Traffic 2011; 12: 1659–1668 © 2011 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2011.01225.x

Review

Exosome Secretion: Molecular Mechanisms and Rolesin Immune Responses

Angelique Bobrie1,2, Marina Colombo1,2,3,

Graca Raposo1,3,∗ and Clotilde Thery1,2,∗

1Institut Curie, Paris, France2INSERM U932, Paris, France3CNRS UMR144, Paris, France*Corresponding authors: Clotilde Thery,clotilde.thery@curie.fr and Graca Raposo,graca.raposo@curie.fr

Exosomes are small membrane vesicles, secreted by

most cell types from multivesicular endosomes, and

thought to play important roles in intercellular communi-

cations. Initially described in 1983, as specifically secreted

by reticulocytes, exosomes became of interest for immu-

nologists in 1996, when they were proposed to play a role

in antigen presentation. More recently, the finding that

exosomes carry genetic materials, mRNA and miRNA, has

been a major breakthrough in the field, unveiling their

capacity to vehicle genetic messages. It is now clear that

not only immune cells but probably all cell types are able

to secrete exosomes: their range of possible functions

expands well beyond immunology to neurobiology, stem

cell and tumor biology, and their use in clinical applica-

tions as biomarkers or as therapeutic tools is an extensive

area of research. Despite intensive efforts to understand

their functions, two issues remain to be solved in the

future: (i) what are the physiological function(s) of exo-

somes in vivo and (ii) what are the relative contributions

of exosomes and of other secreted membrane vesicles

in these proposed functions? Here, we will focus on the

current ideas on exosomes and immune responses, but

also on their mechanisms of secretion and the use of this

knowledge to elucidate the latter issue.

Key words: antigen presentation, immune responses,

intracellular trafficking, RNA, secreted membrane vesicles

Received 11 April 2011, revised and accepted for pub-

lication 3 June 2011, uncorrected manuscript published

online 6 June 2011, published online 30 June 2011

In pluricellular organisms, cells communicate with eachother via extracellular molecules, such as nucleotides,lipids, short peptides or proteins. These molecules arereleased extracellularly by cells and bind to receptorson other cells, inducing intracellular signaling and modi-fication of the physiological state of the recipient cells.In addition to these single molecules, eukaryotic cellsalso release membrane vesicles into their extracellular

environment. Vesicles contain numerous proteins, lipidsand even nucleic acids, and can affect the cells thatencounter these structures in much more complex ways.Although known to exist for several decades, for instancein blood where they are generally called ‘microparticles’ orin seminal fluid where they are called ‘prostasomes’ (1),membrane vesicles have long been thought to be specificto a unique organ, or to represent only cell debris, or signsof cell death. Their potential role as general intercellularmessengers has become a scientific hypothesis withinthe past decade.

Exosomes represent a specific subtype of secreted mem-brane vesicles (reviewed in 2,3). Exosomes are formedinside the secreting cells in endosomal compartmentscalled multivesicular bodies (MVBs). Endosomes aregenerally considered as an intermediate compartmentbetween the plasma membrane, where endocytosis takesplace, and lysosomes where degradation occurs. About25 years ago the groups of Stahl (4) and of Johnstone (5)described, by very elegant pulse-chase and electronmicroscopy experiments, that in reticulocytes undergoingmaturation into red blood cells, multivesicular endosomescould fuse with the plasma membrane rather than withlysosomes, and release their content extracellularly. In1987, the term ‘exosomes’ was proposed to designatethe extracellularly released intraendosomal vesicles (6).Initially described as a means to extrude obsolete compo-nents by a very specific cell type, exosomes remained min-imally investigated for the following 10 years. In addition,most cell biologists remained skeptical about the actualexistence of this ‘weird’ secretion pathway, and were con-vinced that exosomes were merely membrane fragmentsartificially released upon in vitro cell handling. Owing tothe original findings that they could stimulate adaptiveimmune responses (7,8), renewed interest in exosomesarose in the immunology field. Nowadays, exosomes havebeen described in mammals and invertebrates, and appearto be involved in many different processes.

Exosomes and Other Secreted MembraneVesicles

Large-scale protein analyses of exosomes secreted bydendritic cells (DCs) and B lymphocytes (9–11), and thenon numerous other different sources of exosomes [com-piled in Ref. (12)], confirmed that exosomes representa specific subcellular compartment and are not randomcell debris. Exosomes do not contain a random array ofintracellular proteins, but a specific set of few protein

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families arising from the plasma membrane, the endo-cytic pathway and the cytosol. Exosomes contain limitedamounts of proteins from other intracellular compart-ments (nucleus, endoplasmic reticulum, Golgi apparatus),and are clearly different from membrane vesicles releasedby apoptotic cells (9). These observations establish thatexosomes are actively secreted by live cells, and supporttheir proposed intraendosomal origin. The limitation ofproteomic studies, however, is that they do not providequantitative information on the level of each protein iden-tified in exosomes, and thus on the major versus minorconstituents of these vesicles: it is important to stressthat isolated vesicles meet the criteria of exosomes whenquantitatively characterized as bearing enriched amounts(as compared to the secreting cells) of some specificmarkers, especially those of endosomal origin [CD63,major histocompatibility complex (MHC) class II, etc.].

The mean size of exosomes, 40 to 100 nm in diameter,corresponds to that of the internal vesicles of MVBs fromwhich they originate. Exosomes are commonly purified byserial steps of centrifugation and ultracentrifugation (7),and are recovered in 100 000 × g pellets (13). Some cellsalso release other types of membrane vesicles in theirenvironment, which are generated by budding at theplasma membrane toward the outside of the cell. Thesize of these vesicles varies between 50 nm and 1 μmin diameter. The terminology used to name these vesi-cles is wide, including ‘ectosomes’ (14), ‘shed vesicles’ or‘microvesicles’ (this latter term has also been used morewidely for any vesicles whether intra- or extracellular,and whatever their intracellular origin). In addition, vesi-cles budding from subdomains of the plasma membraneof T- and erythroleukemia cell lines, spontaneously orupon human immunodeficieny virus (HIV) gag or Nef pro-tein expression (15,16), or after cross-linking of surfacereceptors (17) have also been called exosomes. Thesevesicles are enriched in classical markers of exosomes(CD63, CD81), display similar density on sucrose gradi-ents and size by electron microscopy, and thus cannotbe distinguished from endosome-derived classical exo-somes. It is unclear yet whether this so-called directpathway of exosome generation is used by other celltypes, and relative proportion of endosome versus plasmamembrane-derived exosomes may also differ in differentcells. The release of large plasma membrane-derived vesi-cles is quickly induced after stimuli, such as fresh fetal calfserum on tumor cells (18), or complement deposition orrise in intracellular Ca2+ in neutrophils (19). The larger vesi-cles are generally purified by ultracentrifugation at a lowerspeed than for the exosomes (i.e. 10 000 × g), but giventheir heterogeneity, the smallest of them are not pelletedat this speed and will instead be copurified with exosomesat 100 000 × g. Importantly, re-examination of the patternof exosomal proteins after floatation on sucrose gradientsshows that different proteins can be differently enrichedin single fractions. The previously defined characteristicsof exosomes, displaying a density of 1.13–1.19 g/mL insucrose (13), could be reinterpreted and possibly lead to

definitions of subpopulations of vesicles according to anarrower range of densities.

Thus far, proteomic studies of microvesicles or ectosomeshave not been as extensive as for exosomes, but theyare underway and they will better our understanding ifand how specific molecules are targeted to these vesi-cles. While comparative studies of exosomes and othervesicles released by the same cells have very recentlybeen performed, understanding the functional specificityof each type of vesicles will certainly be feasible in thenext few years.

One should mention, however, that in many currently pub-lished studies, no effort is made to discriminate betweenthe different types of vesicles: as stressed above, merepurification by differential ultracentrifugation is not suf-ficient to qualify as exosomes, and a combination ofquantitative protein composition, morphological (electronmicroscopy) and physical (floatation on sucrose gradients)criteria should be provided for definite characterization. Asdifferent laboratories use different criteria on their experi-mental models, a consortium of several groups worldwideis currently trying to establish a consensus on modes ofpurification, and required characteristics for future exo-some research, which should hopefully be published inthe next few months. In the meantime, readers can referto our view of these questions in our detailed methodsarticle (13).

Intracellular Mechanisms of ExosomeBiogenesis and Secretion

To address the functions of exosomes, attempts tospecifically inhibit their secretion have been repeat-edly performed. Chemical inhibitors of various molecules[inhibitors of sphingomyelinase (20), of Na+/H+ and Na+/Ca2+ channels (21) or of H+ pump (22)] have beenreported to decrease exosome secretion in the modelcell lines under study. All these inhibitors, however,act on molecules with pleiotropic functions within thecell, and induce major non-exosome-related changes inthe cells, which make them inadequate for the purposeintended. Therefore, specific inhibitors are still needed.Several groups are currently trying to identify such tools,especially by means of deciphering the molecular mecha-nisms involved in the formation of intracellular vesicles ofMVBs and in the fusion of these compartments with theplasma membrane (Figure 1). A consensus has not yetbeen reached on these mechanisms.

Formation of intraluminal vesicles of MVBs, involved inthe silencing and degradation of endocytosed receptors,is known to require ubiquitination of the cytosolic tail ofthe receptors. This post-translational modification leads tosequential interaction of multisubunit protein complexes,collectively designed as the ESCRT (endosomal sortingcomplex required for transport) machinery, that targets the

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Figure 1: Intracellular molecules involved in exosome biogenesis and secretion. Exosomes originate from internal multivesicularcompartments called MVBs which are also late endosomes (LEs). Formation of the internal vesicles of MVBs has been shown to requireESCRT proteins, tetraspanins and the lipid LBPA, but the role of all these molecules in exosome biogenesis is still unclear. The lipidceramide has also recently been shown to allow formation of internal vesicles and to be required for exosome secretion. Several Rabproteins, RAB11, RAB27, RAB35, known to be involved in trafficking of vesicles between intracellular compartments, have been shownto play a role in exosome secretion. The final step required for exosome secretion, i.e. fusion of MVBs with the plasma membrane,most probably involves a complex of SNARE proteins, but the nature of this complex is not known.

receptor to the intraluminal vesicles (reviewed in 23,24).Ten years ago, the identification by proteomic analysis oftwo members of the ESCRT machinery, AIP1/Alix/Vps31and Tsg101/Vps23 (9), in DC exosomes suggested thatexosome secretion could be dependent on the ESCRTs. Afew studies have addressed the function of ESCRTs in thebiogenesis/secretion of exosomes: one report proposedthat Hrs (an ESCRT-0 member) promotes exosome secre-tion by DCs (25), whereas two others did not find anyrole for Tsg101, Alix or Vps4 in exosomal secretion of aglycosyl-phosphatidylinositol (GPI)-anchored protein (pro-teolipid protein, PLP) by oligodendroglial cells (20), or ofVps4B in release of exosomes by direct budding from theplasma membrane (17). Our own unpublished results, bycontrast, show that inhibition of Vps4B increases secretionof exosomes (but also of soluble proteins) by MHC class II-expressing HeLa cells (M. C., G. R. and C. T., unpublishedobservations). It is therefore still unclear whether the dif-ferent members of the ESCRT machinery are involvedat any given step of the exosome biogenesis process,and it may also depend on the cellular and/or intracellu-lar origin of the exosomes studied. On the other hand,ubiquitinated proteins are found in DC exosomes (26), butonly soluble proteins from the lumen of exosomes turnedout to be ubiquitinated, suggesting that they arose fromactivity of the cytosolic ubiquitination rather than of theESCRT machinery. In addition, targeting of transmem-brane proteins, such as MHC class II molecules, to thesevesicles does not require ubiquitination and probably doesnot rely on the ESCRT machinery. Targeting of MHC classII to DC exosomes may rather require their sequestration

in lipid domains enriched in the tetraspanin CD9 (27). Inoligodendroglial cells, secretion of PLP in association withexosomes was shown to require a lipid (ceramide) andthe enzyme responsible for its formation (neutral sphin-gomyelinase) (20). Another lipid, lysobisphosphatidic acid(LBPA), has been shown to allow formation of intralumi-nal vesicles of MVBs for eventual degradation (28), butits involvement in exosome formation has never beenreported. In melanocytes, sequestration of the preme-lanosomal protein Pmel17 in internal vesicles of MVB pre-cursors of melanosomes also depends on a luminal deter-minant and not on ESCRT function (29). Recent studiesindicate that CD63, a tetraspanin known to be enriched inMVBs and used as a hallmark of exosomes, is instrumen-tal in formation of the internal vesicles (G. Van Niel, G. R.et al., in revision). Altogether, these observations suggestthat there are certainly subpopulations of MVBs using dif-ferent machineries for their biogenesis and also leading todifferent types of exosomes. At the current state of theart, it still cannot be excluded that depending on the celltype and/or the conditions (mature/immature, stimulated/unstimulated, etc.) different machineries may be required.

Once formed, MVBs must fuse with the plasma mem-brane to release their content and secrete exosomes.Intracellular trafficking and fusion of compartments classi-cally involve small GTPases of the Rab family. Here also,different molecules have been described in different cells:RAB11 is required for Ca2+-induced exosome secretion bythe K562 erythroleukemia cell line (30), Rab35 is involvedin secretion of PLP-enriched exosomes by oligodendroglial

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cells (31), and we have shown that RAB27A and RAB27Bplay complementary roles in spontaneous secretion ofMHC class II-bearing exosomes by HeLa-CIITA cells (32).As Rab27 is known to play a role in regulated exocytosisof lysosome-related organelles, our work shows that, inHeLa cells, exosomes are of endosomal origin. It remainsto be determined whether these different Rabs act at dif-ferent steps of exosome biogenesis and secretion or areused differently by distinct cell types. It is also possiblethat the secreted vesicles analyzed in the different stud-ies come from different intracellular compartments (alongthe endocytic pathway or even outside this pathway). Ofnote, when analyzing the respective roles of Rab27a andRab27b in exosome secretion by different mouse tumors,we observed variations in the expression of each isoform,and consequent differences in the amount and possiblenature of exosomes secreted when either one or the otherRab27 was extinguished (A. B., C. T., unpublished obser-vations). In any case, targeting one or the other of theseRab proteins to affect exosome secretion will require care-ful evaluation of the consequences (in terms of exosomes,but also other secretions) in each different model systemanalyzed. In addition, the final step of fusion of MVBs withthe plasma membrane for exosome secretion probablyinvolves a specific combination of SNARE proteins. Thiscombination has not yet been identified, and althoughthe SNARE machinery involved in regulated secretionof specialized lysosomes is characterized (33), it is prob-ably not identical to the SNARE complex(es) involvedin plasma membrane fusion of exosome-containingMVBs.

Finally, similar cell biology studies on the generation ofother secreted membrane vesicles have to be performedto allow for proper discrimination of the roles as messen-gers of the different types of vesicles. Other approaches,for instance interfering with specific targeting of physiolog-ically active components of exosomes and/or other vesi-cles, would be another means to address the physiologicalfunctions of exosomes, and are currently underway.

Exosomes and Genetic Materials

In the last 4 years, the presence of an unexpected cargoon exosomes has opened up new avenues in the field. In2007, the group of Lotvall reported the presence of mRNAand miRNA in the cytosolic moiety of exosomes secretedby mast cells (34). In vitro, using large amounts of con-centrated exosomes, they showed that some selectedmRNAs present in exosomes could be translated intoproteins in target cells, thus suggesting a transfer ofgenetic information by exosomes. Interestingly, not allmRNAs present in a cell end up in exosomes, and thereis apparently a specificity of targeting of some mRNAsequences into the released vesicles (34), thereby refutingthe idea that mRNA in exosomes results from a randomcontamination of secreted vesicles by mRNA releasedextracellularly by dying cells. However, it is still unclear,

from the few published studies (which often analyzemixed populations of exosomes and larger microvesicles),whether one can, like for exosomal proteins, find a setof exosomal mRNAs that would be consistently targetedto exosomes of any cell type, possibly in addition to celltype-specific mRNA sequences. Understanding the mech-anisms of mRNA targeting to these vesicles should openthe way to understand the reason and the function of RNAdelivery to secreted vesicles. Small non-coding RNAs,such as miRNA, are also found in exosomes from mastcells (34). Exosomes secreted by EBV-infected B cellswere shown to contain miRNA from the virus and couldaffect the expression of known viral miRNA-target genesonce captured by monocytes (35). Finally, a very recentstudy has shown that transfer of exosomes containing anmiRNA and inhibition of reporter target genes can occurfrom a T cell to a DC, but only at the immune synapse uponspecific MHC–T cell receptor cognate interaction (36). As,conversely, cognate DC–T-cell interaction promotes exo-some secretion by DCs (27), these studies show that theexchange of information via exosomes between DCs andT cells occurs both ways. It is still too early, though, tostate whether such miRNA transfer via exosomes cantake place in physiological situations, but this idea is elic-iting considerable interest. It is also still not clear whetherspecific miRNA sequences, rather than the whole set ofintracellular miRNAs, are targeted to exosomes, becausethe approaches used to characterize exosome-associatedmiRNA are too diverse to be comparable between differ-ent studies. To address these questions, future studiesare needed to carefully assess that the miRNA and mRNAcopurified with exosomes are actually enclosed in exo-somes, for instance by showing resistance to RNAsedigestion and flotation on sucrose gradients: such controlshave been only performed in some published reports, butnot all.

mRNA and miRNA have also been described in microvesi-cles (or mixed exosome/microvesicle preparations)released by tumors or embryonic stem cells (37–39),making it important to compare sequences of RNA tar-geted to either type of vesicles and determine whetherthere is a distinct targeting to each of them. The recentobservations that miRNA, depending on their sequences,can be released extracellularly by cultured cells eitheras free miRNA or encapsulated into exosomes or intolarger secreted membrane vesicles (40), and that miRNAsare present in biological fluids either as complexes withthe protein Ago2, or inside microvesicles (41), suggests aselectivity of miRNA targeting to vesicles. It will be cru-cial to identify the different sequences and determine therespective functions of free and vesicle-encapsulated RNAas found in biological fluids. Finally, a recent publicationshows the presence of short DNA sequences of retro-transposons in exosomes and/or microvesicles secretedby glioblastomas (42).

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Exosomes and Immune Responses

Immunologist’s interest in exosomes came from thediscovery in 1996 (7) that cells of the immune system,Epstein-Barr virus-transformed B lymphocytes, were alsoable to secrete exosomes by fusion of MVBs withthe plasma membrane. Exosomes secreted by thesecells harbor MHC class II dimers bound to antigenicpeptides, molecules essential for the adaptive immuneresponse. These exosomes were also shown to presentthe MHC–peptide complexes to specific T cells, sug-gesting that they could play a role in adaptive immuneresponses. Two years later, the groups of Raposo,Amigorena and Zitvogel (8) analyzed DCs, the immunecells that initiate adaptive immune responses by present-ing MHC–peptide complexes to naïve T lymphocytes.They showed that DCs also secrete exosomes bearingfunctional MHC class I–peptide complexes, which couldpromote induction of CD8+ T-lymphocyte-dependent anti-tumor immune responses in mice in vivo. These resultsset the basis for the hypothesis that exosomes could playactive roles in intercellular communications, at least in theimmune system, and prompted the first attempt at usingthem in clinic as a new type of anti-cancer therapy inhumans [after phase I trials held between 1999 and 2002,a phase II trial is currently ongoing at the Gustave Roussyand Curie Institutes in France (43)].

Various analyses of T-cell activation by exosomes haveshown that exosome-borne MHC–peptide complexes candirectly bind to their cognate T-cell receptor and activateprimed CD4+ and CD8+ T cells [e.g. T-cell lines (7,44,45),memory T cells (46)] (Figure 2). By contrast, exosomesmust be captured by DCs to activate naïve T lympho-cytes: these DCs themselves do not necessarily expressthe right MHC molecules, but can present the exosomalMHC–peptide complexes to specific T cells (46,47). Thisdifference is probably due to the activated conformation oflymphocyte function-associated antigen (LFA-1) integrins

at the surface of primed (but not naïve) T lymphocytes,which allows efficient binding of interellular adhesionmolecule-1 (ICAM1)-bearing exosomes to these T lym-phocytes (45) as it does to LFA-1-expressing DCs (48). Italso probably reflects the need of cytokines secreted byDCs, in addition to the TCR-dependent signal, to activatenaïve T cells. In a recent publication, however, direct acti-vation of the sensitive ovalbumine-specific CD8+ OT-I Tcells by DC-derived exosomes was reported (49), and DCspresent in the exosome T-cell coculture decreased, ratherthan promoted, T-cell activation. In this experimental sys-tem, DCs capture exosomes and degrade exosome-borneMHC–peptide complexes to load the peptide on their ownMHC molecules.

The outcome of T-cell activation by exosomes dependson the physiological state of the cells which secrete them.For instance, exosomes secreted by mature DCs aremore efficient to induce T-cell activation in vitro thanthose of immature DCs (44,50,51), and induce in vivoeffector T-cell (50) and antibody responses (52). Theseimmune effects can be used, for instance, in the contextof anti-cancer therapies (53). By contrast, exosomessecreted by immature DCs can only induce effector anti-tumor responses when coinjected with adjuvants (54) orpreloaded on recipient DCs (55). When injected alone,exosomes secreted by immature DCs (56), or by DCs sub-jected to immunosuppressive treatments or modified toexpress immunosuppressive cytokines (57,58), promoteinstead tolerogenic immune responses, and could poten-tially be useful as treatments of autoimmune diseases.

Exosomes also carry antigens from the cells from whichthey originate, antigens which, after exosome captureby DCs, can be degraded into peptides and associ-ated to MHC molecules for eventual presentation toT lymphocytes. For instance, exosomes secreted bypathogen-infected cells, such as Mycobacterium tubercu-losis- or Mycobacterium bovis-infected macrophages (59),

Figure 2: Proposed antigen-present-

ing functions of exosomes. Exosomessecreted by antigen-presenting cells(APCs) bear MHC–peptide complexes,which can be directly recognized by pre-activated CD4+ and CD8+ T cells, butwhich must be captured and representedby DCs to activate naïve T cells. Exo-somes can also bear ligands for activationof NK cells.

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Figure 3: Proposed immunological

functions of exosomes secreted by

tumors. Exosomes secreted by tumorsdisplay many immune activities. Theycan promote immune responses (‘acti-vating effects’) by transferring antigensto DCs to allow activation of specificT cells, and exposing activating ligandsfor NK cells and macrophages, such asthe inducible heat-shock protein hsp70exposed at their surface upon stress ofthe exosome-secreting tumor cells. Con-versely, they bear various signals able toinhibit different immune cells, as listed in‘inhibitory effects’. Exosomes also carryRNA and protein cargoes which they cantransfer to cells that capture them.

or cytomegalovirus-infected endothelial cells (60), bearantigens from the pathogens and allow induction ofpathogen-specific CD4 and CD8 T-cell responses. Exo-somes secreted by tumor cells (Figure 3) also representa source of tumor antigens for capture and presentationby DCs in vitro (61). When secreted by heat-stressedtumor cells (62) or by cells expressing inflammatorycytokines (63), tumor exosomes can induce efficient anti-tumor immune responses after injection in host mice.

In summary, the proposed role of exosomes in antigen-specific immune responses is to spread antigens orMHC–peptide complexes in order to increase the numberof DCs presenting them, or to directly interact with mem-ory T cells. The outcome of this spreading dependsboth on the state of the DCs which capture exosomes(especially for exosomes from non-stressed or non-infected immature DCs or tumors) and on moleculescarried by exosomes (e.g. proinflammatory signals frommature/infected/stressed cells or immunosuppressive sig-nals from some non-stressed tumors).

On the other hand, exosomes secreted by sometumors also bear various immunosuppressive molecules(Figure 3), which can, in vitro, decrease proliferation ofCD4 and CD8 T lymphocytes (64–67) or natural killer (NK)cells (68,69), or promote the differentiation of immuno-suppressive cells such as regulatory T lymphocytes (70)or myeloid cells (69,71). In in vivo mouse models of mam-mary carcinoma and melanoma, injection of exosomesfrom cultured tumor cells promoted tumor growth andmetastasis by increasing the differentiation of inhibitorymyeloid cells and decreasing NK cell activity (69). Thein vivo net result of contradictory immune effects oftumor-derived exosomes is not yet established, andit is still unclear whether it could be related to the

heterogeneity of the exosome population (see above).By forcing secretion by tumor cells growing in vivo of anantigen on their exosomes (and possibly on other mem-brane vesicles), we obtained efficient anti-tumor immuneresponses (72). This shows that in conditions of artificialoverexpression of an antigen, the antigen-shuttle func-tion of exosomes overcomes their inhibitory effects onimmune cells. By contrast, several groups favor the ideathat in vivo secretion of exosomes by tumors promotestheir growth by inhibiting anti-tumor immune responsesor by promoting angiogenesis or migration outside thetumor bed to form metastases. This hypothesis haseven resulted in depletion of membrane vesicles fromthe blood circulation of patients as a rather bold, in ouropinion, proposed anti-cancer treatment (73). Althoughincreased amounts of exosomes bearing tumor markersare observed in cancer patients with large tumors (74), thisobservation could simply be the result of tumor expansion,rather than a sign of active involvement of the vesiclesin tumor progression. Therefore, the function of exosomesecretion by tumors is still an unresolved issue.

Other tissues and cells secrete exosomes bearingimmunosuppressive molecules. For instance, placenta-derived vesicles are found in the mother’s blood circula-tion (75) and women delivering at term displayed higheramounts of these vesicles, with higher Fas ligand (FasL)-mediated T-cell inhibiting properties than women deliver-ing preterm. Prostasomes in semen (76) and placentalexplants also secrete exosomes that inhibit NK lym-phocytes (77), possibly preventing immune attack of thefetus. Extensive analyses of other components of thesevesicles should help identify their role in the mother’stolerance to fetus. Exosomes displaying immunosuppres-sive effects on T cells are present in milk and colostrum aswell (78). Depending on the state of the host, exosomes

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(or vesicles) present in the bronchoalveolar fluid of the lungcan bear tolerizing molecules (e.g. in mice tolerized foran allergen) (79), or conversely increase proinflammatorycytokine secretion by airway epithelial cells (in sarcoidosishuman patients) (52). Tolerosomes secreted by intestinalepithelium and promoting oral tolerance have also beendescribed in rat (80), but in some other models intestinalexosomes can instead promote antigen presentation (81).Finally, the secretion of exosomes by eukaryotic parasites(Leishmania major) (82) has also been recently describedto dampen immune responses to the parasite and con-tribute to tolerance of the host.

Exosomes thus carry numerous messages betweenimmune cells themselves, or between targets andimmune cells, resulting in very pleiotropic possible func-tions [a more detailed review on all immune functions canbe found in Ref. (43)], but which (if any) of these functionsare really important in vivo remains to be elucidated.

Interaction of Exosomes with Target Cells

To perform all these functions, exosomes must inter-act with target cells in different ways. To be used as asource of antigens, they should be phagocytosed and theircontent degraded in phagosomes into peptides. Indeed,several fluorescent microscopy studies have providedevidence for the capture of these vesicles and accumu-lation in internal endocytic or phagocytic compartments,especially in phagocytic cells (83). It is important, how-ever, to keep in mind that vesicles smaller than 200 nmin diameter cannot be generally detected by conventionalfluorescent microscopy techniques, and that only electronmicroscopy allows visualization of exosomes: the reso-lution threshold of fluorescent microscopes is routinely200 nm and it is impossible to determine whether a flu-orescent dot corresponds to strongly fluorescent single50–100-nm vesicles, or to aggregates of these vesicles,or even to protein aggregates of the antibody used forstaining. Hence, the way individual vesicles interact withrecipient cells is still not known. It could involve bindingat the cell surface via specific receptors (which would beenough to present MHC–peptide complexes to primedT cells or NKG2D ligands to NK lymphocytes), internal-ization by endocytosis or micropinocytosis, and possiblyfusion with the plasma membrane or with the limitingmembrane of endocytic organelles. Labeling exosomeswith lipids whose fluorescence is quenched when con-centrated on small vesicles, but visible when diluted bymixing with a larger recipient membrane (84), has recentlyshown their fusion with recipient cells. This fusion is moreefficient in conditions of an acidic microenvironment,possibly mimicking the situation inside tumor masses.Whether such fusion occurs at the plasma membraneor in internal endocytic compartments is not clear (theauthors propose both sites), but in any case this articleprovides a strong experimental basis to explain the func-tional transfer of genetic materials between immune cells

via exosomes (34,35). It could also help explain other stud-ies showing the transfer of RNA (38) or of an oncogenicmembrane receptor (85) between tumor cells. In theselatter articles, however, the authors did not extensivelycharacterize the secreted vesicles, which probably containa mixture of exosomes and/or other membrane vesicles.

Physiological Functions of Exosomes

Membrane vesicles have been described in numerousbiological fluids, such as sperm (1), blood (serum and/orplasma) (75), milk (44) and urine (86) among others. Thesefluids may contain different types of membrane vesicles,but detection of markers of the endocytic pathway in thesevesicles indicates that they include bona fide exosomes.Moreover, vesicles with the size of exosomes, bearingMHC class II molecules and exosome markers, have beenobserved by electron microscopy at the surface of follicularDCs in human tonsils (87). Thus, exosomes are likely tobe secreted in vivo.

Despite accumulating knowledge on what exosomes (andother secreted membrane vesicles) can do in vitro, orin vivo when injected into animals, the data were obtainedwith vesicles purified and concentrated in vitro, from cellculture supernatants, or from biological fluids. The effi-ciency of the purification and quantification procedures isunknown, and it is likely that ultracentrifugation does notallow for 100% recovery of the vesicles secreted at anygiven time, and part of the secreted vesicles are unacces-sible to purification because they are recaptured by cellsrather than released in the culture medium or fluid. It istherefore very difficult to know whether the amounts ofmembrane vesicles used to observe the described effectscan correspond to physiological amounts of what can besecreted in vivo, or not. In addition, a recent study com-paring functional effects of exosomes secreted by in vitropropagated tumor cells versus tumor cells grown in vivobefore short-term in vitro culture suggests differencesbetween these two populations (88), hence possible dif-ferent properties of in vivo secreted exosomes. Indeed,the looming question in the exosome field is whether theyactually have any physiological functions in vivo. Answer-ing this question will hopefully become possible soon withthe identification of means to inhibit or increase specificallyexosome secretion and/or their content in physiologi-cally active components, without affecting secretion ofother membrane vesicles, general secretion of proteins orlipid mediators or other intracellular physiological functionssuch as apoptosis or autophagy.

Conclusion

Exosomes represent a subclass of secreted membranevesicles with numerous specific immune functions anddiverse potential applications in pathologies.

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We have focused this review on the immune system,but exosomes probably affect many other physiologicalfunctions. Exosomes are secreted by neural, epithelial,muscle and stem cells, and their range of proposedfunctions include contribution to tissue repair (exosomessecreted by mesenchymal stem cells modify host cardiactissue) (89), communication within the nervous system(exosomes are secreted by neurons, Schwann and oligo-dendroglial cells and microglia, and exosomes from eachsource can affect other neural cells) (90–92), and for-mation/transfer of pathogenic proteins responsible forneurodegeneration (such as prions, beta-amyloid peptidesand α-synuclein) (93,94). With no doubt, other systemsand functions will be deciphered in the coming years.

However, many questions remain concerning their phys-iological relevance, but the current expansion of thescientific community working on these fascinating vesi-cles will hopefully help address these issues and lead, inthe next years, to major advances in understanding theirfunctions. One major challenge will be to provide a propercomparison of the properties and functions of exosomesand other classes of secreted membrane vesicles. AnInternational Workshop on Exosomes (IWE-2011) recentlyheld in Paris, France, allowed fruitful exchange of ideasbetween long-term exosome experts and newcomers inthe field, and addressed current challenges in purificationmethods, definitions and characterizations. The next IWEwill take place in Gothenburg, Sweden, in April 2012. AFacebook page has also been set up to assist colleaguesinterested in exosomes (and other secreted membranevesicles) to exchange informations. Hopefully, these newtools will facilitate the combined efforts to address themajor issues of this field.

Acknowledgments

The authors thank Heidi Schreiber for critical reading of the manuscript,and Institut Curie, INSERM, CNRS, ANR, INCa, ARC and Fondation deFrance for funding their work. A. B. is supported by a fellowship fromFrench Ministry of Education, and M. C. by a grant from INCa to C. T.and G. R.

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