Blood diffusion and Th1-suppressive effects of galectin-9–containing exosomes released by Epstein-Barr virus–infected nasopharyngeal carcinoma cells by Jihène Klibi, Toshiro Niki, Alexander Riedel, Catherine Pioche-Durieu, Sylvie Souquere, Eric Rubinstein, Sylvestre Le Moulec, Joël Guigay, Mitsuomi Hirashima, Fethi Guemira, Dinesh Adhikary, Josef Mautner, and Pierre Busson Blood Volume 113(9):1957-1966 February 26, 2009 ©2009 by American Society of Hematology

Specific detection of galectin-9–containing exosomes in the plasma of mice xenografted with NPC tumor lines. Specific detection of galectin-9–containing exosomes in the plasma of mice xenografted with NPC tumor lines. (A) A significant amount of galectin-9 is detected by ELISA in crude plasma samples from mice xenografted with NPC tumor lines (C15, C17, and C666-1), but not control mice either without xenografts (tumor-free) or xenografted with a non-NPC carcinoma tumor line (non-NPC). Assays were performed in triplicate on 3 plasma samples from 3 different mice. (B) A series of plasma samples were collected from 6 mice carrying C15 tumors of various sizes. Galectin-9 concentration in these samples is proportional to the tumor volume (y = 3.2× + 1.7, R2 = 0.994). (C) Captures with anti–HLA class II beads were performed on low-density vesicles (110-kg pellets) derived from pools of murine plasmas. Samples collected with anti–class II beads are designated nos. 3 and 6 (class II). Control samples collected with beads carrying nonspecific Ig are nos. 2 and 5 (NS Ig). In addition to plasma material, protein extracts from the C17 and the non-NPC xenografted tumors were used as controls for Western blot analyses (nos. 1 and 4). (D) Numerous vesicles approximately 70 nm in diameter are captured by HLA class II beads and visualized by electron microscopy when these procedures are applied to plasma vesicles from C17-xenografted but not control mice. Scale bar represents 500 nm. In the inset, 2 vesicles at original magnification (× 4). (E) Western blot analysis detects galectin-9, CD9, CD63, and the DR-α chain in C17 plasma vesicles. In contrast, none of these molecules are detected when the capture is performed on control mouse plasma. Note that CD63 is much more concentrated in C17 exosomes than in the tumor extract. Gp96, which is a cytoplasmic membrane protein, is detected in the C17 and non-NPC tumor extracts (lanes 1 and 4) but not in C17 exosomes (lane 3). Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

Specific detection of galectin-9–containing exosomes in the plasma of NPC patients. 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-density vesicles (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 a non-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) were used 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 class II 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 α chain of the DR molecule in 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 with non-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-NPC additional plasma samples (see Tables S1, S2, and S4 and Figures S1 and S2). Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

Intact exosomes protect galectin-9 against trypsin digestion. Intact exosomes protect galectin-9 against trypsin digestion. (A) Western blot detection of DR-α, CD63, and galectin-9 in exosomes released by C17 cells and purified on a sucrose gradient. Gp96 is a cytoplasmic protein typically absent 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 by comparison 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 μg exosome proteins. (C) Trypsin digestion assay, the same amount of C17 exosomes was used for each condition (35 μg total exosome proteins). They were subjected either to Triton lysis or mock treatment before incubation with trypsin. Two different trypsin concentrations, 15 μg/mL (high trypsin) or 0.01 μg/mL (low trypsin), were used in distinct experiments. For samples treated with low amounts of trypsin, short and long exposures 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, no trypsin, 37°C), and lane 9 (Triton lysis without trypsin addition and incubation at 37°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 trypsin digestion) as compared with lanes 2, 3, and 4 (mock treatment followed by trypsin digestion). After Triton lysis, undigested galectin-9 remained detectable only for the shortest time of incubation (5 minutes) with the smaller concentration of trypsin (0.01 μg/mL; lane 6). In lane 9, a substantial decrease of galectin-9 is observed despite the absence of trypsin, suggesting an effect of endogenous proteolytic enzymes after Triton lysis. Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

Galectin-9 CRD2 is presented at the surface of NPC exosomes. 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 were incubated with magnetic beads coated with the 9M1-3 monoclonal antibody directed to the CRD2 of galectin-9. Beads coated with purified isotype-matched nonspecific Ig were 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 by electron microscopy. (Middle panel) Western blot analysis detects the DR-α and galectin-9 proteins in C17 (C) and C666-1 (D) exosomes, whereas no similar proteins are recovered from control beads coated with irrelevant IgG. Positive controls are provided 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 the surface of live C17 cells by flow cytometry (E), whereas it is absent at the surface of C666-1 (F) cells. Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

Recombinant galectin-9 induces apoptosis in EBV-specific CD4+ T cells. Recombinant galectin-9 induces apoptosis in EBV-specific CD4+ T cells. (A) Intense membrane expression of Tim-3 is detected on 4 EBV-specific CD4+ T cell clones of Th1 subtype but not on polyclonal CD4+ T cells sorted directly from PBMCs (CD4+ PBMC; gray curve, control staining). EBV-specific clones are directed against the EBV-proteins EBNA3C (GB3C), gp350 or BLLF1 (JM1H2), EBNA1, and BZLF1. (B) A recombinant modified form of galectin-9 with increased stability (Gal-9 NC) induces apoptosis in JM1H2 CD4+ T cells with an ID50 of approximately 100 pg/mL (apoptosis was assessed according to the percentage of annexin V–positive cells). (C) Recombinant wild-type galectin-9 (gal-9 WT) at a concentration of 100 pg/mL induces apoptosis in GB3C T cells. Induction of apoptosis is suppressed by preincubation of the T cells with 10 μg/mL blocking anti–Tim-3 monoclonal antibody, but not a control IgG. Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

NPC exosomes induce apoptosis in EBV-specific CD4+ T cells through galectin-9/Tim-3 interaction. NPC exosomes induce apoptosis in EBV-specific CD4+ T cells through galectin-9/Tim-3 interaction. (A) NPC exosomes (C17) containing galectin-9 induce apoptosis in EBV-specific CD4+ T cells (GB3C clone, anti-EBNA3C) without significant effects on polyclonal CD4+ T cells sorted directly from PBMCs (CD4+ PBMC). Tested cells were incubated for 5 hours with purified C17 exosomes at a final concentration of 100 μg/mL exosomal proteins (corresponding approximately to 10 ng/mL galectin-9; Exo C17). 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 by preincubating 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, apoptosis induced by NPC exosomes is not prevented by a nonspecific Ig (NS IgG) or a monoclonal antibody directed to the extracellular portion of the DR-α chain (anti–DR-α, 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 in the 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 apoptosis in 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 more prevalent 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, the percentage 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 is almost entirely reversed by anti–Tim-3 or anti–galectin-9 antibodies (Exo+ anti–Tim-3, Exo+ anti–Gal-9). Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology

Exosomes from HeLa cells induce apoptosis in EBV-specific CD4+ T cells only when they contain galectin-9. Exosomes from HeLa cells induce apoptosis in EBV-specific CD4+ T cells only when they contain galectin-9. (A) Purified exosomes released by transfected HeLa cells expressing either GFP or galectin-9 (l-isoform) were analyzed by Western blot. The first 2 lanes on the left contain total extracts from cells transfected with GFP and galectin-9 (Cell prot). Exosome proteins are analyzed in the next 2 lanes (Exo prot). CD63, which is barely detectable in total cell extracts, is much more abundant in purified exosomes. In contrast to CD63, galectin-9 is detected in exosomes only when these are derived from cells transfected with the galectin-9 gene. (B,C) There is no induction of apoptosis in CD4+ T cells (EBNA1 and JM1H2 clones) treated with control HeLa exosomes (Exo Hela-GFP). In contrast, treatment with galectin-9–positive exosomes induces apoptosis in a large majority of the EBNA1-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 rate observed with C17 exosomes at the same concentration. It is entirely prevented by preincubation of target cells with the anti–Tim-3 antibody. Flow cytometry graphs representative of some experiments summarized in panel B are displayed in panel C. Jihène Klibi et al. Blood 2009;113:1957-1966 ©2009 by American Society of Hematology