TCR signalling network organization at the immunological synapses of murine regulatory T cells.

Eu . J. Immunol. 2017. 0: 1–16 Ma co an Ham e al. DOI: 10.1002/eji.201747041 1 Basic Molecula immunology and signaling Resea ch A icle TCR signalling ne wo k o ganiza ion a he immunological synapses o mu ine egula o y T cells Ma co an Ham∗1,Ren ´ eTeich ∗2, La s Philipsen3, Jana Niemz2, Nicole Amsbe g1, Jose Wissing1, Man ed Nim z1,Lo ha G ¨ obe2, S e anie Kliche3,NadineThiel 2, F ank Klawonn1,5,Ma ioHubo 4, Helmu Jonulei 4, Pe e Reicha d 3,And easJ.M ¨ ulle 3,6, Jochen Huehn∗2and Lo ha J¨ ansch∗1 1Cellula P o eomics, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 2Expe imen al Immunology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 3Ins i u e o Molecula and Clinical Immunology, O o- on-Gue icke Uni e si y, Magdebu g, Ge many 4Depa men o De ma ology, Johannes Gu enbe g-Uni e si y Mainz, Mainz, Ge many 5Depa men o Compu e Science, Os alia Uni e si y o Applied Sciences, Wol enbue el, Ge many 6In a i al Mic oscopy o In ec ion and Immuni y, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many Regula o y T (T eg) cells equi e T-cell ecep o (TCR) signalling o exe hei immunosup- p essi e ac i i y, bu he p ecise o ganiza ion o he TCR signalling ne wo k compa ed o con en ional T (Tcon ) cells emains elusi e. By using accu a e mass spec ome y and mul i-epi ope ligand ca og aphy (MELC) we cha ac e ized TCR signalling and ec ui - men o TCR signalling componen s o he immunological synapse (IS) in T eg cells and Tcon cells. Wi h he excep ion o Themis which we de ec ed in lowe amoun s in T eg cells, o he majo TCR signalling componen s we e ound equally abundan , howe e , hei phospho yla ion-s a us no ably disc imina es T eg cells om Tcon cells. O e - all, his s udy iden i ied 121 T eg cell-speci ic phospho yla ions. Sho - e m igge ing o T cell subse s ia CD3 and CD28 widely ha monized hese a ia ions wi h he excep ion o ele en TCR signalling componen s ha mainly egula e cy oskele on dynamics and molecula anspo . Acco dingly, conjuga ion wi h B cells indeed caused a ian cellula mo phology and e ealed a T eg cell-speci ic ec ui men o TCR signalling componen s such as PKCθ, PLCγ1 and ZAP70 as well as B cell-de i ed CD86 in o he IS. Toge he , esul s om his s udy suppo he exis ence o a T eg cell-speci ic IS and sugges T eg cell-speci ic cy oskele on dynamics as a no el de e minan o he unique unc ional p ope ies o T eg cells. Keywo ds: Immunological synapse MELC Phosphop o eome TCR signalling T eg Addi ional suppo ing in o ma ion may be ound in he online e sion o his a icle a he publishe ’s web-si e In oduc ion CD4+ egula o y T (T eg) cells play an essen ial ole in main- aining immune homeos asis and in p e en ing au oimmune eac- i i y o po en ially sel - eac i e lymphocy es ha ha e escaped Co espondence: D . Lo ha J¨ ansch e-mail: [email p o ec ed] cen al ole ance mechanisms [1]. I is widely accep ed ha bo h Foxp3+T eg cells as well as hei e ec o coun e pa s, Foxp3− con en ional CD4+T (Tcon ) cells, equi e ac i a ion ia he T-cell ecep o (TCR) o ull acquisi ion o hei immunosup- p essi e and e ec o unc ions, espec i ely [2–5]. TCR signalling ∗Sha ed i s and senio au ho ship C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 2Ma co anHame al. Eu . J. Immunol. 2017. 0: 1–16 in T eg cells, he eby, seems o be di e en ially o ganized as in Tcon cells: T eg cells show educed Ca2+ lux and ERK1/2 phos- pho yla ion upon TCR s imula ion [6–8], and downs eam sig- nalling molecules such as Lck, LAT and PLCγ1 a e essen ial o T eg cell supp essi e capaci y [9, 10]. In his line, a no el TCR- media ed ADAP/in eg in-independen PLCγ1 ac i a ion pa hway was desc ibed o be equi ed o supp ession o Tcon cells by T eg cells [11]. Fu he mo e, T eg cells exhibi educed S473 phospho yla ion o Ak which seems a p e equisi e o supp es- sion [12]. Al hough he enzyma ic ac i i y o he y osine kinase ZAP70 seems o be dispensable o he supp essi e pheno ype o Foxp3+T eg cells [13], a single mu a ion wi hin he SH2-domain o ZAP70 leads o impai ed supp essi e ac i i ies, which indica ed i s impo ance a he immunological synapse (IS) [14]. Finally, i has only ecen ly been ecognized ha he ec ui men o TCR sig- nalling componen s in o he IS is di e en ially o ganized in T eg cells and Tcon cells. The p o ein kinase PKCθis ec ui ed o he IS in Tcon cells, in T eg cells, howe e , his kinase was ound o be seques e ed away om he IS, bu s ill o impo ance o con ol supp ession [15]. The spa ial ec ui men o signalling componen s du ing IS o ma ion c i ically depends on cy oskele on dynamics, which in u n is con olled by TCR ac i a ion and subsequen phospho y- la ion o p o eins egula ing cy oskele on eo ganiza ion [5]. As pa o hese p ocesses he mic o ubule-o ganizing cen e (MTOC) is apidly ansloca ed in p oximi y o he IS. MTOC eposi ioning depends on LAT, ZAP70 and SLP76 [16] and is egula ed by a cascade o dis inc iso o ms o he amily o no el p o ein kinase C (nPKC) [17]. The MTOC se es as a pla o m o coo dina e molec- ula mo emen s om and o he IS h ough he suppo o mic o- ubule (MT) mo o s [18]. Fo ins ance, TCR mic oclus e s mo e along MTs owa ds he cen e o he IS in a dynein-dependen manne [19], and hind ance o MTOC pola iza ion, molecula anspo and cy oskele on dynamics p e en p ope p opaga ion o TCR signals [20]. I is now emp ing o specula e ha he localiza ion o sig- nalling modules wi hin he IS may be ins umen al o he o ma- ion o a T eg cell-speci ic IS and he supp essi e pheno ype o T eg cells. A his momen , howe e , he knowledge on how TCR signalling, p o ein ec ui men and IS o ma ion a e di e en ially o ganized in T eg cells is a om comple e. In he p esen s udy, we ha e now sys ema ically s udied he ac i i y and spa ial o ga- niza ion o TCR signalling componen s in ex i o isola ed T eg cells and Tcon cells by employing accu a e mass spec ome y and mic oscopy suppo ed by mul i-epi ope ligand ca og aphy (MELC) [21, 22]. Toge he , ou da a e eal ha TCR engagemen ha monizes he ac i i y s a us wi hin he TCR signalling ne wo k, bu in pa allel induces a dis inc di e ging signalling pa e n a egula o s o cy oskele al dynamics. This was accompanied by a a ian ec ui men o TCR signalling componen s in o he IS upon T cell/B cell conjuga ion as well as a ian CD86 up ake. Thus, esul s o his s udy subs an ia e he concep o a T eg cell-speci ic IS and sugges subse -speci ic cy oskele on dynamics as a no el de e minan o hei supp essi e pheno ype. Resul s Majo TCR signalling componen s a e equally abundan in T eg cells and Tcon cells Al hough a ious TCR signalling componen s ha e al eady been iden i ied ha con ol di e en ia ion and unc ion o T eg cells [23], a comp ehensi e pic u e o he TCR signalling ne wo k o ga- niza ion is s ill missing. Ou i s goal was o analyse he o e all p o ein abundances o TCR signalling componen s compa a i ely in ex i o isola ed, highly pu e mu ine CD4+CD25+T eg cells and CD4+CD25−Tcon cells (Suppo ing In o ma ion Fig. 1A). These T cell subse s we e spo -checked o majo cha ac e is ics, i.e. exp ession o he ansc ip ion ac o Foxp3 in T eg cells and IL-2 p oduc ion in Tcon cells (Suppo ing In o ma ion Fig. 2). Nex , o al p o eomes we e ex ac ed om so ed T eg cells and Tcon cells and quan i a i e pep ide sequencing was pe o med using ex i o iTRAQ labelling and accu a e Fou ie ans o m mass spec ome y (FTMS). Da abase sea ches we e hen es ic ed o high quali y MS da a and iden i ied >4000 p o eins in mu ine T eg cells and Tcon cells (Suppo ing In o ma ion Table 1). Reg- ula o y da a o CD3ε,PLCγ1, Foxp3, CD4 and CD25 (IL2Rα) we e ound in ull acco dance wi h li e a u e, i.e. equal le els o CD3ε and PLCγ1 in bo h subse s and sligh ly educed le els o CD4 and high abundances o Foxp3 and CD25 (IL2Rα) in T eg cells (Fig. 1A, Suppo ing In o ma ion Fig. 3 and Suppo ing In o - ma ion Table 1). C oss-inspec ion o ou da a wi h o me pub- lished p o eomic s udies [24–26] could now subs an ia e a se ies o p o eins wi h T eg cell-speci ic abundances, al hough hese p o eins a e no desc ibed o be di ec ly in ol ed in TCR sig- nalling. This includes Helios, Aiolos, So in nexin-18, E gic1, Annexin A4, CAPG, S im2, NDRG1, Ladinin-1, Niban, S100A4 and Re iculocalbin-1 (Suppo ing In o ma ion Table 1). Wi h espec o TCR signalling we mapped ou p o eome da a wi h he suppo o in e ac ome da abases (GeneGo, Me aCo e and IPA) and con i med ha he canonical TCR signalling pa h- way was comple ely co e ed. No ably, all TCR signalling compo- nen s we e ound wi h simila abundances in bo h T cell sub- se s wi h he excep ion o Themis. In T eg cells, we de ec ed Themis wi h educed p o ein le els (Fig. 1A, Suppo ing In o - ma ion Fig. 3 and Suppo ing In o ma ion Table 1), a inding which was con i med by Wes e n blo ing and a he mRNA le el by quan i a i e RT-PCR (Suppo ing In o ma ion Fig. 4A). Themis ac s downs eam o LAT and is equi ed o IL-2 esponses in Tcon cells [27]. Hence, we specula ed ha educed Themis exp es- sion in T eg cells migh be causal o known ane gic signalling and impai ed IL-2 esponses. Howe e , e o i al o e exp ession o Themis in ex i o isola ed T eg cells did nei he es o e IL-2 p oduc ion no ab oga e supp essi e capaci y o T eg cells (Sup- po ing In o ma ion Fig. 4B and 4C), sugges ing ha educed Themis exp ession is no c i ical o he unc ional p ope ies o T eg cells. In summa y, ou MS analysis e ealed ha majo TCR signalling componen s a e equally abundan in bo h T-cell subse s. C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu Eu . J. Immunol. 2017. 0: 1–16 Molecula immunology and signaling 3 Figu e 1. Quan i a i e mass spec ome y de e mines he abundances and esponsi eness o TCR signalling componen s. iTRAQ-based quan i a- i e in o ma ion o selec ed TCR signalling componen s and phospho yla ion e en s in T eg cells and Tcon cells. Rep esen a i e MS/MS-spec a and pep ide sequences a e gi en in Suppo ing In o ma ion Fig. 3. (A) Abundance: T eg cells and Tcon cells we e eshly isola ed om spleen and lymph nodes o emale BALB/c mice and pooled. P o eomes we e ex ac ed and de i ed pep ides om bo h subse s we e di e en ially labelled by iTRAQ and quan i a i ely cha ac e ized by MS in wo independen s udies (32 mice/s udy). Only in o ma ion o high-con iden pep ides we e accep ed (Suppo ing In o ma ion Table 1). Mean iTRAQ in ensi ies o T eg cells and Tcon cells a e p esen ed; s anda d de ia ions o ela i e di e ences a e CD3ε±0.080, PLCγ1±0.011, Foxp3 ±0.607 and Themis ±0.027 (colou codes a e gi en in he le panels). (B,C) Responsi eness: T eg cells and Tcon cells we e eshly isola ed om spleen and lymph nodes o emale BALB/c mice (580 mice). Cells we e CD3/CD28-s imula ed o 5 min in indi idual pools o cells de i ed om 25 o 30 mice each. Phosphopep ides om bo h subse s and s imula ed/non-s imula ed condi ions we e di e en ially iTRAQ labelled ollowed by 15 quan i a i e LC-MS expe imen s in one independen s udy. Only in o ma ion o high-con iden phosphopep ides we e accep ed (Suppo ing In o ma ion Table 2). (B) Phospho yla ion si es o p e-ac i a ed TCR componen s (CD3ε,CD3ζ) and CD3/CD28- esponsi e down-s eam componen s (ZAP70, SLP76) in T eg cells. (C) Phospho yla ion si es a PKCθ, DBNL, MAPK14 and TIAM1, which disc imina e T eg cells and Tcon cells (colou codes a e gi en in he le panels). Iden i ica ion o di e ging TCR signalling componen s We nex asked, whe he equally abundan TCR signalling compo- nen s migh ha e a di e en ial ac i i y in T eg cells and Tcon cells. P o ein phospho yla ions cons i u e he key mechanism in signalling and we he e o e aimed o p o ile he TCR signalling ne - wo k by using quan i a i e phosphop o eomics. Ou pi o al aim was o s udy he esponsi eness o he TCR signalling componen s unde apid and obus ac i a ion condi ions. Fo ha , T eg cells and Tcon cells we e ex i o isola ed and ba ch-wise ei he le un ea ed o s imula ed o 5 min using an i-CD3 and an i-CD28 an ibodies (Suppo ing In o ma ion Fig. 1B). Phospho yla ed pep- ides o hese ou biological samples we e hen isola ed and compa a i ely analysed by quan i a i e pep ide sequencing using iTRAQ labelling and accu a e FTMS. Da a e alua ion only con- side ed high-con iden phosphopep ides ha iTRAQ-FTMS unam- biguously de ec ed in all ou biological samples. This app oach iden i ied 3756 phospho yla ions in eshly isola ed Tcon cells and T eg cells (Suppo ing In o ma ion Table 2). Abou one- hi d o hese phospho yla ion si es could be assigned o majo signalling pa hways co e ing he TCR signalling ne wo k. To he bes o ou knowledge, we ound all phospho yla- ions o he canonical TCR signalling pa hway pe ec ly in line wi h he li e a u e. Fo ins ance, phospho yla ions a immuno ecep o y osine-based ac i a ion mo i s (ITAMs) o Tcon cells (CD3ε, CD3ζ), as well as on p oximal signal componen s (e.g. ZAP70 and SLP76) we e ound up- egula ed a e TCR engagemen as expec ed (Fig. 1B and Suppo ing In o ma ion Fig. 3B). In e es - ingly, he p o iles o he co esponding y osine phospho yla ions a CD3εand CD3ζin T eg cells e ealed i s di e ences be ween bo h T cell subse s. They we e ound no inducible and wi h sim- ila high in ensi ies in non-s imula ed and s imula ed T eg cells C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu 4Ma co anHame al. Eu . J. Immunol. 2017. 0: 1–16 Figu e 2. Phosphop o eomic p o iling iden i ies con e ging and di e ging TCR esponses. Fo quan i a i e phosphop o eomics, ex i o isola ed T eg cells and Tcon cells we e CD3/CD28-s imula ed o 5 min o le non-s imula ed and analysed by using quan i a i e MS. All egula ion ac o s a e gi en in log2 scale. (A) Box plo s show egula ions o all phospho yla ion si es o p o eins om s imula ed T eg cells o Tcon cells in ela ion o non-s imula ed cells. Numbe s indica e he signi ican ly up- o down- egula ed phospho yla ion si es when assuming a no mal dis ibu ion. (B) Compa ison o up- and down- egula ed phospho yla ion si es in T eg cells and Tcon cells ( om A). (C) Sca e plo depic ing egula ions o all phospho yla ed pep ides in T eg cells (x-axis) and Tcon cells (y-axis). One hund ed and wen y one phosphopep ides ha a e signi ican ly di e en ially egula ed be ween T eg cells and Tcon cells (see Suppo ing In o ma ion Fig. 5) a e indica ed by ed do s. (D) Hea C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu Eu . J. Immunol. 2017. 0: 1–16 Molecula immunology and signaling 5 (Fig. 1B and Suppo ing In o ma ion Fig. 3B). This indica es a p e-ac i a ed pheno ype o he TCR signalling ne wo k in eshly isola ed T eg cells, which likely is a consequence o onic in e - ac ions wi h an igen-p esen ing cells in i o [28]. Using Wes e n blo ing analysis we could indeed con i m he enhanced le el o phospho yla ed CD3ζin non-s imula ed T eg cells in compa ison o non-s imula ed Tcon cells (Suppo ing In o ma ion Fig. 5). Ne e heless, his p e-ac i a ion did no impai he esponsi e- ness o he TCR signalling ne wo k in T eg cells as illus a ed by he induced Y492 phospho yla ion o ZAP70 (Fig. 1B and Sup- po ing In o ma ion Fig. 3B). In a global iew, TCR igge ing in T eg cells causes simila numbe s o induced and ep essed phos- pho yla ion e en s, whe eas Tcon cells showed a highe num- be o signi ican ly up- egula ed phospho yla ions (Fig. 2A). Se - e al TCR componen s exhibi ed simila phospho yla ion esponses in bo h T cell subse s, al hough he numbe o join ly up- and down- egula ed p o eins in T eg cells and Tcon cells is limi ed (Fig. 2B). This includes he a o emen ioned ZAP70 as well as known TCR sca old p o eins SASH3/Sly, a lin, Lad/TSAd and LIME1, indica ing an o e all simila a chi ec u e o he signal ne wo ks. The as majo i y o CD3/CD28- esponding phospho- yla ions exhibi ed subse -speci ic egula ions and a compa a i e inspec ion e ealed a o al o 121 phospho yla ions (Suppo ing In o ma ion Fig. 6) ha disc imina e T eg cells and Tcon cells in he i s i e min o signalling (Fig. 2C and Suppo ing In o - ma ion Table 2, shee 3). F om a mechanis ical angle hese T eg cell-speci ic egula ions can be g ouped in six classes (Fig. 2C and D). Fo ins ance, he deubiqui inase CYLD, which plays a ole in he IS ec ui men o Lck, was induced a i s inhibi o y S414 si e only in Tcon cells (class I). Likewise, class II phospho yla ions a e p edominan ly induced only in T eg cells; comp ising he ac i- a ion si e T538 o PKCθ(Fig. 1C) as well as componen s o gene egula ion (ISW1 and ROA1) and esicle a icking (AP3δ1and α-PIX). In a side-by-side inspec ion o bo h subse s, he majo i y o 121 phospho yla ions (>60 %), showed an opposi e egula o y beha iou unde he same s imula ion condi ion (Fig. 2D, classes V and VI). Howe e , when non-s imula ed Tcon cells se ed as he e e ence o no maliza ion, p o ein-speci ic phospho yla ions became appa en ly simila a e i e min s imula ion (Fig. 2E, com- pa e lanes i e min). This con e gence o phospho yla ions is mos appa en bu no es ic ed o class V and VI p o eins. Fo ins ance, he abundance o phospho yla ed T538 o PKCθ(class II) was a i s lowes in eshly isola ed T eg cells, bu con e ged o simila ac i a ion le el a e TCR engagemen in bo h s imula ed sub- se s. Thus, equal ex i o TCR s imula ion o T eg cells and Tcon cells no ably ha monize he pos - ansla ional s a us o he TCR signalling ne wo k. None heless, a dis inc se o ele en TCR signalling compo- nen s we e no ha monized by TCR engagemen and indica ed an e en di e ging signalling pa e n in T eg cells and Tcon cells (Fig. 2F and MS da a o DBNL, MAPK14 and TIAM in Fig. 1C). This g oup is cons i u ed by known TCR signalling componen s and, om a sys ema ical iew, is en iched o one unc ional class (Table 1): Beside he hema opoie ic lineage-speci ic phospha ase PTPN18 and he T ansducin-like enhance o spli 3 (TLE3), bo h in ol ed in T cell di e en ia ion [29–32], all o he nine com- ponen s a e associa ed wi h cy oskele on dynamics [19, 33–39]. Many o hose p o eins we e ound wi h induced phospho yla ions only in Tcon cells, whe eas hese esponses a e e en ep essed in T eg cells. This includes DC1L1/LIC1, a subuni o he dynein complex, ha is supposed o de e mine ca go load [40]. In con- as , T eg cell-speci ic induced phospho yla ions we e iden i ied a SMCR8 and TIAM, o which he la e is equi ed o LFA-1 in eg in ac i a ion [41]. In conclusion, ex i o T eg cells exhibi ed a p e-ac i a ion TCR pheno ype. Thei phospho yla ion s a us disc imina es hem om Tcon cells, bu can be la gely ha monized unde p ecise TCR ac i a ion condi ions ex i o. Fo ha , phospho yla ions showed opposi e egula ions in T eg cells and Tcon cells and he global phospho yla ion signa u es widely con e ge in only 5 min. S ill, a se o ele en componen s ha a e mos ly epo ed as egula o s o he cy oskele on iden i ied di e ging nodes in he wo subse - speci ic TCR signalling ne wo ks. In his con ex , p o ein ne wo k analyses based on induced phospho yla ed p o eins ac ually con- i med he close in e connec i i y be ween TCR signalling and he cy oskele on (Suppo ing In o ma ion Fig. 7A). The eby, he esponding Tcon cell ne wo k e ealed he supe io en ichmen o “TCR signalling componen s”, whe eas he esponding T eg cell ne wo k e ealed he supe io en ichmen o “cy oskele on dynamics”. Indeed, ne wo k analyses es ic ed o he 121 subse - speci ic phospho yla ions s eng hened he hypo hesis ha mainly TCR signalling and cy oskele on dynamics disc imina e T eg cells om Tcon cells (Suppo ing In o ma ion Fig. 7B). T eg cells e eal unique cell pola iza ion and MTOC posi ioning bu do no ec ui PKCθ o he IS Di e ging signalling componen s indica ed a a ian egula ion o cy oskele al o ganiza ion in he wo T cell subse s ha may o ches a e T cell mo ili y, conjuga ion, IS o ma ion as well as  maps showing egula ions o all 121 signi ican ly egula ed phospho yla ion si es in Tcon cells and T eg cells. Hea maps a e o de ed acco ding o boxes I–VI in (C). (E) Hea map illus a ing ela i e abundances o he 121 signi ican ly egula ed phospho yla ion si es in non-s imula ed and CD3/CD28-ac i a ed Tcon cells and T eg cells wi h non-s imula ed Tcon cells as baseline. No e he gene ally con e ging signalling in bo h subse s esul ing in almos compa able abundances a e 5 min o s imula ion. (F) Hea map illus a ing 16 phospho yla ion si e in ensi ies ha disc imina e T eg cells om Tcon cells a e i e min o s imula ion (le panel; Tcon cells minus T eg cells; >1 on he log2 base). O hose, ele en TCR signalling componen s showed di e ging phospho yla ion si e egula ions ( igh panel and Table 1). Da a is pooled om en independen g oups o each 25–30 mice o nons imula ed and 12 independen pools o each 25–30 mice o CD3/CD28-s imula ed T eg cells and Tcon cells. Phosphopep ides we e isola ed and di e en ially labelled ollowed by quan i a i e MS o 15 ac ions in one independen s udy. In o al, 580 mice, 4.4 ×108cells and 1.1 ×108cells/condi ion we e analysed (see also Fig. 1 and Suppo ing In o ma ion Table 2). C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu 6Ma co anHame al. Eu . J. Immunol. 2017. 0: 1–16 Table 1. In insic T eg cell-speci ic phospho esponses Unip o T i ial names Si e T eg cells Tcon cells Func ion Node STMN1 S a hmin S16 − ++++ Cy oskele al eo ganiza ion ubulin / MTOC [33] CaMKII [53] TLE3 TLE3 S217 − ++++ T ansc ip ional co- ep ession TCF / Le [31] FoxA1 / HDAC1/2 [32] DC1L1 LIC1 S412 − ++++ Vesicle anspo Dynein [19] TCR mic oclus e dynamics ubulin/MTOC [34] RHG15 A hGAP15 S51 − ++++ cy oskele al eo ganiza ion small GTPases (Rac) [78] Pak1/2 [54] PTN18 PTPN18 Y381 −− +++ y osine phospha ase Lck / ZAP70 [29] FLP-1/PTP-HSCF di e en ia ion immune cells Lyn [30] M3K3 MAP3K3 S337 − +++ T cell di e en ia ion / ac i a ion IFN-γ[79] / NFκB [80] MEKK3 PLCγ[55] DBNL D eb in-like S277 −− +++ T cell ac i a ion / IS o ma ion ZAP70 / p38 / PLCγ[35, 36] HIP-55 cy oskele al eo ganiza ion HPK1 / NFAT [37] MK14 MAPK14 Y182 −−−− ++ induced T eg cell gene a ion NFAT / Ak / ERK [81] p38αcy oskele al eo ganiza ion TGFβ/ ILs [82] ML12B MLC20 S20 −−− ++ cy oskele al eo ganiza ion PKC / ILK [83] Myl12b ac in / p38 [38] SMCR8 SMCR8 S416 ++ −− esicle anspo RABs [39] TIAM1 TIAM1 S1437 ++ −−− in eg in signalling 5-1 / 14-3-3 / Rac [41] cy oskele al eo ganiza ion PKC / Rac [70] Lis o 11 TCR signalling componen s ha we e no ha monized upon CD3/CD28-s imula ion and ha e ealed an e en di e ging signalling pa e n in T eg cells and Tcon cells. Regula ions o phospho yla ion si es in T eg cells and Tcon cells a e gi en by “+” and “−” signs (−/+: 0.00–0.50; −−/++: 0.50–1.00; −−−/+++: 1.00–1.50; −−−−/++++:>1.50 (log2 scale)), known unc ional oles in T cells and majo nodes a e lis ed. e ec o unc ions. He e, we ocused on conjuga ion and IS o - ma ion. Fo ha , T eg cells and Tcon cells we e isola ed om o albumin (OVA)-TCR ansgenic mice (DO11.10), IS o ma ion was induced by co-incuba ion wi h OVA pep ide-loaded ex i o isola ed B cells, and pola iza ion dynamics o BT-pai s we e s ud- ied by mic oscopy. Global inspec ion o BT-pai s al eady demon- s a ed a subse -speci ic mo phology pheno ype. Along cell conju- ga ion he shape o Tcon cells was ound o be mo e elonga ed as hose om T eg cells, which exhibi ed a mo e ounded mo - phology upon conjuga ion (Fig. 3A). We nex conside ed only hose BT-pai s, which had es ablished a ma u e ac in-en iched IS as indica ed by phalloidin. Cells we e addi ionally s ained o he mic o ubule ne wo k and we ound he dis ance be ween MTOC and IS signi ican ly diminished in conjuga ed T eg cells as com- pa ed o Tcon cells. To sco e di e ences wi hin he eposi ioning o he MT ne wo k and especially he MTOC in T eg cells and Tcon cells, we de e mined he pola iza ion index [42] upon BT- pai ing and con i med a signi ican T eg cell-speci ic pola iza ion pheno ype and MTOC posi ioning (Fig. 3B). MTOC posi ioning is egula ed by PKC amily membe s [16], and PKCθwas al eady epo ed o delocalise om he T eg cell- IS [15]. In e es ingly, PKCθwas iden i ied as a T eg cell-speci ic componen ollowing TCR engagemen in ou phosphop o eomic app oach (class II). Thus, we nex in es iga ed PKCθlocaliza ion dynamics in ela ion o MTOC posi ioning (Suppo ing In o ma- ion Fig. 1C). IS o ma ion was s udied by using an i-CD3/an i- CD28-coa ed co e slips, and con ocal spinning disk mic oscopy con i med he MTOC posi ioning pheno ype. In T eg cells, his MTOC posi ioning pheno ype was no accompanied wi h a no able ec ui men o PKCθ o he IS in he i s 30 min (Fig. 3C and D). Thus, PKCθis no ac i ely delocalized away om he T eg cell- IS bu ins ead is no ec ui ed a all. In con as , o Tcon cells we could con i m IS ec ui men o PKCθ ha became mo e ob i- ous a e 3D econs uc ion (Fig. 3C and D). In e es ingly, PKCθ ec ui men in o he Tcon cells-IS was only ound ansien ly a e en min, and al eady 30 min a e TCR ac i a ion he majo i y o cells los he pola ized PKCθdis ibu ion (Fig. 3C). Thus, MTOC posi ioning in Tcon cells is accompanied by a ansien IS ec ui - men o PKCθ, whe eas an e en mo e pola ized posi ioning o he MTOC owa ds he IS in T eg cells could no a ac PKCθin o he synap ic compa men . T eg cells es ablish a unique anspo pheno ype o TCR signalling ne wo k componen s a he IS Since we (i) e ealed T eg cell-speci ic and di e ging signalling o cy oskele on egula o s and (ii) cha ac e ized a a ian MTOC posi ioning and impai ed PKCθ ec ui men in T eg cells, we nex aimed o s udy he spa ial o ganiza ion o selec ed TCR signalling componen s in s imula ed T eg cells and Tcon cells. Fo his, we employed he mul i-epi ope ligand ca og aphy (MELC) echnol- ogy [21, 22], and e alua ed a o al o abou 300 OVA-speci ic BT-pai s using a panel o 25 an ibodies (see Ma e ial and Me h- ods, Suppo ing In o ma ion Fig. 1C and Suppo ing In o ma ion Video 1). This app oach allowed subcellula localiza ion analyses C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu Eu . J. Immunol. 2017. 0: 1–16 Molecula immunology and signaling 7 Figu e 3. Subse -speci ic cy oskele al ea angemen and PKCθdis i- bu ion in T eg cells and Tcon cells. (A) B cells om BALB/c mice we e loaded wi h OVA323-339 pep ide and co-incuba ed o 10 min wi h T eg cells o Tcon cells om DO11.10 mice and analysed by immuno luo- escence mic oscopy. Rep esen a i e images om one ou o wo inde- penden expe imen s using six mice each; s aining: ubulin (g een) and F-ac in ( ed). Dashed lines show dis ances om MTOC o IS. Scale ba s de ine 5 μm. (B) Quan i ica ion o he pola iza ion index e ealed al e ed pola iza ion o T eg cells a e B cell engagemen . Pola iza ion index was calcula ed as ollows: dis ance o MTOC o IS di ided by he dis- ance o he u opod o he IS [42] (n=16 o T eg cells and n=20 o Tcon cells; median is indica ed by he ho izon al line; *p<0.05, Mann– Whi ney U es ). Each do ep esen s one analysed cell and ep esen a- i e da a o one o wo independen expe imen s using six mice each is shown. (C, D) PKCθis ansien ly ec ui ed o he IS only in Tcon cells. Pu i ied T eg cells and Tcon cells we e seeded on an i-CD3/an i-CD28 an ibodies coa ed co e slips, ixed a e indica ed ime poin s and anal- ysed using spinning disk mic oscopy (n=3; ou mice pe expe imen ). (C) PKCθdis ibu ion was calcula ed by di iding p oximal localiza ion by dis al localiza ion (pooled da a: n=101 o T eg cells and n=136 o Tcon cells; median is indica ed by he ho izon al line; *p<0.05, Mann– Whi ney U es ) Each do ep esen s one analysed cell and da a shown is pooled om h ee independen expe imen s using ou mice each. (D) Rep esen a i e 3D images o one T eg cell (uppe panel) and one Tcon cell (lowe panel) 10 min a e seeding on co e slips. PKCθis shown in g een, and nuclei (blue) and F-ac in ( ed) we e used o coun e s ain- ing; subs a e a ea is indica ed by a g ay dashed line. Scale ba s de ine 5μm. o key TCR signalling componen s as well as hei speci ic phos- pho a ian s. Following 30 and 120 min a e BT-pai o ma ion, obus luo escence in ensi ies could be de ec ed and quan i ied o calcula e he abundances o componen s in se en dis inc spa ial egions. Ou segmen a ion s a egy, which was based on he posi- ion o pSLP76 in he ac in- ich IS (g een do , Fig. 4A and B), could de ine he SMAC and cSMAC as well as he en i e IS ha spanned he wid h o he SMAC and p o uded i e pixels in o bo h he B and T cell (Fig. 4A and B, and Suppo ing In o ma ion Fig. 8). S able BT-pai s we e de ined based on signals om CD3ε, CD45R, F-ac in, pSLP76 and pLAT and allowed eco ding he posi ion and size o a ma u e IS (Fig. 4A). A i s glance, he basic IS a chi ec u e o BT eg- and BTcon - pai s appea ed simila as indica ed by he global in ensi y pa e n o he inspec ed TCR signalling componen s (Fig. 4C, compa e T eg cells e sus Tcon cells a 30 and 120 min). The MELC analy- ses also con i med he accumula ion o ac i a ed p oximal TCR sig- nalling componen s, i.e. pCD3ζ, pZAP70, pLck, pLAT, pSLP76 and pPLCγ1 a he IS, SMAC and cSMAC. This accumula ion was ound o u he inc ease a e 120 min and coincided wi h he en ich- men o he co esponding o al p o ein le els wi hin hese egions as well (Fig. 4C). S a is ical analyses o he BT-pai s e ealed a numbe o s iking di e ences in IS o ganiza ion: (i) T eg cells we e no ably impai ed in ec ui ing addi ional CD3ε,Lck,SLP76 and PLCγ1 a e he o ma ion o a ma u e IS a 30 min. In con as , Tcon cells we e able o con inue his ec ui men o e a pe iod o 30 o 120 mins mo e e icien ly (Fig. 4D, ela i e di e ences o e ime). In e es ingly, bo h subse s likely con inue signalling along his pe iod as obse ed by he consecu i e accumula ion o he co esponding phospho a ian s. (ii) Signi ican ly lowe amoun s o PLCγ1, CD3εand pZAP70 we e de ec ed a he T eg cell-IS (Fig. 4E, ela i e di e ences in subse s). The eby, pZAP70 was mos ly a ec ed a e 30 min bu emained educed in he cSMAC, and PLCγ1andCD3εwe e ound signi ican ly educed only a e 120 min (Fig. 4E, and ep esen a i e images in Fig. 4F and G). (iii) Ins ead, signi ican ly highe amoun s o CD45, Lck, SLP76 and Fyn as well as pERK, pPLCγ1 and pLAT we e localized a he T eg cell-IS 30 min a e conjuga ion (Fig. 4E). This a gues o a mo e apid ma u a ion o he T eg cell-IS compa ed o he Tcon cell-IS. (i ) Only T eg cells we e able o ec ui CD86 and CD80 o hei IS compa men (Fig. 4E and Suppo ing In o ma ion Fig. 6), as desc ibed p e iously [43]. Thus, CD86 ansendocy osis, as a pa o he T eg cell supp essi e pheno ype, was now demons a ed 30 min a e IS o ma ion and did no indica e any exhaus ion a e 120 min. In conclusion, MELC analyses showed di e en ec ui men o TCR signalling componen s in o he IS o T eg cells e sus Tcon cells a ea ly ime poin s, which, howe e , ul ima ely esul ed in a mo e alike dis ibu ion a 120 min. S ill, he key signalling com- ponen s CD3ε, ZAP70, PLCγ1 and CD86 emained di e en ially dis ibu ed a he IS and could explain T eg cell- and Tcon cell- speci ic pheno ypic esponses. Discussion TCR signalling con ols di e en ia ion and unc ion o T eg cells [23]. Al hough p e ious s udies al eady had desc ibed some di - e ences in TCR signalling be ween T eg cells and Tcon cells [44], a comp ehensi e pic u e o he TCR signalling ne wo k C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu 8Ma co anHame al. Eu . J. Immunol. 2017. 0: 1–16 Figu e 4. Ac i a ion-dependen ec ui men o TCR signalling componen s o he IS. BT- pai s we e gene a ed as in Fig. 3, and pai s we e ixed a e 30 o 120 min and analysed by sequen ial s aining (mul i-epi ope ligand ca og aphy, MELC). (A) Rep esen a i e image showing a BT-pai wi h pSLP76 (g een), CD3ε ( ed) and F-ac in (g ay) s aining ha was used o de ine he di e en compa men s o he IS (IS ci cled by do ed line, SMAC amed by dashed line and cSMAC amed by solid line). (B) Segmen a ion s a egy o he IS com- pa men s as de e mined by he posi ion o pSLP76 s aining (g een do ), which was used o de ine a h ee pixel (0.86 μm) wide SMAC egion s e ched o e he synapse as de ined by F-ac in. The cSMAC was de ined as he mid- dle 33 % o he wid h o he SMAC. The en i e IS a ea was de ined as an o al spanning he wid h o he SMAC and p o uding i e pixel (1.43 μm) beyond he SMAC in o ei he cell. (C) Rela i e in ensi ies o all analysed (phos- pho)p o eins in BT eg- and BTcon -pai s 30 and 120 min a e pai o ma ion de e mined in he B cell (B) and T cell (T) wi hou he IS, he B cell (Bs) and T cell (Ts) pa o he IS wi hou SMAC, he en i e IS, he comple e SMAC (S) and he cSMAC (cS). No e he g adual en ichmen o bo h o al TCR-signalling p o eins and hei phospho yla ed a ian s in he IS (IS, S and cS). (D,E) Rela i e di e ences o all analysed (phospho)p o eins in BT-pai s in he IS (i.e. IS, S and cS). Only s a is ically signi ican di - e ences a e shown (p<0.05, wo- ailed - es ). The ela i e di e ences and p- alues o all BT-pai compa men s a e gi en in Suppo ing In o ma ion Fig. 8. (F,G) Rep esen a i e images showing he localiza ion o p o eins used o de ine he IS compa men s o BT eg- (F) and BTcon - (G) pai s (uppe images; CD3ε( ed) and pSLP76 (g een), Foxp3 (blue) ma ks only T eg cells) and ep esen a i e images showing di e ences be ween BT eg- and BTcon -pai s ega ding educed PLCγ1 (g een) and pZAP70 ( ed) localiza ion wi hin he IS and Ts, and he cSMAC, espec i ely (lowe images). F-ac in is shown in g ay. A pho omon age o BT-pai s (BT eg s. BTcon ) ha includes in o ma ion o all in es iga ed p o eins and phosphop o- eins ob ained by MELC is gi en in Suppo ing In o ma ion Video 1. Rep esen a i e images gi en in A,F,G a e om one o wo indepen- den expe imen s wi h eigh mice each and o 51 BT eg- e sus 130 BTcon -pai s a 30 min and 28 BT eg- e sus 62 BTcon -pai s a 120 min analysed, hea maps shown in C-E ep e- sen pooled da a om all analysed BT-pai s. o ganiza ion was missing. Resul s o his s udy now sugges a T eg cell-speci ic ac i a ion o cy oskele on egula o s and a subsequen T eg cell-speci ic ec ui men o TCR signalling componen s o he ma u e IS. This cons i u es a mechanis ic model o how unc ion- ally dis inc TCR signalling is ealized in bo h T cell subse s. Fi s , he p o eome analyses de e mined la gely iden ical abun- dances o TCR signalling componen s wi hin T eg cells and Tcon cells. This obse a ion is widely in line wi h ecen da a om p o eome s udies o human and mu ine T eg cells, al hough hose s udies did no speci ically ocus on TCR signalling [24–26]. The p esen p o eomic app oach iden i ied low abundance o Themis in T eg cells, a molecule known o play a ole in he ea ly LAT sig- nalosome in Tcon cells [27]. O e exp ession o Themis, howe e , could nei he es o e IL-2 exp ession no educe he supp essi e C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu Eu . J. Immunol. 2017. 0: 1–16 Molecula immunology and signaling 9 capaci y o T eg cells in i o. Ne e heless, Themis le els play a ole in T cell de elopmen and su i al [45–48], and Themis has been epo ed o modula e IL-10 exp ession [49], aspec s ha we e no add essed in his s udy. Thus, p o ein abundances seemed no o be he key de e minan o unc ionally dis inc TCR signalling ne wo ks. In con as , his i s phosphop o eome s udy on p ima y mu ine T cells de ec ed nume ous T eg cell-speci ic phospho y- la ions in he TCR signalling ne wo k. Ex i o T eg cells dis- played a p e-ac i a ed phospho yla ion s a us o key TCR sig- nalling molecules (e.g. CD3ε,CD3ζand SLP76). Ini ially, his appea ed coun e in ui i e since a diminished y osine phospho- yla ion o CD3ζin pe iphe al human T eg cells has been epo ed [7, 8]. Howe e , T cells esiding in seconda y lymphoid o gans - as used in ou s udy - con inuously ecei e “ onic” ecep o s im- uli [50] ha can esul in p iming he down-s eam signal ne - wo k as desc ibed o Tcon cells [28], and his now could be con i med he e o T eg cells as well. The unc ional ole o his p iming needs u he in es iga ion, bu i did no ab oga e T eg cells’ esponsi eness since we ound Y492 in ZAP70 equally well phospho yla ed in ex i o isola ed T cell subse s and equally well inducible upon TCR engagemen . This si e is a known subs a e si e o Lck and se es as an au ophospho yla ion si e o ac i a ed ZAP70 as well [51]. The CD3εimmuno ecep o y osine-based ac i a ion mo i (ITAM), howe e , was al eady ound phospho- yla ed in ex i o isola ed, non-s imula ed T eg cells. Hence i is ai o specula e ha he signal ampli ica ion mechanism in ol - ing he dissocia ion o ZAP70 om he TCR complex, as e y ecen ly desc ibed o Tcon cells [52], is al eady induced in non-s imula ed T eg cells. This would suppo ZAP70 unc ion in inside-ou signalling and adhesion o T eg cells, which did no equi e i s kinase ac i i y [13]. In o al, 121 T eg cell-speci ic phospho yla ions de ine known and no el down-s eam componen s o he TCR signalling ne wo k. Iden ical s imula ion condi ions he eby con e ge he phosphopa e ns signi ican ly wi hin i e min. Mo e no ewo - hy, ha monizing o bo h TCR signalling ne wo ks is p edom- inan ly ealized by coun e - egula ed signalling. This in ol ed well-known p oximal TCR signalling componen s like PKCθ,SLP76 and GRP2/CalDAG-GEFI, and subs an ia es he model o T eg cells ha ing a a ian ac i a ion s a us in i o. Fu he mo e, his demons a es ha T eg cells in i o in eg a e signals om mul- iple ecep o s and ha e he capaci y o e e se he di ec ion o TCR-media ed egula ions. I was exci ing o de ec e en a di e g- ing TCR signalling unde he applied s imula ion condi ions in a se o ele en p o eins. These componen s ha e been ela ed p e iously o dis al pa s o he TCR signalling ne wo k. Nine o hese a e egula o s o cy oskele on dynamics and molecula anspo [19, 33, 37, 53–55] and indeed cellula pola i y, MTOC posi ioning and signal componen ec ui men s hen con i med a cell-pola iza ion as well as an IS assembly pheno ype o T eg cells. Such an IS pheno ype is s ill mos ly de ined by PKCθ ha plays a pi o al ole in MTOC dynamics and IS o ma ion in T cells [56]. Zanin-Zho o and colleagues ecen ly obse ed PKCθ o be delo- calized six min a e T eg cell-IS o ma ion and ga e he i s e i- dence o a T eg cell-speci ic IS o ganiza ion [15]. He e, we could now con i m his pheno ype and u he demons a e ha in con- as o Tcon cells, PKCθis no ec ui ed a all o he T eg cell-IS, whe eas o he TCR componen s we e ound e icien ly ec ui ed. Ac ually, in Tcon cells PKCθis ec ui ed only ansien ly o he IS by so a unknown an agonis ic ansloca ion mechanisms. This inding mus be u he elucida ed e.g. in bi-cellula conjuga ions, bu likely is unde con ol o pos - ansla ional modi ica ions. Du - ing TCR-induced signalling, PKCθin eg a es co-s imula o y signals om CD3 and CD28, and GLK (ge minal cen e kinase- ela ed p o- ein kinase) can phospho yla e PKCθa T538, which coo dina es i s ac i i y and localiza ion [57, 58]. In e es ingly, we ound his si e solely inducible in T eg cells. Fu he mo e, PKCθlocaliza ion migh depend on sumoyla ions [59], which was no add essed in he p esen s udy. Bu wha is he ole o delocalized PKCθin he con ex o T eg cell-IS unc ions? In Tcon cells, upon TCR s imula- ion PKCθsuppo s NF-κB ac i a ion [60] and can main ain phos- pho yla ed RelA, he p65 subuni o NF-κB, in he nucleus [61]. In e es ingly, T eg cells we e ound o accumula e phospho yla ed p65 a he la e IS, al hough i s unc ional ole he e emains elu- si e. In he same pa hway, PKCθis desc ibed as an in e ac ing an agonis o he deubiqui inase CYLD, which ac s on Lck o sup- po i s IS localiza ion [62, 63]. We ound CYLD only esponding in Tcon cells, indica ing ha he PKC/CYLD axis is di e en ially o ganized in T eg cells. A mo e sys ema ic inspec ion o he T eg cell-IS was co ob- o a ed by MELC analyses. When compa ed o Tcon cells, abou one- hi d o he he e-inspec ed componen s displayed signi ican ly di e en ec ui men dynamics o he ma u e IS. Phospho a ian s o TCR signalling componen s we e equally ec ui ed in o he IS o T eg cells and Tcon cells as expec ed, howe e , Tcon cells could ec ui TCR signalling componen s mo e e icien ly. I is emp ing o specula e ha a limi ed ec ui men o hese compo- nen s can dampen TCR signalling in T eg cells. Indeed, ZAP70 phospho yla ed a Y318, a si e unambiguously needed in T cell ac i a ion [64], was educed in he IS in BT eg-pai s a 30 min, and his holds ue o he cSMAC e en a e 120 min. Fu he - mo e, we obse ed educed le els o PLCγ1 in he T eg cell-IS 120 min a e BT-pai ini ia ion. Mechanis ically his could be explained by he di e en ially egula ed S210 in he P1 domain o SLP76 ha acili a es he in e ac ion wi h he SH3 domain o PLCγ1 and is equi ed o TCR-media ed ac i a ion [65]. LAT compe es o PLCγ1 in e ac ion ia his SH3 domain, and his PLCγ1/LAT complex is es ic ed o he cell memb ane in es ing Ju ka T cells [66]. Thus in e e ing wi h SLP76/PLCγ1 in e ac ion h ough S210 phospho yla ion could esul in abe an complex ec ui men ia LAT o he T eg cell-IS. He e, one should no e ha , due o limi a ions in he an ibody panel used in he MELC analyses, we mos p obably unde es ima e he numbe o p o- eins ha show di e ences in IS ec ui men . Fo ins ance also Lymphocy e Cy osolic P o ein 1 (LCP1 o L-plas in/PLSL) migh be di e en ially ec ui ed in o he T eg cell-IS. Phospho yla ion a S5 suppo s i s ansloca ion o he IS and he ewi h i s con- ibu ion o cell mo ili y in Tcon cells [67]. This si e was indeed induced in Tcon cells as expec ed, howe e , in T eg cells he S5 C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu 16 Ma co anHame al. Eu . J. Immunol. 2017. 0: 1–16 77 G ubbs, F. E., Sample c i e ia o es ing ou lying obse a ions. Ann. Ma h. S a . 1950. 21: 27–58. 78 Cos a, C.,Ge mena, G.,Ma in-Con e, E. 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Immunol. 2015. 194: 5781–5788. 83 Xu, J.,Li, T.,Yang,G.M.andLiu,L.M., P o ein kinase C iso o ms espon- sible o he egula ion o ascula calcium sensi i i y and hei ela- ionship o in eg in-linked kinase pa hway a e hemo hagic shock. J. T auma. 2010. 69: 1274–1281. Abb e ia ions: BT-pai : B cell and T cell pai ·BT eg-pai : B cell and T eg cell pai ·BTcon -pai : B cell and Tcon cell pai ·FTMS: Fou ie ans o m mass spec ome y ·IS: immunological synapse ·ITAM: immuno ecep o y osine-based ac i a ion mo i ·iTRAQ: isoba ic ags o ela i e and absolu e quan i ica ion ·MELC: mul i-epi ope lig- and ca og aphy ·MS: mass spec ome y ·MT: mic o ubule ·MTOC: mic o ubule-o ganizing cen e ·OVA: o albumin ·P1 domain: p oline- ich domain ·ROI: egion o in e es ·SH domain: S c homology domain · SMAC: sup amolecula ac i a ion clus e ·cSMAC: cen al-SMAC ·TCR: T cell ecep o ·Tcon cell: con en ional T cell ·T eg cell: egula o y T cell Full co espondence: D . Lo ha J¨ ansch, Cellula P o eomics, Helmhol z Cen e o In ec ion Resea ch, Inho ens asse 7, 38124 B aunschweig, Ge many Fax: +49-831-6181-2655 e-mail: [email p o ec ed] Recei ed: 15/3/2017 Re ised: 28/6/2017 Accep ed: 14/8/2017 Accep ed a icle online: 17/8/2017 C 2017 The Au ho s. Eu opean Jou nal o Immunology published by WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. www.eji-jou nal.eu