU.1, rel B,thehelix-loop-helix(HLH) transcriptionfactorinhibitorofDNAbindingordifferentiation2(Id2),interferonregulatoryfactor(IRF)4 and8,theEts-domaintranscriptionfactorSpi-B,and theNotchfamilyofproteins[14,16].Inaddition,growth factorssuchasFlt3L,KL,TPO,TNF a ,GM-CSF,IL-3, IL-4,andIL-6havebeenshowntopromotedevelopmentandmaturationofDCs[17-20]. *Correspondence:lchang@mgm.ufl.edu 1 DepartmentofMolecularGeneticsandMicrobiology,UniversityofFlorida, Gainesville,Florida,32610USA Fulllistofauthorinformationisavailableattheendofthearticle Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 2010Hanetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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GrowthfactorssuchasKLandFlt3Lappeartobe strictlyrequiredforthegenerationofDCprogenitors fromHPCsinculture[21].I nthelaboratory,GM-CSF andIL-4areroutinelyusedtogenerateDCsfrom adherentPBMCs,andGM-CSFandTNFa caninduce differentiationofHPCsintointerstitialDCsandLangerhan Â’ scellsin12-14days[22].GM-CSFandIL-15,on theotherhand,driveDCdifferentiationfrommonocytes andbonemarrow(BM)buttheroleofIL-15inmyeloid lineagedevelopmentremainspoorlyunderstood[23,24]. IL-15isamemberofthe g Creceptorfamilyofcytokineswhichisexpressedbyavarietyofcelltypes importanttothesurvivaloffibroblasts,Tcellsandnaturalkillercells.IL-15ha sbeenshowntopromotethe survivalofmatureDCsthroughanautocrineantiapoptoticmechanism[25,26],andIL-15-derivedDCsare reportedtodisplayLangerhanscell-likefeatureswith strongTcellactivationpotential[23,24,27,28]. AlthoughDCscanbederivedfromPBMCs,BMor embryonicstemcells,thesourceandtheamountof theseprogenitorcellsarerestricted.While exvivo DC developmentandexpansionapproacheshavebeen attempted,onlyamoderatenumberofDCscanbegeneratedwiththemostefficientsystemreportingabout94 foldexpansionofDCsfromBMcells[29,30].ThescarcityandthevariabilityofthevariousDCsubsetshave significantlyhinderedfundamentalstudiesofthisimportantlineageofimmunecells.Innovativestrategiesthat canreproduciblygeneratealargeamountoffunctional DCsfromalimitednumberofprogenitor/stemcellsare urgentlyneeded.Herewereportanovel exvivo culture systemthatcombinesexpansionofHPCsanddifferentiationofauniquelineageofDCprogenitors(DCPs). Thissystemsupportsexpansionanddevelopmentof bothhumanandmouseHPCsandDCs.ThetotalnumberofDCsgeneratedunderthissystemreachedmore thanfiveordersofmagnitudein30-40days,andthe ex vivo differentiatedDCsdisplayedantigencapture,Tcell activationandtumorsuppressionfunctionssimilarto thatoftheperipheralbloodandBM-derivedDCs.Thus, alargenumberofautologousHPCsandDCscannow beroutinelygeneratedfromasmallnumberofCD34+HPCsforthestudyofimmunecelldevelopmentwith potentialoftranslationalapplications.MethodsCellsandmiceCD34+cellsusedinthisstudywerepurifiedfromBM, mobilizedperipheralblood(MPB)orcordblood(CB) usingmagneticbeads(MiltenyiBiotec)followingthe manufacturer Â’ sinstructionsorpurchasedfromAllCell Inc.(SanMateo,CA),Cambrex(Baltimore,MD)and NationalDiseaseResearchInterchange(Philadelphia, PA).Buffycoatsofperipher albloodofhealthydonors werepurchasedfromCivitanBloodCenter(Gainesville, FL).PBMCswereisolatedfrombuffycoatsofhealthy donorsorfrombloodofcancerpatientswithapproval oftheInstitutionalReviewBoardofUniversityofFlorida.Blymphoblastoidcelllines(BLCLs)weregenerated bytransformingperipheralbloodBlymphocyteswith EBVasdescribedpreviously[31].TheBLCLswerepropagatedincompleteRPMI-1640medium(Gibco,Grand Island,NY)supplementedwith2mML-glutamine,100 ug/mlstreptomycin,100IU/mlpenicillinand10%heat inactivatedfetalbovineserum(FBS)at37Cwith5% CO2.Themousefetalstromalcellswereculturedin MinimalEssentialMediumAlpha(Gibco,GrandIsland, NY)supplementedwith2mML-glutamine,100ug/ml streptomycin,100IU/mlpenicillinand20%heatinactivatedFBS(Gibco)at37Cwith5%CO2.CT26mouse coloncarcinomacelllinewaspurchasedfromATCC (catalogueno.CRL-2638) andculturedinDulbecco ModifiedEagle Â’ sMedium(Gibco,GrandIsland,NY) supplementedwith2mML-glutamine,100ug/mlstreptomycin,100IU/mlpenicillin and10%heatinactivated FBS(Gibco)at37Cwith5%CO2.BALB/cmicewere obtainedfromJacksonLaboratory(BarHarbor,ME) withapprovalfromtheInstitutionalAnimalCareand UseCommitteeofUniversityofFlorida.AntibodiesandreagentsFluoresceinisothiocyanat e(FITC)-conjugatedAbsto CD4,CD8,CD11c,CD33,CD34,CD38,CD86,IFNg andHLA-DR,phycoerythrin(PE)-conjugatedAbsto CD4,CD8,CD11c,CD34,CD83,CD90,CD123,HLADR,IFNg ,andTNF a ,PerCPCy5.5-conjugatedAbto CD8,CD33,PE-Cy7-conjugatedantibodiestoCD8, CD11b,CD11c,CD34,CD62L,CD40andCD80,and allophycocyanin(APC)-conjugatedAbstoCD1a,CD3, CD11c,CD14,CD33,CD69,CD83,CD90,CD133,IFNg andTNFa werepurchasedfromBDPharmingen (SanDiego,CA),eBioscience(SanDiego,CA),Miltenyi Biotec(Auburn,CA),andCal tagLaboratories(Invitrogen,Carlsbad,CA)aslistedi nAdditionalfile1,Table S1.Isotype-matchedantibodieswereincludedascontrols.HLA-A2restricted ,EBVBMLF1GLC-peptide (aminoacid280-288,GLCTLVAML)pentamerwaspurchasedfromProimmune(Springfield,VA).LentivectorpreparationandgenetransferLentivectors(LVs)wereconstructedasdescribedpreviously[32-34].ThegrowthfactorcDNAswereamplified byPCRusingprimersdesignedtocontainanoptimized translationinitiationsequence(-CCACC-5 Â’ totheinitiationcodon).Theprimersusedinthisstudyarelistedin Additionalfile2,TableS2.TheamplifiedcDNAswere clonedintotheself-inactivatingpTYFplasmidbehindthe EF1 a promoter.Togeneratefeedercells,mousefetalHan etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page2of12

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stromalcellsweremultiplytransducedwithLVsat10-50 infectiousunit/cellin12-wellplatesinaminimalvolume of0.3mlperwell.After2h,0.5mloffreshmediawas addedandcellswereincubatedat37Covernight.The infectedcellswerecontinuouslypropagatedformorethan 50passagesandstablelentiviraltransgeneexpressionwas confirmed.ThemouseCT26tumorcellsandDCswere transducedwithLVsencodingacodon-optimizedhuman HPVE6-E7fusionproteinandthechaperoneproteincalnexinaspreviouslydescribed[35].RNAextraction,RT-PCRandmicroarrayanalysisRNAwasextractedusingTri-reagent(MRCInc.,Cincinnati,OH)andoligo(dT)15-primedcDNAwasmadewith MMLVreversetranscriptase (PromegaInc.,Madison, WI).Forsemi-quantitativePCR,allreactionsusedthe sameseriallydilutedcDNAnormalizedtothemouse GAPDH(mGAPDH).ThePCRamplificationconditions wereasfollows:denaturingtemperature,95C;annealing temperature,55-62C;extensiontemperature,72C;the amplificationcycleswere25-35cycles.Productswere resolvedbyagarosegelelectrophoresisandvisualizedby ethidiumbromidestaining.ThePCRprimersusedinthis studyarelistedinAdditionalfile2,TableS2. Forgeneexpressionmicroarrayanalysis,RNAsamples wereharvestedfrompurifiedCD34+HPCs, exvivo culturedDCPs,andadherentPBMC-derivedIL-4andIL15DCs.TheseRNAsampleswereanalyzedusingIlluminaHumanRefSeq-8ExpressionBeadChips.RNA quantitywasdeterminedwiththeAgilentRNA6000 NanoKitandBioanalyzer. Allsamplesdisplayed28S and5.8SpeaksindicatingintactfulllengthRNA.Synthesisofdouble-strandedcDNAand invitro transcription wereperformedwiththeAmbionIlluminaTotalPrepkit accordingtomanufacturers Â’ instructions.Foreachsample,inputquantityforthefirststrandsynthesiswasnormalizedto200ng.After invitro transcriptionreaction, yieldofpurifiedcRNAwasassessedwiththeRiboGreen assayandqualitywasassessedwiththeAgilentBioanalyzer.BeadChiphybridizat ion,stainingandscanning wereperformedaccordingtoIlluminawholegenome expressionforBeadStation.Foreachsample,inputof cRNAwasnormalizedto1500ng.Ascontrol,StratageneUniversalHumanReference(SUHR)RNAwas labeledwiththeAmbionTotalPrepkit.Thelabeled cRNAwasusedasinterchiphybridizationreplicatesand showedstrongcorrelation.B iologicalreplicatepairs wereanalyzedandforunnormalizeddata,thelinearr2wasgreaterthan0.94forallreplicates.GenerationofmatureDCsandantigen-specificimmune cellsPBMCswereisolatedafterFicoll-Hypaquedensitycentrifugation(SigmaAldrich,StLouis,MO).Afterplastic adherence,theadherentcellswereculturedin50ng/ml GM-CSFand25ng/mlIL-4(eBiosourceInternational, Inc.Camarillo,CA)inserum-freeAIM-Vmedium (Invitrogen,SanDiego,CA)togenerateimmatureDCs. ImmatureDCsweretransducedwithLVs,andtreated withLPS(1ug/ml)andTNF a (20u/ml)for24hrto inducematuration.ThematureDCswereloadedwith5 ug/mlofspecificpeptides.Thenon-adherentPBMCs werecoculturedwithirradiated(10Gy)matureDCs,at a20:1ratio,inAIM-VsupplementedwithIL-2(12.5U/ ml)andIL-7(10ng/ml)in24-wellplates.Atday12of coculture,theTcellswererestimulatedorharvested foranalysisaspreviouslydescribed[36,37].TheDCs ofBALB/cmiceweregeneratedfromBMoftumorbearingmiceandtransducedwithLVsaspreviously described[38].Quantitativecytokineandchemokinemultiplexed enzyme-linkedimmunosorbentassay(ELISA)arraysDCswerewashedtwicewithPBSandculturedinAIMVwithoutgrowthfactorsandothersupplementsata densityof106cells/mlfor24hr.Thesupernatantswere collectedanddeliveredtoQuansysBiosciences(Logan, UT)forcustommultiplexedsampletestingaspreviously described[35].Eachsamplewastestedintriplicate.The listofcytokinesandchemokinestestedincluded:IL-1a, IL-1b,IL-2,IL-4,IL-5,IL-6, IL-8,IL-10,IL-12p70,IL13,IL-17,IL-23,IFNg ,TNFa ,TNFb ,Eotoxin, growth-relatedoncogene-alpha(GROa ),monocytechemotacticprotein1(MCP-1),MCP-2,regulatedupon activation,normalT-cellexpressedandsecretedcytokine(RANTES),I-309andthymusandactivation-regulatedchemokine(TARC).AntibodyandpentamerstainingandflowcytometryForantibody(Ab)staining,single-cellsweresuspended inPBScontaining2%FBSand0.05%sodiumazideand pre-incubatedwithanti-CD16/CD32Abfor10minto blockFcRs.Expressionofcellsurfacemarkerswasanalyzedbystandardfour-colorstainingusingFITC-,PE-, PE-Cy7orPerCPandAPC-conjugatedprimaryAbs.To evaluatetheexpressionofintracellularmolecules,cells werewashedandrestimulatedfor5hrinthepresence ofbrefeldinA(1ug/ml)duringthelast2.5hrofculture.Thestimulatedcellswerestainedwithanti-surface markerAbs,washedandpermeabilizedwiththeCytoFix-Cytopermkit(BDPharmingen),accordingtothe manufacturer Â’ sinstruction,thenstainedwithanti-intracellularmarkerAbs,andanalyzedbyflowcytometry. Formultimerstaining,therestingTcellswerestained withPE-labelledpentamer(Proimmune)for12minat roomtemperature,followedbyFITC-labeledorPECy7labelledanti-CD8antibodyfor30minoniceandanalyzedbyflowcytometry.Dat aacquisitionandanalysisHan etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page3of12

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weredoneonaFACSCaliburandFACSAriausingCellQuestandFACSDivasoftware,respectively(BDBiosciences,SanJose,CA),orFlowjosoftware(TreeStar, Inc.Ashland,OR).AnalysisofantigenuptakeImmatureDCswereharvestedandwashedwithAIM-V twiceandre-suspendedinAIM-Vataconcentrationof 5x105cellsperml.DCswereincubatedwithDextranFITCorOVA-FITC(MolecularProbes,Inc.,Eugene, OR)at37Cfor1h;aparallelcontrolwasincubatedat 4Cfor1h.Cellswerewashedthreetimeswithcold FACSbuffer,resuspendedin100ulofcoldFACSbuffer,stainedwithAPC-conjugatedanti-CD11cAb(BD Biosciences,CA)andanalyzedbyflowcytometry.Invivo DCvaccinetumormodelBALB/cCT26coloncancercellsweretransducedwith LV-optE6E7encodingafusionproteinofHPV16E6/E7 togeneratetheCT26-E6E7 cellline.TheBALB/cmice wereinoculatedwith1x105CT26-E6E7tumorcellssubcutaneously.Sevendayslaterthemicewerevaccinated with2-5x105immatureDC/LV-optE6E7orDC/LVoptE6E7/LV-calnexinderivedfromtumor-bearingmice, weeklyfor2weeks(n=5pergroup).Tumorsizewas measuredovertimeusingcalipersandmeantumor volume(inmm3)wasdetermined.StatisticalanalysisThestatisticalanalysiswasperformedusingStudent Â’ sttestandGRAPHPADPRISM4software.ResultsExpansionofCD34+HPCsanddevelopmentofDC progenitors(DCPs)HPCscanexpandinculturebuthavelimitedpotential ofmaintaininghematopoieticstemcellphenotypeand function[39].Weestablishedaseriesoflentivectormodifiedstromalcell(LSC)linestoprovidecell-free andcell-associatedsignalsthatcansupportcontinuous expansionofHPCsanddifferentiationofDCs(Fig.1). TheLSClinesincludeLSC-KFT(KL,Flt3L,TPO),LSCKFTb(KL,Flt3L,TPOandbFGF),LSC-KFT63(KL, Flt3L,TPO,IL-6,andIL-3)andLSC-KFT63b(KT, Flt3L,TPO,bFGF,IL-6,IL-3andbFGF)fortheexpansionofHPCs,andLSC-KFT-GM15(KL,Flt3L,TPO, GM-CSFandIL-15)andLSC-KFT-mGM15(KL,Flt3L, TPO,mouesGM-CSF,andIL-15)forthedifferentiation andexpansionofhumanandmouseDCs,respectively. LSC-KFT,LSC-KFTb,LSC-KFT63andLSC-KFT63b supportedHPCexpansiontosimilarextents,andthe totalexpansionfoldvaried withindividualdonors. Underthisculturecondition,HPCsconsistently expandedtwentytoonehundred-foldintwentydays, followedbymorethanonethousand-foldexpansionand differentiationintoDCPsinthirtydays(Fig.1A).This dualculturesystemsupportsexpansionanddevelopmentofDCsfrombothhumanandmouseHPCs. Toverifyexpressionofthevariousgrowthfactorsin LSCs,RNAsharvestedfromLSCswereanalyzedby semi-quantitativeRT-PCR(Figure1Band1C).Weconfirmedthatlentivectorexpressionwasstableevenafter 50passagesinthesecelllines(datanotshown).Highly enrichedhumanCD34+HPCsderivedfromadultmobilizedperipheralbloodandBMexpressedhighlevelof hematopoieticprogenitormarkerCD133andlowlevel ofCD33(Figure2A).Thisculturesystemsupported HPCexpansionforbothhealthydonorsandcancer patients;forexample,adultperipheralblood(PB)HPCs expandedinLSC-KFT63btoaboutonehundred-foldin twotothreeweeks(Figure2A).Surfacephenotypeanalysisindicatedthatthe exvivo expandedHPCsgradually lostprogenitormarkers(CD34,CD90,andCD133), whichwasaccompaniedbyincreasedexpressionofmyeloiddifferentiationmarkersCD38andCD33(Figure 2B).SimilarresultshavebeenobtainedwithmouseBM Sca1+Lin-(lineage-minus)HPCs(datanotshown).DifferentiationandexpansionofDCPstowardmeyloid DC-likephenotypeToseeiftheLSCculturesystemcangeneratefunctional DCs,wefirstexpandedCD34+HPCsinLSC-KFT63b. Afteraninitial20-40-foldexpansion,thecellswere transferredtoLSC-KFT-GM15.TheDCPscontinuedto expandseveralordersofmagnitudein30days;they werethentransferredtofeeder-freeculturesupplementedwithGM-CSFandIL-15togeneratefunctional immatureDCsasillustratedinFigure3A. AnalysisofmyeloidandDClineagedifferentiation markersincludingPU.1,Langerin,Id2,hIL7R-a,CCL17, hCCR6,andE-cadherin(E-CAD)oftheDCPsfromday 0,4,9,13,23and39bysemi-quantitativeRT-PCR revealedagradualincrea seinmyeloid(PU.1)andDC differentiationmarkers(Id2,hIL7R-a,CCL17,and hCCR6),andastochasticexp ressionofdifferentiating Langerhanscellmarkers(LangerinorCD207andECAD)ascomparedwithmonocyte-derivedimmature DCs(imDC,Figure3B).After35days,theDCPsdisplayedadifferentiationprofilesimilartothatofmonocyte-derivedimDCs,exceptforE-CAD,whichwas down-regulated.KineticanalysisofmonocyteandDC markersincludingCD14,CD11c,CD1a,CD11b,HLADR,CD83,CD40,CD123,andCD86byflowcytometry showedthatthe exvivo -expandedDCPsgraduallydifferentiatedtowardmatureDCsw ithincreasedexpression kineticsofcostimulator ymoleculesCD40,CD86,and DCmaturationmarkerCD83(Figure3C).ArepresentativeflowcytometryanalysisofDCmarkersofday39Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page4of12

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DCPsisshowninFigure3D;atthistimepoint,DCPs displayedincreasedactivationandmaturationmarkers resemblingconventionalma tureDCs.Similarresults wereobservedwithmouseDCPsexpandedintheLSCKFT-mGM15culture(notshown). Wenextexaminedthegeneexpressionprofileofthe DCPsatdifferenttimepointsafterdifferentiationfrom HPCsusingIlluminaBeadChipHumanRefSeq-8arrays. RNAsampleswereharvestedfromanearlytimepoint (day4)andalatetimepoint(day23),andcomparedto RNAsharvestedfromCD34+HPCsandadherent PBMC-derivedIL-4andIL-15DCs.Clusteranalysisof unnormalizedsampledatashowedthatallbiological replicates(twoDCPs,twoIL-4DCsandthreeCD34+HPCs)sortedintothesamegroups(Figure4).Sample dendogramrevealedthattheday4differentiatedDCPs displayedgeneexpressionprofileresembledCD34+HPCs,whereastheday23differentiatedDCPsdisplayedexpressionprofileresembledIL-4DCs(Figure4).Cytokineandchemokinesecretionprofilesofthe exvivo generatedDCsAsthemorphologyandsurfacemarkerexpressionpatternoftheHPC-DCPsresembledmyeloidDCs,we furtherexaminedtheexpressionprofileofinflammatory cytokinesandchemokines.Forcomparison,weincluded theconventionalIL-4DCsandIL-15DCsgenerated fromadherentPBMCs.HPC-DCPsfromadultBM CD34+HPCswerekeptinfeeder-freeculturesupplementedwithGM-CSFandIL-15togenerateimmature DCsandthenweretreatedwithTNFa andLPSto inducematuration,andafterextensivewashes,thecells wereincubatedinserum-freeAIM-Vmediumatadensityof1x106cellspermlfor24hr.Thesupernatants Figure1 Exvivo HPCtoDCexpansionanddevelopmentsystem (A) SchematicrepresentationoftheLSCculturesystemandthelentivector constructs.ThetwoLSClines,LSC-KFT63bandLSC-KFT-GM15,producethespecifiedhematopoieticgrowthfactors,whichsupporttheexpansion ofHPCsandDCPs. (B) and (C) Semi-quantitativeRT-PCRanalysesoflentiviraltransgeneexpressionintheLSClines.Allofthegrowthfactor genesarehumanoriginexceptforthemouseGM-CSF(mGM-CSF);thecontrolendogenousmouseGAPDH(mGAPDH)geneexpressionis shownatbottom. Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page5of12

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werecollectedandanalyzedusingamultiplexELISA array,whichsimultaneouslymeasuresapanelof23 cytokinesandchemokines[35].Theresultsfromtwo donorsaresummarizedinTable1.Wenotedthat HPC-DCPsdisplayedatrendofupregulationofinflammatorycytokinesandchemokines,withmarkedincrease inIL-1b,IL-6,GROa (CXCL1),I-309(CCL1),MCP-1 (CCL2),andMCP-2(CCL8)comparedwiththetraditionalIL-4DCs,suggestingthattheDCPshavepotent proinflammatoryleukocytechemotacticandactivating functions.Theoverallcytokineandchemokineprofileof DCP-derivedDCsmimickedthoseoftheIL-15DCs exceptthattheDCP-derivedDCsproducedreduced levelsofIFNg andTNF a ,atlevelssimilartothoseof theIL-4DCs,yetwithsubstantiallyincreasedexpression ofGROa andMCP-1.AntigencaptureandTcellactivationfunctionsofthe ex vivo generatedDCsProfessionalantigenpresentingcellscanuptakeand processantigensandstimulateTcells.Toexamine thesefunctions,wecomparedthe exvivo generatedDCs withimmatureadherentPBMC-derivedDCs(PBMCDCs)fortheirantigenuptakefunctionbyfeedingthem withfluorescentDQ-OVA(OVA-FITC)anddextranFITCparticles.ThePBMC-DCscapturedfluorescent particlesat37Cbutnotat4C,asdemonstratedwith flowcytometry.Theday37DCP-DCs,whichcontained alargenumberofCD11c-posi tivecells,capturedantigensasefficientlyasdidthePBMC-DCs(Figure5A). ToseeiftheDCP-derivedDCswerecapableofactivatingantigen-specificTcells,wesetupaDC/Tcellcoculturesystemaspreviouslydescribed[31,35,37].TheDCs weretransducedwithLVsencodingaviralantigen,EBV BMLF(LV-BMLF),oratrunc atedself-antigentNGFR (LV-tNGFR).Aftermaturation,theDCswerecocultured withautologousmonocytedepletedPBMCsfor12days togenerateantigen-specificTcells.TheactivatedTcells wererestimulatedwiththecorrespondingDCsandincubatedwithaBMLFpeptide-specific(GLCTLVAML, HLA-A2*-restricted)MHCpentamertodetectantigenspecificresponse.BoththeDCP-DCsandthePBMCDCsinducedantigen-specificTcellresponsewhentransducedwithLV-BMLF,butnotLV-tNGFR(Figure5B,left panel).IntracellularstainingforIFNg expressioninCD4 andCD8TcellsconfirmedthattheDCP-DCsactivated BMLF-specificTcellsaseffectivelyasdidthePBMCDCs(Figure5B,rightpanel).Incontrast,theself-antigen tNGFRdidnotregisterasubstantialresponse.Invivo tumorsuppressionmediatedbyDCP-DCsderived fromtumor-bearingmiceTheaboveassaysdemonstratedthattheHPC-derived DCsdisplayedantigenpresentationandTcellactivation functionssimilartothatofmonocyte-derivedDCs.To examinetheirtherapeuticpotential,wedesignedan experimentusingaprevious lyestablishedsyngeneic mousetumormodel.BALB/cmiceimplantedwith CT26coloncancercellsexpressingacodon-optimized HumanPapillomaVirus16(HPV16)E6andE7fusion Figure2 ExpansionofCD34+HPCsandphenotypeanalysis (A)Exvivo expansionkineticsofCD34+HPCsinLSCculture.HPCswere purifiedusinganti-CD34AbmagneticbeadsandtheexpansionkineticsonLSC-KFT63bofHPCsfromfourdonorsareplottedatbottom.The purifiedCD34+cellswereanalyzedwithanti-CD34,anti-CD133,andanti-CD33Absbyflowcytometryandrepresentativeflowgraphsof mobilizedPBCD34+HPCsfromthreedonorsareillustrated. (B) Flowcytometryanalysisofhematopoieticprogenitoranddifferentiationmarkers afterHPCexpansioninLSCculturefor9days.TheCD34+HPCsofcordbloodandadultPBwereanalyzedandpresented. Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page6of12

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protein(optE6E7)areprot ectedthroughimmunization withDCstransducedwithLVsencodingoptE6E7and calnexin,achaperoneprotein[31,35].Tomimicsituationinacancerpatient,wederivedDCPsfromBMof tumor-bearingmicetoseeifaprotectiveanti-cancer immuneresponsecanbeinduced.Figure6Aillustrates thestrategyofgeneratingDCPsfromBMoftumorbearingmice.CT26/optE6E7tumorswerefirstestablishedinBALB/cmice,afterconfirmingtumorgrowth, Sca1+Lin-HPCswerepurifiedfromBMofthetumorbearingmice.TheHPCswereexpandedintheLSCculturesystemasdescribed;arepresentativemouseDCP expansioncurveisillustrated(Figure6A).Themouse DCPsexpandedmorethan6ordersofmagnitude within30days.TogenerateDCvaccines,thetumor mouse-derivedDCPsweredifferentiatedfortwodaysin IL-15DCmediumasdescribedinMaterialsandMethodsandtransducedwithLV-optE6E7orLV-optE6E7 plusLV-calnexin.BALB/cmi cebearingCT26/optE6E7 tumorsweredividedintotwogroupsoffivemiceeach group,andreceivedtwoDCvaccinationsataone-week interval.WeobservedthatmiceinjectedwithDCsmodifiedbyLV-optE6E7plusLV-calnexindisplayed increasedsurvivalthanthosemodifiedbyLV-optE6E7 alone(Figure6B),aresultconsistentwithpreviousfindingsusingBM-derivedIL-4DCs[35].DiscussionTheaccesstogoodqualityandsufficientamountoffunctionalDCsiscriticaltothesuccessofimmunotherapy Figure3 Exvivo generationoffunctionalDCs (A) Schematicillustrationofexpansionofhu manandmouseDCPsinculture.Thehuman (CD34+)ormouse(Sca1+/Lin-)HPCswereculturedonLSC-KFT63bfor10days,andthentransferredtoLSC-KFT-GM15(LSC-KFT-mGM15for mousecells)toexpandfor30days.TheresultingDCPswerefurtherculturedinmediumsupplementedwithGM-CSF,IL-15andgrowthfactors toinduceimmatureandmatureDCs.Theaveragefoldofexpansionineachstageisindicated. (B) Kineticanalysisofmyeloidcelldifferentiation markers.TheexpressionkineticsofmolecularmarkersformyeloidcellsincludingPU.1,Langerin,Id2,hIL7Ra ,CCL17,hCCR6,andE-cadherin(ECAD)inthedevelopinghumanDCPswereexaminedbyRT-PCR. (C) ExpressionkineticsofDCsurfacemarkersoftheDCPsbasedonflow cytometryanalysis. (D) Surfacephenotypeofday37DCPsfromtheLSC-KFT-GM15culture.Thenumberinsideeachoftheflowgraphrepresents percentageofpositivecells. Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page7of12

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againstcancerandinfections.Insuchsettings,itisdesirabletogeneratealargenumberofDCscapableofactivatingantigen-specificeffectorTcellsbutnotT regulatorycells(Tregs).Forexamples,infusionofmyeloidDCsandsystemicadmini strationofIL-2havebeen showntoinduceandexpandCD4+FoxP3+Tregsinmyelomaandrenalcancerpatients[31,40-42].Herewepresentareliableandhighlyreproduciblestrategybasedon expansionofCD34+HPCsaswellasdifferentiationofa uniquelineageoffunctionalDCprogenitors(DCPs) usingstromalcellsengineeredwithlentivectorsencoding multiplegrowthfactors.The exvivo generatedDCs exhibitedcanonicalantigenpresentationfunctions includingantigenuptake,processingandactivationofT cellsand invivo anti-cancereffects. TheCD34+HPCsconstituteaheterogeneouscell populationthatcangeneratevariouslineagesofDCs [10,15,19,43,44].Weadoptedacultureconditionwhich supportsexpansionofCD34+HPCsandDCdifferentiationthroughthecombinationofcell-freeandcell-associatedsignalsincludingthos esupportinghematopoietic stemcellproliferation(KL,FL,TPO,IL-3,IL-6,bFGF), myeloidDCdifferentiation(GM-CSF),aswellasIL-15 whichisknowntopromoteleukocytesurvivaland expansion.Thisunique exvivo cultureconditionsupportsexpansionofanovellineageofDCsthataredifferentfromtheconventionalmyeloidDCs.Itis plausiblethatthecontinuedrenewalofdifferentiating DCPsinsuchasystemwasduetothelackofIL-4and TNFa ,thetwocommongrowthfactorsusedinmany reportedmethodsandknowntoinduceDCmaturation Figure4 Microarraydendogramanalysisof exvivo differentiatedDCPs .GeneexpressionprofilesofCD34+HPCderivedDCPsatearly(day4)andlate(day23)timepointsafter differentiationinLSC-KFT-GM15wereexaminedusingIllumina humanwhole-genomeRefSeq8expressionBeadChipcontaining 24,000genes.Therelatedcelltypesaregroupedinclustersinthe dendogram,includingday4andday23DCP,IL-4DC(asingle donorandamixoffivedonors),IL-15DCandthreeCD34+HPC specimens.StratageneUniversalHumanReferenceRNA(SUHRR) wasincludedforqualitycontrol. Table1AnalysisofcytokinesandchemokinessecretedbymatureDCsCytokines/ChemokinesIL-4DCs(pg/ml)IL-15DCs(pg/ml)DCPs(pg/ml) IL-1 a 135(32)332(1,228) 1,202(239) IL-1 b 338(92)601(2,696) 8,582(2,440) IL-26(5)7(7)6(4) IL-43(3)2(4)2(1) IL-52(1)4(7) 9(6) IL-6652(53)1,664(8,036) 43,607(4,605) IL-8154,440(55,237)155,499(297,615) 303,222(286,234) IL-1068(53)7(114)110(162) IL-12p706(3)16(25) 8(6) IL-136(5)170(148) 10(11) IL-1525(23)2,359(248) 87(81) IL-178(4)12(13) 5(6) IL-2317(30)79(90) 215(36) IFNg <1(<1)2,331(1,905) <1(1) TNF a 149(72)1,439(3,152) 226(70) TNF b <1(2)87(67) 3(6) Eotaxin2(3)3(3)4(2) GROa 8,394(2,158)2,142(15,744)131,310(19,241) I-3091,989(533)15,086(28,758) 18,141(42,805) MCP-1342(571)547(4,971) 184,457(127,001) MCP-211(9)339(2,512) 2,381(46) RANTES1,588(179)2,552(1,875) 1,666(3,085) TARC469,352(337,109)1,783(1,636) 20,739(14,855) Resultsareaveragesoftriplicateanalysesofcytokinesandchemokines(pg/ml/106cells/24hr)secretedbymatureDCsoftwodonors(the2nddonorshownin parenthesis)usingmultiplexELISAarrays.Up-( )anddown-regulations( )aredepictedbyarrows,anddoublearrowsindicateadifferencemorethanten-fold fromtheIL-4DCs.Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page8of12

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andblockproliferationthus limitingtheirexpansion [30].ThisnovellineageofDCsisdifferentfromthe commonlyknownIL-4DCsorIL-15DCs.Multiplex cytokineandchemokinearr ayanalysissuggeststhat DCPsresembleIL-15DCsexceptthatDCPsexpressed lessTNF a andIFNg ,butmuchhigherlevelsof inflammatorycytokines(IL-1a,IL-1b,andIL-6)associatedwithhighlevelsofchemotacticfactors(GROa andMCP-1)ascomparedwithIL-15DCs(Table1). Thebeadchipmicroarrayan alysisofgeneexpression profileindicatesthatthe exvivo differentiatedDCPs shareacommonDCprogenitorbutbranchedin Figure5 Functionalanalysesofthe exvivo expandedDCPs (A) Analysisofantigencapturefunctionofthe exvivo derivedDCPs.Antigen capturewasdemonstratedusingdextran-FITCorOVA-FITCparticleinternalizationfollowedbyflowcytometryanalysis.ExamplesofFITC-positive controlPBMC-derivedDCsareshownattop;antigencapturewasdetectedat37Cbutnotat4C. (B) Analysisofantigen-specificTcell stimulationfunction.DCsweretransducedwithLVsencodingacontroltruncatedNGFR(tNGFR)proteinorBMLFproteinofEBV,andincubated withautologousTcells(fromthesameHLA-A*0201donor)for10-12days.TheBMLF-specificA*0201TCRbearingTcellsweredetectedusinga PE-conjugatedMHC-peptidepentamerbyflowcytometry(leftpanel).Antigen-specificeffectorfunctionwasanalyzedbasedonintracellular expressionofIFNg asdescribedinMaterialsandMethods. Figure6 DCPsderivedfromBMHPCsoftumor-bearingmicesuppresstumorgrowth invivo (A)Exvivo expansionofDCPsfromBM HPCsoftumor-bearingBulb/cmice.Balb/cmicewereinjectedwithCT26/optE6E7tumorcells,andafter14days,BMHPCs(Sca1+/Lin-)were harvestedandculturedonLSC-KFTcells.Aftertendays,theexpandedHPCsweretransferredtoLSC-KFT-mGM15togenerateDCPs.A representativemouseDCPexpansiongrowthcurveisshown. (B) SuppressionoftumorgrowthinBalb/cmiceimmunizedwithLV-modified DCPs.CT27/optE6E7tumorcellswerefirstestablishedinBalb/cmice.Themicewereimmunizedwiththe exvivo expandedDCPs,whichwere derivedfromtheBMHPCsofCT26/optE67tumor-bearingBalb/cmice.TwotypesofLV-modifiedDCsweretested:DCstransducedeitherbyLVoptE6E7aloneorbyLV-optE6E7plusLV-calnexin.Thepercentagesofsurvivalofthetwogroupsofmiceareillustrated.Survivalwasbasedon tumorsizesmallerthan1cm3withoutlesions. Han etal JournalofImmuneBasedTherapiesandVaccines 2010, 8 :8 http://www.jibtherapies.com/content/8/1/8 Page9of12

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betweentheperipheralbloodadherentcell-derivedIL-4 DCsandIL-15DCs(Fig.4).Functionalanalysisshows thatDCP-DCsarefullycapableofantigenpresentation andstimulationofantigen-specificTcells.Weconclude thatthe exvivo derivedDCP-DCsrepresentaunique lineageofDCsdisplayingphenotypeandfunction betweenIL-4DCsthathaveprominentadaptive immunefunctions,andIL-15DCsthathaveprominent innateimmunefunctions(manuscriptinpreparation). Furtherdevelopmentalstudymayelucidatethe invivo identityoftheHPC-derivedDCPs. SeveralstudieshavereportedthatinadditiontoKL,FL andTPO,othergrowthfactorsincludingbFGF,bonemorphogeneticprotein4(BMP4) ,IL-3,IL-6orstromalcell derivedfactor-1(SDF-1orCXCL12)canhelpincrease CD34+HPCexpansionandmaintaintheirundifferentiated state[45-47].EpigeneticmodificationusingDNAmethyltransferase(DNMT)inhibitor5-azacytidine(5-aza)and/or histoneacetylaseinhibitortrichostatinA(TSA)canblock differentiationofhematopoieticstemcellsandmoderately promotetheirexpansion[48].Supplementationofthese factorsintheLSCsystem,however,doesnotfurther increaseHPCexpansionpotential(datanotshown). Nevertheless,this exvivo systemoffersaconvenientand reproducibletwo-dimensionalculturesystemforthestudy ofself-renewalanddevelopmentofhumanHSCandDC. Analysisofadditionalregulatoryfactorscanbeeasilyintegratedintothisculturesystem. ThedevelopmentandmaturationofDCsincancer patientsmaybefunctionallydefective,resultingin reducedexpressionofclassIIMHCanddiminished antigencross-primingactiv ity[7,49-51].Inaprevious report,wehaveshownthatIL-4DCsfrommultiple myelomapatientscanbefunctionallyimprovedthrough upregulationofthechaperoneproteincalnexin,which substantiallyincreasesthe secretionofinflammatory cytokinesandchemokinesaccompaniedbyastrong memoryTcellresponse[31,35];DCP-DCs,asillustrated here,mayaccomplishthesamewithoutfurthermodifications.AsIL-15hasbeenshowntoreduceTregactivitiesandincreaseantigen-s pecificCD8Tcellresponse invitro and invivo ,the exvivo generatedDCP-DCs havepotentialofovercomingDCdysfunctionsincancer patients[26,52,53].DCPsfromcancerpatientsincluding multiplemyeloma,acutemyeloidleukemia,acutelymphoblasticleukemia,Hodgkin Â’ slymphomaandglioblastomapatientshavebeensuccessfullygeneratedfroma smallnumberofBMCD34+HPCsoverseveralorders ofmagnitude(>106)(unpublished).Thus,this exvivo approachavoidspotentialimmunesuppressivemicroenvironmentofDCdevelopmentinpatients.Further effortsinvalidatedGMPprocessdevelopmentandstandardizationofthefeeder culturesystemareneeded beforeDCP-DCsarereadyforclinicaltrials.ConclusionsThis exvivo DCdevelopmentsystemsupportsarobust expansionofanovelDClineageinculturefromasmall numberofCD34+HPCs,whichprovidesacriticalsolutiontoproblemsoftenencounteredinimmunotherapy.AdditionalmaterialAdditionalfile1:TableS1 .Antibodiesandtheirspecificclones: AntibodieswerepurchasedfromBDPharMingen,InvitrogenCALTAG laboratories,eBiosciencesandCellSignaling. Additionalfile2:TableS2 .PrimersequencesforcDNAcloningandRTPCR. Listofabbreviations BM:bonemarrow;PB:peripheralblood;HPC:hematopoieticprogenitorcell; PBM:peripheralbloodmonocyte;LSC:lentivector-modifiedstromalcell;LV: lentiviralvector;TPO:thrombopoietin;KL:kit-ligand;FL:Flt3-ligand;bFGF: basicfibroblastgrowthfactor;DC:dendriticcell;DCP:DCprogenitor;tNGFR: truncatednervegrowthfactorreceptor;BLCL:Blymphoblastoidcellline; ICCS:intracellularcytokinestaining. Acknowledgements WethankL.Lien,L.Guo,W.ChouandG.Eubanksfortechnicalassistance. ThisstudywassupportedbyYonglingFoundationandNIH.Publicationof thisarticlewasfundedinpartbyUniversityofFloridaOpen-Access PublishingFund. Authordetails1DepartmentofMolecularGeneticsandMicrobiology,UniversityofFlorida, Gainesville,Florida,32610USA.2VectoriteBiomedicaInc.,Taipei,Taiwan.3DepartmentofPathology,ImmunologyandLaboratoryMedicine,University ofFlorida,Gainesville,Florida,32610USA.4DepartmentofMedicine, UniversityofFlorida,Gainesville,Florida,32610USA. Authors Â’ contributions Allauthorshavereadandapprovedthefinalmanuscript,andare accountablefortheintegrityoftheresearchandanalysisofthedata;LJCis responsiblefortheconceptionandexecution;SH,YW,EP,SOandBWare responsiblefordatacollectionananalysis;LJCisresponsibleforinitial draftingofthemanuscriptandallareresponsibleforrevisionsofthe manuscript. Competinginterests YichenWangwasapaidemployeeofVectoriteBiomedicaInc.The UniversityofFloridaholdspatentsonmodifieddendriticcellsandrelated inventions.Dr.Changisentitledtoashareofthesalesroyaltyreceivedby theUniversity.Thetermsofthesearrangementshavebeenreviewedand approvedbytheUniversityinaccordancewithitsconflictofinterest policies. Received:23July2010Accepted:24November2010 Published:24November2010 References1.FarkasA,ConradC,TonelG,BorbenyiZ,KemenyL,DobozyA,NestleFO: Currentstateandperspectivesofdendriticcellvaccinationincancer immunotherapy. 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