Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral Latent Membrane Protein 1 and the immunomodulatory protein galectin 9
© Keryer-Bibens et al; licensee BioMed Central Ltd. 2006
Received: 14 September 2006
Accepted: 08 December 2006
Published: 08 December 2006
Nasopharyngeal carcinomas (NPC) are consistently associated with the Epstein-Barr virus (EBV). Their malignant epithelial cells contain the viral genome and express several antigenic viral proteins. However, the mechanisms of immune escape in NPCs are still poorly understood. EBV-transformed B-cells have been reported to release exosomes carrying the EBV-encoded latent membrane protein 1 (LMP1) which has T-cell inhibitory activity. Although this report suggested that NPC cells could also produce exosomes carrying immunosuppressive proteins, this hypothesis has remained so far untested.
Malignant epithelial cells derived from NPC xenografts – LMP1-positive (C15) or negative (C17) – were used to prepare conditioned culture medium. Various microparticles and vesicles released in the culture medium were collected and fractionated by differential centrifugation. Exosomes collected in the last centrifugation step were further purified by immunomagnetic capture on beads carrying antibody directed to HLA class II molecules. Purified exosomes were visualized by electron microscopy and analysed by western blotting. The T-cell inhibitory activities of recombinant LMP1 and galectin 9 were assessed on peripheral blood mononuclear cells activated by CD3/CD28 cross-linking.
HLA-class II-positive exosomes purified from C15 and C17 cell supernatants were containing either LMP1 and galectin 9 (C15) or galectin 9 only (C17). Recombinant LMP1 induced a strong inhibition of T-cell proliferation (IC50 = 0.17 nM). In contrast recombinant galectin 9 had a weaker inhibitory effect (IC50 = 46 nM) with no synergy with LMP1.
This study provides the proof of concept that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. It confirms that the LMP1 molecule has intrinsic T-cell inhibitory activity. These findings will encourage investigations of tumor exosomes in the blood of NPC patients and assessment of their effects on various types of target cells.
Nasopharyngeal carcinoma (NPC) is a human epithelial malignancy which represents a major threat for public health in several areas of the world . Very high incidence foci are found in south-China, especially in Guandong and Guangxi provinces (25 to 40/100,000/year) but also in other populations of South-East Asia, for example in the Sarawak people of Borneo island . Intermediate risk areas include Philippines, Vietnam, Indonesia and several countries of North and West Africa (incidence of 4 to 8/100,000/year). Most NPCs have minimal epithelial maturation and are classified as undifferentiated (WHO type III) or poorly differentiated (WHO type II). A few cases are differentiated (WHO types I). EBV association is constant regardless of patient origin and tumor differentiation except for some rare cases of differentiated NPC (type I) in Western countries . Another striking feature of NPC is the presence of a massive lymphoid infiltrate in the primary tumor. This infiltrate contains mostly T lymphocytes and a minority of B-cells, monocytes, dendritic cells and eosinophils. The abundant production by malignant NPC cells of inflammatory cytokines, including interleukin 1 alpha, Macrophage-Inhibitory-Protein 1 (MIP1) and CXCL10 is likely to favour the leucocyte infiltrate [4–7].
EBV-infection in NPC cells is predominantly latent. Several copies of the EBV genome (about 170 kb) are contained in the nuclei of malignant cell. Most of the about 80 EBV genes are silenced but several immunogenic viral proteins are consistently expressed in NPCs, including EBNA1 (Epstein-Barr nuclear antigen 1), LMP1 (latent membrane protein 1), LMP2 and the BARF1 protein [8–10]. Most of these viral proteins are immunogenic in humans. Precursors of HLA-restricted CD8 cytotoxic T-cells (CTLs) directed to LMP1, LMP2 and EBNA1 are present in the peripheral blood of healthy carriers . Anti-LMP1 and LMP2 CTLs are detected in NPC patients' peripheral blood [12, 13].
So far the mechanisms of local immune escape in NPCs have remained poorly understood. One clue has been provided by studies on EBV-transformed B-cells which release exosomes containing the EBV-encoded LMP1 in the extra-cellular medium [14, 15]. Both recombinant extra-cellular LMP1 and exosomes from EBV-transformed B-cells have inhibitory effects on T-cell activation and proliferation [14, 15]. However, until the present study, there were no data regarding the production of exosomes by NPC cells. Our attention was drawn to this issue by our recent findings on the interaction of LMP1 with the cellular protein galectin 9 in NPC cells . Galectin 9, a β-galactoside-binding protein, was originally characterized in Hodgkin's lymphoma cells and has various immunomodulatory properties [17–20]. It is secreted by a mechanism which is so far not well understood . However, other galectins – namely galectins 1 and 3 – are known to be secreted in association with exosomes [22, 23].
This study was designed to address the hypothesis of a possible production of exosomes containing LMP1 and/or galectin 9 by malignant NPC cells. We report that LMP1-positive NPC cells release HLA-class II-positive exosomes containing both LMP1 and galectin 9 whereas LMP1-negative NPC cells release exosomes containing only galectin 9. Since both LMP1 and galectin 9 are known to have effects on immune response mechanisms, these observations are likely to improve our understanding of host-tumor relationships in NPC and possibly in Hodgkin's disease.
NPC tumor lines and preparation of NPC cell conditioned media
C15 and C17 are EBV-positive NPC tumor lines permanently propagated by subcutaneous passage into nude mice . Suspensions of NPC cells were obtained by dispersion of xenografted tumors using type II collagenase as previously described . Residual cell aggregates were removed by filtration through a nylon cell strainer with 100 μm pores. Dispersed cells were incubated for 48 h in 24-well plates at 106 cells/well in 1.5 ml RPMI culture medium supplemented with 1.5% fetal calf serum and 5 mM Hepes. Collected supernatants were clarified by centrifugation at 300 g for 10 min and frozen at -80°C prior to differential centrifugation. Collected 300 g cell pellets were also stored at -80°C.
CS1-4 (DakoCytomation, Denmark) is a pool of 4 MoAbs directed to the C-terminal part of LMP1; it was used under the form of hybridoma culture supernatant, as provided by the manufacturer . Galectin 9 was detected using an affinity-purified polyclonal antibody raised against the C-terminal Carbohydrate Recognition Domain (CRD) of human galectin 9 whose central motif maps at residues 287–293 [16, 27]. Western blot detections of the DR α chain and CD63 protein were performed using the DA6.147 and TS 63 murine monoclonal antibody respectively (both kindly provided by E. Rubinstein)[28, 29].
Cell protein extraction and Western blot analysis
Cell pellets were dissolved in pre-chilled RIPA buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5% NP 40, 0.1% SDS) supplemented with Complete protease inhibition mixture (Roche Molecular, Meylan, France) and sonicated. Extracts were then clarified by centrifugation for 15 min at 10,000 g at 4°C. Protein concentration was assayed by the Lowry method using a detergent-compatible micro-assay system (Biorad, Marnes-la-Coquette, France). Western blotting was performed on PVDF membranes (Immobilon P, Millipore, St Quentin en Yvelines, France) according to standard protocols, using HRP-conjugated secondary antibodies and the ECL system (Amersham, Les Ulis, France).
Differential centrifugation of extra-cellular microparticles and vesicles
NPC cell conditioned media were subjected to sequential centrifugations. Following each centrifugation step, the pellet was collected for further analysis, and the supernatant was used for subsequent centrifugation. In addition to the initial 300 g step made prior to freezing and storage, culture supernatants were centrifuged twice at 1200 g (10 min) and then once at 10,000 g (30 min), 40,000 g (60 min) and 100,000 g (60 min) using a Beckman XL-80 ultracentrifuge with a SW41 or SW27 rotor. Pellets were named according to their order in the separation process from P1 (300 g) to P6 (100,000 g). When required, several procedures of differential centrifugation were performed in parallel. All pellets collected at the same step were resuspended in 500 μl serum free culture medium, pooled and pelleted again by ultracentrifugation using a TL-100 Beckman rotor (100,000 g, 60 min). Using this approach, as much as 120 ml of culture supernatant were routinely processed in one experiment yielding approximately 100, 80 and 180 μg proteins for P4, P5 and P6 respectively. For Western blot analysis, representative pellets of each separation step were dissolved in RIPA buffer (30 to 100 μl), sonicated and clarified as indicated for cell protein extraction. An aliquot of each protein extract was used to assay protein concentration prior to gel separation. For electron microscopy analysis, pellets P4, P5 and P6 were submerged and aggregated in the glutaraldehyde solution. For immunomagnetic isolation of exosomes, pellet P6 was resuspended in 400 μl culture medium.
Immunomagnetic isolation of exosomes produced by NPC cells
A resuspended P6 pellet derived from 60 ml conditioned medium was incubated for 5 h at 4°C, with 3.5 × 107 magnetic beads carrying an anti-HLA class II monoclonal antibody in 500 μl serum-free culture medium, under mild agitation (Dynabeads-HLA class II, Dynal-Invitrogen). The same type of magnetic beads carrying an irrelevant monoclonal IgG were used as control beads (Dynabeads Pan Mouse IgG, Dynal Invitrogen). Following the capture step, magnetic beads were washed 4 times in 1 ml PBS. For electron microscopy analysis, loaded beads were resuspended in the fixative solution. For Western blot analysis of captured material, loaded beads were boiled 5 min in Laemmli buffer in order to release proteins for gel separation.
Cell or microparticle pellets were fixed 1 h with 1.6% glutaraldehyde at 4°C, washed and fixed again in aqueous 2% osmium tetroxide, then dehydrated and embedded in epon resin. Ultrathin sections were cut on an LKB-III ultra-microtome, stained for contrast with uranyl acetate and lead citrate and examined with a Zeiss EM 902 transmission electron microscope.
For visualisation of exosomes following immunomagnetic purification, loaded beads were fixed for 1 hour at 4°C in 1.6% glutaraldehyde in phosphate Sörensen buffer 0.1 M, pH 7.3, and washed 3 × 20 min in Phosphate buffer. They were subsequently resuspended in 4% agar, fixed 1 hour at room temperature with 2% osmic acid (Carlo Erba, France) and washed in water. Samples were then dehydrated using increasing percentages of ethanol : 70% (30 min), 80% (20 min), 95% (30 min), 100% (1 hour). Finally they were included in Epon by progressively mixing Epon with ethanol. Polymerisation was made at 60°C for 48 hours. Ultrathin sections were cut with a Reichert Ultramicrotome III and counterstained with uranyl acetate and lead citrate.
Full length LMP1 (LMP1) was produced in recombinant baculovirus-infected Sf9 cells and purified by immuno-affinity chromatography as previously described [14, 30]. His-tagged LMP1dTM1 deleted of amino-acids 24–78 was similarly produced in baculovirus and purified by nickel-affinity chromatography. Human galectin-9 (M isoform) was produced in E. Coli by using the pET-11a vector as previously reported . For all 3 recombinant proteins, the absence of degradation was checked prior to functional experiments by western blotting using appropriate antibodies (data not shown).
Assessment of peripheral blood T-cell inhibition
The ability of several recombinant proteins to antagonize peripheral blood T-cell activation and proliferation was assessed on PBMCs (peripheral blood mononuclear cells) stimulated with anti-CD3/anti-CD28 beads. These beads are potent activators of peripheral blood resting T-cells (Dynabeads CD3/CD28 T cell Expander, Dynal-Invitrogen). PBMCs were mixed with stimulating beads and candidate inhibitory proteins and then seeded in 96-well round-bottom culture plates. In each well, 1 × 105 cells were mixed with 17 000 beads in 200 μl RPMI medium with 10% FCS. During the last 8 h, 3.7 × 104 Bq [3H] thymidine was added per well. The cells were harvested onto fibreglass filters and [3H] thymidine incorporation was determined by liquid scintillation counting.
LMP1 and galectin 9 associate with extra-cellular particles and vesicles released by NPC cells
Small NPC vesicles carrying LMP1 and/or galectin 9 display essential characteristics of exosomes
Impact of recombinant LMP1 and galectin 9 on peripheral blood T-cell proliferation induced by CD3-CD28 cross-linking
Malignant as well as non-malignant cells can release several types of vesicles and microparticles [33, 34]. Most of these elements are thought to be shed directly from the plasma membrane. In contrast typical exosomes derive from late endosomal structures called multivesicular bodies and have distinctive morphological and biochemical characteristics, including a high content of HLA class II molecules and tetraspanins like CD63 . By differential centrifugation and immunomagnetic capture, we have isolated vesicles derived from NPC cells displaying two major features of exosomes : a diameter in the range of 30–90 nm (70 nm) and surface expression of HLA class II molecules. We have demonstrated that exosomes derived from LMP1-positive NPC cells contain both LMP1 and galectin 9, whereas those derived from LMP1-negative cells contain only galectin 9. For technical reasons we chose to capture exosomes only from the P6 fraction (100,000 g pellet), but obviously galectin 9 and LMP1 were abundant in the P4 (10 000 g) and P5 (40 000 g) fractions. They are probably associated to additional types of extra-cellular vesicles possibly including exosomes of larger size and – in the case of the C15 supernatant – retroviral particles. Nevertheless this is the first demonstration that NPC cells release LMP1 and galectin 9 and that these proteins are at least in part associated with typical exosomes. Release of galectin 9 in the extra-cellular medium has long been known in other cellular models but, to our knowledge, this is the first demonstration that galectin 9 can be carried by exosomes as previously reported for galectins 1 and 3 [21–23].
Both LMP1 and galectin 9 have proven immunomodulatory properties. Soluble recombinant LMP1 has strong inhibitory effects on the activation of human resting T-cells in vitro. The same inhibition is obtained using short peptides containing a critical inhibitory motif derived from LMP1 first transmembrane segment (LALLFWL – amino-acids 34–40). This motif has a strong homology with a retroviral motif also known to antagonize T-cell activation . In figure 3, the lack of T cell inhibition by LMP1dTM1 which is deleted of the critical inhibitory motif is consistent with these previous observations. Several immunomodulatory activities of galectin 9 have been reported. Recombinant galectin 9 induces maturation of human dendritic cells with enhanced differentiation of Th1 lymphocytes . On the other hand, galectin 9 induces apoptosis of human T-lymphocytes, especially pre-activated CD4 lymphocytes . Finally, in mice, galectin 9 has been shown to induce apoptosis of mature Th1 cells through binding and activation of the Tim-3 ligand . In our experimental system, we have found no synergy between soluble LMP1 and galectin 9. Our data are consistent with the idea that LMP1 has a broad anergic effect on various T-cell populations whereas galectin 9 can induce inhibitory effects on more restricted populations. In any event, in the future, it will be necessary to investigate the effects of LMP1 and galectin 9 in the context of exosomes and not only as soluble molecules.
Because of the immunomodulatory properties of LMP1 and galectin 9, our observations are expected to have a major impact in the elucidation of host-tumor relationships in NPC patients. Indeed the emergence of a malignant process producing several immunogenic viral proteins in a context of local inflammation and heavy leucocytic infiltration remain one major paradox of this disease. It is even more surprising since previous reports indicate that NPC cells retain a functional antigen presenting machinery [12, 35]. This suggests that NPC immune escape is promoted by specific features of the tumor microenvironment. To address this problem several groups have investigated local production of immunosuppressive cytokines but without significant results. For example production of Fas ligand by NPC cells is not frequently observed at early stages of the disease . TGF-beta gene is not expressed at a higher level in tumor tissue compared to normal adjacent mucosa . Studies on IL-10 have yielded conflicting results [5, 37, 38]. In this context, it will be extremely important to confirm that exosomes carrying LMP1 and/or galectin 9 are released by malignant NPC cells in situ. Moreover, alterations of systemic immunity has been reported in NPC patients [39, 40]. It will be important to investigate the presence of LMP1 and/or galectin 9 containing exosomes in the biological fluids of these patients. Similar hypotheses might also be relevant to Hodgkin lymphomas where galectin 9 expression has been initially reported and which is associated to EBV in about 50% of the cases with consistent and intense expression of LMP1 [17, 41].
This study demonstrates that NPC cells can release HLA class-II positive exosomes containing galectin 9 and/or LMP1. Along with galectins 1 and 3, galectin 9 should now be included in the list of galectins which can be carried by exosomes. This report confirms that recombinant LMP1 has intrinsic T-cell inhibitory activity although no synergy between recombinant galectin 9 and LMP1 were found in T-cell inhibition experiments. One important aim in the future will be to assess on various target cells the biological activity of extra-cellular galectin 9 and LMP1 when they are inserted in exosomes. Another important aim will be to investigate the presence of galectin 9/LMP1-positive exosomes in the blood and biological fluids of NPC patients.
Cécile Keryer-Bibens was supported by a fellowship from the French Ministère de la Recherche et de la Technologie. This work was supported by a grant from the Ligue Nationale contre le Cancer (comité du Val de Marne), the Fondation de France (n° 2001004522) and Dutch Cancer Foundation (KWF-2001-2511). We thank Jeffrey Klarenbeek for LMP1 purifications and Martine Heyman, Emilie Viey, Gérard Pierron and Marc Lipinski for helpful discussions.
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