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.
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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 .
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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).
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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.
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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,

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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).
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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
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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).
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