Continuous T cell receptor signaling required for synapse maintenance and full effector potential

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1 Continuous T cell receptor signling required for synpse mintennce nd full effector potentil Johnnes B Hupp 1,2, Michel Gleimer 1, Cenk Sumen 1,3 & Mrk M Dvis 1,2 Although signls through the T cell receptor (TCR) re essentil for the initition of T helper cell ctivtion, it is uncler wht function such signls hve during the prolonged T cell ntigen-presenting cell contct. Here we simultneously trcked TCR-CD3 complex nd phosphoinositide 3-kinse ctivity in single T cells using three-dimensionl video microscopy. Despite rpid internliztion of most of the TCR-CD3, TCR-dependnt signling ws still evident up to 1 h fter conjugte formtion. Blocking this interction cused dissolution of the synpse nd proportionl reductions in interleukin 2 production nd cellulr prolifertion. Thus TCR signling persists for hours, hs cumultive effect nd is necessry for the mintennce of the immunologicl synpse. T cell ctivtion involves considerle reordering of cell surfce nd cytoplsmic components into n immunologicl synpse 1,2. Synpse formtion is dependent on TCR-medited signls tht, in concert with costimultory signls, cuse the cellulr polriztion of the T cell cytoskeleton, memrne receptors nd selected cytoplsmic signling effectors towrds the T cell ntigen-presenting cell (APC) interfce. Synpse-ssocited signling lso results in the sptil segregtion of the TCR, CD28, lymphocyte-function-ssocited ntigen 1 (LFA-1) nd other surfce molecules into suprmoleculr ctivtion clusters 3. The exct function of the immunologicl synpse in T cell ctivtion is not well understood nd proly depends on the physiologicl context in which it forms. Although not required for TCR signl initition 4, synpse formtion hs een ssocited with the induction of T cell prolifertion, cytokine production, negtive thymocyte selection nd lytic grnule relese 5 8. Although the mechnisms tht couple TCR-lignd-inding events to T cell ctivtion re only poorly understood in the context of the immunologicl synpse, signling pthwys hve een defined in considerle detil through iochemicl nd genetic experimenttion. One of the erliest signling events in T cell ctivtion is the phosphoryltion of CD3 molecules, followed y the ssemly of intrcellulr signling complexes 9. These complexes constitute rnching points for numerous signling pthwys tht ultimtely led to ctivtion-dependent chnges in gene expression nd cell cycle progression. Despite the longevity of T cell APC interctions, recent report suggests tht TCR signls my e required only in the erly stges of nive T cell ctivtion 4. This is lso indicted y the fct tht T cells internlize up to 9% of their surfce TCR pool within the first 3 6 min of cell contct 1,11. Additionlly, one study indicted tht mture T cell lsts re mximlly stimulted y only 6 min of exposure to peptide mjor histocomptiilty complex (MHC) nd ntiodies to CD28 on solid sustrte 12. In this study we exmined the involvement nd function of TCRdriven signling in the context of the immunologicl synpse etween helper T cells nd B cells from its formtion to its dissolution 1 24 h lter. Using dul-color, three-dimensionl video microscopy, we monitored simultneously the rpid redistriution of TCR-ssocited CD3ζ nd the locl genertion of 3 -phosphoinositides, one of the erliest second messengers downstrem of TCR enggement. Despite the rpid internliztion of CD3ζ into synpse-proximl vesicles in the first few minutes, we still detected TCR-dependent signling ctivity 1 h fter conjugte formtion. We lso show tht locking the TCR lignd even mny hours fter conjugte formtion resulted in the rpid dissolution of the synpse structure, cell detchment nd lessening of the T cell s effector potentil. Continuous ntigenic stimultion is thus necessry for oth the integrity of the immunologicl synpse s well s full T cell ctivtion. RESULTS CD3ζ-CFP AND expression in T cell lsts A key stumling lock t present in efforts to understnd the sptil reltionships etween different molecules involved in T cell recognition hs een the simultneous expression of different green fluorescence protein (GFP) colors in the sme T cell. Although this hs een chieved in T cell hyridoms nd Jurkt T cells y electroportion 13,14, this hs proven extremely difficult in freshly isolted T cells, which require lterntive gene expression methods such s retrovirl infection. In our experience, simultneous infection of cells with retroviruses encoding seprte fusion proteins invrily gives rise to cells tht express only one of the reporter constructs, despite the correltion etween the retrovirl titer during infection nd the cellulr 1 Stnford University School of Medicine, Deprtment of Microiology nd Immunology nd 2 Howrd Hughes Medicl Institute, Beckmn Center B221, 279 Cmpus Drive, Stnford, Cliforni 9435, USA. 3 Present ddress: Center for Blood Reserch, Hrvrd Medicl School, 2 Longwood Avenue, Boston, Msschusetts 2115, USA. Correspondence should e ddressed to M.M.D. (mdvis@pmgm2.stnford.edu). NATURE IMMUNOLOGY VOLUME 4 NUMBER 8 AUGUST

2 Prolifertion ( 1 4 c.p.m) Not infected CD3ζ-GFP MCC concentrtion (µm) mount of reporter expression (J.B.H. & M.M.D, unpulished dt). We hypothesized tht successful infection could e due to virl ggregtes, nd thus we cultured different virus pckging lines together to promote the formtion of mixed ggregtes. This procedure permitted expression of mny comintions of fusion proteins in CD4 + nd CD8 + T cells (this mnuscript nd M. Purhoo, unpulished results). Here we sought to exmine the reltionship etween the TCR-CD3 complex nd erly signling ctivity t the synpse. To monitor TCR- CD3 molecules, we used CD3ζ fused to cyn fluorescent protein (CFP; fusion, CD3ζ-CFP), GFP vrint tht is good mrker for synpse formtion 15. To detect TCR-derived signls, we used the pleckstrin homology (PH) domin of the serine/threonine kinse Akt, lso known s protein kinse B (PKB), fused to yellow fluorescence protein (). This nd similr pproch using GFP fusion of the PKB PH domin were shown to e well suited for detection of PI3 kinse ctivity in mcrophge cell line 16 nd, more recently, in T cells 17,18. For our studies, we used primry lymph node T cell lsts from 5c.c7 αβ TCR trnsgenic mice retrovirlly trnsduced to express PH(AKT)- YFP s well s CD3ζ-CFP. Infected T cells expressed moderte mounts of the reporter constructs nd were indistinguishle from uninfected T cells in their response to moth cytochrome C (MCC) peptide ound to the mouse clss II molecule IE k (Fig. 1). We were le to imge the dynmics of oth fluorophores nerly simultneously, seprted y less thn 1 ms within ech imge plne. We were le to see ntigen-dependent genertion of 3 -phosphoinositides y the rpid recruitment of from cytoplsmic nd nucler loctions to the interfce fter B cell contct (Fig. 2). As shown efore 17 19, this reloction ws strictly dependent on the presence of ntigen. At the time of B cell contct, CD3ζ-CFP ws lredy present in detectle clusters t the interfce, then ccumulted rpidly t sites of incresed PI3K ctivity (Fig. 2d,e nd Supplementry video online). This colocliztion indicted cusl reltionship etween lignd recognition through the TCR nd PI3K ctivtion nd ws indictive of n erly urst in TCR-medited signling ctivity. Susequently, most of the TCR-ssocited CD3ζ-CFP t the interfce ws internlized into dense centrl cluster of vesicles minutes fter APC contct. Mesurement of TCR surfce expression y flow cytometry showed tht 5c.c7 T cells hd internlized 7% of their TCRs 3 min fter B cell contct (Supplementry Fig. 1 online). Despite this loss, recruitment to the interfce remined high. The locliztion of the PH domin nd, y inference, P13K ctivity continued throughout the durtion of the ssy (Fig. 2g) nd for mny hours (discussed elow). coloclized with the memrne-proximl ut not the memrne-distl suset of internl CD3ζ-CFP, indicting grdul dissocition of PI3K Prolifertion ( 1 4 c.p.m) Not infected MCC concentrtion (µm) Figure 1 Retrovirl expression of CD3ζ-CFP nd did not ffect the degree of T cell prolifertion in response to ntigen. (,) 5c.c7 TCRαβ T cell lsts expressing CD3ζ-CFP () nd () on dy 9 were pooled with irrdited CH27 B cells tht hd een loded with MCC peptide (concentrtions, horizontl xis) nd pulsed for 16 h with [ 3 H]thymidine 48 h fter cell pooling. Error rs represent stndrd devitions from triplicte dt sets. Dt re representtive of two independent experiments. ctivity from internlized TCR destined for endosoml destruction or recycling 11,2. In summry, removl of ctivted TCRs from contct interfce hd no pprecile effect on the mount of synpse-ssocited PI3K ctivity. TCR signls propelled sustined PI3K ctivity Although the rpid onset of PI3K ctivity is ntigen dependent nd is t lest in prt directly linked to TCR-medited ctivtion of the trnsmemrne dptors LAT (linker for ctivtion of T cells) nd TRIM (TCR-intercting molecule), costimultion through CD28 nd ctivtion of chemokine receptors lso cuses PI3K ctivtion 21. In ddition, PI3K remins ctive for severl hours fter initil B cell contct 17. To determine just how long PI3K ctivity persists nd to wht extent it depends on TCR-lignd enggement, we used monoclonl ntiodies specific for I-E k (ma ) or I-E k /MCC (ma D4), or isotypemtched control ntiodies, to lock TCR-medited signling in T cell APC conjugtes t 1, 3, 6 nd 1 h fter the experiment egn. Typiclly, more thn 9% of the T cells hd mde B cell contct within 15 min of cell pooling (dt not shown). The rtio of cytoplsmic nd memrne ssocited YFP fluorescence intensity served s mesure of 3 -phosphoinositide production. T cells continued to generte 3 -phosphoinositides throughout the entire time course of the experiment, though in somewht smller mounts in the older conjugtes (Fig. 3). This ctivity ws strictly dependent on TCR-medited signling t ll times, s ntiody lockde of TCR lignds led to complete dissiption of locliztion t the interfce. We showed in rel time the collpse of PI3K ctivity s result of ntigen neutrliztion in -expressing 5c.c7 TCRαβ trnsgenic T cell tht hd een in contct with n MCC-loded CH27 B cell for 5 h. Before ddition of the locking ntiody, YFP fluorescence ws confined minly to the T cell plsm memrne contcting the B cell, ut ws lso detectle to minor extent outside the contct re. Withdrwl of stimultory peptide-mhc lignds resulted in loss of surfce-ssocited PI3K ctivity only 2 min fter ntiody ddition. We thus infer tht neither the synptic rchitecture nor the inding sttus of stimultory lignds provided pprecile protection ginst the locking ntiody. This result ws consistent with dynmic nd permele memrne pposition, llowing fst ccess of the ntiody to the TCR lignds. It is lso likely tht fewer TCRs were ound to lignd t this stge. In seprte control experiment, we lso included two monoclonl ntiodies inding to I-A k, which re present on CH27 B cells ut irrelevnt for ntigen recognition (Supplementry Fig. 2 online). Trgeting nonntigenic MHC clss II molecules y these ntiodies hd no effect on the PI3K signl. The lockde of ntigenic stimuli led to 75 VOLUME 4 NUMBER 8 AUGUST 23 NATURE IMMUNOLOGY

3 Time fter initil cell contct _ 1 min min 1 min 2 min 6 min 16 min DIC 1 µm g c d e f CD3ζ-CFP Merge Interfce Interfce CD3ζ-CFP Figure 2 Antigen-induced PI3K ctivity coloclized with TCR-CD3 complexes within the nscent immunologicl synpse nd remined minly synpse ssocited t lter stges despite sustntil TCR internliztion. T lymphocytes were isolted from 5c.c7 αβ TCR trnsgenic mice nd infected with two tches of retroviruses expressing nd CD3ζ-CFP. Usully 15% of the T cells were positive for the expression of oth constructs t the time of imging (dy 6). CH27 B cells hd een pulsed with the MCC peptide (.4 µm) nd were pooled with trnsduced T cells. () Differentil interference contrst (DIC) cquisitions. ( d) Epifluorescent midplne cquisitions of (), CD3ζ-CFP (c) nd their corresponding overlys (d). (e,f) Three-dimensionl interfce reconstructions of (e) nd CD3ζ-CFP (f). (g) A close-up view of the re of contct t the 16-min time point (white rectngle, fr right pnel of ) of (red) nd CD3ζ-CFP (green) nd their corresponding overly. To improve imge qulity, out-of-focus light ws removed from fluorescent imge stcks using lind deconvolution lgorithm. The white r (fr left pnel of ) indictes oject size; the flse-color look-up tle (ottom right) indictes intensity vlues for interfce reconstructions (high-low representtion for nd fold increse (left mrgin) over verge surfce intensity for CD3ζ-CFP) CD3ζ-CFP Merge - High - Low rekup of the T cell APC conjugte in 2% of cses. Thus, continuous stimultion through the TCR is required not only for the PI3K signl ut lso for cell dhesion tht involves multiple signls. TCR-driven clcium moiliztion An increse in cytoplsmic intrcellulr clcium is essentil for nd is n erly mrker of T cell ctivtion 22. It cn thus serve s nother erly indictor of the T cell response. Using the clcium-sensitive dye fur-2, we monitored T cell clcium nd, nlogous to the ntiody lockde experiments descried ove, its dependence on continuous TCRderived stimuli t vrious times fter conjugte formtion. A previous study successfully used this pproch to provide cusl link etween TCR occupncy nd the clcium signl 1 min fter conjugte formtion 23. Rtiometric nlysis of fur-2-loded T cells showed tht the clcium signl persisted for more thn 1 h fter conjugte formtion (Fig. 4). As with PI3K, the incresed clcium depended on continuous TCR signling, s it could e suppressed t ll time points y the nti- IE k lockde. Sustined TCR signls mintin immunologicl synpse Withdrwl of stimultory ntigenic stimuli resulted in the rekup of out 2% of the T cell B cell couples (Fig. 3 nd dt not shown). The T cell integrin LFA-1 promotes T cell dhesion in n ctivtion-dependent wy y incresing its ffinity for intrcellulr dhesion molecule 1 (ICAM-1) fter stimultion 24,25. One hllmrk of synpse formtion is the ccumultion of ICAM-1 LFA-1 lignd pirs in ring-like pttern 3, with n occsionl centrl enrichment within the T cell B cell interfce. We nlyzed the extent to which mintennce of the ICAM-1 ring requires continuous TCR-medited signling in ntiody-lockde experiments (Fig. 4). We incuted MCC-loded CH27 B cells expressing ICAM-1 fused to GFP (ICAM-1 GFP) with 5c.c7 T cells for up to 1 h nd ssessed ICAM-1 GFP distriution y fluorescence NATURE IMMUNOLOGY VOLUME 4 NUMBER 8 AUGUST

4 Rtio (I synpse / I cytoplsm ) Blocking ntiody: Isotypecontrol Anti-IEk 1 h 3 h 5 h 1 h Anti- IEk/MCC Figure 3 PI3K ctivity remined synpse-ssocited for more thn 1 h in n ntigen-dependent wy. () Dy-6 5c.c7 TCRαβ trnsgenic T cells expressing were pooled with CH27 B cells tht hd een pulsed with the MCC peptide (.4 µm). Aout 9% of the T cells were found in conjugtes 15 min fter cell pooling (dt not shown). Monoclonl ntiodies were dded to conjugtes (ddition times, in key) nd T cells were inspected for locliztion of PH(AKT)- YFP 15 min lter. The rtio of synpse-ssocited fluorescence intensity to cytoplsmic fluorescence intensity (I synpse /I cytoplsm ) served s reltive mesure for the mount of PI3K ctivity. Error rs represent the stndrd devition of intensity rtios derived from 25 T cells tht were inspected in ech group. Blockde of stimultory lignds resulted in detchment of out 2% of the T cells independently of the time fter cell pooling. Anti-, ntiody to. () Addition of ntiody to I-E k / MCC (D4) led to rpid cesstion of synptic PI3K ctivity nd cused T cell detchment. Dt represent the time course of the response of 5-hour-old conjugte to ntiody tretment. Times ove the differentil interference contrst (DIC) nd midplne imges refer to the time of D4 ddition. microscopy. Without ntiody lockde, B cells showed the chrcteristic ring-like ICAM-1 ccumultion pttern. After out 1 h, this chnged to disk-like pttern in out one third of the conjugtes, presumly reflecting of grdul termintion of T cell B cell communiction. Addition of the ntiody to I-E k (ma ) induced dissiption of the ICAM-1 ring t ll time points fter B cell encounter. Continuous TCR-medited signls re therefore lso required to mintin functionl cell dhesion medited y LFA-1 ffinity up-regultion. Continued TCR signling shpes effector potentil We exmined the importnce of continuous T cell ntigen recognition for the secretion of interleukin 2 (IL-2) nd cell prolifertion y T helper cells (Fig. 5). To enle rpid nd synchronized conjugte Anti-I-E k DIC 3 s 1 µm 3 s 9 s 15 s Time fter ddition of ntiody 21 s 27 s formtion, we pooled T cells with irrdited nd ntigen-loded B cells t rtio of 1:2 nd gently centrifuged them in 96-well roundottomed pltes. We determined the mount of IL-2 secreted into the medi y enzyme-linked immunosorent ssy (ELISA) 48 h lter (Fig. 5).The detection of IL-2 mounts ove ckground mounts required 4 h of uninterrupted T cell ntigen recognition, yet more thn 1 nd less thn 24 h were required for mximl IL-2 production. T cell prolifertion, s mesured y [ 3 H]thymidine uptke 48 h fter cell pooling, lso required continuous ntigenic stimultion, though to lesser degree (Fig. 5c). Agin, etween 1 nd 24 h of TCR stimultion were necessry for full prolifertive response. To ovite the possiility tht we hd missed erlier or lter prolifertive events in our nlysis, we determined the totl numer of cell Anti-I-E k Conjugtes (%) Clcium signl: Elevted (i 2) Moderte (1.2 < i < 2) Low (i 1.2) Conjugtes (%) ICAM-1 distriution: Ring Disk Fint ring 1 h 3 h 5 h 1 h 1 h 3 h 5 h 1 h No pttern Figure 4 Continued clcium moiliztion nd the integrity of the immunologicl synpse depend on TCR signls. () Clcium moiliztion continued for more thn 1 h fter conjugte formtion nd ws fold enrichment dependent on persistent TCR signls. Dy-6 5c.c7 TCRαβ trnsgenic T cell lsts were pooled t rtio of 1:2 with CH27 B cells tht hd een pulsed with.4 µm MCC peptide. Cells were loded with the clcium-sensitive dye fur-2 15 min efore nd imges were otined 15 min fter the ddition of ntiody to I-E k (Anti I-E k ; +, with;, without) fter cell pooling (ntiody ddition times, elow grph). T cells could e esily identified y their smller size. Bsed on the rtiometric nlysis of fur-2 fluorescence, the increse (i) in clcium signl ws clssified s elevted (more thn 2-fold), moderte (etween 1.1-fold nd 2-fold) or low (etween 1-fold nd 1.2-fold). n = 5. () The integrity of the immunologicl synpse depended on persistent TCR signls. Dy-6 5c.c7 TCRαβ trnsgenic T cell lsts were pooled t rtio of 1:2 with ICAM-1 GFP expressing CH27 B cells tht hd een pulsed with.4 µm MCC peptide. Conjugtes were inspected 15 min fter the ddition of ntiody to I-E k (Anti I-E k ; +, with;, without) fter cell pooling (ntiody ddition times, elow grph). Synptic ICAM-1 GFP distriutions were clssified s ring (ring-shped enrichment of 1.4 verge surfce density with n occsionl centrl enrichment), disk ( 1.4 verge density spred over the entire contct re), fint ring (ring-shped ICAM-1 enrichment of <1.4 verge surfce density) or no pttern (rndom pttern with mximl enrichment of 1.4 verge density). n = VOLUME 4 NUMBER 8 AUGUST 23 NATURE IMMUNOLOGY

5 Positive control (%) Positive control (%) Isotype control Positive control Isotype control Positive control IL-2 h 1 h 2 h 4 h 6 h 1 h 24 h [ 3 H]thymidine Isotype control No ntiody Numer of divisions: 321 CFSE h 1 h 2 h 4 h 6 h 1 h 24 h , Figure 5 Sustined TCR signling exerted cumultive effect on T cell ctivtion. (,) Antiody lockde interfered with the IL-2 nd the prolifertive response in time-dependent wy. Here, 5c.c7 TCR αβ trnsgenic T cell lsts were pooled with irrdited MCC-pulsed (.4 µm) CH27 B cells (-h time point) nd ntiody to IE k /MCC ws dded fter cell pooling (ntiody ddition times, elow grphs). The positive control ws left untreted. The isotype-mtched control ntiody (ma Tü36, mouse IgG2) ws dded t the -h time point. () Secreted IL-2 ws determined from medi superntnt y ELISA 48 h fter cell pooling. () Cells were then pulsed for 16 h with [ 3 H]thymidine nd nlyzed for thymidine uptke. Negtive controls (T cells lone, B cells lone, nd B cells nd T cells pooled in the sence of MCC peptide) showed ckground mounts (dt not shown). [ 3 H]thymidine incorportion of the -h smples cn e ttriuted to residul prolifertion of irrdited B cells (<5% of positive control). Error rs refer to the stndrd devition derived from quintuple dt sets. The dt shown re representtive of three independent experiments. (c) Anlysis of the numer of cell divisions showed cumultive effect of persistent TCR signls on the prolifertive response. Here 5c.c7 TCR αβ trnsgenic T cell lsts were leled with CFSE nd pooled with irrdited MCC-pulsed CH27 B cells (-h time point). Antiody to IE k /MCC (D4) ws dded fter cell pooling (ddition times, elow grphs). The positive control ws left untreted (no ntiody; ottom). The isotype-mtched control ntiody (Tü36) ws dded t the -h time point. Cells were nlyzed y flow cytometry 12 h fter cell pooling. Dt re representtive of three independent experiments. divisions y stining the T cells with croxyfluorescein dicette succinimidyl ester (CFSE) 26 (Fig. 5d). As is typicl for effector nd memory T cells, most T cells underwent three cell divisions fter 5 d. In contrst, ddition of the D4 ntiody (to I-E k MCC) t ny time point up to 1 h resulted in reduction in the numer of cell divisions. This ws most evident t the -h time point (no division) nd ws rely detectle t the 24-h time point (three divisions). Notly, 1 h of c h 1 h 2 h 4 h 6 h 1 h 24 h uninterrupted TCR signling gve rise to only two cell divisions nd ws thus insufficient for the T cells to proliferte to their full cpcity. DISCUSSION In this report we demonstrted tht TCR signls were sustined throughout the lifetime of the T cell APC interction nd tht this signling ctivity ws required for the mintennce of the immunologicl synpse nd for full commitment to T cell ctivtion. At its lowest point, the TCR density ws pproximtely 3% of its originl mount, consistent with previous reports 1,11,2. Here we showed tht TCR-CD3 internliztion ws well underwy s erly s 6 min fter T cell APC contct nd seemed to loclize to one centrl cluster of vesicles. Even with this loss, there were still mny TCRs t the interfce. Similrly, it seems likely tht the filure to find TCR-proximl tyrosine phosphoryltion events 3 6 min fter initil cell contct 4 reflects the limittions of ville detection methods rther thn the sence of these events. Here, we ccessed loclized ctivtion of PI3K s well s the moiliztion of intrcellulr clcium to ssess the contriution of TCR signling. Although oth signls could derive from signling ctivities other thn TCR-proximl tyrosine phosphoryltion, the ntiody-lockde studies confirmed their dependence on TCR signls t ll times. The requirement of persisting TCR signls for the progression of the T cell ctivtion progrm sheds light on the physiology of T cell help ut leves mny questions unnswered. Why re these conjugtes long lived? Signl integrtion itself might require time nd certinly concludes in time-consuming cellulr processes such s gene trnscription, trnsltion, protein secretion nd induction of mitosis. Although continued TCR signls seem to fuel these processes, their requirement might exert proofreding mechnism to void the ctivtion of ystnder APCs. Conversely, APCs might e given the necessry time to process T cell derived stimuli. CD4 + effector T cells ecome committed fter only 1 h of stimultion y plte-ound MHC-peptide complexes nd ntiodies to CD28; prolonged ntigenic stimultion (>5 h) led to ctivtion-induced cell deth rther thn T cell prolifertion 12. The disprity etween those results nd ours reported here my indicte the importnce of signls not provided y the plte-ound method of stimultion (such s cytotoxic T lymphocyte ntigen 4 (CTLA-4)-medited ttenution). The ctivtion of effector or memory T cells nd the priming of nive T cells re distinct events in the immune response; they involve T cells of different developmentl stges, engge different APCs, occur t different ntomicl sites nd fulfill different physiologicl functions 27,28. Nonetheless, oth types of ctivtion re TCR-signl dependent. Although there is considerle controversy over the lifespn of individul APC T cell interctions 29 31, there is strong evidence tht T cell priming is influenced y the durtion of TCR stimultion. A recent report suggested tht only 2 h of cell interction re required for the priming of nive CD4 + T cells through contct with T cell nd B cell depleted splenocytes 4. Consistent with the results reported here, those conditions gve rise to only one cell division, which proly represents premture termintion of priming. Indeed, prolonged TCR stimultion hd cumultive effect on the prolifertive cpcity of nive CD4 + T cells tht divide up to nine times 32. A previous study indicted tht the numer of cell divisions in the T helper cell type 1 versus type 2 polriztion of the effector T cell comprtment might e involved 33. The durtion of TCR signling might influence the development of helper T cell susets, s suggested efore 34. Antigen-dependent initition of integrin-medited cell dhesion is well chrcterized; however, much less is known out the mechnisms tht led to synpse termintion. Representing key feture of NATURE IMMUNOLOGY VOLUME 4 NUMBER 8 AUGUST

6 the mture immunologicl synpse, the ring-shped locliztion of the ICAM-1 dhesion molecule requires continuous TCR signling, finding with rod implictions for cell-contct-dependent communiction in dptive immunity. A decrese in TCR signls my lso e centrl to the physiologicl dissolution of T cell B cell conjugtes. Downregultion of surfce TCR might constitute one ut proly not the only mechnism contriuting to signl ttenution. After n initil decrese in TCR surfce density, equilirium is proly reched through ongoing lignd-induced TCR internliztion nd replenishment of the cell surfce pool with newly synthesized TCRs 32. However, s shown here, TCR-medited signling continues for mny hours even t reduced receptor surfce expression. Signl ttenution might lso result from the inhiitory function of CTLA-4, which competes with the costimultory receptor CD28 for inding to B7-1 nd B7-2, nd disrupts TCR-proximl tyrosine phosphoryltion events through recruitment of the intrcellulr SHP-2 phosphtse 35. Undetectle in nive CD4 + T cells, CTLA-4 protein expression is upregulted in effector T cells, yet is confined minly to endosoml comprtments in the sence of ntigenic stimuli 36. CTLA-4 requires trnsport to the immunologicl synpse to exert its inhiitory function, nd its ccumultion there seems proportionl to the strength of the ntigenic signl 37. Further studies might show reltionship etween CTLA-4 recruitment nd synpse termintion. Proly reflecting their distinct function in cquired immunity, very different requirement for TCR signls pplies to the ctivtion of nive nd effector or memory CD8 + cytolytic T cells (CTLs). Without the need for costimultion, effector CTLs cn kill ny ntigenic MHC clss I expressing nucleted cell during comprtively short interction through Fs-induced poptosis or y delivery of lytic grnules 38. In the sence of clerly identifile regultory function of CTLs, the min rison d être of the CTL trget cell synpse might e to help verify ntigenicity nd provide sptil orienttion for swift delivery of lethl hit. A rief period of ntigenic stimultion is sufficient to trigger clonl expnsion nd differentition during CTL priming 39,4. This notly different ehvior might underlie the distinct regultion of the CTL response to pthogenic chllenge, ut should lso e considered in the physiologicl context of cross-priming through CD4 helper T cells. APCs must up-regulte costimultory lignds s precondition for CTL priming, in process tht often involves much longer ntigen-dependent interctions with nive CD4 helper T cells In summry, our findings provide insight on the interdependence etween signl reception through the immunologicl synpse nd cellulr signl integrtion. Antigenic lignds not only drive synpse formtion ut re lso eqully importnt for its mintennce. The need for some TCR signling t ll times demonstrtes the rpid reversiility of the cellulr mechnisms required to preserve n ordered cell-cell interfce. Such requirement lso seems to represent regultory feedck mechnism, which would help to determine the response to n ntigenic chllenge. METHODS Plsmids nd cell lines. DNA encoding PH(AKT) s cloned into peyfp-c1 (ref. 16) ws provided y T. Meyer (Stnford University). PCR using the primers JH3/1 (5 -CCATCGATGGAGTGAACCGTCAGATCCGCTAGCGCTACCGG TC-3 ) nd JH4/1 (5 -GGATCGATCCTCTACAAATGTGGTATGGCTGATTA TGATCA-3 ) gve rise to 1.2-k frgment tht ws inserted through ClI (underlined sequence) into the retrovirl expression vector pmg-1b ( gift from G. Noln, Stnford University). DNA encoding full-length mouse CD3ζ-CFP (provided y M. Krummel, University of Cliforni t Sn Frncisco) ws cloned into the retrovirl expression vector pib II using the 5 XhoI nd 3 NotI restriction sites. Both retrovirl expression vectors re derived from the MFG series of retrovirl vectors nd hve full-length Moloney long-terminl repets, n extended Ψ sequence nd n internl riosoml entry site upstrem of the lsticydin-s deminse gene. For virus production, the ecotropic virus pckging line Phoenix E ( gift from G. Noln, Stnford University) ws stly trnsfected with these expression vectors. Antiodies nd mice. Antiodies (to IE k ) 44 nd D4 (to IE k -MCC) 45 were purified from hyridom culture superntnt. RM4-5 (ntiody to mouse CD4) conjugted to Cy5-phycoerythrin or fluorescein isothiocynte 46, Tü36 (mouse IgG2, κ ntiody to HLA DR), Tü39 (mouse IgG2, κ ntiody to HLA DP, DQ, DR) 47 nd 2.4G2 (ntiody to mouse CD16/CD32, FcγIII/II) 48 were purchsed from BD PhrMingen. The 5c.c7 TCRαβ trnsgenic mice (on B1A ckground) were purchsed from Jckson lortories. Retrovirl trnsduction. Virus-producing lines were grown t 32 C for 2 d in 15-cm culture dishes. For dul-color experiments, pckging lines (producing CD3ζ-CFP nd expressing viruses) were pooled nd cultured together. Collected medi superntnt ws filtered through syringe filters with pore size of.45 µm (Schleicher nd Schuell) nd centrifuged overnight t 1, r.p.m. (Sorvll, RC5B centrifuge, SA6 rotor). At 24 h efore infection (dy ), lymphocytes were isolted from lymph nodes of 5c.c7 trnsgenic mice nd stimulted in 24-well pltes in the presence of 2 µm HPLC-purified MCC peptide (ANERADLIAYLQADK) t density of cells per well nd per milliliter of full medium (RPMI contining 1% FCS (Gemini), 2 mm L-glutmine (Irvine Scientific), 1 U/ml penicillin/streptomycin, 5 µm β-mercptoethnol (Sigm-Aldrich) nd 1 mm sodium pyruvte (Irvine Scientific)). For infection, the virus-contining pellets from the overnight centrifugtion nd 5 µg/ml polyrene (Sigm-Aldrich) were dded to cells (one pellet per milliliter of cell culture), which were thn sujected to 1-h centrifugtion in 24-well pltes t 2,5 r.p.m. (Allegr 6KR; Beckmn Coulter); this ws considered dy 1. Cells were cultured t 37 C. On dy 2, 1 U/ml IL-2 ws dded, nd on dy 3, 4 µg/ml lsticydin-s (Invitrogen) ws dded. On dy 5, ded cells were removed y centrifugtion through Histopque-1119 (Sigm-Aldrich) cushion (7 min t 2, r.p.m. in Beckmn Allegr 6KR tletop centrifuge). Cells were used for experiments on dy 6 nd dy 9. Microscopy. Experiments were done using Zeiss Axiovert S1TV microscope (Crl Zeiss) nd 4 Flur (NA 1.3) ojective. The microscope ws fitted with high-speed piezo-driven Z-dimension motor (Physik Instrumente). A Sutter DG-4 high speed opticl chnger (Sutter Instruments) equipped with 3-W Xenon light ul served s right excittion source nd supports chnges in excittion wvelengths within 1 ms. For dul-color experiments, emission ws opticlly split using Dul-View Micro-Imger (Opticl Insights) nd nlyzed synchronously using CFP nd YFP emission filters (Chrom Technology). Imges were cquired with cooled interline Coolsnp hq cmer (Roper Scientific). Z-stcks usully contined 21 imge plnes tht were 1 µm prt tht were cquired in the cmer streming mode to minimize exposure time. Imging experiments were done t 37 C nd 7% CO 2 in imging uffer (full RPMI medium without phenol red) using n ojective heter (Bioptechs) nd Zeiss environmentl control system. For clcium imging, T cells were pulsed for 3 min t room temperture with 5 µg/ml fur-2-m (Moleculr Proes) nd wshed once in imging uffer. The rtio of the fur-2 emission intensity t 51 nm resulting from 34-nm nd 38-nm excittion ws determined to quntify the reltive intrcellulr clcium concentrtion. Dt cquisition nd nlysis were done in Metmorph 5. (Universl Imging). To remove out-of-focus light, threedimensionl fluorescence imge stcks were sujected to 4 itertions of lind deconvolution lgorithm (Delur 8.; AutoQunt Imging). IL-2 secretion ELISA nd [ 3 H]thymidine incorportion. T cell lsts (1 1 5 ; dy 9) were pooled with irrdited (12, rd) CH27 B cells (2 1 5 ) tht hd een pulsed over night with.4 µm MCC peptide, in 96-well round-ottomed pltes in volume of 2 µl nd spun t 5 r.p.m. in Beckmn Allegr 6KR tletop centrifuge for 2 min to enforce cell contct formtion. After 48 h, 5 µl culture superntnt were removed nd ssyed for IL-2 y ELISA s descried efore 49. Ech well ws then pulsed for 16 h with 1 µci [ 3 H]thymidine/2 µl. Incorported [ 3 H]thymidine ws quntified s descried efore VOLUME 4 NUMBER 8 AUGUST 23 NATURE IMMUNOLOGY

7 CFSE ssy. T cells (dy 9) were wshed twice in Hnk s lnced slt solution plus 2 mm clcium nd djusted to concentrtion of cells per ml, then leled for 1 min t 37 C with 1 µg CFSE (Moleculr Proes). Cells were wshed twice in full medi. T cells (1 1 5 ) were pooled with irrdited ( rd) nd MCC-pulsed (.4 µm) CH27 B cells (2 1 6 ) in 96-well roundottomed pltes in volume of 2 µl nd spun t 5 r.p.m. in Beckmn Allegr 6KR tletop centrifuge for 2 min to enforce cell contct formtion. After 3 d, cells were trnsferred to 48-well pltes nd 3 µl fresh medi ws dded to ech smple. At 12 h fter cell pooling, cells were stined with monoclonl ntiody to mouse CD4 conjugted to Cy5-phycoerythrin nd nlyzed y flow cytometry (Epics Elite ELP; Beckmn-Coulter). Smples were nlyzed using Flow-Jo softwre (Treestr). Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk M.F. Kuhns, M.F. Krummel, R. Scimms nd L.C. Wu nd for dvice nd discussions. We thnk P.J. Eert, C. Gerke, M. Krogsgrd, B.F. Lillemeier, Q.-J. Li nd M. A. Purhoo for comments on this mnuscript. We thnk N. Prdo for technicl ssistnce nd S.M. Wheton for finl preprtion of the mnuscript. J.B.H. ws fellow of the Cncer Reserch Institute nd M.G. is predoctorl fellow of the Howrd Hughes Medicl Institute. This work ws supported y grnts from the Howrd Hughes Medicl Institute nd the Ntionl Institutes of Helth. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Received 15 April; ccepted 3 June 23 Pulished online 13 July 23; doi:1.138/ni Grkoui, A. et l. The immunologicl synpse: moleculr mchine controlling T cell ctivtion. Science 285, (1999). 2. Dustin, M.L., Bromley, S.K., Dvis, M.M. & Zhu, C. Identifiction of self through twodimensionl chemistry nd synpses. Annu. Rev. Cell. Dev. Biol. 17, (21). 3. Monks, C.R., Freierg, B.A., Kupfer, H., Sciky, N. & Kupfer, A. Three-dimensionl segregtion of suprmoleculr ctivtion clusters in T cells. Nture 395, (1998). 4. 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