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Keywords:

  • antigen presentation;
  • immune responses;
  • intracellular trafficking;
  • RNA;
  • secreted membrane vesicles

Abstract

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists 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 applications 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 exosomes 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.

In pluricellular organisms, cells communicate with each other via extracellular molecules, such as nucleotides, lipids, short peptides or proteins. These molecules are released extracellularly by cells and bind to receptors on other cells, inducing intracellular signaling and modification of the physiological state of the recipient cells. In addition to these single molecules, eukaryotic cells also release membrane vesicles into their extracellular environment. Vesicles contain numerous proteins, lipids and even nucleic acids, and can affect the cells that encounter these structures in much more complex ways. Although known to exist for several decades, for instance in blood where they are generally called ‘microparticles’ or in seminal fluid where they are called ‘prostasomes’(1), membrane vesicles have long been thought to be specific to a unique organ, or to represent only cell debris, or signs of cell death. Their potential role as general intercellular messengers has become a scientific hypothesis within the past decade.

Exosomes represent a specific subtype of secreted membrane vesicles (reviewed in 2,3). Exosomes are formed inside the secreting cells in endosomal compartments called multivesicular bodies (MVBs). Endosomes are generally considered as an intermediate compartment between the plasma membrane, where endocytosis takes place, and lysosomes where degradation occurs. About 25 years ago the groups of Stahl (4) and of Johnstone (5) described, by very elegant pulse-chase and electron microscopy experiments, that in reticulocytes undergoing maturation into red blood cells, multivesicular endosomes could fuse with the plasma membrane rather than with lysosomes, and release their content extracellularly. In 1987, the term ‘exosomes’ was proposed to designate the extracellularly released intraendosomal vesicles (6). Initially described as a means to extrude obsolete components by a very specific cell type, exosomes remained minimally investigated for the following 10 years. In addition, most cell biologists remained skeptical about the actual existence of this ‘weird’ secretion pathway, and were convinced that exosomes were merely membrane fragments artificially released upon in vitro cell handling. Owing to the original findings that they could stimulate adaptive immune responses (7,8), renewed interest in exosomes arose in the immunology field. Nowadays, exosomes have been described in mammals and invertebrates, and appear to be involved in many different processes.

Exosomes and Other Secreted Membrane Vesicles

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

Large-scale protein analyses of exosomes secreted by dendritic cells (DCs) and B lymphocytes (9–11), and then on numerous other different sources of exosomes [compiled in Ref. (12)], confirmed that exosomes represent a specific subcellular compartment and are not random cell debris. Exosomes do not contain a random array of intracellular proteins, but a specific set of few protein families arising from the plasma membrane, the endocytic pathway and the cytosol. Exosomes contain limited amounts of proteins from other intracellular compartments (nucleus, endoplasmic reticulum, Golgi apparatus), and are clearly different from membrane vesicles released by apoptotic cells (9). These observations establish that exosomes are actively secreted by live cells, and support their proposed intraendosomal origin. The limitation of proteomic studies, however, is that they do not provide quantitative information on the level of each protein identified in exosomes, and thus on the major versus minor constituents of these vesicles: it is important to stress that isolated vesicles meet the criteria of exosomes when quantitatively characterized as bearing enriched amounts (as compared to the secreting cells) of some specific markers, 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 from which they originate. Exosomes are commonly purified by serial steps of centrifugation and ultracentrifugation (7), and are recovered in 100 000 ×g pellets (13). Some cells also release other types of membrane vesicles in their environment, which are generated by budding at the plasma membrane toward the outside of the cell. The size of these vesicles varies between 50 nm and 1 µm in diameter. The terminology used to name these vesicles is wide, including ‘ectosomes’(14), ‘shed vesicles' or ‘microvesicles’ (this latter term has also been used more widely for any vesicles whether intra- or extracellular, and whatever their intracellular origin). In addition, vesicles budding from subdomains of the plasma membrane of T- and erythroleukemia cell lines, spontaneously or upon human immunodeficieny virus (HIV) gag or Nef protein expression (15,16), or after cross-linking of surface receptors (17) have also been called exosomes. These vesicles are enriched in classical markers of exosomes (CD63, CD81), display similar density on sucrose gradients and size by electron microscopy, and thus cannot be distinguished from endosome-derived classical exosomes. It is unclear yet whether this so-called direct pathway of exosome generation is used by other cell types, and relative proportion of endosome versus plasma membrane-derived exosomes may also differ in different cells. The release of large plasma membrane-derived vesicles is quickly induced after stimuli, such as fresh fetal calf serum on tumor cells (18), or complement deposition or rise in intracellular Ca2+ in neutrophils (19). The larger vesicles are generally purified by ultracentrifugation at a lower speed than for the exosomes (i.e. 10 000 ×g), but given their heterogeneity, the smallest of them are not pelleted at this speed and will instead be copurified with exosomes at 100 000 ×g. Importantly, re-examination of the pattern of exosomal proteins after floatation on sucrose gradients shows that different proteins can be differently enriched in single fractions. The previously defined characteristics of exosomes, displaying a density of 1.13–1.19 g/mL in sucrose (13), could be reinterpreted and possibly lead to definitions of subpopulations of vesicles according to a narrower range of densities.

Thus far, proteomic studies of microvesicles or ectosomes have not been as extensive as for exosomes, but they are underway and they will better our understanding if and how specific molecules are targeted to these vesicles. While comparative studies of exosomes and other vesicles released by the same cells have very recently been performed, understanding the functional specificity of each type of vesicles will certainly be feasible in the next few years.

One should mention, however, that in many currently published studies, no effort is made to discriminate between the different types of vesicles: as stressed above, mere purification by differential ultracentrifugation is not sufficient to qualify as exosomes, and a combination of quantitative protein composition, morphological (electron microscopy) and physical (floatation on sucrose gradients) criteria should be provided for definite characterization. As different laboratories use different criteria on their experimental models, a consortium of several groups worldwide is currently trying to establish a consensus on modes of purification, and required characteristics for future exosome research, which should hopefully be published in the next few months. In the meantime, readers can refer to our view of these questions in our detailed methods article (13).

Intracellular Mechanisms of Exosome Biogenesis and Secretion

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

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

image

Figure 1. Intracellular molecules involved in exosome biogenesis and secretion. Exosomes originate from internal multivesicular compartments called MVBs which are also late endosomes (LEs). Formation of the internal vesicles of MVBs has been shown to require ESCRT proteins, tetraspanins and the lipid LBPA, but the role of all these molecules in exosome biogenesis is still unclear. The lipid ceramide has also recently been shown to allow formation of internal vesicles and to be required for exosome secretion. Several Rab proteins, RAB11, RAB27, RAB35, known to be involved in trafficking of vesicles between intracellular compartments, have been shown to 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.

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Formation of intraluminal vesicles of MVBs, involved in the silencing and degradation of endocytosed receptors, is known to require ubiquitination of the cytosolic tail of the receptors. This post-translational modification leads to sequential interaction of multisubunit protein complexes, collectively designed as the ESCRT (endosomal sorting complex required for transport) machinery, that targets the receptor to the intraluminal vesicles (reviewed in 23,24). Ten years ago, the identification by proteomic analysis of two members of the ESCRT machinery, AIP1/Alix/Vps31 and Tsg101/Vps23 (9), in DC exosomes suggested that exosome secretion could be dependent on the ESCRTs. A few studies have addressed the function of ESCRTs in the biogenesis/secretion of exosomes: one report proposed that Hrs (an ESCRT-0 member) promotes exosome secretion by DCs (25), whereas two others did not find any role for Tsg101, Alix or Vps4 in exosomal secretion of a glycosyl-phosphatidylinositol (GPI)-anchored protein (proteolipid protein, PLP) by oligodendroglial cells (20), or of Vps4B in release of exosomes by direct budding from the plasma membrane (17). Our own unpublished results, by contrast, show that inhibition of Vps4B increases secretion of exosomes (but also of soluble proteins) by MHC class II-expressing HeLa cells (M. C., G. R. and C. T., unpublished observations). It is therefore still unclear whether the different members of the ESCRT machinery are involved at any given step of the exosome biogenesis process, and it may also depend on the cellular and/or intracellular origin of the exosomes studied. On the other hand, ubiquitinated proteins are found in DC exosomes (26), but only soluble proteins from the lumen of exosomes turned out to be ubiquitinated, suggesting that they arose from activity of the cytosolic ubiquitination rather than of the ESCRT machinery. In addition, targeting of transmembrane proteins, such as MHC class II molecules, to these vesicles does not require ubiquitination and probably does not rely on the ESCRT machinery. Targeting of MHC class II to DC exosomes may rather require their sequestration in lipid domains enriched in the tetraspanin CD9 (27). In oligodendroglial cells, secretion of PLP in association with exosomes was shown to require a lipid (ceramide) and the enzyme responsible for its formation (neutral sphingomyelinase) (20). Another lipid, lysobisphosphatidic acid (LBPA), has been shown to allow formation of intraluminal vesicles of MVBs for eventual degradation (28), but its involvement in exosome formation has never been reported. In melanocytes, sequestration of the premelanosomal protein Pmel17 in internal vesicles of MVB precursors of melanosomes also depends on a luminal determinant and not on ESCRT function (29). Recent studies indicate that CD63, a tetraspanin known to be enriched in MVBs and used as a hallmark of exosomes, is instrumental in formation of the internal vesicles (G. Van Niel, G. R. et al., in revision). Altogether, these observations suggest that there are certainly subpopulations of MVBs using different machineries for their biogenesis and also leading to different types of exosomes. At the current state of the art, it still cannot be excluded that depending on the cell type and/or the conditions (mature/immature, stimulated/ unstimulated, etc.) different machineries may be required.

Once formed, MVBs must fuse with the plasma membrane to release their content and secrete exosomes. Intracellular trafficking and fusion of compartments classically 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 by the K562 erythroleukemia cell line (30), Rab35 is involved in secretion of PLP-enriched exosomes by oligodendroglial cells (31), and we have shown that RAB27A and RAB27B play complementary roles in spontaneous secretion of MHC class II-bearing exosomes by HeLa-CIITA cells (32). As Rab27 is known to play a role in regulated exocytosis of lysosome-related organelles, our work shows that, in HeLa cells, exosomes are of endosomal origin. It remains to be determined whether these different Rabs act at different steps of exosome biogenesis and secretion or are used differently by distinct cell types. It is also possible that the secreted vesicles analyzed in the different studies come from different intracellular compartments (along the endocytic pathway or even outside this pathway). Of note, when analyzing the respective roles of Rab27a and Rab27b in exosome secretion by different mouse tumors, we observed variations in the expression of each isoform, and consequent differences in the amount and possible nature of exosomes secreted when either one or the other Rab27 was extinguished (A. B., C. T., unpublished observations). In any case, targeting one or the other of these Rab proteins to affect exosome secretion will require careful evaluation of the consequences (in terms of exosomes, but also other secretions) in each different model system analyzed. In addition, the final step of fusion of MVBs with the plasma membrane for exosome secretion probably involves a specific combination of SNARE proteins. This combination has not yet been identified, and although the SNARE machinery involved in regulated secretion of specialized lysosomes is characterized (33), it is probably not identical to the SNARE complex(es) involved in plasma membrane fusion of exosome-containing MVBs.

Finally, similar cell biology studies on the generation of other secreted membrane vesicles have to be performed to allow for proper discrimination of the roles as messengers of the different types of vesicles. Other approaches, for instance interfering with specific targeting of physiologically active components of exosomes and/or other vesicles, would be another means to address the physiological functions of exosomes, and are currently underway.

Exosomes and Genetic Materials

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

In the last 4 years, the presence of an unexpected cargo on exosomes has opened up new avenues in the field. In 2007, the group of Lötvall reported the presence of mRNA and miRNA in the cytosolic moiety of exosomes secreted by mast cells (34). In vitro, using large amounts of concentrated exosomes, they showed that some selected mRNAs present in exosomes could be translated into proteins in target cells, thus suggesting a transfer of genetic information by exosomes. Interestingly, not all mRNAs present in a cell end up in exosomes, and there is apparently a specificity of targeting of some mRNA sequences into the released vesicles (34), thereby refuting the idea that mRNA in exosomes results from a random contamination of secreted vesicles by mRNA released extracellularly by dying cells. However, it is still unclear, from the few published studies (which often analyze mixed populations of exosomes and larger microvesicles), whether one can, like for exosomal proteins, find a set of exosomal mRNAs that would be consistently targeted to exosomes of any cell type, possibly in addition to cell type-specific mRNA sequences. Understanding the mechanisms of mRNA targeting to these vesicles should open the way to understand the reason and the function of RNA delivery to secreted vesicles. Small non-coding RNAs, such as miRNA, are also found in exosomes from mast cells (34). Exosomes secreted by EBV-infected B cells were shown to contain miRNA from the virus and could affect the expression of known viral miRNA-target genes once captured by monocytes (35). Finally, a very recent study has shown that transfer of exosomes containing an miRNA and inhibition of reporter target genes can occur from a T cell to a DC, but only at the immune synapse upon specific MHC–T cell receptor cognate interaction (36). As, conversely, cognate DC–T-cell interaction promotes exosome secretion by DCs (27), these studies show that the exchange of information via exosomes between DCs and T cells occurs both ways. It is still too early, though, to state whether such miRNA transfer via exosomes can take place in physiological situations, but this idea is eliciting considerable interest. It is also still not clear whether specific miRNA sequences, rather than the whole set of intracellular miRNAs, are targeted to exosomes, because the approaches used to characterize exosome-associated miRNA are too diverse to be comparable between different studies. To address these questions, future studies are needed to carefully assess that the miRNA and mRNA copurified with exosomes are actually enclosed in exosomes, for instance by showing resistance to RNAse digestion and flotation on sucrose gradients: such controls have been only performed in some published reports, but not all.

mRNA and miRNA have also been described in microvesicles (or mixed exosome/microvesicle preparations) released by tumors or embryonic stem cells (37–39), making it important to compare sequences of RNA targeted to either type of vesicles and determine whether there is a distinct targeting to each of them. The recent observations that miRNA, depending on their sequences, can be released extracellularly by cultured cells either as free miRNA or encapsulated into exosomes or into larger secreted membrane vesicles (40), and that miRNAs are present in biological fluids either as complexes with the protein Ago2, or inside microvesicles (41), suggests a selectivity of miRNA targeting to vesicles. It will be crucial to identify the different sequences and determine the respective functions of free and vesicle-encapsulated RNA as found in biological fluids. Finally, a recent publication shows the presence of short DNA sequences of retrotransposons in exosomes and/or microvesicles secreted by glioblastomas (42).

Exosomes and Immune Responses

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

Immunologist's interest in exosomes came from the discovery in 1996 (7) that cells of the immune system, Epstein-Barr virus-transformed B lymphocytes, were also able to secrete exosomes by fusion of MVBs with the plasma membrane. Exosomes secreted by these cells harbor MHC class II dimers bound to antigenic peptides, molecules essential for the adaptive immune response. These exosomes were also shown to present the MHC–peptide complexes to specific T cells, suggesting that they could play a role in adaptive immune responses. Two years later, the groups of Raposo, Amigorena and Zitvogel (8) analyzed DCs, the immune cells that initiate adaptive immune responses by presenting MHC–peptide complexes to naÏve T lymphocytes. They showed that DCs also secrete exosomes bearing functional MHC class I–peptide complexes, which could promote induction of CD8+ T-lymphocyte-dependent anti-tumor immune responses in mice in vivo. These results set the basis for the hypothesis that exosomes could play active roles in intercellular communications, at least in the immune system, and prompted the first attempt at using them in clinic as a new type of anti-cancer therapy in humans [after phase I trials held between 1999 and 2002, a phase II trial is currently ongoing at the Gustave Roussy and Curie Institutes in France (43)].

Various analyses of T-cell activation by exosomes have shown that exosome-borne MHC–peptide complexes can directly bind to their cognate T-cell receptor and activate primed CD4+ and CD8+ T cells [e.g. T-cell lines (7,44,45), memory T cells (46)] (Figure 2). By contrast, exosomes must be captured by DCs to activate naÏve T lymphocytes: these DCs themselves do not necessarily express the right MHC molecules, but can present the exosomal MHC–peptide complexes to specific T cells (46,47). This difference is probably due to the activated conformation of lymphocyte function-associated antigen (LFA-1) integrins at the surface of primed (but not naÏve) T lymphocytes, which allows efficient binding of interellular adhesion molecule-1 (ICAM1)-bearing exosomes to these T lymphocytes (45) as it does to LFA-1-expressing DCs (48). It also probably reflects the need of cytokines secreted by DCs, in addition to the TCR-dependent signal, to activate naÏve T cells. In a recent publication, however, direct activation of the sensitive ovalbumine-specific CD8+ OT-I T cells by DC-derived exosomes was reported (49), and DCs present in the exosome T-cell coculture decreased, rather than promoted, T-cell activation. In this experimental system, DCs capture exosomes and degrade exosome-borne MHC–peptide complexes to load the peptide on their own MHC molecules.

image

Figure 2. Proposed antigen-presenting functions of exosomes. Exosomes secreted by antigen-presenting cells (APCs) bear MHC–peptide complexes, which can be directly recognized by preactivated CD4+ and CD8+ T cells, but which must be captured and represented by DCs to activate naÏve T cells. Exosomes can also bear ligands for activation of NK cells.

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The outcome of T-cell activation by exosomes depends on the physiological state of the cells which secrete them. For instance, exosomes secreted by mature DCs are more efficient to induce T-cell activation in vitro than those of immature DCs (44,50,51), and induce in vivo effector T-cell (50) and antibody responses (52). These immune effects can be used, for instance, in the context of anti-cancer therapies (53). By contrast, exosomes secreted by immature DCs can only induce effector anti-tumor responses when coinjected with adjuvants (54) or preloaded on recipient DCs (55). When injected alone, exosomes secreted by immature DCs (56), or by DCs subjected to immunosuppressive treatments or modified to express immunosuppressive cytokines (57,58), promote instead tolerogenic immune responses, and could potentially be useful as treatments of autoimmune diseases.

Exosomes also carry antigens from the cells from which they originate, antigens which, after exosome capture by DCs, can be degraded into peptides and associated to MHC molecules for eventual presentation to T lymphocytes. For instance, exosomes secreted by pathogen-infected cells, such as Mycobacterium tuberculosis- or Mycobacterium bovis-infected macrophages (59), or cytomegalovirus-infected endothelial cells (60), bear antigens from the pathogens and allow induction of pathogen-specific CD4 and CD8 T-cell responses. Exosomes secreted by tumor cells (Figure 3) also represent a source of tumor antigens for capture and presentation by DCs in vitro(61). When secreted by heat-stressed tumor cells (62) or by cells expressing inflammatory cytokines (63), tumor exosomes can induce efficient anti-tumor immune responses after injection in host mice.

image

Figure 3. Proposed immunological functions of exosomes secreted by tumors. Exosomes secreted by tumors display many immune activities. They can promote immune responses (‘activating effects') by transferring antigens to DCs to allow activation of specific T cells, and exposing activating ligands for NK cells and macrophages, such as the inducible heat-shock protein hsp70 exposed at their surface upon stress of the exosome-secreting tumor cells. Conversely, they bear various signals able to inhibit different immune cells, as listed in ‘inhibitory effects'. Exosomes also carry RNA and protein cargoes which they can transfer to cells that capture them.

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In summary, the proposed role of exosomes in antigen-specific immune responses is to spread antigens or MHC–peptide complexes in order to increase the number of DCs presenting them, or to directly interact with memory T cells. The outcome of this spreading depends both on the state of the DCs which capture exosomes (especially for exosomes from non-stressed or non-infected immature DCs or tumors) and on molecules carried by exosomes (e.g. proinflammatory signals from mature/infected/stressed cells or immunosuppressive signals from some non-stressed tumors).

On the other hand, exosomes secreted by some tumors also bear various immunosuppressive molecules (Figure 3), which can, in vitro, decrease proliferation of CD4 and CD8 T lymphocytes (64–67) or natural killer (NK) cells (68,69), or promote the differentiation of immunosuppressive cells such as regulatory T lymphocytes (70) or myeloid cells (69,71). In in vivo mouse models of mammary carcinoma and melanoma, injection of exosomes from cultured tumor cells promoted tumor growth and metastasis by increasing the differentiation of inhibitory myeloid cells and decreasing NK cell activity (69). The in vivo net result of contradictory immune effects of tumor-derived exosomes is not yet established, and it 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 an antigen on their exosomes (and possibly on other membrane vesicles), we obtained efficient anti-tumor immune responses (72). This shows that in conditions of artificial overexpression of an antigen, the antigen-shuttle function of exosomes overcomes their inhibitory effects on immune cells. By contrast, several groups favor the idea that in vivo secretion of exosomes by tumors promotes their growth by inhibiting anti-tumor immune responses or by promoting angiogenesis or migration outside the tumor bed to form metastases. This hypothesis has even resulted in depletion of membrane vesicles from the blood circulation of patients as a rather bold, in our opinion, proposed anti-cancer treatment (73). Although increased amounts of exosomes bearing tumor markers are observed in cancer patients with large tumors (74), this observation could simply be the result of tumor expansion, rather than a sign of active involvement of the vesicles in tumor progression. Therefore, the function of exosome secretion by tumors is still an unresolved issue.

Other tissues and cells secrete exosomes bearing immunosuppressive molecules. For instance, placenta-derived vesicles are found in the mother's blood circulation (75) and women delivering at term displayed higher amounts of these vesicles, with higher Fas ligand (FasL)-mediated T-cell inhibiting properties than women delivering preterm. Prostasomes in semen (76) and placental explants also secrete exosomes that inhibit NK lymphocytes (77), possibly preventing immune attack of the fetus. Extensive analyses of other components of these vesicles should help identify their role in the mother's tolerance to fetus. Exosomes displaying immunosuppressive effects on T cells are present in milk and colostrum as well (78). Depending on the state of the host, exosomes (or vesicles) present in the bronchoalveolar fluid of the lung can bear tolerizing molecules (e.g. in mice tolerized for an allergen) (79), or conversely increase proinflammatory cytokine secretion by airway epithelial cells (in sarcoidosis human patients) (52). Tolerosomes secreted by intestinal epithelium and promoting oral tolerance have also been described in rat (80), but in some other models intestinal exosomes can instead promote antigen presentation (81). Finally, the secretion of exosomes by eukaryotic parasites (Leishmania major) (82) has also been recently described to dampen immune responses to the parasite and contribute to tolerance of the host.

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

Interaction of Exosomes with Target Cells

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

To perform all these functions, exosomes must interact with target cells in different ways. To be used as a source of antigens, they should be phagocytosed and their content degraded in phagosomes into peptides. Indeed, several fluorescent microscopy studies have provided evidence for the capture of these vesicles and accumulation in internal endocytic or phagocytic compartments, especially in phagocytic cells (83). It is important, however, to keep in mind that vesicles smaller than 200 nm in diameter cannot be generally detected by conventional fluorescent microscopy techniques, and that only electron microscopy allows visualization of exosomes: the resolution threshold of fluorescent microscopes is routinely 200 nm and it is impossible to determine whether a fluorescent dot corresponds to strongly fluorescent single 50–100-nm vesicles, or to aggregates of these vesicles, or even to protein aggregates of the antibody used for staining. Hence, the way individual vesicles interact with recipient cells is still not known. It could involve binding at the cell surface via specific receptors (which would be enough to present MHC–peptide complexes to primed T cells or NKG2D ligands to NK lymphocytes), internalization by endocytosis or micropinocytosis, and possibly fusion with the plasma membrane or with the limiting membrane of endocytic organelles. Labeling exosomes with lipids whose fluorescence is quenched when concentrated on small vesicles, but visible when diluted by mixing with a larger recipient membrane (84), has recently shown their fusion with recipient cells. This fusion is more efficient in conditions of an acidic microenvironment, possibly mimicking the situation inside tumor masses. Whether such fusion occurs at the plasma membrane or in internal endocytic compartments is not clear (the authors propose both sites), but in any case this article provides a strong experimental basis to explain the functional transfer of genetic materials between immune cells via exosomes (34,35). It could also help explain other studies showing the transfer of RNA (38) or of an oncogenic membrane receptor (85) between tumor cells. In these latter articles, however, the authors did not extensively characterize the secreted vesicles, which probably contain a mixture of exosomes and/or other membrane vesicles.

Physiological Functions of Exosomes

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

Membrane vesicles have been described in numerous biological fluids, such as sperm (1), blood (serum and/or plasma) (75), milk (44) and urine (86) among others. These fluids may contain different types of membrane vesicles, but detection of markers of the endocytic pathway in these vesicles indicates that they include bona fide exosomes. Moreover, vesicles with the size of exosomes, bearing MHC class II molecules and exosome markers, have been observed by electron microscopy at the surface of follicular DCs in human tonsils (87). Thus, exosomes are likely to be secreted in vivo.

Despite accumulating knowledge on what exosomes (and other secreted membrane vesicles) can do in vitro, or in vivo when injected into animals, the data were obtained with vesicles purified and concentrated in vitro, from cell culture supernatants, or from biological fluids. The efficiency of the purification and quantification procedures is unknown, and it is likely that ultracentrifugation does not allow for 100% recovery of the vesicles secreted at any given time, and part of the secreted vesicles are unaccessible to purification because they are recaptured by cells rather than released in the culture medium or fluid. It is therefore very difficult to know whether the amounts of membrane vesicles used to observe the described effects can correspond to physiological amounts of what can be secreted in vivo, or not. In addition, a recent study comparing functional effects of exosomes secreted by in vitro propagated tumor cells versus tumor cells grown in vivo before short-term in vitro culture suggests differences between these two populations (88), hence possible different properties of in vivo secreted exosomes. Indeed, the looming question in the exosome field is whether they actually have any physiological functions in vivo. Answering this question will hopefully become possible soon with the identification of means to inhibit or increase specifically exosome secretion and/or their content in physiologically active components, without affecting secretion of other membrane vesicles, general secretion of proteins or lipid mediators or other intracellular physiological functions such as apoptosis or autophagy.

Conclusion

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

Exosomes represent a subclass of secreted membrane vesicles with numerous specific immune functions and diverse potential applications in pathologies.

We have focused this review on the immune system, but exosomes probably affect many other physiological functions. Exosomes are secreted by neural, epithelial, muscle and stem cells, and their range of proposed functions include contribution to tissue repair (exosomes secreted by mesenchymal stem cells modify host cardiac tissue) (89), communication within the nervous system (exosomes are secreted by neurons, Schwann and oligodendroglial cells and microglia, and exosomes from each source can affect other neural cells) (90–92), and formation/transfer of pathogenic proteins responsible for neurodegeneration (such as prions, beta-amyloid peptides and α-synuclein) (93,94). With no doubt, other systems and functions will be deciphered in the coming years.

However, many questions remain concerning their physiological relevance, but the current expansion of the scientific community working on these fascinating vesicles will hopefully help address these issues and lead, in the next years, to major advances in understanding their functions. One major challenge will be to provide a proper comparison of the properties and functions of exosomes and other classes of secreted membrane vesicles. An International Workshop on Exosomes (IWE-2011) recently held in Paris, France, allowed fruitful exchange of ideas between long-term exosome experts and newcomers in the field, and addressed current challenges in purification methods, definitions and characterizations. The next IWE will take place in Gothenburg, Sweden, in April 2012. A Facebook page has also been set up to assist colleagues interested in exosomes (and other secreted membrane vesicles) to exchange informations. Hopefully, these new tools will facilitate the combined efforts to address the major issues of this field.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References

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

References

  1. Top of page
  2. Abstract
  3. Exosomes and Other Secreted Membrane Vesicles
  4. Intracellular Mechanisms of Exosome Biogenesis and Secretion
  5. Exosomes and Genetic Materials
  6. Exosomes and Immune Responses
  7. Interaction of Exosomes with Target Cells
  8. Physiological Functions of Exosomes
  9. Conclusion
  10. Acknowledgments
  11. References
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