Exosomes bearing HLA-G are released by melanoma cells

Exosomes Bearing HLA-G are Released by Melanoma Cells Be´atrice Riteau, Florence Faure, Catherine Menier, Sophie Viel, Edgardo D. Carosella, Se`bastian Amigorena, and Nathalie Rouas-Freiss ABSTRACT: Tumor cells release membrane vesicles, named exosomes, capable of specific cytotoxic T-lymphocyte activation by transferring tumor antigens to dendritic cells. By contrast, the nonclassical human leucocyte antigen (HLA)-G class I molecule displays immunotolerant properties and can be ectopically expressed by tumor cells, thereby allowing their escape from immunosurveillance. We describe here that a melanoma cell line, named Fon, established from an HLA-G–positive melanoma biopsy, spontaneously expressed high levels of the HLA-G1 membrane-bound isoform. Exosomes released by Fon cells were purified and analyzed both for their density on sucrose gradient and their protein composition by Western blotting and flow cytometry. Besides the expression of wellABBREVIATIONS APC antigen-presenting cell CTL cytotoxic T lymphocyte HLA human leucocyte antigen

INTRODUCTION Exosomes are small membrane vesicles secreted by a multitude of cell types, including reticulocytes [1–3], platelets [4], mast cells [5], B and T lymphocytes [6 –9], and dendritic cells [10]. Tumor cells were also recently described as releasing exosomes [11, 12]. These 60- to 90-nm vesicles originate from fusion of late multivesicular endosomes/lysosomes with the plasma membrane. Of particular interest is the recent finding that exosomes released by tumor cells contain and transfer tumor antigens to dendritic cells allowing cytotoxic T lymphocyte

From the Service de Recherches en He´mato-Immunologie, CEA-DSVDRM, Hoˆpital Saint-Louis, IUH, Paris, France (B.R., C.M., E.D.C., N.R-F.), and INSERM U520, Institut Curie, Paris, France (F.F., S.V., S.A.). Address reprint requests to: Nathalie Rouas-Freiss, Service de Recherches en He´mato-Immunologie, CEA-DSV-DRM, Hoˆpital Saint-Louis, IUH, 1 avenue C. Vellefaux, 75010 Paris, France; Tel: ⫹33 (1) 5372-22 27; Fax: ⫹33 (1 ) 4803-1960; E-mail: [email protected]. Received July 11, 2003; accepted August 14, 2003. Human Immunology 64, 1064 –1072 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Inc.

described proteins such as Lamp-2, notably, these melanoma-derived exosomes bore HLA-G1. In addition, exosomes harboring HLA-G1 were secreted by the HLAG–negative M8 melanoma cells transfected with the HLA-G1 cDNA. Thus, the presence of tolerogenic HLA-G molecules on melanoma-derived exosomes may provide a novel way for tumors to modulate host’s immune response. Human Immunology 64, 1064 –1072 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc. KEYWORDS: HLA-G; exosomes; tumor; immunosurveillance; tolerance; melanoma

Lamp mAb NK

lysosomal associated membrane protein monoclonal antibody natural killer

(CTL) cross-priming [11, 12]. Thus, tumor-derived exosomes could be useful for antitumoral treatment. Tumor cells have developed a number of strategies that enable them to escape from immune detection. One of them is the expression of inhibitory ligands such as the nonclassical human leucocyte antigen (HLA) class I molecule HLA-G that mediates a negative signal through interaction with inhibitory receptors present on immunocompetent cells [13]. HLA-G is normally absent on healthy tissues except for trophoblast [14] and thymus [15]. Interestingly, both its transcription and protein expression are upregulated on some tumors cells, as demonstrated in biopsies from patients with melanoma [16 –18], breast cancer [19], renal carcinoma [20, 21], primary cutaneous lymphoma [22], lung cancer [23], glioma [24], epithelial cutaneous malignant lesions [25], and colorectal cancer [26]. In contrast to the data obtained with surgically removed tumor lesions, the detection of HLA-G on in vitro established tumor cell lines has 0198-8859/03/$–see front matter doi:10.1016/j.humimm.2003.08.344

Melanoma-Derived Exosomes Bear HLA-G

been mainly unsuccessful [27–30]. This is probably because of the loss of HLA-G expression during long-term cell culture. Consequently, up to now, one renal carcinoma cell line [20] and four glioma cell lines [24] have been found to display low HLA-G–membrane-bound expression. Recently, short-term ovarian carcinoma cell lines were described as exhibiting constitutive HLA-G surface expression that was gradually lost upon longterm in vitro propagation [31]. HLA-G can be expressed as seven HLA-G protein isoforms, four of which are membrane-bound (HLA-G1, -G2, -G3, and -G4) and three being soluble (HLA-G5, -G6 and -G7) [32]. These HLA-G isoforms exhibit immunomodulatory properties such as inhibition of natural killer (NK) cytolysis and CTL responses [33–37]. The full-length HLA-G1 isoform, which exhibits a structure similar to that of classical HLA class I molecules, interacts with at least three inhibitory receptors, immunoglobulin (Ig)-like transcript-2 (ILT-2 or CD85j) present on NK cells, T cells and antigen-presenting cells (APC) [38, 39], ILT-4 (CD85d) on APC [40], and p49/ KIR2DL4 (CD158d) on NK cells [41, 42]. In the present report, we investigated whether HLA-G was present in exosomes of HLA-G–positive tumor cells, thus representing a potential additional pathway for these tumors to escape from immunosurveillance. We studied a melanoma cell line, named Fon, which was derived in vitro from an HLA-G–positive tumor lesion [43]. To our knowledge, this is the first description of a melanoma cell line expressing HLA-G1 at its cell surface and at a high level. Interestingly, this melanoma cell line secreted exosomes that also contained HLA-G1. The expression of HLA-G on both tumor cells and tumor-derived exosomes may have important insight in tumor progression and metastasis. MATERIALS AND METHODS Cell Lines and Antibodies Fon (i.e., T1Fon, according to the initial description [43]) and G2 Fon are melanoma cell lines derived from the primary lesion and the metastatic lymph node, respectively, as previously described [43]. M8 is an HLAG–negative melanoma cell line, as previously described [44]. Cells were maintained in RPMI 1640 medium (Sigma, St Quentin Fallavier, France) containing 10% FCS (Biological Industries). The M8-pcDNA cells (transfected with the control vector alone) and the M8HLA-G1 cells (transfected with the vector containing the HLA-G1 cDNA) were obtained as previously described and selected in media containing 100 ␮g/ml hygromicin (Sigma) [45]. The cells used were routinely tested for and found to be free of mycoplasma. The following mAbs (monoclonal antibodies) were

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used: W6/32, a mouse IgG2a anti-HLA class I heavy chain associated with ␤2-microglobulin (Sigma); 87G, a mouse IgG2a recognizing native HLA-G1 membranebound protein (kindly provided by D. Geraghty, Fred Hutchinson Cancer Research, Seattle, WA); 4H84, a mouse IgG1 anti–HLA-G free heavy chain (kindly provided by S. Fisher and M. McMaster, University of California, San Francisco, CA); HC10, a mouse IgG2a, anti–HLA-B, -C free heavy chain (kindly provided by H. Ploegh, Harvard University, Cambridge, MA); HMB45, a mouse IgG1 antimelanoma gp100 (Interchim); antilysosomal proteins CD107b (Lamp-2) (Pharmingen); antiCD63 (MoF11 clone kindly provided by Dr. Vincendau, University of Bordeaux II, Bordeaux, France); and anti-ER resident gp96 (SPA 850, Stressgen). Both 4H84 and 87G mAbs had been previously validated during the HLA-E, -F, and -G International Preworkshop [45]. Exosome Purification M8 transfectants and Fon cells were grown for 48 h in RPMI medium supplemented with 10% exosome-free FCS. Exosomes were then prepared from these supernatants and purified, as previously described [7]. Briefly, three successive centrifugations at 300 g (5 minutes), 1200 g (20 minutes), and 10,000 g (30 minutes) were performed to eliminate cells and debris, followed by two successive centrifugations for 1 hour at 70,000 g and one centrifugation at 100,000 g for 1 hour. The exosome pellet was washed at each time point in a large volume of phosphate buffered saline (PBS). The amount of exosomal proteins recovered in PBS was measured by Bradford assay (Bio-Rad). Western Blot Analysis Exosome proteins or total cell lysates were solubilized in Laemli sample buffer at 95 °C, under reducing conditions, and separated in 8% sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. Then, Western blot analysis was conducted as previously described [35]. Immunohistochemistry Analysis Deparaffinized tissue sections were subjected to epitope retrieval treatment by high temperature in 10 mM sodium citrate buffer (pH 6.0) using a commercial microwave to optimize immunoreactivity. Slides were then rehydrated for 5 minutes in PBS containing 0.05% saponin and 10 mM HEPES buffer. Endogenous peroxidase activity was quenched by treating sections for 5 minutes at room temperature with 3% hydrogen peroxide in water. Nonspecific binding was prevented by applying 30% human serum for 20 minutes before staining with the primary mAb for 30 minutes at room temperature. An isotype-matched antibody was used under similar conditions to control nonspecific staining.

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Immunostaining was evaluated on tissues using the DAKO EnVision ⫹ System, Peroxidase (AEC) (Dako, Trappes, France), as previously described [45]. Flow Cytometry Analysis Cells were washed in PBS and stained with the corresponding primary mAb in PBS 2% heat-inactivated fetal calf serum for 30 minutes at 4 °C. After washing, cells were subsequently stained with an F(ab⬘)2 goat antimouse IgG antibody conjugated with phycoerythrin (PE) (Beckman Coulter, Villepinte, France) for 30 minutes at 4°C. Control aliquots were stained with an isotypematched antibody to evaluate nonspecific binding to target cells. For flow cytometry analysis, exosomes were coupled to latex beads, as previously described [9]. From the dot plot representation of forward and side scatter, only single beads were gated for fluorescence analysis. Data acquisition and analysis were performed either on an EPICS XL flow cytometer using Expo-32 software (Beckman-Coulter) or a FACScan flow cytometer using Cell Quest software (Becton Dickinson). Flotation of Exosomes on a Continuous Sucrose Gradient Flotation of exosomes on a continuous sucrose gradient was performed as previously described, but in a SW41 rotor [7]. Fractions of the gradient were diluted in PBS and ultracentrifuged for 1 hour at 100,000 g. The pellet of each fraction was loaded on an 8% SDS gel for Western blot analysis. RESULTS AND DISCUSSION Whereas HLA-G proteins are detected in various primary and metastatic melanoma cells ex vivo, no HLA-G expression was observed at the surface of melanoma cell lines established in vitro after long-term culture. In spite of the difficulties to maintain HLA-G expression on tumor cell lines, we here describe that a melanoma cell line, called Fon, expresses a high level of cell surface HLA-G1 molecules. Indeed, by carrying out flow cytometry analysis, the Fon melanoma cell line was positively stained by the 87G mAb specific for the full-length HLA-G1 membrane-bound isoform [46] (Figure 1A). As a control, we used the HLA-G–negative M8 melanoma cell line that was transfected either with the vector alone (M8-pcDNA) or the HLA-G1 cDNA (M8-HLA-G1) [35]. As with the Fon melanoma cell line, M8-HLA-G1 cells exhibited HLA-G1 cell surface expression, whereas M8-pcDNA did not. HLA-G1 protein expression was confirmed by Western blot analysis using the 4H84 mAb, specific for an epitope located in the ␣1 domain common to all HLA-G isoforms [47]. A band at 39 kDa corresponding to the predictive molecular weight of

FIGURE 1 Analysis of human leukocyte antigen (HLA)-G protein expression in Fon cells. (A) primary melanoma Fon (Fon) cells, metastatic ganglionary Fon (G2 Fon) cells, M8HLA-G1 cells, and M8-pcDNA (negative control) cells were analyzed by flow cytometry after staining with the 87G mAb specific for the membrane-bound HLA-G1 isoform (bold histograms; light histograms are negative control obtained with isotypic irrelevant antibody). Fon cells spontaneously expressed HLA-G1 at a high level, similar to that of M8-HLAG1. The metastatic Fon cells also expressed HLA-G1 molecules. (B) Same amount of proteins from Fon, M8-HLA-G1, and M8-pcDNA whole cells were loaded on an 8% SDS gel followed by Western blotting using the 4H84 anti-HLA-G mAb. HLA-G1 was expressed in Fon cells and M8-HLA-G1 cells, but not in M8-pcDNA cells.

HLA-G1 was revealed in both Fon and M8-HLA-G1 cells, whereas no HLA-G was detected into the M8pcDNA control cell line (Figure 1B). To our knowledge, this is the first description of a melanoma cell line that constitutively expresses a high level of HLA-G1 at its cell surface. Notably, the level of ectopic expression of HLA-G1 in Fon cells is comparable with that of the HLA-G1-transfected M8 melanoma cell line. This patient developed a lymph node metastasis, and another tumor cell line, called G2 Fon, was established [43]. Flow cytometry analysis indicated that, as observed

Melanoma-Derived Exosomes Bear HLA-G

FIGURE 2 Immunohistochemical analysis of HLA-G, gp100, and HLA-B and -C expression in the cutaneous melanoma biopsy of Fon patient. Paraffin-embedded sections of cutaneous melanoma lesion from the Fon patient were stained either with the 4H84 mAb (anti–HLA-G), the HMB45 mAb (anti-gp100), the HC10 mAb (anti–HLA-B and -C), or a mouse immunoglobulin-G1 as negative control. Original magnification: (A) ⫻200; (B) ⫻100.

in the Fon cells, G2 Fon cells displayed HLA-G1 molecules at their cell surface (see Figure 1A). This result reveals that, in vivo, HLA-G expression may be maintained in time and space on melanoma cells developing outside their initial localization. This stable expression of HLA-G in both the primary and metastatic sites may be due to an intrinsic property of the malignant cells. For instance, HLA-G gene repression may be reversed by demethylation, leading to HLA-G protein expression [48]. Concomitantly, microenvironmental factors common to both tumor sites may also promote HLA-G protein expression on tumor cells. This observation is in accordance with a previous study showing HLA-G expression in autologous primary and metastatic melanoma biopsies [16]. In addition, immunohistochemical analysis of tissue sections of the cutaneous melanoma biopsy of Fon patient indicated that classical HLA-B and -C molecules were expressed in situ by all the different cell types, whereas HLA-G was only expressed by melanoma cells. Both HLA-G and PMel 17/gp100 stainings colocalized in melanoma cells (Figure 2). This result clearly shows the in vivo expression of HLA-G in the melanoma malignant cells. It is of note that healthy tissue surrounding the tumor lesion was negative for HLA-G (data not shown). The role of HLA-G in protecting tumor cells from host’s immunosurveillance was first demonstrated with human melanoma cells, in which the expression of HLA-G isoforms confers protection from NK lysis [44].

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This finding was supported by the recent description of several glioma cell lines in which HLA-G1 is cell surface– expressed either in a constitutive manner or after induction by interferon (IFN)-␥, and protects these tumors from lysis of alloreactive peripheral blood mononuclear cells [24]. Moreover, we recently showed that the MHC class I-like (MICA) triggering signal for NK cell tumor lysis can be counteracted by HLA-G1-mediated inhibitory signal [49]. Tumor cells such as melanomas are known to secrete exosomes that contribute to antitumor responses [11]. In the present study, we searched for the presence of the immunosuppressive HLA-G molecule on tumor-derived exosomes. Because HLA-G1 was expressed at the cell surface of Fon cells and M8-HLA-G1 cells, we investigated whether the exosomes secreted by these melanoma cells also contained HLA-G1 molecules. For this purpose, exosomes produced by Fon cells, M8-HLA-G1 cells, and by M8-pcDNA cells used as negative control, were analyzed by Western blot using the 4H84 anti– HLA-G mAb. As shown in Figure 3, the 4H84 mAb revealed a band at 39 kDa in both Fon- and M8-HLAG1– derived exosomes, but not in M8-pcDNA– derived exosomes, demonstrating that HLA-G1 proteins were present in exosomes secreted by HLA-G1–positive melanoma cell lines. To check the exosomal nature of the tumor-secreted vesicles, we investigated the presence of certain proteins known either to accumulate (i.e., Lamp-2, a lysosomal associated membrane protein), or to be absent (i.e., gp96, an ER-derived glycoprotein) in exosomes [11]. For this purpose, the same Western blots were hybridized with an anti-gp96 and then with an anti-CD107b (Lamp-2) mAbs. As shown in Figure 3, although exosomes secreted by Fon cells and M8 transfectants were enriched in Lamp-2, they were devoid of gp96 protein in contrast to the corresponding cell ly-

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FIGURE 3 Western blot analysis of human leukocyte antigen (HLA)-G, gp96, and Lamp-2 in Fon cells and M8 transfected cells and in the corresponding derived exosomes. Same amount of proteins from whole-cell lysates (Cells) or exosomes (Ex) (3 ␮g) from Fon cells, M8-HLA-G1 cells, and M8-pcDNA cells were run on a 8% SDS gel and then, analyzed by Western blot using the 4H84 mAb specific for HLA-G and the anti–ER-resident protein gp96 mAb, and then with the an anti–Lamp-2 mAb. HLA-G and Lamp-2 were present in M8HLA-G1 and Fon cell lysates and exosomes, whereas HLA-G was absent in M8-pcDNA. Gp96 was only present in cell lysates.

sates. This pattern of expression is in good agreement with the known protein composition of dendritic cellderived exosomes [50]. Moreover, the exosomes released by Fon cells accumulated classical HLA class I molecules and the MART-1 tumor antigen (data not shown). To further investigate whether HLA-G accumulated in these tumor-derived exosomes, M8-pcDNA–and M8HLA-G1– derived exosomes were subjected to a continuous sucrose gradient followed by Western blot analysis using sequentially the anti–HLA-G 4H84, the antigp96, and the anti-CD107b (Lamp-2) mAbs. Both HLA-G1 and Lamp-2 were detected in exosomes released by M8-HLA-G1 cells at a density ranging 1.11 to 1.17 g/ml, which corresponds to known exosome density (FigFIGURE 4 Western blot analysis of human leukocyte antigen (HLA)-G and Lamp-2 in M8-HLA-G1 exosomes. A total of 25 ␮g of M8-HLA-G1 exosomes were loaded on a continuous sucrose gradient (0.25-2 M sucrose, corresponding to densities ranging 1.05 to 1.31 g/ml) followed by Western blot analysis using sequentially, the anti– HLA-G 4H84, anti-gp96, and anti-Lamp-2 mAbs. HLA-G and Lamp-2 colocalized in 1.11 to 1.17 g/ml density fractions of M8-HLA-G1– derived exosomes.

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ure 4) [7, 11, 51]. This result confirms that HLA-G molecules are indeed expressed by exosome-like vesicles. No gp96 protein was detected in any fraction of the sucrose gradient (see Figure 4). Exosomes were also analyzed by electron microscopy further supporting their morphology as membrane vesicles homogenous in size (approximatively 80 nm) (data not shown). Finally, we examined surface expression of the HLA-G1 protein on melanoma-derived exosomes previously coated on latex beads by conducting flow cytometry experiments using the 87G anti–HLA-G1 mAb. This technique allows detection of protein expressed at the surface of limiting membrane of exosome [9]. As shown in Figure 5, exosomes derived from both the transfected M8-HLA-G1 and Fon cells were positively stained by the 87G mAb, whereas those produced by the M8-pcDNA control cell line were not. The classical HLA class I molecules and the tetraspan protein CD63, a lysosome-resident protein such as Lamp-2, were also present at the surface of these exosomes, as attested by their staining with the W6/32 and the anti-CD63 mAbs, respectively. Altogether, our results demonstrate that HLA-G–positive melanoma cells release exosomes bearing lysosomal associated membrane proteins, classical HLA class I molecules, as well as the nonclassical HLA-G class I molecule. The properties of dendritic cell-derived exosomes to boost T-cell response led to propose and start the use of tumor peptide-loaded dendritic cell-derived exosomes as a new cell-free strategy for cancer immunization [10, 52]. Recently, tumor-derived exosomes have been described as a novel pathway of antigen transfer from tumor cells to dendritic cells, allowing cytotoxic T lymphocyte–mediated antitumor responses in mice and the rejection of established tumors [11, 12, 53]. It is yet unknown whether the interaction of HLA-G1–positive exosomes with immunocompetent cells, such as NK cells, T cells, and APCs that bear HLA-G–inhibitory

Melanoma-Derived Exosomes Bear HLA-G

FIGURE 5 Flow cytometry analysis of human leukocyte antigen (HLA)-G, HLA class I, and CD63 molecules in Fon-, M8-HLA-G1–, and M8-pcDNA– derived exosomes. After coating to latex beads, exosomes staining was achieved with the anti–HLA-G1 87G mAb, the W6/32 pan class I mAb, and the antitetraspan lysosomal protein CD63 mAb. Exosomes released by Fon and M8-HLA-G1 cells harbored HLA-G together with HLA class I and CD63 molecules, whereas those from M8-pcDNA did not contain HLA-G.

receptor counterparts (i.e., CD85d, CD85j, and CD158d) is functional and can transduce an inhibitory signal. Moreover, during the interaction of HLA-G–positive exosomes with dendritic cells, HLA-G may have putative effect on antigen transport and presentation. Similarly, whether a negative signal can be transduced or not after the interaction of HLA-G–positive exosomes with T cells or NK cells will determine the fate of these effector lymphocytes. If this interaction is efficient, the exosomes would “spread” the HLA-G– dependent tolerogenicity, and if this interaction cannot deliver a negative signal, the exosomes would inhibit cell-cell interaction and con-

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sequently counteract the HLA-G inhibitory effect. Further investigations are now required to determine whether the release of HLA-G–positive exosomes by HLA-G–positive malignant cells may constitute a novel way for tumors to escape from immunosurveillance. ACKNOWLEDGMENTS

We are grateful to Drs. D.E. Geraghty, H. Ploegh, S. Fisher, and M. McMaster for providing us with antibodies. We thank Drs. M-F Avril and A. Spatz (Institut Gustave Roussy, Villejuif, France) for providing us with tumor sample biopsies. We thank Ire`ne Krawice-Radane for technical assistance and Danielle Lankar for electron microscopy analysis. B.R. was supported by the Association pour la Recherche contre le Cancer. Be´ atrice Riteau and Florence Faure contributed equally to this work.

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