Reevaluation of NOD/SCID Mice as NK Cell-Deficient Models

Moreover, the NKG2D and Ly49A levels in NK cells from NOD/SCID mice were higher ... The mAbs APC anti-mouse CD49b (clone DX5) and Annexin V/7-AAD kit were ... BioMedResearchInternational+JournalMenuPDFJournaloverviewForauthorsForreviewersForeditorsTableofContentsSpecialIssuesSubmitBioMedResearchInternational/2021/ArticleArticleSectionsOnthispageAbstractIntroductionMaterialsandMethodsResultsDiscussionConclusionDataAvailabilityConflictsofInterestAcknowledgmentsReferencesCopyrightRelatedarticlesResearchArticle|OpenAccessVolume2021|ArticleID8851986|https://doi.org/10.1155/2021/8851986MiaoMiao,HenryMasengere,GuangYu,FengpingShan,"ReevaluationofNOD/SCIDMiceasNKCell-DeficientModels",BioMedResearchInternational,vol.2021,ArticleID8851986,11pages,2021.https://doi.org/10.1155/2021/8851986ShowcitationReevaluationofNOD/SCIDMiceasNKCell-DeficientModelsMiaoMiao,1,2HenryMasengere,2GuangYu,2andFengpingShan11DepartmentofImmunology,SchoolofBasicMedicalScience,ChinaMedicalUniversity,Shenyang110122,China2DepartmentofImmunology,SchoolofBasicMedicalScience,JinzhouMedicalUniversity,Jinzhou121001,ChinaShowmoreAcademicEditor:ZiqingLiReceived04Sep2020Revised21Jul2021Accepted21Oct2021Published10Nov2021Objective.Naturalkiller(NK)cell-deficientmiceareusefulmodelsinbiomedicalresearch.NOD/SCIDmicehavebeenusedasamodelofthistypeinresearch.However,theactualstatusofNKcellsinNOD/SCIDmiceandCB17/SCIDmiceincomparisonwiththatinBALB/cmicehasnotbeensufficientlyevaluated.Methods.Splenocytesfromnaïveorpoly(I:C)-treatedmicewereisolatedforphenotypingandanalysisofcytotoxicity-relatedmoleculesandinhibitoryreceptors;forcytotoxicityassay,purifiedNKcellswerealsoused.Results.TheproportionofsplenicNKcellsdidnotdiffersignificantlybetweenNOD/SCIDandCB17/SCIDmice.TheperforinlevelsinNKcellsweresimilarbetweenthepoly(I:C)-treatedCB17/SCIDandNOD/SCIDmice,whilethegranzymeBandNKG2A/C/ElevelsinNKcellsfromNOD/SCIDmiceweresignificantlylowerthanthosefromCB17/SCIDmice.Moreover,theNKG2DandLy49AlevelsinNKcellsfromNOD/SCIDmicewerehigherthanthosefromCB17/SCID.ThesplenocytesfromCB17/SCIDmiceshowedhighercytotoxicitythanthosefromNOD/SCIDmice,whilethecytotoxicityofpurifiedNKcellsbasicallydidnotdifferbetweenthetwostrains.Afterinvitrostimulationwithcytokines,thesplenocytesfromCB17/SCIDmiceshowedhigherIFN-γproductionthanthosefromNOD/SCIDmice;however,NKcellsdidnot.Conclusion.TherewasnosignificantdifferenceintheproportionofsplenicNKcellsbetweenCB17/SCIDandNOD/SCIDmice,andthefunctionofNKcellswasonlypartiallycompromisedinNOD/SCIDmice.CautionshouldbetakenwhenconsideringtheuseofNOD/SCIDmiceasanNK-deficientmodel.1.IntroductionAsauniquememberoftheinnateimmunecells,NKcellsplayimportantrolesinvariousimmuneresponses,includingtumorimmunity[1–3],transplantrejection[4],andautoimmunediseases[5–7].ResearchtoobtainabetterunderstandingofthecharacteristicsofNKcellsisunderway.SomeNKcellshavebeenshowntoexertadaptiveimmuneresponseandarenamedmemoryoradaptiveNKcells[8,9].Inthefieldofbiomedicalresearch,NKcell-deficientmicearewidelyusedandeffectivetools[10–14].CB17/SCIDmice,sincefirstbeingreportedbyBosmaetal.[15]in1983,areknowntolackadaptiveimmunefunctionbutexpressnormalNKcells[16–18].NOD/LtSz-scid/scidmice(NOD/SCID)weregeneratedasanewmurinemodelin1995byShultzetal.[19]anddescribedashavingmultipledefectsininnateimmunity,aswellaslackingbothTandBcells.Sincethen,thesemicehavebeenusedasanNK-deficientmodelinmanystudies[20–25].Basedonpublisheddata[26–28]andourunpublishedexperimentalobservations,itappearsthatNKcellsinNOD/SCIDmicemaynotbeasdeficientaspreviouslythought.Afterreviewinganoriginalpaper[19],werealizedthattheproportionofNKcellsfromtheNOD/SCIDsplenocyteswasdetectedusingNK1.1antibody,andNKcellactivitywasassessedusingtotalsplenocytesaseffectorcells.NK1.1isanantigenthatislimitedlyexpressedonlyinsomeinbredmousestrains(e.g.,C57BL/6orC57BL/10);mostotherinbredmousestrains(e.g.,BALB/c)expressNK1.1atalowlevelornotatall[29].AlthoughanotherresearchgroupinvestigatedtheNKcellsintheplacentaofpregnantNOD/SCIDmiceusingDX5asanNKcellmarker[30],acleartechnicaldefectunderminedthecredibilityoftheobtaineddata.Moreover,mostofthedatareportedtodatewereobtainedusingsplenocytesaseffectorcellstoevaluatethecytotoxicityofNKcellsinNOD/SCIDmice.ToclarifythisdiscrepancyandrevealtheactualstatusofNKcellsinNOD/SCIDmice,inthisstudy,wereevaluatedtheNKcellactivityofNOD/SCIDincomparisonwiththatofCB17/SCIDandBALB/cmice.2.MaterialsandMethods2.1.ReagentsThemAbsAPCanti-mouseCD49b(cloneDX5)andAnnexinV/7-AADkitwereobtainedfromBDPharMingen(SanDiego,CA,USA).FITCanti-mouseCD3ε(clone145-2C11),PE/CY7anti-mouseCD19(clone1D3),andPEanti-mouseNK1.1(clonePK136)wereobtainedfromSungeneBiotech(Tianjin,China).PE/Cy7anti-mouseCD69(cloneH1.2F3),PE/Cy7anti-mouseNKp46(clone29A1.4),andFITCanti-mouseNKG2A/C/E(clone20d5)werefromeBioscience(SanDiego,CA,USA).FITCanti-mousegranzymeB(cloneGB11),PEanti-mouseNKG2D(cloneCX5),PEanti-mouseperforin(cloneS16009A),PEanti-mouseIFN-γ(cloneXMG1.2),PEanti-mouseFasL(cloneMFL3),PerCP/Cy5.5anti-mouseTRAIL(cloneN2B2),FITCanti-mouseLy49A(cloneYE1/48.10.6),andbuffersforstaining,fixation,andpermeabilization,aswellasCFSECellDivisionTrackerKit,werefromBioLegend(SanDiego,CA,USA).MouseIFN-γELISAkitwasfromDakeweBiotechCo.,Ltd.(Shenzhen,China).RecombinantmurineIL-2,IL-12,andIL-15werefromPeproTechInc.(Rockhill,NJ,USA).Poly(I:C)wasfromInvivoGen(SanDiego,CA,USA).EasySep™mouseNKcellisolationkitwasfromSTEMCELLTechnologies,Inc.(Cambridge,UK).2.2.MiceSix-toeight-week-oldNOD/SCID,CB17/SCID,BALB/c,andC57BL/6micewereobtainedfromBeijingVitalRiverLaboratoryAnimalTechnologyCo.,Ltd.(Beijing,China)andmaintainedatJinzhouMedicalUniversityinaSpecificPathogenFree-levelLaboratoryAnimalRoom.AllexperimentswithanimalswereperformedinaccordancewiththeGuidefortheCareandUseofLaboratoryAnimalsasapprovedbyChinaNationalInstitutesofHealth.2.3.AnalysisofCellSurfaceMoleculesbyFlowCytometry(FCM)Splenocytesfromeachsampleweresuspendedin100 μlofstainingbuffer.Acocktailofantibodies(combinationoffluorescent-conjugatedantibodiesselectedfromthoselistedinSection2.1)wasaddedtothecellsuspension,andthen,thestainedsampleswereincubatedonicefor20 min.Afterwashingwithphosphate-bufferedsaline(PBS),thestainedcellswereanalyzedbyFCM(BDBiosciencesFACSCantoII,USA).2.4.AnalysisofIntracellularMoleculesbyFCMCells(insomeexperiments,cellswerestainedforsurfacemarkersfirstandthenwashedtwiceinPBS)werefixedandpermeabilizedfor20 minatroomtemperature.AfterwashingtwiceinPBS,cellsweresuspendedinstainingbufferandstainedwithPE-perforin(orPE-IFN-γ)andFITC-granzymeBantibodiesatroomtemperaturefor20 mininthedark.Aftertwomorewashes,thestainedcellswereanalyzedbyFCM.2.5.InVivoStimulationofNKCellswithPoly(I:C)NOD/SCIDandCB17/SCIDmicewereinjectedintraperitoneally(i.p.)withpoly(I:C)atadosageof100 μg/20 gbodyweight[31]at0and40 h.Splenocyteswereisolatedfrommiceat64 h.2.6.InVitroStimulationofNKCellswithCytokinesSeveralcytokinesrelatedtoNKcellfunctionwereselectedfromthosereportedearlier[32,33]forNKcellactivationexperiments.Thecellswerestimulatedwithcytokinesinaccordancewithpublishedprotocols[34–36]withsomemodifications.Briefly,splenocytes()fromnaïveorpoly(I:C)-treatedmicewereculturedwithIL-2(1000 U/ml),IL-12(10 ng/ml),andIL-15(20 ng/ml)for24–48 hinanincubatorat37°Cwitha5%CO2humidifiedatmosphere.Monensin(×1,BioLegend)wasaddedtotheculturemediumforthelast5 hofincubation.2.7.AssessmentofNKCellCytotoxicityYAC-1cellswerelabeledwith5 μMCFSEfor20 minat37°CandthenwashedtwiceinPBS.Miceweretreatedwithpoly(I:C)asmentionedabove(Section2.5).Splenocytesweretakenfromthemiceat64 h.NKcellswerepurifiedfromthesplenocyteswithEasySep™mouseNKcellisolationkit,inaccordancewiththemanufacturer’sinstructions.SplenocytesorpurifiedNKcellswerestimulatedwithcytokinesfor24 hasmentionedabove(Section2.6).Afterstimulation,thesplenocyteswerecoculturedwiththeCFSE-labeledYAC-1cellsateffector-to-targetratios(E:T)of100 : 1,25 : 1,and6.25 : 1,whilethepurifiedNKcellswerecoculturedwiththeCFSE-labeledYAC-1cellsateffector-to-targetratios(E:T)of10 : 1,5 : 1,2.5 : 1,and1.25 : 1.After6 hofincubationat37°C,cellswerecollectedandwashedtwiceinPBSandthenstainedwith7-AADanddetectedbyFCM.2.8.AssayofCytokineProductioninCultureSupernatantbyELISAThesupernatantsfromthecytokine-stimulatedsplenocytes(Section2.6)werecollected,andIFN-γwasanalyzedusingamouseIFN-γELISAkit,inaccordancewiththemanufacturer’sinstructions.Briefly,dilutedsamples(100 μl/well)wereaddedtothemicroplatethathadbeenprecoatedwithanantibodyspecificforIFN-γandincubatedwithabiotinylatedantibodyagainstIFN-γ.Eachsamplewasassayedinduplicate.After90 minofincubationat37°C,themicroplatewaswashedandincubatedwithstreptavidin-HRPconjugateat37°Cfor30 min,followedbywashingandincubationat37°Cfor10–20 mininasubstratesolutiontodetectperoxidaseactivitybeforetheadditionofthestopsolution.Theabsorbancewasmeasuredat450 nm.2.9.StatisticalAnalysisAllstatisticalanalyseswereperformedusingSPSS16.0statisticalsoftwarepackage(SPSS,Inc.,Chicago,IL,USA).Alldatawerefromatleastthreeindependentexperiments,andthepresenteddataareexpressedas.Statisticalanalysiswascarriedoutbyone-wayANOVA.Valuesofandwereconsideredstatisticallysignificant.3.Results3.1.VerificationofNK1.1ExpressioninDifferentMouseStrainsToverifytheexpressionofNK1.1inNOD/SCIDmice,wecomparedtheexpressionofNK1.1andDX5onsplenocytesindifferentmousestrainsasindicated.TheresultsshowedthatNK1.1+DX5+cellswereprominentinC57BL/6mice.Incontrast,almostnoNK1.1+DX5+cellswereobservedinBALB/c,CB17/SCID,andNOD/SCIDmice(Figure1).(a)(b)(a)(b)Figure1 TheexpressionofNK1.1indifferentmousestrains.Thesplenocyteswereisolatedfromnaïvemice,stainedwithfluorescence-conjugatedantibodiestoNK1.1andDX5,andanalyzedbyFCM.(a)RepresentativeFCMhistogramshowsthepercentagesofNK1.1+DX5+cellsgatedonCD3-splenocytes.(b)StatisticalanalysisofthepercentagesofNK1.1+DX5+cells.Resultsarepresentedas.micepergroup.Valuesofwereconsideredstatisticallysignificant.Threeindependentexperimentswereperformed,andrepresentativedataareshown.3.2.AssessmentofT,B,andNKCellsinNaïveMiceT-andB-celldeficiencieswereconfirmedbycomparingthepercentagesofCD3+andCD19+cellsinthesplenocytesfromBALB/cwiththosefromCB17/SCIDandNOD/SCIDmice.ThepercentagesofNKcellsinCB17/SCIDandNOD/SCIDmicewerehigherthanthoseinBALB/cmice,asdeterminedbycomparingDX5+cells,buttherewerenosignificantdifferences()inthesepercentagesbetweenCB17/SCIDandNOD/SCIDmice(Figure2).(a)(b)(a)(b)Figure2 AnalysesofT,B,andNKcellsinsplenocytesfromnaïvemice.Thesplenocyteswerestainedwithfluorescence-conjugatedantibodiestoCD3,CD19,andDX5andanalyzedbyFCM.(a)RepresentativeFCMhistogramsshowthepercentagesofCD3-,CD19-,andDX5-positivecellsgatedonallsplenocytes.(b)StatisticalanalysisofthepercentagesofCD3-,CD19-,andDX5-positivecells.Resultsarepresentedas.micepergroup.Valuesofwereconsideredstatisticallysignificant.Threeindependentexperimentswereperformed,andrepresentativedataareshown.3.3.AnalysesofCytotoxicity-RelatedMoleculesandInhibitoryReceptorsin/onSplenicNKCellsfromNaïveMiceSplenocytesfromnaïveBALB/c,CB17/SCID,andNOD/SCIDmicewerestainedforsurfaceorintracellularmoleculesandanalyzedwithFCMgatingonDX5+splenicNKcells.TheexpressionofCD69,FasL,TRAIL,andNKp46andtheproductionofgranzymeBandperforindidnotshowsignificantdifferencesamongthethreemousestrains,buttheexpressionofNKG2DonthesplenicNKcellsfromCB17/SCIDwassignificantlylowerthanthatfromBALB/candNOD/SCIDmice.TheexpressionofinhibitoryreceptorNKG2A/C/EonthesplenicNKcellsfromCB17/SCIDandBALB/cwassignificantlyhigherthanthatfromNOD/SCIDmice,whiletheexpressionofLy49AonthesplenicNKcellsofNOD/SCIDmicewassignificantlyhigherthanthatofCB17/SCIDandBALB/cmice(Figures3(a)and3(b)).(a)(b)(a)(b)Figure3 Theexpressionofcytotoxicity-relatedmoleculesandinhibitoryreceptorsin/onsplenicNKcellsfromnaïvemice.(a)RepresentativeFCMdatashowingtheCD69,FasL,TRAIL,perforin,granzymeB,NKp46,NKG2D,NKG2A/C/E,andLy49AexpressiongatedonsplenicDX5+NKcells.(b)HistogramshowsthestatisticalanalysisofFCMdataforCD69,FasL,TRAIL,perforin,granzymeB,NKp46,NKG2D,NKG2A/C/E,andLy49Aexpression.Resultsarepresentedas.–8micepergroup.Valuesofwereconsideredstatisticallysignificant.Threeindependentexperimentswereperformed,andrepresentativedataareshown.3.4.AssessmentofKillingCapacityandCytotoxicity-RelatedMoleculesandInhibitoryReceptorsExpressionin/onNKCellsfromPoly(I:C)-TreatedMiceSplenocytesandpurifiedNKcellsfrompoly(I:C)-treatedmicewerestimulatedinvitrowithcytokinesfor24 handthenmixedwithtargetcellsatdifferentE:Tratios(Section2.7).AsshowninFigure4,thecytotoxicityofsplenocytesfromtheCB17/SCIDmicewassignificantlyhigherthanthatfromtheNOD/SCIDmice;thatis,thepercentagesofthetargetcellslysedbytheeffectorcellsfromNOD/SCIDmicewereaboutone-halftotwo-thirdsofthatfromCB17/SCIDmiceateachE:Tratio(Figure4(a)).However,thecytotoxicityofpurifiedNKcellsshowednosignificantinterstraindifferenceinthepercentagesoflysedtargetcellsatalloftheE:Tratiosexceptforthelowestone(Figure4(b)).Intheanalysisofcytotoxicity-relatedmoleculesbygatingonsplenicDX5+NKcells,nosignificantdifferencesintheexpressionofNKp46ortheproductionofperforinwereobservedbetweenthetwomousestrains,whereastheproductionofgranzymeBwassignificantlyhigherinthesplenicNKcellsfromCB17/SCIDthanthatfromNOD/SCIDmice.Inaddition,theexpressionofNKG2DwasmuchhigheronthesplenicNKcellsfromNOD/SCIDthanthatfromCB17/SCIDmice.TheexpressionofinhibitoryreceptorNKG2A/C/EonthesplenicNKcellsfromCB17/SCIDwassignificantlyhigherthanthatfromNOD/SCIDmice,whiletheexpressionofLy49AonthesplenicNKcellsfromNOD/SCIDwassignificantlyhigherthanthatfromCB17/SCIDmice(Figures4(c)and4(d)).(a)(b)(c)(d)(a)(b)(c)(d)Figure4 Theexpressionofcytotoxicity-relatedmolecules,inhibitoryreceptors,andkillingcapacityofNKcellsfrompoly(I:C)-treatedmice.SplenocytesorpurifiedNKcellsfrompoly(I:C)-treatedmicewerestimulatedwithcytokinesandthenmixedwithCFSE-labeledYAC-1cellsatdifferentE:Tratiosasindicated.(a)Thekillingactivityofthesplenocytes.(b)ThekillingactivityofthepurifiedNKcells.(c)FCManalysesofperforin,granzymeB,NKp46,NKG2D,NKG2A/C/E,andLy49AexpressiongatedonsplenicDX5+NKcells.Histogramsshowrepresentativedatafromthreeindependentexperiments.(d)HistogramshowsthestatisticalanalysisofFCMdatafortheexpressionofperforin,granzymeB,NKp46,NKG2D,NKG2A/C/E,andLy49A.Resultsarepresentedas.micepergroup.Valuesofwereconsideredstatisticallysignificant.3.5.AnalysisofIFN-γInVitroSplenocytesfromnaïveorpoly(I:C)-treatedmicewerestimulatedwithcytokines(IL-2,IL-12,IL-15)for48 hbeforetheanalysisofIFN-γincellsbyFCMandinsupernatantbyELISA.AsshowninFigures5(a)and5(b),theproductionofIFN-γandthepercentageofDX5+cellsamongthecytokine-stimulatedsplenocytesfromnaïveCB17/SCIDmicewerehigherthanthosefromnaïveNOD/SCIDmice.TheanalysisofIFN-γintheculturesupernatantwasinlinewiththatinthesplenocytes(Figure5(c));thatis,thelevelfromCB17/SCIDwassignificantlyhigherthanthatfromNOD/SCIDmice.Meanwhile,theexpressionlevelofIFN-γinsplenicNKcells(gatedontheDX5+populationofsplenocytes)showednostatisticallysignificantinterstraindifference.(a)(b)(c)(d)(e)(f)(g)(a)(b)(c)(d)(e)(f)(g)Figure5 AssayofIFN-γincellsandcellculturesupernatantinvitro.Thesplenocytesfromnaïve(a–e)orpoly(I:C)-treated(f,g)micewereculturedwithcytokinesfor48 h.(a)RepresentativeFCMdatashowIFN-γinandtheexpressionofDX5onthesplenocytesfromCB17/SCIDandNOD/SCIDmice.(b)StatisticalanalysisoftheFCMdataforIFN-γandDX5expression.(c)StatisticalanalysisoftheELISAdataforIFN-γintheculturesupernatant.(d)RepresentativeFCMdatashowtheIFN-γexpressiongatedonDX5+populationsofthesplenocytesfromnaïvemice.(e)StatisticalanalysisoftheFCMdatafortheIFN-γexpressiononsplenicDX5+populations.(f)RepresentativeFCMdatashowtheIFN-γexpressiongatedonDX5+populationsofthesplenocytesfrompoly(I:C)-treatedmice.(g)StatisticalanalysisoftheFCMdatafortheIFN-γexpressiononDX5+populationsofthesplenocytesfrompoly(I:C)-treatedmice.FCMhistogramsshowrepresentativedataofthreeindependentexperiments.Resultsarepresentedas.micepergroup.Valuesofwereconsideredstatisticallysignificant.4.DiscussionInthisstudy,weinvestigatedtheproportionandactivityofsplenicNKcellsinBALB/c,CB17/SCID,andNOD/SCIDmiceinordertoreevaluateNOD/SCIDmiceasanNKcell-deficientmodel.CB17/SCIDmiceareaBALB/cstockcongenicforaC57BL/KaIghallotypeanddeficientinadaptiveimmunity[19].NOD/SCIDmiceweredevelopedinordertotransferthescidmutationfromCB-17congenicmicetodiabetes-susceptiblenonobesediabetic(NOD)mice[37].Therefore,atleastinpart,bothCB17/SCIDandNOD/SCIDmicehaveaBALB/cbackground,andNOD/SCIDmiceacquireimmunologicaldefectsfromboththeirCB17/SCIDandNODbackgrounds.AlthoughthelackofexpressionofNK1.1ontheNKcellsinBALB/cmiceiswellknown[29,38,39],consideringthatNOD/SCIDmiceweregeneratedbyShultzetal.[19]andtheyevaluatedNKcellsinNOD/SCIDmicebytargetingNK1.1,weconfirmedthestatusofNKcellsbytargetingDX5andNK1.1inBALB/c,C57BL/6,CB17/SCID,andNOD/SCIDmice.Asweexpected,aclearNK1.1+cellpopulationwasdetectedonlyinC57BL/6mice,whiletheDX5+populationsweredetectedinallofthefourmousestrains(Figure1),indicatingthatthedatafromtheflowcytometryanalysesofNOD/SCIDsplenicNKcellsreportedbyShultzetal.[19]areinadequate.TofurtherascertaintheproportionofNKcellsinNOD/SCIDmiceandensurethatthemicehadno“leaky”SCID[40]inthisassay,weassessedthesplenicNKcellsbyFCMusingDX5asanNKcellmarkerandconfirmedtheexistenceofadaptiveimmunecellsusingBALB/cmiceasacontrol.AlthoughlowerpercentagesofDX5+cellswereobservedinthesplenocytesfromsomeNOD/SCIDmice,thestatisticalanalysisshowednosignificantdifferenceintheproportionofsplenicDX5+cellsbetweentheNOD/SCIDandCB17/SCIDmice.ThesignificantlylowCD3andCD19signalsinthesplenocytesfromNOD/SCIDandCB17/SCIDmiceconfirmedthereliabilityoftheresults(Figure2).Notably,areporthasshownthat32%ofthesplenicpopulationfromCB17/SCIDmicewereNK1.1-positivefortheNKcellscomparedwith20%ofthatfromNOD/SCIDmice[41].Wedonotknowthereasonforthisdiscrepancy,exceptthatthemicethattheyusedwereproducedfromtheirownbreedingcolony,andthemouseanti-mouseNK1.1antibodywasphycoerythrin-conjugatedandobtainedfromSerotecLtd.(Kidlington,UK).ToevaluatethefunctionalactivityofNKcellsinNOD/SCIDmice,wedetectedtheexpressionofcytotoxicity-relatedmoleculeson/inNKcellsbyFCM(Figure3).OurdatashowedthattheexpressionlevelsofCD69,FasL,TRAIL,granzymeB,andperforinon/inthesplenicNKcellsfromnaïvemicewereallsimilaramongthethreemousestrains,exceptthattheNKcellsfromNOD/SCIDmiceandBALB/cmiceevenexpressedahigherlevelofNKG2DthanthosefromCB17/SCIDmice.ThissuggestedthatthecytotoxicityofNKcellsinNOD/SCIDmicecouldbenolessthanthatinCB17/SCIDmice.BasedonthesefindingsandconsideringthattheNKcellcytotoxicitywasassessedbyShultzetal.usingsplenocytesfrompoly(I:C)-treatedmice,weadoptedtheirprotocolandcomparedthecytotoxicitywiththatofpurifiedNKcells.ThecytotoxicityofsplenocytesfromtheCB17/SCIDmicewassignificantlyhigherthanthatfromtheNOD/SCIDmice(Figure4(a)).ThisisinlinewithareportbyShultzetal.[19].However,thecytotoxicityofpurifiedNKcellsshowednosignificantinterstraindifferenceinthepercentagesoflysedtargetcellsatalloftheE:Tratiosexceptforthelowestone(Figure4(b)).Toobtainabetterunderstandingofthisdiscrepancy,wecheckedtheexpressionofcytotoxicity-relatedmoleculeson/inthecytokine-stimulatedNKcellsfromthepoly(I:C)-treatedmice(Figure4(c)).StatisticalanalysisoftheFCMdatashowednosignificantdifferenceintheproductionofperforinamongthetwomousestrains,whilesignificantlyhigherlevelsofgranzymeBwereobservedinNKcellsfromCB17/SCIDmicethanthatfromNOD/SCIDmice.Meanwhile,theexpressionofNKG2DonNKcellswassignificantlyhigherinNOD/SCIDthanthatinCB17/SCIDmice(Figure4(d)).Aspoly(I:C)caninducemacrophagesanddendriticcellstoproducecytokinesthatsubsequentlyactivateNKcells[42,43],andthemacrophagesanddendriticcellsinNOD/SCIDmicearefunctionallylessmaturethanthoseinCB17/SCID[19],wepostulatethatthediscrepancybetweenthecytotoxicitiesoftotalsplenocytesandthepurifiedNKcellscouldbeexplainedbythecontributionofcytotoxicfactorsotherthanNKcellsinsplenocytes.AsforthesimilarityinthecytotoxicityofpurifiedNKcellsfromthetwostrains,multiplefactorsmightbeinvolved.ThemajormechanismofgranzymeBinNKcellkillingisthroughcooperationwithperforin.Therefore,alowlevelofperforincouldlimitthefunctionofhigh-levelgranzymeB.Accordingtopublisheddata,NKG2AandLy49smayactsynergisticallyinregulatingNKcellfunction.MembersoftheLy49familyarecriticalforNKcelleducation,whileNKG2AisrequiredforsucheducationparticularlywhentheLy49familyisabsent.Ly49signalingmayinfluencetheNKcellreceptorrepertoire,whileNKG2AdeficiencymildlyimpairsNKcellactivity[44].Therefore,althoughthereisasignificantlyhigherlevelofgranzymeBandalowerlevelofLy49AexpressioninNKcellsfromCB17/SCIDmice,itispossiblethatthesignificantlyhigherlevelofNKG2DandthelowerlevelofNKG2A/C/EinNOD/SCIDmiceaswellasthesimilarexpressionofkillerreceptorNKp46andtheproductionofperforinmayworktogethertominimizeorbalancethedifferencesoftheabovefunctionalfactorsbetweenthetwomousestrains.However,consideringthecomplexityofthefunctionalmoleculesinNKcellregulation,wecannotruleoutthepossibilitythatotherfactorsmaybeinvolvedinthecytotoxicitythatweobserved.Thus,wecannotdrawdefinitiveconclusionsonthisissuebasedonourcurrentexperimentaldata.WeidentifiedthediscrepancyinthekillingactivityofthepurifiedNKcellsathighervs.lowerE:TratiosandspeculatethatthekillingabilitymaybemoredependentongranzymeBwhentherearefewereffectorcells.Ofcourse,morefactorsmaycontributetotheactivationandfunctionofNKcells,andaninterestingremainingquestionishowthosefunctionalmoleculesareintegratedduringNKcellkilling,whichneedsfurtherinvestigation.ToobtainabetterunderstandingofthefunctionofNKcellsinthetwomousestrains,weevaluatedthemajorcytokineproductionoftheNKcells.Earlystudiesshowedthatpoly(I:C)wasabletostimulateIL-12productionwithsubsequentNKcellactivationandimprovedIFN-γproduction[45],andthatcytokinessecretedbyNKL-IL15cells,includingIFN-γ,couldinducetheexpressionofNKG2DligandsontargetcellsandthusincreasethesusceptibilityofleukemiacellstoNKcell-mediatedcytolysis[23].Therefore,weassessedtheIFN-γproductionoftotalsplenocytesandsplenicNKcellsfromnaïveandpoly(I:C)-treatedmiceforstraincomparison.AsshowninFigure5,whenthetotalsplenocytesfromnaïvemicewerestimulatedwithcytokines(IL-2,IL-12,IL-15),boththeproportionofNKcellsandIFN-γproductionweresignificantlyhigherinthesplenocytesfromCB17/SCIDthanthosefromNOD/SCIDmice;however,whenwefocusedontheNKcellsbygatingontheDX5+population,IFN-γproductionshowednosignificantdifferenceamongthesethreemousestrains.ThesefindingsfurthersuggestthatthefunctionofNKcellsinNOD/SCIDmiceisacomplextraitundermultigeniccontroland,althoughitisnotfullycomparablewiththatinBALB/c,itisonlypartiallycompromised.WenoticedthereporteddatathattheproportionofDX5+NKcellsintheplacentasfromNOD/SCIDmicewassignificantlylowerthanthatfromBALB/cmice,andtheNKcellsstrikinglyincreasedafterthepoly(I:C)treatmentofNOD/SCIDmice[30].SupposingthattheirFCMdataarestillvalidafteracompensationcorrection,basedonourdata,apossibleexplanationforthiscouldbethatthereweremorefunctionalmoleculesinBALB/cmicethatcouldassistintheactivationandmigrationofNKcellsandthusbedetectedinsomespecificlocationatahighlevel.However,investigationofthedistributionofNKcellsinorgansotherthanthespleenwasoutsidetheremitofthiswork,soitisnotpossibletoaddressthismatterinthecurrentstudy.Anothernotablepointisthat,asindicatedinarecentreportbyFlavelletal.[24],inpreviousstudies,anti-asialoGM1antibodywasusedtodepleteNKcellsinvivotocomparativelyevaluatethecytotoxicityofNKcellsinNOD/SCIDvs.CB17/SCIDorothermousestrains.However,theauthorsassertedthatNKcellsarelikelytobethemajor,butnotnecessarilythesole,participatingcytotoxiceffectorcell.Ourfindingsinthecurrentstudy,togetherwiththefunctionallycompromisedmacrophages,complement,andsomeotheradaptiveimmunocomponentsinNOD/SCIDmice,furtherinvalidatethoseevaluationmodels.5.ConclusionThereisnosignificantdifferenceintheproportionofsplenicNKcellsbetweenCB17/SCIDandNOD/SCIDmice,andthefunctionofNKcellsisonlypartiallycompromisedinNOD/SCIDmice.Thus,oneshouldbeverycautiouswhenusingNOD/SCIDmiceasanNKcell-deficientmodel.DataAvailabilityThedatageneratedoranalyzedduringthisstudyareincludedinthisarticle.Rawdatasupportingthefindingsofthisstudyareavailablefromthecorrespondingauthoruponreasonablerequest.ConflictsofInterestTheauthorsdeclarethattherearenoconflictsofinterestregardingthepublicationofthispaper.AcknowledgmentsThisstudywassupportedbytheNationalNaturalScienceFoundationofChina(No.81373162).ReferencesA.V.VladimirJurišić,K.M.Martinović,andG.Konjević,“TheroleofNKcellsincancer,”inCancerImmunology,N.Rezaei,Ed.,pp.133–146,Springer,2020.Viewat:PublisherSite|GoogleScholarM.Abdel-LatifandR.A.Youness,“Whynaturalkillercellsintriplenegativebreastcancer?”WorldJournalofClinicalOncology,vol.11,no.7,pp.464–476,2020.Viewat:PublisherSite|GoogleScholarJ.A.MyersandJ.S.Miller,“ExploringtheNKcellplatformforcancerimmunotherapy,”NatureReviews.ClinicalOncology,vol.18,no.2,pp.85–100,2021.Viewat:PublisherSite|GoogleScholarB.Toth,L.Zhu,H.Karakizlisetal.,“NKcellsubsetsinidiopathicrecurrentmiscarriageandrenaltransplantpatients,”JournalofReproductiveImmunology,vol.138,2020.Viewat:PublisherSite|GoogleScholarS.J.Lin,C.Y.Hsu,M.L.Kuo,P.T.Lee,H.S.Hsiao,andJ.Y.Chen,“Phenotypicandfunctionalcharacterizationofnaturalkillercellsinrheumatoidarthritis-regulationwithinterleukin-15,”ScientificReports,vol.10,no.1,p.5858,2020.Viewat:PublisherSite|GoogleScholarU.C.Kucuksezer,E.AktasCetin,F.Esenetal.,“Theroleofnaturalkillercellsinautoimmunediseases,”FrontiersinImmunology,vol.1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