The E3 ligases Itch and WWP2 regulate autoimmune neuroinflammation by controlling T_H2 to T_H17 cell conversion via interleukin-4-STAT5 axis in mice

Domain: neuroscience
Published: 2026-01-23
Editorial outcome: minor_revision

Abstract
Multiplesclerosis(MS)isaneurodegenerativeautoimmunediseaseprimarilymediatedbyThelper17(T 17)cells.WepreviouslyshowedthatItch/WWP2doubleknockout(DKO)Tcellsproducehighlevelsoftype2cytokines,driving
spontaneousautoinflammation.Here,wereportthatDKOT 2-highcarryingautoantigen-specificTCR(2D2)developatypicalspontaneousexperimentalautoimmuneencephalomyelitis(EAE),withCD4+TcellssimultaneouslyproducingIL-4andGM-CSF,directlycausingneuroinflammation.Unexpectedly,IL-4deletioninDKOT 2-high2D2miceexacerbatesT 17-drivenclassicalEAE,indicatingaT 2toT 17conversion.Furthermore,weshowthattheJAK3/STAT5signalingpathwayiscriticalformaintainingT 2lineagestabilitybymodulatingBlimp1andc-MaftherebysuppressingT 17differentiation.Importantly,wefindthatthisphenomenoncanalsobeobservedin
dupilumab-treatedpatientswithatopicdermatitiswhodeveloppsoriasis.Thus,ourfindingsuncoverthemolecularantagonismandplasticityintheT 2andT 17cellprogramsandidentifypotentialtherapeutictargetsformodulatingT 2andT 17cellresponsesinautoimmunediseases.

Introduction
Uponactivation,naïveCD4+TcellsdifferentiateintovariousThelper(T )cellsubsets,includingT 1,T 2,T 17,andT cells,eachplayingdistinctrolesinresponsesagainstpathogensandtumorsaswellasthedevelopmentofinflammatorydiseases1,2. T 2 cells secrete interleukin-4 (IL-4), IL-5, and IL-13, thereby promoting B cell proliferationandIgEproduction3,whereasT 17cellsproduceIL-17andareimportantfordefendingagainstfungiandbacteriainfection4.IthaslongbeenrecognizedthatT 17cellsareinvolvedinautoinflammation,whileT 2cellsareresponsiblefordrivingallergicdiseasessuchasasthmaandatopicdermatitis.ThepotentialinvolvementofT 2cellsinautoimmunepathogenesisrequiresfurtherinvestigation.

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for the human inflammatory demyelinating disease, multiple sclerosis (MS)5. Extensive evidence supportsacentralroleforautoreactiveCD4+T cellsinEAEpathogenesis,inparticularT 1andT 17cells,whichinfiltrateintothecentralnervoussystem(CNS)6,7.IntheCNS,thepathogenicT 17cellssecrettheinflammatorycytokinesIL-17,IFN-γ,andGM-CSF,leadingtotheactivationandmaturationofCNS-residentmicrogliaandastrocytes,therecruitmentofCNS-invadingmonocytes,andultimatelyneuroinflammationanddemyelination8–10.However,thespecificroleofT 2cellsinEAEremainsunclear.Althoughitwasreportedthatmyelinbasicprotein-specificT 2cellsinduceEAEinimmunodeficientmice11,itisgenerallythoughtthatT 2cellsplayaprotectiveroleinautoimmunediseasesbysuppressingT 1celldifferentiation12,13.Ontheotherhand,T 17cellsarealsoinvolvedinthedevelopmentofallergy14,15. Aspreviouslyreported, the frequency of T 17 cells was higher in the blood of patients with atopic dermatitis16. Moreover, treatment with an anti-IL-4R antibody in allergic mice significantly induced T 17 differentiation in the lungs, indicating a transition from T 2 cells to T 17 cells17. However, the cellular and molecular mechanisms that regulate the T 2 and T 17 cell plasticity remain to be elucidated.

Thesignaltransducerandactivatoroftranscription(STAT)familyproteinsarecytoplasmictranscriptionfactors,whichbecomephosphorylatedandactivatedbymultipleupstreamkinasesuponthecytokinereceptorengagement,andthentranscriptionallydrivethedifferentiationofvariousCD4+ Tcells18,19.IL-4iscrucialforT 2celldifferentiation.IL-4interactswiththetypeIIL-4receptor,consistingofIL-4Rα and common γ chain, and then activates the intracellular kinasesJAK1andJAK3,whichnotonlyinducethephosphorylationofSTAT6butalsoenhancethephosphorylationofSTAT520,21.ThephosphorylatedSTAT6upregulatesGATA-3,themasterregulatorofT 2cells,whichthenenhancesIL-4production,promotingthedevelopmentoftheT 2lineage22. STAT5 also plays a key role in T 2 cell differentiation by regulating IL-4 production23. Moreover,ithasbeenreportedthatSTAT5isalsoinvolvedinT 17celldifferentiation,althoughitsroleiscontroversial24–26.InadditiontoGATA-3andSTATproteins,severalothertranscriptionfactors,includingIRF4,c-Maf,andJunB,playsignificantrolesduringT 2celldifferentiation27–29. WepreviouslyfoundthatdeletionofItchandWWP2(DKO)leadsto spontaneousdifferentiationofT 2cellsandtype2autoinflammation30. In this study, we investigate the roles of T 2 cells and IL-4 in the pathogenesisofneuroinflammation.WefindthatDKO2D2miceexhibitspontaneousatypicalEAEdrivenbyIL-4+andGM-CSF+T 2cells.However,deletionofIL-4inDKO2D2miceexacerbatesneuroinflammationandcausesclassicalT 17-drivenEAE.WefurtherestablishthatIL-4promotesSTAT5phosphorylation,leadingtoBlimp1upregulationandsubsequentsuppressionofT 17celldifferentiation.ThesefindingsuncoverapreviouslyunrecognizedimmunoregulatoryroleforT 2cellsinneuroinflammationandofferinsightsintotargetingtheT 2–T 17axisfortreatingautoimmuneconditions.

Results
We have previously demonstrated that CD4+ T cells lacking the E3 ligases Itch andWWP2 secrete excessive type 2cytokines and cause spontaneous lung inflammation in B6 mice30. However, it remained unknown whether these cells could recognize self-antigen and drive autoimmuneresponses.Toaddressthisissue,wecrossbredtheDKO-B6mice with myelin oligodendrocyte glycoprotein (MOG) autoantigen-specificTCRtransgenic2D2mice,whicharecommonlyusedtostudythepathogenesisofMS11.At16weeksofage,theWwp2−/−Itchf/fCd4-Cre2D2 mice (called “DKO mice” here) exhibited markedly lower body weightcomparedtotheirwild-typecounterparts(Fig.1a).DKOmicealsoshowedhighertitersofserumIgEthanwild-typemice,whileIgG1levelswerecomparablebetweengroups(Fig. 1b). Additionally, DKO mice displayed splenomegaly and enlarged inguinal lymph nodes(Fig.1c).FlowcytometryanalysisofthesplenocytesrevealedalowerfrequencyofCD4+Tcells(Fig.1dandSupplementaryFig.1a)andanincreasedconversionofnaïveTcellsintoeffectorTcellsinDKOmicecomparedtowild-typemice(Fig.1e).SplenicCD4+TcellsfromDKOmiceproducedmoreIL-4thanthosefromwild-typemice,implyinga potentialcontributionofT 2cellstoautoinflammationinDKOmice(Fig.1f).However,GM-CSFproductionshowednosignificantdifferenceinsplenicCD4+Tcells(Fig.1f and Supplementary Fig. 1b).

We subsequently observed that over 80% of the DKO mice exhibitedspontaneousatypicalEAEsymptoms,includingataxia,lossofbalance,splayedlegs,andtailrigidity(Fig.1g,h).HistologicalanalysisrevealedthatthecerebellumoftheDKOmiceexhibitedneuroinflammation,characterizedbymphocyteinfiltrationanddemyelination(Fig.1i).TheinfiltrationofCD45hiCD11b+myeloidcellsandthemicroglia(CD45midCD11b+)wereincreasedintheCNSofDKOmicewithatypicalEAEsymptoms(Fig.1j–l).AlthoughpreviousstudieshavegenerallylinkedatypicalEAEdevelopmentwithneutrophilinfiltrationintheCNS31,32,wefoundthatinfiltratingneutrophillevelsweresimilar between DKO and wild-type mice (Supplementary Fig. 1c). Additionally,theproportionofinfiltratingeosinophilswashigherintheCNSofDKOmicethanwild-typecontrols(Fig.1m),indicatingthatthetype2immunitymightbeinvolvedinthedevelopmentofatypicalEAE.

T 2 cells directly induce type 2 immune responses through the secretionofcytokinessuchasIL-4,IL-5,andIL-13,andindirectlybyfacilitatingantibodyproductionbyBcells.ToelucidatewhetherT 2cellswereinvolvedinthedevelopmentofspontaneousatypicalEAEinDKOmice,weanalyzedtheCD4+TcellsintheCNSofDKOandwild-typemice.WeobservedasignificantelevationinboththeproportionandtotalnumberofCD4+TcellsinDKOmicerelativetotheirwild-typecounterparts(Fig.2a).Moreover,IL-4productionwasmarkedlyelevatedinCD4+TcellsfromDKOmice(Fig.2b),indicatingthatT 2cellshaddirectlypenetratedtheCNS.Surprisingly,theT 2-likeCD4+TcellsinDKOmiceexhibitedenhancedGM-CSFproduction,withanIL-4andGM-CSFco-producingT 2populationdetectedintheCNS(Fig.2b).However,IL-17AproductioninCD4+Tcellswascomparablebetweenthegroups,thoughIFN-γproductionwasincreasedinDKOCD4+Tcells(Fig.2c),possiblyasasecondaryeffectofneuroinflammation.

ToinvestigatewhetherT 2cellsinDKOmiceplayanintrinsicroleinatypicalEAEdevelopment,weemployedthepassiveEAEmodel(Fig.2d).WeisolatednaïveCD4+TcellsfromDKOandwild-typemiceandculturedtheminvitrowithanti-CD3andanti-CD28antibodiesfor48h. Activated CD4+ T cells from DKO mice exhibited significantly higherlevelsofIL-4andGM-CSFproductioninvitro,comparedtothosefromwild-typemice(SupplementaryFig.2a).We then adoptively transferred the stimulated T cells into immunodeficient Rag1−/− mice. Rag1−/−micebearingCD4+TcellsfromDKOmicedevelopedatypicalEAEandexhibitedsignificantlylowerbodyweightcomparedtothecontrolgroupreceivingwild-typeCD4+Tcells(Fig.2e,f).ConsistentwiththeoriginalphenotypesinDKOmice,infiltratingDKOT 2cellsinRag1−/−micesignificantlyincreasedIL-4andGM-CSFproductionaswellasIFN-γproductionintheCNS(Fig.2g,h).ToelucidatethepathologicalroleofGM-CSFinatypicalEAE,weadministeredananti-GM-CSFneutralizingantibodyinapassiveEAEmodel.Althoughanti-GM-CSFtreatmentdidnotaffecttheonsetofEAE,iteffectivelyalleviatedthediseasesymptomscomparedtothecontrolgroupbyday24,indicatingthatGM-CSFexacerbatesatypicalEAEseverity(SupplementaryFig.2b,c).Furthermore,GM-CSFinfluencedtheinfiltrationandactivationofmicrogliaandmyeloidcellsintheCNS(SupplementaryFig.2d,e),buthadnodetectableeffectoneosinophilorneutrophilpopulations(SupplementaryFig.2f,g).Collectively,thesefindingsdemonstrateanintrinsicroleofT 2-likeCD4+TcellsinthedevelopmentofatypicalEAE.

TofurtherconfirmtheroleT 2cellsinthedevelopmentofneuroinflammation, we deleted IL-4 expression in DKO mice (called “TKO mice” here) by crossing DKO mice with Il4 KO mice. Contrary to our expectations, TKO mice showed significantly lower body weightcomparedtoDKO,Il4KO,andwild-typemiceasearlyas8-week-oldandexhibitedhighermortalityrates,withdeathsoccurringasearlyas11weeksofage(Fig.3a,b).BothTKOandDKOmicedevelopedsplenomegaly and lymphadenopathy compared to Il4 KO and wild-type mice (Fig. 3c). Additionally, the frequencies of splenic CD4+ T cells in TKOandDKOmicewerelowerthaninwild-typeandIl4KOmice,butwerecomparablebetweenDKOandTKO mice (Fig. 3d and Supplementary Fig. 3a). In splenic CD4+ T cells, the proportion of effector CD4+ T cells was higher in TKO mice compared with DKO mice,whilenaïveCD4+TcellswerecomparablebetweenDKOandTKO mice(Fig.3e).

WesubsequentlyevaluatedtheclinicalscoresforEAEsymptomsandfoundthatdeletionofIL-4inDKOmiceexacerbatedtheprogressionofEAE,asTKOmiceexhibitedtailparalysisasearlyas6-week-oldandunderwentascendingparalysis,andtheclassicalclinicalscoresofTKOmicewereconsistentlyhigherthanDKOmice(Fig.3f).Histologicalanalyses showed lymphocyte infiltration, demyelination, and mononuclearcuffinginthespinalcordofTKOmice(Fig.3g).Compared with DKO mice, demyelination in the spinal cord was more severe in TKOmice(Fig.3h).IntheCNS,thefrequencyofCD4+TcellswashigherinTKOmicecomparedwithwild-typeandIl4KOmice,thoughnosignificantdifferencewasobservedbetweenDKOandTKO groups(Fig.3i).Additionally,theexpressionoftheT 17cell-associatedmastertranscriptionfactorRORγtwassignificantlyincreasedinTKOCD4+Tcells,suggestingthatIL-4-deficientDKOTcellshadconvertedintoRORγt+T 17-likecells,particularlywithintheCNS(Fig.3j,k).Tovalidatethesefindings,weemployedthepassiveEAEmodel(Fig.2d).After induction, the body weight significantly decreased in mice of TKOgroupcomparedtoDKOgroupbyday26(Fig.3l).TheclinicalscoresrepresentingclassicalEAEwerealsosignificantlyhigherintheTKOgroupthaninDKO,Il4KO,andwild-typegroups,whiletheatypicalEAEscoresweredetectedintheDKOgroup(Fig.3m).Thesedatacollectivelyindicate that IL-4 negatively regulates classical EAE development.

ToelucidatehowIL-4deficiencyinfluencesthepathogenesisofEAE,weexaminedcytokineproductionofCD4+TcellsintheCNSacrossvariousspontaneousEAEmice.WeobservedasignificantreductioninIL-4andGM-CSFproductioninTKOmicecomparedtoDKOmice,withamarkedincreaseinIL-17AproductioninTKOmice(Fig.4a,b).ThesefindingsindicatethatdeficiencyofIL-4promotestheconversionofItch/WWP2-deficientCD4+TcellsfromIL-4+T 2cellsintoIL-17A+T 17cells.Additionally,no significant differences were observed in the frequencies of IFN-γ+CD4+TcellsandIFN-γ+IL-17A+CD4+Tcellsamongwild-type, Il4 KO, DKO, and TKO mice (Fig. 4b), which effectively excludedtheinfluenceofT 1cells.Consequently,lossofIL-4inCD4+TcellsfacilitatestheshiftfromT 2toT 17cells,exacerbatingtheprogressionofneuroinflammationfromatypicaltoclassicalEAE.

To investigate whether the effect of IL-4 on the T 2 to T 17 transitionistissue-specific,wealsoanalyzedcytokineproductioninCD4+Tcellsfromvarioustissues.Consistentwithpreviousdata,splenicCD4+TcellsfromDKOmiceshowedincreasedIL-4productioncomparedtoothergroups,whileGM-CSFandIFN-γproductionweresimilarinthesefourgroups(Fig.4c,d).SplenicCD4+TcellinTKOmiceproducedslightlymoreIL-17AthanthoseinDKOmice(Fig.4c).Given that T 17 cells drive mucosal inflammation, we also analyzed the CD4+Tcellsinthecolon.Over10%ofcolonCD4+TcellsproducedIL-17A;however,therewerenosignificantvariationsinIL-17Aproductionamongthefourgroups(Fig.4e),whichmightbeduetotheimmunemicroenvironment.Additionally,IL-4andGM-CSFproductioninCD4+TcellswereelevatedinDKOmicecomparedwithothermice(Fig.4f).SinceT 2cellsmediateallergicinflammation,weanalyzedcytokineproductioninlungandskin.ThefrequencyofCD4+Tcellswassimilarinthelungsof6-to8-week-oldwild-type,Il4KO,DKO,andTKOmice(SupplementaryFig.3b).DKOmiceshowedenhancedproductionofIL-4and GM-CSF in CD4+ T cells,while IL-17A production remained unchanged(SupplementaryFig.3c,d).Inmiceolderthan12weeks,theproductionofIL-17AwasincreasedinlungTKOCD4+TcellscomparedtoDKOmice(SupplementaryFig.3e,f),aphenomenonweattributetoanindirecteffectoftheinflammatorymilieu inaged mice. Inpost-auricularskin,productionofcytokinesassociatedwithtype2inflammation,suchasIL-4,IL-13,andIL-25,wasincreasedinDKOmicecomparedtothewild-typegroup(SupplementaryFig.3g–i).Additionally,IL-17AproductionwasslightlyincreasedinTKOmicecomparedtoDKOmiceandwascomparabletothewild-typegroup(SupplementaryFig.3j).Collectively,these results indicate that the function of IL-4 in controlling T 2 or T 17 cell polarization is specific to the CNS.

Tofurther confirmthese in-vivofindings,we establishedanin-vitroT 0toT 17-likecellculturesystem.NaïveCD4+Tcellsfromwild-type,DKO,andTKOmicewereculturedwithanti-CD3andanti-CD28antibodiesfor48h,followedbysupplementationwithanti-IFN-γ,IL-6,andTGF-β. Cells were harvested on day 4 (Fig. 5a). An upregulated expressionofGATA3wasobservedinDKOcellscomparedwiththewild-typeandTKOcellsundertheT 0,T 2,andT 0toT 17polarizingconditions(Fig.5b).However,thisphenomenonwasabsentunderT 17conditions(Fig.5b).Furthermore,deficiencyofIL-4rescuedRORγt expression in TKO cells under both T 0 and T 0 to T 17 polarizingconditions(Fig.5c).FurtheranalysisofcytokineproductionshowedthatIL-4productionwassignificantlyhigherinDKOCD4+Tcellgroupthaninwild-typeandTKOunderT 0,T 2,andT 0toT 17conditions(Fig.5d).Conversely,TKO CD4+ T cells produced substantially more IL-17A under both T 17 and T 0 to T 17 conditions(Fig.5d).Given the relevance of pathogenic T 17 (pT 17) cells to neuroinflammation,wealsoinvestigated the differentiation ofCD4+T cells both in pT 17 and T 0 to pT 17 conditions (Supplementary Fig. 3k). In TKO mice, the naïve CD4+ T cells also exhibited enhanced IL-17A production (Supplementary Fig. 3l). Similar findings were also detected in T 2 to T 17 conditions (Supplementary Fig. 3l). Collectively, these findings indicate that IL-4 regulates the bifurcation of naïve CD4+ T cells into T 2 or T 17 under the T 0 to T 17 conditions.

To better understand how IL-4 controls T 17 polarization, we conductedRNAsequencing(RNA-seq)toanalyzethetranscriptomesofnaïveCD4+TcellsandT 17-likeCD4+TcellsculturedinvitrounderT 0toT 17conditionsacrossfourgroups(Fig.5a).InnaïveCD4+Tcells,weobserved110upregulatedand106downregulatedgenesinTKOcellscomparedtoDKOcells(SupplementaryFig.4a).InpolarizedT 17cellsfromtheTKOgroup,therewere1694upregulatedand1393downregulatedgenesrelative to DKO T 17-like cells (Fig. 6a), demonstrating significant transcriptional changes. Further, gene expression profiles of TKO and DKO groups at boththenaïveandT 17-likestagesshowedthatdifferentiallyexpressedgeneswereprimarilyenrichedinpathwaysrelatedtocytokineresponses,T 17celldifferentiation,IL-17signaling,andtheJAK-STATpathway(SupplementaryFig.4b,c).ToelucidatethebiologicalprocessesinfluencedbyIL-4inDKOandTKOCD4+TcellsunderT 0toT 17cultureconditions,weconductedgeneontology(GO)enrichmentanalysis.As expected, the differentially expressed genes were enriched in pathways related to T cell activation and differentiation, inflammatory response, and the cytokinesignalresponses(Fig.6b).

Additionally,genesencodingtranscriptionfactorscriticalforT 2celldifferentiation,suchasGata3andMaf,wereexpressedathigherlevelsinDKOpolarizedCD4+Tcellscomparedtowild-type,Il4KO,andTKOcells(Fig.6c).Conversely,transcriptionfactorsassociatedwithT 17celldifferentiation,suchasRorc,Myc,andTgif1,wereelevatedintheTKOcellgroup(Fig.6c).We further analyzed the cytokine and cytokine receptor genes that showed different expression between TKOandDKOcellgroup(Fig.6d).TheexpressionofIl4andIl4rawasupregulated in DKO cells, while Il17a, Il17f, and Il22 showed higher levelsinTKOcells(Fig.6d).Collectively,thesefindingsindicatethatdeletionofIL-4inT 2cellssubstantiallyreducesT 2signaturegeneexpressionandpromotesT 17signaturegeneexpression.ThetrendofT 2 to T 17 conversion was progressive following IL-4 deletion. Additionally, Il6ra gene expression was significantly higher in both DKOandTKOcellsthaninwild-typeandIl4KOcells(Fig.6d).Furthermore,geneslinkedtopathogenicT 17cells,suchasIl23r,Ifng,andIl1r1,werealsoelevatedintheTKOgroup(Fig.6d).Tovalidatethesefindings, we conducted Gene Set Enrichment Analysis (GSEA) using RNA-seqdatafromTKOandDKOpolarizedT 17-likecellsagainstT -specificgenelists(Fig.6e).ThegenesupregulatedinT 2cellsrelativetoT 17cells(GSE:11924)werenotablyenrichedintheDKOcellgroup.Notably,asimilarshiftfromT 2toT 17-inducedinflammationinatopicdermatitisunder IL-4Rα blockade hasalsobeen reported,as shownintheonsetofpsoriasissubsequenttodupilumabtherapy33–36. Re-analysisofapreviouspublishedhumanpatientdatarevealedthattheexpressionofgenesassociatedwithpathogenicT 17cells,includingIl17a,Il22,Il1β,Il23r,andRorc,waselevatedinskinsamplesfromdupilumab-inducedpsoriasis,whereastheexpressionofIl4andGata3 was increased in atopic dermatitis patients (Supplementary Fig. 4d, e)37.

GOanalysesfurtherindicatedenrichmentoftheTCRsignalingpathway and the MAPK signaling pathway in polarized T 17-like cells (Fig. 6b).We assessed ERK1/2activationusing westernblotanalysis and found that the phosphorylation levels of ERK1/2 were similar across all four groups (Supplementary Fig. 4f). Given our previous findings that Itch and WWP2 cooperate to regulate TCR signaling strengthandinfluenceCD4+Tcelldifferentiation,weexploredwhetherIL-4regulatesT 17celldifferentiationviaTCRsignaling.WeevaluatedtheactivationofTCR-proximalsignalingcomponentsandobservedthatIL-4deficiencydidnotaffectthephosphorylationofZAP70,LAT,andPLCγ1inCD4+Tcellsuponanti-CD3plusanti-CD28stimulation(SupplementaryFig.4g).

MultiplestudieshaveshownthattheJAK/STATsignalingpathwayplaysacriticalroleinTcellpolarization38.STAT5andSTAT6promoteT 2celldifferentiationandSTAT3isthemajorpromotorofT 17celldifferentiation39–42.OurgeneenrichmentanalysisshowedthattheJAK/STATsignalingpathwaywasenrichedinbothnaïveCD4+TcellsandpolarizedT 17-likecellsfrom TKO mice compared with those from DKOmice(Fig.6bandSupplementaryFig.4b,c).WenextexploredwhetherIL-4modulatesSTATactivationinCD4+Tcells.WestimulatedthesplenocyteswithMOG for 48h in vitro and detected the phosphorylationofSTAT3,STAT5,andSTAT6inCD4+Tcellsusingflowcytometry.UnstimulatedDKOCD4+Tcells exhibited higher phosphorylationofSTAT6thaninwild-type,Il4KOandTKOTcells(Fig.6f).However,thephosphorylationlevelofSTAT6inDKOcellswassimilartothatofwild-typecounterpartsuponstimulation(Fig.6f).IL-4deletionsignificantlyreducedSTAT6phosphorylationinIl4KOandTKO CD4+ T cells, consistent with the previous findings that IL-4 is criticalfor STAT6 activation (Fig. 6f)43. We observed that the phosphorylation levels of STAT5 was similar across all four groups in unstimulated CD4+ T cells (Fig. 6g). However, DKO CD4+ T cells exhibitedhigherphosphorylationofSTAT5thanTKOcellsafterstimulation(Fig.6g),indicatingthatIL-4regulatesSTAT5phosphorylation,eitherdirectlyorindirectly.SincephosphorylationofSTAT3istheindicatorofT 17differentiation,wefoundthatphosphorylationofSTAT3washigherinTKOCD4+TcellsthaninIl4KOandDKOcellsafterstimulationwiththeMOG peptide(SupplementaryFig.4h),consistentwiththeabove-mentionedfindingsthattheTKOCD4+TcellspreferentiallydifferentiatedintoT 17-likecells.Overall,thesefindingssuggestthatIL-4regulatestheactivationofSTAT3,STAT5,andSTAT6.

ToinvestigatewhetherphosphorylationofSTATproteinsisthedirectdownstreamregulatorofIL-4thatrestrictsT 17differentiation,wetreatednaïveCD4+TcellsfromDKOandTKO mice with STAT protein inhibitors under T 0 to T 17 conditions (Fig. 5a). Asexpected,treatmentwithspecificSTAT6inhibitorAS1517499blockedIL-4productioninDKOCD4+Tcellsinadose-dependentmanner,whereaspromotedIL-17Aproduction(SupplementaryFig.4i).However,AS1517499treatmentinDKOCD4+TcellssignificantlyincreasedIL-17AproductionrelativetoTKOcells(SupplementaryFig.4i),suggestingthatSTAT6isnotthedirectdownstreamregulatorofIL-4incontrollingT 17celldifferentiation.

BesidespromotingT 2celldifferentiation,STAT5playsacrucialregulatory role in T 17 cell differentiation24. We treated naïve CD4+ TcellswithSTAT5-IN,apotentandhighlyspecificSTAT5βinhibitor44. Increasing concentrations of STAT5-IN treatment led to a dramatic upregulationofIL-17AproductioninbothDKOCD4+TcellsandTKOcellscomparedtothecontrolgroup(Fig.6h).Furthermore,Atlowerconcentrations (1μM), STAT5-IN moderately increased IL-17A productioninbothcellgroups(Fig.6h).Inthe1μMand10μMSTAT5-INtreatedgroups,TKO CD4+ T cells produced more IL-17A than DKO counterpart(Fig.6h).Additionally,highconcentrationsofSTAT5-INtreatmentcounteractedtheIL-4-mediatedinhibitionofIL-17AproductioninbothDKOandTKOcells(Fig.6h).STAT5-INalsocausedasignificant,dose-dependentreductioninIL-4productioninDKOCD4+Tcells(Fig.6h).Takentogether,thesefindingsdemonstratethatIL-4reducesIL-17AproductioninDKOCD4+TcellsunderT 0toT 17cultureconditionsbydirectlyregulatingSTAT5activation,ratherthanSTAT6.WhilethetreatmentwithSTAT5-INdidnotaffectGATA3expression,RORγt expression increased only at high inhibitor concentrationsinbothDKOandTKOCD4+Tcells(SupplementaryFig.4j),indicatingthatthefunctionofSTAT5isRORγt and GATA3 independent.

Cytokine-induced receptor dimerization leads to trans-phosphorylation of JAK kinases and subsequent STAT activation45. WefirstinvestigatedwhetherIL-4deficiencyaffectscytokinereceptor expressiononthecellsurface.SplenicCD4+TcellsfromIl4KOandTKOmicedisplayedreducedIL-4Rα(CD124)expressioncomparedtowild-typecounterparts,whileDKOCD4+TcellsexhibitedhigherexpressionofIL-4Rαthanwild-typecells(Fig.7a),consistentwithpreviousfindingsthatIL-4regulatestheexpressionofIL-4Rα46.ItisgenerallyacceptedthatIL-2isone of the major cytokines inducing STAT5phosphorylationinCD4+Tcells47.WeassessedtheexpressionofIL-2RαsubunitCD25toexamineifIL-4regulatesIL-2signalingandfoundthattherewasnodifferenceamongthesefourgroups(Fig.7b),indicatingIL-4deficiencydoesnotimpactIL-2Rexpression.IL-4initiallybindstoIL-4RαandrecruitsthecommonγchaintoformthetypeIIL-4receptor,initiatingtheactivationofthedownstreamJAK3/STAT5signalingpathway21. Wethenfoundthatcommonγ-chain(CD132)expressionwasslightlyhigherinDKOandTKOsplenicCD4+Tcellscomparedtotheircounterparts(Fig.7c),whichwasassociatedwiththeactivationofCD4+Tcells.However,deletionofIL-4didnotaffectCD132expressioninTKOcellscomparedtoDKOcells(Fig.7c).Furthermore,toinvestigatewhetherdeletionofIL-4affectstheactivationofJAK3,weisolatedtheCD4+Tcellsfromthespleensofwild-type,Il4KO,DKO,andTKOmiceandstimulatedinvitrowithanti-CD3andanti-CD28.JAK3phosphorylationwassimilarinunstimulatedCD4+Tcellsacrossallfourgroups(Fig.7dandSupplementaryFig.5a).Followingstimulation,DKOCD4+TcellsexhibitedhigherJAK3phosphorylationcomparedtowild-type,Il4KO,andTKOcells(Fig.7d).Overall, these findings suggestthat deletion of IL-4does not affect common γ chain expression but regulates IL-4Rα expression and type I IL-4receptorcomposition,thusinfluencingJAK3andSTAT5activationinDKOCD4+Tcells.

ToconfirmtheroleofIL-4onSTAT5phosphorylationinDKOCD4+Tcells,weactivatedsplenocyteswithMOG peptide,blockedIL-2signalingwithanti-IL-2andanti-CD25,andaddedIL-4toinduceIL-4signalingactivation.BeforeMOG peptide stimulation, STAT5 phosphorylation was comparable in wild-type, DKO, and TKO splenocytes,butincreasedovertimeafterstimulation(Fig.7e).Consistentwithpreviousreports48, anti-IL-2and anti-CD25 treatment markedly reduced STAT5 phosphorylation in all three groups relativetoMOG stimulation alone(Fig.7e),indicatingthatIL-2signalingiscrucialforSTAT5phosphorylation.Additionally,incubationofIL-4totheanti-IL-2andanti-CD25treatedgroupsmarkedlyenhancedSTAT5phosphorylation(Fig.7e),suggestingthatIL-4signalingisalsoimportanttoinducetheSTAT5phosphorylation.Inparticular,STAT5phosphorylationinDKOCD4+Tcellstreatedwithanti-IL-2andanti-CD25wassignificantlyhigherthaninwild-typeandTKOcells(Fig.7e),indicatingthatIL-4signalingpredominantlyinducesSTAT5phosphorylationinDKOcells.Insummary,theseresultshighlightanessentialroleofIL-4indrivingSTAT5phosphorylationinDKOCD4+Tcells,throughtheγ chainofthetypeIIL-4receptorandJAK3signalingpathway.

SincetheJanuskinaseJAK3istheupstreamofSTAT5,weinvestigatedwhetherJAK3inhibitionpromotesIL-17Aproduction.WetreatednaïveCD4+TcellsfromthespleensofDKOandTKOmicewithtofacitinib,aJAK3inhibitor49,underT 0toT 17polarizingconditions(Fig.5a).TofacitinibtreatmentsignificantlyenhancedIL-17AproductioninbothDKOandTKOCD4+Tcellsinadose-dependentmannerandsimultaneouslyinhibitedIL-4productioninDKOcells(Fig.7f).ThesestudiesindicatethatthedeletionofIL-4regulatestheformationoftypeIIL-4receptorandtheactivationofJAK3/STAT5signalingpathway,therebycontrollingT 17cellpolarization.

GiventhecriticalroleofIL-6signalinginT 17cellpolarization,wealsoinvestigatedwhetherthedeletionofIL-4affectedtheIL-6signalingpathway,CD126 (IL-6Ra)expression onCD4+ Tcellswassimilaramongthesefourgroups(SupplementaryFig.5b).Moreover,JAK2phosphorylationwasundetectableinbothunstimulatedandstimulatedcellsamongthesegroups(SupplementaryFig.5c),indicatingthatIL-4deletiondoesnotdirectlyaffectIL-6signaling.WealsocheckedJAK1phosphorylation.TheunstimulatedDKOandTKOCD4+TcellsshowedslightlylowerJAK1phosphorylation(SupplementaryFig.5d).Afterstimulation,DKOCD4+TcellsdisplayedhigherJAK1phosphorylationcomparedtowild-type,Il4KO,andTKOcells,duetoanincreaseinIL-4receptorαsignaling(SupplementaryFig.5d).

ToexplorehowSTAT5inhibitsthedifferentiationofT 17cellsinDKOT 2-likecells,weperformedgenome-wideanalysisofSTAT5occupancyinCD4+TcellsunderT 0toT 17cultureconditionsusingcleavageundertargetsandtagmentation(CUT&Tag).WeintegratedRNA-seqdatacomparingTKOandDKOCD4+TcellswithregionsboundbySTAT5fromtheCUT&Tagdata,identifying167genesinducedbySTAT5anddifferentiallyexpressedbetweenDKOandTKOcells(Fig.8a).GivenpreviousreportsthatBlimp1andc-MafhelpcontrolT 17cellpolarization50–52,weexaminedtheirgenomiclociandobservedsignificantSTAT5bindingatthePrdm1andMafpromotersinDKOcells(SupplementaryFig.6a,b),butnotattheRorc or Il17a promoters(SupplementaryFig.6c,d).Consistently,weanalyzedtwoSTAT5bindingmotifsassociatedwitheachgene,andCUT&Tag-qPCRconfirmedstrongSTAT5occupancyatthePrdm1andMafpromotersinDKOT 17-likeCD4+Tcells(Fig.8b,c).Furthermore,therelativeRNAlevelsofPrdm1andMafwerealsosignificantlyincreasedinDKOCD4+Tcellscomparedwithwild-type,Il4KO,andTKOcells(Fig.8d,e).FlowcytometricanalysisalsoshowedsignificantlyhigherexpressionofBlimp1andc-MafinDKOCD4+TcellscomparedwiththeTKOcounterparts(Fig.8f,g),whilesingledeletionofIL-4dramaticallyreducedBlimp1expression(Fig.8f).TheseresultscollectivelydemonstratethatIL-4-STAT5axispotentlysuppressesT 17celldifferentiation,throughdirectbindingofSTAT5tothePrdm1andMafpromotersanddrivingBlimp1andc-MafexpressioninDKOCD4+Tcells.

To further confirm the role of STAT5 in suppressing T 17 cell differentiationinDKOCD4+Tcells,weisolatedthenaïveCD4+Tcellsfromthefourgroupsandinfectedthemwithretroviruscarryingeitherwild-typeSTAT5orSTAT5-CA,aconstitutivelyactivatedformofSTAT5,theninduceddifferentiationoftheseinfectedcellsunderT 17conditions(Fig.8h).Asexpected,STAT5-CAexpressiondramaticallysuppressedIL-17Aproductioninthesefourgroups(Fig.8i),whileIL-17Aproductionslightlydecreasedinthewild-typeSTAT5group(Fig.8i),consistentwithourfindingthatIL-4modulatesIL-17AproductionbyregulatingSTAT5phosphorylation.Furthermore,duetothestrongT 17-polarizedconditions,GM-CSFproductionwaslimited(Fig.8i)andIL-4productionwasundetectable(SupplementaryFig.7).TofurtherelucidatetheroleofSTAT5phosphorylationinsuppressingT 17celldifferentiationinvivo,weutilizedthe2D2EAEadaptivetransfermodel(Fig.8j).MicereceivingSTAT5-CA-expressingTcellsdevelopedsignificantlymilderdiseasethanthosereceivingwild-typeSTAT5oremptyvector-transducedTcells(Fig.8k,l).STAT5-CA-infectedTcellsinfiltratingtheCNSexhibitedsignificantlylowerfrequenciesofIL-17Aproduction(Fig.8m),indicatingthesuppressiveroleofSTAT5inIL-17Aproductioninvivo.Conversely,theproductionofGM-CSFwassignificantlyincreased(Fig.8m).TherewasnodifferenceintheproductionofIFN-γorinthefrequencyofIL-17A+IFN-γ+TcellsintheCNS(Fig.8m).Together,theseresultscollectivelyvalidateSTAT5asanegativeregulatorofT 17cellpolarizationinDKOTcellsinvivo.

Methods
Sexasabiologicalvariable
6–8week-oldmaleandfemalemicewereexaminedinthisstudy,withsimilarfindingsobservedinbothsexes.FemaleRag1−/−micewereutilizedasrecipientmiceduetotheirincreasedsusceptibilitytoEAE.Nevertheless,ourfindingsareexpectedtoberelevanttobothsexes,sincemalesdevelopEAEwithasimilarphysiopathology.

MiceandTcelllines
Wwp2−/−Itchf/fCd4-Cre C57BL/6 mice have been previously characterized30.The2D2TCRtransgenicmicewerekindlyprovidedbyDr. Chen Dong of Tsinghua University11, Beijing, China. Il4−/− mice (#002253)andRag1−/−mice(#002216)wereacquiredfromtheJacksonLaboratory.Allmicewerebredandhousedunderspecificpathogen-freeconditionsinatemperature-controlledenvironmentat22‒24°Cwitha12-hlight/darkcycle.TheanimalexperimentswereperformedfollowingprotocolsapprovedbytheAnimalCareandUseCommittee(numberF16-00228-A5061-01toTsinghuaUniversity)andInstitutionalReviewBoard(20-LYC-1 to Liu Lab) of Tsinghua University. Experimentalanimalsandcontrolanimalswerebredseparately.Foreuthanasia,themicewereeuthanizedbyCO2accordingtotheapprovedprotocols. The primary CD4+ T cells involved in the in-vitro assays, retrovirus infection experiments, and passive EAE model were all derivedfrom2D2transgenicmice.

Antibodies
ForTcellculture,antibodiestomouseCD3ε(clone145-2C11,#BE0001-1, 3µg/ml), CD28 (clone 37.51, #BE0015-1, 2µg/ml), IFN-γ (clone R4-6A2, #BE0054, 10µg/ml), IL-2(clone JES6-1A12, # BE0043, 20ng/ml) werefromBioXCell;purifiedanti-mouseCD25antibodywerefromBio Legend (clone 3C7, # 101902, 1µg/ml). For flow cytometry and ELISA, anti-mouse CD44-FITC (clone IM7, #45-0441-82, 1:500), anti-mouse Blimp-1-PE (5E7, #150005, 1:100), anti-mouse/human CD11b-BV421(cloneM1/70,#101236,1:500),anti-mouseCD11c-PE/Cy7(cloneN418,#117318,1:500),anti-mouseLy6G/Ly6C(Gr1)-AF700(clone1A8,#108422, 1:500), Purified anti-mouse IgE (clone RME-1, #406902, 1:2000), Purified anti-mouse IgG1 (clone RMG1-1, #406602, 1:4000) were from Bio Legend; anti-mouse CD4-eF450 (clone RM4-5,#48-0042-82, 1:500), anti-mouse CD25-PE (PC61.5, #25-0251-82, 1:500), anti-mouse CD62L-PE/Cy7 (clone MEL-14, # 25-0621-82, 1:500), anti-mouseCD126-PE(cloneD7715A7,#12-1261-80,1:500),Anti-Mouse/Rat IL-17A-PE (clone 17B7, #12-7177-81, 1:400), anti-mouse IFN-γ-eF450 (cloneXMG1.2,#48-7311-82,1:200),anti-mouseGM-CSF-PE/Cy7(cloneMP1-22E9,#25-7331-82,1:100),anti-mouseRORγt-PerCP-Cy5.5(cloneB2D,#46698182,1:100),anti-mouse/humanc-Maf-PE(clonesym0F1,#12-9855-41,1:100)werefromeBioscience;andanti-mouseSiglec-F-PE-CF594(cloneE50-2440,#562757,1:1000),anti-mouseCD124-PE(clone mIL4R-M1, #552509, 1:500), anti-mouse IL-4-FITC (clone11B11, #557728, 1:100), anti-mouse GATA-3-FITC (clone L50-823, #560163, 1:100)wereobtainedfromBDBiosciences.ForPhosflowanalysis,anti-mouse/human pSTAT3 at Tyr705-PE (eBioscience, clone LUVNKLA, #12-9033-42, 1:100), anti-mouse pSTAT5 at TyrY649-APC (BD Biosciences,clone47,#612599,1:100),anti-mousepSTAT6atTyrY641-PE(BDBiosciences,cloneJ71-773.58.11,#558252,1:100).Forimmunoblotanalysis,antibodytomouseLck(clone3A5,#sc-433,1:2000)wasfromSanta Cruz Biotechnology; antibody to mouse ZAP70/Syk phosphorylatedatTyr319andTyr352(clone65E4,#2717,1:1000),ZAP70(clone D1C10E, #3165, 1:2000), PLCγ1 phosphorylated at Tyr783 (#2821, 1:1000), PLCγ1 (clone D9H10, #5690, 1:2000), Jak1 phosphorylated at Tyr1034 and Tyr1035 (clone D7N4Z, #74129, 1:1000), Jak1(cloneD1T6W,#50996,1:2000),Jak2phosphorylatedatTyr1008(cloneD4A8,#8082,1:1000),Jak2(cloneD2E12,#3230,1:2000),Jak3phosphorylatedatTyr980andTyr981(cloneD44E3,#5031,1:1000),Jak3 (clone D7B12, #8863, 1:2000), p44/42 MAPK (Erk1/2) phosphorylated at Thr202 and Tyr204 (clone D13.14.4E, #4370, 1:1000), p44/42 MAPK (Erk1/2) (clone 137F5, #4695, 1:2000) were from Cell Signaling Technology; antibodies to mouse LAT phosphorylated at Tyr191(#07-278,1:1000)andLAT(#06-807,1:2000)werefromMillipore; antibodies to Actin (clone AC-74, #A2228, 1:5000) was from Sigma-Aldrich; antibody to mouse Lck phosphorylated at Tyr394(#PA5-37628,1:1000)wasfromThermoFisherScientific.ForCUT&Taganalysis,antibodyto mouseSTAT5(D2O6Y, #94205,1:50) was from Cell Signaling Technology; antibody to Rabbit IgG H&L(#ab6702, 1:100) was from Abcam. For anti-GM-CSF treatment, antibody to mouseGM-CSF(cloneMP1-22E9,#HY-P99134,20mg/kg)andisotypecontrolRatIgG2akappa(#HY-P990679,20mg/kg)werefromMCE.

Tcelldifferentiationinvitro
2D2TCRtransgenicCD4+Tcellswereisolatedfromthespleenandperipherallymphnodesusinganti-CD4microbeads(MiltenyiBiotec,clone REA604, #130-117-043, 1:10) according to the manufacturer’s instructions.Subsequently, naïve CD4+ T cells (CD4+ CD25− CD62Lhi CD44low)weresortedusingtheBDFACSAria™Fusioncellsorter(BD Biosciences).ThecellswereculturedinRPMI1640medium(Gibco,#C11875500BT)supplementedwith10%FBS(Gibco,#10091-148,1:10),penicillin-streptomycin(Hyclone,#30010,1:100),β-mercaptoethanol(Gibco,#21985023, 1:1000), and NEAA(Gibco,#11140050,1:100) (T cellmedium),andactivatedwithplate-boundanti-CD3(3µg/ml)andanti-CD28(2µg/ml).T 2celldifferentiationwasinducedwithrecombinant human IL-2 (Pepro Tech, #200-02-50, 10ng/ml), mouse IL-4 (PeproTech,#214-14,20ng/ml),andanti-mouseIFN-γ(10µg/ml).T 17cellsweregeneratedusingacombinationofhumanTGF-β(R&DSystems,#240-B-002,1ng/ml),mouseinterleukin6(mIL-6)(PeproTech,#216-16,20ng/ml),andanti-mouseIFN-γ(10µg/ml).TopromotethedifferentiationfromT 0toT 17conditions,naiveCD4+Tcellswereinitiallyactivatedwithanti-CD3andanti-CD28for48h,andthenchanged the medium with T 17-polarizing conditions. Molecular inhibitors, including AS1517499 (#S8685) and Tofacitinib (#S2789) fromSelleck,aswellasSTAT5-IN(HY-101853)fromMCE,wereaddedtotheculturemediumasearlyastheTcellswerestimulatedwithanti-CD3/CD28and/orcytokines.Fourdayslater,cellswereharvestedforsubsequentanalyses.

RetroviruspreparationandTcellinfection
STAT5anditsmutantswereclonedintoapMIG-IRES-GFPretroviralvector61.TheplasmidandpCL-ECOpackagingvectorweretransfectedintoPLAT-E (Platinum-Eco)cells using TransIT (Mirus, #MIR 2154) accordingtothemanufacturer’sinstructions62.Approximately4–6hpost-transfection,themediumwasreplacedwithTcellmedium,andtheculturesupernatantcontainingretroviralparticleswascollectedat24and48h,thenfilteredthrough45-μmfilters.2D2TCRtransgenicnaïveCD4+Tcellsweresortedaspreviouslydescribedandactivatedwithanti-CD3/CD28for20h.Subsequently,thesupernatantcontaining retroviruses, supplemented with polybrene (YEASON,#40804ES86,8µg/ml)andT 17-polarizingcytokines,wasaddedtothepre-activatedCD4+Tcells.Thecellsunderwentspininfectionat500×gfor90minat37°Candwerethenincubatedforanadditional2h. On day 2, the cells were reinfected and subsequently cultured underT 17-polarizingconditionsfor4moredays.Afterthisperiod,theinfectedcellswereharvestedforfurtheranalysis.

AdaptivetransferEAEinduction
NaïveCD4+TcellswereisolatedfromthespleenandlymphnodesofWT,Il4KO,DKO,andTKO2D2TCRtransgenicmiceandwerestimulated in vitro using anti-CD3/CD28 for two days. Subsequently, 2–3×106activated2D2TCRtransgenicCD4+TcellswereintravenouslyadministeredintofemaleRag1−/−recipientmice.Bothbodyweightanddiseasescoresweremonitoreddaily. Classical EAE scores, ranging from0to5,wereassignedaspreviouslydescribed63.Thepresenceofatypicaldiseasemanifestations,specificallybalanceissuesandataxia,wasdocumented. Definitions of ataxia-related behaviors have been previously established64. Ataxic symptoms were evaluated on a cumulative 4-point scale, with each of the following physical manifestationsassignedonepoint:splayedlegsandtailrigidity,draggingofthetrunk,andinstabilityleadingtolateralfalls. For experiments involvingStat5OEcells,thesecellsunderwenttransductionasoutlinedpreviously,followedbyisolationofGFP-positivecells,whichwerethenculturedunderT 17-polarizingconditionsforanadditional24h.Finally,1×106ofthesecellswerecollectedandintravenouslyinjectedintofemaleRag1−/−recipientmice.Foranti-GM-CSFtreatment,aneutralizinganti-mouseGM-CSFantibody(20mg/kgdilutedin0.2mlofPBS)wasinjectedintraperitoneallyintomiceevery2daysstartingonday2followingEAEinduction.ControlmicereceivedratIgG2abythesameinjectionprotocols.

IsolationoflymphocytesfromCNSinEAEmice
At the peak of EAE disease progression, mice were euthanized and perfusedintracardiallywithcoldphosphate-bufferedsaline(PBS).Thebrain and spinal cord were subsequently extracted and washed extensivelywithcoldPBS.TheCNStissueswerefinelymincedbeforebeing mechanically dissociated through a 70µm cell strainer. The resulting cell suspensions were enriched using a 40% Percoll (GE, #17089109)gradient,andthepurifiedsingle-cellsuspensionsfromtheCNSwerepreparedfordownstreamflowcytometryanalysis.

Flowcytometry
Single-cellsuspensionswereinitiallystainedwithviabilitymarkersandsurface antigen-specific antibodies. For intracellular cytokine detection,cells,whetherculturedin-vitroorisolatedfromin-vivoexperiments,wererestimulatedusingphorbol12-myristate13-acetate(PMA;Sigma-Aldrich,#P8319,50ng/ml)andionomycin(Sigma-Aldrich,#407952,500ng/ml),inconjunctionwithGolgiStop(BDBiosciences,#554724, 1:1500) for 3–4h prior to surface antigen staining. Subsequentstepsincludedfixationandpermeabilization(BDBiosciences,#554722) as directed by the manufacturer’s guidelines. The stained cells were then analyzed on a Fortessa5 (BD Biosciences) flow cytometerusingFlowJo10software,excludingdeadcellsviaLIVE/DEAD® FixableVioletDeadCellStain(Invitrogen,#L34955,1:1600).Priortophosphorylation staining (Phosflow), ex vivo CD4+ T cells were stimulatedwithspecificcytokinesfor15min:mouseinterleukin6(mIL-6)(PeproTech,#216-16,100ng/ml)forSTAT3phosphorylation,recombinanthumanIL-2(Pepro Tech, #200-02-50, 100ng/ml) for STAT5 phosphorylation,andmouseIL-4(PeproTech,#214-14,100ng/ml)forSTAT6phosphorylation.Post-stimulation,CD4+Tcellswerefixedwithfixationbuffer(BD,#554655)for30minandpermeabilizedwithmethanolovernightat−20°C.Followingthis,cellswerewashedwithstainingbuffer(PBSwith1%FBS)andincubatedwiththerespectivephospho-STAT(pSTAT)antibodiesasindicatedbefore.ExamplesofgatingstrategiesareprovidedinSupplementaryFig.8.

Brainandspinalcordhistology
Before separating the brain and spinal cord, mice were perfusedintracardially with cold PBS and then with 4% phosphate-bufferedformalin.ThewholeCNSwasfixedin4%phosphate-bufferedformalinovernightandembeddedinparaffin.Subsequentsectioning(10μm)andstainingforhematoxylinandeosin(H&E)orluxolfastblue(LFB)wereperformedbyServicebio.

Immunoblot
PurifiedCD4+Tcells(2×106)fromthespleenandlymphnodesof2D2TCRtransgenicmicebeforeandafterstimulationwerelysedin100µLofRIPAlysisbuffer(YEASON,#20101ES60)onicefor30min,followedbycentrifugationat3217×gand4°Cfor30min.Thesupernatantwasthentransferred to a new tube and heated in SDS-PAGE loading buffer (CWbio,#CW0052,1:5)at98°Cfor10min.Thesamplesweresubsequentlyloadedontoa10%SDS-PAGEgelandelectrotransferredto0.45µmpolyvinylidenedifluoridemembranes(Millipore,#IPVH00010).These membranes were subjected to immunoblotting using specific primaryantibodiesandhorseradishperoxidase–conjugatedsecondaryantibodies. Detection was performed using an enhanced chemiluminescencesystem(Thermo Fisher Scientific, #34087). The grayscale valuesofproteinbandswerequantifiedusingImageJsoftware.

RNAisolationandquantitativereal-timePCR
Forthequantitativeanalysisofgeneexpressionviareal-timePCR,totalRNA was extracted from in vitro cultured cells using TRIzol reagent(Magen,#R4801-02),andcDNAwassynthesizedusingaSuperscriptIIIFirstStrandSynthesisKit(Invitrogen,#18080051)followingthemanufacturer’sinstructions.Thereal-timePCRassayswereconductedintechnicalreplicatesusingTaq Pro Universal SYBR qPCR Master Mix (Vazyme, #Q712-03) on a Light Cycler 2.0 system (Roche). Gene expression levels were normalized to the β-actin gene. The primers designedtoamplifythetargetgeneswereasfollows:forPrdm1,forward(5′-TTCTCTTGGAAAAACGTGTGGG-3′) and reverse (5′-GGAGCCGGAGCTAGACTTG-3′);forMaf,forward(5′-GGAGACCGACCGCATCATC-3′)andreverse(5′-TCATCCAGTAGTAGTCTTCCAGG-3′).

CUT&Tag-qPCRandlibrarygenerationforsequencing
TheCUT&Tagassaywasconductedwithseveralmodificationsusingthe Hyperactive Universal CUT&Tag Assay Kit for Illumina (VazymeBiotech,#TD904).Initially,5×105primaryTcellswereisolatedandcoupledtoConAbeadsfor10min.Subsequently,thesebead-boundcellswereincubatedovernightat4°Cin50µl of antibody buffer containingtheprimaryantibody.Afterward,thesecondaryantibodywasaddedandincubatedforanadditionalhour.Thecellswerewashedandincubatedwith0.04µMbiotinylated-pA/G-Tnpfor1h,followedbysuspensionintagmentationbufferTTBLforanotherhour.ThereactionwashaltedbytheadditionofProteinaseKand10%SDSat55°Cfor15min.Aspike-inmix(1pg/105cells)wasincorporatedforsubsequentdata normalization. Post the stopping step, tagmented DNA was captured and isolated using streptavidin magnetic beads. Library constructionwasperformeddirectlyonthebeadsusingtheNEBNextUltraDNALibraryPrepKitforIllumina(NEB,#E7370S).ThecompletedCUT&Tag libraries were sequenced on the Illumina NovaSeq 6000 platforminPE150modebyNovogene,Beijing,China.
ForCUT&Tag-qPCR,theaforementionedCUT&Tagprotocolwasfollowed,exceptthatafterDNAextraction,theDNA-boundSAbeadswereresuspendedandterminatedwith5µlofStopbufferandheatedat95°Cfor5min.TheqPCRreactionsutilizedthepurifiedDNAastemplatesandwereconductedintriplicate.Gene-specificCUT&Tag-qPCRprimerstargeted150–250bpsequencessurroundingtheputativetranscriptionfactor-bindingsites.Theassayswereaccomplishedwiththefollowingantibodies:anti-STAT5antibodydiluted1:50;Anti-rabbit-IgGdiluted1:100.

ATAC-seqlibrarygenerationandsequencing
ATAC-seqwasperformedonapproximately1×10⁵viablecellsusingtheHyperactiveATAC-SeqLibraryPrepKitforIllumina(VazymeBiotech,#TD711) following the manufacturer’s protocol with minor modifications.Briefly,cellswerepelletedbycentrifugationat500×gfor5minat4°Candlysedinlysisbufferonicefor5mintoisolatenuclei.Followingnucleicollectionbycentrifugationunderthesameconditions,thepelletwasresuspended inthetransposome mixandincubatedat37°Cfor30mintofacilitatetagmentation.Thereactionwasterminatedbyaddingstopbufferandincubatingat37°Cfor5min.ThetagmentedDNAweresubsequentlypurifiedusingmagneticbeads.Thebead-boundDNAwaswashedtwicewith80%ethanolandelutedin20μLofnuclease-freewater.LibraryconstructionwasperformedusingtheNEBNext®Ultra™DNALibraryPrepKitforIllumina®(NEB,#E7370S).Finallibrariesweresequenced on an Illumina NovaSeq 6000 platform (KaiTaibio, Hangzhou,China)utilizingpaired-end150-bp(PE150)reads.

ATAC-seqandCUT&Tagdataanalysis
Rawpaired-endsequencedreadswerefirstcutforadaptorsequencesandtrimmedusingtrimmomatic(v.0.32).Then,cleanedfastqdataweremappedtothemousegenome(mm10)usingBowtie2(v.2.4.2).Picard was used for marking and removing duplication. Samtools(v.1.11) wasused for converting and sorting the SAM files intoBAMformat.Macs2(v.2.1.0)wasusedforpeakcalling.DeeptoolswasusedfornormalizingsignalsfromsortedBAMfilesviabamCoverage,andcomputeMatrixwasusedforcalculatingtheoverallsignaldistributionaroundthepeakcentercalledbymacs2usingthe“reference-point”model. Differential ATAC seq or Cut&Tag peaks were identified by DiffBind (v3.12.0). Data were annotated and visualized using Bioconductor-CHIPseeker(v.1.28.3)andIGV(v.2.11.9).
ForCUT&Tag-qPCR,therelativeproportionoftargetcontentwascalculatedusingtheΔCtmethod.TheCtvaluesofeachCUT&Tagsample,whetherantibodyorIgG,canbenormalizedusingitscorrespondingDNAspike-inascontrol:ΔCt=Ct –Ct .Foldenrichment of the target region in each antibody sample was then determinedrelativetotheIgGcontrol:Foldenrichment=2−ΔCt.

RNA-seqandanalysis
FortheRNAsequencinganalysis,totalRNAwasextractedfromCD4+TcellsfollowingstimulationwithPMAandionomycinusingTRIzolreagent,basedonthemanufacturer’sguidelines.TheresultingRNA-seq libraries were constructed and subsequently sequenced by BGI GenomicsontheDNBSEQplatformutilizing50-bppaired-endreads.Differential expression analysis was performed using the negative binomialmodelimplementedinDr.Tom(v.2.0,BGI),whichintegratesDESeq2’sstatisticalframework.Heatmapsweregeneratedtovisualizethesedifferences,withexpressionvaluescomputedaslog2-transformed reads per kilobase of exon per million mapped reads(RPKM). Additionally, Gene Set Enrichment Analysis was conductedusingtheGseaPrerankedmethodbyGSEA(v.4.4.0),employingaclassicscoringschemetoassessthebiologicalsignificanceofobservedexpressionpatterns.

Publishedmicroarraydatasetsanalysis
ThehumanpatientsskinmicroarraydatasetsareavailablefromtheGeneExpressionOmnibus(GEO):GSE185764.MicroarrayprobeIDwasobtainedfromGEO.TheCELfilesfromAD,DI-PSOpatientsandHCskinmicroarraydatawereimportedintoTranscriptomeAnalysisConsole(v.4.0,ThermoFisherScientific),andthenthegeneexpressionlistandDEGsgeneratedbysetting:LimmaAnova,DABG(<0.05)andthefoldchangeofeverygene(>2or<−2),togetherwiththeircorrespondingpvalueorFDRpvalue(Ben-jamini–Hochbergadjustedp value) (<0.05). Data were analysed in heatmap2 (v.3.2.0) and GraphPadPrism(v.10.0)forgraphicalrepresentations.

Statisticalanalysis
StatisticalsignificancewasassessedusingStudent’st-test,one-wayortwo-wayanalysisofvariance(ANOVA),asappropriate,withGraphPadPrism(v.10.0).Eachexperimentwasconductedaminimumoftwotothreetimes,yieldingconsistentresults.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study by Zhao et al, the authors assessed the role of E3 ligases Itch and WWP2 in Th2 stability during autoimmune neuroinflammation. The authors found that mice containing MOG-specific TCR (2D2) and also lacking the E3 ligases Itch and WWP2 (DKO), developed spontaneous EAE. This EAE susceptibility 2D2-DKO mice was associated with increased IL-4 and GM-CSF producing T cells in the periphery and in the CNS. Interestingly, deletion of IL-4 in 2D2-DKO mice exacerbated EAE severity and this was associated with an elevated frequencies of Th17 cells and reduction in IL-4 producing Th2 cells. Mechanistic work performed by the authors revealed that the JAK3/STAT5 signaling pathway is critical for maintaining Th2 lineage stability by regulating the expression of Blimp1 and c-Maf, thereby preventing CD4+ T cells from differentiating into Th17 cells. The data appear of high quality, and support the conclusions. Overall, this is an interesting study that reveals the role of IL-4 in preserving the lineage stability of Th2 cells during CNS autoimmunity. Although the study is interesting there are some concerns that need to be addressed before the manuscript can be considered for publication.\n---------------------\nTo substantiate their claim that IL-4 limits the ability of DKO Th2 cells to become Th17 cells, the authors should perform Th17 polarization experiments with differentiated Th2 cells from DKO and TKO cells.\nThe authors should perform luxol fast blue staining in the CNS of DKO and TKO mice with EAE to validate their claim that TKO mice exhibit enhanced demyelination.\nIt’s unclear why the profiling of STAT molecules was performed with T cells stimulated under MOG antigen while the gene expression profiling was performed with T cells polarized under Th0 and Th17 conditions.\nBecause RORgT can be expressed by non-Th17 cells, the authors should measure the frequencies of IL-17A+ RORgT double producers in the CNS of DKO and TKO mice with EAE to validate their claim that RORγt+ Th17-like cells are altered.\nTh17 differentiation assays in Fig 4 were performed under non-pathogenic Th17 polarizing conditions. It’s unclear if naïve T cells from TKO mice also exhibit enhanced ability to differentiate into pathogenic Th17 cells, which are relevant to EAE.\nAlthough the work is generally rigorous, the full set of data represented in the figures is often not clear. Many of the panels say “n=#,” but it’s not clear if n refers to biological replicates from a single experiment vs independent experiment. Some figures do not mention the number of biological replicates used. This should be clarified in the figure legends.", "reviewer_2": "Building upon their previous results (Aki D et al., Nat Immunol 2018), Zhao et al. investigated the roles of Itch and WWP2 in CD4+ T cells within the context of neuroinflammation. Utilizing an extensive set of genetic mouse models and in vitro experiments, the authors found that MOG35-55-specific 2D2-TCR transgenic mice lacking Itch and WWP2 in CD4+ T cells exhibited atypical EAE symptoms, with immune responses characterized by a Th2-driven pathology, primarily mediated by IL-4. Concerning downstream signaling of IL-4, the authors propose that activation of the IL-4 signaling pathway alone is sufficient for inducing the phosphorylation of STAT5. Furthermore, they systematically examined the function of STAT5 in Th17 cell differentiation, concluding that by enhancing the expression of Blimp1 and c-Maf, STAT5 suppresses the initiation of Th17 cell differentiation. As a clinical link to human disease, the authors re-analyzed skin sample datasets from dupilumab-associated psoriasis in individuals with atopic dermatitis and confirmed the upregulation of genes related to pathogenic Th17 cells.\nThis well-written manuscript presents a thorough analysis of the interaction between Th2 and Th17 differentiation, providing novel insights into the role of Th2-mediated immune responses in the context of autoimmunity. The authors have addressed a clinically relevant issue, and the methodology appears sound. However, a few points require clarification:\nMajor Criticism:\n- In the introduction, the authors inaccurately describe the current understanding of the pathophysiology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Specifically, the authors assert that Th17 cells are the \"key drivers of MS progression\" (page 3, lines 41-22), which does not align with the current consensus on MS-related disease progression (Kuhlmann T et al., Lancet Neurol 2022). Additionally, they claim that \"In the central nervous system (CNS), TH17 cells secrete the inflammatory cytokines IL-17, IFN-γ, and GM-CSF, which recruit polymorphonuclear cells from peripheral tissues to the CNS and lead to neuroinflammation\" (page 3, lines 42-44). However, polymorphonuclear cells constitute only a minor population in MS lesions (Lassmann H, Cold Spring Harb Perspect Med 2018). Furthermore, the authors state that \"TH2 cells are also implicated in the pathogenesis of MS\" (page 3, lines 44-46). To date, convincing evidence for a significant role of Th2 cells has only been demonstrated in the context of EAE, not in human MS (Attfield KE et al., Nat Rev Immunol 2022). We recommend that the authors frame EAE as an appropriate model for studying the differential roles of T helper cells in shaping the autoimmune response, rather than focusing on MS pathophysiology, which is neither correctly described nor revisited in the discussion.\n- Throughout the methods section, the authors do not consistently specify whether experiments were conducted using 2D2 T cells (e.g., pages 22-24). At times, it is unclear when 2D2 cells were employed. Please clarify when 2D2 cells are used to provide a cohesive statement that acknowledges their use throughout the manuscript, while also noting any exceptions.\n- Additionally, it is crucial to ascertain whether the observed immune phenotype is exclusively mediated by 2D2 cells or also involves bystander cells. What is the percentage of 2D2 cells in the analysis, as shown in Figure 1D-F, Figure 2A-D, Figure 2G/H and Figure 3D/E. It is also unclear how many of the transferred cells are 2D2 cells (Figure 2D). Could these be measured using appropriate antibodies against the TCR alpha and beta chains or MOG-tetramers?\n- In Figure 6D, the authors present an immunoblot analysis of phosphorylated and total JAK3. A quantification using densitometry would be good.\nMinor Criticism:\n- To investigate whether the effect of IL-4 on the Th2 to Th17 transition is tissue-specific, the authors analyzed cytokine production in CD4+ T cells from the colon and spleen (pages 8-10). However, given that other organs, such as the lung and skin, are commonly affected by Th2-mediated diseases (e.g., asthma and atopic dermatitis), it would be valuable to know whether these organs were also analyzed in the study.\n- Considering previous data on the role of IL-4 in the context of neuroinflammation (Ponomarev ED et al., J Neurosci 2007), the authors tend to overstate the novelty of their findings in some instances. We advise them to moderate the wording, e.g., on page 19, lines 407-408 (\"Unexpectedly, deletion of IL-4 exacerbated EAE symptoms and led to the development of classical EAE\").", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript entitled “The E3 ligases Itch and WWP2 regulate autoimmune neuroinflammation by controlling Th2 to Th17 cell conversion via interleukin-4-STAT5 axis” by Zhao et al investigates the roles of Itch, WWP2, and IL-4 in experimental autoimmune encephalomyelitis (EAE) using genetically deficient mouse models. The authors showed that deletion of Itch and WWP2 (DKO) leads to atypical EAE, characterized by neuroinflammation. Further deletion of the Il4 gene in the DKO mice (creating TKO mice) resulted in the conversion of Th2 cells to Th17 cells, driving the development of classical EAE. Mechanistically, they identify the IL-4R/JAK3/STAT5 signaling pathway as a key regulator that promotes Blimp1 expression, which subsequently inhibits Il17a expression. Although the study sheds some light on the phenomenon of atypical neuroinflammation and its molecular mechanism, I have the following concerns regarding the results presented.\n1. The concept of “atypical EAE” remains unclear. While the authors attribute the neuroinflammation observed in DKO mice to IL-4, the co-expression of IL-4 and GM-CSF by DKO CD4+ T cells raises the possibility that GM-CSF, rather than IL-4, is the primary driver of neuroinflammation. GM-CSF is a well-established contributor to EAE pathogenesis, yet the authors have not explored its role in this phenomenon.\n2. Although the mechanisms underlying Th2-to-Th17 plasticity identified in this study are novel, the advancement of knowledge is limited because the concept of CD4+ T cell plasticity is already well established in the field."}, "author_response": "Responses to Reviewers’ Comments\nWe thank the reviewers for their careful reading, insightful comments and suggestions.\nThese are valuable and helpful for us to improve the manuscript. We have addressed the\nquestions point-by-point as described in the followings (in blue font). Furthermore, in the\nrevised manuscript, edited texts are highlighted in yellow and all new or revised figures\nare highlighted with a red box.\nReviewer #1 (Remarks to the Author)\nIn this study by Zhao et al, the authors assessed the role of E3 ligases Itch and WWP2 in\nTh2 stability during autoimmune neuroinflammation. The authors found that mice\ncontaining MOG-specific TCR (2D2) and also lacking the E3 ligases Itch and WWP2\n(DKO), developed spontaneous EAE. This EAE susceptibility 2D2-DKO mice was\nassociated with increased IL-4 and GM-CSF producing T cells in the periphery and in the\nCNS. Interestingly, deletion of IL-4 in 2D2-DKO mice exacerbated EAE severity and this\nwas associated with an elevated frequencies of Th17 cells and reduction in IL-4\nproducing Th2 cells. Mechanistic work performed by the authors revealed that the\nJAK3/STAT5 signaling pathway is critical for maintaining Th2 lineage stability by\nregulating the expression of Blimp1 and c-Maf, thereby preventing CD4+ T cells from\ndifferentiating into Th17 cells. The data appear of high quality, and support the\nconclusions. Overall, this is an interesting study that reveals the role of IL-4 in preserving\nthe lineage stability of Th2 cells during CNS autoimmunity. Although the study is\ninteresting there are some concerns that need to be addressed before the manuscript\ncan be considered for publication.\nWe thank the reviewer #1 for the constructive feedback, which has significantly improved\nthe quality of our revised manuscript. All raised concerns have been systematically\naddressed through expanded methodological validation, including T cell differentiation\nassays to further corroborate our conclusions.\nTo substantiate their claim that IL-4 limits the ability of DKO Th2 cells to become Th17\ncells, the authors should perform Th17 polarization experiments with differentiated Th2\n1\ncells from DKO and TKO cells.\nWe thank the reviewer for this insightful suggestion. Accordingly, we performed the\nrequested experiments.\nAs shown in the red box of Rebuttal Fig.1 (also supplementary Fig. 3j and k in the revised\nmanuscript), we added new data about Th17 polarization experiments using in vitro-\ndifferentiated Th2 cells from DKO and TKO mice. The production of IL-17A was\nsignificantly increased in TKO CD4+ T cells compared to DKO cells.\nRebuttal Fig. 1, The production of cytokines in CD4+ T cells cultured in vitro. a The\nprocedure for in vitro T cell differentiation. b Frequency of IL-4 and IL-17A production in\nthe CD4+ T cells from WT, DKO, and TKO mice, which were cultured in vitro as in (a)\n(key; n= 3 per group). Each symbol represents one individual replicate.\nThe authors should perform luxol fast blue staining in the CNS of DKO and TKO mice\nwith EAE to validate their claim that TKO mice exhibit enhanced demyelination.\nWe also thank the reviewer for raising this critical point. To address the comment, we\nhave now performed Luxol Fast Blue (LFB) staining on spinal cord sections from both\nDKO and TKO mice with spontaneous EAE, specifically analyzing regions of\ndemyelination (Rebuttal Fig. 2). This data has been incorporated into Fig. 3h and\n2\ndescribed in lines 157-158 of the revised manuscript. We appreciate the opportunity to\nstrengthen this aspect of our work.\nRebuttal Fig. 2, LFB staining of spinal cord lesions in DKO and TKO mice. Scale bars,\n100m.\nIt’s unclear why the profiling of STAT molecules was performed with T cells stimulated\nunder MOG antigen while the gene expression profiling was performed with T cells\npolarized under Th0 and Th17 conditions.\nWe appreciate the reviewer’s observation regarding stimulation conditions. First, to mimic\nthe in vivo CD4+ T cell activation upon MOG antigen recognition in the spleen, we\nstimulated splenocytes with MOG for 48 hours to activate naïve T cells and measured\n35-55\nSTAT protein phosphorylation levels. This experimental design eliminated potential\ninterference from exogenous cytokines on STAT phosphorylation levels.\nAdditionally, data from Fig. 5b demonstrate significant enrichment of STAT-associated\nphosphoregulatory proteins in the differentially expressed gene profiles between DKO\nand TKO CD4+ T cells polarized under Th0 to Th17 conditions, which are consistent with\nthe conclusions drawn from both methods.\nTo directly address this reviewer’s comment, we assessed STAT protein phosphorylation\nduring Th0 to Th17 polarization. Phosphorylation levels of STAT5 and STAT6 were\nelevated in DKO CD4⁺ T cells compared to TKO cells, consistent with MOG stimulation\ndata (Rebuttal Fig. 3). We believe this dual approach provides complementary insights\ninto STAT protein phosphorylation in DKO and TKO cells. Due to the nature of this piece\n3\nof information is only additive to the already presented data, this new data will be used\nfor reviewer’s purpose only.\nRebuttal Fig. 3, the phosphorylation of STAT proteins in CD4+ T cells polarized under Th0\nto Th17 conditions (key; n= 5 per group). Each symbol represents one individual\nreplicate.\nBecause RORgT can be expressed by non-Th17 cells, the authors should measure the\nfrequencies of IL-17A+ RORgT double producers in the CNS of DKO and TKO mice with\nEAE to validate their claim that RORγt+ Th17-like cells are altered.\nWe thank the reviewer for raising this important point. We agree that RORγt expression is\nnot exclusive to Th17 cells. To rigorously validate alterations in Th17-like cells, we have\nnow quantified IL-17A⁺ RORγt⁺ double-positive CD4⁺ T cells in the CNS of DKO and TKO\nmice with EAE. The new data are included in Rebuttal Fig. 4 (also Fig. 3k in the revised\nmanuscript) and support our original conclusions.\nRebuttal Fig. 4, Frequency of RORγt+ IL-17A+ CD4+ T cells in the CNS of WT, Il4 KO,\nDKO, and TKO mice (key; n= 4 per group). Each symbol represents an individual mouse.\nTh17 differentiation assays in Fig 4 were performed under non-pathogenic Th17\npolarizing conditions. It’s unclear if naïve T cells from TKO mice also exhibit enhanced\nability to differentiate into pathogenic Th17 cells, which are relevant to EAE.\npathogenic Th17 cells in our study. We agree that evaluating the potential of naïve T cells\nfrom TKO mice to differentiate into pathogenic Th17 cells is critical for understanding\ntheir role in EAE pathogenesis. To address this point, we conducted additional\nexperiments using pathogenic Th17-polarizing conditions and included these data in the\nrevised manuscript (the red box of Rebuttal Fig. 1,also supplementary Fig. 3j and k in the\nrevised manuscript). Our results show that TKO CD4+ T cells exhibited a significantly\nincreased frequency of IL-17A⁺ cells under pathogenic conditions. Compared with the\nWT mice, the production of IL-17A slightly decreased in DKO CD4+ T cells, suggesting\nthat overproduced IL-4 inhibits IL-17A expression. We have added description about this\npart in the revised manuscript (page 11, lines 219-223).\nRebuttal Fig. 1 (same as above), The production of cytokines in CD4+ T cells cultured in\nvitro. a The procedure for in vitro T cell differentiation. b Frequency of IL-4 and IL-17A\nproduction in the CD4+ T cells from WT, DKO, and TKO mice, which were cultured in vitro\nas in (a) (key; n= 3 per group). Each symbol represents one individual replicate.\nAlthough the work is generally rigorous, the full set of data represented in the figures is\noften not clear. Many of the panels say “n=#,” but it’s not clear if n refers to biological\nreplicates from a single experiment vs independent experiment. Some figures do not\n5\nmention the number of biological replicates used. This should be clarified in the figure\nlegends.\nWe appreciate this observation. The relevant figure legends have been clarified in the\nrevised manuscript.\n===================================================================\nBuilding upon their previous results (Aki D et al., Nat Immunol 2018), Zhao et al.\ninvestigated the roles of Itch and WWP2 in CD4+ T cells within the context of\nneuroinflammation. Utilizing an extensive set of genetic mouse models and in vitro\nexperiments, the authors found that MOG35-55-specific 2D2-TCR transgenic mice\nlacking Itch and WWP2 in CD4+ T cells exhibited atypical EAE symptoms, with immune\nresponses characterized by a Th2-driven pathology, primarily mediated by IL-4.\nConcerning downstream signaling of IL-4, the authors propose that activation of the IL-4\nsignaling pathway alone is sufficient for inducing the phosphorylation of STAT5.\nFurthermore, they systematically examined the function of STAT5 in Th17 cell\ndifferentiation, concluding that by enhancing the expression of Blimp1 and c-Maf, STAT5\nsuppresses the initiation of Th17 cell differentiation. As a clinical link to human disease,\nthe authors re-analyzed skin sample datasets from dupilumab-associated psoriasis in\nindividuals with atopic dermatitis and confirmed the upregulation of genes related to\npathogenic Th17 cells.\nThis well-written manuscript presents a thorough analysis of the interaction between Th2\nand Th17 differentiation, providing novel insights into the role of Th2-mediated immune\nresponses in the context of autoimmunity. The authors have addressed a clinically\nrelevant issue, and the methodology appears sound. However, a few points require\nclarification:\nWe thank this Reviewer for the complimentary remarks in recognizing the quality of our\nstudy.\nMajor Criticism:\n- In the introduction, the authors inaccurately describe the current understanding of the\npathophysiology of multiple sclerosis (MS) and experimental autoimmune\nencephalomyelitis (EAE). Specifically, the authors assert that Th17 cells are the \"key\ndrivers of MS progression\" (page 3, lines 41-22), which does not align with the current\nconsensus on MS-related disease progression (Kuhlmann T et al., Lancet Neurol 2022).\nAdditionally, they claim that \"In the central nervous system (CNS), TH17 cells secrete the\n7\ninflammatory cytokines IL-17, IFN-γ, and GM-CSF, which recruit polymorphonuclear cells\nfrom peripheral tissues to the CNS and lead to neuroinflammation\" (page 3, lines 42-44).\nHowever, polymorphonuclear cells constitute only a minor population in MS lesions\n(Lassmann H, Cold Spring Harb Perspect Med 2018). Furthermore, the authors state that\n\"TH2 cells are also implicated in the pathogenesis of MS\" (page 3, lines 44-46). To date,\nconvincing evidence for a significant role of Th2 cells has only been demonstrated in the\ncontext of EAE, not in human MS (Attfield KE et al., Nat Rev Immunol 2022). We\nrecommend that the authors frame EAE as an appropriate model for studying the\ndifferential roles of T helper cells in shaping the autoimmune response, rather than\nfocusing on MS pathophysiology, which is neither correctly described nor revisited in the\ndiscussion.\nWe appreciate the thorough comment of our manuscript. We have carefully considered\nthe raised issues and have made corrections to address the inaccuracies in the\ndescriptions. As the reviewer mentioned, the critical focus of this project was on the\nfunction of helper T cells in autoimmune neuroinflammation (EAE) and the trans-\ndifferentiation between Th2 and Th17 cells, the description of human MS may not be\nappropriate. We agree that our original assertion oversimplified the complex\npathophysiology of MS. As recommended, we focused on the description about the\ndysregulation of T cells in EAE rather than MS. We added the description of T cells in\nEAE in Introduction part (page 3, lines 38-49).\nAdditional, as suggested, we have added in the Discussion part about the usefulness of\nEAE and its limitations (page 23, lines 500-505).\n- Throughout the methods section, the authors do not consistently specify whether\nexperiments were conducted using 2D2 T cells (e.g., pages 22-24). At times, it is unclear\nwhen 2D2 cells were employed. Please clarify when 2D2 cells are used to provide a\ncohesive statement that acknowledges their use throughout the manuscript, while also\nnoting any exceptions.\nWe thank this reviewer for the valuable comments. Most of the studies used 2D2 T cells.\nTo further clarify this issue, in the methods section, we have added T cell lines statement\nin the revised manuscript (page 25, lines 535-537).\n8\n- Additionally, it is crucial to ascertain whether the observed immune phenotype is\nexclusively mediated by 2D2 cells or also involves bystander cells. What is the\npercentage of 2D2 cells in the analysis, as shown in Figure 1D-F, Figure 2A-D, Figure\n2G/H and Figure 3D/E. It is also unclear how many of the transferred cells are 2D2 cells\n(Figure 2D). Could these be measured using appropriate antibodies against the TCR\nalpha and beta chains or MOG-tetramers?\nWe thank the reviewer for highlighting this essential methodological consideration.\nConsistent with prior findings (Bettelli E et al., J. Exp. Med. 2003), transgenic\nVα3.2+ Vβ11+ T cells represented >98% of CD3+ T cells in 2D2 transgenic mice. In our\nanalysis, we quantified the proportion of Vα3.2+ Vβ11+ CD4+ 2D2 cell among these four\ngroups and observed that more than 90 percent of CD4+ T cells are 2D2 cells (Rebuttal\nFig. 5). These findings indicate that neuroinflammation in DKO and TKO 2D2 mice is\nspecifically mediated by 2D2 cells, not bystander C57BL/6 cells.\nRebuttal Fig. 5, Flow cytometry plots show Va3.2 and Vb11 co-expression of CD4+ T cells\nin spleen.\n- In Figure 6D, the authors present an immunoblot analysis of phosphorylated and total\nJAK3. A quantification using densitometry would be good.\nWe thank this reviewer for the kind notice. An analysis of grayscale values for various\nproteins in WB has been incorporated into Fig. 6d, and corresponding updates were\nmade to the figure legend and Methods part (page 29, line 624; page 45, lines 958-959).\nMinor Criticism:\n- To investigate whether the effect of IL-4 on the Th2 to Th17 transition is tissue-specific,\nthe authors analyzed cytokine production in CD4+ T cells from the colon and spleen\n9\n(pages 8-10). However, given that other organs, such as the lung and skin, are\ncommonly affected by Th2-mediated diseases (e.g., asthma and atopic dermatitis), it\nwould be valuable to know whether these organs were also analyzed in the study.\nWe thank the reviewer for this insightful suggestion regarding the tissue-specific effects\nof IL-4 on Th2-to-Th17 plasticity. We agree that extending our analysis to organs beyond\nthe colon and spleen—particularly those implicated in Th2-driven pathologies, such as\nthe lung (asthma) and skin (atopic dermatitis)—would strengthen the study’s translational\nrelevance. To address this point, we conducted additional experiments using lung and\nskin samples from our mouse models. These data are now included in the Rebuttal Fig. 6\n(also supplementary Fig. 3a to i in the revised manuscript) and associated description\nwas added in revised manuscript (page 10, lines 192 to 204). Below, we summarize the\nkey results and their implications.\nLung CD4⁺ T cells:\nFirst, we examined CD4+ T cells in the lungs of 6- to 8-week-old WT, Il4 KO, DKO, and\nTKO mice. The TKO mice already exhibited classical EAE symptoms. The frequency of\nCD4+ T cells was similar among these four groups (Rebuttal Fig.6a). DKO mice showed\nenhanced production of IL-4 and GM-CSF in CD4+ T cells, while IL-17A production\nremained unchanged (Rebuttal Fig.6b and c). This suggests that there was no\nconversion from Th2 to Th17 cells in the lungs of DKO and TKO mice as early as 6-8\nweeks of age. Next, we assessed cytokine production in the lungs of mice older than 12\nweeks. The production of IL-17A was increased in TKO CD4+ T cells compared to DKO\nmice (Rebuttal Fig.6d and e). We consider this phenomenon in the lung is an indirect\neffect mediated by the inflammatory environment in older mice. It may be due to\nactivated Th17 cells migrating to the lung, rather than lung-resident T cells recognizing\nthe MOG antigen and differentiating into Th17 cells.\nSkin CD4⁺ T cells:\nWe also analyzed the cytokine levels in the postauricular skin of WT, DKO, and TKO\nmice. The production of cytokines associated with type 2 inflammation, such as IL-4, IL-\n13, and IL-25, was increased in DKO mice compared to the WT group (Rebuttal Fig.6f to\nh). Additionally, IL-17A production was slightly increased in TKO mice compared to DKO\n10\nmice and was comparable to the WT group (Rebuttal Fig.6i).\nRebuttal Fig. 6, Cytokine production in lung and skin from WT, Il4 KO, DKO, and TKO\nmice. a Flow cytometry of lung CD4+ T cells from 6- to 8-week-old WT, Il4 KO, DKO, and\nTKO mice. b, c Flow cytometry plots illustrating the production of IL-4, GM-CSF, IL-17A,\nand IFN-γ by CD4+ T cells in the lungs of mice described in (a). d, e Flow cytometry plots\nshowing the production of IL-4, GM-CSF, IL-17A, and IFN-γ by CD4+ T cells in the lungs\nof 12-week-old WT, Il4 KO, DKO, and TKO mice. f to i Relative mRNA expression levels\n11\nof Il4 (f), Il13 (g), Il25 (h), Il17a (i) in the postauricular skin of mice as in (d, e).\nWe thank the reviewer for highlighting the importance of tissue context in Th2/Th17\nbalance. Our new data confirm that the phenomenon of Th2-to-Th17 cell trans-\ndifferentiation predominantly occurs within the CNS. This observation may be linked to\ninflammatory environments, indicating the critical influence of microenvironmental cues\non T-cell plasticity. Additionally, this phenomenon could be attributable to the use of 2D2\nmice in our experimental model, underscoring the importance of TCR-mediated antigen\nrecognition. We hope that these revisions have fully addressed the raised concerns.\n- Considering previous data on the role of IL-4 in the context of neuroinflammation\n(Ponomarev ED et al., J Neurosci 2007), the authors tend to overstate the novelty of their\nfindings in some instances. We advise them to moderate the wording, e.g., on page 19,\nlines 407-408 (\"Unexpectedly, deletion of IL-4 exacerbated EAE symptoms and led to the\ndevelopment of classical EAE\").\nWe appreciate this kind notice and have thus made changes in the revised manuscript to\nmake the wording more appropriate (page 21, line 438). As the reviewer commented that\nprevious studies have documented the role of IL-4 in neuroinflammation, we initially\nhypothesized that additional IL-4 deletion would ameliorate EAE symptoms in DKO mice.\nHowever, the fact is contrary to our speculation: the absence of IL-4 leads to a shift from\nTh2 to Th17 cell trans-differentiation, thereby promoting the occurrence of more severe\nclassical EAE symptoms.\n===================================================================\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nThe manuscript entitled “The E3 ligases Itch and WWP2 regulate autoimmune\nneuroinflammation by controlling Th2 to Th17 cell conversion via interleukin-4-STAT5\naxis” by Zhao et al investigates the roles of Itch, WWP2, and IL-4 in experimental\nautoimmune encephalomyelitis (EAE) using genetically deficient mouse models. The\nauthors showed that deletion of Itch and WWP2 (DKO) leads to atypical EAE,\ncharacterized by neuroinflammation. Further deletion of the Il4 gene in the DKO mice\n(creating TKO mice) resulted in the conversion of Th2 cells to Th17 cells, driving the\ndevelopment of classical EAE. Mechanistically, they identify the IL-4R/JAK3/STAT5\nsignaling pathway as a key regulator that promotes Blimp1 expression, which\nsubsequently inhibits Il17a expression. Although the study sheds some light on the\nphenomenon of atypical neuroinflammation and its molecular mechanism, I have the\nfollowing concerns regarding the results presented.\nWe thank the Reviewers for their thoughtful and insightful comments on our manuscript.\nThe suggestions help us to improve the clarity and presentation of our work, as detailed\nin the point-by-point responses below.\n1. The concept of “atypical EAE” remains unclear. While the authors attribute the\nneuroinflammation observed in DKO mice to IL-4, the co-expression of IL-4 and GM-CSF\nby DKO CD4+ T cells raises the possibility that GM-CSF, rather than IL-4, is the primary\ndriver of neuroinflammation. GM-CSF is a well-established contributor to EAE\npathogenesis, yet the authors have not explored its role in this phenomenon.\nWe thank the reviewers for highlighting the need to clarify GM-CSF's pathological role in\natypical EAE. Atypical EAE, characterized primarily by ataxia, was first described in 1996.\nWhile its pathogenesis is linked to inflammatory changes in the brainstem and\ncerebellum, the underlying mechanisms remain elusive. It has been reported that CNS-\ninfiltrating neutrophils are critical in the pathogenesis of atypical EAE, producing CXCL2\nand recruiting themselves to the brainstem (Yudong Liu et al., J Immunol 2024). Notably,\nIFN- and IL-17 are dispensable for the development of atypical EAE (Mark A Kroenke et\nal., Eur J Immunol 2010; Ti-Ara J Turner et al., J Immunol 2023).\nproduction contributes to atypical EAE (John R. Lukens et al., Immunity 2015). While\ntheir work linked neuroinflammation to neutrophil infiltration, our data revealed\ncomparable neutrophil frequencies and numbers in DKO and WT mice (supplementary\nFig. 1). Moreover, we observed that eosinophils represented an increased fraction of the\ninfiltrating myeloid cell population in DKO mice. This aligns with clinical observations,\neosinophilic vasculitis was also detected in patients with MS (Raphael Schneider et al.,\nAmerican Academy of Neurology 2015; Kongsak Tanphaichitr et al., JAMA Neurology\n1980). Collectively, we hypothesize that Th2 cell-mediated type 2 immunity orchestrates\neosinophil recruitment and pathogenic antibody production via IL-4, thereby promoting\natypical EAE.\nSince GM-CSF can be transiently expressed by several Th subsets (Wanqiang Sheng et\nal., Immunome Res 2015), we propose that elevated GM-CSF production in DKO cells\nmay be related to the increased IL-4-induced STAT5 phosphorylation. GM-CSF is a well-\nestablished mediator of neuroinflammation, recruiting circulating myeloid cells and\nactivating phagocytes, especially microglia, in the CNS (Burkhard Becher et al., Immunity\n2016; Sabine Spath et al., Immunity 2017; Eugene D Ponomarev et al., J Immunol 2017).\nNotably, our data demonstrate that both the proportion and number of microglia are\nsignificantly increased in the CNS of DKO mice, although the role of microglia in atypical\nEAE remains controversial (Alejandro Montilla et al., Cell Death Dis 2023; Stephen J.\nRubino et al., Nat Commun 2018). The potential roles of CNS-elevated GM-CSF in DKO\nmice, particularly regarding neutrophil infiltration and microglia activation, need further\nexploration. Obviously, this is beyond the scope of the current work. We indeed\nappreciate the reviewer’s comment and further discussed this issue in the Discussion\nsection of the revised manuscript (pages 21-22, lines 454-461).\n2. Although the mechanisms underlying Th2-to-Th17 plasticity identified in this study are\nnovel, the advancement of knowledge is limited because the concept of CD4+ T cell\nplasticity is already well established in the field.\nWe thank the reviewers to raise the novelty issues. First, we appreciate the reviewer’s\ncomment that our findings on Th2 to Th17 plasticity are novel. Second, regarding the\ngeneral advancement of knowledge in this field, we provide the following reasonings:\nTh1 and Th2 subsets based on cytokine profiles in 1986, there has been a rapidly\nexpanding literature surrounding CD4+ T cell subsets and making the case for broad\nplasticity among these subsets. Whereas we take for granted the general theory for Th\ncell subset plasticity, key spatial-temporal questions remain unresolved: e.g., does the\nplasticity prefer in a certain direction? or the bifurcation occurs at any stage of T cell\ndifferentiation? Importantly, does such conversion bear biological/clinical relevance?\nSpecifically, Th17 cells have been extensively studied and are considered to have a great\ndegree of plasticity, capable of converting into Th1-like or Treg-like Th17 cells by\nsecreting cytokines such as IFNγ and IL-10. Recent findings have shown their conversion\ninto T follicular helper (Tfh) cells. On the other hand, Th2 cells, as the major players in\ntype 2 immune responses, are widely regarded as a terminally differentiated and stable\nsubset. Currently, apart from studies exploring the association between Th9 and Th2\ncells, research on the conversion of Th2 cells to other subsets remains sparse,\nparticularly regarding the relationship to Th17 cells. Prior work revealed that under in vitro\nculture conditions, classical Th2 effector/memory cells had the potential to produce IL-17\nafter stimulation with proinflammatory cytokines IL-1β, IL-6, and IL-21 (Yui-Hsi Wang et\nal., J. Exp. Med. 2010). In 2022, another group demonstrated that RORγt deletion drives\nthe conversion of mature Th17 cells into Th2-like cells (Xinxin Chi et al., Sci. Adv. 2022).\nHowever, both studies demonstrated terminal-stage T cell plasticity. In our manuscript,\nwe demonstrated that during early-stage of naïve T cell differentiation, loss of IL-4 directs\nthe T cells to differentiate from Th2 into Th17 lineage. Moreover, given the mutual\nantagonism between Th2 and Th1 cells during differentiation, it would be logical to\nhypothesize that IL-4 deficiency promotes a shift from Th2 to Th1 cells, thereby\nenhancing IFN-γ production. Unexpectedly, however, our findings reveal a novel pathway\nfor Th2 cell conversion. More importantly, clinical cases have been reported that patients\ntreated for atopic dermatitis with the IL-4Rα inhibitor may experience the onset of\npsoriasis (cited in the manuscript). However, the underlying mechanisms remained\nunclear. Our work thus provides molecular insights relevant to human diseases.\nTherefore, the current study provides two new findings: (1) The IL-4/JAK3-STAT5 axis\nacts as a checkpoint limiting Th2-to-Th17 conversion, and (2) IL-4 governs Th2/Th17\nplasticity during the initial activation of naïve T cells in inflammatory environment. Such\nfindings advance our knowledge and provide new insights into the “text-book” concept of\nT helper cell plasticity. Notably, these discoveries elucidate paradoxical clinical outcomes\nof biologics targeting IL-4/IL-13 and provide actionable strategies to optimize JAK/STAT\nmodulation across immune disorders. To further address the raised issue, we added\nmore discussion on page 23, lines 485-491 in the Discussion part.\nIn sum, we do appreciate the reviewer’s positive comments about our work, and at the\nsame time, the philosophic point about the general theory of T helper cell plasticity.\nObviously, the plasticity issue is far beyond the scope of this manuscript or probably any\nother single paper. We sincerely believe that the above-described responses have\nsufficiently addressed the reviewer’s concerns.\n15"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed my concerns. The manuscript can be accepted for publication.", "reviewer_2": "All criticism has been appropriately and satisfactorily addressed. I have no further comments and congratulate the authors.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors properly addressed my concern."}, "author_response": ""}]

Decision letter