Biophysical mechanism of T-cell receptor triggering in a reconstituted system

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1 ARTICLE oi:1.138/nture1122 Biophysil mehnism of T-ell reeptor triggering in reonstitute system John R. Jmes 1 & Ronl D. Vle 1 A T-ell-meite immune response is initite y the T-ell reeptor () interting with peptie-oun mjor histoomptiility omplex () on n infete ell. The mehnism y whih this intertion triggers intrellulr phosphoryltion of the, whih lks kinse omin, remins poorly unerstoo. Here, we hve introue the n ssoite signlling moleules into non-immune ell n reonstitute lign-speifi signlling when these ells re onjugte with ntigen-presenting ells. We show tht signlling requires the ifferentil segregtion of phosphtse n kinse in the plsm memrne. An rtifiil, hemilly ontrolle reeptor system genertes the sme effet s, emonstrting tht the ining energy of n extrellulr proteinprotein intertion n rive the sptil segregtion of memrne proteins without trnsmemrne onformtionl hnge. This generl mehnism my exten to other reeptors tht rely on extrinsi kinses, inluing, s we emonstrte, himeri ntigen reeptors eing evelope for ner immunotherpy. In ition to interellulr ommunition meite y solule moleules, two ells n trnsmit signls through memrnessoite reeptors n ligns. Aptive immunity represents suh system, in whih the MHC protein on the surfe of ntigenpresenting ells (APCs) interts with the on T lymphoytes. If the ins of the right omplementrity, the intertion results in tyrosine phosphoryltion of the (herein referre to s triggering ) n the initition of signls tht tivte the T ell 1. The hs no intrinsi kinse tivity, unlike mny other reeptors 2, n inste relies on T-ell-speifi kinse lle (ref. 3). Also istint from other systems, the phosphoryltle tyrosine resiues of the (the immunoreeptor tyrosine-se tivtion motifs (ITAMs)) 4 o not resie on the polypepties tht ontt the (, ) ut inste re ontine on tightly ssoite CD3 suunits (,, e 2, f 2 ). The phosphorylte ITAMs then in seon kinse,, whih is susequently tivte n rives ownstrem signlling 5. Despite onsierle work, the mehnism y whih ining les to triggering remins poorly unerstoo (reviewe in ref. 6). Some moels propose tht ining evokes onformtionl hnge in the tht mkes its ytoplsmi ITAM omins more essile to kinse 7. Alterntive triggering hypotheses inlue tivtion through the ggregtion of moleules 6, n kineti segregtion 8, where phosphoryltion is fvoure y its prtitioning into plsm memrne omins tht ontin kinse ut re eplete of (lso known s PTPRC), n unnt trnsmemrne phosphtse. However, lthough lustering 9 n the segregtion of wy from the hve een oserve 1, it hs not een estlishe whether suh events re neessry or suffiient for signl trnsution ross the plsm memrne. In ition, the physil sis of protein segregtion within the plsm memrne is unler. Reonstitution of iologil phenomenon with efine omponents hs proven to e powerful mens for isseting moleulr mehnisms. We hve me use of this pproh y introuing the genes enoing the n other proteins require for regulting its phosphoryltion into non-immune ell n repitulting triggering when this ell forms onjugte with n APC. Beuse eh protein n e introue seprtely n is genetilly engineere, this system hs llowe us to test moels of triggering n the roles of iniviul proteins in mnner tht is iffiult to hieve with ntive T ells. Reonstitution of regulte triggering We first sought to reonstitute -meite phosphoryltion in non-immune ell n then etermine whih ftors re neee to keep the quiesent (Fig. 1). As the sis of our reonstitution, we expresse 11 the omplete set of protein hins of the 1G4 (ref. 12) in the plsm memrne of HEK ells (herefter referre to s HEK-1G4) (Supplementry Methos n Supplementry Fig. 1). The expresse omplex i not show etetle phosphoryltion (ssye y phospho-speifi ntioy to the CD3f hin, n essentil suunit require for signlling 13,14 ) unless n were o-expresse (Fig. 1). kinse tivity, s etete y mesuring levels of tivting (Tyr 394) n inhiitory (Tyr 55) phosphoryltion 3, seeme to e unffete y the presene of the or (Fig. 1). However, tivity, s mesure y inrese Tyr 493 phosphoryltion 5, ws only etetle in the presene of oth n the (Fig. 1), whih is in greement with previous t suggesting tht this kinse is intive until it ins to phosphorylte CD3f ITAMs 15 (Fig. 1). We onfirme the tivity of y emonstrting phosphoryltion of o-expresse LAT, its ownstrem sustrte n ritil ptor protein for T-ell signlling (Supplementry Fig. 2). To estlish quiesent system tht oul e tivte y, we next sought to restrin the kinse tivity of. CSK inues n intive onformtion of y phosphorylting its roxy terminus (Tyr 55) (ref. 16). However, o-expressing CSK n CSK-ining protein (CBP, lso known s PAG1), whih lolizes CSK to the plsm memrne (Fig. 1), ws insuffiient to repress phosphoryltion of CD3f (Fig. 1). is tyrosine phosphtse tht moultes T-ell signlling in omplex mnner y ephosphorylting the inhiitory Tyr 55 n tivting Tyr 394 of (refs 17, 18) n the ITAM tyrosines of the (Fig. 1). Co-expression of with severely 1 Howr Hughes Meil Institute n Deprtment of Cellulr n Moleulr Phrmology, University of Cliforni, Sn Frniso, 6 16th Street, Sn Frniso, Cliforni 94158, USA. 64 NATURE VOL JULY Mmilln Pulishers Limite. All rights reserve

2 ARTICLE RESEARCH Reeptor n kinses Moultors CBP CSK CSK CBP with short peptie (ESO9V) (ref. 19) n expresse in the Rji B-ell line s the APC (Fig. 2 n Supplementry Methos). Beuse HEKs n Rji B ells hve no intrinsi ffinity for eh other (Supplementry Fig. 3), we expresse the immune-ell hesion proteins CD2 n ICAM1 on the HEK-1G4 ells (Fig. 2), whih n intert with their respetive ounter-reeptors n LFA-1 expresse enogenously on Rji ells (Supplementry Fig. 1). This strongly inrese the onjugtion etween the two ell types, with the LFA-1ICAM1 intertion eing the preominnt river (Supplementry Fig. 3). py394 py55 APC py493 LFA-1 CD3ζpY 1 CD2 ICAM1 CBP HEK-1G4 :41s iminishe -inue tivtion ut only moestly inhiite phosphoryltion of the CD3f hin of the (Fig. 1). However, simultneous expression of CSK, CBP n onsierly reue CD3f phosphoryltion (Fig. 1). This synergy epene on CBP (Fig. 1), initing tht memrne reruitment of CSK is require for its poteny. Thus, the two mjor tivities known to repress phosphoryltion in T ells re suffiient, when omine, to keep the in quiesent stte in this reonstitute ell system. is normlly ytosoli ut ins to phosphorylte ITAMs. The trnslotion of green fluoresent protein (GFP) from the ytosol to the plsm memrne therefore provies mirosopy-se ssy of triggering. Inee, expression in HEK-1G4 ells use GFP to umulte t the plsm memrne (Fig. 1, e), wheres o-expression of CSK, CBP n elolize to the ytoplsm (Fig. 1e). Furthermore, GFP trnslote rpily to the plsm memrne upon phosphtse inhiition y pervnte (Fig. 1f n Supplementry Movie 1). Thus, this visul ssy provies ynmi reout of triggering. Reonstitution of T ellapc onjugte Hving ientifie miniml set of omponents tht oul e expresse in HEK-1G4 ells to mimi the sl or off stte of their T-ell ounterprt, we next ttempte to trigger signlling y hving the 1G4 intert with its lign, the MHC lss-i omplex oun Distne (μm) e 1 CD2ExInt 5 Int k In ol V ntr O9 o L Ex ζ D45 S R N TC Ex C E 2 CD t Co ESO9V Figure 1 Regultle triggering in n engineere HEK ell line., Shemti representtion of moleules trnsfete into HEK ells., Western lot of phosphorylte proteins fter trnsfetion of HEK ells with selete moleules (green irles). py, phosphorylte tyrosine., Cells trnsfete with the, n were trnsfete with itionl moleules (green irles), showing the synergisti tion of moultory proteins CSK, CBP n to restrin. The erese phosphoryltion etween lnes five n seven is ue to enogenous CSK reruitment., Confol imges of HEK-1G4 showing GFP reruitment to the plsm memrne in the presene of. e, Quntifition of reloliztion with inite moleules trnsfete in HEK-1G4 ells. Dt re men 6 s.e.m. of three inepenent experiments (,3 ells per experiment). f, Aition of 1 mm pervnte to HEK-1G4 ells (expressing omponents in lne eight of ) use the umultion of memrne-lolize. Sle rs, 5 mm. Peptie presente to 23:49s Triggere ells (%) CSK/CBP CSK Control ESO9V 5 Reltive intensity (%) f Pervnte e Lolize (%) Figure 2 The exlusion of phosphtse is neessry n suffiient for triggering., Proteins expresse in HEK-1G4 for ell onjugtion., HEK1G4 ells, expressing ll omponents shown in, were onjugte with APCs (Rji ells) expressing ognte (ESO9V) or ontrol. Coloure oxes enote protein representtion in the overly imge., A representtive line profile of memrne fluoresene from (re), (lue) n (green) t the onjugte interfe., Quntifition of triggering (efine s unmiguous reruitment of to onjugte region) for ll onjugtes esrie in text. Dt re men 6 s.e.m. of inepenent experiments (n 5 4 or 5, 315 onjugtes per experiment). e, Foring into the onjugte region y fusing to CD2 (CD2ExInt) loks triggering ( remins ytosoli). Quntifition is shown in. Sle rs, 5 mm. 5 J U LY VO L N AT U R E Mmilln Pulishers Limite. All rights reserve

3 RESEARCH ARTICLE APC FRB CD3ζInt FKBP Rpmyin HEK-1G4 FRBCD3ζInt Rpmyin 1 Reltive intensity (%) Triggere ells (%) We foun tht the HEK-1G4 ells forme up-shpe ontt roun the APC expressing ntigeni (Supplementry Movie 2), whih ws notly similr to T ellapc onjugtes2. ws onentrte t the site of ellell intertion (Fig. 2, ), n importntly, in the HEK-1G4 ells trnslote to the plsm memrne in this region, showing tht triggering h ourre (Fig. 2; onfirme y immunolotting in Supplementry Fig. 2). Triggering ws rpi n oul e oserve within 1 min of ell ontt (Supplementry Movie 3). Memrne trnslotion of i not our when HEK-1G4 ells were onjugte with Rji ells expressing ontrol peptiemhc omplex (Fig. 2, ) or in HEK1G4 ells lking trnsfete (Fig. 2 n Supplementry Fig. 2). We lso foun tht the ITAMs of CD3f were suffiient for lignspeifi trnslotion y using miniml omplex with the intrellulr sequenes of CD3, n e trunte fter the trnsmemrne omin (ExfInt, where Ex enotes the extrellulr n trnsmemrne prts of the moleule n Int enotes the intrellulr prt; Fig. 2). Although the APC use in these experiments expresse the ognte t high levels (,5 per mm2), reruitment ws still oservle t lower, more physiologil, levels of ntigeni (,3 per mm2) (refs 21, 22) (Supplementry Figs 1 n 4). In summry, n APC expressing the pproprite is le to eliit speifi -triggering response from our reonstitute HEK-1G4 ell. Disrupting the tin ytoskeleton severely inhiits T-ell tivtion23, ut its role in triggering is unertin. Depolymeriztion of tin filments in HEK-1G4 ells efore mixing with APCs signifintly erese the numer of onjugtes (Supplementry Fig. 3), ut ells tht i intert still showe reruitment (Supplementry Fig. 3). This result inites tht the ytoskeleton filittes the initil ellell intertions ut is not essentil for triggering in this reonstitute ell system. Mehnisti insight into how the ws triggere y me from the oservtion tht phosphtse ws exlue from the ellell interfe where the ws oun to its ognte lign (Fig. 2, ), wheres remine inlue (Supplementry Fig. 5). As omplete repression of phosphoryltion of the require, CSK n CBP (Fig. 1), the segregtion of wy from the woul e preite to shift the stey-stte lne towrs phosphoryltion. Previous stuies hve lso foun tht most moleules re exlue from the interfe of rel T ellapc onjugtes24, n oul e involve in triggering26,27. However, it hs een iffiult to sertin whether exlusion is responsile for, or onsequene of, triggering. To show more efinitively tht exlusion hs usl role, we reirete phosphtse tivity into the ellell interfe y fusing the intrellulr phosphtse omins of to the extrellulr n trnsmemrne omins of CD2 (terme CD2ExInt), euse CD2 ws lolize within the onjugte interfe (Supplementry Fig. 5). The CD2ExInt onstrut ws oserve lerly within the interfe, s expete (Fig. 2e), n ws no longer reruite to this region, initing tht triggering ws olishe (Fig. 2, e). This result shows tht exlusion is require for triggering in our reonstitute ell system. To show tht exlusion n triggering were not epenent on eh other, we temporlly seprte the formtion of the signlling zone from triggering y engineering lking its ITAMs ut inste hving n intrellulr reruitment omin (FKBP) (ExFKBPInt; Fig. 3). We expete this reeptor to lolize within the ellell interfe through its extrellulr intertion with, ut to e unle to reruit in the sene of ny signlling motifs. To initite phosphoryltion n reruitment, n FKBP-ining omin (FRB) ws fuse to the ytoplsmi region of CD3f (FRBCD3fInt), whih n e inue to imerize with ExFKBPInt upon ition of the rug rpmyin (Fig. 3). n were exlue n onentrte from the 5 Rpmyin: Peptie: Control ESO9V , 1,5 Time (s) Figure 3 ining n triggering n e physilly n temporlly unouple., Shemti of rpmyin-inue triggering ssy, with oxe region expne in right pnels showing tht rpmyin joins ytosoli FRBCD3fInt to the ExFKBPInt onstrut., Rpmyin ition uses the umultion of (n FRBCD3fInt) t the HEK ExFKBPInt ell interfe. Sle r 5 mm., Quntifition of rpmyininue triggering. Dt re men 6 s.e.m. over five experiments)., Normlize fluoresene intensity of the reruitment of (green) n FRBCD3fInt (re) with time. Dt re men 6 s.e.m. for four ells. onjugte interfe, respetively, in the sene of rpmyin. However, ws reruite to the memrne in the zones of exlusion only when the seprte omponents of the (ExFKBPInt n FRB-CD3fInt) were rought together with rpmyin (Fig. 3, ). Time-lpse imging showe no isernile ely etween the trnslotion of FRBCD3fInt to the plsm memrne n the susequent reruitment of (Fig. 3 n Supplementry Movie 4). The mehnism of segregtion We next wishe to explore wht fores proue exlusion. MHC oun with ontrol peptie i not eliit segregtion (Fig. 2), therefore the hesion pirs (CD2 n LFA-1ICAM1) must e inple of riving exlusion (Supplementry Fig. 5). We next teste the intertion lone, n foun tht it ws suffiient for onjugtion (Supplementry Fig. 3) n segregtion (Fig. 4). Fluoresene reovery fter photolehing (FRAP) n fluoresene loss in photolehing (FLIP) experiments showe tht the remine moile fter ining n free to espe the onjugte region, showing tht the segregte zone ws not forme y n immoile ggregte n remine ontiguous with the rest of the ell surfe (Supplementry Fig. 6). Thus, the intertion 6 6 N AT U R E V O L J U LY Mmilln Pulishers Limite. All rights reserve

4 ARTICLE RESEARCH mcherry 6 GFP APC HEK-1G Intensity ( 1 3 ) nm 5 1,5 2,5 Distne (nm) CD86 Ex Int β 2 AR is neessry n suffiient for exlusion, with no requirement for ownstrem triggering/signlling. Next, we exmine how the intertion ffete the sping of the two plsm memrnes of the interting ells. Using suiffrtion-resolution metho, we mesure the seprtion etween GFP-tgge in the HEK ells n mcherry in the APC (Fig. 4 n Supplementry Methos). The mesure istne of,31 nm (Fig. 4) suggests ellell seprtion of,15 nm, whih grees well with the 13 nm ell-to-ell istne etween T ells n onjugte APCs mesure y eletron mirosopy (ref. 28). By ontrst, the memrne seprtion for Rji ells expressing ontrol onjugte through hesion moleules lone (ICAM1LFA-1 or ICAM1LFA-1 n CD2) ws muh greter (Fig. 4). These results show tht the intertion rings the memrnes muh loser together thn ours with the hesion moleules. Proteins with extene extrellulr omins (suh s ) might e prevente from entering regions of lose memrne CAAX Men intensity rtio 1..5 Cellell istne (nm) CD86 Ex Int ICAM1 LFA-1 CD2/ ICAM1LFA-1 Interting moleules β 2 AR CAAX Figure 4 The intertion rives protein exlusion t onjugte regions., A shemti n representtive imge t set showing tht the intertion is suffiient to rive exlusion n its own lustering. Sle r, 5 mm., The inter-memrne istne etween the onjugtes (see Supplementry Methos) ws mesure, shown shemtilly s the seprtion etween the two fluorophores over norml line (white line) verge ross the onjugte region (otte ox). This proeure ws performe for the ognte intertion (n 5 2 ells), LFA-1 ICAM1 (n 5 23 ells) n CD2 1 LFA-1ICAM1; n 5 2 ells) intertions in the presene of ontrol. Dt represent men 6 s.e.m., HEK ells were trnsfete with GFP n inite moleule (fuse to mcherry) n onjugte with APCs ( phosphtse omins shown in ornge). Representtive imges of the onjugte region re shown, with quntifition of the rtio of fluoresene insie n outsie of the interfe. Dt re men 6 s.e.m. (n 5 2) for eh onstrut. pposition, s suggeste y the kineti-segregtion moel 8. We teste this hypothesis y fusing the intrellulr phosphtse omins of to n extrellulr omin (from CD86) of omprle size to the (terme CD86 Ex Int ). CD86 Ex Int ws lso exlue from the ellell interfe, lthough its exlusion (Fig. 4) n triggering (Supplementry Fig. 7, ) were somewht lower thn tht seen with the lrge onstrut (Fig. 4). Beuse of this unntiipte exlusion of protein with smll extrellulr omin, we next teste mcherry fusion proteins of series of memrne proteins with ifferent properties:, trnsmemrne protein with n equivlently size extrellulr omin to CD86; 2 -renergi reeptor ( 2 AR, lso known s ADRB2), seventrnsmemrne protein with smll extrellulr loops; n prenylte version of the fluorophore linke to the inner leflet of the ilyer y using short trgeting sequene (CAAX) (Fig. 4). ws exlue to similr level s CD86 Ex Int, showing tht the segregtion of the ltter onstrut ws not ue to the intrellulr phosphtse omins of (Fig. 4). 2 AR, protein with lrge lterl footprint, ws lso prtilly exlue (Fig. 4), possily through rowing effet rising from the high ensity of in the onjugte region. However, the prenylte mcherry ws istriute evenly throughout the ell memrne, showing no exlusion y (Fig. 4). The ove experiments nnot explin why CD2 (Supplementry Fig. 5) n CD2 Ex Int (Fig. 2e), whih hs extrellulr omins similr in size to CD86, were not exlue from the ellell ontt zone. We speulte tht ining of CD2 to its lign () expresse enogenously on the APC provie ounterting fore to onstrin CD2 within the onjugte region. If this were true, then expressing CD28, the ining prtner for CD86, on the APC shoul iminish the -meite segregtion of CD86 Ex Int. Inee, when CD86 Ex Int -expressing HEK-1G4 ells were onjugte with APCs o-expressing CD28, CD86 Ex Int lolize t the ellell interfe n triggering ws gretly erese (Supplementry Fig. 7, ). In summry, the intertion lone is ple of exluing plsm memrne proteins with extrellulr extensions n tht exlusion n e overome y the energy provie y ining to protein prtner on the APC. Triggering of rtifiil reeptors The preeing experiments suggeste tht triggering results from segregtion, whih is riven y the ining intertion etween the n. If this is true, then intertions etween extrellulr omins of memrne proteins with the proper sping n ffinity might eliit omprle effets to, s hs een shown in T ells 28. To explore this ie, we engineere hemilly ontrolle, ell-surfe-reeptor system onsisting of trnsmemrne protein with extrellulr FKBP n the intrellulr CD3f ITAM omins expresse in the HEK ell (FKBP Ex f Int ; mimiking the ) n trnsmemrne protein with n extrellulr FRB expresse in the APC (mimiking the ) (Fig. 5). FKBP Ex f Int n FRB Ex will only intert in the presene of rpmyin, forming omplex tht spns similr istne to. In the sene of rpmyin, onjugtes forme (through the LFA-1 ICAM1 intertion) ut ws not reruite to the memrne (Fig. 5, ). However, with rpmyin, FKBP Ex f Int reeptor lusters were oserve t the ellell interfe, even with low levels of FRB Ex lign tht re equivlent to physiologil ensities of ntigen (,5 moleules per mm 2 ; Supplementry Fig. 1). Furthermore, ws exlue from n ws reruite to these reeptor lusters, initing tht triggering h ourre (Fig. 5). Our rtifiil reeptor is struturlly nlogous to himeri ntigen reeptors (CARs), whih hve n extrellulr single-hin ntioy frgment fuse to trnsmemrne sequene n ytoplsmi CD3f ITAMs. When the CD19-speifi CAR (ref. 29), whih hs 212 Mmilln Pulishers Limite. All rights reserve 5 JULY 212 VOL 487 NATURE 67

5 RESEARCH ARTICLE shown promise in reent linil trils s gene therpy tretment of leukemi29, ws expresse in our reonstitute HEK ells n these ells were onjugte with Rji B ells (whih re CD191), we oserve CAR lustering, reruitment n exlusion (Fig. 5e, f). This triggering ws lign speifi, s onjugtion with CD192 ells i not proue these effets (Fig. 5e, f). Interestingly, the interfe of mny CD192CAR HEK ell n CD191 Rji B-ell onjugtes showe highly onvolute memrne surfe (Supplementry Movie 5), whih ws not seen for ells interting through either or FKBPExfInt2FRBEx. This memrne effet oul e prout of high-ffinity ntioy ining n might wrrnt further investigtion for its relevne to CAR poteny or potentil sie effets. 5 Conlusion FRBEx Rpmyin FKBPExζInt CD3ζ FKBPExζInt 1 Rpmyin Triggere ells (%) e CAR CD19 1 f Triggere ells (%) Rpmyin 5 CD19 Figure 5 Artifiil reeptor systems n use exlusion n triggering., Shemti of the hemilly inuile reeptor system. FRBEx reples on the APC n FKBPExfInt reples the. Rpmyin inues FKBPExfIntFRBEx intertion. Aitionl moleules hve een omitte for lrity., Rpmyin ition uses umultion, enoting reeptor triggering (gmm orretion pplie to lower imges). FRBEx-expressing ells re shown y otte lines. Sle r, 5 mm., Quntifition of rpmyin-inue triggering. Dt re men 6 s.e.m. over four experiments., A eonvolve three-imensionl renering of the reonstitute ell interfe shown in. Sle r, 2 mm. e, Reonstitute HEK ells with the reple y CAR speifi for CD19 (see text) were onjugte with either CD192 (Jurkt) or CD191 (Rji) ells, mrke y otte lines. Sle r, 5 mm. f, Quntifition of CAR-meite triggering. Dt re men 6 s.e.m. over three experiments. Our reonstitution experiments show physil mehnism for triggering tht iffers from imeriztion or onformtionl-hnge moels propose for mny ell-surfe reeptors. We fin tht the ining energy of the intertion genertes n exlusion fore for memrne proteins with lrge n/or unligte extrellulr omins, even in the sene of ownstrem signlling. By linking n inhiitory phosphtse tivity to trnsmemrne protein () tht is sujet to the exlusion fore n n tivting kinse () to the inner leflet of the memrne tht is not, the intertion n shift the kinsephosphtse lne n thus trigger the, s first suggeste y the kineti-segregtion moel8. Beuse the FKBPrpmyinFRB or ntioyntigen moules n reple the extrellulr intertion, onformtionl hnges in the re unlikely to e ritil for the funmentl mehnism of triggering. Co-reeptors n tin lso o not seem to e essentil for trnsuing ining ross the plsm memrne, lthough it is very prole tht they re neessry for hieving the high sensitivity n full seletivity of T-ell tivtion. This oul e teste in the future y pushing our reonstitute system to respon to very low ntigen ensities using more sensitive re-outs of triggering. The preise mehnism y whih the intertion results in protein exlusion remins elusive, lthough our t provie ertin lues. Exlusion oring to the kineti-segregtion moel is se on the greter size of the extrellulr omin of ompre to the omplex. However, lthough size influenes the extent of exlusion, our t show tht memrne proteins with smll extrellulr omins re lso exlue, suggesting tht itionl fores must e ting on the system. We speulte upon the riving fore in the following moel, whih inorportes previous finings334. Within the initil hesion meite y LFA-1ICAM1 etween the T ell n APC, trnsient flututions ring the two memrnes in loser pposition, llowing n moleules to intert (Fig. 6). The ining energy of this intertion must e suffiient to overome unfvourle memrne ening n ompression of the lrge proteins tht onstitute the Figure 6 A moel for steps in -meite segregtion se on memrne ening n energy minimiztion. The shemti uses equivlent moleule representtions to previous figures (only extrellulr omins re shown for simpliity) n oxe regions highlight fetures of eh pnel., After initil hesion riven y lrge reeptors suh s LFA-1, trnsient flututions in the inter-memrne istne permit enounters etween n ; the ining intertion overomes energetilly CD2 Consolition Initition Enhnement unfvourle memrne ening (re regions)., Aitionl moleules (suh s CD2 n ) provie itionl ining energy tht stilizes regions of lol memrne ening n my enhne the lol exlusion of lrger moleules y intertions., Consolition of isrete ontt regions serves to minimize unfvourle memrne ening n les to the exlusion of smller proteins tht o not provie ny ountering lignining free energy. See text for etils. 6 8 N AT U R E V O L J U LY Mmilln Pulishers Limite. All rights reserve

6 ARTICLE RESEARCH glyolyx, suh s, whih our when the two memrnes regions re rought lose together (Fig. 6). In ritil next step, seprte regions of lose memrne pposition onsolite pssively into lrger, ontiguous regions, resulting in net erese in memrne ening, s hs een previously suggeste 35. Proteins tht o not provie ny ining energy will e exlue over time, euse, s they iffuse from the region, interting moleules will e further lustere s the re of lose pposition is minimize (Fig. 6). These sme priniples my explin how miroomins initite when T ells intert with on supporte lipi ilyer 3638 (note the smll exlusion zones in our experiments t low lign ensities (Fig. 5 n Supplementry Fig. 4)). Overll, our t suggest how the ining energy ssoite with speifi moleulr reognition events tht tke ple etween two interting ells n e trnsue into intrellulr iohemil retions tht hnge ell ehviour. This moel provies plusile mehnism to explin how CARs trigger T ells to kill nerous ells 29 n my pply to other ell types tht signl using memrneoun reeptors n ligns. METHODS SUMMARY Multiple proteins were expresse in HEK ells using omintion of trnsient n stle expression y lentivirl trnsution; proteins were expresse t lose to physiologil levels n in the orret loliztion s esrie in Supplementry Methos. To rete ell onjugtes, the two ell types were entrifuge, resuspene t high ensity n ple on glss-ottome ishes for live-ell imging t 37 uc using spinning is onfol mirosopy. Imge nlysis, inluing the intermemrne istne lgorithm, ws performe using ImgeJ n Mtl (Supplementry Methos). Full methos n supplementry mteril ompny this pper. Reeive 14 Novemer 211; epte 8 My 212. Pulishe online 27 June Smith-Grvin, J. E., Koretzky, G. A. & Jorn, M. S. T ell tivtion. Annu. Rev. Immunol. 27, (29). 2. Lemmon, M. A. & Shlessinger, J. Cell signling y reeptor tyrosine kinses. Cell 141, (21). 3. Plios, E. H. & Weiss, A. Funtion of the Sr-fmily kinses, n Fyn, in T-ell evelopment n tivtion. Onogene 23, 7998 (24). 4. Love, P. E. & Hyes, S. M. ITAM-meite signling y the T-ell ntigen reeptor. Col Spring Hr. Perspet. Biol. 2, 2485 (21). 5. Au-Yeung, B. B. et l. The struture, regultion, n funtion of ZAP-7. Immunol. Rev. 228, 4157 (29). 6. vn er Merwe, P. A. & Dushek, O. Mehnisms for T ell reeptor triggering. Nture Rev. Immunol. 11, 45 (211). 7. Xu, C. et l. Regultion of T ell reeptor tivtion y ynmi memrne ining of the CD3e ytoplsmi tyrosine-se motif. Cell 135, (28). 8. Dvis, S. J. & vn er Merwe, P. A. The kineti-segregtion moel: triggering n eyon. Nture Immunol. 7, 8389 (26). 9. Lillemeier, B. F. et l. n Lt re expresse on seprte protein islns on T ell memrnes n ontente uring tivtion. Nture Immunol. 11, 996 (21). 1. Vrm, R., Cmpi, G., Yokosuk, T., Sito, T. & Dustin, M. L. T ell reeptor-proximl signls re sustine in peripherl mirolusters n terminte in the entrl suprmoleulr tivtion luster. Immunity, (26). 11. Szymzk, A. L. et l. Corretion of multi-gene efiieny in vivo using single selfleving 2A peptie-se retrovirl vetor. Nture Biotehnol. 22, (24). 12. Aleksi, M. et l. Depenene of T ell ntigen reognition on T ell reeptor peptie MHC onfinement time. Immunity 32, (21). 13. Holst, J. et l. Slle signling meite y T ell ntigen reeptor-cd3 ITAMs ensures effetive negtive seletion n prevents utoimmunity. Nture Immunol. 9, (28). 14. Irving, B. A. & Weiss, A. The ytoplsmi omin of the T ell reeptor f hin is suffiient to ouple to reeptor-ssoite signl trnsution pthwys. Cell 64, (1991). 15. Deinl, S. et l. Struturl sis for the inhiition of tyrosine kinse tivity of ZAP- 7. Cell 129, (27). 16. Bergmn, M. et l. The humn p5sk tyrosine kinse phosphoryltes p56lk t Tyr-55 n own regultes its tlyti tivity. EMBO J. 11, (1992). 17. Hermiston, M. L., Xu, Z. & Weiss, A. : ritil regultorof signling threshols in immune ells. Annu. Rev. Immunol. 21, (23). 18. Suners, A. E. & Johnson, P. Moultion of immune ell signlling y the leukoyte ommon tyrosine phosphtse,. Cell. Signl. 22, (21). 19. Chen, J. L. et l. Struturl n kineti sis for heightene immunogeniity of T ell vines. J. Exp. Me. 21, (). 2. Monks, C. R., Freierg, B. A., Kupfer, H., Siky, N. & Kupfer, A. Three-imensionl segregtion of suprmoleulr tivtion lusters in T ells. Nture 395, 8286 (1998). 21. Altn-Bonnet, G. & Germin, R. N. Moeling T ell ntigen isrimintion se on feek ontrol of igitl ERK responses. PLoS Biol. 3, e356 (). 22. Mnz, B. N., Jkson, B. L., Petit, R. S., Dustin, M. L. & Groves, J. T-ell triggering threshols re moulte y the numer of ntigen within iniviul T-ell reeptor lusters. Pro. Ntl A. Si. USA 18, (211). 23. Vlitutti, S., Dessing, M., Aktories, K., Gllti, H. & Lnzvehi, A. Sustine signling leing to T ell tivtion results from prolonge T ell reeptor oupny. Role of T ell tin ytoskeleton. J. Exp. Me. 181, 5784 (1995). 24. Johnson, K. G., Bromley, S. K., Dustin, M. L. & Thoms, M. L. A suprmoleulr sis for tyrosine phosphtse regultion in sustine T ell tivtion. Pro. Ntl A. Si. USA 97, (2).. Leupin, O., Zru, R., Lrohe, T., Muller, S. & Vlitutti, S. Exlusion of from the T-ell reeptor signling re in ntigen-stimulte T lymphoytes. Curr. Biol. 1, (2). 26. Irles, C. et l. etoomin ontrols intertion with GEMs n tivity for optiml signling. Nture Immunol. 4, (23). 27. He, X., Woofor-Thoms, T. A., Johnson, K. G., Shh, D. D. & Thoms, M. L. Trgeting of protein tyrosine phosphtse tivity to lipi miroomins on the T ell surfe inhiits signling. Eur. J. Immunol. 32, 7887 (22). 28. Chouhuri, K., Wisemn, D., Brown, M. H., Goul, K. & vn er Merwe, P. A. T-ell reeptor triggering is ritilly epenent on the imensions of its peptie-mhc lign. Nture 436, (). 29. Porter, D. L., Levine, B. L., Klos, M., Bgg, A. & June, C. H. Chimeri ntigen reeptormoifie T ells in hroni lymphoi leukemi. N. Engl. J. Me. 365, 7733 (211). 3. Shw, A. S. & Dustin, M. L. Mking the T ell reeptor go the istne: topologil view of T ell tivtion. Immunity 6, (1997). 31. Qi, S. Y., Groves, J. T. & Chkrorty, A. K. Synptiptternformtionuring ellulr reognition. Pro. Ntl A. Si. USA 98, (21). 32. Weikl, T. R. & Lipowsky, R. Pttern formtion uring T-ell hesion. Biophys. J. 87, (24). 33. Cooms, D., Demo, M., Wofsy, C. & Golstein, B. Equilirium thermoynmis of ellell hesion meite y multiple lignreeptor pirs. Biophys. J. 86, (24). 34. Alkoskel, J. M. et l. Mehnisms for size-epenent protein segregtion t immune synpses ssesse with moleulr rulers. Biophys. J. 1, (211). 35. Burroughs, N. J. & Wulfing, C. Differentil segregtion in ellell ontt interfe: the ynmis of the immunologil synpse. Biophys. J. 83, (22). 36. Yokosuk, T. et l. Newly generte T ell reeptor mirolusters initite n sustin T ell tivtion y reruitment of Zp7 n SLP-76. Nture Immunol. 6, (). 37. Cmpi, G., Vrm, R. & Dustin, M. L. Atin n gonist MHCpeptie omplexepenent T ell reeptormirolusters s sffols for signling. J. Exp. Me. 22, (). 38. Bunnell, S. C. et l. T ell reeptor ligtion inues the formtion of ynmilly regulte signling ssemlies. J. Cell Biol. 158, (22). Supplementry Informtion is linke to the online version of the pper t Aknowlegements We thnk A. vn er Merwe n V. Cerunolo for the 1G4 sequene, A. Weiss for ell lines n vie, C. June for the CD19 CAR onstrut, N. Stuurmn n K. Thorn for mirosopy help n memers of the Vle lortory for isussions. R.D.V. is Howr Hughes Meil Institute investigtor n J.R.J. is fellow of the Jne Coffin Chils Memoril Fun. Author Contriutions J.R.J. oneive the stuy, ollete the t n onute the nlyses. J.R.J. n R.D.V. esigne the experiments n wrote the mnusript. Author Informtion Reprints n permissions informtion is ville t The uthors elre no ompeting finnil interests. Reers re welome to omment on the online version of this rtile t Corresponene n requests for mterils shoul e resse to R.D.V. (vle@mp.usf.eu). 212 Mmilln Pulishers Limite. All rights reserve 5 JULY 212 VOL 487 NATURE 69