Nature Immunology
3, 1156 - 1162 (2002)
Published online: 11 November 2002; | doi:10.1038/ni854
Indirect activation of naïve CD4+ T cells by dendritic cell−derived exosomesClotilde Théry1, 3, Livine Duban1, 2, 3, Elodie Segura1, Philippe Véron1, Olivier Lantz1, 2, 3
& Sebastian Amigorena1, 31 INSERM U520, Institut Curie, 12 rue Lhomond, 75005 Paris, France. 2 Laboratoire d'Immunologie Préclinique, Institut Curie, 26 rue d'Ulm, 75005 Paris, France. 3 These authors contributed equally to this work.
Correspondence should be addressed to Sebastian Amigorena sebastian.amigorena@curie.frDendritic cells (DCs) secrete vesicles of endosomal origin, called exosomes, that bear major histocompatibility complex (MHC) and T cell costimulatory molecules. Here, we found that injection of antigen- or peptide-bearing exosomes induced antigen-specific naïve CD4+ T cell activation in vivo. In vitro, exosomes did not induce antigen-dependent T cell stimulation unless mature CD8 - DCs were also present in the cultures. These mature DCs could be MHC class II−negative, but had to bear CD80 and CD86. Therefore, in addition to carrying antigen, exosomes promote the exchange of functional peptide-MHC complexes between DCs. Such a mechanism may increase the number of DCs bearing a particular peptide, thus amplifying the initiation of primary adaptive immune responses.Exosomes are membrane vesicles that form within late endocytic compartments, which are called multivesicular bodies (MVB), and are secreted upon fusion of these compartments with the plasma membrane1,
2. Various hematopoietic and nonhematopoietic cell types secrete exosomes, including reticulocytes, B lymphocytes, platelets, mastocytes, several tumor cells, intestinal epithelial cells, T lymphocytes and dendritic cells (DCs). At present, it is unclear whether exosomes have any biological function and, if so, what these functions are.
The molecular composition of exosomes from different cellular sources has been analyzed. DC-derived exosomes contain several proteins that are potentially involved in their biogenesis, targeting and putative immunological function3,
4. Proteins that may be involved in exosome biogenesis in MVB include tsg101, several annexins, Rab GTPases and several signal transduction molecules (for example, 14-3-3, a heterotrimeric G protein and Alix). Exosomes also accumulate different members of the tetraspanin family, including CD9, CD63 and CD81. Although their precise function is uncertain, tetraspanins are enriched in membrane microdomains and form networks with several other membrane proteins5. Exosomes also bear integral and membrane-associated adhesion molecules, such as  M 2 and milk fat globule−EGF factor VIII (MFG-E8). These molecules are most likely involved in addressing exosomes to target cells6,
7. DC-derived exosomes also accumulate several proteins involved in T cell stimulation, including major histocompatibility complex (MHC) class I and II, and CD86 (a potent T cell costimulatory molecule). The heat shock protein hsc73, involved in peptide delivery to antigen-presenting cells (APCs)8, is also enriched in exosomes.
The presence of molecules in exosomes that are involved in T cell stimulation has prompted different groups to examine the immunostimulatory capacities of these vesicles. Exosome-associated MHC molecules are functional: exosomes produced by Epstein-Barr virus−transformed B cells incubated with a mycobacterial antigen stimulate MHC class II−restricted T cell clones in vitro9; and exosomes produced by tumor peptide−pulsed DCs induce T cell−dependent tumor rejection in vivo10. These observations suggest that exosomes secreted by APCs induce antigen-specific MHC-restricted T cell responses.
To analyze the mechanism of antigen-specific T cell stimulation by exosomes in vivo and in vitro, we used CD4+ T cell receptor (TCR)−transgenic T cells specific for the male antigen H-Y11. We found that exosomes secreted by male DCs induced stimulation of naïve CD4+ T cells in vivo and in vitro. In vitro DC-derived male exosomes were used by mature DCs as a source of antigen to stimulate T cells. Unexpectedly, however, male exosomes could also stimulate T cells in the presence of MHC class II−deficient DCs, which cannot represent H-Y antigens or peptide. Indeed, exosomes bearing complexes of MHC class II and H-Y peptide, in the absence of the intact antigen, also stimulated specific T cells in vitro in the presence of mature DCs. MHC class II−deficient DCs stimulated T cells with exosomal peptide-MHC (pMHC) complexes. Exosomes therefore mediate the transfer of pMHC complexes between different DC populations.
Results
Male exosomes activate T cells
To analyze antigen-specific, MHC-restricted T cell stimulation by exosomes, we used a mouse strain called Marilyn. These mice contain a monoclonal population of CD4+ T cells specific for a peptide from Dby, a male H-Y antigen, presented by I-Ab (ref. 12). Marilyn mouse lymph nodes contain 93−98% CD4+ V 6+ T cells (Fig. 1a), which are negative for CD69 and CD44 and can therefore be considered to be fully naïve T cells11 (and data not shown). Subcutaneous injection of male cells into Marilyn mice induces T cell activation in the draining lymph nodes, as shown by changes in surface expression of T cell activation markers11 (and our unpublished observations).
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Exosomes were purified from culture supernatants obtained from the growth factor−dependent DC line D113 as described3 (female exosomes) or from bone marrow−derived DCs (BMDCs) from male C57BL/6 (B6) mice (male exosomes). Exosomes were injected into the footpads of female CD45.2+ B6 mice into which carboxyfluorescein diacetate succinimidyl ester (CFSE)−stained CD45.1+ Marilyn lymph node cells had been transferred previously. Marilyn T cells represented 0.1−0.2% of the total cell population in the lymph nodes from the host (Fig. 1b). The expression of CD69 (Fig. 1b) and CD44 (Fig. 1c) in the draining lymph nodes was analyzed 2 and 6 days after injection, respectively. Male exosomes induced specific activation of Marilyn T cells (gate R2 in Fig. 1b,c), but not of the endogenous CD4+ T cells (gate R3 in Fig. 1b). Activated Marilyn T cells underwent several divisions, as shown by the loss of CFSE staining (Fig. 1d). Female exosomes produced by D1 cells (Fig. 1b−d) or by female BMDCs (data not shown) did not activate Marilyn T cells. Therefore, DC-derived exosomes bearing male Dby antigen induced activation of naïve Dby-specific T cells in vivo.
By analogy with observations made on tumor exosomes, which carry tumor antigens14, these results suggested that male exosomes carried male antigen, which could be captured by the endogenous dermal DCs and presented to T cells in the lymph node. In the absence of available antibodies specific for Dby, we used an indirect approach to test this hypothesis. Male exosomes were incubated with female BMDCs and Marilyn T cells in vitro, and T cell proliferation was measured 3 days later. Marilyn T cells were efficiently activated by as little as 100 ng of male exosomes (Fig. 1e). Higher male exosome concentrations (up to 1  g) induced similar Marilyn T cell proliferation amounts to those induced by DCs in the presence of nanomolar concentrations of H-Y peptide (see the plateau in Fig. 1f). This proliferation was antigen-dependent, as it was not observed in the presence of up to 10 g of female exosomes (data not shown). To confirm that Marilyn T cell activation was also MHC-restricted, we cultured male exosomes with MHC class II−deficient BMDCs obtained from I-A-/- mice. In the absence of male exosomes, MHC class II−deficient BMDCs did not induce Marilyn T cell proliferation, even in the presence of 20 nM H-Y peptide (Fig. 1f). However, when fed with male exosomes, these DCs induced Marilyn T cell proliferation (Fig. 1e). Although this proliferation was weaker than that obtained with wild-type (WT) BMDCs, it was reproducibly observed, whereas peptide never induced any proliferation. Because DC-derived exosomes contain MHC class II10, this observation suggested that exosomes from male BMDCs could carry, in addition to the male antigen, H-Y−I-Ab complexes that could directly bind to the Marilyn TCR.
Female H-Y−exosomes activate T cells
To analyze the presence and function of pMHC complexes on exosomes in the absence of intact antigen, we used exosomes produced by DCs from female mice pulsed with the H-Y peptide (H-Y−exosomes). These exosomes, or D1 cells pulsed with the H-Y peptide (H-Y−DCs), were injected in the footpads of female B6 mice that contained adoptively transferred CD45.1+ Marilyn T cells. Two days after injection, H-Y−exosomes induced CD69 up-regulation in CD4+ Marilyn T cells (gate R2, Fig. 2a), but not in CD4+ T cells from the B6 host (gate R3, Fig. 2a), which established the antigen specificity of T cell activation by H-Y−exosomes. Quantification of the percentage of CD69-expressing Marilyn T cells in four independent experiments showed similar activation by H-Y−exosomes and H-Y−DCs (31% and 29%, respectively) (Fig. 2b). Control exosomes obtained from unpulsed DCs from female mice and unpulsed D1 cells did not induce up-regulation of CD69. Thus, we concluded that exosomes bearing the H-Y peptide induce specific T cell activation in vivo.
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To rule out the possibility that the effect of H-Y−exosomes was due to residual H-Y peptide used to pulse the DCs, rather than H-Y−I-Ab complexes carried by exosomes, we prepared exosomes from MHC class II−deficient (I-A-/-) BMDCs. When analyzed by whole-mount electron microscopy, such exosomes were identical to WT BMDC-derived exosomes (data not shown).They bore MHC class I (Fig. 2c) and other markers of exosomes, such as hsc70 and MFG-E84 (data not shown), but no MHC class II molecules (Fig. 2c). In two independent experiments, exosomes produced by H-Y−pulsed WT DCs induced CD69 up-regulation in the lymph nodes of Marilyn mice after footpad injection, whereas exosomes purified in parallel from H-Y−pulsed MHC class II−deficient DCs did not (Fig. 2d). Therefore, T cell activation by exosomes requires the presence of peptide-loaded I-Ab molecules on the surface of exosomes.
To determine whether stimulation of naïve T cells by exosomes resulted in full activation and proliferation, we analyzed the draining lymph nodes of adoptively transferred B6 mice 6 days after exosome injection. In H-Y−exosome-injected mice, >70% of the Marilyn T cells had up-regulated the late activation marker CD44 (Fig. 3a,b). Injection of H-Y−DCs induced slightly higher ranges of Marilyn T cell activation than H-Y−exosomes (Fig. 3b). Injection of H-Y−exosomes also increased the number of Marilyn cells (that is, CD45.1+ cells) in the lymph nodes: we found 1% Marilyn cells upon injection of H-Y−exosomes compared to 0.1% in untreated mice (Fig. 3c). Injection of H-Y−DCs induced a higher (2.5%) accumulation of Marilyn T cells (Fig. 3c). Both treatments also induced the recruitment of nonspecific T cells in the draining lymph nodes, as shown by the increase in lymph node size (data not shown). This increase in Marilyn T cell numbers after H-Y−exosome injection could be correlated with T cell proliferation, as shown by loss of CFSE staining. Six days after injection with control DCs (Fig. 3d) or exosomes (Fig. 1c), all Marilyn cells had retained full CFSE fluorescence intensity. In mice that had received H-Y−exosomes, the Marilyn cells had undergone from one to six rounds of division (Fig. 3d). T cell proliferation induced by H-Y−exosomes was slightly less efficient than that observed with H-Y−DCs, where a majority of Marilyn T cells found in the draining lymph nodes were in their 5th or 6th division cycle. Although exosomes and DCs loaded with saturating doses of peptide initially induced equivalent CD69 up-regulation 2 days after injection (Fig. 2), exosomes were less efficient at inducing CD44 up-regulation and proliferation 6 days after injection (Fig. 3). Therefore, exosomes induced T cell activation and proliferation in vivo.
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Exosomes require DCs to stimulate T cells
The experiments presented thus far suggested that, after footpad injection, exosomes migrate to the lymph node where they directly activate T cells. To test this possibility, Marilyn T cells were cultured in the presence of various doses of H-Y−exosomes and proliferation was measured 3 days later. No T cell proliferation was observed with any of the concentrations of H-Y−exosomes used, which suggested that exosomes cannot stimulate naïve T cells directly in vitro (Fig. 4a).
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One of the key differences between the in vivo and in vitro conditions used here is that after footpad injection, exosomes may be taken up by DCs residing in the skin, which could participate in exosome-induced T cell priming. To examine the possible involvement of DCs in naïve T cell priming by exosomes, immature or mature D1 cells were incubated with varying concentrations of H-Y−exosomes for 3−5 h and then Marilyn lymph node cells were added. When mature D1 cells were present, exosomes induced effective proliferation of naïve T cells, whereas in the presence of immature D1 cells, the proliferative response was much lower (Fig. 4a). T cell proliferation was detected with as little as 300 ng of H-Y−exosomes (Fig. 4a). Control exosomes that did not bear the H-Y peptide did not induce any proliferation (data not shown). Mature D1 cells were also much more efficient than immature D1 cells at stimulating Marilyn T cell proliferation in the presence of H-Y peptide (Fig. 4b). Therefore, H-Y−exosomes activate T cells only in the presence of mature DCs.
To test the possibility that Marilyn T cell stimulation by H-Y−exosomes was due to residual H-Y peptide present in the exosome preparation, we prepared exosomes from H-Y−pulsed I-A-/- BMDCs or their WT B6 counterparts. Mature D1 cells cultured with H-Y−pulsed WT exosomes induced strong Marilyn T cell proliferation, whereas the MHC class II−deficient exosomes only induced weak T cell proliferation (Fig. 4c). In this assay, three independent preparations of MHC class II−deficient exosomes induced, at most, T cell proliferation responses comparable to 20 pM of H-Y peptide; this suggested that exosome preparations were contaminated by, at most, 20 pM H-Y peptide. This contamination could not account for the T cell stimulation induced by exosomes in vitro, as H-Y−exosomes induced T cell proliferation responses equivalent to 620  106 pM H-Y peptide (in 15 independent exosome preparations).
These results, together with our in vivo results, suggested that exosomes become competent for naïve T cell activation after uptake by DCs, which present the exosome-derived pMHC complexes. To test this possibility directly, we incubated the D1 cells with H-Y−exosomes for different time periods and then washed the D1 cells before adding Marilyn T cells. Under these conditions, exosomes preincubated for 4 h with D1 cells induced similar T cell proliferative responses to exosomes incubated with DCs and T cells for the entire duration of the culture (Fig. 4d). Shorter exosome and D1 cell preincubation periods (1 h) reduced T cell proliferation (Fig. 4d). These experiments showed that exosomes "arm" mature DCs for the induction of T cell proliferation.
Presentation of exosomes by other APCs
We determined whether exosomes could be used by other APCs to activate naïve T cells. Macrophages and B lymphocytes were compared to D1 cells for naïve T cell activation with H-Y peptide or H-Y exosomes. D1 cells and activated—that is, lipopolysaccharide (LPS)-treated—B cells and macrophages were characterized by fluorescence-activated cell scanning (FACS) with markers for DCs (CD11c+, Mac-1+), macrophages (CD11c-, Mac-1+) and B cells (B220+) (Fig. 5a). Activated B cells and D1 cells expressed similar amounts of costimulatory molecules (including CD40 and CD86) and MHC class II (Fig. 5a). Macrophages expressed less MHC class II and CD86, but more CD40, than immature D1 cells (Fig. 5a). Neither LPS-treated B cells nor macrophages induced proliferation of Marilyn T cells in the presence of H-Y peptide, whereas immature D1 cells induced a proliferative response (Fig. 5b). Similarly, when pulsed with H-Y−exosomes, neither LPS-treated B cells nor macrophages stimulated T cell proliferation, whereas immature D1 cells induced T cell proliferation (Fig. 5c), although not as efficiently as mature D1 cells (Fig. 4). DCs are therefore the only APCs that stimulate naïve T cell proliferation efficiently in vitro both with H-Y peptide and with H-Y−exosomes.
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Different DC subpopulations induce qualitatively different immune responses and stimulate CD4+ and CD8+ T lymphocytes with different efficiencies15,
16. We examined, therefore, whether exosomes could be presented to naïve T cells by different DC subpopulations. CD8+ and CD8- DCs were purified from the spleens of female mice. The two DC populations expressed similar amounts of CD11c, MHC class II and CD40. The CD8+ cells expressed slightly more CD86 (Fig. 6a). In agreement with published data17, the CD8- DC population stimulated naïve CD4+ T cells more effectively than the CD8+ DC population in the presence of H-Y peptide (Fig. 6b). H-Y−exosomes derived from CD8- D1 cells activated Marilyn T cells more effectively in the presence of CD8- DCs than CD8+ DCs (Fig. 6c). Therefore, the specificity of naïve T cell priming is dictated by the DC subtype that presents the exosomes and not by the DC subpopulation that produces them (at least for exosomes produced by CD8- DCs).
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Roles of MHC and costimulation molecules
To test whether the exosomes were also a source of T cell costimulatory molecules, we used MHC class II−deficient or costimulatory molecule−deficient DCs as exosome recipients in the Marilyn in vitro assay. BMDCs were generated, in parallel, from female WT, MHC class II−deficient (I-A-/-) or CD80-/- CD86-/- (B7-/-) B6 mice and treated for 24 h with LPS to induce maturation. BMDCs cultures contained >75% CD11c+ cells (Fig. 7a). All cell types expressed similar amounts of CD40. As expected, DCs from I-A-/- mice did not express any I-Ab molecules (Fig. 7a), but expressed normal amounts of the costimulatory molecules CD80 (also known as B7-1) and CD86 (also known as B7-2); in contrast, DCs from B7-/- mice expressed normal amounts of I-Ab molecules, but no CD80 nor CD86.
| | Figure 7. H-Y−I-Ab complexes harbored by H-Y−exosomes and CD80 and CD86 costimulatory molecules present on recipient DCs are necessary to induce Marilyn T cell proliferation in vitro. | | |  | BMDCs were generated in vitro from female B6 mice (WT DC), their MHC class II−deficient counterparts (I-A-/- DC) (a,b,d) or their B7-/- counterparts (a,c,e). (a) Characterization by FACS of the mature cell cultures obtained after overnight LPS treatment. Expression of the DC-specific marker CD11c is shown as an indication of cell purity. Expression of T cell stimulatory molecules—MHC class II, CD40, CD80 and CD86—is shown. Shaded histograms, negative controls; open histograms, specific antibodies. [3H]thymidine incorporation in cultures of Marilyn T cells with (b,c) H-Y peptide or (d,e) H-Y−exosomes from D1 cells, in the presence of WT DCs, I-A-/- DCs or B7-/- DCs. One representative of ten (b,d) or four (c,e) experiments is shown.
 Full Figure and legend (49K) |
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H-Y peptide−pulsed I-A-/- DCs did not stimulate Marilyn T cell proliferation at any of the peptide doses used (Fig. 7b). B7-/- DCs, however, stimulated Marilyn T cells, but the peptide concentration required was tenfold higher than that required for stimulation by WT DCs (Fig. 7c). The situation was reversed when H-Y−exosomes were used as source of antigen: I-A-/- DCs incubated with exosomes from H-Y−loaded D1 cells—that is, exosomes bearing H-Y−I-Ab complexes—induced Marilyn T cell proliferation (Fig. 7d), whereas B7-/- DCs were unable to do so (Fig. 7e). In ten experiments, the maximal T cell stimulation induced by exosomes in the presence of I-A-/- DCs was 30−100% of the stimulation induced by the same exosome preparations in the presence of WT DCs. These results suggested that irrelevant MHC class II expressed on mature DCs contribute to naïve T cell stimulation by exosomal MHC class II.
Therefore, MHC class II−deficient DCs that are unable to present the H-Y peptide to Marilyn T cells can stimulate T cells with H-Y−I-Ab complexes derived from exosomes. In contrast, although they retain some stimulation ability in the presence of high peptide concentrations, DCs lacking CD80 and CD86 cannot stimulate T cells after sensitization by exosomes. Exosomes are therefore vehicles for pMHC complexes and require costimulatory molecules expressed by recipient DCs to stimulate T cells.
Discussion
Cells can secrete different types of membrane vesicles in vitro1,
2. DC-derived exosomes are the best characterized population of secreted vesicles, both in terms of structure and function. We analyzed here the mechanisms of immune stimulation by DC-derived exosomes and showed that exosomes activate naïve T lymphocytes in vivo and in vitro. Naïve T cell stimulation by exosomes in vitro required two distinct DC populations. One population of DCs is required to produce exosomes bearing intact antigen or pMHC class II complexes. After having taken up these exosomes, the other population of DCs either reprocessed the antigen contained in exosomes or acquired pMHC class II from exosomes and stimulated T lymphocytes. In this latter situation, MHC class II molecules are absolutely necessary on the DC population that produces exosomes (and hence on the exosomes themselves), but the second DC population can be devoid of MHC class II molecules. Therefore, in vitro, exosomes mediate the transfer of pMHC class II complexes between two different DC populations.
Is there any evidence, then, for the exchange of membrane proteins between different cells in vivo? Several studies have reported the heterologous acquisition of membrane proteins in vivo. Follicular DCs bear abundant surface MHC class II molecules in situ, but do not synthesize them18. Other studies have also reported the in vivo acquisition of membrane molecules by different cell types, including thymocytes19 and natural killer cells20,
21. The modes of membrane exchange in vivo (after direct cell-cell contact, through secreted membranes or after phagocytosis, for example) were not addressed and in none of these studies were any biological functions directly associated with the exchange of membrane proteins.
Two studies have reported the transfer of membrane proteins associated with specific functions. Human peripheral blood mononuclear cells release membrane microparticles that contain CCR5, a receptor for macrophage-tropic HIV22. These vesicles transferred CCR5 to CCR5-deficient cells, which became sensitive to HIV infection. Spreading of proteins called "morphogens" through several cell layers, which allows the establishment of gradients of these morphogens, is necessary for anterio-posterior and dorso-ventral patterning of various organs during embryogenesis. Secreted membranes are involved in spreading between adjacent cells of the membrane-associated "Wingless" morphogen in Drosophila wing buds cultured ex vivo23. Whether or not these membranes correspond to exosomes (that is, are derived from multivesicular endosomes) was not addressed. Nevertheless, these two studies have established that the exchange of membrane proteins between different cells may have functional consequences.
Could exosomes be involved physiologically in the functional exchange of membrane proteins? Although direct evidence for exosome function in vivo is scarce, several studies show that they can be produced in vivo. Electron microscopic analysis of human tonsils showed the presence of membrane vesicles of the right size and shape, which also bore MHC class II and CD6324. Exosomes were therefore proposed to account for MHC class II presence on follicular DCs (which do not synthesize MHC class II). Vesicles bearing several exosome markers, including CD63 and MHC class II, can be purified from human serum (M.-P. Caby and C. Bonnerot, unpublished data). In addition, exosomes can be purified from ascitic fluid from cancer patients25. These exosomes transfer tumor antigens to DCs and promote cross-presentation. Whether or not the exosomes produced in vivo serve any physiological function remains to be addressed. Their distinct mode of biogenesis and protein composition, which is conserved between human and mouse as well as between different cell types, argues for some specific cellular function. This function could have originally been related to the elimination of lysosomal proteins. We propose that the immune system has "learned" how to use these secreted membranes as a source of antigens or membrane proteins for immunoregulation.
A critical aspect, when considering exosome function, is their efficacy for T cell activation compared to DCs. We found that 5 g of male exosomes and 5  105 male DCs induced CD69 up-regulation on Marilyn T cells with comparable efficiencies. We have observed some variability in exosome production by DCs, but, on average, 5 g corresponds to exosome production by roughly 107 DCs over 24 h. This number is a minimal estimate, as we do not know how many exosomes are lost or inactivated during the purification procedures. The total amounts of MHC class II actually injected into the mice are similar when 5 105 DCs and 5 g of exosomes are used; 5 105 DCs represent >30 g of total protein. Therefore, depending on whether one considers cell numbers, MHC molecules or total protein amounts, exosomes are less, equally or more efficient than DCs for T cell priming in vivo.
A key aspect of our study is the in vitro analysis of how exosomes function. Our observation that exosomes bearing the relevant pMHC complex do not stimulate specific T cells directly was unexpected. Indeed, exosomes from B lymphocytes incubated with mycobacterial heat shock protein 65 (hsp65) induce proliferation of human antigen−specific T cell clones9. The naïve T cells we used here, which can only be stimulated by mature DCs, may account for this discrepancy. It is possible that exosomes mediate a low degree of TCR cross-linking that may be sufficient for the activation of T cell clones, but not of naïve T cells. Alternatively, the T cell clones themselves may serve as "exosome-presenting cells", whereas the activation of naïve T cells requires mature DCs. Indeed, in the experimental system we used here, exosomes could not transfer T cell−stimulating abilities to APCs that lack the ability to activate naïve T cells by themselves. Neither macrophages nor B lymphocytes induced Marilyn T cell proliferation, whatever the source of antigen provided (up to 10 nM H-Y peptide or H-Y−exosomes). Similarly, CD8+ splenic DCs showed a reduced capacity to activate naïve T cells after incubation with either peptide or exosomes. Therefore, the APC and not the cellular source of exosomes dictates the specificity of T cell stimulation. The low efficiency of naïve T cell stimulation by exosome-treated B lymphocytes, macrophages or CD8+ DCs could of course be due to either the low efficiency of naïve T cell stimulation by these cells or to the lack of appropriate exosome binding or uptake capacity. In any case, exosomes do not overcome the specificity of naïve T cell stimulation by incompetent APCs.
Transfer by exosomes of pMHC complexes between DCs was demonstrated here with the use of MHC class II−deficient DCs as recipients. Such APCs are unable to stimulate Marilyn T cells with H-Y peptide, but they do stimulate naïve T cells in the presence of exosomes bearing the relevant pMHC complexes. We consistently observed that, in the presence of exosomes, MHC-deficient DCs induced slightly less efficient T cell activation than WT DCs. This difference is most likely due to the absence of endogenously loaded MHC at the surface of the MHC II−deficient DCs. These "irrelevant" MHC class II molecules facilitate initial DC−T cell interactions and favor specific T cell stimulation in vitro26. We observed that Marilyn T cells adoptively transferred into MHC class II−deficient hosts can be activated by H-Y−exosomes, but with less efficacy than in WT hosts (unpublished observations). These results suggested that transfer of pMHC complexes to the host DC also occurs in vivo.
In addition to transferring pMHC complexes, exosomes may also transfer antigens: melanoma tumor cells secrete exosomes carrying melanoma antigens that recipient DCs can use for cross-presentation to CD8+ T lymphocytes14,
25. We had similar findings for MHC class II cross-presentation of the endogenous H-Y antigen. The transfer of peptides associated with hsp from macrophages or tumor cells to APCs has been reported8,
27. Whether exosomes, which bear abundant hsp70 molecules4, mediated the exchange of intact antigen or peptide-loaded hsp70 is still unclear. Nevertheless, we have shown here that in addition to antigen transfer, DC-derived exosomes bear additional antigenic information in the form of pMHC complexes.
Our results are consistent with a role for exosomes in spreading antigen-specific signals by exchanging both antigens and pMHC complexes between different DCs. We propose that after encountering antigen, immature DCs may produce exosomes carrying intact antigen, peptide-loaded hsp70 and/or peptide-loaded MHC. These antigen-bearing exosomes could be taken up by other DCs, which have not themselves encountered antigens. Upon stimulation by an adequate maturation signal, these "recipient" DCs would then become competent for naïve T cell stimulation.
It is tempting to speculate that exosomes represent preformed microdomains that contain both MHC and costimulatory molecules. The idea that MHC class II and costimulatory molecules are delivered from endocytic compartments to the cell surface within preformed functional microdomains has been proposed28. Our results suggest that exosomes could also represent preformed microdomains, which—instead of being delivered from the inside of the cell—could bind to the DC surface from the external milieu. Like "internal" microdomains, exosomes form intracellularly and contain both specific pMHC class II and costimulatory molecules. Exosomes could remain organized in clusters at the surface of the recipient mature DCs, thus preventing dilution of the specific pMHC complexes and promoting the formation of functional immune synapses.
Whether exosomes are produced in peripheral tissues or after the migration of DCs to lymph nodes, or both, remains to be addressed. If they are produced in the periphery, exosomes may either traffic through the lymph to draining lymph nodes or diffuse locally, being captured by immature DCs in the periphery, and eventually migrate to the lymph nodes. Alternatively, exosomes could be produced after migration of peripheral DCs to the lymph nodes where they may sensitize lymph node−resident DCs with specific pMHC complexes. Whether any of these mechanisms actually contribute to T cell priming in vivo remains to be addressed.
Regardless of their physiological role and mode of action, we have shown that exosomes stimulate naïve CD4+ T cells in vivo by the transfer of pMHC complexes to DCs. These results encourage the clinical use of exosomes, as the stimulation of CD4+ responses is critical for the efficacy of vaccines aimed at inducing CD8+ cytolytic T cell responses in vivo.
Methods
Mice.
Marilyn mice on a B6 (CD45.2+) RAG-2−deficient genetic background that were expressing the TCR (V 1.1-J35) and TCR (V6-J2.3) chains from Marilyn—a CD4+ T cell clone specific for the complex of a male antigen (H-Y) peptide with I-Ab—have been described11. These mice were crossed with CD45.1+ B6 mice to obtain CD45.1+ Marilyn mice. B6 mice were from IffaCredo (L'abresle, France), I-A-/- mice were from Centre de Distribution, Typage et Archivage (CNRS UPS44, Orléans, France) and B7-/- mice were a gift of B. Salomon (CNRS/UPMC ESA 7087, Hôpital La Pitié-Salpêtrière, France). Live animal experiments were done in accordance with the guidelines of the French Veterinary Department.
Cells.
The DC line D1 (generated from the spleen of a female B6 mouse13) was cultured as described3. BMDCs were generated by 2−3 weeks of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF)−containing conditioned medium3,
4 and were used when FACS analysis of the cell population revealed >75% CD11c+ cells. Maturation was induced by 24-h treatment with 20 g ml-1 of LPS (Sigma Chemical Co, St., MO). D1 cells and BMDCs were CD8- DCs. CD8+ and CD8- DCs were purified from spleen as described29 with anti-CD11c−coupled magnetic beads (N418 clone, Miltenyi Biotec, Paris, France). This was followed by cell sorting on a FACS DiVa (BD-PharMingen, Le Pont de Claix, France) after staining with anti-CD11c (HL-3 clone) and anti-CD8. B cells were purified from spleen with anti-B220 magnetic beads (Miltenyi Biotec) and cultured for 48 h in the presence of 20 g ml-1 of LPS. Macrophages were generated from bone marrow by culture for 5 days in medium supplemented with 30% M-CSF−containing L929-conditioned medium as described30 and treated with 20 g ml-1 of LPS for 48 h.
Exosome purification.
Exosomes were purified from the supernatant of D1 cells or BMDCs cultured in medium depleted of fetal calf serum−derived exosomes by overnight centrifugation at 100,000g (depleted medium)3. To prepare exosomes bearing H-Y−I-Ab complexes, DCs were pulsed for 2−3 h with 500 nM H-Y peptide (NAGFNSNRANSSRSS), the culture medium was replaced with fresh depleted medium without H-Y peptide and the supernatant was collected 24 h later. Exosomes were purified by filtration on 22 m pore filters, followed by ultracentrifugation at 100,000g, as described3. In each exosome preparation, the concentration of total proteins was quantified by a Bradford assay (BioRad, Marnes la Coquette, France). In 24 independent exosome preparations from D1 cells and BMDCs, the average amount of recovered exosomal proteins was 0.31 0.02 g/1 106 cells/24 h. The amount of MHC class II in each exosome preparation was compared to that of a known number of immature D1 cells by immunoblotting. We found that 1 g of exosomes contains the same number of MHC class II molecules as 0.19 106 0.04 106 D1 cells.
Antibodies and reagents.
Antibodies used for FACS analysis were as follows (all from BD-PharMingen): fluorescein isothiocyanate−conjugated anti−mouse CD11c, I-Ab, H-2Kb, CD40, CD86, CD80 and CD8 and the corresponding isotype controls; phycoerythrin-conjugated anti−mouse V6, CD45.1, I-Ab, CD40 and CD86; allophycocyanin-conjugated anti−mouse CD4 and CD11c; and biotin-conjugated anti−mouse CD69 and CD44. Tricolor-conjugated streptavidin was from Caltag (Burlingame, CA). Antibodies used for exosome characterization by immunoblotting were rabbit antisera to the mouse MHC class II chain COOH terminus4 and to mouse MHC class I (P8, provided by H. Ploegh, Boston, MA). CFSE was from Molecular Probes (Eugene, OR).
In vivo injection in mice.
Exosomes or DCs were injected subcutaneously in the hind footpads of Marilyn mice or female B6 mice that had been injected intravenously 24 h earlier with 106 lymph node cells from CD45.1+ Marilyn mice. In some experiments, Marilyn lymph node cells were labeled with CFSE (5 M in PBS−0.1% bovine serum albumin for 8 min at 37 °C) before injection into B6 mice. Cells from the draining lymph nodes (that is, pooled ipsilateral popliteal and inguinal lymph nodes) were analyzed 2 and 6 days later. For in vivo injection, D1 cells were pulsed with 50−500 nM H-Y peptide for 1.5−3 h before injection (both peptide concentrations gave the maximal Marilyn T cell stimulation in vitro, see Figs. 1, 4 and 6). A total of 3 105 − 5 105 cells were injected, which represented 15−30 g of total nonnuclear proteins (on average a DC postnuclear supernatant contained 6 g of proteins per 105 cells). Injected exosomes ranged between 5−40 g total protein.
In vitro T cell stimulation assay.
Exosomes or H-Y peptide were incubated in 96-well plates with or without 1 104 − 2 104 APCs in 50 l culture medium for 3−5 h at 37 °C. The plates were irradiated (5000 rad) and 5 104 cells from Marilyn mice lymph nodes were added in 100 l of culture medium. For experiments that used splenic DCs, 3 104 were used, and incubation in 50 l was done for 1 h before irradiation and addition of Marilyn cells. [3H]thymidine (0.5 Ci/well) was added 72 h later for 18 h and incorporation was measured by liquid scintillation counting after collection of the cells on glass fiber filters with an automatic cell harvester (Tomtec, CA). In some experiments, DCs were washed twice with prewarmed culture medium after preincubation with exosomes, then irradiated before Marilyn T cells were added. In these experiments, control conditions involved D1 cells washed before addition of exosomes, irradiation and addition of Marilyn T cells.
Received 1 July 2002; Accepted 1 October 2002; Published online: 11 November 2002.
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