Unidirectional transfer of microrna-loaded exosomes from T cells to antigen-presenting cells

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1 Reeived 9 Aug Aepted Mr Pulished 9 Apr DOI:.8/nomms8 Unidiretionl trnsfer of mirorna-loded exosomes from T ells to ntigen-presenting ells Mrí Mittelrunn,, Cristin Gutiérrez-Vázquez,, Crolin Villrroy-Beltri, Susn González, Fátim Sánhez-Co, Mnuel Ángel González, Antonio Bernd & Frniso Sánhez-Mdrid, The immune synpse is n exquisitely evolved mens of ommunition etween T ells nd ntigen-presenting ells (APCs) during ntigen reognition. Reent evidene points to the trnsfer of RNA vi exosomes s novel mode of interellulr ommunition. Here we show tht exosomes of T, B nd dendriti immune ells ontin mirorna (mirna) repertoires tht differ from those of their prent ells. We investigte whether mirnas re exhnged during ognte immune intertions, nd demonstrte the existene of ntigen-driven unidiretionl trnsfer of mirnas from the T ell to the APC, medited y the delivery of CD6 + exosomes on immune synpse formtion. Inhiition of exosome prodution y trgeting neutrl sphingomyelinse- impirs trnsfer of mirnas to APCs. Moreover, mirnas trnsferred during immune synpsis re le to modulte gene expression in reipient ells. Thus, our results support mehnism of ellulr ommunition involving ntigen-dependent, unidiretionl interellulr trnsfer of mirnas y exosomes during immune synpsis. Centro Nionl de Investigiones Crdiovsulres, Melhor Fernández Almgro,. 89, Mdrid, Spin. Serviio de Inmunologí, Hospitl Universitrio de l Prines, Instituto de Investigión Snitri Prines, Diego de León, Mdrid, Spin. These uthors ontriuted eqully to this work. Correspondene nd requests for mterils should e ddressed to F.S.-M. (emil: fsnhez.hlpr@slud.mdrid.org). nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

2 nture ommunitions DOI:.8/nomms8 To mount n effetive immune response, different immune ell types need to ommunite with eh other. Cell synpses re highly speifi mens of interellulr ommunition. During the formtion of the immunologil synpse (IS), trnsmemrne nd memrne-ssoited moleules re reorgnized into highly segregted struture t the T ell ntigen-presenting ell (APC) ontt site,. The tin ytoskeleton reorgnizes to provide physil pltform to support the IS struture, wheres the tuulin ytoskeleton is direted towrds the IS, where the mirotuuleorgnizing entre (MTOC) lolizes,. The trnslotion of the MTOC is n erly event during IS formtion, nd llows loliztion of the seretory omprtments the Golgi pprtus nd the ytotoxi grnules in lose pposition to the APC. The polriztion of the seretory pprtus to the IS provides the sis for polrized seretion of ytokines, nd the exoytosis of lyti grnules y ytotoxi T ells 6. An lterntive vesiulr trffiking, depending on the endoyti pthwy, hs lso een reported to e ritil for IS funtion. Trnsport of the T ell reeptor (TCR) 7 nd lymphoytespeifi tyrosine kinse 8 to the IS depends on omponents of the endosoml omprtments, nd endosoml trnsport is essentil oth to trget TCRs nd other moleules to the APC ontt site nd for signl downmodultion y ontrolling TCR endoytosis. Finlly, lysoisphosphtidi id, mrker of lte endosomes (multivesiulr odies; MVBs), lolizes very lose to the entre of the IS of helper T ells 9, suggesting tht MVBs lso polrize to the IS. The IS my thus serve s fous for oth exoytosis nd endoytosis 6,. On exoyti fusion of MVBs with the plsm memrne, ells relese exosomes; nd these nm vesiles re inresingly reognized s signifint vehiles for interellulr ommunition,. The genertion of MVBs is well-defined event in the endosoml pthwy nd it is evident tht they hve dul role, s in ddition to their involvement in exosoml relese MVBs lso temporrily store proteins nd lipids destined for lysosoml degrdtion. The role of exosomes in diverse physiologil nd pthologil settings is still inompletely understood; however, evidene hs een reported for their involvement in importnt proesses, suh s ntigen presenttion, tumour immunity nd the trnsmission of infetious gents. Exosomes ontin hrteristi omposition of proteins, nd express ell reognition moleules on their surfe tht filitte their seletive trgeting of nd uptke y reipient ells. Reent reports indite tht exosomes lso hrour vriety of mrnas nd mirornas (mirna),, whih n e trnsferred to reipient ells nd modulte their funtion 8. These findings hve inresed interest in the role of exosomes in ell-to-ell ommunition, nd support the ide tht exosomes might onstitute n exquisite mehnism for lol nd systemi interellulr trnsfer not only of proteins ut lso of geneti informtion in the form of RNA,,9. MiRNAs re lrge fmily of smll ( nuleotides long), nonoding RNAs tht downregulte gene expression y preventing the trnsltion of speifi mrna into protein. The emergene of mir- NAs s potent post-trnsriptionl regultors of gene expression hs rod implitions in ll res of ell iology, inluding the immune system. For exmple, speifi mirnas suh s mir- nd mir-8 regulte oth the immune response nd immune system development 6. In ddition, geneti ltion of the whole mirna mhinery or speifi mirnas severely ompromises immune development nd n led to utoimmune disorders nd ner 7,8. Here we present evidene tht exosomes medite ntigen-driven unidiretionl trnsfer of mirnas from the T ell to the APC during T ell APC ognte immune intertions. Moreover, our dt indite tht mirnas trnsferred on immune synpsis n e funtionl in the reipient ell. Results MiRNA profiles of immune ells nd their exosomes. To determine the mirna repertoires of exosomes sereted y immune ells, we isolted exosomes from ell superntnts of the Rji B-ell line, the Jurkt-derived J77 T ell line, nd primry dendriti ells (DCs) derived from humn monoytes. Exosomes were isolted y series of mirofiltrtion nd ultrentrifugtion steps 9, nd exosome identity ws ssessed y extensive protein nlysis with liquid hromtogrphy with tndem mss spetrometry (LC MS/MS) tehnology. Aout 6% of proteins found in the nlysed exosome smples hve een previously found in other types of exosomes; these inlude the tetrspnins CD6, CD8 nd CD9, proteins involved in memrne trnsport nd fusion (R GTPses, nnexins nd fotillin), nd other exosoml mrkers suh s Tsg (dt not shown). Moreover, exosomes derived from T lymphoytes nd from APCs oth ontined RNA. Profiling of RNA isolted from exosomes nd their donor ells indites tht exosomes re highly enrihed in smll RNA speies (Supplementry Fig. S). Agilent mirorna mirorry nlysis (Agilent) showed tht ertin mirnas re expressed t higher levels in exosomes thn in their donor ells nd vie vers (Fig. nd Supplementry Dt ). The hierrhil lustering nd the prinipl omponent nlysis of the rry dt grouped the smples ording to their ellulr or exosoml origin (Fig.,). Severl mirnas (for exmple, mir-76, mir-6, mir-6-p nd mir-67-p) were signifintly more undnt in exosoml smples from ll ell types; others, for exmple mir-, were found only in exosomes derived from the primry DCs; in ontrst, others (for exmple, mir-, mir- nd mir-) were more highly represented in ells thn in exosomes (Fig. ). These dt indite tht speifi mirna popultions re seletively sorted into exosomes. Consistently, there ws lower overll similrity etween the mirna repertoire in exosomes nd their orresponding ells thn etween the exosomes of different ellulr origin (Fig. d). Multivesiulr odies from T lymphoytes polrize towrds the IS. To nlyse the pity of ells to tke up immune exosomes, we generted Rji B nd J77 T ells stly expressing the exosoml mrker CD6 fused to green fluoresent protein (GFP). The tetrspnin CD6 is very undnt in exosomes, nd inside ells lolizes minly to MVBs nd lysosomes, with only smll pool present t the plsm memrne. Cytometry nd western lot nlyses onfirmed the presene of CD6-GFP in exosomes relesed y these ells (Fig. nd Supplementry Fig. S). The purified CD6-GFP exosomes were then inuted with non-trnsfeted J77 ells or Rji ells (reipient ells) for 6 h. Flow ytometry nlysis reveled tht oth J77 T nd Rji B ells hve the pity to tke up immune exosomes (Fig. ). It is importnt to highlight tht Rji B ells tke up T ell-derived exosomes to greter extent thn their own exosomes nd vie vers. Moreover, CD6-GFP ws deteted t the surfe of reipient ells y onfol mirosopy (Fig. ), suggesting tht exosomes re not internlized ut remin tthed to the reipient plsm memrne. To ddress whether exosomes medite the trnsfer of mirna during ognte immune intertions, we first studied the intrellulr distriution of MVBs the omprtments from whih exosomes rise during the formtion of n IS. In these experiments, Rji B ells were pulsed with Stphyloous enterotoxin superntigen-e (SEE) nd then inuted with TCR-Vβ8 + J77 T ells. MVB loliztion ws ssessed y immunofluoresene nlysis of CD6 nd two omponents of the ESCRT (endosoml sorting omplex required for trnsport): Hrs, omponent of the ESCRT- omplex; nd the lte ting ESCRT mhinery omponent VPS. In the presene of SEE, whih promotes formtion of fully funtionl IS, the MVBs of the J77 T lymphoyte lost their rndom ytoplsmi distriution nd ongregted ner the IS (identified y CD nd tin stining); in ontrst, the loliztion of MVBs in the Rji B ell remined unhnged (Fig., nd Supplementry Fig. S). Similr results were otined in experiments in whih CH7C7 T ells, ering n influenz hemgglutinin (HA) peptide-speifi TCR, were nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

3 nture ommunitions DOI:.8/nomms8 PC Hierrhil lustering J77 ells DC Rji ells DC Rji ells Rji exosomes PC J77 ells J77 exosomes J77 Exo DC exosomes Rji Exo DC exo. :. hs-mir- hs-mir-76 hs-mir-6 hs-mir-6-p hs-mir-67-p hs-mir-9 hs-mir- hs-mir- hs-mir- onjugted to HA peptide-pulsed Hom B ells, thus onfirming tht ntigen-indued formtion of n IS polrizes T-ell MVBs to the ontt site (Fig.,). Live ell imging of CD6-GFP-expressing J77 T ells enountering SEE-pulsed Rji B ells reveled tht the MVBs of the T ells move to the IS during the first min (Fig. d, nd Supplementry Movie ). The IS promotes the trnsfer of exosomes from the T ell to the APC. To investigte whether the IS promotes the trnsfer of exosomes from T ells to APCs, CD6-GFP T ells were ultured with Rji ells (stined lue with hloromethyl derivtive of minooumrin DC exosomes J77 exosomes Rji exosomes J77 exosomes r =. r =. r =. r =.7 DC J77 ells Rji ells Rji exosomes Figure mirorna profiles of exosomes nd their prentl ells. () Mirorry nlysis of exosoml mirnas versus the mirnas of their respetive donor ells. Exosomes were isolted y seril entrifugtion nd filtrtion steps from superntnts of donor ells ultured in RPMI-6 supplemented with exosome-depleted FBS (%). Totl RNA, inluding mirorna, ws isolted from exosomes nd their donor ells nd mirna profiles were ssessed y mirorry tehnology. The pnel shows the hierrhil lustering of the VSN-normlized rry dt in the log sle verged per iologil replite for eh origin (exosomes per ells) nd ell type. DC, dendriti ells; J77, Jurkt-derived J77 T ell line; Rji, Rji B ell line). () Prinipl omponent nlysis (PCA) of ll normlized rry dt. Eh point represents hyridized smple: tht is, different iologil smples per origin nd ell type. x xis, first prinipl omponent (PC); y xis, seond prinipl omponent (PC). Cell smples, red; exosoml smples, lue. () Hetmp of the VSN-normlized dt for seleted mirnas. For visuliztion purposes the expression profiles were entred on the medin of the profile. The sle r ross the ottom depits stndrd devition hnge from the men. (d) Stter plots of the exosome versus ell verged rry dt in eh ell type nd of Rji exosomes versus J77 exosomes. The orreltion is shown. d ARTICLE (CMAC)) for 6 h, y whih most stge onjugtes will hve seprted. The oulture ws nlysed y flow ytometry for the trnsfer of CD6-GFP. SEE-pulsed Rji ells quired CD6-GFP from the T ells, wheres trnsfer in the sene of SEE ws negligile (Fig. ). No trnsfer of GFP signl ws deteted when the ssy ws performed in the opposite diretion (from Rji-CD6-GFP to J77 ells; Fig. ) or when using J77 ells overexpressing non-exosoml memrne or ytoplsmi proteins (CD69-GFP nd GFP; Fig. ). In ddition, we lso deteted trnsfer of other moleules relted to exosomes nd vesiles, suh s CD8 (ref. ) nd LAT (Supplementry Fig. S). To define the requirement for ell ell ontt, we seprted donor nd reipient popultions y. µm pore-size Trnswell memrne. J77-CD6-GFP T ells in trnswell ssys were treted with nti-cd plus nti-cd8 to tivte them nd therefore enhne exosome relese (Supplementry Fig. S). Although T ell CD69 levels indited tht ells hd een tivted to the sme extent s fter formtion of n IS, no CD6-GFP ws deteted on APC reipient ells, whether or not these were loded with SEE (Fig. nd Supplementry Fig. S). Also, in ontt (non- Trnswell) oultures, stimultion with CD nd CD8 As in the sene of SEE did not support CD6-GFP trnsfer (Supplementry Fig. S,). Moreover, CD6-GFP trnsfer in stndrd (in the presene of SEE) ontt oultures ws olished y ddition of the tin ytoskeleton inhiitors ytohlsin-d nd ltruulin-a, whih disrupt the IS, wheres the mirotuule inhiitor noodzol hd no effet (Fig. ). These results indite tht ell ell ontt nd T-ell tivtion y themselves do not support exosoml trnsfer. To onfirm tht the exosoml trnsfer requires funtionl IS, we oultured J77-CD6-GFP T ells with mix of Rji B ells nd BLS- B ells, whih lk HLA lss II nd nnot form n IS. After 6 h, GFP signl ws deteted on SEE-primed Rji B ells (Fig. d). In ontrst, BLS- B ells did not trigger CD6 trnslotion (Fig d, right pnel) or CD lustering. These findings indite tht lthough T nd APCs n oth tke up immune exosomes, ognte immune intertions promote the trnsfer of exosomes from the T lymphoyte to the APC nd their unidiretionl quisition y the onjugted APC. We next nlysed the mehnism of exosome uptke y APCs. In post-onjugted Rji ells, whih hd quired CD6-GFP on IS formtion, the CD6-GFP o-lolized t the plsm memrne with endogenous mjor histoomptiility omplex (MHC)-II moleules, inditing tht the exosomes either remined tthed or hd fused with the ell surfe. Intensity profiles round the ell perimeter indite similr distriutions of oth moleules in the plsm memrne (Fig. e). In ontrst, in Rji ells tht hd quired CD6-GFP y diret ddition of exosomes isolted from J77-CD6-GFP ells (non-synpti uptke), the GFP signl ppered s ggregtes tthed to the plsm memrne nd did not oinide with endogenous plsm memrne MHC-II (Fig. e). To find out whether T ell exosomes re fused or only dhered to the APC plsm memrne, we treted the Rji ells with trypsin. Trypsin tretment did not derese the GFP signl quired y SEEloded Rji ells from onjugted J77-CD6-GFP, inditing tht exosomes trnsferred on immune synpsis n fuse with the APC plsm memrne or e internlized (Fig. f). In ontrst, trypsin deresed the fluoresene signl of Rji ells tht quired CD6- GFP y non-synpti uptke (Fig. f). These results suggest tht the IS might not only promote the seretion of exosomes from T ell to APC, ut might lso indue the fusion of these exosomes with the plsm memrne of the reipient ell. The IS promotes the trnsfer of exosoml mirna. To demonstrte delivery of exosoml mirna from T ell to APC fter IS formtion, we stly overexpressed mir- in J77-CD6-GFP T ells (Fig. ). This mirna is not normlly expressed in the donor (J77) or reipient (Rji) ells, ut is sorted to the exosomes of primry immune nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

4 nture ommunitions DOI:.8/nomms8 GFP J77 Q Q Q Q J77-CD6GFP Q Q Q Q Q Q Q Q Isotype ontrol Anti-CD6PE CD6-GFP + reipient ells (%) Rji J77 Anti-CD6 Reipient ells Projetion Setion DIC +J77-exo Control Figure Uptke of CD6-GFP exosomes y immune ells. () Untrnsfeted J77 T ells nd J77 T ells stly expressing CD6-GFP (J77-CD6-GFP ells) were ultured in exosome-depleted medium for h nd exosomes were purified from superntnts y ultrentrifugtion. Exosomes were lelled with nti-cd6-phyoerythrin nd nlysed y flow ytometry. () Uptke of CD6-GFP exosomes y T ells nd B ells (reipient ells). Untrnsfeted ells were inuted with CD6-GFP exosomes for 6 h nd nlysed y flow ytometry. Dt represent the perentge of GFP-positive ells ( ± s.e.m.) of three independent experiments. Open rs, no exosomes; striped rs, Rji exosomes; filled rs, J77 exosomes. () Confol mirosopy detetion of CD6-GFP (green) on the surfe of reipient ells (Rji) fter inution with J77-CD6-GFP exosomes. Cell memrnes were stined for the ellsurfe moleule CD (red) nd nulei were stined with HOESCHT (lue). Imges show mximl projetions of onfol imges (projetion), one representtive onfol setion (setion) nd the DIC imges. Sle r, μm. ells (DCs nd T lympholsts; Figs nd, nd Supplementry Dt ). J77-CD6-GFP ells expressing mir- (J-) were oultured with unprimed or SEE-primed Rji B ells (stined lue with CMAC), nd fter h the Rji ells were sorted y flow ytometry (Supplementry Fig. S6) nd their mir- ontent nlysed y reverse trnsription PCR. mir- ws trnsferred to Rji ells only in the presene of SEE (Fig. ). Rji ells tht quired high mounts of CD6-GFP ontined orrespondingly high mounts of mir-, demonstrting orreltion etween the trnsfer of mir- nd exosoml proteins (Fig. ). To demonstrte tht mir- is not expressed de novo in Rji ells fter IS formtion, we repeted the experiment with J77-CD6-GFP ells stly overexpressing mir- (J-); onjugtion with these ells did not indue expression of mir- in Rji ells (Fig. ). The ility of peptide ntigen-speifi IS to trnsfer mirna ws further demonstrted in HA-loded CH7C7 ells overexpressing mir- (C-) nd onjugted to Hom- ells (Fig. ), demonstrting ntigen-speifi diretionl trnsfer of exosoml mirna from T ell to APC. We lso investigted the trnsfer of endogenous mirnas y primry SEE-speifi T lymphoytes. These ells trnsferred endogenous mir- nd mir- 9 to Rji APCs in SEE-speifi mnner (Fig. d). To onfirm tht mirna trnsfer is medited y exosomes, we loked exosome prodution in J- ells. Cermide, iosynthesis of whih is regulted y neutrl sphingomyelinse- (nsmse), triggers the udding of exosomes into MVBs, nd inhiition of nsmse therefore redues the seretion of CD6-ontining exosomes nd mirnas 6. Aordingly, the seretion of exosomes y J77 ells ws impired when nsmse tivity ws redued either y ddition of the inhiitor mnumyin-a (Fig. 6) or y smll hirpin RNA (shrna) silening (Fig. 6 nd Supplementry Fig. S7). Trgeting of nsmse tivity inhiited the IS-dependent trnsfer of CD6-GFP (Fig. 6,d) nd mirna- (Fig. 6e,f). Trnsfer of CD6-GFP nd mir- ws lso loked y refeldin (Fig. 6,e), whih inhiits the gunine nuleotide-exhnge protein BIG nd regultes the onstitutive relese of exosome-like vesiles 7. Exosome seretion nd CD6-GFP trnsfer y J- ells ws lso inhiited y shrna silening of the R GTPse R7, whih is implited in the exosoml relese pthwy 8 (Supplementry Fig. S7 d). In ontrst, trnsfer of CD6-GFP ourred normlly from Hrsinterfered J77 T ells (Supplementry Fig. S7e nd Fig. 6d), in greement with previous studies tht demonstrted tht the ESCRT system is unneessry for the relese of exosomes nd mirna,6. Trnsferred mirnas regulte gene expression on the APC. To determine whether synptilly trnsferred mirna- is funtionl in the reipient APC, we rried out luiferse reporter ssys nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

5 nture ommunitions DOI:.8/nomms8 ARTICLE HRS DIC CD6 DIC CD CD HA CD CD Atin +HA Atin d min min 6 min 9 min min 8 Trnslotion t the IS (%) 6 CH7-C7 CD6 Hrs CD CD6 Hrs HOM- Figure MVBs in T ells trnslote to the IS. () J77 T ells were onjugted with SEE-pulsed or non-pulsed Rji B ells loded with CMAC (lue). After min, ells were fixed nd stined for the MVB mrker Hrs (red), CD nd tin (oth green). Pltes show mximl projetions of onfol imges nd the DIC imges. () CH7C7 T ells were onjugted with HA-loded or non-loded HOM B ells (lue). After min, ells were fixed nd stined for CD6 (green), CD nd tin (oth red). Pltes show mximl projetions of onfol imges nd the DIC imges, sle rs pplies to nd. () Perentge of T nd APC ells in whih CD6, Hrs nd CD relolized to the T ell APC ontt re in the presene of HA peptide (filled rs) or its sene (open rs). Dt re the rithmeti mens ± s.e.m. of four experiments. P <. ompred with the sene of ntigen (Mnn Whitney test). (d) Live ell imging of J77-CD6-GFP ells seeded on fironetin-oted overslips nd onjugted with SEE-primed Rji ells (lue). Cells were monitored y time-lpse onfol mirosopy t s intervls. Pltes show mximl projetions of onfol imges. Sle rs, μm. with full-length -UTR onstruts of the mir- trget SOX nd the mir--insensitive UBEF 9. Rji ells trnsfeted with luiferse onstruts were oultured with J- ells nd luiferse tivity ws ssessed. Expression from the SOX -UTR ws signifintly redued in SEE-primed Rji ells tht hd een in ontt with J- ells, wheres expression from the UBEF -UTR ws unffeted (Fig. 7). Expression from these -UTR reporters ws not ffeted y oulture of Rji ells with J- ells, inditing tht the proess is not generl effet of IS formtion, ut rther involves the diret trnsfer of the speifi mirna. Redution in luiferse tivity fter onjugte formtion ws lso deteted in Rji ells expressing se pirs 9 9 of the SOX -UTR, whih enompsses the mir- seed sequene; nd inhiition of luiferse expression ws olished y muttion of this sequene (Fig. 7). The IS thus guides the trnsfer of funtionl mirna from the T ell to the APC. Contrsting with IS-dependent mirna trnsfer, ddition of exosomes isolted from J- ells to trnsfeted Rji ells hd no effet on luiferse tivity (Fig. 7,), inditing tht ny mir- quired in this wy ws non-funtionl. Disussion The dt presented here estlish highly effiient interellulr mehnism for the trnsfer of regultory geneti informtion exlusively in the miroenvironment of the IS. Unlike other exmples of RNA trnsfer vi mirovesiles, where non-synpti relese potentilly llows the exhnge of geneti mteril t distne, our dt indite tht during ognte immune intertions there is unidiretionl trnsfer of mirna from the T to the APC. This geneti ommunition is ntigen driven nd ppers to e linked to the formtion of the IS. The importne of exosome relese in this proess is demonstrted y the orreltion of mirna trnsfer with tht of CD6 nd y its lokde with inhiitors of exosome prodution. Cells relese different types of vesiles into the extrellulr spe. Two types of vesiles n e distinguished depending on their ellulr origin. Shedding vesiles re generted y udding from the plsm memrne into the extrellulr spe,, wheres exosomes hve n endosoml origin in MVBs: MVBs either fuse with lysosomes for degrdtion or with the plsm memrne to relese their intrluminl vesiles (ILVs) s exosomes. Mixed popultions, ontining shedding vesiles nd exosomes, re referred s mirovesiles. Mirovesiles re inresingly reognized s importnt meditors of ell-to-ell ommunition. They n trnsfer reeptors, proteins, mrna nd mirna to trget ells vi intertion with speifi reeptors. Mirovesiles derived from emryoni stem ells hve een reported to reprogrmme hemtopoieti progenitors through the delivery of mrna 6. The trnsfer of RNA speies vi mirovesiles to endothelil ells indues ngiogenesis, nd progenitor moiliztion 7. In the immune system, exosoml nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

6 nture ommunitions DOI:.8/nomms8 CMAC CD6-GFP CD6-GFP + reipient ells (%) 6 P <. Rji/ J77-GFP J77/Rji-GFP CMAC CMAC GFP CD69-GFP. 8 BLS.8. CD6-GFP + Rji ells (%) 6 Control Lt A Cyt D NCD Twell d CMAC CD6-GFP Rji.7 BLS 8.7 Rji. CD6-GFP + reipient ells (%) 6 P <. e DIC Projetion Setion f IS-dependent uptke Non-synpti uptke MHC-ll CD6-GFP (J77ell) MHC-ll CD6-GFP (J77 exo) (ARU) (ARU) Intensity,,,,,,,, 6 7(µm) (µm) CD6-GFP + Rji ells (%) 8 6 IS-dependent uptke 8 6 Non-synpti uptke Control J77-CD6GFP Exo Figure Ag reognition indues trnsfer of exosomes from T ell to APC. () Left: J77-CD6-GFP donor ells were onjugted with SEE-primed or unprimed Rji ells (reipient, in lue). Right: Rji-CD6-GFP donor ells ( ± SEE) were onjugted with lue-leled J77 reipients. After 6 h, oultures were nlysed y flow ytometry. Br hrt shows the perentge ± s.e.m. of positive reipient ells (n = 9, P <., Student s t-test). () J77 ells trnsfeted with CD69-GFP or GFP were onjugted with SEE-primed Rji ells (lue) nd nlysed s in. () FACS nlysis of the perentge of GFPpositive Rji ells fter inution with J77-CD6-GFP ells in ontt with oulture s in (ontrol), in the presene of inhiitors of tin (ltrunulin-a, LtA or ytohlsin-d, CytD) or tuulin (noodzol, NCD), or fter inution with seprtion of donors nd reipients y. µm pore-size trnswell memrne (Twell). T ells in trnswells were tivted with CD + CD8 As nd Rji ells were loded with SEE. (d) J77-CD6-GFP donor ells were onjugted with : mix of two B ell lines: Rji ells (lk) nd BLS- ells (lue) nd nlysed s in. Br hrt shows perentges ± s.e.m. of positive reipient ells (n = 6, P <., Mnn Whitney test). Mximl projetions of onfol imges nd the DIC of triple onjugted formed y J77-CD6- GFP (green), Rji ells (lk sterisk) nd BLS- (CMAC stined nd lue sterisk), stined for CD (red) re shown, sle r shown in e. (e) Confol nlysis of Rji ells tht quired CD6-GFP diretly from J77-CD6-GFP fter IS formtion (IS-dependent trnsfer) or fter externl dministrtion of CD6-GFP exosomes isolted from J77-CD6GFP superntnts (non-synpti uptke). Cells were stined for MHC-II (red). Imges show mximl projetions of onfol imges, one representtive onfol setion, nd the DIC imges. Plots show ell perimeter fluoresene intensity profiles of the green nd the red signls. ARU: ritrry reltive units. Sle r, μm. (f) Rji ells ( ± SEE) were treted s in e. FACS nlysis of the effet of trypsin on the CD6-GFP ontent of Rji ells tht quired exosomes y IS-dependent trnsfer or non-synpti uptke. White rs, ontrol; grey rs, min trypsin; lk rs, min + min trypsin. Br hrt shows perentges ± s.e.m. of positive ells (n = ). trnsfer of mrna ours etween mst ells, nd virl mirnas sereted y EBV-infeted ells n e tken up y uninfeted reipient ells 8. Syntheti mirna mimetis nd virl mirnas hve een reported to e trnsferred etween leukoytes, lthough the diret involvement of exosomes/mirovesiles in this se ws not demonstrted. Exhnge of proteins etween immune ells hs een extensively reported, ut the mehnism of this trnsfer remins unler. This phenomenon, whih hs een lled trogoytosis, is onsidered to e n ntigen-speifi event tht requires the formtion of n IS. Our dt suggest tht the mehnism for the trnsfer of ertin proteins during immune synpsis is the diretionl relese of exosomes. In ordne with other reports, our dt show tht exosomes n e trnsferred etween ells t distne. However, in the se of immune ells, the presene of speifi ntigen indues the formtion of n IS, nd enhnes this exosoml trnsition. ISs re highly orgnized ell ell ontts where, on ntigen reognition, T-ell tivtion is initited nd tuned. The genertion of n IS involves the polriztion of severl T-ell orgnelles towrds the APC. Among them, the MTOC, the Golgi pprtus nd ytotoxi grnules re required for effetor funtions, suh s exoytosis of lyti grnules y ytotoxi T ells or polrized seretion of ytokines y helper T ells 6. However, not ll ytokines re relesed diretly t the IS; some, suh s tumour nerosis ftor or interleukin (IL)-, re delivered vi multidiretionl pthwy,. The formtion of n IS indues the polriztion of T-ell MVBs towrds the IS nd enhnes the dishrge of exosomes (see ref. nd the present report). This loliztion suggests tht the fusion of MVBs with the plsm memrne for exosome relese might our t the IS zone. Moreover, when CD6-GFP T ells were oultured simultneously with two B ell lines, only B ells presenting SEE were le to uptke the CD6-GFP signl, strongly inditing polrized, IS-dependent trnsfer. Further experimenttion will e needed, however, to unequivolly determine whether exosoml trnsport ours in polrized mnner through the IS or is multidiretionl. Interestingly, CD6 ws not trnsferred in T B ontt oultures in the presene of stimuli tht tivte T ells without forming n IS. These results indite tht ell ell ontt nd T-ell tivtion re not suffiient for the oserved exosoml trnsfer, nd tht the formtion of n IS is ritil. For exmple, ntigen might t on the reipient B ell to filitte exosome uptke. Moreover, only exosomes quired on IS formtion pper to e funtionl in the reipient ell. These dt suggest tht, in ddition to exhnge of exosomes t distne, the formtion of n IS enhnes this trnsfer nd promotes the diretionl nd funtionl relese of T-ell exosomes to the APC. Complex mehnisms underlie the sorting of rgo into different popultions of ILVs. Uiquitinted proteins require the tion of the ESCRT mhinery to medite degrdtive protein sorting, wheres other rgos suh s CD6 re sorted in ermide-dependent pthwy tht genertes nother popultion of ILVs destined for seretion s exosomes. Our dt nd previous reports 8,6 indite tht not ll mirnas n e inorported into exosomes, nd therefore tht the pkging of speifi mirna popultions into exosomes is seletive. However, the mehnisms ontrolling this sorting remin unler. mirnas inorporte into exosomes nd re relesed vi nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

7 nture ommunitions DOI:.8/nomms8 ARTICLE 8 6 T lympholst DC 6 SEE: J77 Rji + + Donor ells: ( ) J- J- 8 6 DC J J- J- CD6 lo CD6 hi J- 8 6 J- exo J- exo e Exosomes Control Brefel Mnu..6. CD6-GFP + Rji ells (%) 8 6 Control Brefel Mnu kd d f CD6-GFP + Rji ells (%) shrna ontrol Exosomes shrna nsmse kd shcontrol shnsmse sicontrol sihrs Donor ells: HA: ermide-dependent pthwy independent of the ESCRT mhinery 6. Moreover, s proteins of the RNA-indued silening omplex hve een deteted in exosomes 7, it is fesile tht ssoition with RNA-indued silening omplex omponents ontrols the pkging of mirnas in exosomes. Bloking MVB formtion y ESCRT depletion results in impired mirna silening 7,8, thus suggesting tht MVBs ould e mirna rossrod to seretory pthwy nd gene silening. Other potentil mehnisms for the pkging of speifi mirnas in exosomes re modifition nd their ssoition with trget mrna. Our mehnisti studies with hemil d mir-9 (AU) + + SEE: + + ( ) C- C- T lympholst Figure Exosoml mirna- is trnsferred from T ell to APC in n Ag-speifi mnner. () Levels of mir- were ssessed y quntittive reverse trnsription PCR (qrt PCR) in primry dendriti ells nd T lympholsts, nd in Rji nd J77 ells. J77-CD6-GFP ells were stly trnsdued with mir- (J- ells) or mir- (J- ells), nd mir- levels were determined y qrt PCR in ells nd derived exosomes. Dt re representtive of three experiments (men nd s.e.m.) () mir- levels in SEE-primed Rji ells sorted h fter onjugtion with J- ells. J- were used s ontrol donor ells. Left pnel, Dt re representtive of seven independent experiments (men nd s.e.m), P =. (one-smple t-test). Right pnel, n = independent experiments; P =. (one-smple t-test); error rs represent s.e.m. () mir- levels in HA-primed HOM- ells sorted h fter onjugtion with CH7C7 ells overexpressing mir- (C-). CH7C7 ells overexpressing mir- (C-) were used s ontrol donor ells. Dt re representtive of five experiments (men nd s.e.m.). (d) mir- nd mir-9 levels in SEE-primed Rji ells sorted h fter onjugtion with primry T lympholsts-expressing mir- nd mir-9 endogenously. Dt re representtive of three experiments (men nd s.e.m.), P =.6 (one-smple t-test) AU, ritrry units. Control Brefel Mnu inhiitors nd nsmse sirna show tht exosoml mirnas re exhnged during IS formtion vi ermide-dependent, ESCRTindependent pthwy. In ordne with previous reports, we shcontrol shnsmse Figure 6 Inhiition of exosome iogenesis impirs trnsfer of exosoml mirnas nd proteins through the IS. () J77 ells were ultured in exosome-depleted medium for h, in the presene of inhiitors of nsmse (mnumyin-a, Mnu) or BIG (refeldin, Brefel). Purified exosomes sereted y equl numers of ontrol or treted ells were nlysed y immunolotting for the presene of the exosome mrker CD8. Densitometri nlyses were performed, nd the rtio etween the treted nd the ontrol ondition is shown. () CD8 immunolot of exosomes purified from equl numers of ells trnsdued with ontrol or nsmse shrna. Densitometri nlyses were performed, nd the rtio etween the silened nd the ontrol ondition is shown. () FACS nlysis of the CD6-GFP ontent of Rji reipients ells fter onjugtion with J- ells in the presene of mnumyin or refeldin. Dt re the perentge ± s.e.m. of Rji-GFP-positive reipient ells reltive to the SEEloded ontrol ondition. n = independent experiments; P. (onesmple t-test). (d) FACS nlysis of the CD6-GFP ontent of Rji reipient ells fter oulture with J77-CD6GFP ells expressing shnsmse or sihrs. Dt re the perentge ± s.e.m. of Rji-GFP-positive reipient ells reltive to the SEE-loded ontrol ondition. n = 8 independent experiments, P =. (one-smple t-test). (e) Quntittive reverse trnsription PCR (qrt PCR) nlysis of mir- in Rji ells sorted fter onjugtion with J- ells in the presene of mnumyin or refeldin. n = independent experiments; P =. (unpired t-test). (f) qrt PCR nlysis of mir- in Rji ells sorted fter onjugtion with J- trnsdued with shnsmse or shcontrol. n = independent experiments; P =. (unpired t-test); error rs represent s.e.m. nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

8 Luifese tivity (reltive level) Luifese tivity (reltive level) SOX -UTR (full-length). J- J- J- J- SOX -UTR seed sequene WT UBEF -UTR SOX -UTR seed sequene MUT. J- J- J- J- Donor ells Donor ells Figure 7 Synptilly trnsferred mir- downregultes trget gene expression in the APC. () Rji ells were trnsfeted with reporter onstruts onsisting of the luiferse sequene pled upstrem of the full-length -UTR of either the mir--trget gene SOX or the ontrol gene UBEF. Luiferse tivity ws ssyed h fter oulture of SEE-loded Rji with J77-miR- ells or ontrol J- ells (synpti trnsfer) or fter ddition of derived exosomes to SEE-loded Rji ells (non-synpti uptke). () Experiments s in performed with Rji reipient ells expressing the wild-type of mutted seed sequene for mir- from the SOX -UTR. Luiferse tivities re shown reltive to ontrol inutions with untreted Rji ells. Dt re mens ± s.e.m.; n = independent experiments, P <. (Newmn Keuls multiple omprison test). White rs, ontrol; grey rs, synpti trnsfer; lk rs, non-synpti trnsfer oserved tht knoking down memers of the R GTPses fmily inhiits exosome seretion 8 nd the trnsfer of exosoml ontent during immune synpsis, wheres Hrs silening hs no effet. Our results show tht the trnsfer of CD6 orreltes with the trnsfer of one mirna, mir-, from the T ell to the APC. mirna- is espeilly suitle for our tehnil pproh, s it is expressed in primry leukoytes, is highly enrihed in exosomes, nd its expression is negligile in Rji B ells. It is fesile tht other exosoml mirnas n lso e trnsferred during immune synpsis. Our dt show tht levels of mir-9, nother mirna undnt in exosomes, inrese in Rji ells fter onjugtion with T lympholsts. However, s this mirna is endogenously expressed y B ells, we nnot distinguish etween endogenous upregultion nd horizontl trnsfer. Tody there is no dout tht mirnas re importnt modultors of the immune system,,9. Modifition of gene expression in reipient ells y trnsferred geneti mteril ould ount for severl exosome funtions. This exosoml trnsfer of regultory RNAs is potentilly powerful mens of orhestrting gene expression during the genertion of the immune response, nd inreses the omplexity of ommunition etween ells. Our dt demonstrte tht mirnas trnsferred during immune synpsis n e funtionl in the reipient ell, nd suggest tht T ell-derived mirnas regulte speifi trgets in APCs. As n exmple, we demonstrte tht mirna- downregultes trnsltion of SOX- mrna. This mirna- trget gene hs reently een reported in rest ner ells 9. In ontrst, Rji ells tht were exposed to isolted exosomes without ellulr ontt showed no mirna tivity, nd the exosoml protein signl ould e removed y trypsin tretment. nture ommunitions DOI:.8/nomms8 How exosomes deliver their ontent to reipient ells remins unler. It hs een proposed tht, depending on the origin of exosomes nd the identity of the reipient ells, exosomes might e internlized y endoytosis, phgoytosis, or y diret fusion with the plsm memrne. Whtever the mehnism, the differenes we oserved etween IS-dependent exosoml trnsfer nd uptke without ell ontt support the notion tht the immune synpse promotes the trnsfer of mirna-loded exosomes y T ell nd filittes funtionl delivery of the mirna into the APC. Methods Cells nd regents. The humn Jurkt-derived T-ell lines J77l (TCR Vαl. Vβ8) nd CH7C7 (Vβ TCR speifi for HA peptide) nd the lympholstoid B-ell lines Rji (Burkitt lymphom), HOM- (HLA-DR EBV-trnsformed) nd BLS- (HLA lss II-null B-LCL, generted from ells of ptients with type II Bre lymphoyte syndrome) were ultured in RPMI (Sigm) ontining % fetl ovine serum (Invitrogen). Stle ell line lones overexpressing CD6-GFP were generted y trnsfetion nd seletion with G8 ( mg ml ). The CD6-GFP ell lines were susequently trnsdued y lentivirl infetion to overexpress mir- or mir- nd seleted with lstiidin ( µg ml ) nd G8 ( mg ml ); the resulting ell lines were designted s follows: J77-CD6-GFP-miR (J-), J77-CD6-GFP-miR (J-), CH7C7-CD6-GFP-miR (C-) nd CH7C7-CD6-GFP-miR (C-). Humn peripherl lood mononuler ells were isolted from uffy ots from helthy donors y seprtion on iooll grdient (Biohrom). After min of dhesion step t 7 C, non-dherent ells were ultured dys in the presene of phytohemgglutinin ( µg ml ) to indue lymphoyte prolifertion. To otin T lympholsts, IL- ( U ml ) ws dded to the medium every dys for time period of 8 dys. To otin SEE-speifi T lympholsts, ells were ultured dys in the presene of SEE. After dhesion step, dherent monoytes were ultured in the presene of IL- nd grnuloyte mrophge olony-stimulting ftor to indue their differentition into DCs. Mturtion of DCs ws promoted y dding lipopolyshride 6. The fluoresent ell trkers CMAC nd BCECF (is-roxyethyl-roxyfluoresein) were from Moleulr Proes. Ltrunulin-A (LtA), mnumyin-a, noodzol (NCD), ytohlsin-d (CytD) nd reominnt humn fironetin nd poly-l-lysine were from Sigm. The following nti-humn ntiodies were produed in the lortory: nti-cd6 ma (Te /8), nti-cd ma (t), nti-cd (D/9) nd nti-mhc-ii (DCIS/). Phyoerythrin-nti-humn CD6 ws from BD Biosienes, nti-mouse CD6 ma (NKI/C-) nd nti-hrs were from HGS Am, nd nti-cd8 ma (A6) from Snt Cruz. Lentiviruses-expressing shrna were otined from the Open Biosystems Expression Arrest RNAi onsortium (TRC) lirry. Clone ID of the shrna used were: TRCN9 for RAB7A, nd TRCN897, TRCN896, TRCN89, TRCN89, TRCN89 for nsmase (SMPD). Hrs ws knoked down with pir of RNA sequenes ( -CGACAAGAACCCACACGUCtt-, together with - AAGCGGAGGGAAAGGCCACUUTT- ) from Amion. Control sequenes were On-Trget plus non-trgeting sirna no. nd (Dhrmon) nd the sequene -UUCUCCGAACGUGUCACGUtt-. Cell trnsfetion. J77 or Rji ells were trnsfeted with CD6-GFP, CD-GFP, EGFP, LAT-GFP or CD8-GFP plsmids y eletroportion 7. Cells were resuspended in Opti-MEM (GIBCO; 7 ells per ml) with µg of DNA plsmid nd eletroported with Gene Pulser Xell (Bio-Rd) t µf, mv during ms t mm Bio-Rd uvettes (Bio-Rd). CD6-GFP-positive ells were FACS sorted, loned nd ultured in RPMI ontining mg ml G8 (Invitrogen). Lentivirl infetion. J77-CD6-GFP ells overexpressing mirna- or mirna- were generted y lentivirl infetion. HEK9T ells were o-trnsfeted (Lipofetmine; Invitrogen) with mirve plsmids enoding the desired mirna (Geneservie) nd pcl-ampho plsmid (RetroMx). Superntnts were olleted fter 8 7 h, filtered (. µm) nd dded to J77-CD6-GFP ells. Cells were entrifuged (, g, h) nd inuted for h t 7 C. Medium ws repled with RPMI-ontining lstiidin ( µg ml ) nd G8 ( mg ml ). To silene RAB7A nd nsmse, HEK9T ells were o-trnsfeted with orresponding shrna plko system plsmids (Open Biosystems) nd pcmv- R8.9-(Delt 8.9) nd pmd.g-vsv-g. Superntnts were dded to J77-CD6-GFP-miR- (J-) ells, whih were ultured in RPMI ontining µg ml puromyin. Exosome purifition. Donor ells were ultured in RPMI-6 supplemented with % FBS (depleted of ovine exosomes y overnight entrifugtion t, g), nd exosomes were prepred from ell superntnts y severl entrifugtion nd filtrtion steps 9. Briefly, ells were entrifuged ( g for min) nd the superntnt filtered through. µm memrnes. Exosomes were pelleted y ultrentrifugtion t, g for 6 min t C (Bekmn Coulter Optim L- XP, Bekmn Coulter). RNA isoltion. Totl RNA ws extrted with TRIzol regent (Invitrogen) nd the mirnesy mini kit (Quigen), nd ws sreened for purity nd onentrtion nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.

9 nture ommunitions DOI:.8/nomms8 ARTICLE in Nnodrop- Spetrophotometer (Thermo Sientifi). RNA integrity ws ssessed y ethidium romide lelling on.% grose gel. MiroRNA nlysis. The Agilent Bionlyzer (Agilent) for totl RNA (RNA nno hips) nd for smll RNA (smll RNA hips) were used to ssess the lrge nd smll RNA profiles in ells nd exosomes. Mirorry experiments were performed using the humn mirorna mirorry from Agilent. Arrys were performed on three different RNA preprtions from Rji nd J77 ells nd their exosomes, nd two preprtions eh from humn DCs nd their exosomes. Quntittive rel-time-pcr. Mture mirna nd mrna quntifition ws performed y TqMn rel-time PCR (Applied Biosystems) ording to the mnufturer s instrutions. The following TqMn mirna Assys nd TqMn Gene expression Assys (Applied Biosystems) were used: hs-mir- (6), hs-mir-9 (), RAB7A (Hs68_m) nd SMPD (nsmse) (Hs87_m). HY () nd RNU9 () were used s endogenous ontrols for mirna, wheres GAPDH (Hs7899_g) nd HPRT (Hs67_ m) were used s endogenous ontrols for mrna (ll from Applied Biosystems). Retions were performed in triplite in µl volumes. Quntittive mirna or mrna expression dt were quired nd nlysed using the ABI Prism 79HT Sequene Detetion System (Applied Biosystems). Dt were further nlysed using Biogzelle QBsePlus softwre (Biogzelle), nd results re expressed in ritrry units. Non-synpti uptke experiments. Exosomes were purified s desried ove nd dded to reipient ells in rtio : (reipient:donor ells). After 6 h, reipient ells were nlysed y flow ytometry. Conjugte formtion. To distinguish B ells from T ells, B ells (Rji or Hom) were loded with the lue fluoresent trker CMAC 8. B ells were inuted with SEE (Toxin Tehnology) or HA peptide (New Englnd Peptide) s pproprite, nd mixed with J77 ells (ntive or expressing CD6-GFP ± mir- or mir-) or CH7C7 ells t rtio of :8. Conjugtion t 7 C ws ontinued for times indited in eh se. Where indited, J77 ells were preinuted with mnumyin-a ( µm), refeldin ( mg ml ), noodzol ( µm), ytohlsin-d ( µm) or ltrunulin-a ( µm). In experiments with Rji-CD6-GFP ells, J77 ells were lelled with CMAC. Where indited, Rji nd J77 ells were prevented from oming into diret ontt y seprting with. µm pore-size trnswell memrne (Costr). This llows the pssge of exosomes ut preludes diret ell ell intertion. When indited, T ells were tivted with phorol myristte ette ( ng ml ) plus Ionomyin (. µg ml ), nti-cd plus nti-cd8 (oth t µg ml ) or SEE (. µg ml ). Fluoresene onfol mirosopy. For immunofluoresene ssys, ells were plted onto slides oted with poly-l-lysine ( µg ml ), inuted for min, fixed, loked nd stined with the indited primry ntiodies ( µg ml ) followed y lex88- or Rhodmine Red X-leled seondry ntiodies ( µg ml ). For live-ell imging, Rji (preloded with CMAC nd SEE) nd J77-CD6-GFP ells were dded to overslips oted with fironetin ( µg ml, Sigm), mounted in Attofluor open hmers (Moleulr Proes), nd mintined t 7 C in % CO tmosphere. Smples were exmined with Lei SP onfol mirosope (Lei) fitted with 6 ojetive, nd imges were proessed nd ssemled using Lei softwre. Flow ytometry nlysis nd sorting. Cell smples were nlysed with FAC- SCnto flow ytometer nd FACSDiv softwre (BD Biosienes). Vile ells were identified y propidium iodide exlusion. Singlet ells were diserned with stringent multiprmetri gting strtegy sed on FSC nd SSC (pulse width nd height). T ells nd APCs were distinguished y CMAC stining nd GFP fluoresene. Cells were sorted on FACSAri flow ytometer (BD Biosienes). Rji ells were isolted fter oulture with T ells y sorting for CMAC-positive singlet ells. Where indited, Rji ells were lso sorted sed on CD6-GFP levels. Immunolotting. Cells or exosomes were lysed in RIPA (% NP,.% deoxyholte,.% SDS in TBS), with oktil of protese inhiitors (Complete, Rohe). Proteins were seprted in % rylmide/isrylmide gel nd trnsferred to nitroellulose memrne. Proteins were visulized with LAS- fter memrne inution with speifi ntiodies ( µg ml ) nd seondry ntiodies onjugted to peroxidse ( µg ml ). Bnd intensities were quntified using WCIF Imge J softwre nd results re expressed reltive to the ontrol ondition. UTR reporter ssys. Psihek dul luiferse reporter vetors (Promeg) loned with the full-length -UTRs of SOX nd UBEF nd the SOX -UTR segment (se pirs 9 9) ontining the wild-type or mutted (se pir 8) mir- seed sequene were gift from Dr Mssgué 9 (Memoril Slon-Kettering Cner Center, New York). Rji ells were trnsfeted y eletroportion nd, 6 h lter, oultured with J- or J-. After h, ells were lysed nd the rtio of Renill nd Firefly luiferse tivities ws mesured y the dul luiferse ssy (Promeg). Sttistil nlysis. Mirorry dt were normlized y the vrine stiliztion normliztion (VSN) method 9, nd sttistis were nlysed with liner models s implemented in the limm Bioondutor pkge 6. For sttistil nlysis of other experiments, dt were nlysed y unpired t-test or one-smple t-test s stted in eh se nd the P-vlue ws lulted. Referenes. Monks, C. R. F., Freierg, B. A., Kupfer, H., Slky, N. & Kupfer, A. Threedimensionl segregtion of suprmoleulr tivtion lusters in T ells. Nture 9, 8 86 (998).. Dustin, M. L. A novel dptor protein orhestrtes reeptor ptterning nd ytoskeletl polrity in T-ell ontts. Cell 9, (998).. Kupfer, A. & Dennert, G. Reorienttion of the mirotuule-orgnizing enter nd the Golgi pprtus in loned ytotoxi lymphoytes triggered y inding to lysle trget ells. J. Immunol., (98).. Snho, D. et l. Regultion of mirotuule-orgnizing enter orienttion nd tomyosin ytoskeleton rerrngement during immune intertions. Immunol. Rev. 89, 8 97 ().. Huse, M., Lillemeier, B. F., Kuhns, M. S., Chen, D. S. & Dvis, M. M. 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Cutting edge: dynmi redistriution of tetrspnin CD8 t the entrl zone of the immune synpse in oth T lymphoytes nd APC. J. Immunol. 69, (). 9. Huer, W., von Heyderek, A., Sultmnn, H., Poustk, A. & Vingron, M. Vrine stiliztion pplied to mirorry dt lirtion nd to the quntifition of differentil expression. Bioinformtis 8 (Suppl ), S96 S (). 6. Smyth, G. K. Liner models nd empiril yes methods for ssessing differentil expression in mirorry experiments. Stt. Appl. Genet. Mol. Biol., Artile (). Aknowledgments We re grteful to Dr J. Mssgué nd Dr F. Pérez-Vizíno for providing regents. We thnk Dr M. Yñez-Mó, Dr E. Veig, Dr H. de l Fuente, F Bixuli, Dr V. Roh- Perugini, Dr M. Izquierdo, Dr N. Mrtin-Cofrees nd Dr A Cruz-Adli for their invlule ontriution to this work. We lso thnk the CNIC Genomis nd Cellomi Units for tehnil support nd thnk Simon Brtlett for help with English editing. This work ws supported y grnts SAF-8-6, INSINET 96 CAM, Red RECAVA RD6/- nd FIPSE 668/7. The CNIC is supported y the Spnish Ministry of Helth nd Consumer Affirs nd the Pro-CNIC Foundtion. M.M. ws supported y the Instituto de Slud Crlos III nd C.G.-V. y the Comunidd de Mdrid. Author ontriutions M.M. nd F.S.-M. oneived nd plnned the study. M.M. nd C.G.-V. designed the experiments; M.M. isolted smples for rrys, nd performed GFP nd mirna trnsfer experiments nd onfol mirosopy ssys. C.G.-V. generted stle lones, nd performed infetions for shrna silening, onjugte experiments, western lot nlysis nd UTR luiferse experiments. C.V.-B. performed mirna trnsfer experiments, reverse trnsription PCR ssys nd western lot nlysis. F.S.-C. nlysed mirorry dt; S.G., M.A.G. nd A.B. ontriuted mterils/nlytil tools nd sientifi disussion; M.M., C.G.-V. nd F.S.-M. wrote the pper. Additionl informtion Aession odes: The mirorry dt hve een deposited in NCBI s Gene Expression Omnius nd re essile through GEO Series ession numer GSE7997. Supplementry Informtion ompnies this pper t ntureommunitions Competing finnil interests: The uthors delre no ompeting finnil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to ite this rtile: Mittelrunn, M. et l. Unidiretionl trnsfer of mirornaloded exosomes from T ells to ntigen-presenting ells. Nt. Commun. :8 doi:.8/nomms8 (). Liense: This work is liensed under Cretive Commons Attriution-NonCommeril- NoDerivtive Works. Unported Liense. To view opy of this liense, visit retiveommons.org/lienses/y-n-nd/./ nture ommunitions :8 DOI:.8/nomms8 Mmilln Pulishers Limited. All rights reserved.