Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4 þ effector T cells

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1 Received 4 Jul 15 Accepted 9 Fe 16 Pulished 18 Apr 16 DOI: 1.138/ncomms116 OPEN Ndfip-medited degrdtion of Jk1 tunes cytokine signlling to limit expnsion of CD4 þ effector T cells Clire E. O Lery 1, Christopher R. Riling 1, Lynn A. Spruce, Hu Ding, Suresh Kumr 3, Guoping Deng, Yuhong Liu 4, Steven H. Seeholzer & Pul M. Oliver 1, Nedd4 fmily E3 uiquitin ligses hve een shown to restrict T-cell function nd impct T-cell differentition. We show here tht Ndfip1 nd Ndfip, ctivtors of Nedd4 fmily ligses, together limit ccumultion nd function of effector CD4 þ Tcells. Using three-prt proteomics pproch in primry T cells, we identify stiliztion of Jk1 in Ndfip1/-deficient T cells stimulted through the TCR. Jk1 degrdtion is orted in ctivted T cells tht lck Ndfips. In wild-type cells, Jk1 degrdtion lessens CD4 þ cell sensitivity to cytokines during TCR stimultion, while in Ndfip-deficient cells cytokine responsiveness persists, promoting incresed expnsion nd survivl of pthogenic effector T cells. Thus, Ndfip1/Ndfip regulte the cross tlk etween the T-cell receptor nd cytokine signlling pthwys to limit inpproprite T-cell responses. 1 Perelmn School of Medicine, University of Pennsylvni, Phildelphi, Pennsylvni 1914, USA. Deprtment of Pthology nd Lortory Medicine, Cell Pthology Division, The Children s Hospitl of Phildelphi, Phildelphi, Pennsylvni 1914, USA. 3 Progenr Inc, Mlvern, Pennsylvni, 19355, USA. 4 Deprtment of Peditrics, The Children s Hospitl of Phildelphi, Phildelphi, Pennsylvni 1914, USA. Correspondence nd requests for mterils should e ddressed to P.M.O. (emil: pulo@mil.med.upenn.edu). NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms

2 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 Integrtion of signls from T-cell receptor (TCR), co-receptors nd cytokine receptors directs prolifertion, survivl nd differentition of T cells. Cross tlk mong these pthwys is essentil to prevent errnt T-cell responses. One exmple of such cross tlk is TCR-induced downregultion of cytokine receptor signlling to limit cytokine responses 1 4. Uiquityltion of protein sustrtes y E3 uiquitin ligses cn regulte oth TCR nd cytokine receptor signlling. Severl memers of the Nedd4 fmily of E3 ligses hve known roles in T cells, including limiting T H differentition, regulting ctivtion, nd promoting nergy 5 9. However, s unised screens for identifiction of E3 ligse sustrtes, prticulrly in primry cells, re rre, only hndful of protein trgets for Nedd4 E3 ligses hve een identified using trgeted pproches. To dte, pulished sustrtes of these E3 ligses include TCR signlling intermedites nd TCR-ctivted trnscription fctors 5 9. In mice, loss of function of the Nedd4 fmily memer Itch results in CD4 þ T-cell hyperctivtion nd T H cytokine production, leding to spontneous inflmmtion 5,1. Similr immunopthology is oserved in humns with loss of function muttion in Itch 11. In vitro, ctlytic ctivity of Itch nd relted ligses is potentited y, or requires interction with, Nedd4 fmily intercting protein 1 (Ndfip1) nd Ndfip (ref. 1). Loss of function muttions in mice support tht Ndfip1 ctivtes Itch in vivo to limit T cell ctivtion nd T H differentition In vitro inding nd uiquityltion ssys suggest tht Ndfip1 nd Ndfip re oth sufficient to ctivte the ctlytic function of Nedd4-fmily E3 ligses 1,16 19 ; however, n in vivo role for Ndfip is unknown. Here we estlish role for Ndfip in regulting immune responses. Although Ndfip / mice show no overt immunopthology, nimls with T cells lcking oth Ndfip1 nd Ndfip hve drmticlly incresed pool of ctivted CD4 þ T cells compred with mice with T cells lcking only Ndfip1. To determine the mechnism wherey loss of Ndfip1/Ndfip drives expnsion of effector cells, we developed n unised nd reproducile proteomic workflow for use in primry T cells. Consistent with pulished dt, this proteomic pproch confirmed tht the E3 ligses Itch nd Nedd4- (lso clled Nedd4L) re ctive in n Ndfip-dependent mnner in stimulted effector T cells 8,1,13,19. We identified severl cndidte sustrtes for Ndfip-dependent degrdtion, nd vlidted tht Jk1 is errntly degrded in T cells lcking Ndfip1/Ndfip. Jk1 is uiquitylted on multiple lysine residues nd degrded following TCR stimultion of cells; this degrdtion is orted in Ndfip-deficient cells. Consistent with incresed stility of Jk1, T cells lcking Ndfip1/Ndfip fil to undergo TCR-medited downregultion of cytokine responsiveness, Jk1-dependent process, s mesured y STAT5 phosphoryltion following exposure to IL-. Incresed Jk1 signlling led to incresed survivl nd prolifertion of these cells in vitro, while in vivo this drives n expnded popultion of pthogenic effector T cells. Our dt revel tht TCR-induced cytokine non-responsiveness requires Ndfip-dependent degrdtion of Jk1. This is previously unknown function for Ndfips in restricting cytokine signlling to limit expnsion, nd, consequently, pthogenicity, of CD4 þ effector T cells. Results Genertion of Ndfip knockout/gfp knock-in mice. Given tht deficiency in either Itch or Ndfip1 leds to hyperctive T cells nd T H -medited pthology 5,13,15, nd knowing tht Ndfip1 nd Ndfip hve similr functions in vitro 1,16 19, we investigted whether Ndfip might lso ply role in T cells. We generted Ndfip knockout mice y insertion of GFP into exon of the Ndfip gene, putting susequent exons out of frme (Supplementry Fig. 1 c). We oserved Mendelin frequencies of Ndfip / mice (Supplementry Fig. 1d), in contrst to the su-mendelin frequency of Ndfip1 / mice (Supplementry Fig. 1e), t wening. Using Ndfip þ / mice, which express one copy of GFP under the Ndfip promoter, we nlysed GFP s reporter of Ndfip expression. In splenocytes, we oserved the highest GFP expression in T cells (Supplementry Fig. ). In stimulted Ndfip þ / CD4 þ T cells, GFP high cells incresed in undnce over the course of stimultion nd corresponded with CD44 expression, high CD5 expression nd cell division (Supplementry Fig.,c). Ndfip / mice do not show signs of inflmmtion. As our GFP reporter indicted high Ndfip expression in T cells, we focused our nlysis of Ndfip / mice on the T-cell comprtment. Ndfip / mice hd norml thymic popultions nd norml CD4 þ nd CD8 þ T cell percentges in lymph nodes nd spleens (Fig. 1; Supplementry Fig. 3). Ndfip / mice hd no increse in percent of CD44 þ T cells or cytokine production upon ex vivo nlysis (Fig. 1,c; Supplementry Fig. 3). Helper T-cell differentition in vitro, mesured y expression of linege defining trnscription fctors nd cytokine production, ws similr etween Ndifp / nd control cells (Supplementry Fig. 3,c). If Ndfip1 nd Ndfip hve overlpping moleculr functions, expression of Ndfip1 might msk effects of Ndfip in immune cells. Ndfip1 mrna expression is incresed on T-cell ctivtion, consistent with its role limiting errnt ctivtion nd cytokine production in stimulted nive CD4 þ T cells 13,. Compring expression of Ndfip1 nd Ndfip mrna during CD4 þ T cell stimultion reveled tht Ndfip1 ws more roustly induced on initil stimultion thn Ndfip (Fig. 1d). However, Ndfip1 nd Ndfip oth incresed in expression following re-stimultion. Together with our GFP reporter dt, these dt support tht Ndfip expression is incresed in newly ctivted CD4 þ T cells, ut more strongly induced during stimultion of previously ctivted T cells. Ndfip deficiency excertes inflmmtion in Ndfip1 mice. To test whether Ndfip1 expression in Ndfip / mice msked effects of Ndfip deficiency, we generted mice douly deficient in Ndfip1 nd Ndfip. The numer of doule knockout (DKO) pups ws unexpectedly low t wening, nd foetl nlysis indicted su-mendelin frequencies (Supplementry Tle 1). We next generted mice douly deficient for Ndfip1 nd Ndfip in T cells y crossing Ndfip / mice to Ndfip1 fl/ þ CD4 Cre þ mice. Ndfip1 fl/fl CD4 Cre þ mice () exhiit T-cell-medited, T H -ised inflmmtion similr to tht oserved in Ndfip1 / mice. We hypothesized tht loss of Ndfip in Ndfip1 mice would worsen the Ndfip1 phenotype if Ndfip cts to prevent errnt T-cell responses. We oserved tht Ndfip / Ndfip1 fl/fl CD4 Cre þ (referred to s ) mice showed incresed inflmmtion t rrier surfces, incresed spleen size nd decresed ody weight reltive to ge-mtched, Ndfip / nd control mice (Fig. c). Splenic CD4 þ T cells from mice were more likely to express CD44 (Fig. d) nd produce IL-4 (Fig. e,i). We oserved significntly incresed numer of CD4 þ T cells in spleens from mice (Fig. f) due lrgely to n incresed numer of CD44 þ CD4 þ T cells (Fig. g); the numer of CD6L þ CD4 þ T cells in spleens ws not sttisticlly different from nd control mice (Fig. h). NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms116

3 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 ARTICLE c Thymus IL Spleen CD4 CD CD4 IFNγ d 1 Ndfip1 Ndfip CD4+ CD8+ CD6L 71 CD Reltive expression ex vivo 6 h 4 h 48 h Rest 6 h restim 4 h restim Figure 1 Ndfip / mice do not show signs of inflmmtion. (,) Representtive flow cytometry nlysis of T-cell popultions from thymus nd spleen of 5 7-week-old Ndfip / nd ge-mtched control mice: () CD4 þ nd CD8 þ cells, () CD44 nd CD6L expression on these cells, s noted. (c) Intrcellulr cytokine stining for IL-4 nd IFNg in CD4 þ T cells from Ndfip / nd spleens stimulted ex vivo with PMA nd ionomycin in the presence of BFA. Representtive of t lest five mice per genotype, 5 7 weeks of ge. (d) CD4 þ Tcells were stimulted in vitro for the indicted time periods with CD3/CD8. Ndfip1 nd Ndfip expression ws nlysed y qpcr. Ndfip1/Ndfip expression reltive to Act ws normlized to expression in unstimulted CD4 þ T cells. Representtive of minimum of three independent experiments. To seprte the oserved increse in IL-4 producing T cells from the expnded popultion of ctivted CD4 þ T cells in mice, we exmined cytokine production y CD44 þ cells. CD44 þ CD4 þ T cells were somewht more likely to produce IL-4 thn CD44 þ CD4 þ T cells (Fig. j), ut this ws not sttisticlly significnt. Among, nd control mice there ws no difference in the likelihood of CD44 þ CD4 þ T cells to produce IFNg (Fig. k). Together, these dt indicte tht loss of Ndfip in mice with Ndfip1-deficient T cells drives expnsion of effector CD4 þ T cells with pthogenic T H ctivity. Thus, Ndfip, like Ndfip1, negtively regultes T-cell immune responses. Ndfip deficiency cuses intrinsic CD4 þ effector T cell expnsion. Our phenotypic nlysis of Ndfip / Ndfip1 fl/fl CD4Cre þ mice suggested tht loss of oth Ndfip1 nd Ndfip in T cells could led to expnsion of pthogenic T H effector T cells, or tht loss of Ndfip in non-t-linege cells could drive incresed expnsion nd pthogenicity of Ndfip1-deficient T cells. To differentite etween these possiilities, we generted mixed foetl liver chimers. These were nlysed 6 weeks fter reconstitution due to inflmmtory disese in DKO/ cell recipients. In these chimers, DKO CD4 þ T cells were more likely to e CD44 þ compred with CD4 þ T cells within the sme host. The likelihood of eing CD44 þ ws significntly higher for DKO CD4 þ T cells compred with cells in the sme host, ut only trended to e higher for Ndfip1 / CD4 þ T cells reltive to their counterprts (Fig. 3,). CD44 þ DKO CD4 þ T cells were significntly more prolifertive, s indicted y Ki67 stining (Fig. 3c). This ws lso true for Ndfip1 / CD4 þ T cells, though to lesser extent. As previously pulished, loss of Ndfip1 ws sufficient to drive IL-4 production 15 ; further loss of Ndfip resulted in n incresed proportion of CD4 þ T cells producing IL-4, even mong CD44 þ cells, reltive to Ndfip1-deficient cells (Fig. 3d f). Thus, T cells lcking oth Ndfip1 nd Ndfip re much more likely thn either their counterprts or T cells lcking only Ndfip1 to e ctivted in vivo, nd, once ctivted, re more prolifertive nd more likely to produce cytokine. The incresed percent of ctivted Ndfip-deficient CD4 þ T cells suggested competitive dvntge of Ndfip deficiency mong ctivted cells. We nlysed this y determining the reltive frequencies of CD45./CD45.1 cells in different T-cell popultions, normlizing to the chimerism oserved in IgM þ B cells from one mrrow. We found significnt increse in the reltive frequency of CD44 þ CD4 þ DKO T cells isolted from spleen or lung (Fig. 3g). We oserved similr pttern for Ndfip1 / nd, surprisingly, Ndfip / CD4 þ T cells, lthough this did not chieve sttisticl significnce. Thus, the NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms

4 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 Control d Lung Oesophgus e CD6L CD Skin CD4 IL-4 Body weight (g) c Cell count ( 1 6 ) Body weight, 5 7 wks of ge *** *** Spleen cellulrity, 5 7 wks of ge NS ** f g #CD #CD44+ CD3+ CD4+ ( 1 6 ) #Totl CD4+ T cells in spleen #CD44+ CD4+ T cells in spleen * *** *** h i #CD6L+ CD #IL-4+ CD3+ CD4+ ( 1 6 ) #Nive CD4+ T cells in spleen #IL-4+ CD4+ T cells in spleen NS *** j k IL-4+CD44+/CD44+CD4+ IFNγ+CD44+/CD44+ CD %IL-4+ cells of CD44+ CD4+ T cells in spleen NS ** NS %IFNγ+ cells of CD44+ CD4+ T cells in spleen Figure Ndfip deficiency excertes inflmmtion in Ndfip1 fl/fl CD4Cre þ mice. () H&E-stined sections of oesophgus, lung nd skin from representtive 8-week-old control, Ndfip1 fl/fl CD4 Cre þ () nd Ndfip / Ndfip1 fl/fl CD4 Cre þ () mice (r represents 1 mm). (,c) Body weight () nd (c) spleen count of 5 7-week-old,, Ndfip / nd mice. Men±s.e.m. n ¼ 5 15 mice. (d,e) Representtive flow cytometry nlysis of splenic CD3 þ CD4 þ T cells, showing (d) expression CD44 nd CD6L nd (e) intrcellulr levels of IL-4 fter ex vivo stimultion with PMA/ ionomycin in the presence of BFA. (f i) Quntifiction of (f) the numer of CD4 þ T cells, (g) nive CD6L high CD4 þ T cells, (h) CD44 þ CD4 þ T cells nd (i) IL-4þ CD4s from spleen nlysed y flow cytometry. Men±s.e.m., n ¼ 7 1 mice. (j,k) Quntifiction of percent IL-4 þ or IFNg þ CD4 þ T cells mong CD44 high T cells. Men±s.e.m., n ¼ 4 7 mice. P vlues clculted y one-wy ANOVA with Holm Sidk test for multiple comprisons: *Po.5, ** Po.1, *** Po.1, Po.1. popultion of previously ctivted CD4 þ T-effector cells is significntly expnded when oth Ndfip1 nd Ndfip re lcking. Ndfip1/Ndfip-deficient CD4 þ T cells cuse incresed colitis. Our dt indicte tht oth Ndfip1 nd Ndfip control effector cell numers nd pthogenicity. To test this we trnsferred nive, Ndfip /, Ndfip1 nd CD4 þ T cells into Rg1 / recipients to induce colitis. CD4 þ T cells cused severe pthology: recipients hd high spleen/ody weight rtios, contrcted colons nd incresed colon crypt depth (Fig. 4 d). Recipients of Ndfip / CD4 þ T cells showed equivlent pthology to Ndfip1 recipients oth cohorts developed more severe inflmmtion thn cell recipients s mesured y inflmmtion index, lthough colon histopthology, s quntified y crypt depth, ws similr. Ndfip / T cells, like cells, were IL-17A producers, while cells, like Ndfip1-deficient T cells, produced IL-4 (Fig. 4e). Thus, while Ndfip1 plys dominnt role in limiting T H differentition, fter initil ctivtion oth Ndfip1 nd Ndfip limit the pthogenic potentil of ctivted CD4 þ T cells y limiting their expnsion nd function. Ndfip1/Ndfip-deficient T cells outcompete cells in vitro. We then sought to model these phenotypic findings in vitro.when nive CD4 þ T cells were exmined fter 5 dys of in vitro stimultion, cells showed incresed GATA3 expression nd prolifertion reltive to control cells (Supplementry Fig. 4 c). We lso oserved n increse in cell viility reltive to experiment-mtched control cells (Supplementry Fig. 4d). We next tested whether Ndfip-deficient CD4 þ T cells could outcompete cells within the sme cytokine environment. cells co-cultured with CD4 þ T cells hd incresed GATA3 expression nd prolifertion, ut this ws significntly 4 NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms116

5 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 ARTICLE CD6L (CD45.1) (CD45.1) CD44 Ndfip1 / (CD45.) DKO (CD45.) d CD4 (CD45.1) Ndfip1 / (CD45.) (CD45.1) DKO (CD45.) IL-4 e IL-4+ CD3+CD4+ f %IL-4+/CD3+CD4+CD IL-4 production in spleen Ndfip1 / DKO Competitor genotype IL-4+ CD44+ CD4 T cells in spleen 1 ** 8 CD45.1+ CD * 4 Ndfip1 / DKO Competitor genotype CD45.1+ CD45.+ %CD44+ CD3+CD4+ %CD44+ CD4+ T cells in spleen 1 * * 8 CD45.1+ CD Ndfip1 / DKO Competitor genotype c %Ki67+/CD3+CD4+ CD44+ %Ki67+ CD44+ CD4+ T cells 5 8 ** CD45.1+ CD *** 4 Ndfip1 / DKO Competitor genotype g Fold chnge in competitor cells/bm 3 1 Ndfip1 / DKO A B C D E A B C D E A B C D E A B C D E Figure 3 Ndfip deficiency cuses intrinsic CD4 þ effector T-cell expnsion. ( g) Mixed foetl liver chimers using CD45., Ndfip1 /, Ndfip / or Ndfip1/Ndfip DKO foetl liver were nlysed 6 weeks following reconstitution. () Representtive CD44 nd CD6L stining of splenic CD4 þ T cells from Ndfip1 / mixed chimer (top) nd DKO mixed chimer (ottom), previously gted on live, singlet CD3 þ CD4 þ CD45.1 or CD45. þ cells. (,c) Flow cytometry nlysis of CD45.1 þ nd CD45. þ splenic T cells from chimers showing () percentges of CD44 þ cells nd (c) percentges of CD44 þ tht re Ki67 þ.(d f) Ex vivo stimulted splenocytes were stined for IL-4. (d) Representtive flow plots of IL-4 þ CD4 þ T cells, (e) comined dt from d, (f) percentges of CD44 þ CD4 T cells tht re IL-4 þ.(g) Percentges of CD45. cells mong vrious T-cell susets, normlized for reconstitution, s determined y the rtio for CD45.:CD45.1 IgM þ B þ B cells in the one mrrow. Comprtments nlysed re s follows: A ¼ IgM þ B cells in one mrrow, B ¼ doule positive thymocytes, C ¼ single positive CD4 þ thymocytes, D ¼ CD44 þ CD4 þ T cells in spleen, E ¼ CD44 þ CD4 þ T cells in lung. Dt shown in nd e were pooled from two experiments, 7 8 chimers per group; (c,f,g) hve 4 5 chimers per group. Quntifictions re verge ±s.e.m. P vlues clculted y two-wy ANOVA (,c,e,f) or repeted mesures one-wy ANOVA (g), with Holm Sidk test for multiple comprisons: *Po.5, ** Po.1, *** Po.1, Po.1. decresed reltive to cells within the sme culture (Supplementry Fig. 4e i). cells continued to show enhnced viility on dy 5 CD4 þ T cells significntly out-numered cells in the sme culture (Supplementry Fig. 4h,i). Thus, incresed cytokine production in the sence of Ndfips is insufficient to explin incresed viility nd prolifertion. These dt suggest tht loss of oth Ndfip1 nd Ndfip leds to incresed survivl nd prolifertion of ctivted CD4 þ T cells, resulting, in vivo, in n expnded popultion of previously ctivted effector cells. Furthermore, Ndfip-deficient CD4 þ T cells outcompete cells within the sme cytokine milieu, suggesting tht Ndfip-deficient T cells re more competent to respond to cytokines promoting division nd survivl. Identifiction of differentil uiquityltion y proteomics. Hving determined tht Ndfip1 nd Ndfip oth negtively regulte ctivted effector CD4 þ T cells, we wnted to understnd the mechnism underlying the incresed prolifertion nd survivl of Ndfip-deficient CD4 þ T cells. Both Ndfip1 nd Ndfip hve een shown in vitro to ind nd ctivte severl memers of the Nedd4 fmily of E3 uiquitin ligses. While puttive sustrtes of Nedd4 fmily E3 ligses hve een descried in trgeted studies, in primry lymphocytes unised screens to identify sustrtes of these nd other uiquitin ligses re lcking. To ddress this need, we developed three-prt proteomic workflow to identify Ndfip-dependent uiquityltion in ctivted CD4 þ T cells (Fig. 5). We first used stle isotope lelling of mino cids in cell culture (SILAC) in comintion with tndem uiquitin inding entities (TUBEs) 1 to enrich polyuiquitylted proteins from mixed nd Ndfip-deficient cell lystes. We reproducily otined SILAC rtios (/DKO) for B,5 proteins (Fig. 5). In prllel, we performed whole proteome nlysis of DKO nd CD4 þ T cells. We then clculted the unenriched input NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms

6 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 Spleen weight/ody weight Inflmmtion index Colon length Crypt depth * ** * NS.6 *** 7 3 *** Ctrl Colon length (mm) Ctrl c Crypt depth (μm) Ctrl d Control Colon 1 μm e IL IL-4 Figure 4 Ndfip1/Ndfip-deficient CD4 þ T cells cuse incresed colitis. ( e) sorted nive CD4 þ T cells from, Ndfip /, nd mice were trnsferred into 6-week-old Rg1 / recipients. Mice were weighed twice weekly nd killed 6 weeks fter trnsfer when % weight loss ws oserved in multiple mice. Spleen weight nd ody weight were compred to generte n inflmmtion index () nd colons were mesured (). H&E-stined sections of the distl colon were imged on the ojective, nd crypt depth ws quntified (c,d). Splenocytes were stined for intrcellulr IL-4 nd IL-17 fter ex vivo stimultion with PMA/ionomycin in the presence of BFA, nd nlysed y flow cytometry (e). Previously gted on live singlets, CD4 þ, dump gte-. Quntifictions shown ±s.e.m. n ¼ 5 7 mice. Control (ctrl) mice did not receive T cells. P vlues clculted y ordinry one-wy ANOVA with Holm Sidk test for multiple comprisons: *Po.5, ** Po.1, *** Po.1, Po.1. rtio (DKO/) for ech protein nd pplied this correction to the TUBE-enriched SILAC rtio for the sme protein cross ech iologicl replicte (Fig. 5c; Supplementry Fig. 5, nd Supplementry Dt 1). Peptides derived from trypsin clevge of uiquitylted proteins contin di-glycine remnnt (K-e-GG) on modified lysine residues. The vst mjority of such diglycine peptides re derived from uiquitin linkges 3. To limit our nlysis to proteins directly modified y uiquitin, we utilized n ntiody ginst the K-e-GG motif 4,5 to enrich modified peptides from ctivted CD4 þ T cells (Supplementry Dt ). Anlysing proteins with t lest three TUBE SILAC rtios nd t lest one modified lysine (B1, proteins, Fig. 5d) indicted good reproduciility cross replictes (Fig. 5e; Supplementry Dt 3). Thus, despite technicl limittions imposed y nlysing primry lymphocytes, this pproch yields reproducile dt with good depth of proteome coverge. Ndfip-dependent function of Nedd4 fmily ligses in T cells. Ndfip1 nd Ndfip ind nd ctivte the enzymtic function of severl Nedd4 fmily E3 uiquitin ligses in vitro; once ctive, these ligses promote their own uiquityltion 1,16 18,6. Thus, in effector T cells K-e-GG peptides should e oserved for ctive Nedd4 fmily ligses; if these sme ligses depend on Ndfips for function, incresed uiquityltion in cells would result in high /DKO TUBE SILAC rtios. We therefore sought to vlidte our proteomics screen y identifying Nedd4 fmily E3 ligses with Ndfip-dependent function in effector T cells. Among Nedd4 fmily memers, we consistently oserved Itch, Nedd4- nd WWP fter TUBE immunoprecipittion; nlysis of the whole proteome reveled incresed undnce of Itch nd Nedd4- in DKO T cells, while WWP ws not consistently identified (Supplementry Dt 1). Of the Nedd4 fmily ligses, only Itch nd Nedd4- were oserved following diglycine remnnt immunoprecipittion (Supplementry Dt ). Correcting the TUBE SILAC rtios (/DKO) for oth Itch nd Nedd4- to ccount for the incresed undnce of these ligses in Ndfip-deficient T cells indicted significntly more uiquityltion in cells compred with Ndfip-deficient cells (Fig. 5c; Supplementry Fig. 5c). Itch is known to interct with, nd e ctivted y, Ndfip1 in T cells, while Nedd4- hs een shown to work with either Ndfip1 or Ndfip (refs 8,13). In Ndfip douly deficient T cells, decresed ctivity of Itch/Nedd4- could indicte E3 ligse dependence on oth Ndfip1 nd Ndfip, or either Ndfip1 or Ndfip lone. We first ssessed inding of Ndfips to Nedd4 fmily E3 ligses using the cytoplsmic domins of either Ndfip1 or Ndfip to isolte the endogenous E3 ligses from T-cell lystes. Both Ndfip1 nd Ndfip were cple of isolting Itch nd Nedd4- from CD4 þ T cells in this pulldown ssy (Fig. 6). Anlysis of Ndfip1/- deficient cells yielded similr results (Fig. 6). Next, we tested the ility of Ndfip1 nd Ndfip to ctivte Itch enzymtic function, which is restrined through 6 NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms116

7 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 ARTICLE Stimulte cells, rest, restimulte: lyse nd quntify protein Ndfip1 / 1 C CD4+ T cells TUBE enrichment Pixelte gel, digest, LC-MS/MS,5 e Corrected SILAC rtios (/DKO) CD4+ T cells 13 C CD4+ T cells Trypsin digest, nti-k-ε-gg IP LC-MS/MS c Avg corrected TUBE SILAC rtio (log) Nedd4- Itch Avg # unique peptides per protein d Corrected TUBE rtios K-ε-GG proteins 1, Figure 5 Identifiction of differentil uiquityltion y proteomics. () Schemtic of the three proteomics methods used. () Are proportionl Venn digrm illustrting the reproduciility of proteins identified s hving SILAC rtios fter TUBE enrichment in three out of four iologicl replictes. (c) SILAC TUBE rtios (/DKO) for ech protein were corrected using input rtio (DKO/) s clculted y lel-free quntifiction of whole proteome DKO nd dt sets. The verge corrected rtio is plotted ginst the verge numer of unique peptides oserved per protein cross four whole proteome LC-MS/MS experiments. Plot limited to 4 verge unique peptides for clrity. Itch nd Nedd4- (indicted) hve log trnsformed corrected rtio 41 t the protein level. Itch ¼ 1.57±.17 nd Nedd4- ¼ 3.18±.6. (d) Overlp of proteins with corrected SILAC TUBE rtios (oserved in t lest three experiments) nd proteins identified with t lest one K-e-GG peptide (in t lest one of the three K-e-GG immunoprecipittion experiments). (e) Het mp illustrting reproduciility of corrected SILAC TUBE rtios (/DKO, log trnsformed, oserved in t lest three of four experiments) for proteins identified with K-e-GG peptides. Reverse ¼ SILAC leling swpped. Reverse Expt1 Expt Expt3 utoinhiition 1,7,8. Using time-resolved fluorescence resonnce energy trnsfer (TR-FRET) ssy 1, we oserved Itch uiquityltion ctivity only in the presence of Ndfip1 or Ndfip (Fig. 6). While unequl purity of the GST fusion Ndfips mde quntittive comprisons of ctivtion difficult (Supplementry Fig. 5d), we cn conclude tht oth Ndfip1 nd Ndfip re sufficient to ctivte the E3 ligse function of Itch. We recently determined tht Ndfip1 ctivtes Itch ctlytic ctivity y promoting uiquitin chrging of Itch 9. However, the role of Ndfip in this process is unknown. We found tht either Ndfip1 or Ndfip is sufficient for E-medited uiquitin chrging of Itch (Fig. 6c), indicting tht Ndfip1 nd Ndfip ctivte Itch vi the sme moleculr mechnism. Thus, oth Ndfips could promote the uiquityltion ctivity of Itch oserved in our proteomic nlysis. We then ssessed whether Nedd4- is ctivted y Ndfips. Under our cell-free in vitro conditions using the humn homologue of Nedd4-, Nedd4L, we oserved tht Nedd4L ws not utoinhiited; nevertheless, n incresed rte of Nedd4L ctivity ws evident when Ndfip1/Ndfip were included in the rection (Fig. 6d,e). Thus, while Ndfips re not required for function of Nedd4L in these ssys, they potentite its ctlytic ctivity. Both Itch nd Nedd4-/Nedd4L hve een pulished to work with Ndfips in T cells, nd re known to utouiquitylte when ctivted, indicting tht our ssy cn identify Ndfipdependent ligse ctivity nd chnges in Ndfip-dependent uiquityltion in n unised fshion 8,1,13,15,19,9. Jk1 degrdtion is dependent on Ndfip1/Ndfip. Hving vlidted tht our screen cn identify Ndfip-dependent uiquityltion, we turned to sustrte identifiction. Cndidte sustrtes of Ndfip-dependent uiquityltion (Supplementry Dt 4) hd positive corrected TUBE SILAC rtios (/DKO) in ll replictes, low error cross replictes, nd multiple K-e-GG peptides. We identified Jk1 nd Jk s differentilly uiquitylted in Ndfip-deficient cells, nd oserved K-e-GG peptides from ll Jk fmily memers (Supplementry Fig. 6,). Anlysis of Jk, which is known to e monouiquitylted, nd for which suppressor of cytokine signlling (SOCS)-medited degrdtion hs een demonstrted 3, reveled tht Jk is not roustly degrded during TCR stimultion (Supplementry Fig. 6c,d). Although Jk1 uiquityltion hs never een shown in T cells, it is known to hve short hlf-life 3.Consistentwith uiquityltion of Jk1, we oserved tht severl distinct lysines in Jk1 hd the K-e-GG motif. Jk1 is key component in signlling vi common g chin contining cytokine receptors, nd cn drive survivl, prolifertion nd differentition of NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms

8 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 IP: Blot CD4+ T cells Input GST Ndfip1cyto Ndfipcyto DKO CD4+ T cells Input GST Ndfip1cyto Ndfipcyto Itch Nedd4- TRF rtio Ndfip1 + Itch Ndfip + Itch Itch Coomssie Ndfipcyto Ndfip1cyto GST Repet c d e IB: SA (itoin-ub) Coomssie Itch +Ndfip1 +Ndfip Itch~U E1~U Itch Ndfip Ndfip1 TRF rtio Repet Ndfip1 + Nedd4L Ndfip + Nedd4L Nedd4L TRF rtio Repet Ndfip1 + Nedd4L Ndfip + Nedd4L Nedd4L Figure 6 Ndfip1 nd Ndfip promote Itch nd Nedd4- ctivity. () Immunolot of Itch nd Nedd4- isolted from ctivted or Ndfip1/Ndfip DKO CD4 þ T-cell lystes y GST pulldown of Ndfip1 nd Ndfip cytosolic domin GST fusion proteins. Coomssie stin for the GST fusion proteins reveled full-length products for ech construct s well s GSTclevge product. () Itch ctivity, in the sence or presence of Ndfip1 or Ndfip, ws nlysed vi TR-FRET polyuiquityltion ssy. (c) E/E3 trnsthioltion ssy ws used to test whether Ndfip promotes uiquitin chrging of Itch. Biotinylted uiquitin non-covlently ound to Itch ws nlysed y western lot using fluorescent streptvidin (top); totl protein ws visulized y Coomssie stin (ottom). (d,e) As in, humn Nedd4L ctivity ws ssessed lone or in the presence of Ndfip1 or Ndfip y TR-FRET. Dt for ech pnel is representtive of minimum of three independent experiments. ctivted T cells Therefore, we hypothesized tht Jk1 degrdtionincd4þ T cells depends on Ndfip1 nd Ndfip. Jk1 ws roustly degrded in TCR-stimulted cells treted with cycloheximide, trnsltion inhiitor (Fig. 7). Ndfip-deficient CD4 þ T cells showed significntly less degrdtion (Fig. 7,). We lso oserved incresed phosphoryltion of STAT5/ fter stimultion of Ndfip-deficient cells compred with cells (Fig. 7c,d). We did not oserve ny chnge in Jk1 stility or p-stat5/ signl in CD4 þ T cells from Ndfip1 mice, suggesting tht loss of Ndfip1 lone is not sufficient to ffect Jk1 stility nd STAT5 ctivity (Fig. 7 d). Levels of totl STAT5 were not significntly incresed in CD4 þ T cells, nd STAT5 did not show cycloheximide sensitivity (Fig. 7e,f), consistent with pulished dt 37. Thus incresed levels of p-stat5 re indictive of incresed Jk1 ctivity in Ndfipdeficient cells. A lnce of protein synthesis nd degrdtion controls Jk1 levels during T cell ctivtion, Jk1 trnsltion is hlted nd existing Jk1 is degrded 3. For oth nd Ndfip-deficient cells, reltive levels of Jk1 remining fter stimultion were not ltered y cycloheximide (Fig. 7,,g). Ndfip-deficient cells showed incresed Jk1 fter stimultion in either condition, indicting tht Jk1 protein synthesis is terminted normlly in stimulted cells. Furthermore, this indictes tht Jk1 stility in T cells is not impcted y new protein synthesis (e.g., production of cytokines). Overll, Jk1 degrdtion ws significntly fster in CD4 þ T cells compred with cells (Fig. 7h). Jk1 degrdtion during the first hours of TCR stimultion ws intct in Ndfip-deficient cells; however, further Jk1 degrdtion ws orted (Supplementry Fig. 7,). As with Ndfip1, loss of Ndfip lone ws not sufficient to ffect Jk1 stility during TCR stimultion (Supplementry Fig. 7c). If Jk1 degrdtion is promoted y Ndfip-dependent E3 uiquitin ligses, either directly or indirectly, then unstimulted cells, with ongoing Jk1 synthesis, should show incresed Jk1 over time in the sence of Ndfips. In unstimulted cells, we found tht Jk1 in cells ws stle, ut in the sence of Ndfip1/Ndfip Jk1 levels stedily incresed (Fig. 7i). After 4 h in the sence or presence of TCR fter trnsltionl recovery occurs in TCR-stimulted cells Jk1 ws significntly incresed in CD4 þ T cells s compred with cells (Fig. 7j). Ndfip-deficient T cells show persistent cytokine signlling. High Jk ctivity cn led to incresed cell viility nd prolifertion through STAT5 ctivtion nd susequent trnscription of STAT5 trget genes Consistent with this, we oserved incresed expression, ut not errnt degrdtion, of the STAT5 trget cyclin D (refs 31,43,44) in stimulted Ndfip1/Ndfip-deficient CD4 þ T cells (Supplementry Fig. 7d). Similrly, surfce expression of the IL- receptor (IL-R, k CD5), nother STAT5 trget 45, ws incresed on stimulted cells compred with CD4 þ T cells, even when these cells were co-cultured (Supplementry Fig. 7e,f). To determine if Jk1 stiliztion medites the oserved in vitro nd in vivo hyperctivtion nd hyperviility of Ndfip-deficient CD4 þ T cells, we tested whether Jk inhiition could normlize levels of prolifertion/survivl. CD4 þ T cells stimulted in vitro in the presence of Jk inhiitor showed reduced prolifertion, down to levels, in 8 NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms116

9 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 ARTICLE αcd3/8 ±CHX: %Jk1 remining reltive to c αcd3/8 hours: d p-stat5/ reltive to CD4+ CD CD4+ CD h 6 h Jk1 GAPDH f STAT5 reltive to h p-stat5 Tuulin ctrl e αcd3/8 ±CHX: g Jk1 reltive to rest CD4+ CD TCR stimultion (h) h TCR CHX j STAT5 Tuulin 4 h Jk1 reltive to rest i Jk1 reltive to rest h Rte of Jk1 degrdtion (RU per hour) * No stimultion (h) * +TCR * TCR Figure 7 Jk1 degrdtion is dependent on Ndfip1/Ndfip. ( j) Immunolotting of restimulted nd CD4 þ T cells. () Cycloheximide ws dded h fter CD3/CD8 stimultion; cells were then incuted for n dditionl 4 h. () Level of Jk1 ws normlized to GAPDH. The stility of Jk1 ws determined y normlizing the percent Jk1 remining in or cells to the percent remining in experiment-mtched control cells. Dt shown re verge ±s.e.m. from three to five iologic replictes in more thn three experiments. (c,d) p-stat5 ws quntified reltive to tuulin. Reltive levels of p-stat5 in nd CD4 þ T cells were normlized to experiment-mtched control cells t nd 6 h of restimultion. Dt shown is verge ±s.e.m. for two iologic replictes. (e,f) Immunolotting of totl STAT5 in restimulted nd CD4 þ T cells. Cells were stimulted for h, nd cycloheximide ws dded for n dditionl h of stimultion. (f) Level of STAT5 ws normlized to GAPDH. The stility of STAT5 ws determined y normlizing the reltive STAT5 remining fter stimultion to the mount of STAT5 t h. Dt shown re verge ±s.e.m. from three iologic replictes. (g j) Levels of Jk1, normlized to GAPDH, t vrious timepoints following restimultion of nd CD4 þ T cells reltive to Jk1 in IL--rested cells t time. (h) Rte of Jk1 degrdtion in reltive units per hour over 6 h of stimultion. (i,j) Levels of Jk1, normlized to GAPDH, t vrious timepoints following rest in the sence of IL-/TCR, of nd CD4 þ T cells reltive to Jk1 in IL--rested cells t time. (j) Asing nd i, levels of Jk1, normlized to GAPDH, 4 h ±TCR stimultion of nd CD4 þ T cells reltive to Jk1 in IL--rested cells t time. Dt shown in g j verge ±s.e.m. from two to four iologic replictes in more thn three experiments. P vlue clculted y two smple, unpired t-test *Po.5. dose-dependent mnner; cell prolifertion ws responsive to Jk inhiition ut did not show dose dependence, suggesting complete Jk inhiition occurred t lower dose (Fig. 8,). CD5 decresed in oth nd CD4 þ T cells in dosedependent mnner (Supplementry Fig. 7g). Strikingly, CD4 þ T cells showed roust decline in viility, down to levels, when treted with the Jk inhiitor (Fig. 8,c). To determine if sensitivity to Jk inhiition ws dependent on incresed cytokine vilility, we co-cultured nd CD4 þ T cells nd treted with the Jk inhiitor. This reveled distinct regultion of viility nd prolifertion. cells cultured with cells now showed dose-dependent decreses in prolifertion in the presence of Jk inhiition, lthough they still proliferted less thn cells (Supplementry Fig. 8,). In contrst, the effect of Jk inhiition on viility ws independent of incresed cytokine production: the enhnced viility of cells ws lost upon tretment with the Jk inhiitor, which hd limited effect on cell viility (Supplementry Fig. 8c). Therefore, following ctivtion, the incresed survivl nd prolifertive cpcity of CD4 þ T cells is due to incresed Jk-dependent cytokine signlling. Incresed cell viility is independent of incresed cytokine production, nd cn e normlized y Jk inhiition. During cute TCR signlling, cytokine responsiveness decreses; Jk1 degrdtion is thought to e required for this desensitiztion 1 3. In TCR stimulted CD4 þ T cells, decresed induction of STAT5 phosphoryltion downstrem of the IL- receptor limits effector cell expnsion y inducing growth rrest nd poptosis 1. To test whether Jk1 stiliztion in Ndfipdeficient T cells llows persistent cytokine signlling, we exmined STAT5 phosphoryltion fter IL- tretment in cells tht were or were not receiving TCR signls (Fig. 8d,e). In unstimulted cells, p-stat5 ws significntly incresed in CD4 þ T cells reltive to cells on IL- tretment, consistent with the incresed levels of Jk1 oserved in cells t this time (Fig. 7j). Strikingly, CD4 þ T cells stimulted through NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms

10 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 Ded DMSO CFSE Jk inhiitor %Divided 4 or more times reltive to untreted *** DMSO ** *** Jki(nM) c Viility reltive to untreted DMSO Jki(nM) d TCR pretret +IL- No TCR +IL- No TCR e p-stat5 MFI 6 NS *** ** 4 *** ctrl IL p-stat5 CD3/CD8 pretreted Figure 8 Ndfip-deficient T cells show persistent cytokine signlling. ( c) Sorted nive CD4 þ T cells from nd mice were stimulted with CD3/CD8 in the presence of Jk inhiitor I (JAKi) nd nlysed on dy 5 y flow cytometry. () Representtive plots of CFSE dilution nd viility stining in nd CD4 þ T cells ±Jk inhiitor (31. nm). (,c) Quntifiction of () percent of nd cells divided four or more times reltive to DMSO-treted experiment-mtched cells nd (c) viility of nd CD4 þ T cells normlized to DMSO-treted experimentmtched cells. Dt shown is verge ±s.e.m. from six iologic replictes. P vlues clculted y multiple t-test with Holm Sidk correction. (d,e) p-stat5 stining in nd CD4 þ T cells rested in the sence of IL- overnight then treted ±CD3/CD8 eds efore ddition of IL-. (d) Representtive flow cytometry histogrms. (e) Quntifiction of p-stat5 MFI for eight to nine iologic replictes nd four independent experiments, showing verge ±s.e.m. Ptterned rs indicte ddition of exogenous IL-. P vlues clculted y two-wy ANOVA with Holm Sidk test for multiple comprisons. *Po.5, ** Po.1, *** Po.1, Po.1. their TCR filed to phosphorylte STAT5 in response to IL-, while in cells there ws no difference in the p-stat5 response to IL- in the presence or sence of TCR signlling (Fig. 8d,e). Indeed, cells exhiited strong STAT5 phosphoryltion under TCR stimultion lone, without ddition of exogenous IL- (Fig. 8e). This ws significntly decresed when IL- ws locked in TCR stimulted CD4 þ T cell cultures, indicting tht cells persistently signl through IL- in n utocrine or juxtcrine fshion while ctively signlling through the TCR (Supplementry Fig. 8d). Thus, Ndfip1 nd Ndfip re required for TCR-induced cytokine desensitiztion, nd, in the sence of Ndfips, stiliztion of Jk1 cn drive persistent IL- signlling to promote cell survivl nd prolifertion. Discussion Fine-tuned control of signlling downstrem of the TCR, co-receptors nd cytokine receptors is criticl in ctivted T cells. Pertured signlling cn lter T cell function, differentition, survivl nd prolifertion with life-thretening consequences. One lyer of control is degrdtion of key signl trnsducers vi uiquityltion to ttenute ctivting signls. Consistent with this, prior work hs detiled the errnt CD4 þ T-cell responses tht occur in the sence of the E3 ligse Itch nd one of its known ctivtors, Ndfip1 (refs 5,9,13 15,46). Here we uncover role for Ndfip, nother ctivtor of Itch nd relted ligses, in limiting effector T-cell immunopthology y controlling effector cell expnsion. Unlike Ndfip1, Ndfip does not impct nive T-cell ctivtion or differentition; however, together Ndfip nd Ndfip1 limit the expnsion/persistence of effector CD4 þ T cells. In previously ctivted CD4 þ T cells, Ndfip1 nd Ndfip re cting in coordinted, ut perhps non-redundnt, mnner to prevent errnt T cell responses. While expression of oth Ndfip1 nd Ndfip is incresed during T-cell stimultion our dt indicte distinct mgnitude nd kinetics, providing one possile explntion for why Ndfip knockout mice do not phenocopy mice lcking Ndfip1. Nothing is known out how Ndfips intrinsiclly regulte T-effector cells. To investigte this, we developed proteomics workflow in primry CD4 þ T cells for unised identifiction of differentil uiquityltion in primry T cells. Using whole cell proteome nlysis, polyuiquitin pulldown nd diglycine remnnt nlysis, we exmined differentil uiquityltion in CD4 þ T cells sufficient or deficient for Ndfips. Supporting the roustness of this proteomic pproch, our nlysis identified tht, of nine Nedd4 fmily ligses, Nedd4- nd Itch re likely enzymticlly ctive during stimultion of effector CD4 þ T cells nd utouiquitylted in n Ndfip-dependent mnner. This is consistent with previously pulished work 8,13. Biochemiclly, we vlidte tht ctlytic function of oth Itch nd Nedd4- cn e ctivted or potentited y Ndfip1 nd Ndfip, indicting tht our screen successfully identifies Ndfip-dependent uiquityltion events. Using this proteomics pproch we identified Jk1 s errntly uiquitylted in the sence of Ndfips. Jk1 hs multiple uiquitylted lysines, nd ws more stle in stimulted Ndfip-deficient CD4 þ T cells. The hyperviility nd hyperprolifertion of Ndfip-deficient T cells ws locked y Jk inhiition. These inhiitor experiments reveled tht, while hyperviility 1 NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms116

11 NATURE COMMUNICATIONS DOI: 1.138/ncomms116 ARTICLE ws dependent on incresed Jk signlling independent of cytokine production, the role of Jk in prolifertion ws proportionte to cytokine vilility. Consistent with Jk1 stiliztion, Ndfip-deficient cells filed to undergo TCR-medited cytokine non-responsiveness stimulted Ndfip-deficient cells remined sensitive to IL-, ctivting signlling cscdes downstrem of Jk1 tht promote survivl nd prolifertion. These dt support tht Ndfip-dependent Jk1 degrdtion plys n importnt role in limiting cytokine responsiveness during TCR stimultion, lthough whether this is direct effect of Ndfip-medited ctivtion of Jk-trgeting E3 ligses remins to e seen. Notly, in the sence of Ndfips, Jk1 degrdtion did occur fter TCR stimultion, ut ws limited temporlly. Thus, initil Jk1 degrdtion is Ndfip-independent. If Ndfip-dependent ligses (for exmple, Nedd4 fmily ligses) trget Jk1 for degrdtion directly, then one possiility is tht these ligses re ctivted y n Ndfip-independent mechnism downstrem of erly TCR signlling, llowing them to uiquitylte Jk1. Activtion of these ligses cn occur vi phosphoryltion (Itch, Nedd4, Nedd4-) nd clcium inding (Smurf, Nedd4, Nedd4-) However, fter termintion of erly signlling events tht could relieve ligse utoinhiition in clcium or phosphoryltion-dependent mnner, ligse ctivity my depend on Ndfips. Alterntively, Nedd4 fmily E3 ligses my e dispensle for erly Jk1 degrdtion. Nevertheless, Ndfipindependent degrdtion of Jk1 in CD4 þ T cells is clerly not sufficient to terminte cytokine signls, s Ndfip-deficient cells fil to undergo TCR-medited cytokine non-responsiveness. Single deficiency in either Ndfip1 or Ndfip ws not sufficient for Jk1 stiliztion. These dt suggest tht Ndfip1 nd Ndfip compenste for one nother in promoting Jk1 degrdtion in restimulted effector CD4 þ T cells. In these previously ctivted cells, expression of oth Ndfips is induced eqully well. One intriguing ide is tht incresed sensitivity of previously ctivted T cells to TCR restimultion necessittes incresed ction of Ndfip-dependent E3 ligses, nd therefore incresed overll expression of Ndfips is required for efficient ctivtion of lrger pool of these E3s. In ddition, the sequence of Ndfip1 contins solvent lysine residues in close proximity to its PY motifs, which my e preferentilly uiquitylted when Ndfip1 intercts with nd ctivtes Nedd4 fmily E3 ligses 5. Ndfip does not hve these lysines while this my negtively impct its ility to ctivte Nedd4 fmily E3 ligses 5, it my lso extend its hlf-life, therey providing longer lsting, lthough less efficient, ctivtion signl for Nedd4 E3 ligses. While our work directly reltes to IL- promoting survivl nd prolifertion, incresed IL- signlling hs other effects on T cells. Incresed phosphoryltion of STAT5 downstrem of IL- receptor signlling promotes T H differentition 33,53 nd limits T FH differentition 54. Previously pulished sustrtes of Ndfipdependent ligses lso ply roles in promoting T H differentition nd inhiiting T FH development 5,8,9. While degrdtion of Nedd4 fmily sustrtes could promote T FH nd limit T H differentition, regultion of Jk1 levels, either directly or indirectly y Ndfip-dependent E3 ligses, nd downstrem cytokine signlling could ugment these pthwys in coopertive or synergistic mnner. We propose model in which Ndfip1 nd Ndfip work together to limit cytokine signlling y promoting Jk1 degrdtion (Supplementry Fig. 9). In effector T cells not undergoing TCR stimultion, Ndfip1 nd Ndfip mintin Jk1 levels in lnce with protein synthesis. When T cells re ctivted vi their TCR, Ndfip1 nd Ndfip enforce period of cytokine non-responsiveness, y promoting Jk1 degrdtion, to restrict the effector pool. Promoting TCR-medited downregultion of cytokine signlling is previously unknown role for Ndfip1 nd Ndfip nd their cognte E3 ligses. We propose tht Ndfip-dependent degrdtion of Jk1 is criticl in determining effector CD4 þ T cell numers tht, when disrupted, leds to pthologic ccumultion of effector CD4 þ T cells. Methods Mouse strins. Ndfip1 / nd Ndfip1 fl/fl CD4Cre þ mice hve een descried 13,. Ndfip1 / mice hve een ckcrossed to the C57BL/6 for more thn 1 genertions; Ndfip1 fl/fl mice were derived from insertion of loxp sites into C57BL/6 emryonic stem cells nd re mintined on C57BL/6 ckground. CD45.1 þ (C57BL6.SJL-Ptprc Pepc/BoyJ) nd Rg1 / (B6.19S7- Rg1tm1Mom/J) re mintined in house. Ndfip knockout/gfp knock-in mice were generted y Tconic Biosciences, Inc. (TconicArtemis) s descried in Supplementry Fig. 1 using C57BL/6 emryonic stem cells, nd re mintined on C57BL/6 ckground. Animls were used t 5 7 weeks of ge with the exception of the 16-week-old Ndfip / nd control mice used for phenotypic nlysis in Supplementry Fig. 1. Rg1 / hosts for chimer experiments were 8 weeks old. Control nimls were Ndfip1 fl/fl Cre-, Ndfip1/Ndfip þ / þ, or CD45.1 s pproprite. Ndfip1/ DKO mice were generted y reeding Ndfip / Ndfip1 þ / femles to Ndfip þ / Ndfip1 þ / mles. mice were generted y reeding Ndfip / Ndfip1 fl/fl CD4Cre- mice to Ndfip / Ndfip1 fl/ þ CD4Cre þ mice. We oserved no sex differences in Ndfip / or mice experiments were performed on oth femle nd mle mice using pproprite sex/ge-mtched controls, nd dt shown re pooled mle nd femle mice. Mice were mintined in rrier fcility t the Children s Hospitl of Phildelphi. All procedures were pproved y the Institutionl Cre nd Use Committee of the Children s Hospitl of Phildelphi. Ndfip KO/KI lleles were detected using the following primers on genomic DNA: _F 5 -CCCTGT GCCACCTCCGTACAGTG-3 ; _R 5 -GCTGAGGCAGTGCGCAGACTTA C-3 ; KO/KI_F 5 -CTTCAAGCAGACCTACAGCAAG-3 ; KO/KI_R 5 -CCTG TTATCCCTAGCGTAACG-3. The FLP trnsgene ws detected using the following PCR primers on genomic DNA: FLP_F 5 -GACAAGCGTTAGTA GGCACAT-3 ; FLP_R 5 -GGCAGAAGCACGCTTATCG-3. All nimls used in experiments descried were FLP negtive. Genotyping primers for Ndfip1 knockout lleles, Ndfip1 flox lleles nd CD4Cre hve een descried previously. Flow cytometry nd immunolot ntiodies. All flow cytometry ntiodies were used t 1: unless otherwise noted. The following flow cytometry ntiodies were purchsed from Biolegend: CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), IL-17A (TC11-18H1.1), CD3e (17A), B (RA3-6B), CD45. (14) nd CD45.1 (A). Antiodies ginst IFNg (XMG1.) nd Ki67 (B56) were purchsed from BD Biosciences. The remining ntiodies for flow cytometry were purchsed from ebioscience: IgM (II/41), CD5 (PC61.5), CD6L (MEL-14), GATA3 (TWAJ), IL-4 (11B11), IgM (II/41), T-et (4B1), FoxP3-iotin (FJK-16s, 1:15), CD93/AA4.1 (AA4.1). Biotinylted FoxP3 ws detected with fluorophoreconjugted streptvidin (S3354 Invitrogen, 1:5). p-stat5/ for flow cytometry ws purchsed from BD (p Y694, 47). The following primry ntiodies were used for immunolotting: rit Jk1 (333; Cell Signling Technologies, 1:5), monoclonl rit Jk (DE1, Cell Signling Technologies, 1:5), rit p-stat5/ (C11C5; Cell Signling Technologies, 1:5), monoclonl mouse tuulin (B-5-1-; Sigm, 1:1,), monoclonl mouse gpdh (m374; Millipore, 1:,), monoclonl mouse Itch (3; BD Biosciences, 1:5), rit Nedd4- (413, Cell Signling Technologies, 1:5), rit Cyclin D (M-, Snt Cruz Biotechnology, 1:1,) nd monoclonl rit STAT5 (E89, Acm, 1:5). GST pulldown. Ndfip cytoplsmic domin constructs were prepred s previously descried 9. Plsmids were trnsformed into BL1(DE3) Escherichi coli nd purified y glutthione sephrose 4B (GE Life Sciences). nd Ndfip1/Ndfip knockout CD4 þ T cells were cultured s descried elow. Rested cells were restimulted for 4 h with PMA/ionomycin s descried elow, lysed in 1% NP4 (1% NP4, 15 mm NCl, 5 mm TrisHCl with phosphtse nd protese inhiitors), preclered with GST, nd then incuted with 1 mg recominnt Ndfip1, Ndfip or GST protein. Protein ws collected on glutthione sephrose eds, nd eluted y ddition of 4 Lemmli smple uffer with oiling. Bound protein ws nlysed y SDS polycrylmide gel electrophoresis (SDS PAGE) nd western lotting s descried elow. Polyuiquityltion ssy nd uiquitin chrging ssy. Polyuiquityltion ws monitored using homogeneous E3 ligse TR-FRET ssy (Progenr, Inc.) s descried 1. Briefly, uiquityltion Mix contining Itch or Nedd4L ws comined with vrying doses of Ndfip1/ nd HTRF Detection Mix. TR-FRET ws monitored in rel time using PerkinElmer Envision plte reder (Ex 34 nm; Em1 5 nm; Em 48 nm); TR-FRET rtio ws clculted s Em 5 /Em 48. Uiquitin chrging ssy nd expression of recominnt Itch were descried y Riling et l. 9 : recominnt E, E1, nd uiquitin were incuted on ice in the presence of ATP nd mgnesium to generte pre-chrged uiquitinbe conjugtes. This chrging rection ws quenched y ddition of EDTA, fter which E3 ligse (Itch) ws NATURE COMMUNICATIONS 7:116 DOI: 1.138/ncomms