Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins

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1 Clcineurin imposes T cell unresponsiveness through trgeted proteolysis of signling proteins Vigo Heissmeyer, Fernndo Mcián,5, Sin-Hyeog Im,5, Rjt Vrm 2, Stefn Feske, K Venuprsd 3, Hu Gu 4, Yun-Ci Liu 3, Michel L Dustin 2 & Anjn Ro Sustined clcium signling induces stte of nergy or ntigen unresponsiveness in T cells, medited through clcineurin nd the trnscription fctor NFAT. We show here tht C 2 -induced nergy is multistep progrm tht is implemented t lest prtly through proteolytic degrdtion of specific signling proteins. Clcineurin incresed mrna nd protein of the E3 uiquitin ligses, Cl- nd GRAIL nd induced expression of Tsg0, the uiquitin-inding component of the ESCRT- endosoml sorting complex. Susequent stimultion or homotypic cell dhesion promoted memrne trnsloction of nd the relted protein Nedd4, resulting in degrdtion of two key signling proteins, nd. T cells from - nd Cl-deficient mice were resistnt to nergy induction. Anergic T cells showed impired clcium moiliztion fter TCR triggering nd were unle to mintin mture immunologicl synpse, insted showing lte disorgniztion of the outer ring contining lymphocyte functionssocited ntigen. Our results define complex moleculr progrm tht links gene trnscription induced y clcium nd clcineurin to prdoxicl impirment of signl trnsduction in nergic T cells. In ddition to ctivting signling pthwys tht hve positive effect, receptor stimultion induces negtive feedck pthwys tht ttenute or terminte positive signling. One of the est-documented mechnisms of countering productive responses involves removl of phosphte groups or other ctivting modifictions from proteins tht medite signl trnsduction (receptors, kinses, dpter proteins nd trnscription fctors ). In nother mechnism, positive signls increse the mounts or ctivities of negtive regultors or inhiitory proteins; indeed, mny genes tht re rpidly induced y ctivtion of signling pthwys encode proteins tht hve negtive effects in the sme pthwy 2. In third mechnism, ctivted signl trnsducers re selectively trgeted for degrdtion, terminting ongoing signls nd lso interfering with susequent stimultion. Cytoplsmic signling proteins nd nucler trnscription fctors tend to e polyuiquitinted nd trgeted for protesoml degrdtion 3, wheres lignd-ctivted surfce receptors, including receptor tyrosine kinses, G proteincoupled receptors nd the T cell receptor (TCR) re more often degrded y endocytosis nd trfficking to the lysosome 4,5. Induced endocytosis of ctivted receptors t the plsm memrne nd sorting of the receptors into multivesiculr odies t the endosoml memrne is regulted through tgging of receptor or dptor proteins with uiquitin 4. Together these mechnisms ensure lnced response to extrcellulr signls nd protect cells from the deleterious effects of chronic ctivtion. In reltively common scenrio, single second-messenger or signling molecule simultneously medites oth positive nd negtive outcomes downstrem of surfce receptors. This point hs een well illustrted in T cells, in which C 2 signling is essentil not only for prolifertion nd effector function ut lso for imposition of n nergic stte in which positive signls cnnot e initited or re sustntilly delyed or ttenuted 6. Sustined signling through C 2 nd clcineurin results in sustined ctivtion of the trnscription fctor NFAT, which in turn induces mny genes encoding effector cytokines, chemokines nd other products in the productive immune response 7. However the sme trnscription fctor, when prectivted in the sence of its trnscriptionl prtner AP- (Fos-Jun), induces different set of genes encoding known or presumed negtive regultors of T cell signling, thus mediting n opposing progrm of T cell nergy or tolernce 8. Among the negtive molecules induced in these conditions re severl tyrosine phosphtses tht would e expected to downregulte TCR signling y opposing the effects of tyrosine kinses such s Zp70, Lck nd Itk; dicylglycerol kinse-α, which metolizes the second messenger dicylglycerol; nd genes encoding severl proteses nd E3 uiquitin ligses, which might induce T cell nergy y promoting degrdtion of downstrem signling molecules in T cells. The interfce etween the T cell nd the ntigen-presenting cell (APC) is n importnt site for regultion of TCR signling. In the Center for Blood Reserch nd Deprtment of Pthology, Hrvrd Medicl School, 200 Longwood Avenue, Boston, Msschusetts 025, USA. 2 Progrm in Moleculr Pthogenesis nd Deprtment of Pthology, Skirll Institute of Moleculr Medicine, New York University School of Medicine, New York, New York 006, USA. 3 Division of Cell Biology, L Joll Institute for Allergy nd Immunology, Sn Diego, Cliforni 922, USA. 4 Deprtment of Microiology, Columi University, College of Physicins nd Surgeons, 70 West 68th Street, HHSC, New York, New York 0032, USA. 5 Present ddresses: Deprtment of Pthology, Alert Einstein College of Medicine, Bronx, New York 046, USA (F.M.) nd Deprtment of Life Science, Kwngju Institute of Science nd Technology, Oryong-dong, Puk-ku, Kwngju , Kore (S.-H.I.). Correspondence should e ddressed to A.R. (ro@cr.med.hrvrd.edu). Pulished online 5 Ferury 2004; doi:0.038/ni047 NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH

2 CD3ζ Zp70 Lck Fyn MEKK-2 6 h High density h LAT Gds Sos PLC-γ2 Vv- Wsp p85 PI3K CD45 SHP- SHP-2 PTP-B Clcineurin NFAT NFAT5 T cellapc interfce, dhesion molecules nd T cell receptor signl trnsduction mchinery re orgnized into distinct suprmoleculr ctivtion clusters in the mture immunologicl synpse 9,0. T cell prolifertion is strongly correlted with stility of the immunologicl synpse over period of mny hours 9, ut prdoxiclly, the time frme of formtion of the mture immunologicl synpse (minutes) corresponds temporlly to period of T cell receptor degrdtion nd downregultion of tyrosine kinse signling 2. These findings re not necessrily contrdictory: the erly nd lte phses of signl trnsduction re well known to hve different iologicl consequences 3, nd the immunologicl synpse is clerly dynmic structure tht is cple of influencing T cell responses over period of severl hours y integrting signls from diverse cell surfce receptors in ddition to the TCR 4. Here we hve investigted the mechnism of T cell unresponsiveness (nergy) induced y C 2 nd clcineurin, specificlly testing the hypothesis tht T cell nergy is in prt consequence of proteolytic ctivtion. We show tht sustined C 2 -clcineurin signling leds to trnscriptionl upregultion of t lest three E3 uiquitin ligses, 5, Cl- 6 nd GRAIL 7, nd lso induces incresed expression of Tsg0, the receptor involved in sorting monouiquitinted proteins to the lysosoml degrdtion pthwy 8. Restimultion of these sensitized cells results in memrne locliztion of nd the relted E3 ligse Nedd4, which trget the key signling proteins phospholipse C-γ () nd protein kinse C-θ () for monouiquitintion nd lysosoml degrdtion. Anergic T cells form immunologicl synpses normlly, ut consistent with downregultion of signling proteins fter restimultion, the synpses re unstle nd norml structures ccumulte rpidly. Mice lcking either or Cl- develop utoimmune disese 5,92, nd we show tht T cells from these mice re resistnt to C 2 -induced nergy, consistent with function for oth E3 ligses in T cell nergy nd tolernce. Our findings define multistep progrm in which sustined signling through C 2 nd clcineurin imposes T cell unresponsiveness y promoting enhnced expression nd memrne locliztion of molecules involved in uiquitintion Akt α-cd3 α-cd28 PMA c d p65 p05 p50 IKKγ IKKβ [ 3 H]thymidine incorportion (c.p.m. 0 3 ) Unstimulted α-cd3 nd α-cd28 Untreted -pretreted Figure Decresed levels correlte with T cell nergy. () Chnges in signling proteins in nergic T cells. T cell nergy ws induced y tretment of the T H cell clone D5 with () or without () µm ionomycin for 6 h. The cells were wshed to remove ionomycin nd were incuted t higher cell density for 2 h t 37 C. Whole-cell extrcts were nlyzed y immunolotting. (d) Two signls re required for mximl loss of. D5 cells were nergized y tretment with µm ionomycin () for 6 h, then were wshed to remove ionomycin nd were incuted t higher cell density for h t 37 C. Extrcts were ssyed for y immunolotting. () Extrcts were prepred either directly (lnes nd 2) or fter resuspension t high cell density nd incution for h (lnes 3 nd 4). (c) Cells were pretreted for 6 h with ionomycin nd restimulted for h with nti-cd3, nti-cd3 plus nti-cd28, ionomycin, or PMA plus ionomycin. (d) The extent of nergy induction in prolifertion ssy nd the extent of decrese in fter the step of incution t high cell density were evluted in prllel in single culture of untreted () nd ionomycin-pretreted () D5 cells. α-, nti-. nd lysosoml degrdtion of key signling proteins downstrem of the TCR. RESULTS Sustined C 2 signls promote degrdtion Sustined C 2 nd clcineurin signling induces stte of T cell unresponsiveness or nergy, chrcterized y upregultion of mny nergy-ssocited genes 6,8. Incresed intrcellulr C 2 concentrtions ([C 2 ] i ) in resting conditions, decresed C 2 moiliztion in response to B cell receptor stimultion nd upregultion of tolerncessocited genes hve lso een documented in tolernt B cells tht hve een exposed continuously to ntigen in vivo 22,23. The trnscriptionl profile of nergic T cells included severl genes encoding proteses nd E3 uiquitin ligses 8, leding us to hypothesize tht T cell nergy ws in prt consequence of proteolytic ctivtion in cells. To test this, we ssessed the mounts of mny signling proteins in T cells nergized y sustined exposure to ionomycin (Fig. ). We noted limited numer of chnges, mong them reproducile decrese in intensity of the nd. This decrese occurred minly during susequent step of cell stimultion or homotypic cell dhesion, s decresed fter incution of the ionomycin-treted cells t high cell density for n hour, s well s fter restimultion with ntiody to CD3 (nti-cd3), nti- CD3 plus nti-cd28, or phorol 2-myristte 3-cette (PMA) plus ionomycin (Fig.,c). We did not find the decrese in fter restimultion with ionomycin lone, ruling out the possiility of direct effect of C 2 -clcineurin signling (Fig. c). The decrese reflected lowered mounts of protein, nd ws not due to cell deth, post-trnsltionl modifiction of, relocliztion of to different intrcellulr comprtment or decresed PLCγ gene trnscription (dt not shown). In experiments done with optimized conditions, there ws strong correltion etween loss of nd extent of nergy induction in prllel prolifertion ssy (Fig. d). Thus, nergic T cells degrde in two seprle stges. A period of sustined C 2 -clcineurin signling is required to initite the degrdtion progrm, ut degrdtion is 256 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

3 CsA * RsGAP RsGRP α-cd3 α-cd PLC-γ2 ctully implemented during susequent step of cell stimultion or homotypic cell dhesion. Clcineurin is required for nd degrdtion We used the clcineurin inhiitor cyclosporin A (CsA) to evlute the involvement of clcineurin in degrdtion in nergic T cells (Fig. 2). D5 T cells sujected to 6 h of ionomycin pretretment followed y cell-cell contct showed sustntil decrese in, nd RsGTPse-ctivting protein (RsGAP), ut no chnges in severl other signling proteins, including RsGRP, Lck, Zp70 nd PLC-γ2 (Fig. 2, lnes nd 2). Degrdtion ws completely locked y inclusion of CsA during the ionomycin tretment step (Fig. 2, ottom right, lne 3). mycin lso induced n increse of out twofold in totl protein uiquitintion (Fig. 2, ottom right, lnes nd 2), which ws locked y CsA (Fig. 2, lne 3). Primry T cells nergized y stimultion with nti-cd3 lone showed decresed,, RsGAP nd, to lesser extent, Lck, ut not PLC-γ2, compred with cells productively stimulted with oth nti-cd3 nd nti-cd28 (Fig. 2). The dt re consistent with the hypothesis tht clcineurin ctivtes uiquitin-dependent proteolytic pthwys tht promote protein degrdtion. We ssessed the functionl consequences of degrdtion in nergic T cells y exmining C 2 moiliztion (Fig. 2c). We rendered primry T helper type (T H ) cells nergic y ionomycin pretretment, then leled them with the C 2 indictor fur-2. We induced C 2 moiliztion y TCR-CD3 crosslinking, fter which we stimulted the cells with ionomycin to identify helthy, responsive cells. Consistent with the ide of centrl function for (ut Lck Zp70 Uiquitin Rs-GAP Lck PLC-γ2 c [C 2 ]i, nm,400,200, ,400,200, Untreted pretreted TCR Time (s) Figure 2 Decresed nd impired C 2 moiliztion correlte with T cell nergy. () Clcineurin-dependent degrdtion of trget proteins in nergic T cells. D5 T cells were treted for 6 h with ionomycin (), CsA or oth, then were wshed nd incuted t incresed cell density for h. Cell extrcts were prepred nd nlyzed y immunolotting for proteins (mrgins) or for the extent of uiquitin modifiction of totl protein in the lystes (ottom right lot). The fster-migrting nd in the immunolot (*) is the originl Zp70 signl on the sme lot, which ws reproed without prior stripping. mycin tretment reproducily upregultes protein in mnner insensitive to CsA; therefore, the pproprite comprison in this cse is etween lnes 3 nd 2, not lnes nd 2. () Signling proteins in primry T H cells ctivted y complete stimultion with nti-cd3 nd nti-cd28 (left lnes) or nergized y incomplete stimultion with nti-cd3 lone (right lnes). Equl numers of T cells were nlyzed y immunolotting for proteins (right mrgin). (c) Primry T H cells from 2B4 mice were left untreted (top) or were pretreted with ionomycin for 6 h (ottom) efore fur-2 leling, incution with iotinylted nti-cd3 nd [C 2 ] i imging. After n oservtion period of 00 s (left downwrd rrows), streptvidin ws dded to induce TCR crosslinking (TCR); t 600 s (right downwrd rrows), ionomycin () ws dded to identify responsive cells. C 2 moiliztion ws monitored y time-lpse video microscopy. Dt represent individul (gry) nd verged (lck) trces from 00 CD4 nd ionomycin-responsive single cells. α-, nti-. TCR not PLC-γ2) in C 2 moiliztion nd T cell ctivtion 24, nergic T cells responded very poorly to TCR stimultion compred with untreted T cells. As reported efore 25,26, we lso noted strong impirment of C 2 moiliztion in T cells from DO.0 trnsgenic mice tht were orlly tolerized y ovlumin feeding (dt not shown). Thus, the degrdtion oserved in nergic T cells correltes with pronounced impirment of C 2 moiliztion in response to TCR triggering. Becuse this effect occurs without ovious dely, degrdtion my e initited during the fur-2 leling step s result of cell-cell contct. Alterntively, other mechnisms such s dephosphoryltion y tyrosine phosphtses upregulted during the step of sustined C 2 -clcineurin signling 8 my contriute to loss of ctivity in nergic T cells (these possiilities re not mutully exclusive). is sustrte for Nedd4 nd All three trgets of the C 2 -clcineurindependent degrdtion progrm,, nd RsGAP, hve C2 domins (Supplementry Fig. online). These domins cn medite C 2 - dependent phospholipid inding or my serve s C 2 -dependent or C 2 -independent protein interction domins 27. C2 domins re lso found in the nd Nedd4 fmily of E3 uiquitin ligses 28 (Supplementry Fig. online), leding us to test the hypothesis tht these E3 ligses re involved in degrdtion. coimmunoprecipitted with oth Nedd4 nd (Fig. 3) nd ws sustrte for uiquitintion nd degrdtion y nd Nedd4 (Fig. 3, c). In 293 cells, ionomycin induced uiquitintion (Fig. 3, lnes 4 nd 5), nd much of the uiquitinted 600 NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH

4 ARTICLES Myc-Nedd4 Myc- IP α-myc Blot α- e High cell density MG32 U Nedd4 Lyste Blot α-au IP α-au Blot α-au Lyste Blot α-myc Nedd4 2 3 Lyste Blot α-myc Lyste Blot α-au d DN Mock Nedd4 Nedd4 Long exposure Nedd4 8 Nedd4 3 5 f Short exposure c IP α-au Blot α-ha IP α-myc Blot α-myc HA-uiquitin Myc- AU IP α- Blot α-u IP α- Blot α- 5 6 U LAT 7 Cytoplsm Detergent Detergent solule insolule Figure 3 E3 uiquitin ligses of the HECT type induce uiquitintion nd degrdtion of. () Physicl interction of Nedd4 nd with. AU. epitopetgged ws coexpressed in HEK 293 cells with Myc-tgged or Myc-tgged Nedd4. Anti-Myc immunoprecipittes (top two pnels) or whole-cell lystes (ottom two pnels) were nlyzed y immunolotting (proteins, right mrgin). in immunoprecipittes ws detected with cocktil of monoclonl ntiodies. () induces mono-, di- nd polyuiquitintion of. HEK 293 cells were trnsfected in duplicte with expression vectors encoding hemgglutinin (HA)-tgged uiquitin, AU.-tgged nd/or Myc-tgged (ove lnes), nd one culture of ech pir ws stimulted with 3 µm ionomycin () for 30 min efore cell extrction. Cell extrcts were immunoprecipitted with nti-au. nd nlyzed for uiquitin-modified (U) or totl immunoprecipitted (top two pnels) or were directly nlyzed for nd expression y immunolotting (ottom two pnels). (c) nd Nedd4 promote degrdtion. HEK 293 cells were mock-trnsfected (Mock) or were trnsfected with, Nedd4 or ctlyticlly inctive Nedd4 mutnt (DN Nedd4) nd AU nd were stimulted with ionomycin ( ) or were left unstimulted ( ). Top, cell lystes were nlyzed for expression y immunolotting with ntiodies to the AU tg. Bottom, immunolotting of the lystes from unstimulted cells (lnes, 3, 5 nd 7) with ntiodies to endogenous nd Nedd4 shows tht the mounts of ectopiclly expressed nd endogenous Nedd4 were similr, wheres the expression of introduced ws out fourfold greter thn tht of endogenous. (d) D5 cells were left untreted () or were stimulted with ionomycin () for 6 h, then were wshed nd incuted t higher cell density for 2 h. Cytoplsmic, detergent-solule nd detergent-insolule memrne frctions were nlyzed y immunolotting. (e) The protesome inhiitor MG32 promotes ccumultion of modified form of. D5 cells were left untreted ( ) or were pretreted with ionomycin ( ), then were wshed nd incuted in the sence () or presence () of MG32 (tretments ove lnes). Extrcts were immunolotted for nd. (f) ecomes monouiquitinted in cells sujected to sustined C2 signling. D5 cells were left untreted () or were pretreted with ionomycin (), nd cell lystes were immunoprecipitted with nti-. Immunoprecipittes were nlyzed for uiquitin modifiction (U) y immunolotting. α-, nti-; IP, immunoprecipittion. migrted s doulet corresponding to mono- nd diuiquitinted forms (Fig. 3, rrow, top). Coexpression of strongly enhnced uiquitintion, incresing the mounts of mono-, di- nd polyuiquitinted forms, ut the ionomycin dependence of uiquitintion ws less notle in these conditions (Fig. 3, lnes 2 nd 3). When we cotrnsfected 293 cells with nd Nedd4 or expression vectors nd treted the cells with ionomycin, we noted loss of (Fig. 3c, top, lnes 3, 4 nd 7, 8). This decrese ws locked y coexpression of dominnt negtive Nedd4 protein ering n lnine sustitution t the ctive site cysteine of the HECT domin (Fig. 3c, top, lnes 5 nd 6). The sucellulr locliztion of nd Nedd4 ws ltered in nergic T cells, s the comintion of ionomycin tretment nd homotypic cell dhesion cused strong trnsloction of oth proteins to the detergent-insolule 258 memrne frction (Fig. 3d). In the sme conditions, the memrne dpter LAT loclized to oth detergent-solule nd detergentinsolule memrne frctions nd ws eqully undnt in these frctions in resting nd nergized cells. Thus, the C2 domincontining E3 ligses nd Nedd4 re strong cndidtes for mediting degrdtion in T cells nergized y sustined C2 signling. Although C2 domins do not necessrily engge in C2dependent protein-protein interctions27, they re likely to promote colocliztion of trget nd effector proteins t the plsm memrne, possily through interctions with specific phospholipids or dpter proteins such s the nnexins29. Indeed, nnexins I nd VI were mong the proteins most highly induced in proteomic nlysis of tolernt T cells in vivo26, indicting tht the mechnism for loclizing E3 ligses to the memrne frction in nergic T cells is VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

5 CsA Reltive intensity Tsg lso induced nd/or ctivted s prt of the nergy progrm, either during the first step of sustined C 2 -clcineurin signling or in response to T cellapc contct. is monouiquitinted in nergic T cells The protesome inhiitor MG32 did not prevent degrdtion, nor did it inhiit the loss of protein noted in ionomycinpretreted D5 T cells sujected to homotypic dhesion (Fig. 3e). Insted, MG32 slightly incresed the ccumultion, only in nergized T cells, of modified form of visile in long exposure of the imging film to the immunolot. This species migrted with n pprent moleculr weight of out 0 kd greter thn tht of itself, indicting tht it represented monouiquitinted form. The monouiquitin modifiction is trnsient nd techniclly difficult to detect: sorting of monouiquitinted proteins into the internl vesicles of multivesiculr odies involves n oligtory deuiquitintion step 8, nd therefore the proteins reside in the uiquitinted stte for only very short time. Moreover, in contrst to polyuiquitintion, the monouiquitin modifiction presents only one epitope for detection with the ntiodies to uiquitin compred with mny more epitopes for polyuiquitintion. Nevertheless, we were le to demonstrte unmiguously tht ws monouiquitinted in nergic T cells, y immunoprecipitting PKCθ nd immunolotting for uiquitin (Fig. 3f). Untreted T cells showed no uiquitin modifiction, wheres ionomycin-pretreted T cells tht were llowed to interct homotypiclly showed distinct nd t moleculr weight corresponding to tht of monouiquitinted, with no pprent signl t higher moleculr weights. These results indicted tht degrdtion of signling proteins in nergic T cells ws not ccomplished through the protesome, which inds with high ffinity only to proteins tgged with four or more uiquitin moieties 3, ut rther through the lysosoml pthwy, in which monouiquitintion promotes sorting of proteins ssocited with the limiting memrne of endosomes into smll internl vesicles tht ccumulte in the lumen s the endosomes mture 8,30. Cl Nedd4 Reltive mrna expression 20 0 Resting CsA Plcg Cl Rnf28 Figure 4 Upregultion of E3 ligses in T cells sujected to sustined C 2 signling. () Upregultion of, Cl- nd Tsg0 in nergic T cells. D5 cells were left resting () or were stimulted () with ionomycin (), CsA or oth. Cell extrcts were evluted for, Tsg0, Cl- nd Nedd4 y immunolotting, nd reltive protein expression ws quntified (elow lnes). () D5 cells were left untreted or were stimulted with ionomycin () or ionomycin plus CsA for 0 h, nd expression of, Cl, Rnf28 (GRAIL) nd Plcg mrna ws evluted y rel-time RT-PCR, normlized to mounts of mrna encoding the riosoml protein L32. Dt represent the verge ± s.d. of the rtio of mrna expression in ionomycin-treted or ionomycin nd CsAtreted to tht in untreted cells. Results re representtive of t lest two independent experiments. Clcineurin induces E3 ligses nd Tsg0 expression We sked whether C 2 -induced nergy ws ssocited with upregultion of the protein mchinery involved in nd degrdtion. In yest, endosoml sorting is ccomplished y the endosome-ssocited endosoml sorting complex required for trnsport (ESCRT- complex), which inds monouiquitin- nd diuiquitin-tgged trnsmemrne proteins nd sorts them into the invginting structures tht form the internl vesicles 3 ; the resulting multivesiculr odies fuse with lysosomes nd deliver their contents for degrdtion 8,30. The criticl uiquitin-inding component of the yest ESCRT- complex is Vps23p, the mmmlin homolog of which is Tsg0 (ref. 8). Tsg0 is essentil for downregultion of the ctivted EGF-receptor, which is uiquitinted y the E3 ligse Cl 30. Cl proteins re known to diminish proximl TCR trnsduction y downregulting the TCR 6 s well s y uiquitinting nd inducing degrdtion of TCR-coupled tyrosine kinses 32. Bsed on these considertions, we sought to determine whether E3 ligses of the Cl nd Nedd4 fmilies nd the uiquitin receptor Tsg0 were upregulted during the development of T cell nergy. Tsg0, nd Cl- (the min Cl fmily memer in mture T cells 6 ) incresed in C 2 - nd clcineurin-dependent wy during the priming step of nergy (Fig. 4). nd Tsg0 incresed out threefold in ionomycin-treted D5 cells, nd the increse ws locked y CsA. Cl- ws induced even more highly, nd its induction ws prtly locked y CsA. There ws no chnge in the mount of Nedd4 protein under these conditions, despite the memrne relocliztion of Nedd4 shown in Figure 3d. Upregultion of the E3 ligses reflected n nergy-ssocited trnscriptionl progrm: mrna expression remined constnt, ut the mounts of mrna encoding, Cl- nd GRAIL (n nergy-ssocited E3 ligse 7 encoded y Rnf28) incresed y 8- to -fold in ionomycin-treted T cells, nd this increse ws mostly locked y CsA 7 (Fig. 4). Thus, sustined C 2 nd clcineurin signling is ssocited not only with nd degrdtion ut lso with upregultion of severl molecules with involvement in protein monouiquitintion, endosoml sorting nd lysosoml degrdtion: the E3 ligse, linked here to the monouiquitintion nd downregultion of nd ; the Cl- E3 ligse, linked together with Cl to the monouiquitintion nd downregultion of the TCR 6 ; the endosome-ssocited E3 ligse GRAIL 7 ; nd Tsg0, the receptor component of the endosoml ESCRT- complex, which medites sorting of monouiquitinted proteins into multivesiculr odies trgeted for lysosoml fusion nd degrdtion 8. Disintegrtion of immunologicl synpses in nergy Becuse TCR signling occurs t the interfce (immunologicl synpse) etween the T cell nd the APC 9,0, we monitored the formtion nd susequent ctivity of the immunologicl synpse in NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH

6 c Stle synpses (%) DMSO Wek inhiitor Strong inhiitor Figure 5 mycin-nergized T cells show decresed stility of the immunologicl synpse. (,) Primry T H cells from 2B4 TCR trnsgenic mice were left untreted (top rows; control) or were pretreted with ionomycin (ottom rows), then were incuted for 40 min on plnr phospholipid ilyers contining Oregon greenleled I-E K gonist MCC peptide complexes nd indocrocynine-leled ICAM-. The distriution of ICAM- (red) nd I-E k MCC (green) molecules in T cellilyer contct zones ws cptured t different times. The gry pnels in re interference reflection microscopy imges in which cellilyer contcts pper s drk res. We hve otined similr results in more thn four independent experiments. (c) Involvement of in synpse stility. Mture T cell synpses were llowed to form, then wek (U73343) or strong (U7322) PLC-γ inhiitors were dded. Right, percentge of cells with mture synpses reltive to the sme cells efore the ddition of inhiitors, s shown for one representtive experiment of three totl. untreted nd nergic T cells. In oth cses, the immture immunologicl synpse, chrcterized y peripherl TCRmjor histocomptiility complex (MHC)peptide contcts nd centrl lymphocyte function-ssocited ntigen- nd intercellulr dhesion molecule (LFA-ICAM-) contcts, developed quickly into the mture structure with core TCR-MHC-peptide contct region nd peripherl LFA-ICAM- ring (Fig. 5,, 5- nd 6-min time points). Thus, nergic cells show no impirment of the memrne nd cytoskeletl interctions necessry for synpse mturtion. However, the mture synpse, which persisted stly in the untreted T cells for t lest n hour fter the initil contct 02, ws unstle in nergic T cells. Anergic T cells showed prtil or, occsionlly, complete rekdown of the outer LFA- ring within 020 min fter the mture synpse ws estlished, nd often lso showed errnt morphology of the inner TCR core (Fig. 5,, 0 min nd lter). Prllel nlysis of fluorescence nd contct re ptterns showed tht nergic T cells hd migrtory phenotype, in which the outer LFA-ICAM- ring ecme disorgnized, pulling wy from nd distorting the TCR-MHC clusters, which were drgged ehind the moving T cells (Fig. 5). In this respect, nergic T cells (t 0 min nd lter) ct like cells tht do not receive TCRmedited stop signl 0,33,34. Migrtion of nergized T cells would mke strong contriution to unresponsiveness ecuse the T cellapc contct would not e stly mintined. We sought to determine whether synpse instility could e ttriuted to the loss of function. We llowed T cells to estlish mture synpses for 60 minutes, fter which we otined imges of fields contining stle immunologicl synpses with centrl MHC clusters nd complete ICAM- rings nd recorded the loctions of the synpses. We then treted the stle synpses sequentilly with the wek nd strong phospholipse inhiitors U73343 nd U7322, respectively, nd evluted the effects of the drugs on the previously imged synpses. Wheres the wek inhiitor only hd slight effect on synpse integrity, the strong inhiitor effectively olished the LFA- contct ring (Fig. 5c), n effect tht resemled the phenotype of disintegrtion of the outer LFA- ring oserved in nergic T cells (Fig. 5,). These dt emphsize the requirement for in mintennce of the mture immunologicl synpse nd confirm previous reports tht oth nergic T cells nd T cells treted with phospholipse inhiitors do not ind fironectin efficiently 25. -dependent dicylglycerol production is required for effective inside-out signling 25,35, wheres ctivtion, which is downstrem of dicylglycerol production y, hs een linked to efficient 260 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

7 3 H incorportion (c.p.m. 0 3 ) c Activted Anergized Brekdown product Actin (ng/ml) WT / Cl / WT / Cl / α-cd3 Actin WT formtion of the immunologicl synpse (T.N. Sims, T. Soos, D.R. Littmn nd M.L.D., unpulished oservtions). These results indicte strongly tht the errnt synpse morphology of nergic T cells cn e ttriuted to reduced signling through nd nd consequent reduced LFA- ctivity. Genetic evidence for involvement of nd Cl- in nergy Mice deficient in either or Cl- hve utoimmune phenotypes 5,92, indicting tht these E3 ligses re importnt in suppressing immune responses to self ntigens. To evlute the prticiption of nd Cl- in C 2 -induced T cell nergy, we tested T cells from / (y) nd Cl / mice (Fig. 6). We treted the cells with incresing doses of ionomycin, then stimulted them with nti-cd3 plus nti-cd28, fter which we ssyed prolifertion ([ 3 H]thymidine incorportion). / nd Cl / T cells were resistnt to nergy induction t low doses of ionomycin, nd this effect ws prtilly overcome t higher doses of ionomycin (Fig. 6). We lso ssessed the ility of / nd Cl / T cells to degrde nd in response to nergy-inducing conditions. As expected, protein decresed in wild-type T cells fter the cells were nergized with nti-cd3 stimultion in the sence of costimultion, ut / nd Cl / T cells did not show this decrese (Fig. 6). Likewise, wild-type T cells showed the expected decrese in protein fter ionomycin pretretment followed y restimultion with nti-cd3, ut we did not find this effect in T cells from / nd Cl / mice (Fig. 6c). Finlly, we compred the kinetics of synpse disintegrtion in control nd Cl / T cells tht hd een nergized y pretretment with ionomycin (Fig. 6d). As expected, control 5CC7 TCR trnsgenic T cells exposed to peptide-loded MHC nd LFA- molecules in lipid ilyers formed d Time (min) CC7 5CC7 nergized Cl / Cl / nergized Stle synpses (%) n = n = 748 n = 50 n = 347 / Cl / 5CC7 5CC7 Cl / Cl / nergized nergized Figure 6 Cl- nd -deficient T cells re resistnt to nergy induction. () CD4 T cells from C57BL/6 (WT), Cl / nd / mice were stimulted with nti-cd3 nd nti-cd28 for 2 d nd were left resting for 5 d. Cells were then left untreted or were treted for 6 h with 2500 ng/ml of ionomycin (), fter which prolifertive responses to nti-cd3 nd nti-cd28 stimultion were mesured y [ 3 H]thymidine incorportion. () T H cells from C57BL/6 (WT), Cl / nd / mice were llowed to differentite for week, then were stimulted with plte-ound nti-cd3 in the presence of CTLA4-Ig (Anergized) or with nti-cd3 nd nti- CD28 (Activted) for 2 d, then were llowed to rest for 3 d in medi without interleukin 2. Cell extrcts were nlyzed for nd ctin y immunolotting. (c) T H cells from C57BL/6 (WT), / nd Cl / mice were left untreted () or were treted for 6 h with ionomycin (), were wshed, then were restimulted () or not () with plte-ound nti-cd3 (α-cd3). Cell extrcts were nlyzed for nd ctin y immunolotting. (d) Formtion of immune synpses ws evluted s descried in Figure 5, with T H cells from wild-type or Cl / 5CC7 TCRtrnsgenic mice nd lipid ilyers displying ICAM- nd I-E k pigeon cytochrome C (PCC) molecules. Top, individul representtive cells (genotypes, left mrgin) oserved over time course of 50 min. Bottom, quntifiction of results, showing the percentge of cells with stle synpses t 35 min fter synpse formtion ws initited. synpses tht were stle throughout the oservtion period of 50 min (Fig. 6d), wheres 5CC7 T cells tht were pretreted with ionomycin for 6 h formed the mture synpse quickly (<5 min) on contct with the ilyer ut then showed synpse disorgniztion nd developed the migrtory phenotype. Synpses formed y untreted Cl / T cells were s stle s those formed y wild-type T cells, ut synpses formed y ionomycin-pretreted Cl / T cells were mostly protected from synpse disintegrtion, s judged y their stility for up to 35 min of oservtion. Thus, Cl- contriutes sustntilly to the erly disintegrtion of the immunologicl synpse in nergic T cells. However, the synpses rek down t lter times in ionomycin-pretreted Cl / T cells (50 min), indicting tht other fctors re lso involved. DISCUSSION Bsed on our dt, we propose tht T cell nergy is initited nd implemented through complex multistep negtive signling process, t lest one fcet of which involves the coordinte ctions of severl E3 ligses. The progrm is initited y sustined C 2 - clcineurin signling nd culmintes in proteolytic degrdtion of nd, two centrl prticipnts in the TCR signling cscde 24,36. The first step of the progrm involves clcineurinmedited upregultion of three E3 ligses,, Cl- nd GRAIL, s well s Tsg0, the uiquitin-inding component of the endosoml sorting complex. This process my e medited through NFAT in the sence of AP- coopertion 8. Degrdtion of signling proteins is implemented during second step of T cellapc contct, during which the immunologicl synpse forms normlly nd the E3 ligses, Nedd4 nd Cl- move to detergent-insolule memrne frctions, where they my coloclize with ctivted sustrte NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH

8 proteins. The memrne frction my include rft memrnes, endosoml memrnes or oth, consistent with previous findings tht, RsGAP, Tsg0 nd GRAIL re ll found ssocited with endosomes 4,7. As result, the ctive, memrne-proximl pool of signling proteins ecomes monouiquitinted nd cple of stle interction with Tsg0. This interction in turn results in sorting of the monouiquitinted proteins into multivesiculr odies nd their trgeting for lysosoml degrdtion. In the third step, degrdtion of ctive nd leds to diminished TCR nd LFA- signling. Once this hppens, the mture synpse cnnot e mintined nd the inility to sustin stle APC contct further reduces the ntigen responses of nergic T cells. Overll, therefore, stle difference in gene expression profile etween norml nd nergic T cells is trnsformed through uiquitin modifiction into trnsient increse in turnover of ctivted signling proteins, therey ltering the migrtion ctivity of T cells nd estlishing persistent unresponsive stte. The ttrctive feture of such downregultory progrm is tht signling molecules would e trgets for degrdtion only when they re ctivted. In normlly ctivted T cell, signling through the TCR results in mintined phosphtidylinositol-3,4,5 triphosphte production, -dependent production of second messengers nd C 2 -moiliztion, continuing for severl hours. In n nergic T cell in which the, Cl-, Nedd4 nd GRAIL E3 ligses re upregulted nd/or prectivted for memrne locliztion, nd ctivtion would e rpidly followed y E3-medited monouiquitintion t rft or endosoml memrnes, nd this, through Tsg0, would immeditely sequester the ctive enzymes within endosomes where they cnnot e rectivted. Thus, imposition of T cell nergy would e loclized nd efficient process in which nd would e eliminted only in the context of ctive, memrne-loclized signling complexes, nd mssive depletion of the ulk of cellulr would not e required (nd is not found). Consistent with this hypothesis, nergic T cells show no pprecile downregultion of PLC-γ2, which despite hving the sme domin orgniztion s is not prt of the signling pthwy downstrem of the TCR 24. Our dt indicte tht lck of nergy induction is the moleculr mechnism underlying the utoimmune phenotypes of Cl--deficient nd -deficient (y) mice. y mice show splenomegly nd lymphocyte infiltrtion in severl tissues nd chronic inflmmtion in the skin 5,9, wheres Cl ltion is ssocited with spontneous T cell ctivtion nd utontiody production 2 nd enhnced experimentl utoimmune encephlomyelitis 20. Furthermore, Cl is principl gene linked to susceptiility to type I dietes in rts 37. Becuse / nd Cl / T cells hve very similr phenotypes of resistnce to C 2 - induced nergy (impired prolifertion nd impired degrdtion of nd ), it is likely tht these two proteins cooperte to evoke T cell nergy, either in the context of multiprotein complex 38 or y cting sequentilly in pthwy of nergy induction. Cl fmily memers re essentil for internliztion nd downregultion of the T cell receptor 6, possily through their ility to ind the CIN85 prlog CD2AP 39 ; thus, one likely scenrio is tht Cl- regultes TCR internliztion through monouiquitintion nd sorting into internl vesicles of lte endosomes, wheres functions to monouiquinte nd, routing them into the sme lysosoml degrdtion pthwy s the TCR. The E3 ligse GRAIL, which resides in the endosoml memrne nd is upregulted in nergic T cells 7, could synergize with these effectors to further enhnce protein uiquitintion nd degrdtion. Consistent with severl previous studies, our findings point to complex function for integrins in T cell nergy. Anergic T cells (especilly T cell clones) show n initil tendency to interct homotypiclly, ut implementtion of T cell nergy results in reduced inding of LFA- to its lignd ICAM-. Anergic T cells show incresed trnscription of the gene encoding LFA-α 40 ; upregultion of the cofctor GRP- (cytohesin 3), overexpression of which leds to incresed ctivity of LFA- (refs. 4,42); nd incresed GTP loding of Rp (ref. 43), which increses integrin dhesiveness nd is crucil for ntigendependent synpse formtion Anergic T cells lso show upregultion of CD98 (ref. 8), which induces LFA- dhesion through ctivtion of the smll GTPse Rp (ref. 48). Prdoxiclly, however, T cells from mice trnsgenic for constitutively ctive Rp did not show n unresponsive phenotype, ut insted showed incresed prolifertion in response to APC-peptide stimultion tht correlted with incresed vidity of β nd β 2 integrins 45. A recent report my resolve this prdox, ecuse it showed tht much higher mounts of ctivted (GTP-loded) Rp thn in the constitutively ctive Rp trnsgenic model could e chieved y inctivtion of the gene encoding SPA-, the min GTPse-ctivting protein for Rp in peripherl T cells 49. T cells from young SPA--deficient mice tht hd een immunized with ntigen in djuvnts developed n unresponsive phenotype resemling the phenotype tht develops in wild-type mice injected with solule ntigen 49. We postulte tht the incresed mount of Rp-GTP in nergic T cells leds to prectivtion of integrins 45, which in turn co-opts TCR signling y premturely ctivting the E3 ligse-ssocited nergy progrm tht we hve defined here. However, the resulting degrdtion of ctive nd interferes with LFA- ctivity, thus conferring the phenotype of synpse disorgniztion tht we note in nergic T cells. There is controversy out the precise function of the immunologicl synpse in TCR signling. The kinetics of formtion of the mture immunologicl synpse correspond to period in which there is sustntil T cell receptor internliztion nd degrdtion s well s decrese from the erly pek of tyrosine kinse signling 2. However, stle immunologicl synpse seems to e required for ongoing C 2 moiliztion, phosphtidylinositol 3 kinse ctivtion nd interleukin 2 production 9. Our findings offer synthesis of these two opposing views, showing tht the synpse is dynmic structure whose stility requires ongoing signling through the TCR nd which in turn modultes the overll level of T cell responses y integrting positive nd negtive signls from vriety of surfce receptors including the TCR, costimultory receptors nd integrins. In summry, we hve shown tht C 2 -clcineurin signling, which is essentil for the productive immune response, lso promotes T cell nergy through progrm of uiquitin modifiction nd trgeted degrdtion of signling proteins. Thus, our studies confirm tht T cell nergy involves n ctive process tht precedes unresponsiveness to ntigen. It is notle tht two min prticipnts in this re E3 uiquitin ligses whose involvement in negtive signling hs lredy een confirmed in mouse models of utoimmune disese. Further iochemicl nlysis of the C 2 - nd clcineurin-induced nergy progrm should uncover dditionl prticipnts, potentilly encoded y the mny genetic loci tht predispose to utoimmune disese. Note dded in proof: The y cells used in these studies show T H 2 is tht hs een ttriuted to incresed JunB, postulted trget 50, rising the possiility tht the utoimmune phenotype of y mice is due to T cell hyperprolifertion in response to incresed interleukin 4. However, this explntion is not comptile with the finding tht ckcrossing y mice to the B0 ckground elimintes their T H 2 is in vivo, ut the mice still develop utoimmune disese (V. Prrvicini nd R. Zmoysk, personl communiction). 262 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

9 METHODS Mice. BALB/cJ mice nd DO.0, 2B4, AND nd 5CC7 TCR-trnsgenic mice were otined from Jckson lortories nd were mintined nd red in pthogen-free conditions in rrier fcility. Cell culture, cell stimultion nd nergy induction ex vivo. The mouse D5 (Ar-5) T H cell clone ws grown s descried 8. CD4 cells were isolted from spleens nd lymph nodes of mice with positive selection with nti-cd4 mgnetic eds (Dynl) nd were differentited into T H cells for 2 weeks with stndrd protocols 8. For most experiments, nergy ws induced y tretment of primry T H cells or the D5 T H clone ( 0 6 cells/ ml) with µm ionomycin for 6 h. This method hs the dvntge of providing homogeneous popultion of nergic T cells tht hve synchronously experienced the sme intensity of prior C 2 signls nd so re suitle for iochemicl investigtion. CsA ws included in some experiments t concentrtion of 2 µm. Cells were wshed to remove ionomycin nd were incuted t higher cell density (3 0 6 cells/ ml) for 2 h t 37 C. In the experiment in Figure, the highdensity incution step ws included ut hd not een plnned. For the experiment in Figure c, D5 cells were restimulted with µg/ml of nti-cd3 with or without 2.5 µg/ml of nti-cd28, or with 20 nm PMA, µm ionomycin or oth. For the experiment in Figure 2, primry T H cells were stimulted with plte-ound nti-cd3 to induce nergy or with comintion of nti-cd3 nd nti-cd28 to induce productive ctivtion. In oth cses the cells go through phse of ctive prolifertion, ut cells tht received only nti-cd3 stimultion responded much less well to susequent restimultion thn cells previously stimulted with oth nti-cd3 nd nti-cd28. For the experiments of Figure 6, T H cells from C57BL/6, Cl / or / mice tht hd een differentited in vitro for week were stimulted for 2 d with plteound nti-cd3 (3 µg/ml) in the presence of CTLA4-Ig (0 µg/ml) to induce nergy or with nti-cd3 (0.25 µg/ml) nd nti-cd28 (2 µg/ml) to induce ctivtion, then were left resting for 3 d in medi without interleukin 2. The extent of nergy induction ws evluted y intrcellulr cytokine stining or in stndrd prolifertion ssys 8. HEK 293 cells were grown nd trnsfected with C 2 phosphte with stndrd protocols. Antiodies. Antiodies to Zp70 (Z24820), Lck (L5620), (60089), (I84520) nd clcineurin (60259) were otined from BD Trnsduction Ls. Antiodies to Fyn (06-33), RsGAP (05-78), SOS (06-246), Vv- (05-29) nd Nedd4 (07-049) were purchsed from Upstte Biotechnologies. Snt Cruz Biotechnology ntiodies were used to detect CD3ζ (sc-239), Mekk-2 (sc-088), RsGRP (sc-8430), uiquitin (sc-807), PLC-γ2 (sc-407), Cl- (sc-705), NF-κB p65 (sc-09), NF-κB p50 (sc-92), IKKγ (sc-8330) nd Myc-tgged (sc-789) nd hemgglutinin-tgged (sc-805) proteins. Antiody to the AU. (MMS-30P) epitope tg ws purchsed from Covnce; nti-akt (9272), from Cell Signling; nti-tsg0 (83), from Genetex; nd nti-ikkβ (AHO0362), from Biosource. Antiodies to NFAT nd NFAT5 were produced in the Ro l. Antiodies to Gds nd LAT were gifts from E. Clrk (University of Wshington, Settle, WA) nd L. Smelson (Ntionl Cncer Institute, Bethesd, MD), respectively. Antiodies to p85 phosphtidylinositol 3 kinse were otined from the lortory of L. Cntley; nd ntiodies to SHP-, SHP-2 nd PTP-B were provided y B. Neel (oth from Beth Isrel Deconess Medicl Center, Hrvrd Medicl School, Boston, MA). Endogenous ws detected with polyclonl ntiserum provided y A. Toker (Beth Isrel Deconess Medicl Center, Hrvrd Medicl School, Boston, MA), which ws rised ginst the epitope APRRTRVNGDNR, representing the finl C-terminl mino cids of the protein. The rectivity of this ntiserum with is unlikely to e ffected y phosphoryltion, s proposed erlier 5, s the epitope does not contin ny serine or tyrosine residues nd its single threonine residue is not prt of ny predictle phosphoryltion motif, s judged y the Scnsite computer progrm. Furthermore, commercil ntiody source, comprising pool of four different monoclonl ntiodies (05-63; Upstte Biotechnologies), lso llowed us to visulize the differences in protein mounts in untreted nd nergic T cells when the ntiody ws used t fivefold higher dilution thn recommended. We hve lso used second monoclonl ntiody to (E-7; Snt Cruz Biotechnology) to immunoprecipitte in prtilly denturing conditions from resting nd nergized primry T cells. These experiments confirmed tht nergized T H cells sujected to restimultion showed reduction in protein compred with their counterprts tht were not restimulted; however, corresponding nti-cd3 stimultion of resting T H cells did not result in such decrese (dt not shown). Expression plsmids. Nedd4 (KIAA0093) cdna nd cdna 5 were inserted vi SlI-NotI into prk5 vectors contining n N-terminl sequence encoding the Myc epitope. R. Arhm (Duke University, Durhm, NC) nd J. Huiregtse (University of Texs t Austin, Austin, TX) provided expression plsmids for nd Nedd4, respectively. Cell extrcts, immunoprecipittions nd immunolots. D5 cells were extrcted t concentrtion of 0 6 cells/0 µl in rdioimmunoprecipittion ssy (RIPA) uffer (20 mm Tris, ph 7.5, 250 mm NCl, mm DTT, 0 mm MgCl 2, % Nonidet P-40, 0.% SDS nd 0.5% sodium deoxycholte) supplemented with protese nd phosphtse inhiitors ( mm phenylmethyl sulfonyl fluoride, 25 µg/ml of protinin, 25 µg/ml of leupeptin, 0 mm NF, 8 mm β-glycerophosphte nd 0. mm sodium orthovndte). Re-extrction of the resulting DNA pellet with hot SDS smple uffer followed y immunolotting did not show ny residul in the SDS frction of either resting or ionomycin-nergized D5 cells, confirming the efficiency of extrction with RIPA uffer (dt not shown). For ssessment of proteins in cell extrcts, µl of RIPA extrcts were seprted y 92% SDS-PAGE, nd proteins were electrotrnsferred onto nitrocellulose memrnes. For immunoprecipittion, 500,000 µl of RIPA cell extrcts were used. For coimmunoprecipittion from lystes of trnsfected HEK 293 cells, cells from one 0-cm dish were lysed in 50 mm HEPES, ph 7.5, 00 mm NCl, mm EDTA, 0,5% Nonidet P-40 nd 0% glycerol plus phosphtse nd protese inhiitors. Lystes were preclered with either protein A or protein GSephrose, immunoprecipittion proceeded for 4 h nd the resulting precipittes were wshed three to four times with the uffer used for cell extrction. For detection of monouiquitinted, cell lystes from 0 8 untreted or ionomycin-pretreted D5 cells were immunoprecipitted with nti-. Immunolots used ntiody solutions in 5% milk nd TBS (0 mm Tris-HCl, ph 8.0, nd 50 mm NCl) nd wshes used TBS contining 0.05% Tween-20. Reltive nd intensities were quntified y NIH IMAGE Qunt nd corrected for ckground within ech lne. Cell frctiontion. Cell frctiontion ws done essentilly s descried 52 with D5 cells. Cells were swollen for 5 min in hypotonic uffer E (0 mm Tris, ph 7.4, 0 mm KCl,.5 mm MgCl 2 nd mm DTT supplemented with protese nd phosphtse inhiitors) nd lysed y Dounce homogeniztion. Lystes were centrifuged t 00,000g for 30 min, yielding superntnt (cytosol) nd pellet tht ws resuspended in uffer E contining % Nonidet P-40 nd ws recentrifuged t 00,000g for 30 min to seprte the detergent-solule frction in the superntnt from the detergent-insolule frction (pellet). The pellet ws resuspended y soniction in RIPA uffer nd clered y centrifugtion. One fourth of the superntnt from ech centrifugtion step (cytoplsm, detergent solule nd detergent insolule frctions) ws nlyzed for y immunolotting for Nedd4, nd LAT. [C 2 ] i imging nd immunocytochemistry. Intrcellulr clcium ws mesured in primry T H cells from 2B4 mice. Cells were loded with µm fur-2 ceto-methyl ester (Moleculr Proes) for 30 min t 20 C, wshed nd resuspended in loding medium (RPMI 0% FCS), incuted with 2.5 µg/ml of iotinylted nti-cd3 (2C, Phrmingen) for 5 min t 20 C nd ttched to poly-l-lysinecoted coverslips mounted in RC-20 closed th chmer (Wrner Instrument). The fur-2-loded cells were perfused with Ringer s solution contining 2 mm clcium (55 mm NCl, 4.5 mm KCl, 0 mm D-glucose, 5 mm HEPES, ph 7.4, mm MgCl 2 nd 2 mm CCl 2 ) nd were stimulted y crosslinking of the surfce-ound iotinylted nti-cd3 with 2.5 µg/ml of streptvidin (Pierce), fter which helthy cells were identified y their responsiveness to µm ionomycin (Cliochem). Single-cell video imges were otined on Zeiss Axiovert S200 epifluorescence microscope with OpenL imging softwre (Improvision). Fur-2 emission ws detected t 50 nm fter excittion t NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH

10 340 nd 380 nm. The 340/380 rtio imges were cquired every 5 s fter ckground sutrction. Clirtion vlues (R min, R mx nd S f ) were derived from cuvette mesurements with clcium clirtion uffer kit (Moleculr Proes) nd s descried 53. Rel-time PCR nlysis. Totl RNA ws prepred from untreted or ionomycin-pretreted D5 cells with Ultrspec regent (Biotecx). cdna ws synthesized from 2 µg of totl RNA s templte, with cdna synthesis kit (Invitrogen). Quntittive rel-time PCR ws done in n I-Cycler (BioRd) with SYBR Green PCR kit (Applied Biosystems). The sequences of the primer pirs were: L32 sense, 5 -CGTCTCAGGCCTTCAGTGAG-3, nd L32 ntisense, 5 - CAAGAGGGAGAGCAAGCCTA-3 ; sense, 5 -AAGCCTTTGACCCC TTTGAT-3, nd ntisense, 5 -GGTTCAGTCCGTTGTCCACT-3 ; sense, 5 -GTGTGGAGTCACCAGACCCT-3, nd ntisense, 5 -GCT TCTACTTGCAGCCCATC-3 ; Cl sense, 5 -CTTAAATGGGAGGCACAGT AGAAT-3, nd Cl ntisense, 5 -CAGTACACTTTATGCTTGGGAGAA-3 ; Rnf28 sense, 5 -GTAACCCGCACACCAATTTC-3, nd Rnf28 ntisense, 5 - GTGAGACATGGGGATGACCT-3. Therml cycling conditions were 95 C for 5 min, then 40 cycles of 95 C, 65 C, nd 72 C for 30 s ech, terminting with single cycle t 72 C for 5 min. Signls were cptured during the polymeriztion step (72 C). A threshold ws set in the liner prt of the mplifiction curve, nd the numer of cycles needed to rech it ws clculted for ech gene. Melting curve nlysis nd grose gel electrophoresis were done to test the purity of the mplified nds. Normliztion ws done y using mounts of mrna for the riosoml protein L32 s n internl control for ech smple. Formtion of immunologicl synpses in lipid ilyers. Plnr ilyers were prepred essentilly s descried 0, except tht the moth cytochrome C (MCC) peptide ws loded on glycosylphosphtidylinositol (GPI)I- E k for 24 h. Bilyers were prepred with Oregon greenleled GPII-E k nd indodicrocynine-leled GPIICAM- in prllel plte flow cells (Bioptechs). Control nd ionomycin-pretreted T cells from TCR-trnsgenic mice were injected into the flow cell t density of 0 6 cells/ml. Ares of ilyers where cells were forming synpses were imged with fluorescein isothiocynte nd indodicrocynine optics on n Olympus IX-70 inverted microscope equipped with Hmmtsu ORCA-ER digitl cmer nd xenon rc lmp s light source for fluorescence microscopy. The filter wheels, shutters nd the cmer were controlled with IPLAB softwre on Mcintosh pltform. Bright field, interference reflection nd fluorescence imges were collected nd processed with Metmorph softwre. The ckground from the fluorescence imges ws sutrcted with the produce-ckground correction imge function, which is sed on medin filtering to sutrct ckground tht is nonuniform. The percentge of cells dhering ws evluted y compring right field nd interference reflection microscopy imges. Experiments with phospholipse inhiitors were done with AND T cell lsts (dy 8). Cells were llowed to form immunologicl synpses on ilyers contining 80 molecules/µm 2 of Oregon green I-E k MCC nd 200 molecules/µm 2 of indodicrocynineicam- in the presence of 0.0% dimethylsulfoxide (the crrier concentrtion for µm U7322 nd U73343). After 60 min, fields contining stle immunologicl synpses with centrl MHC clusters (green) nd complete ICAM- rings (red) were imged nd the loctions were recorded with n utomted stge nd IPL softwre. The stle synpses were then treted sequentilly with µm U73343 nd µm U7322 (wek nd strong PLC-γ inhiitors, respectively). After ech drug tretment, the sme fields were imged within 0 min so tht the effects of the drugs on mny individul synpses could e determined. The quntittive dt reflect the percentge of intct LFA-ICAM- rings fter crrier or drug tretment on 03 contct res. In seprte experiments, the effects of U73343 nd U7322 were stle for up to h nd U7322-dependent destruction of the LFA- dhesion ring ws not dependent on prior tretment with U We found these effects in three independent experiments with U7322 concentrtions from 0. to µm (dt not shown). Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk memers of the Ro nd Dustin lortories for discussions, nd T. Strr for the preprtion of ICAM- nd I-E k for plnr ilyer experiments. We lso thnk A. Altmn, H. Bnd, J. Brugge, C. Jozeiro, M. Ktn for dvice nd regents. Supported y Ntionl Institutes of Helth grnts RO-AI4823, RO-AI4027 nd RO3-HD39685 (to A.R.), RO-AI50280 nd R2-AI48542 (to Y.-C.L.) nd AI-43542; n Irene Dimond Foundtion grnt (to M.L.D.); EMBO (V.H.); nd the Cncer Reserch Institute (S.-H.I. nd S.F.). COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. 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