Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells

Hélène Vincent-Schneider1, Pamela Stumptner-Cuvelette1, Danielle Lankar1, Sabine Pain1, Graça Raposo2, Philippe Benaroch1 and Christian Bonnerot1

1 INSERM U520, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France 2 CNRS, UMR 144, Institut Curie, 12 rue Lhomond, 75005 Paris, France

Correspondence to: C. Bonnerot; E-mail: Christian.Bonnerot{at}curie.fr
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Exosomes are small vesicles (60–100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1–hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1–HA(306–318) complexes activated HA/DR1-specific T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.

Keywords: antigen presentation, dendritic cell, exosome, mast cell, MHC


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The endocytic pathway of antigen-presenting cells (APC) has several key characteristics accounting for their unique ability to elicit MHC class II-restricted T cell responses. Endosomes and lysosomes from these cells are compartments with an important role in antigen presentation, in which antigenic peptides associate with MHC class II molecules. Antigen-processing compartments were first described in human B cell lines as multilaminar/multivesicular MHC class II compartments (MIIC) (1,2). Resident endosomal proteins, such as CD63, and MHC class II molecules accumulate in these compartments. In addition, the fusion of MIIC with the plasma membrane leads to the plasma membrane expression of peptide–MHC class II complexes and to the secretion of small vesicles, called exosomes, bearing MHC class II–peptide complexes. Although the secretion of exosomes has been reported in various hematopoietic cells including reticulocytes, platelets, B lymphocytes, mast cells and dendritic cells (DC) (38), the function of cell-derived exosomes remains unclear.

The accumulation of MHC class II–peptide complexes on the limiting membranes of exosomes derived from APC, such as B cells or DC, led to evaluation of the role of exosomes bearing MHC class II in the T cell response. High concentrations of B-Epstein–Barr virus (EBV)-derived exosomes have been shown to activate specific CD4 T cell clones directly (5). Dendritic cell-derived exosomes have recently been reported to accumulate MHC class II and I molecules, along with co-stimulatory molecules (8,9). In addition, exosomes produced by DC exposed to tumor-derived antigenic peptides have been shown to induce a cytotoxic T lymphocyte-mediated response leading to the regression of established tumors in mice (8). Thus, exosomes bearing MHC molecules and antigenic peptides induce potent T cell responses, although the stimulation of specific T cells with peptide-pulsed exosomes was consistently less efficient than the stimulation of intact cells by incubation with peptides (5,8,10). This suggests that exosomes do not interact directly with effector cells. The precise mechanism of this phenomenon still remains to be understood.

Exosomes from platelets and mast cells are secreted upon cell activation, suggesting that exosomes may be involved in homeostasis or inflammatory reactions. Furthermore, bone marrow-derived mast cells (BMMC) and the mast cell line rat basophilic leukemia (RBL)-2H3 mostly accumulate MHC class II molecules in small vesicles contained in secretory granules. The exocytosis of mast cell secretory granules may be triggered by antigen-induced aggregation of the high-affinity receptors (Fc{epsilon}RI) for IgE (11) or exposure to a calcium ionophore. The activation of BMMC expressing MHC class II leads, as expected, to the release of exosomes bearing MHC class II.

Homogeneous and reliable preparations of exosomes, bearing a well-defined MHC class II–peptide complex, are a key tool for investigating exosome function. We explored the mechanisms of T cell activation by MHC class II+ exosomes, by expressing HLA-DR1 {alpha} and ß chains alone or in combination with an Ii chain mutant (IiHA), which carries a sequence encoding the antigenic hemagglutinin (HA) peptide (12) in place of the class II invariant chain peptide (CLIP) region (13), in the RBL cell line RBL-2H3. Co-expression of the IiHA construct facilitated the endogenous loading of most of the MHC class II molecules present in RBL DR1IiHA cells with the HA peptide. The accumulation of MHC class II in secretory granules made it possible to induce the release of class II+ exosomes by cross-linking IgE receptors or adding calcium ionophores. RBL DR1-derived exosomes loaded with HA peptide and RBL DR1IiHA-derived exosomes contain functional MHC class II complexes that stimulate specific CD4+ T cells weakly. Coupling exosomes to carrier beads increased the efficiency of T cell stimulation. In contrast, exosomes stimulated DR1–HA-specific CD4+ T cells very efficiently if they were first incubated with murine DC. Thus, exosomes may need to be captured by DC to stimulate specific T cells efficiently.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chemical reagents, antibodies and peptides
All chemicals used in this study were purchased from Sigma (St Louis, MO). RPMI 1640, FCS, PBS, penicillin, streptomycin, sodium pyruvate and L-glutamine were purchased from Gibco (Paisley, UK). We used the following mAb: L243 (22,23) which recognizes HLA-DR complexes devoid of the Ii chain, Tü36 (24) directed against HLA-DR alone or associated with the Ii chain, 1B5 (25) directed against HLA-DR {alpha} AD1 (26) an anti-rat CD63 mAb (a gift from Dr R. P. Siraganian, NIH, Bethesda, MA), LY1C6 an anti-rat lysosome-associated membrane protein (Lamp1) mAb (a gift from Dr W. Hunziker, Epalinges, Switzerland) and 1A12 an anti-rat CD81 mAb (27) (a gift from Dr J. P. Kinet, Harvard University, Boston, MA). Secondary antibodies included horseradish peroxidase-, FITC-, phycoerythrin (PE)- or Texas Red-coupled F(ab')2 fragments and were obtained from Jackson ImmunoResearch (Jackson Laboratory, West Grove, PA). The HA306–318 peptide (PKYVKQNTLKLAT) (12) was synthesized by Synt:em (Nime, France).

Cells
RBL-2H3 (28) and RBL-derived cell lines, HOM2, THA1.7, and Jurkat T cells were grown in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 0.1% ß-mercaptoethanol and 2% sodium pyruvate. The B-EBV cell line HOM2 is homozygous for HLA-DR1 (29). THA1.7 are Jurkat T cells transfected with constructs encoding the {alpha} and ß chains of a DR1–HA(306–318)-specific TCR (30). Jurkat T cells were kindly provided by Dr O. Acuto (Institut Pasteur, Paris, France). RBL cells expressing I-AbIi and TH30 T cell hybridoma were previously described (14). The murine immature DC line D1 (H-2B) and BMD8 (H-2D) were kindly provided by Dr P. Ricciardi-Castagnoli (Centre of Cellular and Molecular Pharmacology, Milan, Italy), and cultured as previously described (15).

DR expression in RBL cells
The cDNAs used for stable transfection in RBL-2H3 cells were inserted downstream from a Sr{alpha} promoter (31) in expression vectors carrying resistance genes for hygromycin B (NTH2), neomycin (NTNeo) and zeocin (NTZeo). The HLA-DR1 {alpha} chain cDNA (32) was subcloned into the NTNeo vector, the HLA-DR1 ß chain cDNA (33) into the NTHygro vector and the IiHA cDNA (13) into the NTZeo vector. The resulting {alpha}, ß and IiHA constructs were then used to transfect the RBL-2H3 cell line as previously described (34). Briefly, cells were electroporated at 260 V, 975 µF with 50 µg of linearized plasmid, using a BioRad (Marnes la Coquette, France) Gene Pulser II. Two days after transfection, the cells were transferred to selection medium. Surviving cells were cloned by limiting dilution. Clones were analyzed for surface MHC class II expression by cytofluorometry with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).

Flow cytometry analysis
Cells were washed in 3% FCS/0.1% NaN3 in PBS (FACS buffer) prior to staining as previously described (14). Flow cytometry analysis was performed with a FACSCalibur machine using CellQuest software (Becton Dickinson). As exosomes are too small for FACScan analysis, we bound the exosomes to surfactant-free white aldehyde/sulfate latex beads (3.8 µm diameter) and concentrated at 1.5 x 104 beads/µl (Interfacial; Dynamics, Portland, OR) (35). We incubated 10 µl beads (corresponding to 1.5 x 105 beads) with 30 µg exosomes for 10–15 min and then for 1 h at room temperature in a final volume of 1 ml PBS. Glycine (100 mM) in PBS was added to the beads, which were then incubated for 30 min at room temperature to saturate the remaining binding sites. Beads were washed twice with FACS buffer, and analyzed with a FACSCalibur and CellQuest software.

Antigen-presentation assays
APC (5 x 104) were incubated with or without the HA peptide 5 µM in the presence of 5 x 104 THA1.7 or Jurkat T cells/well for 24 h. Each experiment was performed in duplicate. We removed 50 µl of supernatant and froze it at –80°C for 1 h. We measured IL-2 release from the T cell hybridoma by monitoring [3H]thymidine incorporation in the IL-2-dependent CTL-L2 cell line. Cells were harvested after an additional 6 h incubation in the presence of 0.25 µCi [3H]thymidine/well. Each point corresponds to the mean of duplicate samples, which differed by <5%.

For presentation assays with exosomes and exosomes cross-linked to beads, 5 x 104 THA1.7 or Jurkat T cells/well were incubated with the corresponding exosome preparation and incubated with or without the HA peptide (0–10 or 50 µM). For exosome antigen-presentation assays in the presence of D1 DC, exosomes derived from RBL DR1 or RBL DR1IiHA were incubated with 5 x 104 THA1.7 cells in the presence or absence of the HA peptide, with or without 5 x 104 D1 cells. For exosome antigen-presentation assays in the presence of BMD8 DC, exosomes derived from RBL I-Ab Ii were incubated with 5 x 104 TH30 cells in the presence of the I-E {alpha} 52–68 peptide (10 µM) with 5 x 104 BMD8 cells. After 24 h, IL-2 release was assayed as described above (36).

Exosome purification
Exosomes were prepared from supernatants of RBL-2H3 cells and derived cell lines, generally degranulated by incubation with a calcium ionophore (1 µM ionomycin or A23187) for 30 min at 37°C (37) or by cross-linking of Fc{epsilon}RI as previously described (38). Briefly, cells were incubated with anti-DNP IgE for 20 h at 37°C and degranulation was then triggered by adding 10 µg/ml DNP-BSA and incubating for various periods of time. Exosomes were isolated as previously described (5). Briefly, cell supernatants were successively centrifuged at 300 g for 10 min, 1200 g for 20 min, 3000 g for 30 min and finally 70,000 g for 1 h. The exosome pellet was washed in PBS, centrifuged at 70,000 g for 1 h and resuspended in PBS and protein concentration determined by Bradford assay (BioRad).

For the immunoisolation of exosomes, 5 µl latex beads (corresponding to 0.75 x 105 beads) were incubated overnight with 15 µg of purified anti-CD63 mAb at 4°C. Binding sites were saturated by incubation in 100 mM glycine in PBS for 30 min at room temperature and in the beads were then washed in FACS buffer. We incubated 10 µl beads with 30 µg purified exosomes (or FCS as negative control) and carried out labeling for FACS analysis as described above for exosomes directly coupled to beads.

Sucrose gradient
RBL DR1 exosomes (100 µg) were resuspended in 2 ml 2.5 M sucrose, 20 mM HEPES/NaOH, pH 7.2. A linear sucrose gradient (2.0–2.5 M sucrose, 20 mM HEPES/NaOH, pH 7.2) was layered on top of the exosome suspension in a SW41 tube (Beckman Instruments, Gagny, France). Gradients were centrifuged for 15 h at 100,000 g and a 1-ml fraction was collected from the top of the tube. Densities were evaluated using a refractometer. Membranes were collected from the collected fractions after centrifugation at 70,000 g for 1 h at 4°C in TLA 100.4 tubes (Beckman Instruments). Exosome pellets were solubilized in reducing Laemmli buffer and heated at 95°C for 5 min before analysis by SDS–PAGE and Western blotting.

Western blotting
Cells were lysed in 0.5% Triton, 300 mM NaCl, 50 mM Tris, pH 7.4 in PBS supplemented with 10 µg/ml each of leupeptin, chemostatin, aprotinin, pepstatin, N-ethyl maleinide and 1 mM PMSF. Cell or exosome lysates were diluted in reducing Laemmli buffer and heated at 95°C for 5 min before analysis on SDS–12% polyacrylamide gels. Proteins were transferred to a PVDF membranes (Millipore, Bedford, MA). The membranes were incubated in blocking solution, then with IB5 [an anti-DR mAb (25)] and finally with horseradish peroxidase-labeled donkey anti-mouse IgG secondary antibody (Jackson Laboratory). Antibody binding was detected by chemiluminescence, using an ECL kit (Boehringer Mannheim, Mannheim, Germany).

Immunoelectron microscopy
RBL DR1 cells (degranulated or not) were fixed by incubation in a mixture of 2% paraformaldehyde in phosphate buffer 0.2 M, pH 7.4 and 0.125% glutaraldehyde for 2 h at room temperature. Fixed cells were processed for ultrathin sectioning and immunolabeling as previously described (6). Cells were washed with phosphate buffer 0.2 M, pH 7.4 and phosphate buffer 0.2 M, pH 7.4/50 mM glycine, and were then embedded in 7.5 % gelatin. Small blocks were infiltrated with 2.3 M sucrose at 4°C for 2 h and then frozen in liquid nitrogen. Ultrathin cryosections prepared with a Leica ultracut FCS (Wien, Austria) were retrieved in a mixture of 2% methylcellulose and 2.3 M sucrose (v/v), and indirectly immunogold labeled by incubation with the rat monoclonal anti-HLA-DR1 antibody L243 followed by a rabbit anti-mouse antibody (Dako, Copenhagen, Denmark). Antibodies were detected with Protein A coupled to 10-nm gold particles (purchased from Dr J. W. Slot, Department of Cell Biology, Utrecht University, The Netherlands). The sections were contrast stained, embedded in a mixture of methylcellulose and uranyl acetate, and viewed under a CM120 Twin Phillips electron microscope (Eindhoven, The Netherlands). For electron microscopy of the isolated exosomes, the pellets from the 70,000 g centrifugation were placed on Formvar carbon-coated electron microscopy grids, fixed as above, immunolabeled and stained using the method described for ultrathin cryosections. Exosomes from RBL I-AbIi cells were labeled in similar conditions using M5/114 mAb (14)


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
RBL cells expressing HLA-DR1 chains together with the IiHA construct are efficient APC
To generate mast cell lines expressing MHC class II–peptide complexes, we transfected the RBL cell line RBL-2H3 with cDNAs encoding HLA-DR1 {alpha} and ß chains with or without a previously described IiHA construct (Fig. 1A). We failed to detect any endogenous MHC class II in our RBL-2H3 clone using the anti-rat MHC class II, OX6 (data not shown). The core CLIP sequence of this human Ii-derived construct has been replaced by the sequence encoding the antigenic HA306–318 peptide of influenza virus HA (13). After selection and cloning, one clone for each combination was selected on levels of surface MHC class II expression. RBL DR1 cells, RBL DR1IiHA cells and HOM2 B-EBV cells displayed similar levels of surface expression of MHC class II molecules, as determined by cytofluorimetry (Fig. 1B).



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Fig. 1. RBL-2H3 cells expressing DR1 with or without the IiHA construct stimulate specific T cells. (A) Schematic representation of wild-type Ii and the IiHA construct (13). The p33 isoform of human wild-type Ii is represented, indicating the transmembrane region (TM) and the two N-linked carbohydrate side chains (positions 114 and 120). The sequence of the CLIP region (residues 81–104) is shown below. The sequence encoding amino acids (87–102) of the CLIP region was deleted and replaced by the sequence encoding the HA (306–318) (12), as shown below. (B). RBL-2H3 cells expressing HLA-DR1 with (RBL DR1IiHA) or without (RBL DR1) the IiHA construct, and the B-EBV cell line HOM2 were tested for surface expression of MHC class II molecules by cytofluorimetry, using Tü36, an anti-HLA-DR mAb (black line). Gray bars correspond to negative control staining obtained with the secondary antibody only. Levels of surface MHC class II were similar in all three cell lines. (C) RBL DR1, RBL DR1IiHA, HOM2 cells and parental RBL cells were assayed for presentation of the HA peptide to the THA1.7 T cell line (black symbols) specific for HLA-DR1/HA (306–318) complexes. Jurkat T cells (gray symbols) with unknown specificity were used as a negative control. T cell stimulation was measured using the CTL-L2 assay and was plotted as a function of peptide concentration. (D) Antigen-presentation assays were carried out as described above, except that we tested the stimulation of THA1.7 by various numbers of RBL DR1, RBL DR1IiHA and HOM2 in the presence (closed symbols) or absence (open symbols) of 10 µM HA peptide. T cell stimulation, measured by CTLL-2 assay, was plotted as a function of the number of APC/well. Representative results from three experiments, performed in triplicate.

 
We then tested the ability of these cells to stimulate Jurkat T cells expressing or not a specific TCR recognizing HLA-DR1–HA306–318, known as THA1.7. The cells were incubated with various concentrations of HA peptide and IL-2 secretion by T cells was measured in the supernatants. Both B-EBV Hom2 and RBL DR1 cells efficiently stimulated the THA1.7 cell line on addition of HA peptide, whereas no stimulation of non-specific Jurkat T cells was observed in the same conditions (Fig. 1C). Parental RBL cells were not able to stimulate THA1.7 cells upon addition of HA peptide (Fig.1C). Interestingly, the concentration of HA peptide required to generate half-maximal stimulation was similar for HOM2 and RBL DR1 cells (~0.1 µM). As expected, RBL DR1IiHA cells stimulated THA1.7-specific T cells in the absence of HA peptide, whereas non-specific Jurkat cells were not stimulated. Furthermore, the addition of HA peptide to RBL DR1IiHA cells did not increase THA1.7 stimulation (Fig. 1C). We further investigated HA loading of RBL DR1IiHA cells by determining the number of presenting cells required to stimulate THA1.7 cells in the presence or absence of a saturating concentration of HA peptide (5 µM). Upon addition of HA peptide, RBL DR1 cells gave half-maximal stimulation with as little as 50 cells/well, whereas HOM2 cells required ~20 times more cells to induce a similar level of THA1.7 stimulation. The ability of RBL DR1IiHA cells to stimulate THA1.7 cells was independent of the presence of peptide and half-maximal stimulation was achieved with ~30 cells/well. Therefore, RBL DR1IiHA cells were efficiently loaded with the endogenous HA peptide from the IiHA construct and these cells were competent for antigen presentation.

RBL cells secrete vesicles bearing HLA-DR1 molecules
As MHC class II molecules accumulated in the secretory granules of mast cells, we analyzed the distribution of HLA-DR molecules on ultrathin cryosections of RBL DR1 cells by immunogold labeling with the anti-DR antibody, L243. MHC class II molecules were mostly detected in intracellular compartments similar to multivesicular bodies (MVB) containing large numbers of vesicles, but faint labeling was also observed at the plasma membrane (Fig. 2A). The segregation of CD63, Lamp1 and serotonin, together with HLA-DR1, in these MVB (data not shown) suggests that human MHC class II molecules accumulated in the secretory granules of RBL2H3 cells, as previously reported by our group (14). We therefore determined whether mast cell degranulation induced the release of small vesicles bearing MHC class II.



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Fig. 2. RBL DR1 cells secrete MHC class II+ microvesicles. (A) Ultrathin cryosections of RBL DR1 were immunogold labeled with mAb L243, recognizing HLA-DR {alpha}ß complexes devoid of the Ii chain, detected with 10-nm gold particles coupled to Protein A (PAG 10). MHC class II molecules were detected in numerous compartments, especially MVB containing numerous internal vesicles. Bar = 200 nm. (B) RBL DR1 cells (107 per point) were degranulated by incubation with IgE anti-DNP (overnight saturation at 37°C) and 10 µg/ml DNP-BSA, or by incubation with 1 µM A23187 or 1 µM ionomycin at 37°C for various periods of time. Supernatants were collected then ultracentrifuged at 70,000 g and analyzed by Western blotting with an anti-HLA-DR {alpha} mAb. Lysates of 2 x 105 to 105 cells were run on the same gel. (C) RBL DR1 cells were stimulated by cross-linking Fc{epsilon}RI as in (B), then ultrathin cryosections were labeled as described in (A). Bar = 200 nm. (D) Supernatants of RBL DR1cells incubated with 1 µM ionomycin for 30 min were ultracentrifuged at 70,000 g and the resulting pellet analyzed by whole-mount electron microscopy. MHC class II molecules were detected as in (A).

 
RBL cell degranulation was induced by cross-linking IgE receptors with IgE anti-DNP/DNP-BSA immune complexes or by adding a calcium ionophore (A23187 or ionomycin). Supernatants were collected after stimulation for various periods of time, ultracentrifuged and the pellets analyzed by Western blotting with an anti-HLA-DR {alpha} mAb. All cell lysates were loaded on the same gel and subjected to electrophoresis to estimate the number of DR molecules associated with these vesicles. The amount of DR1 {alpha} chain present in the supernatant gradually increased over time, reaching a maximum at 30 min (Fig. 2B). The addition of either of the calcium ionophores induced levels of DR1 secretion twice as high as those induced by IgE receptor cross-linking. Comparison of the DR1 {alpha} chain signals observed for vesicle preparations (obtained from 107 cells) with those obtained from cells lysates (1–2 x 105 cells) indicated that almost 2% of total MHC class II molecules were secreted upon mast cell degranulation (Fig. 2B).

To confirm that MHC class II-containing vesicles were secreted, we prepared ultrathin cryosections of RBL DR1 cells following IgE receptor cross-linking and analyzed them by immunogold labeling with the anti-DR antibody, L243. Exocytosis profiles with DR1-labeled vesicles were detected (Fig. 2C). We purified vesicles from the supernatant of stimulated RBL DR1 cells by ultracentrifugation and analyzed them directly by whole-mount electron microscopy. Heterogeneous populations of vesicles 60–80 nm in diameter were detected (Fig. 2D). Most had single membranes, but those with double membranes had denser lumina and frequently displayed positive HLA-DR labeling (Fig. 2D). Contaminations of vesicles preparations by endoplasmic reticulum membranes were excluded by the lack of any endoplasmic reticulum protein (calnexin) in exosomes pellets (data not shown).

Thus, RBL DR1 cells can secrete large numbers of small vesicles carrying HLA-DR1 molecules, which have morphological features in common with previously described exosomes.

Characterization of the secreted vesicles
To determine whether the vesicles secreted by RBL DR1 cells were indeed exosomes, vesicles obtained by ultracentrifugation were subjected to sucrose gradient sedimentation and the MHC class II content of each fraction was analyzed by western blotting. Most of the MHC class II molecules were found in fractions with a sucrose density of 1.16–1.21 g/ml (Fig. 3A). Thus, the vesicles bearing MHC class II sedimented at a slightly higher density than that previously reported for exosomes (1.135 g/ml). As these vesicles originated from MVB, as described for exosomes, we analyzed their endocytic/lysosomal pathway protein content. The vesicles were too small for direct analysis by cytofluorimetry and most of the antibodies against rat lysosomal proteins do not work in Western blotting experiments. We therefore analyzed the protein composition of the vesicles by coupling the vesicles to 3.8-µm latex beads and immunolabeling with FITC-conjugated specific antibodies. FACScan analysis of the vesicles attached to the beads revealed that they contained large amounts of CD63, CD81 and MHC class II, but almost no Lamp1 (Fig. 3B). A key feature of exosomes is the association of MHC class II molecules with tetraspanin on the same vesicle. We investigated whether the vesicles secreted by RBL cells contained CD63 and DR1, by coating beads with the anti-CD63 mAb AD1 and incubating them with vesicles prepared from RBL DR1 cells. We then washed the beads and assessed the amount of DR1 associated with the beads using a PE-conjugated anti-DR mAb. Significant specific labeling was observed, suggesting that a population of vesicles carried both DR and CD63 (Fig. 3C).



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Fig. 3. Microvesicles secreted by RBL DR1 correspond to exosomes. (A) Membranes isolated by ultracentrifugation from supernatants of RBL DR1 cells were loaded onto a linear sucrose gradient. Membranes were collected from the gradient fractions and analyzed by Western blotting for HLA-DR expression with the mAb 1B5 (upper panel). Results were quantified by analyzing scanned films with NIH imaging software. The densities of the various gradient fractions are indicated at the bottom. The lower panel shows the estimated amount of HLA-DR {alpha} chain as a function of sucrose density. (B) We coupled 30 µg of microvesicles or of BSA to 10 µl latex beads and tested for surface expression of CD63 (AD1 mAb), Lamp1 (LY1C6 mAb), CD81 (1A12 mAb), and mature HLA-DR1 (L243 mAb) and total HLA-DR1 (Tü36 mAb). (C) Latex beads pre-incubated with the anti-CD63 mAb AD1 were used to immunoisolate microvesicles secreted from RBL DR1 cells. Beads were further analyzed by flow cytometry for HLA-DR surface expression using PE-coupled L243 and Tü36 mAb. In (B) and (C), gray histograms correspond to the same beads incubated with PE-coupled isotypes controls.

 
Upon stimulation, RBL cells secrete vesicles of 60–80 nm in diameter, which float on sucrose gradients, and bear CD63, HLA-DR and CD81 molecules. We therefore concluded that these vesicles had features similar to those previously described for exosomes in B cells, DC and mast cells.

RBL exosomes carry functionally competent DR molecules
As we were able to produce homogeneous and reliable preparations of exosomes bearing MHC class II molecules from RBL DR1 and RBL DR1IiHA cells, we used these exosomes to investigate the activation of specific T cells in vitro. We investigated whether DR1+ exosomes directly activated T cell by adding various amounts of exosomes to THA1.7 cells or Jurkat T cells and measuring IL-2 secretion into the cell supernatant. Both exosomes from RBL DR1 cells in the presence of saturating amounts of the HA peptide and exosomes from RBL DR1IiHA cells without peptide stimulated THA1.7 cells. No THA1.7 cell stimulation was observed with empty DR1 exosomes and DR1–HA exosomes did not stimulate irrelevant Jurkat T cells (Fig. 4A). However, the stimulation of specific T cells with HA-loaded DR1 exosomes was not efficient because THA1.7 cell stimulation was detectable only upon the addition of a large quantity of exosomes (50 µg/well).



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Fig. 4. Exosomes of RBL DR1 and RBL DR1IiHA cells stimulated specific T cells. A. Various quantities of exosomes from RBL DR1 and RBL DR1IiHA cells were incubated with THA1.7 cells, with (closed symbols) or without (open symbols) 10 µM HA peptide. T cell stimulation was measured by a CTL.L2 assay. (B) Exosomes from RBL DR1 and RBL DR1IiHA cells were cross-linked to beads (30 µg exosomes/10 µl beads) and incubated with THA1.7 or Jurkat T cells in the presence of various concentrations of the HA peptide. T cell stimulation, measured by a CTL-L2 proliferation assay, was plotted as a function of peptide concentration. (C) Various amounts of exosomes derived from RBL DR1 or RBL DR1IiHA cells were cross-linked to 10 µl beads and assayed for their capacity to stimulate THA1.7 T cells in the presence of saturating amounts of HA peptide (10 µM). T cell stimulation was evaluated as described in (B). The results shown are representative of three independent experiments.

 
Soluble MHC class II molecules are known to stimulate specific T cells inefficiently. There might therefore be too few MHC class II molecules on the surface of exosomes to stimulate T cells. DR1 or DR1IiHA exosomes were therefore cross-linked to latex beads (3.8 µm diameter) and used in a similar antigen-presentation assay. After cross-linking to beads, exosomes from RBL DR1 cells efficiently stimulated the THA1.7 cell line only in the presence of saturating amounts of peptide, whereas no stimulation of Jurkat T cells was observed in the same conditions (Fig. 4B, left panel). Similar results were obtained with exosomes from RBL DR1IiHA cells in the absence of peptide (Fig. 4B, right panel), confirming that the HLA-DR molecules carried by these exosomes are effectively loaded with HA peptide. In both cases, 0.3 µg of exosomes was enough to induce the half-maximal stimulation of THA1.7 cells. Finally, we carried out a peptide titration of RBL DR1 exosomes cross-linked to beads. Half-maximal stimulation of the THA1.7 cell line was obtained at a concentration of 1 µM HA peptide (Fig. 4C), which is only 10 times higher than that with RBL DR1 cells (Fig. 1C). These data indicate that exosomes bearing HA/DR1 complexes specifically stimulate T cells. However, the density of MHC class II molecules at the surface of exosomes, the size or composition of exosomes may be limiting for the efficient induction of T cell stimulation in vitro.

Exosomes efficiently stimulate specific T cells after transfer to DC
DC are classical antigen-presenting cells involved in T cell priming. As exosomes bearing MHC class II–peptide complexes did not efficiently stimulate specific T cells, we investigated the role of DC in exosome-dependent T cell stimulation. The murine immature DC line D1 (15) was used in an antigen-presentation assay in which various amounts of DR1 exosomes were added to D1 cells in the presence or absence of the HA peptide. DR1 exosomes efficiently stimulated THA1.7 cells if 10 µM HA peptide was added (Fig. 5, left panel). Similarly, exosomes from RBL DR1IiHA cells efficiently stimulated the THA1.7 cell line independently of the presence of HA peptide (Fig. 5, right panel). Both types of exosomes stimulated T cells if 0.3–0.6 µg of exosome preparation was added to each well, whereas in the absence of DC, 100 times more exosomes were required to achieve similar levels of T cell stimulation (Fig. 4A) and no T cell stimulation was achieve when similar exosomes were incubated in the same conditions with B cells (data not shown). Thus, DC facilitate the induction of efficient exosome-dependent T cell stimulation.



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Fig. 5. Exosomes can transfer MHC class II–peptide complexes to DC. (A) D1, a murine DC line, was incubated overnight with various quantities of exosomes from RBL DR1 or RBL DR1IiHA cells and the THA1.7 cell line. T cell stimulation was evaluated in the presence or absence of 5 µM HA peptide, as described in Fig. 1(C). The results shown are representative of three independent experiments. (B, left panel) Exosomes from RBL I-AbIi cells were analyzed by whole-mount electron microscopy. MHC class II molecules were detected with mAb M5/114, then immunogold labeling was obtained by incubation with rabbit anti-rat IgG antiserum and 10-nm gold particles coupled to Protein A (PAG 10). Bar = 200 nm. (B, right panel) BMD8 DC line (black circles) was incubated overnight with various quantities of I-Ab exosomes, TH30 T cell hybridoma and 10 µM I-Ed {alpha}52–68 peptide. T cell stimulation was evaluated as described in Fig. 1(C). Similar experiments were performed in the absence of BMD8 murine DC (black squares). (C) D1 cells or THA1.7 cells were incubated overnight with 30 µg/ml DR1 exosomes and MHC class II molecules were detected by flow cytometry using L243 mAb.

 
Similar results were obtained with murine MHC class II+ exosomes upon incubation with murine DC. Exosomes were prepared from RBL cells expressing I-Ab Ii complexes and were then characterized for MHC class II expression. By electron microscopy, whole-mount I-AbIi exosomes preparation displayed heterogeneous populations of vesicles similar in size and morphology to those isolated from RBL DR1 cells (Fig. 5B, left panel). Immunolabeling with M5/114 mAb detected 10–20% of class II+ vesicles. Various amounts of I-Ab exosomes were added to murine BMD8 immature DC, expressing I-Ad but not I-Ab class II allele, in the presence of the I-Ed {alpha} 52–68 peptide (10 µM) and TH30 cells, a T cell hybridoma specific for I-Ab/I-Ed {alpha} 52–68 peptide complexes. T cells stimulation was dependent of the addition of I-Ab exosomes on BMD8 DC (Fig. 5B, right panel), whereas no TH 30 stimulation was observed in the absence of I-Ed {alpha} 52–68 peptide or BMD8 DC (Fig. 5B, right panel). T cells stimulation by class II+ exosomes required therefore incubation of exosomes with DC.

These results suggested that exosomes were transferred to DC. We investigated this possibility by incubating D1 cells for various periods of time with 50 µg of DR1 exosomes at 37°C. We then detected DR1 molecules by FACScan analysis with L243 mAb. DR1 molecules were first detected at the surface of DC after 1 h (data not shown) and they increased in number thereafter, reaching a maximum at 18 h (Fig. 5C). If THA1.7 cells (Fig. 5C) or mouse B lymphocytes (not shown) were incubated in the same conditions with similar concentrations of DR1 exosomes in the presence of HA peptide, no L243+ MHC class II molecules were detected at the surface of the cells. Thus, the stimulation of T cells by MHC class II+ exosomes requires the incubation of these exosomes with DC.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We show here that RBL cells expressing HLA-DR1 molecules secrete vesicles able to stimulate MHC class II-restricted T cells. These vesicles were probably produced by MVB, as suggested by immunoelectron microscopy profiles, and were 60–80 nm in diameter. They had a density of 1.2 g/ml on a sucrose gradient, and expressed CD63, HLA-DR and CD81 molecules. On the basis of all these criteria, these microvesicles resembled the exosomes produced by human B cells originally described by Raposo et al. (5). One important advantage of our cellular model is that exosome secretion can be induced very efficiently, resulting in reliable exosome production.

To produce exosomes carrying a single type of MHC class II–peptide complex, we made use of our previously characterized construct derived from the Ii chain (13). The core CLIP sequence of this construct has been replaced by a sequence encoding the HA306–318 antigenic peptide. We found that RBL cells transfected with this construct together with constructs encoding the DR {alpha} and ß chains expressed mainly DR1–HA complexes at the cell surface. The addition of exogenous HA peptide did not increase the capacity of RBL DR1IiHA to stimulate specific T cells. A very similar construct has been characterized and used in HLA-DR1-transfected human embryonic kidney cell line 293, generating similar results. Van Bergen et al. also eluted peptides from the DR1 molecules and showed that most contained the HA peptide, with ragged ends corresponding to the flanking sequences of the Ii construct (16).

Both RBL DR1 and RBL DR1IiHA cells were remarkably efficient at stimulating specific T cells. RBL DR1 cells achieved half-maximal stimulation of THA1.7 cells with 1/10 the amount of HA peptide required to achieve the same level of stimulation by HOM2, a B-EBV cell line homozygous for DR1. This suggests that the DR1 molecules in RBL cells were more easily loaded with peptide than those in HOM2 cells, as MHC class II levels at the surface were similar for the two cell types. The loading of MHC class II with exogenous peptide was recently shown to require active recycling of MHC class II. Differences in recycling capacity may account for the observed discrepancy. It is also possible that the expression of human DR molecules in a rat cell line (RBL DR1) may have resulted in a lower level of peptide occupancy or the binding of peptides with lower affinity. Titration of the number of RBL DR1IiHA or RBL DR1 cells in the presence of saturating amounts of HA peptide revealed that as few as 100 cells were able to generate half-maximal stimulation of 5 x 104 T cells, whereas 1000 HOM2 cells were required to generate a similar response. This provides support for the idea that RBL DR1 cells are easily loaded with exogenous peptide. RBL DR1IiHA and RBL DR1 generated similar maximal responses in the presence of HA peptide, supporting the notion that, in both situations, the majority of the DR1 molecules were loaded with HA peptide. We previously showed that murine MHC class II molecules (I-Ab) may be expressed as functional molecules in RBL cells (14). Therefore, RBL cells are a useful model for expressing MHC class II complexes loaded with a given peptide, supplied exogenously or introduced via an Ii chain-derived construct.

We found that MHC class II molecules were present on exosomes secreted by RBL DR1 cells exposed to appropriate stimuli. We studied their capacity to stimulate T cells and observed that the threshold at which T cells responded increased by a factor of 2 log, for a given quantity of exosomes, if the exosomes were chemically cross-linked to beads. This suggests that there are too few MHC class II molecules per exosome to create an immunological synapse on the target T cell. Indeed, it has been established that a minimum of ~10 MHC class II molecules are needed for efficient reorganization of the TCR at the T cell surface, which in turn leads to a signal transduction cascade activating the T cell (17,18).

Interestingly, immature mouse DC, like beads, increased the capacity of exosomes to stimulate T cells by a factor of 2 log. Other cell types, such as B cells and mast cells, had no such effect (not shown). The transfer of vesicles from APC to T cells following specific interaction has been reported before (19). It is unclear whether exosomes play a physiological role in vivo. However, immunization with exosomes proved to be very efficient in a mouse in vivo tumor model (8). The significant enhancement by DC of the stimulatory capacity of MHC class II molecules carried by exosomes in vitro suggests that DC may have a similar function in vivo. They may capture circulating exosomes and present them to T cells. Indeed, follicular DC (FDC) have been demonstrated to bind MHC class II+ microvesicles, which closely resemble exosomes in protein content and morphology, specifically to their surface (20). Such a role would also be compatible with the reported ability of FDC to trap circulating viral particles, similar in size to exosomes, at their surface (21). It is unclear whether DC fuse with exosomes and process them, and the possibility of such presentation in vivo for the maintenance of peripheral tolerance and induction of specific immune responses is also unknown. However, our model should provide a useful tool for addressing these important issues in future research.


    Acknowledgements
 
We would like to thank D. Tenza for technical assistance with electron microscopy, S. Morchoisne for the subcloning of THA1.7 cells and P. Veron for advice on FACScan analysis with latex beads. We would also like to thank Dr C. Hivroz and N. Blanchard for advice concerning Jurkat T cells. Special thanks are addressed to Dr C. Thery and Dr. S. Amigorena for stimulating discussion and helpful comments on exosomes. This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, from Institut Curie, from APCells and from the comité de Paris de la Ligue Nationale contre le Cancer. H. V.-S. holds a fellowship from the Ministère des Universités et de la Recherche. P. S.-C. was supported by a grant from the Agence Nationale de la Recherche contre le SIDA.


    Abbreviations
 
APC—antigen-presenting cell

BMMC—bone marrow-derived mast cell

CLIP—class II invariant chain peptide

DC—dendritic cell

EBV—Epstein–Barr virus

FDC—follicular dendritic cell

HA—hemagglutinin

Lamp—lysosome-associated membrane protein

MIIC—MHC class II compartment

MVB—multivesicular body

PE—phycoerythrin

RBL—rat basophilic leukemia


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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