IMMUNOBIOLOGY

Blood diffusion and Th1-suppressive effects of galectin-9–containing exosomesreleased by Epstein-Barr virus–infected nasopharyngeal carcinoma cellsJihene Klibi,1 Toshiro Niki,2 Alexander Riedel,3 Catherine Pioche-Durieu,1 Sylvie Souquere,4 Eric Rubinstein,5

Sylvestre Le Moulec,6 Joel Guigay,7 Mitsuomi Hirashima,8 Fethi Guemira,9 Dinesh Adhikary,3 Josef Mautner,3 andPierre Busson1

1Universite Paris-Sud, Centre National de la Recherche Scientifique (CNRS)–Unité Mixte de Recherche (UMR) 8126, Paris, France, and Institut deCancerologie Gustave Roussy, Villejuif, France; 2Gal Pharma, Kagawa, Japan; 3Clinical Cooperation Group, Institute for Clinical and Molecular Biology,GSF-National Research Center for Environment and Health, Munich, Germany; 4Unité Propre de Recherche (UPR) 1983 CNRS–Institut Andre Lwoff, Villejuif,France; 5Inserm U602, Institut Andre Lwoff, Villejuif, France; 6Adult Medicine Department, Institut de Cancerologie Gustave Roussy, Villejuif, France;7Cervicofacial Surgery and ENT Department, Institut de Cancerologie Gustave Roussy, Villejuif, France; 8Department of Immunopathology, Faculty of Medicine,Kagawa University, Kagawa, Japan; and 9Clinical Biology Department, Institut Salah Azaiz, Tunis, Tunisia

Epstein-Barr virus (EBV)–associated na-sopharyngeal carcinoma (NPC) is the thirdmost frequent virus-associated humanmalignancy. How this tumor escapes im-mune recognition despite the expressionof several viral antigens has remainedpoorly understood. Our previous in vitrostudies have shown that NPC cells re-lease exosomes containing high amountsof galectin-9, a ligand of the membranereceptor Tim-3, which is able to induceapoptosis in mature Th1 lymphocytes.

Here, we sought to determine whethergalectin-9–carrying exosomes were pro-duced in NPC patients and whether suchexosomes might play a role in the im-mune evasion of NPC cells. We reportthat galectin-9–containing exosomes areselectively detected in plasma samplesfrom NPC patients and mice xenograftedwith NPC tumors. The incorporation intoexosomes protects galectin-9 against pro-teolytic cleavage but retains its Tim-3–binding capacity. Importantly, NPC exo-

somes induce massive apoptosis in EBV-specific CD4� cells used as a model oftarget T cells. This effect is inhibited byboth anti–Tim-3 and antigalectin-9 block-ing antibodies. These results indicate thatblocking galectin-9/Tim-3 interaction invivo might alleviate the Th1-suppressiveeffect of NPC exosomes and sustain anti-tumoral T-cell responses and thereby im-prove clinical efficacy of immunothera-peutic approaches against NPC. (Blood.2009;113:1957-1966)

Introduction

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)–associated malignancy and the third most frequent virus-associatedhuman malignancy after hepatocarcinomas and cervix carcinomas.Each year, approximately 80 000 new cases are diagnosed world-wide. The geographic distribution of NPC is not uniform. It isrelatively rare in European and North American countries. Veryhigh incidence foci are found in South China, especially inGuandong and Guangxi provinces (25-40 per 100 000 per year).1

Areas of intermediate incidence (approximately 3-8 per 100 000per year) include a large number of developing or emergingcountries, especially in North and Central Africa (Tunisia, Algeria,Morocco, Somalia, and Kenya) and in Southeast Asia (Philippines,Vietnam, Indonesia).

NPC is an epithelial malignancy with a complex etiologyinvolving viral, environmental, and hereditary factors. Exceptfor some very rare cases of atypical highly differentiated NPCoccurring in Western countries, the intact EBV genome isalways contained in the nuclei of all malignant cells.2,3 Many ofthe approximately 80 EBV genes are silent, but several viralRNAs and proteins are consistently expressed in NPC andcontribute to the malignant phenotype.4 NPC oncogenesis alsorequires a variable assortment of cellular genetic or epigeneticalterations.5 Another important biologic feature of NPC is the

presence of a massive lymphoid infiltrate in the primary tumor.This infiltrate contains mostly T lymphocytes and a minority ofB cells, monocytes, dendritic cells, and eosinophils. The abun-dant production by malignant NPC cells of inflammatorycytokines, including interleukin-1� (IL-1�), macrophage-inhibitory protein 1 (MIP1), and CXCL10, is likely to favor theleukocyte infiltrate.6-9

Several EBV proteins are consistently expressed in NPCs,including EBNA1, LMP1, LMP2, and BARF1.10-12 In addition,there is indirect evidence that viral latency is occasionallydisrupted in small foci of malignant cells, resulting in transientproduction of lytic cycle proteins.13-15 Production of the earlylytic transactivator BZLF1, which is highly immunogenic, hasbeen formally demonstrated in NPC specimens.16 The emer-gence of a malignant process producing several immunogenicviral proteins in a context of local inflammation and heavyleukocytic infiltration is one major paradox of NPC pathogene-sis. Previous reports have shown that NPC cells retain afunctional antigen-presenting machinery, suggesting that im-mune escape by NPC is brought about by specific features of thetumor microenvironment.17,18 However, for a long time, thesearch for local immunosuppressive factors has been disappoint-ing. For example production of Fas ligand by NPC cells is not

Submitted February 27, 2008; accepted October 28, 2008. Prepublished onlineas Blood First Edition paper, November 12, 2008; DOI 10.1182/blood-2008-02-142596.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

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frequently observed at early stages of the disease, and thetransforming growth factor-� (TGF-�) gene is not expressed at ahigher level in tumor tissue compared with normal adjacentmucosa.7,19

Our recent findings of a consistent galectin-9 production byNPC cells have brought new insights in this field. Galectin-9 is a�-galactoside binding lectin that contains 2 carbohydrate recogni-tion domains (CRD) joined by a “linker” region. It is expressed invarious mammalian tissues, especially by kidney cells, thymusepithelial cells, and M cells of mucosal lymphoid structures.20,21

Galectin-9 expression can be induced in various cell types bycytokines, especially interferon-� and IL-1�.20 It is also inducibleby EBV infection.22 We have shown that galectin-9 is extremelyabundant in NPC cells, in xenografted tumors, as well as in clinicalspecimens.23 Moreover, when NPC cells are grown in vitro,galectin-9 is released into the cell culture medium in associationwith exosomes.24 Meanwhile, galectin-9 has been identified as aspecific agonist of Tim-3, which is a death-inducing receptorexpressed by mature Th1 cells.25 In this study, we intended toinvestigate the structural and physiologic characteristics of NPCexosomes as carriers of galectin-9 and agonists of Tim-3 on Th1T cells. We report that galectin-9–containing exosomes are selec-tively detected in the plasma of NPC patients or mice xenograftedwith NPC tumor lines. In vitro they induce apoptosis in EBV-specific CD4� lymphocytes through galectin-9/Tim-3 interactions.These findings support a model in which NPC exosomes exert Th1suppressive functions at both the tumor and systemic levels.

Methods

Tumor and cell lines

C15 and C17 are xenografted EBV-positive NPC tumor lines permanentlypropagated by subcutaneous passage in nude mice.26 As previouslyreported, nonmalignant cells of human origin were eliminated from thexenografts beyond the first mouse passage. Therefore all human cellscarried by mice grafted with C15 and C17 tumors are malignant NPCcells.26,27 Suspensions of NPC cells were obtained by dispersion ofxenografted tumors as previously described.28 All animal procedures wereperformed by certified personnel according to French and Europeanregulations in the animal facility of the Gustave Roussy Institute ofOncology (Villejuif, France). C666-1, an EBV-positive NPC tumor linepropagated in vitro, was kindly provided by D. P. Huang and K. W. Lo(Chinese University of Hong Kong, Hong Kong).29 It was grown in RPMImedium supplemented with 25 mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid (HEPES) and 7.5% fetal calf serum (FCS). The HeLacervix carcinoma cell line was cultured in Dulbecco modified Eaglemedium (DMEM) with 5% FCS. All experiments with nude mice wereperformed in the Gustave Roussy Institute of Oncology with requiredpermissions and approval of the governing board of our animal facility.

Generation of EBV-specific CD4� T-cell clones

This procedure has been described previously.30-32 Briefly, peripheral bloodmononuclear cells (PBMCs) were obtained from healthy EBV carriers, andCD4� cells were selected over magnetic cell sorting (MACS) columns(Miltenyi Biotec, Bergisch Gladbach, Germany). Initial stimulation wasperformed using autologous PBMCs pulsed with recombinant EBV pro-teins: EBNA1, BZLF1, EBNA3C, and BLLF1 (gp350 envelope glycopro-tein). These proteins were produced as histidine (His)–tagged proteins bytransient transfection of HEK 293T cells and purification over Nickel-NTAagarose beads (QIAGEN, Hilden, Germany). Autologous PBMCs (106/mL)cultured in AIM-V medium (Invitrogen, Karlsruhe, Germany) were pulsedwith 500 ng/mL of the appropriate EBV recombinant protein for 24 hours,washed, irradiated (40 Gy), and mixed with an equal number of 106/mL

autologous CD4� PBMCs in T-cell medium (RPMI 1640 with 10 mMHEPES and 10% human serum). After 48 hours, 10 U/mL recombinant IL-2(Chiron Behring, Marburg, Germany) were added to the cultures. T-cellclones were generated by limiting dilution cloning as described.30 Theywere maintained in culture by restimulation every 2 weeks using mini-lymphoblastoid cell lines (LCLs) pulsed with the appropriate EBV-recombinant protein. These mini-LCLs were established by infection ofprimary B cells with the genetically engineered mini-EBV strain and do notsupport lytic viral replication.31

Transient transfection of cell lines

Human galectin-9 was expressed in HeLa cells after transfection of thefull-length cDNA of galectin-9 (long isoform) in the pBK-CMV plasmid(Stratagene, La Jolla, CA) using the calcium-phosphate method.

Protein extraction from cultured cells

Cell pellets were solubilized in prechilled radio immunoprecipitation assay(RIPA) buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM ethylenedia-minetetraacetic acid [EDTA], 0.5% sodium deoxycholate, 0.5% NonidetP40 [NP40], 0.1% sodium dodecyl sulfate [SDS]) supplemented withcomplete protease inhibitor cocktail (Roche Applied Science, Neuilly-sur-Seine, France) and sonicated on ice. Extracts were clarified by centrifuga-tion for 15 minutes at 16 000g at 4°C. Protein concentration was assayed bythe Lowry method using a detergent-compatible microassay system (Bio-Rad Laboratories, Gif-sur-Yvette, France).

Antibodies

In Western blot analyses, galectin-9 was detected using an affinity-purifiedrabbit polyclonal serum (anti-G9–CT-L1) raised against a recombinantpeptide derived from the C-terminal CRD2.23,24,33 For flow cytometricanalysis, exosome capture, or blocking of ligand-receptor binding, we useda monoclonal antibody directed to residues 208-215 of galectin-9 (9M1-3).This epitope, FSTPAIPP, is located at the N-terminal border of theC-terminal CRD of galectin-9 (CRD2). Monoclonal antibodies against CD9(TS9) and CD63 (TS63) have been previously published.34 The DA6.147was used for Western blot detection of the DR-� chain of the humanleukocyte antigen (HLA) class II antigens.35 The glucose-regulated protein94 (Grp94 or gp96) was visualized with a rat monoclonal antibody(Stressgen, Ann Arbor, MI). For detection of Tim-3 by flow cytometricanalysis and blocking of ligand-receptor binding, we used a monoclonalantibody (2E2) directed to its ligand-binding site.36,37 This reagent waskindly provided by David Anderson and Vijay Kuchroo (Center forNeurologic Diseases, Harvard Medical School, Boston, MA).

Recombinant proteins and galectin-9 detection byenzyme-linked immunosorbent assay

The m-isoform (medium-sized) of the wild-type galectin-9 lacks exon 5 andhas a shorter linker region than the full-length l-isoform (long-sized).20,38

Galectin-9 NC(null) or NC is an artificial deletion mutant lacking the linkerregion, which increases its resistance to proteases (no linker peptidebetween the N- and C-CRD).39 Both the m-isoform and galectin-9 NC wereproduced in Escherichia coli using the pET11a vector and purified byaffinity chromatography on a lactose-agarose column.39 Final preparationswere checked for absence of endotoxin contamination. Detection ofgalectin-9 by enzyme-linked immunosorbent assay (ELISA) was performedaccording to the protocol used for synovial fluid from rheumatoid arthritispatients.40 Due to unidentified factors inhibitory for this ELISA system,human plasmas required substantial dilution (8�), resulting in poorsensitivity. Mouse plasmas did not contain such inhibitory factors.

Exosome production in vitro

C17 NPC cells obtained by dispersion of xenografts were incubated in24-well plates at 106 cells/well in 1.5 mL RPMI medium supplementedwith 1.5% FCS. The in vitro–propagated epithelial cell lines, HeLa andC666-1, were grown to 60% confluence in 75-cm2 flasks and incubated

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in 10 mL appropriate culture medium with 1.5% FCS. Conditionedmedia were collected after 48 hours of incubation at 37°C, centrifugatedat 300g for 10 minutes to remove biggest cell remnants and debris, andfrozen at �80°C.

Preparation of exosomes from plasma samples

Heparinized blood samples were collected from mice carrying variousxenografted tumors by puncture of the nasal venous sinus. Plasmas wereseparated from red cells by centrifugation at 3000g at 4°C for 10 minutesand frozen at �80°C. On the other hand, human plasma samples werecollected from 26 donors. This procedure was done with patient informedconsent obtained in accordance with the Declaration of Helsinki andInstitutional Review Board (IRB) approval from the Institut GustaveRoussy and the Salah Azaiz Institute. Clinical and pathologic data on donorpatients are summarized in Tables S1 through S4 (available on the Bloodwebsite; see the Supplemental Materials link at the top of the online article).A first series of plasma samples were obtained from 12 NPC patients (A toL) diagnosed at the ENT Department of the Salah Azaiz Institute (Tunis,Tunisia). All these patients had nonkeratinizing carcinomas of the undiffer-entiated type and were previously untreated (Table S1). The nonkeratiniz-ing type of NPC is known to be constantly associated to EBV, irrespectiveof the ethnic background.2,3 A second series of plasma samples werecollected prospectively from 5 NPC patients (M to Q) referred to theGustave Roussy Institute of Oncology (Villejuif, France) or to Parishospitals working in collaboration with the Institute (Table S2). For each ofthese 5 patients, EBV association was confirmed by EBV-encoded RNA(EBER) in situ hybridization (Figure S1). Control plasma samples wereobtained from 1 healthy volunteer and 8 patients referred for non-NPCmalignant tumors to the Salah Azaiz (TA, TB, and TC) or the GustaveRoussy (TD to TH) Institute (Tables S3,S4). For exosome preparation,1.5 mL thawed plasma samples were diluted 1:4 times in phosphate-buffered saline (PBS) and subjected to a first centrifugation in a SW41TiBeckman rotor at 12 000g for 45 minutes. The 12-kg pellet wasdiscarded, whereas the supernatant was subjected to a second centri-fugation at 110 000g for 2 hours using the same rotor. The 110-kg pellet,containing low-density vesicles, was washed by resuspension in PBS andcentrifuged in a TL100 Beckman rotor for 1 hour at 110 000g. HLA

class II–positive exosomes were isolated from this second pellet byimmunomagnetic capture.

Purification of exosomes from culture media using a sucrosegradient

This procedure was adapted from the method described by Lamparski etal.41 All steps were performed at 4°C. Thawed culture supernatants (at least75 mL) were clarified by 2 consecutive centrifugations, first at 1900g for15 minutes and then at 12 000g for 30 minutes. Clarified supernatants werethen subjected to ultracentrifugation at 66 000g for 2 hours using a Ti45Beckman rotor, resulting in a pellet of low-density vesicles. Exosomescontained in this pellet were further purified by flotation on a cushion madeof a sucrose solution in deuterium oxide (D2O; Sigma-Aldrich Chimie,Lyon, France). Practically, the low-density pellet was resuspended infiltered PBS (10 mL for an initial volume of a 7-mL supernatant), loaded ontop of a 1-mL sucrose/D2O solution (20 mM Tris, 30% sucrose D2O,pH 7.4) in a SW 41 polycarbonate tube. This 2-phase discontinuousgradient was subjected to ultracentrifugation at 76 000g for 90 minutes on aSW41 Ti Beckman rotor. The cushion containing the exosomes was thencollected without disturbing the pellet. The exosomes were diluted 1:5 andpelleted by ultracentrifugation at 110 000g in a SW41 rotor for 90 minutes.Two additional washing steps were performed in a smaller volume(ultracentrifugation at 110 000g using a TLA100 Beckman rotor). Washedexosomes were then resuspended in PBS, subjected to protein assay, and35-�L aliquots with a protein concentration of 1 to 5 �g/�L were stored at�80°C. When prepared for immunomagnetic capture, low-density vesicleswere directly subjected to the washing steps without prior purification on asucrose cushion.

Immunomagnetic capture of exosomes from conditionedculture media or plasma samples

Materials subjected to capture were low-density vesicles (110-kg pellets)derived either from 50 mL conditioned culture medium or from 1.5-mLplasma samples. Beads used for capture were 4.5-�m-diameter magneticbeads. In most experiments, they were coated with an anti–HLA class IImonoclonal antibody (Dynabeads–HLA class II; Dynal, Invitrogen, Cergy

Figure 1. Specific detection of galectin-9–containingexosomes in the plasma of mice xenografted withNPC tumor lines. (A) A significant amount of galectin-9 isdetected by ELISA in crude plasma samples from micexenografted with NPC tumor lines (C15, C17, and C666-1),but not control mice either without xenografts (tumor-free) orxenografted with a non-NPC carcinoma tumor line (non-NPC). Assays were performed in triplicate on 3 plasmasamples from 3 different mice. (B) A series of plasmasamples were collected from 6 mice carrying C15 tumors ofvarious sizes. Galectin-9 concentration in these samples isproportional to the tumor volume (y 3.2� � 1.7,R2 0.994). (C) Captures with anti–HLAclass II beads wereperformed on low-density vesicles (110-kg pellets) derivedfrom pools of murine plasmas. Samples collected withanti–class II beads are designated nos. 3 and 6 (class II).Control samples collected with beads carrying nonspecific Igare nos. 2 and 5 (NS Ig). In addition to plasma material,protein extracts from the C17 and the non-NPC xenograftedtumors were used as controls for Western blot analyses (nos.1 and 4). (D) Numerous vesicles approximately 70 nm indiameter are captured by HLA class II beads and visualizedby electron microscopy when these procedures are appliedto plasma vesicles from C17-xenografted but not controlmice. Scale bar represents 500 nm. In the inset, 2 vesicles atoriginal magnification (� 4). (E) Western blot analysis de-tects galectin-9, CD9, CD63, and the DR-� chain in C17plasma vesicles. In contrast, none of these molecules aredetected when the capture is performed on control mouseplasma. Note that CD63 is much more concentrated in C17exosomes than in the tumor extract. Gp96, which is acytoplasmic membrane protein, is detected in the C17 andnon-NPC tumor extracts (lanes 1 and 4) but not in C17exosomes (lane 3).

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Pontoise, France). The same type of magnetic beads carrying an irrelevantmonoclonal immunoglobulin (Ig) was used as control beads. In one set ofexperiments (see Figure 2), the 9M1-3 monoclonal antibody directed to thegalectin-9 CRD2 was loaded on magnetic beads (10 �g for 5 � 107 beads)coated with a secondary anti–mouse Ig (Dynabeads Pan Mouse IgG; Dynal,Invitrogen). Control beads were coated with irrelevant purified monoclonalmouse IgG (Sigma-Aldrich Chimie). For the capture process, particlescontained in 110-kg pellets were resuspended in 250 �L serum-free culturemedium and incubated for 5 hours at 4°C, with 3.5 � 107 beads under mildagitation. After the capture step, magnetic beads were washed 4 times in1 mL PBS. Exosome-loaded beads intended for electron microscopyexamination were resuspended in the fixative solution. For Western blotanalysis of captured material, the beads were boiled for 5 minutes inLaemmli buffer to release proteins before gel loading.

Electron microscopy

For visualization of exosomes after immunomagnetic purification, loadedbeads were fixed in glutaraldehyde and embedded in Epon as previouslydescribed.24 Ultrathin sections were cut with a Reichert Ultramicrotome III(Reichert, Depew, NY) and counterstained with uranyl acetate and leadcitrate before being observed with a Tecnai Spirit transmission electronmicroscope (FEI, Hillsboro, OR). Images were recorded with a SISMegaviewIII charge-coupled device (CCD) camera driven by the AnalySIS3.1 image processing software (Silicon Integrated System, Taiwan, Repub-lic of China).

Western blot analysis

Cell or exosome protein extracts were separated on polyacrylamide gels instandard conditions except when planning detection of CD9 and CD63tetraspanins, which require nonreducing conditions. Gels were blotted onpolyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore,

Molsheim, France) according to standard protocols. Specific protein bandswere visualized using horseradish peroxidase–conjugated secondary anti-bodies and appropriate revelation systems, enhanced chemiluminescence(ECL) system (GE Healthcare, Buc, France) or Supersignal Westpico(Pierce, Perbio Europe, Belgium), depending on the required sensitivity.When required, densitometry of the chemiluminescence films was doneusing a GS-710 calibrated imaging densitometer with Quantity Onesoftware (Bio-Rad Laboratories).

Resistance to trypsin digestion assay

To solubilize lipid membrane components, purified C17 exosomes (35 �gproteins in 7 �L PBS) were subjected to lipid membrane lysis by 1% TritonX-100 for 30 minutes at 4°C. Mock-treated exosomes were incubated underthe same conditions in the absence of Triton. Both treated and mock-treatedexosome suspensions were then subjected to trypsin digestion at 37°C with2 enzyme concentrations (15 and 0.01 �g/mL) and increasing times ofincubation (5, 15, and 30 minutes; T4549; Sigma-Aldrich Chimie). Trypsinactivity was only marginally affected by the presence of Triton (data notshown). After completion of trypsin incubation time, the samples wereimmediately mixed with an equal volume of Laemmli buffer and boiled for3 minutes before gel loading.

Membrane immunostaining and flow cytometry

Single-cell suspensions of C17 and C666-1 NPC cells were incubatedwith 10 �g/mL of the anti–galectin-9 antibody 9M1-3 for 30 minutes,washed with PBS supplemented with 3% FCS, and then incubated withfluorescein isothiocyanate (FITC)–conjugated anti–mouse antibody for30 minutes. Propidium iodide (PI; 5 �g/mL) was added before analysis.Similarly, EBV-specific CD4� T cells were first incubated with 3 �g/mLanti–TIM-3 antibody 2E2 for 1 hour, washed, and then incubated withphycoerythrin (PE)–conjugated anti–mouse antibody for 30 minutes.

Figure 2. Specific detection of galectin-9–containing exosomes in the plasma of NPC patients. (A) Captures with anti–HLA class II beads were performed on low-densityvesicles (110-kg pellets) derived from human plasma samples. Initially, plasmas were collected from 2 NPC patients (NPC A and B; samples 2, 3, 4, and 5), 1 patient with anon-NPC head and neck carcinoma (non-NPC TA; samples 6 and 7), and 1 healthy donor (samples 8 and 9). In each case, beads coated with nonspecific Ig (NS Ig) wereused as negative controls. In addition to plasma material, a protein extract from the C17-xenografted tumor was used as a positive control for Western blot detection (no. 1).Clinical and pathologic data on donor patients are provided in Tables S1 and S3. (B) Numerous bilamellar vesicles approximately 70 nm in diameter are visualized by HLA classII capture and electron microscopy when these procedures are applied to plasma vesicles from NPC patients but not control subjects. Scale bar represents 500 nm. In the inset,2 vesicles at original magnification (� 4). (C) In parallel experiments, Western blot analysis revealed expression of galectin-9, CD9, CD63, and the � chain of the DR moleculein NPC plasma vesicles. In contrast, none of these proteins is detected when the same procedure is applied to control plasmas. Gp96 is only detected in the tumor extract.(D) Galectin-9–carrying exosomes are captured by anti–HLA class II beads from 4 additional samples of NPC plasmas (NPC C, D, E, and F) but not from 2 control subjects withnon-NPC tumors (non-NPC TB and TC). Clinical and pathologic data on donor patients are in Tables S1 and S3. Consistent results were obtained for 11 NPC and 5 non-NPCadditional plasma samples (see Tables S1, S2, and S4 and Figures S1 and S2).

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After PI addition, stained cells were analyzed by flow cytometry usingthe CellQuest software (FACSCalibur; BD Biosciences, Erembodegem,Belgium).

Stimulation of the Tim-3 receptor on EBV-specific CD4� T cellsand assessment of apoptosis induction

CD4� T cells from 4 clones specific for EBV proteins were chosen astargets of Tim-3–binding agents. These clones were specific for EBNA3C(GB3C), gp350 or BLLF1 (JM1H2), EBNA1, and BZLF1. These T cells(106 cells/well) were incubated for 5 hours with one of the following agents:(1) recombinant wild-type (WT) or modified (NC) galectin-9 at variousconcentrations, or (2) suspensions of gradient-purified exosomes, adjustedto a final protein concentration of 100 �g/mL. In some experiments,preincubation with blocking antibodies were performed, either on CD4�

target cells (2E2 anti–Tim-3 antibody, 10 �g/mL for 1 hour) or onexosomes (9M1-3 anti–galectin-9 antibody, 10 �g/mL for 1 hour). AfterTim-3 stimulation, target cells were stained with annexin V–FITC and PIusing the Annexin V–FITC Apoptosis Detection kit (Roche AppliedScience). FITC and PI staining were analyzed by flow cytometry using theCellQuest software, FACSCalibur.

Results

Galectin-9–positive exosomes are produced by NPC cellsin vivo

To investigate whether galectin-9–containing exosomes were pro-duced by NPC cells in vivo, plasma samples were collected fromnude mice xenografted with NPC tumors. Control samples wereobtained from tumor-free nude mice and mice xenografted with anon-NPC human tumor. By ELISA, galectin-9 was specificallydetected in the plasma samples from NPC-xenografted but notcontrol mice (Figure 1A). In addition, galectin-9 concentration inmouse plasma samples was proportional to the tumor volume asshown for the C15 xenografts (Figure 1B). In the next step,low-density vesicles contained in plasma samples were collected as110-kg pellets. These pellets were further treated by magneticbeads to isolate vesicles expressing surface HLA class II. As shownin Figure 1C-E, such vesicles were captured from xenografted NPCmouse plasma but not control samples. Upon electron microscopyexamination, captured HLA class II–positive vesicles had size andmorphology typical of exosomes. Western blot analysis of theirproteins readily detected galectin-9 with a predominance of thel-isoform in contrast with the tumor extract containing equalamounts of the l- and m-isoforms.20,38 Identification of thesevesicles as exosomes was substantiated by detection of the CD63and CD9 tetraspanins, which are markers of the endosomalcompartment, and the absence of the cytoplasmic marker gp96. Asimilar procedure was applied on plasma samples collected from aseries of 17 NPC patients and 9 controls, including one healthyindividual and 8 patients affected by various types of malignanciesdistinct from NPC. As shown in Figures 2 and S2, HLA classII–positive exosomes were captured, and exosome-derivedgalectin-9 was detected in 16 of 17 NPC plasma samples. Incontrast, no galectin-9 was detected in 8 of 9 control samplesprocessed according to the same procedure; only 1 of them wasborderline or weakly positive (sample TF from a patient affected byan epithelioid sarcoma). This distribution of positive cases speaksstrongly in favor of a specific relationship between NPC anddetection of galectin-9–containing exosomes in patients’ blood(2-tailored P .001 by Fisher exact test). Because of its very poorsensitivity in the context of human plasma, our galectin-9 ELISAsystem could not be applied to human samples (see “Recombinant

proteins and galectin-9 detection by enzyme-linked immunosor-bent assay”). Nevertheless, data obtained from xenografted miceand NPC patients by exosome capture and Western blot analysisprovide compelling evidence that galectin-9–containing exosomesare consistently produced by NPC cells in vivo and diffuse from thetumor interstitial fluids to the bloodstream.

Relative resistance to proteolysis and surface presentation ofgalectin-9 carried by NPC exosomes

Subsequent experiments were performed on exosomes produced byNPC cells in vitro to investigate their characteristics as galectin-9carriers. Exosomes produced by C17 NPC cells were purified on asucrose gradient and assessed both qualitatively and quantitatively.As shown on Figure 3A, they contained HLA class II and CD63molecules in the absence of detectable gp96. The amount ofgalectin-9 contained in the purified exosome fraction was assessedby Western blot analysis using recombinant galectin-9 as areference (Figure 3B). Approximately 0.4 ng galectin-9 was se-creted in exosomes by 1 million C17 cells within 48 hours. Thisamount is roughly equivalent to 1/10 000 of all exosomal proteinsreleased by C17 cells. Like most proteins released in the extracellu-lar space, galectin-9 is vulnerable to digestion by proteolyticenzymes.39,42 It was hypothesized that it could be protected fromenzymatic degradation by its insertion into exosomes. Therefore,C17 exosomes were subjected to Triton lysis, to dissolve lipidmembrane components, followed by trypsin treatment. Controlexosomes were subjected to the same procedure, except with theabsence of Triton. As shown in Figure 3C, trypsin degradation of

Figure 3. Intact exosomes protect galectin-9 against trypsin digestion.(A) Western blot detection of DR-�, CD63, and galectin-9 in exosomes released byC17 cells and purified on a sucrose gradient. Gp96 is a cytoplasmic protein typicallyabsent from exosomes. (Left side) C17 total extract. (Right side) Exosome proteins.(B) Galectin-9 contained in C17 exosomes is quantified in a Western blot analysis bycomparison to a series of recombinant galectin-9 samples ranging from 1 to 25 ng(rec gal-9). On average, 2 to 5 ng galectin-9 are contained in 50 �g exosomeproteins. (C) Trypsin digestion assay, the same amount of C17 exosomes was usedfor each condition (35 �g total exosome proteins). They were subjected either toTriton lysis or mock treatment before incubation with trypsin. Two different trypsinconcentrations, 15 �g/mL (high trypsin) or 0.01 �g/mL (low trypsin), were used indistinct experiments. For samples treated with low amounts of trypsin, short and longexposures of the blotted membrane are presented. Controls are displayed in lane 1(no Triton lysis, no trypsin addition, and incubation at 4°C), lane 5 (no Triton, notrypsin, 37°C), and lane 9 (Triton lysis without trypsin addition and incubation at37°C). Triton lysis of exosomes greatly enhances trypsin digestion of exosome-bound galectin-9 as shown in lanes 6, 7, and 8 (Triton lysis followed by trypsindigestion) as compared with lanes 2, 3, and 4 (mock treatment followed by trypsindigestion). After Triton lysis, undigested galectin-9 remained detectable only for theshortest time of incubation (5 minutes) with the smaller concentration of trypsin(0.01 �g/mL; lane 6). In lane 9, a substantial decrease of galectin-9 is observeddespite the absence of trypsin, suggesting an effect of endogenous proteolyticenzymes after Triton lysis.

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galectin-9 was considerably enhanced by prior disruption ofmembrane exosomes, thus providing evidence of their protectiverole against proteolytic attack of galectin-9 (Figure 3C). Next wewanted to know whether galectin-9 carried by exosomes couldbehave as an agonist of the Tim-3 receptor. One importantrequirement was the presentation of galectin-9 CRDs at the surfaceof intact exosomes to allow its interaction with the Tim-3 receptor.To address this question, we used the 9M1-3 antibody directed tothe FSTPAIPP epitope of the CRD2 domain of galectin-9. Mag-netic beads coated either with the 9M1-3 antibody or with anantibody directed to the external part of HLA class II moleculeswere used in parallel to capture NPC exosomes. Capture efficiencywas assessed by electron microscopy imaging and Western blotanalysis of the DR-� chain eluted from beads. As shown in Figure4, a significant amount of DR-�–positive exosomes were capturedusing the 9M1-3 antibody, providing evidence that the FSTPAIPepitope of the galectin-9 CRD2 is displayed at the surface of NPCexosomes. Interestingly, surface presentation of galectin-9 CRD2was equivalent for the exosomes derived from the C666-1 and C17NPC cells, although galectin-9 was absent on the plasma mem-brane of C666-1 cells and at a very low level on C17 cells.

Recombinant galectin-9 induces apoptosis in EBV-specificCD4� T cells

To directly assess the immunosuppressive functions of NPCexosomes, we used EBV-specific cytotoxic CD4� T-cell clones as amodel system. These clones were obtained from healthy EBV-

carriers by in vitro stimulation of CD4� cells with recombinantlatent or lytic EBV proteins.31 Some of them have direct cytotoxicactivity.31 The majority preferentially secrete Th1 cytokines.32 Inpreliminary experiments, most of the T-cell clones tested demon-strated intense expression of Tim-3 including GB3C specific forEBNA3C, JM1H2 specific for gp350, one clone specific forEBNA1, and one specific for BZLF1 (Figure 5A). Kinetics studiesdemonstrated a maximal expression of Tim-3 at day 6 afterrestimulation, precisely when they are at their highest level ofanti-EBV activity (data not shown). This was in contrast with theabsence of Tim-3 detection on CD4� T cells sorted directly fromPBMCs given by a healthy donor (Figure 5A). These observationsare consistent with the notion that, among CD4� T cells, Tim-3 isexpressed exclusively on terminally differentiated Th1 cells.43 Tofind out whether the Tim-3 expression on the EBV-specific CD4�

T cells is of physiologic consequence, they were treated withrecombinant galectin-9. Both GB3C and JM1H2 clones were foundto be exquisitely sensitive to apoptosis induced by 2 distinct formsof recombinant galectin-9. Similar results were obtained withgalectin-9 NC, which is a modified artificial species resistant toproteases, and the m-isoform of wild-type galectin-9 (Figure5B,C).38,39 In both cases, the median-inhibitory dose (ID50) wasclose to 100 pg/mL, which is much lower than the 350 ng/mLdescribed for PBMCs or human T-cell lymphotropic virus-1(HTLV1)-infected cells in a previous publication.44

NPC exosomes induce apoptosis in EBV-specific CD4� T cellsthrough galectin-9/Tim-3 interaction

We then set out to address whether galectin-9 in exosomes wouldhave a similar effect. As shown in Figure 6, purified exosomes fromC17 NPC cells induced apoptosis in less than 5 hours in approxi-mately 35% of the GB3C and JM1H2 CD4� T cells (Figure 6A-C).When CD4� cells from the EBNA1-specific clone were treated

Figure 4. Galectin-9 CRD2 is presented at the surface of NPC exosomes.Low-density vesicles (110-kg pellets) released by C17 or C666-1 NPC cells wereincubated with magnetic beads coated with the 9M1-3 monoclonal antibody directedto the CRD2 of galectin-9. Beads coated with purified isotype-matched nonspecific Igwere used for control reactions. (Top panel) Numerous exosomes released by C17(A) or C666-1 (B) are captured by anti-CRD2, but not control beads, and visualized byelectron microscopy. (Middle panel) Western blot analysis detects the DR-� andgalectin-9 proteins in C17 (C) and C666-1 (D) exosomes, whereas no similar proteinsare recovered from control beads coated with irrelevant IgG. Positive controls areprovided by total C17 and C666-1 cell extracts in the left lanes of panels C and D,respectively. (Bottom panel) A small amount of the galectin-9 CRD2 is detected at thesurface of live C17 cells by flow cytometry (E), whereas it is absent at the surface ofC666-1 (F) cells.

Figure 5. Recombinant galectin-9 induces apoptosis in EBV-specific CD4�

T cells. (A) Intense membrane expression of Tim-3 is detected on 4 EBV-specificCD4� T cell clones of Th1 subtype but not on polyclonal CD4� T cells sorted directlyfrom PBMCs (CD4� PBMC; gray curve, control staining). EBV-specific clones aredirected against the EBV-proteins EBNA3C (GB3C), gp350 or BLLF1 (JM1H2),EBNA1, and BZLF1. (B) A recombinant modified form of galectin-9 with increasedstability (Gal-9 NC) induces apoptosis in JM1H2 CD4� T cells with an ID50 ofapproximately 100 pg/mL (apoptosis was assessed according to the percentage ofannexin V–positive cells). (C) Recombinant wild-type galectin-9 (gal-9 WT) at aconcentration of 100 pg/mL induces apoptosis in GB3C T cells. Induction of apopto-sis is suppressed by preincubation of the T cells with 10 �g/mL blocking anti–Tim-3monoclonal antibody, but not a control IgG.

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with C17 exosomes, apoptosis induction was even more massive(Figure 6D). A similar effect was obtained with the BZLF1-specificT cells (data not shown). All these responses were obtained with aconcentration of exosome proteins of 100 �g/mL, equivalent toa galectin-9 concentration of 10 ng/mL (Figure 3B). In contrast,polyclonal CD4� T cells sorted directly from PBMCs, which arenegative for Tim-3, were almost insensitive to the sametreatment (Figure 6A). Because exosomes are rich in membranemolecules, not all of which have been well characterized, it wasimportant to confirm that the induction of apoptosis in theEBV-specific CD4� T cells was the result of direct interactionbetween galectin-9 on the part of the exosomes and Tim-3 on theside of the T cells. Apoptosis induction by C17 exosomes wasalmost entirely inhibited by preincubation of target T cells witha blocking anti–Tim-3 antibody as well as by preincubation ofNPC exosomes with the 9M1-3 antibody directed to galectin-9CRD2 (Figure 6B-D). In control experiments, preincubation ofNPC exosomes with an anti–DR-� antibody had no inhibitoryeffect. To further confirm the specific role of galectin-9 inapoptosis induction by NPC exosomes, CD4� cells, specific foreither gp350 (JM1H2) or EBNA1, were treated with exosomesfrom HeLa cells transfected with either a galectin-9 or a greenfluorescent protein (GFP) expression construct. As expected,galectin-9 was only detectable in exosomes released by galectin-9–transfected Hela cells (Figure 7A). Apoptosis of EBNA1 andJM1H2 CD4� cells was induced by galectin-9–positive exo-

somes, but not by exosomes from control Hela cells. It wasinhibited by the anti–Tim-3 blocking antibody (Figure 7B,C).

Discussion

Galectin-9 plays a key role in the control of immune effector cellsduring various phases of immune responses in mammals. On onehand, it is a proinflammatory factor acting by stimulation ofeffector cells of innate immunity.36,45 For example, it inducesmaturation of monocyte-derived dendritic cells, and through thisprocess, promotes Th1 immune responses.45 On the other hand,galectin-9 has a major role in the self-limitation of the immuneresponse.43 While �-interferon released by Th1 cells inducesgalectin-9 production by various cell types, galectin-9 creates anegative feedback by triggering apoptosis of mature Th1 cellsthrough stimulation of their Tim-3 receptor.25 However, galectin-9,like other galectins, is not a cytokine stricto sensu. Although it isknown to be secreted by various cell types, it has no signalsequence, and it cannot be exported into the extracellular spacethrough the classical biosynthetic pathway.20 We have previouslymade one important step toward the solution of this paradox bydemonstrating that NPC cells cultured in vitro release galectin-9 inthe extracellular medium in association with exosomes.24 Thissuggested that galectin-9–carrying exosomes could be the missinglink between galectin-9–producing cells and T cells expressing

Figure 6. NPC exosomes induce apoptosis in EBV-specific CD4� T cells through galectin-9/Tim-3 interaction. (A) NPC exosomes (C17) containing galectin-9 induceapoptosis in EBV-specific CD4� T cells (GB3C clone, anti-EBNA3C) without significant effects on polyclonal CD4� T cells sorted directly from PBMCs (CD4� PBMC). Testedcells were incubated for 5 hours with purified C17 exosomes at a final concentration of 100 �g/mL exosomal proteins (corresponding approximately to 10 ng/mL galectin-9; ExoC17). Control cells were incubated without exosomes (W/O Exo). Cell clusters visible at the top of the upper right portions of the graphs are related to nonspecific necrosis.(B,C) NPC exosomes (C17) induce apoptosis in EBV-specific CD4� cells from the JM1H2 clone (anti-gp350). Apoptosis of CD4� T cells is almost entirely suppressed bypreincubating the T cells with a blocking anti–Tim-3 antibody or by preincubating the exosomes with the 9M1-3 antibody directed to the galectin-9 CRD2. In contrast, apoptosisinduced by NPC exosomes is not prevented by a nonspecific Ig (NS IgG) or a monoclonal antibody directed to the extracellular portion of the DR-� chain (anti–DR-�, DA6.147).Flow cytometry graphs representative of some experiments summarized in panel B are displayed in panel C. PI-positive/annexin V–negative cells were consistently detected inthe presence of anti–Tim-3 and anti–galectin-9 antibodies even in the absence of NPC exosomes, without satisfactory explanation. (D) NPC exosomes (C17) induce apoptosisin EBNA1-specific CD4� cells. Percentages of annexin V–positive and annexin V/PI-positive cells are presented on the right side of the panel. Spontaneous cell death is moreprevalent in this clone compared with GB3C and JM1H2: 3.8% annexin V and 21.42% annexin V/PI-positive cells in the absence of exosomes (W/O Exo). Nevertheless, thepercentage of annexin V–positive cells is dramatically increased by the incubation with C17 exosomes, in the absence of blocking antibodies (Exo� NS IgG). This effect isalmost entirely reversed by anti–Tim-3 or anti–galectin-9 antibodies (Exo� anti–Tim-3, Exo� anti–Gal-9).

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Tim-3. We have shown here that the same type of galectin-9–carrying exosomes is consistently and specifically detected in theblood of NPC patients. These observations confirm that galectin-9–carrying exosomes are produced by NPC cells in vivo and haveenough stability for long-range diffusion from tumor interstitialfluids into the bloodstream. Some in vitro experiments confirm thatexosomes can behave as efficient carriers of galectin-9: intactexosome structure ensures its protection against proteolytic en-zymes without precluding interactions of its CRDs with externalligands (Figures 3,4).

This study was focused on NPC exosomes as carriers ofgalectin-9. Additional studies will be required to provide moredetails about other components of NPC exosomes with potentiallyimportant roles in host-tumor relationships. We have previously

reported that cells from one NPC xenograft release exosomes invitro containing both galectin-9 and the viral oncoprotein LMP1.24

While this work was in progress, Houali et al reported detection ofLMP1-carrying exosomes in plasma samples obtained from NPCpatients.46 Using our own approach based on immunomagneticcapture of HLA class II–positive exosomes, we have not been ableto detect LMP1 in circulating exosomes from several NPC patients(data not shown). These results suggests that galectin-9 is moreconsistently and more abundantly associated with NPC exosomesthan LMP1. Confirmation of this point will require further investi-gations especially on plasma samples collected at early stages ofthe disease. In this perspective, additional tools like galectin-9ELISA systems with high sensitivity in human plasma samples willalso be important. In another earlier report, Chan et al havepublished observations suggesting that apoptotic malignant cellsrelease apoptotic bodies containing short viral DNA fragments thatare detected in the peripheral blood of NPC patients.47 There isstrong evidence that galectin-9–containing exosomes are distinctfrom these bodies. First, the production of NPC exosomes contain-ing HLA class II molecules and galectin-9 is not dependent onapoptosis (Figure S3). Moreover NPC plasma vesicles captured onclass II beads contain the CD9 protein that was reported to beexcluded from apoptotic bodies48 (Figure 2C).

EBV-specific CD4� T-cell clones have proven to be remarkabletools to investigate the role of galectin-9–positive exosomes asagonists of the Tim-3 receptor. These clones exhibited intenseexpression of Tim-3 and were exquisitely sensitive to apoptosisinduction by recombinant galectin-9, with an ID50 as low as100 pg/mL. The same apoptotic response was induced by NPCexosomes. Using blocking antibodies, we could formally demon-strate that apoptosis induction was mediated by the contact ofgalectin-9 CRDs with the Tim-3 receptor presented at the surface ofexosomes and target T cells, respectively. A similar apoptoticresponse was induced by exosomes derived from HeLa cellstransfected with the galectin-9 but not the GFP gene, confirmingthe key role of galectin-9 in this process. Other CD4� T cellsspecific for non-EBV antigens are probably sensitive to galectin-9–positive exosomes, especially in conditions inducing expression ofthe Tim-3 receptor.

Although relatively neglected for a long time, EBV-specificCD4� T cells increasingly appear to play a key role in the immuneresponse against EBV-infected cells in healthy carriers as well as inpatients affected by posttransplant lymphomas.32,49,50 This prob-ably also applies to NPC and speaks strongly for a role ofgalectin-9–carrying exosomes in NPC immune evasion. Severalstudies have shown that the repertoire of the EBV-specific CD4�

and CD8� T-cell memory is only mildly altered in NPC patients bycomparison with healthy subjects.18,51-53 Moreover, there are indica-tions that precursors of EBV-specific cytotoxic T cells are abundantin the tumor tissue.52 Although they are functionally inactive insitu, they can be reactivated ex vivo.18 These observations suggestthat inhibitory factors contained in the tumor microenvironmenteither block cytotoxic T-cell maturation or prevent their activation/proliferation, potentially by eliminating a subgroup of Th1 cellsthat are required to provide T-cell help. Moreover, Lau et al havereported the presence of large numbers of regulatory T cells(CD4�CD25�FoxP3�) in tumors and peripheral blood of NPCpatients (in addition to a decrease in the absolute count ofperipheral CD4� T cells).54 Because pathways involving galectin-9and or Tim-3 participate in the generation of CD4�CD25�

regulatory T cells, one can speculate that galectin-9 exosomescould contribute to their expansion in tumors and peripheral blood

Figure 7. Exosomes from HeLa cells induce apoptosis in EBV-specific CD4�

T cells only when they contain galectin-9. (A) Purified exosomes released bytransfected HeLa cells expressing either GFP or galectin-9 (l-isoform) were analyzedby Western blot. The first 2 lanes on the left contain total extracts from cellstransfected with GFP and galectin-9 (Cell prot). Exosome proteins are analyzed in thenext 2 lanes (Exo prot). CD63, which is barely detectable in total cell extracts, is muchmore abundant in purified exosomes. In contrast to CD63, galectin-9 is detected inexosomes only when these are derived from cells transfected with the galectin-9gene. (B,C) There is no induction of apoptosis in CD4� T cells (EBNA1 and JM1H2clones) treated with control HeLa exosomes (Exo Hela-GFP). In contrast, treatmentwith galectin-9–positive exosomes induces apoptosis in a large majority of theEBNA1-specific CD4� cells (Exo Hela Gal-9). For JM1H2 cells (specific for gp350),the rate of apoptosis induced by Hela galectin-9 exosomes amount to 60% of the rateobserved with C17 exosomes at the same concentration. It is entirely prevented bypreincubation of target cells with the anti–Tim-3 antibody. Flow cytometry graphsrepresentative of some experiments summarized in panel B are displayed in panel C.

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of NPC patients.55,56 Some data recently obtained in our laboratorysupport this hypothesis (data not shown). The possible influence ofNPC exosomes on Th1 and regulatory T cells is likely to becomean important topic in adoptive immunotherapy. This therapeuticmodality has given promising results in early trials.57 As with otherEBV-associated diseases, CD4� T cells are expected to play a keyrole in the antiviral immune response.49 Monoclonal antibodiesneutralizing the Tim-3–binding domain of galectin-9, as was donewith the 9M1-3 antibody in this study, might be useful to enhancelong-term survival of CD4� EBV-specific T cells infused into NPCpatients.

Beyond the problem of host-tumor relationships in NPCpatients, our findings have important implications for the understand-ing of general aspects of immune regulations in health and disease.There are several cell types producing large amounts of galectin-9in various physiologic or pathologic contexts, including follicle-associated epithelial cells, postnatal thymus in healthy subjects,and astrocytes of brain white matter in plaques of multiplesclerosis.20,21,36 In the future, it will be important to know whetherthese various cell types can produce exosomes carrying galectin-9and whether these exosomes can behave as Tim-3 agonists onCD4� T cells as well as cells of innate immunity, for example,dendritic and natural killer (NK) cells.36

Acknowledgments

We thank David Anderson and Vijay Kuchroo (Harvard MedicalSchool, Boston, MA) for providing the 2E2 antibody, LaurenceZitvogel and Sophie Viaud (Institut Gustave Roussy, Villejuif,

France) for helpful advice and technical assistance in exosomepurification, and Xiaohui Wang and Tadamasa Ooka (Faculte deMedecine Laennec, Lyon, France) for helpful discussions.

This study was supported by grants from the Ligue NationaleContre le Cancer (Comite du Val de Marne), the Agence Nationalede la Recherche (EBV-inter), and the Herpesvirus and CancerNetwork.

Authorship

Contribution: J.K. carried out xenografts, cell cultures, and exo-some purification and was involved in most other studies; T.N.performed ELISA detection of galectin-9 in mouse plasma samples;C.P.-D. was involved in exosome purification and galectin-9detection by Western blot analysis and flow cytometry. A.R., D.A.,and J.M. produced CD4� T-cell clones and were involved inassessment of apoptosis by flow cytometry; S.S. made electronmicroscopy observations. E.R. contributed important reagentsrelated to tetraspanins; M.H. contributed essential reagents relatedto galectin-9; F.G., J.G., and S.L.M. collected plasma samples fromNPC and non-NPC patients; J.M. participated in the design of thestudy and edited the manuscript; and P.B. conceived the study andits design and drafted the manuscript. All authors read andapproved the final manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Pierre Busson, UMR 8126, Institut GustaveRoussy, 39 rue Camille Desmoulins, 94805 Villejuif, France;e-mail: pbusson@igr.fr.

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1966 KLIBI et al BLOOD, 26 FEBRUARY 2009 � VOLUME 113, NUMBER 9

For personal use only.on March 30, 2016. by guest www.bloodjournal.orgFrom

online November 12, 2008 originally publisheddoi:10.1182/blood-2008-02-142596

2009 113: 1957-1966  

Josef Mautner and Pierre BussonRubinstein, Sylvestre Le Moulec, Joël Guigay, Mitsuomi Hirashima, Fethi Guemira, Dinesh Adhikary, Jihène Klibi, Toshiro Niki, Alexander Riedel, Catherine Pioche-Durieu, Sylvie Souquere, Eric carcinoma cells

infected nasopharyngeal−exosomes released by Epstein-Barr virus containing−Blood diffusion and Th1-suppressive effects of galectin-9

 

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