Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes*

Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50–60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.

invariant chain peptide remains temporarily associated with the peptide binding groove of MHC class II but is ultimately displaced by antigenic peptides, a process that is catalyzed by HLA-DM (9 -12).
Immunoelectron microscopy (IEM) studies revealed that in a variety of antigen-presenting cells, the majority of intracellular MHC class II molecules localize to a heterogeneous set of endocytic compartments, collectively termed MIICs (MHC class II-enriched compartments) (Refs. 13 and 14; reviewed in Refs. 15 and 16). MIICs are endocytic vacuoles with internal membrane vesicles and sheets, reminiscent of late endosomes and lysosomes in non-antigen-presenting cells (17). The internal vesicles of MIICs probably originate from inward vesiculation of its limiting membrane (18), analogous to the formation of multivesicular bodies in other cell types (19). MIICs are thought to represent the subcellular site at which MHC class II molecules bind peptides (20 -23). Once formed in MIICs, the ␣␤-peptide complexes are transported to the plasma membrane via largely unidentified pathways. In macrophages, putative transport vesicles, containing peptide-loaded MHC class II molecules, have been described by IEM (24). As an alternative pathway, we and others have shown that multivesicular MIICs can fuse with the plasma membrane directly, thereby inserting MHC class II molecules from the limiting membrane of MIICs into the plasma membrane and releasing the internal vesicles into the cell culture medium (25,26). The released vesicles were termed exosomes (25), analogous to those released by reticulocytes (27,28) and those originating from cytolytic granules released by cytotoxic T-lymphocytes (29,30). Exocytic profiles containing exosomes have also been found on the cell surface of other antigen-presenting cells, such as dendritic cells, tonsil B-cells, monocytes, and macrophages. 2 Exosomes secreted by B-cell lines are capable of effective antigen presentation to T-cells in vitro (25). Except for their relative enrichment in MHC class II and depletion of transferrin receptor (TfR), a marker for plasma membrane and early endosomes, little is known about the protein composition of exosomes. Because interaction of MHC class II-peptide complexes with the T-cell receptor requires appropriate co-stimulatory signals, we anticipated that exosomes might express co-stimulatory molecules at their surface. The main co-stimulatory pathways involve the interaction of CD28 or CTLA-4 on T-cells with CD80 (B7.1) or CD86 (B7.2) expressed on B-cells and other antigen-presenting cells. Here we report that the co-stimulatory molecule CD86 is strongly enriched on internal vesicles of MIICs and on exosomes. Previous IEM studies have shown that the internal vesicles of MIICs also contain the lysosomal membrane protein CD63 (14,31), a member of the tetraspan superfamily. In the present study, we show that several other members of this family, including CD37, CD53, CD81, and CD82, are enriched in exosomes, whereas other MIIC markers, such as Lamp-1, Lamp-2, HLA-DM, and Ii, are not. Tetraspan proteins have shown to be involved in antigen presentation, T-cell signaling, T-cell activation, motility, and adhesion. Also, they are able to form oligomeric complexes (32)(33)(34)(35) and interact with integrins (34,36), HLA-DR (32), and the T-cell co-receptors CD4 and CD8 (37)(38)(39). Because exosomes have been shown to be capable of stimulating T-cells in vitro (25), the selective incorporation of co-stimulatory molecules and tetraspan proteins in exosomes further supports an immunostimulatory role of exosomes in vivo.

EXPERIMENTAL PROCEDURES
Cells and Antibodies-The EBV-transformed human B-cell line RN (HLA-DR15 ϩ ) was maintained as described earlier (25). Rabbit polyclonal anti-MHC class II (13,14)  Isolation of Exosomes-Exosomes were isolated by differential centrifugation as described previously (25). Briefly, RN cells were washed by centrifugation and recultured in fresh medium for 18 h. Cell cultures (35 ml) containing about 5 ϫ 10 7 cells were centrifuged for 10 min at 200 ϫ g (pellet P 1 ). The supernatant was removed and centrifuged for 10 min at 500 ϫ g (pellet P 2 ). This was repeated once (the pooled pellets are referred to as P 2 ). Supernatants were sequentially centrifuged at 2,000 ϫ g twice for 15 min (the pooled pellets are referred to as P 3 ), once at 10,000 ϫ g for 30 min (yielding pellet P 4 ) and once at 70,000 ϫ g for 60 min (yielding pellet P 5 ), using a SW27 rotor (Beckman Instruments, Inc., Fullerton, CA). P 1 contained the cells, whereas P 5 was enriched in exosomes. P 1 -P 5 were directly solubilized in reducing or nonreducing Laemmli sample buffer and incubated for 5 min at 95°C. Samples of each pellet were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (see below). For further purification of exosomes, P 5 was resuspended in 5 ml of 2.6 M sucrose, 20 mM HEPES/NaOH, pH 7.2. A linear sucrose gradient (2.0 -0.25 M sucrose, 20 mM HEPES/NaOH, pH 7.2) was layered on the top of the exosome suspension in a SW27 tube (Beckman Instruments, Inc.). Gradients

FIG. 1. Detection of proteins in P 1 -P 5 pellets.
A, detection of MHC class II, CD86, HLA-DM, Ii, TfR, and ICAM-1 in P 1 -P 5 pellets. Media from cell cultures were sequentially centrifuged for 10 min at 200 g (P 1 ), twice for 10 min at 500 ϫ g, twice for 15 min at 2000 ϫ g (P 3 ), once for 30 min at 10,000 g (P 4 ), and finally once for 60 min at 70,000 ϫ g (P 5 ). P 1 corresponds to cells and P 5 to the fraction enriched in exosomes. P 1 was solubilized in 1 ml and P 2 -P 5 in 50 l of Laemmli sample buffer. 10-l samples were analyzed by SDS-PAGE and Western blotting. Molecular mass markers are indicated in kDa at the left and right. B, detection of Lamp-1, Lamp-2, and tetraspan proteins in P 1 -P 5 pellets. P 1 -P 5 pellets were prepared as above and analyzed under nonreducing conditions. TABLE I Relative enrichment of proteins over TfR in exosomes P 1 (cells) and P 5 (fraction enriched in exosomes) pellets were analyzed as in Fig. 1. The signals from P 1 and P 5 were quantitated with a PhosphorImager, and for each protein, the average ratio P 5 /P 1 Ϯ S.D. is shown in the third column. Averages are presented with number of determinations in parentheses. The relative enrichment of each protein over TfR was then determined by calculating the ratio of signals P 5 /P 1 and normalizing these values to obtain a value of 1 for the TfR. (10) were centrifuged for 15 h at 100,000 ϫ g, after which, 2-ml fractions were collected from the bottom of the tube. To collect membranes from these fractions, they were diluted with 3 ml of phosphate-buffered saline (PBS) and centrifuged for 60 min at 200,000 ϫ g using a SW50.1 rotor (Beckman Instruments, Inc.). The pellets were solubilized in nonreducing SDS-PAGE loading buffer at room temperature for 60 min. Samples of each fraction were then analyzed by SDS-PAGE and Western blotting (see below). Western Blotting-Proteins were separated on 12.5, 10, or 7.5% acrylamide gels (SDS-PAGE) and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membranes were then blocked for 90 min in PBS containing 5% (w/v) nonfat dry milk (Protivar, Nutricia, Zoetermeer, The Netherlands) and 0.1% (w/v) Tween 20 and then reacted for 90 min with primary antibodies followed by labeling with 0.1 g/ml 125 I-labeled protein G (Zymed Laboratories, San Francisco, CA) for 90 min. 125 I Label was visualized and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For detection of monoclonal antibodies, a polyclonal anti-IgG antibody was used as secondary antibody followed by incubation with 125 I-labeled protein G.
Immunoelectron Microscopy-RN cells were prepared for ultrathin cryosectioning and immunogold labeling as described previously (45)(46)(47)(48). For immunoelectron microscopic analysis of exosomes, they were The majority of MHC class II was recovered in a SDS-stable form (C␣/␤) indicating ␣␤-peptide complexes. B, the relative distribution of CD81, CD82, and MHC class II from A. Their distribution was overlapping. C and D, whole-mount IEM of exosomes from sucrose gradient fractions 1.12-1.18 g/ml density prepared as in A. C, double immunolabeling for CD82 (15-nm gold particles) and MHC class II (HLA-DR) (10-nm gold particles) shows their co-localization exosomes. D, co-localization of CD53 (15-nm gold particles) and HLA-DR (10-nm gold particles) on exosomes. Bars, 100 nm purified on sucrose density gradients (see above). Membrane pellets from relevant fractions were resuspended in PBS, pooled in a SW27 tube, and recentrifuged for 60 min at 70,000 ϫ g. The final pellet was then resuspended in PBS/0.02% sodium azide. Droplets of this preparation were directly placed on Formvar carbon-coated grids. Adsorbed membranes were fixed with 2% paraformaldehyde, immunolabeled and stained as above.

RESULTS
Tetraspan Proteins and CD86 Are Enriched on Exosomes-To investigate the presence of specific proteins, exosomes of RN B-cells were collected from the culture media by sequential differential centrifugations, yielding five pellets (P 1 -P 5 ), of which P 1 contained the cells and P 5 was highly enriched in exosomes (25). The pellets were lysed, and equal volume samples were analyzed by SDS-PAGE under either reducing or nonreducing conditions followed by Western blotting. P 1 -P 5 were analyzed for the presence of MHC class II, HLA-DM, and Ii as molecules directly involved in the formation of MHC class II-peptide complexes. The pellets were also analyzed for the presence of the adhesion molecule ICAM-1 (CD54), the co-stimulatory molecule CD86, and the tetraspans CD37, CD63, CD81, and CD82. The relative amount of each protein recovered from P 1 and P 5 was determined, and the ratio P 5 /P 1 was calculated. In order to determine the relative enrichment of proteins in exosomes, their P 5 /P 1 ratio was divided by the P 5 /P 1 value obtained for TfR. TfR, a marker of plasma membrane and early endosomes, served as a reference protein because of its low abundance in exosomes (25) and MIICs (17,22,25). Indeed, exosome preparations contained little TfR (Fig.  1A), excluding major contamination of the exosome preparations by shed plasma membrane. Consistent with previous observations (25), MHC class II was enriched 8-fold over TfR in exosome preparations compared with whole cells (Fig. 1A; Table I). Similarly, the relative enrichment of CD86 over TfR was 9-fold ( Fig. 1A; Table I). Interestingly, exosomes were highly enriched for all tetraspan proteins tested ( Fig. 1B; Table I). Of these, CD37 (36-fold), CD81 (124-fold), and CD82 (41-fold) were present most prominently (Table I). Western blotting could not test the presence of CD53, another tetraspan protein expressed in leukocytes, because the available anti-CD53 antibodies were not suitable for that purpose. However, CD53 was clearly present on vesicles within MIICs by IEM (see below and Fig. 3B). In contrast to the tetraspans, other membrane proteins found in MIICs, HLA-DM, Ii, Lamp-1, and Lamp-2 were not enriched in exosomes ( Fig. 1; Table I). The presence of adhesion molecules was also analyzed. ICAM-1 (CD54) was detected in exosomes with a 4-fold enrichment over TfR (Fig. 1A and Table I). ICAM-1 was also detected on exosomes by IEM (data not shown). LFA-3 (CD58) and ICAM-2 (CD102) were not significantly enriched in exosomes (data not shown). Altogether, these results show that exosomes are selectively and highly enriched in tetraspan proteins.
Colocalization of Tetraspan Proteins and MHC Class II Molecules on Exosomes-In a previous study (25), MHC class IIpositive exosomes displayed an equilibrium buoyant density of 1.10 -1.18 g/ml with a peak at 1.13 g/ml on linear sucrose gradients. To confirm that tetraspan proteins and MHC class II localize to membranes with the same characteristics, exosomes

Ratios of gold particles counted on limiting (l) and internal (i) membranes of multivesicular (MV) and multilaminar (ML) MIICs
Immunogold particles for the four proteins listed were counted on 50 multivesicular and 50 multilaminar MIICs on internal and limiting membranes. The first four columns show the total number of gold particles. The next two columns show the ratios of internal to limiting membranes after correction for the total membrane length measured. isolated from the culture medium by differential centrifugation were floated into a linear sucrose gradient. The distribution of MHC class II, CD81, and CD82 in the gradient was analyzed by Western blotting (Fig. 2A). The distributions of MHC class II, CD81, and CD82 were completely overlapping (Fig. 2B), providing further evidence that CD81 and CD82 are associated with MHC class II-positive exosomes. This conclusion was further strengthened by whole-mount IEM analysis of membranes equilibrating at 1.10 -1.18 g/ml (Fig. 2, C and D). All tetraspan proteins were found to colocalize with MHC class II on these membranes, as illustrated for CD82 (Fig. 2C) and CD53 (Fig. 2D).
Tetraspan Proteins Localize to the Internal Vesicles of MIICs-Because exosomes derive from MIICs, we next studied the subcellular localization of the various tetraspan proteins by IEM on ultrathin cryosections of RN cells. CD82 revealed the highest intracellular labeling and was present in both multivesicular and multilaminar MIICs but most abundantly in the latter (Fig. 3A). CD37 (not shown) and CD53 (Fig. 3B) localized primarily to multivesicular MIICs, whereas CD63 was found in both multivesicular and multilaminar MIICs (Fig. 4A). CD81 was strongly enriched in exosomes (Fig. 4B) but could be found in only a few MIICs. A possible explanation is that the relevant antibody detected intracellular CD81 molecules inefficiently due to epitope masking. Interestingly, all tetraspan proteins were largely confined to the internal membranes of the MIICs. In contrast, other lysosomal proteins, such as Lamp-1 and -2, were located mainly to the limiting membrane of MIICs. Fig.  4A illustrates the distinct distributions of CD63 and Lamp-2 on these membrane domains of MIICs. Exosomes within exocytic profiles were strongly labeled for the tetraspan proteins CD63, CD81, and CD37 (Fig. 4, A, B, and C, respectively), as well as for CD53 and CD82 (not shown).
Differential distributions on internal and limiting membranes were further evaluated by quantifying MHC class II, CD63, CD82, and Lamp-1 labeling on membranes of MIICs. The numbers of gold particles for each protein found over internal and limiting membranes 50 multivesicular and multilaminar MIICs are given in the first four columns of Table II, and the ratios of gold labeling are presented in the last two columns. Both types of values showed a clear enrichment of CD63 and CD82 on internal membranes, which was most prominent in the multilaminar MIICs. In contrast, Lamp-1 was enriched on the limiting membranes, which is in agreement with their low abundance on exosomes. MHC class II labeling of the internal MIIC membranes is the highest of the four proteins measured. Only in the multilaminar MIICs is MHC class II enriched on the internal as compared with the limiting membrane. Thus, the IEM observations are consistent with the biochemical data on isolated exosomes, and they suggest differential protein sorting in MIICs during the formation of internal membranes.

DISCUSSION
In this study, we show that MHC class II, the co-stimulatory molecule CD86, and several tetraspan proteins are enriched on isolated exosomes ( Fig. 1; Table I). MHC class II and CD86 were enriched 8 -9-fold over TfR (Table I). Similar to MHC class II and CD86, the tetraspan CD63, which is known to be present in MIICs (14,49), was enriched 7-fold in exosomes. Other tetraspan proteins, CD37, CD81, and CD82, were even more abundant in exosomes, with relative enrichment factors of 36, 124, and 41, respectively (Table I). The presence of tetraspan proteins on exosomes was confirmed by IEM on whole-mount preparations of exosomes, as well as on ultrathin cryosections of exosomes in exocytic profiles and internal membranes in MIICs, the latter representing the precursors of exosomes. Although CD53 could not be detected by Western blot analyses, IEM demonstrated its presence on the plasma membrane and in MIICs. The labeling pattern of CD53 was similar to that of CD37.
How the tetraspan proteins are transported to MIICs is unclear. Newly synthesized tetraspan proteins may be targeted directly from the trans-Golgi network to the endocytic tract. Alternatively, they may reach MIICs via the plasma membrane through endocytosis. However, they do not contain any of the consensus sorting signals in their cytoplasmic tail. From the differential expression levels of tetraspan proteins at the plasma membrane, distinct rates of transport between the plasma membrane and MIICs can be suspected. The mechanism by which MHC class II, CD86, and the tetraspans are selectively incorporated into the internal vesicles of multivesicular bodies is also not known. A similar phenomenon has been described for the sorting of ligand-bound epidermal growth factor receptor in endosomes, resulting in its downregulation and ultimate degradation in lysosomes (50,51). Phosphorylation of annexin I by this receptor has been suggested to induce inward vesiculation in this process (52).
Even though the precise functions of the tetraspan proteins studied are largely unknown, similarities between their structures suggests closely related functions. They have been described to be important for T-cell development and T-cell activation. CD81 has been implicated in T-cell development (39,53) as a co-inducer molecule during differentiation of immature double positive CD4 ϩ CD8 ϩ thymocytes into single positive CD4 ϩ and CD8 ϩ T-cells in vitro (54). CD81-null mice show a decreased early antibody production in response to antigen, indicating that CD81 is important for the development of humoral immune responses (55). CD53 has been proposed to play a role in thymopoiesis (56,57). Whether CD81 and/or CD53 indeed function as signaling molecules in thymic selection is still theoretical. Alternatively, it can be speculated that these molecules may be important for antigen presentation by MHC class II at the plasma membrane and/or exosomes. Similarly, CD82 has been reported to function as a co-stimulatory molecule for monocytes (58) and to play a role in T-lymphocyte activation (59) and proliferation (60). Tetraspans have also been implicated in cell adhesion. CD63 has been shown to be involved in platelet adhesion to endothelial cells (61), and CD81 is involved in cell adhesion of B-cell, T-cell, and nonlymphoid cell lines (62). Recently, ligation of CD53 has been shown to induce homotypic adhesion of B-cells (63). In this latter case, the authors postulated novel adhesion mechanisms among lymphoid cells modulated by several tetraspans, which represents a novel form of cell communication. Because we found relatively little conventional adhesion molecules, such as ICAM-1 ( Fig. 1A; Table I), ICAM-2, and LFA-3 (data not shown), in exosomes as compared with tetraspan proteins, the presence of tetraspans in exosomes would be consistent with a role in adhesion.
In contrast to mature MHC class II, molecules involved in MHC class II transport and peptide loading, such as HLA-DM and Ii, were not enriched in exosomes. This is consistent with the notion that MHC class II processing occurs in MIICs prior to their fusion with the plasma membrane, resulting in the release of exosomes carrying peptide-loaded MHC class II. This notion was supported by the stability of MHC class II on exosomes in the presence of SDS ( Fig. 2A; Ref. 25). IEM studies have shown that HLA-DM is mainly associated with the limiting membrane of MIICs. This suggests that prior to the recruitment of MHC class II to the internal vesicles of MIICs, the Ii degradation product class II-associated invariant chain peptide is removed from MHC class II and substituted by a pep-tide, a process catalyzed by HLA-DM (9 -12). Interestingly, CD82 has recently been shown to associate selectively with MHC class II/HLA-DM complexes (64). Like HLA-DM, the typical lysosomal membrane proteins, Lamp-1 and Lamp-2, were preferentially localized to the limiting membrane of MI-ICs. For Lamp-2, this observation is not surprising because it has been described as a receptor for the selective import of cytosolic proteins into lysosomes (65).
In conclusion, exosomes are selectively enriched in proteins that play a role in antigen presentation, suggesting their immunological role in vivo. They could be involved in T-cell stimulation and/or in less defined processes, such as the generation of T-cell tolerance or T-cell memory. Further investigations are required to determine the physiological role of exosomes and their potential use in immunotherapy.