an excellent recipient mouse model for engraftment of human ...

The NOD/SCID/β2mnull mouse was developed, and it was lacking NK cell activity and higher engraftment of human cells.16,17 In the present ... SkiptoMainContent Advertisement Close ASHClinicalNews ASHNewsDaily ASH-SAP Blood BloodAdvances Hematology TheHematologist International BloodChineseEdition BloodJapaneseEdition BloodItalianEdition BloodLatinAmericaEdition BloodSpanishEdition ASH ASHHome Research Education Advocacy Meetings Publications ASHStore Cart UserToolsDropdown Cart SignIn SearchDropdownMenu navsearch searchinput Searchinputautosuggest searchfilter AllContentAllJournalsBlood Search ToggleMenuMenu Issues CurrentIssue AllIssues Firstedition Abstracts 2021AnnualMeeting 2020AnnualMeeting 2020LateBreaking 2019AnnualMeeting 2019LateBreaking AllMeetingAbstracts Collections Collections SpecialCollections Multimedia Alerts AuthorCenter Submit AuthorGuide StyleGuide WhySubmittoBlood? About AboutBlood EditorialBoardandStaff Subscriptions PublicAccess Copyright Alerts BloodClassifieds SkipNavDestination ContentMenu Close Abstract Introduction Materialsandmethods Results Discussion References ArticleNavigation HEMATOPOIESIS| November1,2002 NOD/SCID/γcnullmouse:anexcellentrecipientmousemodelforengraftmentofhumancells MamoruIto, MamoruIto 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar HidefumiHiramatsu, HidefumiHiramatsu 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar KimioKobayashi, KimioKobayashi 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar KazutomoSuzue, KazutomoSuzue 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar MarikoKawahata, MarikoKawahata 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar KyojiHioki, KyojiHioki 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar YoshitoUeyama, YoshitoUeyama 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar YoshioKoyanagi, YoshioKoyanagi 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar KazuoSugamura, KazuoSugamura 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar KohichiroTsuji, KohichiroTsuji 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar ToshioHeike, ToshioHeike 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar TatsutoshiNakahata TatsutoshiNakahata 1FromtheCentralInstituteforExperimentalAnimals,DepartmentofPediatrics,GraduateSchoolofMedicine,KyotoUniversity;DepartmentofParasitology,SchoolofMedicine,GunmaUniversity;DepartmentofPathology,SchoolofMedicine,TokaiUniversity;DepartmentsofVirologyandImmunology,GraduateSchoolofMedicine,TohokuUniversity;DivisionofCellularTherapy,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Japan. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar Blood(2002)100(9):3175–3182. https://doi.org/10.1182/blood-2001-12-0207 Articlehistory Submitted: December14,2001 Accepted: April30,2002 Split-Screen ShareIcon Share Twitter LinkedIn ToolsIcon Tools RequestPermissions CiteIcon Cite SearchSite PDF Citation MamoruIto,HidefumiHiramatsu,KimioKobayashi,KazutomoSuzue,MarikoKawahata,KyojiHioki,YoshitoUeyama,YoshioKoyanagi,KazuoSugamura,KohichiroTsuji,ToshioHeike,TatsutoshiNakahata;NOD/SCID/γcnullmouse:anexcellentrecipientmousemodelforengraftmentofhumancells.Blood2002;100(9):3175–3182.doi:https://doi.org/10.1182/blood-2001-12-0207 Downloadcitationfile: Ris(Zotero) ReferenceManager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbarsearch SearchDropdownMenu navsearch searchinput Searchinputautosuggest searchfilter AllContentAllJournalsBlood Search Abstract Toestablishamoreappropriateanimalrecipientforxenotransplantation,NOD/SCID/γcnullmicedoublehomozygousfortheseverecombinedimmunodeficiency(SCID)mutationandinterleukin-2Rγ(IL-2Rγ)allelicmutation(γcnull⁠)weregeneratedby8backcrossmatingsofC57BL/6J-γcnullmiceandNOD/Shi-scidmice.WhenhumanCD34+cellsfromumbilicalcordbloodweretransplantedintothisstrain,theengraftmentrateintheperipheralcirculation,spleen,andbonemarrowweresignificantlyhigherthanthatinNOD/Shi-scidmicetreatedwithanti-asialoGM1antibodyorintheβ2-microglobulin–deficientNOD/LtSz-scid(NOD/SCID/β2mnull)mice,whichwereascompletelydefectiveinNKcellactivityasNOD/SCID/γcnullmice.Thesamehighengraftmentrateofhumanmaturecellswasobservedinasciteswhenperipheralbloodmononuclearcellswereintraperitoneallytransferred.Inadditiontothehighengraftmentrate,multilineagecelldifferentiationwasalsoobserved.Further,even1 × 102CD34+cellscouldgrowanddifferentiateinthisstrain.TheseresultssuggestthatNOD/SCID/γcnullmiceweresuperioranimalrecipientsforxenotransplantationandwereespeciallyvaluableforhumanstemcellassay.ToelucidatethemechanismsinvolvedinthesuperiorengraftmentrateinNOD/SCID/γcnullmice,cytokineproductionofspleencellsstimulatedwithListeriamonocytogenesantigenswascomparedamongthese3strainsofmice.Theinterferon-γproductionfromdendriticcellsfromtheNOD/SCID/γcnullmousespleenwassignificantlysuppressedincomparisonwithfindingsin2otherstrainsofmice.Itissuggestedthatmultipleimmunologicaldysfunctions,includingcytokineproductioncapability,inadditiontofunctionalincompetenceofT,B,andNKcells,mayleadtothehighengraftmentlevelsofxenograftinNOD/SCID/γcnullmice. Subjects: HematopoiesisandStemCells Topics: mice, mice,knockout, severecombinedimmunodeficiency, engraftment, cd34antigens, spleen, scidmice, antibodies, naturalkillercells Introduction Effortstodevelopanimalrecipientsforxenotransplantation,especiallyhumancells,toestablishananimalmodelforhumandiseasesortoinvestigatemechanismsduringthegrowthanddifferentiationofhumanstemcellshavelongbeenpursued.Theepoch-makingdiscoveryofnudemicebyIsaasonandCattanachin19621—micedefectiveinthethymuswithT-celldeficiency—hascontributedtoprogressinthisresearch.Consequently,C.B-17-Prkdcscid(scid)mice,whicharedefectiveinrearrangementsofT-cellreceptor(TCR)andB-cellreceptor(BCR)resultingindefectsoffunctionalTandBcells,werediscoveredin1983.2Afterthesuccessfulengraftmentofhumanperipheralhematopoieticcells,especiallylymphoidcells,3,4andtheestablishmentofHIV-1infectiontoTcellsdevelopedinthesestrains,5,6manyattemptshavebeenmadetodevelopmodifiedseverecombinedimmunodeficiency(SCID)micebygeneticcrossingswithinbredorothermutantstrainsofmicetoobtainamoreefficientmodel.7-10Asaresult,NOD/LtSz-scidmiceestablishedbyGrenieretal11werefoundtobesuperiorrecipientsforhumancells.ThehighengraftmentratesofhumancellsinNOD/LtSz-scidmicehavebeenattributedtoimmunologicalmultidysfunction,includingreductionsinmacrophagefunction,complement-dependenthemolyticactivity,andNKcellactivity.12WealsodevelopedNOD/Shi-scidmiceindependentlyfromNOD/LtSz-scidmiceanddescribedthehighengraftmentrateofhumanhematopoieticcellsinthem.13,14Atpresent,theNOD/SCIDmicehavebeenconsideredanappropriatemodelforanalysisofhumanstemcelldevelopmentandfunction.However,inNOD/LtSz-scidandNOD/Shi-scidmice,theinjectionofanti-NKcellantibodybeforetransplantationamelioratesengraftmentefficiencyfortransplantedhumancells,thusindicatingthatresidualNKcellactivitymightinterferewithengraftmentefficiency.14,15TogeneticallyeliminateNKcellactivity,additionalimpairmentofthegeneresponsibleforNKcelldevelopmentwasintroducedintoNOD/LtSz-scidmice.TheNOD/SCID/β2mnullmousewasdeveloped,anditwaslackingNKcellactivityandhigherengraftmentofhumancells.16,17Inthepresentstudy,weexaminedthenewlydevelopedNOD/SCID/γcnullmousebybackcrossingtheγcnullmousetotheNOD/Shi-scidmouseandthesuperiorengraftmentcapacityoftransferredhumancells.TheγcnullmousewasfoundtolackNKcellactivity.18WhencomparingtheefficienciesofengraftmentlevelsinNOD/SCID/γcnullmice,NOD/SCID/β2mnullmice,andNOD/Shi-scidmiceinjectedwithanti-NKcellantibody,NOD/SCID/γcnullmicewereconfirmedtobesuperiorinengraftmentofhumanhematopoieticcells.Theimmunologicallydetectedseverereductionofinterferon-γ(IFN-γ)productionfromdendriticcellsintheNOD/SCID/γcnullmousespleenandthecomprehensiveimpairmentofimmunologicalfunctionsmayleadtoefficientengraftmentofhumanhematopoieticcells. Materialsandmethods Mice MiceusedinthisstudywereC.B-17/Jic-scid,NOD/ShiJic-scid,NOD/LtSz-scid,NOD/SCID/β2mnullmice(NOD/LtSz-scidwithdeficientβ2-microglobulin),CB57B/6J-γcnullmice,andnewlydevelopedNOD/SCID/γcnull(NOD/ShiJic-scidwithγcnull⁠)mice.C.B-17/Jic-scidmiceweredonatedbyDrM.Bosma(InstituteforCancerResearch,Philadelphia,PA)in1985andweremaintainedinourinstitute.NOD/ShiJic-scidmousewasestablishedby8ormorebackcrossmatingsoftheC.B-17-scidmousetotheNOD/ShiJicmousethatweredonatedbyDrMakino(ShionogiPharmaceutical)andweremaintainedinourinstitute.NOD/LtSz-scidmiceweredonatedbyDrL.D.Shultz(JacksonLaboratory,BarHarbor,ME).NOD/SCID/β2mnullmicewerepurchasedfromJacksonLaboratory.C57B/6J-γcnullmicewereproducedby8backcrossmatingsofγcnullmicetoC57B/6JJicandweremaintainedinourinstitute.NOD/ShiJic-scidwiththeγcnullmousewasdevelopedasfollows:FemaleNOD/Shi-scidmicewerecrossedwithmaleC57BL/6J-γcnullmiceinwhichtheinterleukin-2Rγ(IL-2Rγ)genewasX-chromosome–linked.F1femaleswerematedwithNOD/Shi-scidmales.Malesobtainedwerebackcrossed7timeswithNOD/Shi-scidmice.Miceobtainedby8backcrossingswereintercrossedtoobtainmicehomologousforthescidandγcnullgenes.Miceweregeneticallytypedforpolymerasechainreaction(PCR)todetectwild-typeandγcnullgenesandimmunodiffusiontodetectserumimmunoglobulinM(IgM)forconfirminghomozygosityforthescidgene.Themiceobtained,homologousforbothgenes,werethenmaintainedbysiblingmating.Allmiceweremaintainedormatedinvinylisolatorsunderspecificpathogen-freeconditions.Allmaterialsincludingbedding,food(CL-2;CleaJapan,Tokyo),andtapwaterwereautoclavedat128°Cfor30minutesinalaminarfirm-cappedcontainerandweremovedtothevinylisolatorsinasterilemanner.Forcelltransplantationexperiments,miceweremovedandmaintainedinmicro-isolatorboxeswithlaminarflowhoodsinconventionalanimalSPFfacilitiesofCIEA,TohokuUniversitySchoolofMedicineandKyotoUniversitySchoolofMedicine. Genotypingofγcnullgene MicewerePCRgenotypedusing2setsofprimerstodistinguishbetweenthewild-typeandmutantalleles,asdescribedpreviously.18DNAwasextractedfrommousebloodfromtheorbitalvein,usingnucleicacidpurificationkits(Genome,MagExtractor,MFX-2000;Toyobo,Osaka,Japan).Toidentifythewild-typeallele,CTGCTCAGAATGCCTCCAATTCCwasusedforthe5′primer,andGATCCAGATTGCCAAGGTGAGTAGwasusedforthe3′primer.Toidentifythemutantallele,CTGCTCAGAATGCCTCCAATTCCwasusedforthe5′primer,andCCTGCGTGCAATCCATCTTGTTCAATwasusedforthe3′primer.BothPCRreactionswerecarriedoutfor35cycles(94°C,1minute;55°C,1minute;72°C,1minute)inareactionbuffercontaining1mMMgCl2,0.25mMdNTP,andTaqpolymerase(LifeTechnologies,GrandIsland,NY).Multipliedfragmentstodetectthemutantandwild-typealleleswereapproximately350bpand660bp,respectively. Genotypingofmicrosatelliteloci Forty-sixmicrosatellitemarkers,onemarkerforeachchromosome,werealsoanalyzedtodeterminegeneticbackgrounds.PCRamplificationofmicrosatellitelociwasperformedusingreportedmethods.19Amplifiedproductswereelectrophoresedona3%to4%agarosegel,andethidiumbromidewasusedtofacilitatevisualization. NKcellactivity NKcellactivitywasdeterminedaccordingtomethodsdescribedbyShultzetal,12Micewereintraperitoneallyinoculatedwith100μgpolyinosinic-polycytidylicacid(polyI:C;SigmaChemical,StLouis,MO)tostimulateNKcellactivityfor48hoursbeforeassay.Spleencellswereseparatedfrom4miceofeachstrainofmice,pooledandcoculturedwithchromium51Cr-labeledYAC-1cellsastargetcellsfor4hoursat37°Cin5%CO2in96semi–V-bottomplates(BioTec,Tokyo,Japan)withvariouseffector-target(E/T)cellratios.Eachsamplewasmadeintriplicate,andthesupernatantsharvestedfromeachwellwereassayedonagammacounter(ARC300;Aloka,Tokyo,Japan).Thepresentspecific51Crreleasewascalculatedusingthefollowingformula,whereXisthemeanexperimentalreleasefromtriplicatewells.Totalrelease(T)wasdeterminedfromwellswith51Cr-labeledYAC-1cellsand1HHCl,andspontaneousrelease(S)wasdeterminedfromwellswith51Cr-labeledYAC-1cellsandmedium:PercentSpecificRelease = [(X − S)/(T − S)] × 100. Complement-dependenthemolyticactivity Complement-dependenthemolyticactivityinserafrommicewasalsoassayed,asdescribedpreviously.12Withthemiceanesthetized,bloodwascollectedfromarightaxillaryvein.Whilebloodwaskeptatroomtemperaturefor1hour,serawerecollectedbycentrifugationat2000rpmfor10minutes.Pooledserafrom4to5miceofeachstrainwerestoredat−80°Cuntilassay.Defibrinatedsheepredbloodcells(SRBCs)(NipponBio-TestLaboratories,Tokyo,Japan)werewashed3timeswithRPMI1640bycentrifugationat1500rpmfor5minutesat4°C.FivemilliliterspackedSRBCswasresuspendedinRPMI1640andlabeledwith200μCi(7.4MBq)51Cr(ICNBiochemicals,Irvine,CA)byshakingfor2hoursat37°Cin5%CO2.LabeledSRBCswereagainwashedwithRPMI1640,resuspendedto3%(vol/vol),andincubatedwithrabbitanti-SRBCpolyclonalantiserum(1/30dilution;ICNPharmaceuticals)for30minutesonice.TheSRBC-antibodyconjugateswerewashedtwiceinRPMI1640,resuspendedinRPMI1640at2%vol/vol,andkeptoniceuntiluse.Pooledserawerethawedoniceandseriallydiluted2-foldinRPMI1640.Onehundredmicrolitersdilutedserawasplacedona96-wellV-bottomplate.Immediately,100μL51CrSRBC-antibodyconjugatesuspensionwasaddedtothewell,andtheplatewasincubatedfor2hoursat37°Cin5%CO2.Afterincubation,thecontentswerecentrifuged,andsupernatantswerecollectedandcountedinagammacounter.ThepercentageofspecificreleasewascalculatedusingtheformuladescribedbyShultzetal.12Spontaneousrelease(S)wasdeterminedfromwellswith51CrSRBC-antibodyconjugateinmedia,andtotalrelease(T)wasdeterminedfromwellswith51CrSRBC-antibodyconjugatesand100μL2%sodiumdodecylsulfate:PercentSpecificRelease = [(X × S)/(T × S)] × 100. IL-1productionfrombonemarrowcells AnassayofIL-1productionfrombonemarrowcellsstimulatedwithIFN-γandlipopolysaccharide(LPS)wasperformedasdescribedpreviously.12Bonemarrowcellscollectedfromfemursofmicewereculturedwith500U/mLhumanrecombinantmacrophage–colony-stimulatingfactor(rM-CSF)(Sigma),withandwithout10U/mLratrIFN-γ(Genzyme,Cambridge,MA),andwereculturedfor4daysat37°Cin5%CO2.After4days,themediumwasreplacedwithfreshmediumaloneorwithmediumcontaining10μg/mLEscherichiacoliLPS(LifeTechnologies,GrandIsland,NY).Afteranadditional24-hourincubationperiod,theculturesupernatantswereharvestedandassayedforIL-1αlevelsusingenzyme-linkedimmunosorbentassay(ELISA)kits(AmershamPharmaciaBiotechUnitedKingdom,Buckinghamshire,England).TheamountofIL-1αinthesupernatantswasexpressedasabsorbanceat405nm. Humancellengraftment Umbilicalcordblood(CB)cellswerecollectedduringnormalfull-termdeliveriesafterobtaininginformedconsent.Mononuclearcells(MNCs)wereseparatedbyFicoll-Hypaquedensity-gradientcentrifugationafterdepletionofphagocyteswithSilica(ImmunoBiologicalLaboratories,Fujioka,Japan).CD34+cellswereisolatedusingDynabeadM-450CD34(DynalAS,Oslo,Norway)asdescribedpreviously.20Briefly,MNCsseparatedfromCBweresuspendedat4 × 107cells/mLinphosphate-bufferedsaline(PBS)containing2%bovineserumalbumin(BSA),0.6%citrate,and100IU/mLpenicillinandstreptomycin.TheMNCsuspensionwasincubatedat4°Cfor30minuteswithDynabeadM-450CD34withabead-cellratioof1:1.Beadswithattachedcellswerecollectedusingamagneticparticleconcentrator(MPC;Dynal)andwereincubatedwithDetach-a-beadCD34(Dynal)at37°Cfor15minutestoreleasethecells,andthesewerecollectedbyMPC.Puritywasevaluatedbyflowcytometricanalysis.Approximately95%ofthecellswereCD34+.TodepleteNKcells,NOD/Shi-scidmicewereintraperitoneallygiven400μLPBScontaining20μLanti-asialoGM1antiserum(Wako,Osaka,Japan)shortlybeforethetransplantationofCBCD34+cellsandevery11thdaythereafter.15Allmicewereirradiatedwith2.4Gyusingacobaltradiationsourceshortlybeforecelltransfer.CD34+cells(1 × 105or4 × 104)wereintravenouslyinoculatedintomice.Aftertransplantation,miceweregivensterilewatercontainingprophylacticneomycinsulfate(GibcoBRL).Humanperipheralbloodmononuclearcells(PBMNCs)werecollectedfromadisease-freedonorusingdensity-gradientcentrifugation.Then1 × 107PBMNCswereintraperitoneallyinoculatedintononirradiatedmicewithandwithouttreatmentusinganti-asialoGM1antibody,asdescribedabove.Twoweeksafterinoculation,thecellsinasciteswererecoveredandanalyzedusingflowcytometry. Flowcytometry Todetecthumancellsinmice,multicolorcytometricanalysiswasperformedusingFACScalibur(BectonDickinson[BD],FranklinLakes,NJ),accordingtothemanufacturer'sprotocolbutwithaminormodification.13Peripheralblood(PB)wastakenfromtheretro-orbitalvenousplexusat4,8,and12weeksforcomparisonoftheengraftmentratebetweenNOD/SCID/γcnullandNOD/Shi-scidmicetreatedwithanti-asialoGM1antibodiesorat4,11,and20weeksbetweenNOD/SCID/γcnullandNOD/SCID/β2mnullmiceafterthetransplantationunderetheranesthesia.Bloodwascollectedthroughheparinizedcalibratedpipettes(DrummondScientific,Broomall,PA)andtransferredtoEDTA(ethylenediaminetetraaceticacid)2NacontainingCapiject(TerumoMedical,Somerset,NJ).AcompletebloodcountwasobtainedusingCelltacα(NihonKohden,Tokyo,Japan).At4or5monthsaftertransplantation,themicewerekilledandthefemursandspleenswereremoved.Bonemarrow(BM)andspleencellswerecollectedandsubjectedtoflowcytometry.SamplesweremixedwithrabbitIgGtoblocknonspecificstainingandwereincubatedwithanappropriatevolumeofindicatedantibodiesfor30minutesonice.ThemixturewasdepletedoferythrocytesandwasfixedinLysingSolution(BDPharMingen,SanDiego,CA).Humanwhitebloodcells(WBCs)wereexaminedbydoublestainingwithfluoresceinisothiocyanate(FITC)–conjugatedantihumanCD45antibody(BDPharMingen)andallophycocyanin(APC)-conjugatedantimouseCD45antibody(BDPharMingen).ThepercentageofhumanCD45+cellswascalculatedasfollows:PercentHumanCD45+Cells = No.HumanCD45+Cells/(No.HumanCD45+Cells+No.MouseCD45+Cells) × 100.TodetectmouseNKanddendriticcellsinthespleen,2-colorcytometricanalysiswasalsoperformedusingaflowcytometer(CytronAbsolute,Ortho-ClinicalDiagnostics,Raritan,NJ).Antibodiesusedwerebiotin-conjugatedantimousepanNK(cloneDX5),biotin-conjugatedantimouseCD11b,andFITC-conjugatedantimouseCD11cfromBDPharMingen. CBCD34+celltransplantationinalimitingdose ToevaluatetheefficiencyofNOD/SCID/γcnullmiceashostsforhumanstemcells,CBCD34+cellsweretransplantedinalimitingdose.TheindicateddosesofCBCD34+cellsweretransplantedtoNOD/SCID/γcnullmice,andengraftmentlevelswereexaminedatmorethan3monthsaftertransplantation.Weusedflowcytometricanalysistodeterminehumanhematopoieticcellengraftment.Aprotocolsimilartothatdescribedabovewasused.Briefly,BMcellswereincubatedwithrabbitIgGandstainedwithantihumanCD45-FITC,antimouseCD45-APC,andViaprobe(BDPharMingen).Successfulengraftmentwasdefinedbythepresenceofatleast100humanCD45+andmouseCD45−cellsin1 × 105Viaprobe-negative(live)cells.Therewerefewnonspecificdotswiththismethod.However,toeliminatepossibleoverestimationcausedbynonspecificstaining,wedidnotcountmicewithfewerthan100positivehumancellsasengrafted.Resultsfrom2independentexperimentsonatotalof14micewereanalyzed. AntigenpreparationofListeriamonocytogenes TheLmonocytogenesEGDstrainwasprovidedbyDrM.Mitsuyama(KyotoUniversity)andwasmaintainedasdescribedpreviously.21Inbrief,bacteriawerepassedtwicethroughC57BL/6Jmicetomaintainthevirulence.Single-colonyisolationwasperformedafterplatingthehomogenateofspleensofinfectedmiceonTripto-soybroth(Eiken,Tokyo,Japan)agarplatesandincubatingovernightat37°C.Suspendedbacteriaweregrownovernightat37°CinliquidTripto-soybroth(Eiken)withvigorousshaking,harvested,andwashed3timeswithPBS.AliquotsofbacterialsuspensioninPBSwereused.Heat-killedbacteriawerepreparedbyheatingat74°Cfor90minutes. InvitrocultureofspleencellswithLmonocytogenesantigen Spleencellswereseparatedfrom4ormoremiceineachgroup,asdescribedpreviously.22Onemilliliterof1mg/mLCollagenaseDsolution(RocheDiagnosticsGmbH,Mannheim,Germany)wasinjectedintothespleenthroughasyringeusinga25-gaugeneedle.Afterincubationfor30minutesat37°C,thespleenwasmincedwithascissors,mixedwellwithaPasteurpipette,andpassedthroughanylonmesh.CD11c+cellsweredepletedfromspleencellsfromNOD/Shi-scidmicetreatedwithanti-asialoGM1antiserumusinganti-CD11cantibody-labeledmagneticbeadsbyamagneticcellsorter(MACS;MiltenyiBiotecGmbH,Gladbach,Germany),accordingtothemanufacturer'sprotocol.Twohundredmicroliterscellsuspension(5 × 106/mL)inRPMIsupplementedwith10%fetalbovineserum,10mMmercaptoethanol,andstreptomycinandpenicillin(LifeTechnologies)wascoculturedwith107heat-killedLmonocytogenesin96-wellplatesfor8hoursat37°C.Afterincubation,thesupernatantswerekeptat−80°CuntilELISA. CytokinedetectionbyELISA IFN-γandIL-6inculturesupernatantsweredeterminedusingOptEIAELISAkits(BDPharMingen).Allassayswereperformedinaccordancewithprotocolsrecommendedbythemanufacturer. Statisticalanalysis TheStudentttestwasusedtodeterminestatisticalsignificance,andP