Exosomes and immune response in cancer: Friends or foes?

Ap il 2018 | Volume 9 | A icle 7301 Re iew published: 11 Ap il 2018 doi: 10.3389/ immu.2018.00730 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Ch is oph Kahle , Uni e si ä sklinikum Ca l Gus a Ca us an de Technischen Uni e si ä D esden, Ge many Re iewed by: B uce Milne Hall, Uni e si y o New Sou h Wales, Aus alia Ma in Pichle , Medizinische Uni e si ä G az, Aus ia *Co espondence: Sónia A. Melo [email p o ec ed] Special y sec ion: This a icle was submi ed o Immunological Tole ance and Regula ion, a sec ion o he jou nal F on ie s in Immunology Recei ed: 04Decembe 2017 Accep ed: 23Ma ch2018 Published: 11Ap il2018 Ci a ion: Ba osFM, Ca nei oF, MachadoJC and MeloSA (2018) Exosomes and Immune Response in Cance : F iends o Foes? F on . Immunol. 9:730. doi: 10.3389/ immu.2018.00730 exosomes and immune Response in Cance : F iends o Foes? F ancisco M. Ba os1, Fa ima Ca nei o2,3,4,5, Jose C. Machado3,4,5 and Sónia A. Melo3,4,5* 1 Facul y o Medicine o he Uni e si y o Po o (FMUP), Po o, Po ugal, 2 Depa men o Pa hology, Cen o Hospi ala de São João, Po o, Po ugal, 3 Depa men o Pa hology, Facul y o Medicine o he Uni e si y o Po o (FMUP), Po o, Po ugal, 4 Ins i u e o Resea ch Inno a ion in Heal h (i3S), Po o, Po ugal, 5 Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o (Ipa imup), Po o, Po ugal Exosomes a e a ype o ex acellula esicle whose s udy has g own exponen ially in ecen yea s. This led o he unde s anding ha hese s uc u es, a om being ine was e by-p oduc s o cellula unc ioning, a e ac i e playe s in in e cellula communi- ca ion mechanisms, including in he in e ac ions be ween cance cells and he immune sys em. The deep comp ehension o he c oss alk be ween umo s and he immune sys ems o hei hos s has gained mo e and mo e impo ance, as immuno he apeu ic echniques ha e eme ged as iable op ions o se e al ypes o cance . In his e iew, we p esen a comp ehensi e, upda ed, and elucida i e e iew o he cu en knowledge on he unc ions played by he exosomes in his c oss alk. The oles o hese esicles in umo an igen p esen a ion, immune ac i a ion, and immunosupp ession a e app oached as he ele an in e ac ions be ween exosomes and he complemen sys em. The las sec ion o his e iew is ese ed o he explo a ion o he esul s om he i s phase I o II clinical ials o exosomes-based cell- ee cance accines. Keywo ds: exosomes, cance , immune esponse, ex acellula esicles, clinical ials as opic iNTRODUCTiON The unde s anding o he in e cellula communica ion p ocesses is a key o he de elopmen o mechanis ic insigh s capable o explaining a wide a ie y o bo h physiological and pa hological phenomena. Di ec cell- o-cell con ac , and pa ac ine and endoc ine in e ac ions a e ela i ely well-unde s ood mechanisms ha can accoun o some o hese p ocesses. Howe e , a no el mechanism has eme ged, in ol ing he in e cellula ans e o molecula and gene ic ma e ial h ough ex acellula esicles (EVs), gaining conside able a en ion in ecen yea s (1). Ex acellula esicles a e small phospholipid bilaye esicles, eleased by all p oka yo ic and euka yo ic cells, including cance cells (2–5), which can con ain di e en ypes o RNA, p o eins, mi ochond ial DNA, and bo h single s anded DNA and double s anded DNA, spanning all ch o- mosomes (3, 6–8). The nomencla u e o EVs has been a sou ce o con usion due o he di icul ies posed by he pu i ica ion me hods necessa y o he dis inc ion o he a ious ypes o EVs, and a de ini i e classi ica ion sys em has no been achie ed ye (5, 9). Howe e , EVs can be b oadly classi ied acco ding o hei size and mode o biogenesis in o h ee sub ypes (1): mic o esicles (MV), anging be ween 50 and 1,000nm in diame e , and o igina ing by budding om plasma memb anes (2). Apop o ic bodies (AB), which a e 50–5,000nm in diame e and o igina e om cells unde going p og ammed cell dea h (3). Las , exosomes ange be ween 30 and 150nm in diame e , and o igina e om ea ly endosomes, which a e la e ans o med in o mul i esicula bodies (MVB) by o ma ion o in aluminal esicles (ILV) by budding in o he lumen, ollowed by FigURe 1 | Mechanisms used by exosomes o in luence a ge cells. (A) Di ec in e ac ion be ween su ace ecep o s on he exosome and on he a ge cell. (B) Clea age o su ace ecep o s on he exosome wi h subsequen in e ac ion be ween he ecep o agmen s and ecep o s on he a ge cell. (C) Fusion o he exosome memb ane wi h he plasma memb ane o he a ge cell wi h elease o he exosomal ca go in o he cy oplasm o he cell. (D) In e naliza ion o he whole exosome h ough phagocy osis. 2 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 usion wi h he plasma memb ane and elease o he ILV in o he ex acellula space as exosomes (2, 3, 5, 10). I is wo h no ing ha cance cells end o elease mo e exosomes han heal hy cells, which may be due o enhanced g ow h a e o a esul o s imula ion in esponse o s ess ul condi ions (11). Exosomes ha e been isola ed om all biological luids es ed so a , such as u ine, b eas milk, plasma, sali a, ce eb al spinal luid, amnio ic luid, asci es, bile, semen, b onchoal eola la age luid, and aqueous humo (12–24). Exosomes con ain collec ions o p o eins, some o which show speci ici y o he cell ype ha o igina ed hem, such as MHC class I and II p o eins, while o he s a e p esen in all exosomes, ega dless o he cell o igina - ing hem, sugges ing ha he la e a e ela ed o he common biogenesis pa hway o hese EVs. Indeed, his g oup includes endosomal p o eins, p o eins om he plasma memb ane and om he cy osol. Also, as a consequence o i s genesis, p o eins on he su ace o exosomes ha e he same o ien a ion as he one in hei cell o o igin (2, 5, 25). Exosomes a e also packed wi h di e en ypes o nucleic acids, including DNA in some cases (6, 8, 26), bu mos ly small RNAs, such as ibosomal RNA, ans e RNA, miRNA (mic oRNAs), and messenge RNA (mRNA), ha a e selec i ely loaded in o he esicles. The mechanisms behind he en ichmen in hei ca go a e s ill a om ully unde s ood, bu he p esence o “zipcode” sequence mo i s and pos ansc ip- ional changes a e some o he ways in which speci ic mRNAs and miRNAs can be packaged in o exosomes (26). Exosomes can in luence a ge cells h ough a leas ou di e en mechanisms (Figu e 1) (1): di ec con ac be ween p o eins on he mem- b ane o exosomes and on he plasma memb ane o he ecipien cell, wi h subsequen igge ing o in acellula signaling cas- cades (2). Clea age o he p o eins on he exosomes memb ane ollowed by in e ac ion be ween he p o ein agmen s and mem- b ane ecep o s on he cell (3). Fusion o he exosomes wi h he memb ane o he cell leading o elease o i s ca go (4). Finally, cellula in e naliza ion o he whole esicle by phagocy osis is also possible (2, 27, 28). Immune cells o bo h he adap i e and inna e sys ems a e an impo an componen o he umo mic oen i onmen ha mos imes p esen s pa adoxical oles in umo igenesis. On he one hand, ch onic in lamma o y s a es can se e as agen s o cance ini ia ion and p omo ion and can s imula e angiogenesis and me as asis (29). On he o he hand, he immune sys em is also esponsible o he speci ic iden i ica ion and elimina ion o neo- plas ic cells. This p ocess, known as cance immunosu eillance, is based on he exp ession o umo -speci ic an igens (30–33). The concep o immunosu eillance has been upda ed acco ding o clinical and expe imen al da a o include he no ion ha he immune sys em is no only in ol ed in an i- umo ac i i y, bu also shapes he umo i sel , esul ing in he o mula ion o he cance immunoedi ing hypo hesis. This hypo hesis iews he in e ac ions be ween he immune sys em and neoplasms as a con- inuum consis ing o a leas h ee componen s: (1) elimina ion, FigURe 2 | Pa hways o Tcell ac i a ion by dend i ic cell (DC)-de i ed exosomes. (A) Pep ide MHC class II complexes (pMHC II) can be ans e ed o DC, and hen exposed on he cell su ace (c oss-d essing). (B) Pep ides can be used by DC, which load hem on o hei own endogenous MHC class II molecules, subsequen ly p esen ing hem a hei su ace. Bo h hese pa hways (A,B) allow o he ac i a ion o CD4+ Tcells. (C) Pep ide MHC class I complexes (pMHC I) can di ec ly in e ac and ac i a e CD8+ Tcells in a DC-independen manne . 3 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 oughly co esponding o he concep o immunosu eillance. (2) Equilib ium, a s a e whe e ne umo cell ou g ow h is kep in check by he immune cells, and he e is no clinically appa en disease. (3) Las , he e is escape, when cance cells g ow immu- nologically un es ic ed due o Da winian selec ion p ocesses o he cells mos i o e ade he immune con ol (33). Exosomes ha e ecen ly eme ged as impo an modula o s o he immune esponse in he con ex o cance de elopmen . These EVs can egula e he o ma ion o he immunological synapse be ween T-cells and an igen-p esen ing cells (APC), p omo e he de el- opmen o an immune esponse, and umo -de i ed exosomes a e pa o he immunosupp essi e mechanisms h ough which cance cells inhibi immunosu eillance p ocesses in o de o p og ess and in ade (25, 34–36). This e iew will co e he cu en body o e idence ega d- ing he oles o exosomes in hese biological p ocesses, as well as summa ize hei po en ial ansla ional applica ions, bo h in he apeu ic and diagnos ic p ocedu es. Conclusions om i s phase I and II clinical ials o exosomes-based cell- ee cance accines a e also e iewed, and he in e ac ions be ween exosomes and he complemen sys em will be b ie ly app oached. We hope o p esen a clea , upda ed, and comp ehensi e insigh in o his apidly e ol ing subjec . eXOSOMeS AND ANTigeN PReSeNTATiON An igen p esen a ion cons i u es a undamen al s ep o he immune esponse. This is he p ocess h ough which APC, such as dend i ic cells (DC), mac ophages and B-cells, expose pep ide an igens, bound o MHC class I o class II molecules, o T-cells by o ming a con ac poin be ween he wo cells, e med an immunological synapse (34). MHC class II mol- ecules, speci ic o APC, a e in ol ed in he ac i a ion o CD4+ helpe T-cells h ough he p esen a ion o exogenous pep ides, in e nalized by endocy osis. MHC class I molecules, p esen in all nuclea ed cells, a e necessa y o he widesp ead su eillance o he heal h s a us by CD8+ cy o oxic Tlymphocy es (CTL) and na u al kille (NK) cells (37). They a e also in ol ed in he con ac s es ablished be ween APC and CTL, and he pep ides p esen ed by MHC class I molecules a e mos ly o an endog- enous o igin, being gene a ed by he p o easome. Howe e , he p esen a ion o exogenous an igens in complexes wi h MHC class I is also possible h ough c oss-p esen a ion, a p ocess ha is likely necessa y o he es ablishmen o an an i-cance cellula immuni y (38). Ac i a ion o CD4+ T Cells B cells elease exosomes con aining signi ican amoun s o unc ional newly o med MHC class II molecules associa ed wi h pep ides, along wi h se e al accesso y molecules, such as B7, ICAM-1, and LFA-3. This enables hem o p oduce powe - ul in i o, an igen-speci ic, MHC class II es ic ed, T helpe esponses (39). The impo ance o exosomes in he in e ac ions be ween T helpe cells and Bcells was u he elucida ed by he e idence ha he o me a e powe ul s imula o s o exosomes syn hesis and elease om he la e , namely by ac i a ion o he CD40 and IL-4 ecep o s (40–42). Bcell-de i ed exosomes also con ain MHC class I molecules, and some componen s o he B-cell ecep o (BCR), such as se e al e aspanins, CD19, and immunoglobulin, bu no CD21, a no mal componen o he BCR, p esen in high quan i ies on he su ace o s imula ed Bcells (41, 42). I is wo h no ing ha he BCR is an essen ial piece in he ac i a ion o B cells by an igens, leading o he up ake, deg ada ion, and p esen a ion o an igens (43). Exosomes wi h MHC class II-pep ide complexes, which a e de i ed om pep ide-pulsed DCs can be aken up by MHC class II-de icien DCs, which use he whole exosomal pep ide-MHC complexes o ac i a e T helpe cells, a p ocess e med c oss-d essing ha could con ibu e o ampli y he ini ial adap i e immune esponse (Figu e2) (44–46). This p ocess is as ly mo e e icien when he exosomes a e de i ed om lipopolysaccha ide (LPS)- ea ed ma u e DCs, in compa ison wi h imma u e DCs (45). These di e - ences may be accoun ed o by he signi ican ly smalle amoun s o he adhesion molecule ICAM-1 p esen in imma u e DCs- de i ed exosomes (45). Recipien DCs can also use MHC class II-pep ide complexes om APC-de i ed exosomes as a sou ce o an igens which a e loaded on o hei own MHC class II molecules (Figu e2). This was e idenced by he ac ha exosomes ca y- ing IAb-IEα52–68 complexes can ac i a e CD4+ T-cells in wild- ype animals (WT), bu no in MHC class II−/−KO mice (47) and ha Ma ilyn T-cells, when ans e ed o MHC class II de icien hos s, a e less e icien ly ac i a ed by H-Y exosomes, when compa ed wi h WT animals (48). 4 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 The na u e o he in o ma ion ansmi ed by he exosomes depends also on hei cell o o igin. Fo ins ance, MHC class II-exp essing T-84 in es inal epi helial cells elease exosomes which s ongly ac i a e Tcells, h ough he ans e o pep ide an igens o DC-gene a ed MHC class II molecules, a he han ans e ing he whole MHC class II—pep ide complex (49). On he o he hand, Di-Hwei Hsu and colleagues showed ha DC-de i ed exosomes ans e ed p e-assembled pep ide-MHC class II complexes o APC, which hen go on o ac i a e Tcells (50). APC-de i ed exosomes can also elici esponses by di ec in e ac ion be ween he exosomes and he CD4+ Tcells, in he case o ac i a ed Tcells (40). Howe e , he ac i a ion o naï e Tcells by he exosomes equi es an in e media y, such as MHC class II nega i e DC (40). Bcells can also se e as his in e medi- a y i he exosomes a e eleased om LPS- ea ed ma u e DC, bu no i hey o igina e om imma u e DC (45). Ac i a ion o CD8+ T Cells Since all nuclea ed cells exp ess MHC class I molecules, so do he exosomes sec e ed by mos cells, seemingly gi ing hem he po en ial o ac i a e CTL (7). Howe e , umo -de i ed exosomes can only ac i a e CTL clones a e p ocessing by APC exp essing he co ec MHC haplo ype (24, 51). Tumo -de i ed exosomes con ain cance - ela ed an igens ha may pe mi he ini ia ion o an immune esponse using DC as in e media ies. And e and colleagues we e able o isola e umo -de i ed exosomes om malignan asci es o pa ien s wi h melanoma, and hese we e en iched in melanoma-associa ed an igen (MAGE) ecognized by Tcells (MART-1). These exosomes, once loaded on o DC, pe mi ed in i o c oss-p esen a ion o he an igen, and ac i a- ion o a clone o CTL, which moun ed an e icien in i o an i- umo cellula esponse, as measu ed by he amoun o IFN-γ eleased, and by he p omo ion o speci ic umo cell lysis (24). Fu he mo e, mu ine umo -de i ed exosomes we e shown o con ain sha ed umo an igens which, once loaded on o human DC, can induce e icien c oss-p esen a ion o human CTL leading o in i o c oss-p o ec ion be ween di e en poo ly immunogenic mouse umo s (51). These esul s sugges ha umo exosomes, ei he collec ed om umo cell cul u es o di ec ly om malignan e usions, a e po en ial sou ces o iable an igens o he c ea ion o b oad-spec um immuno he apeu- ic echniques. Exosomes p oduced by DC can also ac i a e CD8+ Tcells indi ec ly h ough c oss-d essing (50). Howe e , APC-de i ed exosomes ha e he addi ional capaci y o di ec ly ac i a ing clones o CTL in a DC-independen manne , by c oss-p esen ing exogenous an igens (Figu e2). Saho U sugi- Kobukai and colleagues demons a ed his by showing ha exosomes om o albumin pep ide-pulsed DCs could s imula e an an igen-speci ic, MHC class I es ic ed, Tcell hyb idoma (52). Resul s om Cha lo e Admy e and colleagues u he con i med his p ocess by showing ha exosomes eleased om monocy e-de i ed DCs can p oduce an igen-speci ic esponses on au ologous CD8+ Tcells om human pe iphe al blood samples (53). They also demons a ed ha , much like he case in exosomes ac i a ion o CD4+ Tcells, his p ocess was mo e e icien when he exosomes came om LPS- ea ed ma u e DC a he han imma u e DC. This di e ence may be accoun ed o by he highe concen a ions o MHC classes I and II and co-s imula o y molecules on he ma u e DC-de i ed exosomes (53). eXOSOMeS iN iMMUNOSUPPReSSiON Exosomes a e pa o he mechanisms cance cells use o c ea e an immunosupp essi e, p o- umo igenic mic oen i onmen , which allows he disease o p og ess (54–59). These mechanisms ha e been obse ed in nume ous cance ypes and se e al di - e en media o s ha e been iden i ied. A ull unde s anding o hese p ocesses may open new a enues o no el he apeu ic modali ies, such as immune-checkpoin blockade he apies, as iable cance he apy op ions. The p oduc ion and elease o exosomes bea ing ac o s capable o inducing apop osis o he su ounding immune cells, such as Fas ligand (FasL) and galec in 9, is one o he mechanisms used by cance cells o induce immunosupp ession (57, 59, 60). Gio anna And eola and colleagues showed ha melanoma cells accumula e in a- cellula FasL, namely wi hin MVB, which in his cance ype a e cha ac e is ically popula ed by melanin- ich melanosomes (59). The melanoma cells we e subsequen ly shown o elease exosomes showing a ma ked posi i i y o FasL ha we e capa- ble o p o oking ecep o -media ed apop osis on Fas-sensi i e Ju ka Tlymphocy es (59). Exosomes induc ion o apop osis in ac i a ed CD8+ T cells was epo ed by Wieckowski and colleagues (54), and immunosupp ession media ed by human colo ec al cance (CRC) cells’ exosomes, bea ing bo h FasL and TNF- ela ed apop osis-inducing ligand (TRAIL), was demons a ed, also ac ing h ough he induc ion o apop osis o ac i a ed human Tlymphocy es (Figu e3) (58). Fu he mo e, pheno ypically simila and p o-apop o ic exosomes we e also p esen in he plasma o CRC pa ien s, demons a ing he in i o elease o hese esicles, hei po en ial ole in modula ing he hos ’s immune en i onmen , and hei possible use as p og- nos ic ma ke s (58). Tcell apop osis induced by FasL-bea ing umo exosomes is signi ican ly inhibi ed by p e iously ea - ing he Tcells wi h IRX-2, a cy okine-based biological agen (61). Ac i a ed Tcells also elease exosomes bea ing FasL and TRAIL, a p ocess dependen on PKD1/2 (62). These esicles can induce apop osis o o he ac i a ed Tcells, in o de o p e en au oimmune damage, in a p ocess called ac i a ion-induced cell dea h (AICD) (63). Pioche-Du ieu and colleagues ha e demons a ed ha Eps ein–Ba i us (EBV)-in ec ed nasopha yngeal ca cinoma (NPC) cells exp ess abundan amoun s o galec in 9 (64), a molecule shown o be an agonis o Tim-3 (65). This TH1- speci ic su ace molecule media es he apop osis o hese cells, a mechanism hough o ha e e ol ed as ano he means o p e en p olonged issue in lamma ion (Figu e 3) (65). Ke ye -Bibens and colleagues showed ha NPC-de i ed exosomes bea bo h galec in 9 and he i al la en memb ane p o ein 1 on hei su aces, also shown o ha e an in insic T-cell inhibi o y abil- i y (60). Mo e ecen ly, Klibi and colleagues ound ci cula ing galec in 9-con aining exosomes in he blood o NPC pa ien s, and epo ed ha hese exosomes had he abili y o induce apop osis in EBV-speci ic CD4+ T lymphocy es h ough he FigURe 3 | Mechanisms used by umo -de i ed exosomes o supp ess immune esponses. (A) Exosomes bea ing apop osis-inducing ligands, such as Fas ligand, TNF- ela ed apop osis-inducing ligand o , in he case o Th1cells, galec in 9, can ini ia e Tcell apop osis. Exosomes also inhibi IL-2-dependen CD8+ Tcell ac i a ion. (B) Exosomes bea ing TGF-β1 can also dis up IL-2 signaling o na u al kille (NK) cells, hus inhibi ing NKcell ac i a ion, cy o oxici y, and p oli e a ion. The exp ession o he NKG2D ecep o on NKcells can also be diminished by exosomes ca ying NKG2D ligands, hus educing NKcell abili y o ecognize malignan cells. (C) Exosomes can educe he exp ession o oll-like ecep o 4 and inhibi he ansc ip ion o MHC class II genes in dend i ic cells, h ough he ans e ence o di e en ypes o mic oRNA. 5 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 galec in 9/Tim-3 pa hway (57). Cance cells can use exosomes o enac o he mechanisms ha modula e he immune en i on- men , such as h ough he in e e ence wi h cy okine-media ed immune-ac i a ion pa hways, like hose ela ed o IL-1, IL-6, and TGF-β (66–69). Human meso helioma exosomes we e shown o al e he way in which immune cells espond o IL-2 by inhibi ing IL-2-d i en p iming o bo h cy o oxic NKcells and CD8+ Tcells, while lea ing he IL-2-dependen ac i a ion o he immunosup- p essi e T eg popula ions una ec ed (Figu e 3). These e ec s we e media ed by a memb ane-associa ed o m o TGF-β1 and show an exosomal “double hi ” mechanism ailo ed o acili a e immune e asion o umo s (66). Tumo -de i ed exosomes also p omo e T eg expansion and inc ease hei immunosupp essi e unc ions. Indeed, Wieck owski and colleagues demons a ed ha umo -de i ed exo somes, bu no DC-de i ed exosomes, induced a subs an ial expansion o he CD4+CD25+FOXP3+ T eg popula ion (54). Szajnik and colleagues u he cla i ied his p ocess by showing ha umo -de i ed exosomes lead o a dose-dependen induc- ion and p omo ion o T eg p oli e a ion. I was also shown ha incuba ing CD4+CD25neg Tcells wi h he same ype o exosomes lead o highe pe cen ages o CD4+CD25+ T cells, indica - ing ha hese esicles media e his cellula con e sion (55). Fu he mo e, pheno ypic changes we e also epo ed, namely an inc ease in he exp ession o immunosupp essi e cy o kines and cy o oxins, such as CTLA-4, FasL, TGF-β1, g anzyme B, pe o in, and IL-10 in he cells co-cul u ed wi h he umo - de i ed exosomes, bu no in he ones in con ac wi h DC-de i ed exosomes (55). Immunosupp essi e ac i i y changes we e also epo ed in his s udy, and he T eg cells p e iously incuba ed wi h he umo -de i ed exosomes showed inc eased capaci y o induce apop osis and inhibi p oli e a ion o he esponde cells. These e ec s we e also shown o be media ed by bo h pe o in and g anzyme B, as he inhibi ion o hese ac o s impai ed he exosomes abili y o induce inc eases in immuno- supp essi e unc ions (55). Wada and colleagues showed ha exosomes de i ed om malignan e usions o human pa ien s we e able o educe he dec ease in T eg numbe s and FOXP3 exp ession le els in a TGF-β1-dependen manne (70). Fu he mo e, umo -de i ed exosomes we e shown o inhibi NK cell immuni y using mu ine mamma y umo cell lines. When he es animals we e submi ed o p e- ea men wi h hese exosomes, accele a ion in he g ow h a e o implan ed umo s was obse ed, as well as a dec ease in he in i o cy o- oxic ac i i y o NKcells incuba ed wi h he exosomes (Figu e3) (67). An inhibi ion o he IL-2-media ed NKcell p oli e a ion, 6 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 la e epo ed o be media ed by TGF-β1 (66), was also obse ed (Figu e3) (67). Mu ine mamma y umo cells’ exosomes we e ound o in e - ac wi h myeloid p ecu so s in he bone ma ow, s imula ing hei elease o IL-6 and hus inhibi ing hei di e en ia ion in o DC (68). Human melanoma and CRC exosomes also ha e he capac- i y o skew he di e en ia ion o monocy es owa d a myeloid line wi h he abili y o supp ess Tcell unc ion h ough he elease o TGF-β (69). This di e en ia ion modula ion is dependen on exosomal Hsp72, on he ansc ip ion ac o S a 3 (71), and on he adap o molecule MyD88, in ol ed in oll-like ecep o (TLR) amily signal ansduc ion (72). La e s udies, howe e , sugges ha mu ine melanoma cells elease exosomes which a e capable o bo h p omo ing ma u a ion o DC and enhancing he Tcell-ac i a ing capaci y o DC (73). Cance cells can also modula e he exp ession o su ace ecep o s on immune cells, such as NKG2D and TLR4 using exosomes. This modula ion can be media ed by p o eins ca ied by exosomes, o i can be accomplished h ough ans e ence o mic oRNAs o a ge cells (56, 74–76). The NKcell NKG2D ecep- o is an impo an componen o cance immunosu eillance, as cance cells o en abe an ly exp ess NKG2D ligands, which ma k hem o NK cell-media ed des uc ion (33). Tumo exosomes bea ing NKG2D ligands and TGF-β1 a e capable o down egula ing he exp ession o NKG2D on NK cells, and educing hei cy o oxic po en ial, hus s opping hem om ec- ognizing and killing malignan cells (Figu e3) (56, 74). Zhou and colleagues showed ha human panc ea ic cance cells’ exosomes a e capable o down egula ing he exp ession o he TLR4 in DCs, h ough he ans e o miR-203, a ype o mic oRNA up egula ed in panc ea ic adenoca cinoma (Figu e3) (75, 77). I was also epo ed ha he DCs did no ha e diminished le els o TLR4 mRNA, sugges ing ha mRNA deg ada ion is no he mechanism behind his ecep o modula ion, which may occu a he ansla ional le el (75). Ding and colleagues, using he same cance model, epo ed he elease o exosomes con aining miR-212-3p which, upon ans e o DC, inhibi ed egula o y ac o X-associa ed p o ein (RFXAP), a ansc ip ion ac o o MHC class II, illus a ing ye ano he DC-supp essing mecha- nism ac ing wi h he help o exosomes (Figu e3) (76). Ying and colleagues demons a ed ha human epi helial o a ian cance cells-de i ed exosomes we e capable o p omo ing he pola i- za ion o mac ophages o he umo -associa ed immunosupp es- si e M2 pheno ype, and ha his ans o ma ion was media ed by miR-222-3p, a ype o miRNA ca ied by hese exosomes which a ge s he SOCS3/STAT3 pa hway. Fu he mo e, hey demons a ed ha hese esicles we e also capable o p omo ing he p oli e a i e and mig a o y capabili ies o o a ian cance cells (78). eXOSOMeS iN iMMUNe ACTi ATiON Apa om he al eady men ioned oles exosomes play in an igen p esen a ion, hese EVs can also con ibu e o he p o- mo ion o bo h inna e and adap i e immuni y h ough o he mechanisms. Mac ophages in ec ed wi h Mycobac e ium a ium, o example, elease exosomes con aining componen s om he bac e ial cell wall which p omo e he ac i a ion o neighbo ing unin ec ed mac ophages (79), and nume ous o he exosomes- media ed p ocesses occu du ing in ec ions wi h di e en ypes o mic oo ganisms o p omo e immune esponses (80). The ole o exosomal immune ac i a ion has also been explo ed in he con ex o au o-immune diso de s, and syno ial ib oblas s o heuma oid a h i is pa ien s we e shown o elease exosomes con aining memb ane-bound TNF-α which could inhibi AICD in CD4+ Tcells (81) and he b onchoal eola la age luid o sa coidosis pa ien s con ained ele a ed le els o exosomes which could s imula e au ologous mononuclea cells (23). Tumo cell-de i ed exosomes bea ing adju an molecules, such as hea shock p o ein (Hsp) 70, can s imula e se e al di e en componen s o bo h adap i e and inna e immune esponses, which moun an an i-cance esponse (82–85). Exosomes de i ed om ca cinoemb yonic an igen-con- aining (CEA) umo cells which we e subjec ed o hea s ess, bea Hsp 70 (82). This molecule is a po en adju an o immune ac i a ion (86, 87), and hese esicles a e capable o inducing a mo e powe ul CEA-speci ic CTL an i- umo esponse when compa ed wi h exosomes de i ed om non-hea s essed umo cells (82). Hea -shocked mouse B lymphoma cells we e also capable o p omo ing an i- umo immune esponses, mos ly media ed by CD8+ T cells, al hough CD4+ T cells we e also necessa y, and he exosomes p omo ed DC ma u a ion (83). Gas pa and colleagues demons a ed ha cells om Hsp70/Bag- 4-posi i e human panc eas and colon ca cinoma lines elease exosomes bea ing hese same molecules on hei su aces, which s imula e NKcell mig a ion and cy oly ic ac i i y (84). Vega and colleagues also showed he ole o Hsp70 posi i e exosomes in he ac i a ion o mac ophages, as measu ed by he amoun o TNF-α eleased, which was ≈260- old highe when compa ed wi h ecombinan Hsp70 s imula ion o he mac ophages (85). DC-de i ed exosomes also bea molecules capable o s imula - ing immune esponses, which ep esen s ano he a gumen in a o o he use o cell- ee DC-based cance accines, which a e explo ed in mo e de ail in he las sec ion o his e iew (88–92). Exosomes de i ed om ma u e DC we e shown o con ain impo an concen a ions o TNF-α. These exosomes we e hen demons a ed o be in e nalized by human al eola epi helial cells, which we e so s imula ed o elease in lamma o y media- o s, such as IL-8, monocy e chemo ac ic p o ein-1, mac ophage in lamma o y p o ein 1β (MIP-1β), egula ed on ac i a ion, no mal Tcell exp essed and sec e ed (RANTES), and TNF-α. These p ocesses seem o be dependen on he TNF-α cascade (88). Phase I clinical ials in ol ing he adminis a ion o DC-de i ed exosomes in pa ien s wi h ad anced non-small cell lung ca cinoma and me as a ic melanoma e idenced an ac i a ion o NKcells in abou hal he pa ien s, showing ha hese exosomes may ope a e an immune ac i a ion o bo h he inna e and adap i e di isions (89–91). This p omo ion o NKcell p oli e a ion and ac i a ion was la e shown o be dependen on he exp ession o memb ane-bound unc ional NKG2D ligands and IL-15Rα, and he inocula ion o DC exosomes was also capa- ble o es o ing he le els o NKG2D on ci cula ing NKcells o ad anced melanoma pa ien s and o inducing umo eg ession in mice (92). 7 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 eXOSOMAL iNTeRACTiONS wiTH THe COMPLeMeNT SYSTeM AND OPSONiNS The complemen sys em, pa o he phylogene ically ancien inna e immune sys em, se es as an unspeci ic ecognize o any in ading pa hogens. I is capable o igge ing he ac i a- ion o he adap i e immune esponse, di ec ly ca alyzing he des uc ion o cells by o ming he memb ane a ack complex (MAC), d i ing sys emic eac ions h ough he elease o ana- phyla oxins, as well as o opsonizing a ge cells o phagocy osis (93). All esicula s uc u es in ci cula ion a e p one o ac i a - ing he complemen sys em, leading o hei deg ada ion. This p ocess has been desc ibed o a i icial liposomes designed o he apeu ic pu poses (94–96) ha , despi e no being di ec ly an igenic, a e capable o ac i a ing he complemen sys em in an an ibody-independen manne h ough elec os a ic in e ac- ions wi h complemen p o eins (97). Hos cells a e na u ally p o ec ed agains he ac i a ion o he au ologous complemen sys em h ough he exp ession o memb ane-bound molecules which inhibi i , such as CD59 which p e en s he o ma ion o he MAC (98, 99) and CD46 and CD55 which ac syne gis ically o s op he o ma ion and deposi ion o C3b and C5b (100, 101). APC-de i ed exosomes, o med in an igen-p ocessing in acel- lula compa men s, a e associa ed wi h an igenic pep ides and should, he e o e, be pa icula ly p one o an ibody-binding and complemen -media ed des uc ion (97). Howe e , Clay on and colleagues demons a ed he exp ession o bo h CD55 and CD59, bu no o CD46, on exosomes o igina ed om human monocy e-de i ed dend i ic cells and cells o B lymphocy e o igin, which we e unc ional in he in i o inhibi ion o complemen -media ed lysis (97). An opsonin can be de ined as a molecule ha binds an igens, ma king hem o phagocy osis. Nume ous molecules can ac as opsonins, including an ibodies and some membe s o he complemen sys em. Milk a globule-EGF- ac o 8 (MFG-E8), a p o ein commonly ound on human milk a globules, was e idenced o ac as an opsonin by binding o phospha idylse - ine on he su ace o dying cells, hus p e en ing he de elop- men o au oimmune diseases by accumula ion o apop o ic cells, which can unde go seconda y nec osis and elease oxic media o s (102, 103). Miksa and colleagues demons a ed ha exosomes de i ed om imma u e DC, bu no ma u e DC, ca ied MFG-E8 and we e able o es o e he e ec i e clea ance o apop o ic cells in sep ic a models, hus supp essing he p o- in lamma o y esponse and p o iding p o ec i e e ec s in he con ex o sepsis (104). This p o ided a demons a ion o he ole o exosomes in p esen ing opsonins (104). CeLL-FRee CANCeR ACCiNeS Dend i ic cells a e he mos e icien cells a p esen ing an igens and a e he only APC capable o ac i a ing naï e Tcells and ini i- a ing he adap i e immune esponse (105). Indeed, i we in e p e cance immunosu eillance as a cycle o s epwise e en s leading o he e ec i e killing o cance cells by Tcells, he cance -immuni y cycle, DC cap u ing and p ocessing o umo neoan igens ac s as he i s s ep, a p ocess which is dependen on he p esence o ce ain molecula signals, such as p o-in lamma o y cy okines, co-s imula o y ligands, molecules eleased om he dying umo cells, and gu mic obiome p oduc s (106). I is hen unde s and- able ha e icien DC-based cance accines ha e been long sough a e , and some encou aging esul s using hese echniques ha e al eady been ob ained, such as wi h he use o Sipuleucel- T Immuno he apy o he ea men o cas a ion- esis an p os- a e cance (107). Howe e , he widesp ead use o DC-based can- ce accines p esen s some impo an limi a ions (108, 109). The use o DC-de i ed exosomes (o en e e ed o in he li e a u e as Dexosomes o simply Dex) cance accines has ecen ly eme ged as an al e na i e which may be capable o o e coming some o hese di icul ies. Fi s , Dex molecula composi ion is easie o de e mine, hus acili a ing he s ic e de ini ion o quali y con ol pa ame e s (46). Dex a e also mo e abundan in pep ide- MHC class II complexes allowing o highe yields (46, 109). Dex, when compa ed wi h DC, also p esen a g ea ad an age du ing long- e m s o age, because hey can sa ely be ozen o up o 6mon hs (109). Adding o hese ad an ages, he immunosup- p essi e umo mic oen i onmen is o en esponsible o inhib- i ing e icien an igen p esen a ion and Tcell s imula ion by DCs, which should no a ec Dex (110, 111). Finally, Dex do no pose mos o he isks in ol ed in he adminis a ion o iable cells, such as he de elopmen o immune dys unc ion, o mic o ascu- la occlusions (112). The unde s anding o dexosomes’ iabili y as immuno he apeu ic agen s depends on he deep comp ehension o hei molecula composi ion. The memb anes o Dex con ain p o eins in ol ed in an igen p esen a ion and Tcell ac i a ion, such as MHC classes I and II and co-s imula o y molecules, like CD86 (B7-2) (113, 114). Molecules in ol ed in Dex a ge ing and docking o ecep o cells, such as ICAM-1, MFG-E8, and membe s o he e aspanin amily o p o eins, such as CD9 and CD81 a e also p esen in Dex (113–116). Mo elli and colleagues desc ibed he mechanisms esponsible o he a ge ing o Dex o DC in e naliza ion, which was shown o be calcium and empe a u e-dependen , and o ely on he p esence o ligands on he su ace o he exosomes, namely MFG-E8, phospha i- dylse ine, CD11a, CD54, CD9, and CD81, and on he su ace o he ecipien DC (α /β3 in eg in, CD11a, and CD54) (116). They also p o ided in i o e idence o bone ma ow Dex up ake no only by splenic DC, bu also by splenic mac ophages and by hepa ic Kup e cells (116). Zi ogel and colleagues p o ided he p oo o concep suppo ing he in i o e icacy o Dex-based immuno he apy (111). Tumo pep ide-pulsed Dex we e capable o inducing in i o CTL p iming, umo g ow h supp ession, and umo emission. Indeed, single in ade mal adminis a ions o hese exosomes p omo ed signi ican umo g ow h supp ession a e a week and, a e 60 days, 40–60% o he animals we e umo - ee (111). Fu he mo e, hese cell- ee immuno he apeu- ic accines we e mo e e ec i e han he di ec adminis a ion o DC accines, which only accomplished a 60 h day umo - ee mice ac ion o 20%. These di e ences may be accoun ed o by he exosomes’ impe iousness o he immunomodula o y e ec s o he umo mic oen i onmen , which can impai he abili y o DC o p esen an igens (111). In he pas decade, se e al clinical ials assessing he easibili y, sa e y, and e icacy o Dex-based cance accines we e pe o med, and he esul s we e gene ally 8 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 encou aging. Two 2,005 phase I ials es ed his immuno he apy app oach, one in ad anced non-small cell lung cance (NSCLC) pa ien s, and he o he in me as a ic melanoma (MM) pa ien s (89, 90). In bo h ials he pa ien s ecei ed ou doses o he accine, consis ing o au ologous Dex loaded wi h se e al di e - en MAGE pep ides. The accine p oduc ion was shown o be easible, and he he apy was well ole a ed by he pa ien s, wi h jus mino g ade 1–2 ad e se e en s (89, 90). Fu he mo e, some in e es ing immunological and clinical esul s we e ob ained: one- hi d o he NSCLC pa ien s showed inc eased sys emic immune esponses agains MAGE, as demons a ed by delayed- ype hype sensi i i y eac i i y, and inc eased NK cell ac i i y was obse ed in hal o he NSCLC pa ien s analyzed and 8/13 o he MM pa ien s, hin ing ha Dex exe hei e ec s in bo h adap- i e and inna e componen s o he immune sys em. Clinically, some o he NSCLC pa ien s also appea ed o show p olonged pos -immuniza ion disease s abili y, and one o he MM pa ien s exhibi ed a mino esponse wi h disappea ance o one ou o h ee subcu aneous lesions, ha ing emained s able a e wa d o up o 24mon hs (89, 90). A mo e ecen phase II clinical ial e alua ed he use o IFN-γ-Dex, Dex de i ed om IFN-γ- s imula ed ma u e DC, as a main enance immuno he apy a e he use o i s line chemo he apy in ad anced NSCLC pa ien s (117). This s udy showed he easibili y o p oduc ion and sa e y o applica ion o IFN-γ-Dex, wi h only one ou o wen y-six o he pa ien s de eloping a g ade 3 hepa o oxici y. Rega ding he clinical ou comes, his ial did no show any objec i e umo esponse, acco ding o he esponse e alua ion c i e ia in solid umo s. Howe e , i did show ha he pa ien s wi h he longes p og ession- ee su i al (PFS) had a signi ican inc ease in NKcell unc ion a e Dex adminis a ion (117). Tumo -de i ed exosomes also wo k as an igen deli e y sys ems, capable o p e en ing umo de elopmen in a CD4+ and CD8+ Tcell-dependen manne (51). Because o his, cell- ee accines based on he use o umo -de i ed exosomes also eme ged as a possibili y. This idea, howe e , p esen ed wi h a big limi a ion, since he isola ion o umo exosomes seemed o equi e he incon enien in i o cul u e o he pa ien s’ umo cells (118). The al eady men ioned indings o And e and col- leagues explains ha malignan e usions o melanoma pa ien s a e exosomes- ich, and ha hese umo exosomes a e capable o ansmi ing umo an igens o DC, which hen go on o ac i a e umo -speci ic CTL capable o moun ing an e icien in i o an i- umo esponse which o e s a solu ion o he abo e-men ioned p oblem (24). Indeed, Dai and colleagues published a phase I clinical ial in which exosomes de i ed om he asci es o ad anced CRC pa ien s we e used as immuno he apy (118). These umo exosomes we e adminis e ed o he pa ien s in combina ion wi h g anulocy e mac ophage colony-s imula ing ac o (GM-CSF), a powe ul adju an which can p omo e he ma u a ion and unc ion o DC (119). Besides demons a ing he easibili y and sa e y o his ea men , wi h only g ade 1–2 ad e se e ec s epo ed, i was also shown ha he combina ion o umo exosomes wi h GM-CSF allowed o a mo e e icien induc ion o sys emic an i- umo immuni y and CTL esponses han he adminis a ion o he isola ed umo exosomes. Rega ding he clinical esul s, he pa ien s ea ed wi h he isola ed umo exosomes showed no he apeu ic esponse, while one pa ien wi h s able disease and one pa ien wi h a mino esponse we e obse ed in he g oup ecei ing asci es-de i ed exosomes plus GM-CSF (118). SUMMARY AND FUTURe PeRSPeCTi eS Since he i s published desc ip ions o exosomes elease om a e iculocy es (120, 121), he ield o exosomes biology g ew explosi ely, and we now know ha hese EVs, a om being a me e cellula mechanism o was e disposal, play coun less oles in in e cellula communica ion. O pa icula in e es o his e iew, exosomes we e desc ibed as key playe s in he c oss alk be ween malignan cells and he immune sys em. Indeed, we know ha exosomes can bo h be p omo e s o umo g ow h and in asion by aiding in he es ablishmen o an immunosup- p essi e mic oen i onmen and agen s a he se ice o cance immunosu eillance, by assis ing an igen p esen a ion and p omo ing e adica ion o umo cells by CD4+ (39) and CD8+ (24) Tcells and by elemen s o he inna e immune sys em, such as NK cells (92). The ansla ional applica ions o exosomes o cance he apy ha e been e ol ing apidly, wi h se e al phase I and II clinical ials e alua ing he sa e y and e icacy o exosomes-based cance accines al eady published showing p omising esul s which will wi hou a ques ion encou age he de elopmen o be e models o s udy in his a ea wi h g ea ansla ional po en ial (89, 90, 117, 118). O he he apeu ic echniques may bene i om he use o exosomes, such as he deli e y o molecules di ec ed agains spe ci ic cance a ge s. Indeed, in collabo a ion wi h o he pee s, we ha e ecen ly shown ha enginee ed exosomes show be e e icacy p o iles, when compa ed wi h a i icial liposomes, in he dis ibu ion o in e e ence RNA speci ic o oncogenic KRAS in panc ea ic cance models. This p ocess is pa ially dependen on he exp ession o CD47 on he exosomes, which allowed o hei escape om CD11b+ monocy es, and consequen ly inc eased hei hal -li e. Imp o ed up ake o he exosomes by cance cells, leading o a mo e po en an i-cance ac i i y, and imp o ed su i als we e also epo ed (122). Gi en he exosomes’ widesp ead a ailabili y in nea ly all body luids, and he p esence o molecules p o iding insigh in o he cons i u ion o he cell ha eleased he esicles, exosomes ha e also eme ged as po en ially good bioma ke s, allowing o cance p o iling and p edic ing ea men esponses (2). Indeed, esea ch in o he alue o plasma exosomal con en in e alua ing esponses o chemo he apy and p edic ing he p obabili y o elapse in acu e myeloid leukemia pa ien s has shown p omising esul s (123, 124). These echniques ake ad an age o he ac ha cance cells elease mo e exosomes han heal hy cells (11). These exci ing ecen ad ances in he ield o exosomes biology will likely b ing p o ound changes o he li es o cance pa ien s. They will pe mi us o use less in asi e ways o ob aining he necessa y in o ma ion abou he disease, and will open up new he apeu ic a enues, mo e e ec i e, and mo e indi idualized, hus minimizing he emendous side e ec s mos pa ien s s ill ha e o cu en ly endu e du ing an i-cance he apy. 9 Ba os e al. Exosomes and Immune Response in Cance F on ie s in Immunology | www. on ie sin.o g Ap il 2018 | Volume 9 | A icle 730 AUTHOR CONTRiBUTiONS SM concep ually designed he manusc ip , edi ed he manusc ip , suppo ed, and supe ised he w i ing p ocess. JM ad ised on he esea ch opic and supe ised he w i ing p ocess. FC supe ised he w i ing p ocess. FB w o e he manusc ip and de eloped he igu es. FUNDiNg The labo a o y is suppo ed by FEDER—Fundo Eu opeu de Desen ol imen o Regional unds h ough he COMPETE 2020—Ope acional P og amme o Compe i i eness and In e na ionaliza ion (POCI), Po ugal 2020, and by FCT— Fundacao pa a a Ciencia e a Tecnologia/Minis e io da Ciencia, Tecnologia e Ino acao in he amewo k o he p ojec s “Ins i u e o Resea ch and Inno a ion in Heal h Sciences” (POCI-01-0145-FEDER-007274), (PTDC/BIM-ONC/2754/2014), and (PTDC/BIM-MEC/2834/2014); and by No e Po ugal Regional P og amme (NORTE 2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (ERDF), in he amewo k o he p ojec NORTE- 01-0145-FEDER-000029. SM is suppo ed by FCT (IF/00543/2013). ReFeReNCeS 1. Raposo G, S oo ogel W. Ex acellula esicles: exosomes, mic o esicles, and iends. J Cell Biol (2013) 200(4):373–83. doi:10.1083/jcb.201211138 2. Sil a M, Melo SA. Non-coding RNAs in exosomes: new playe s in cance biology. 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