The Journal of Immunology

Galectin-9 Increases Tim-3؉ Dendritic Cells and CD8؉ T Cells and Enhances Antitumor Immunity via Galectin-9-Tim-3 Interactions1

Keiko Nagahara,2*† Tomohiro Arikawa,2* Souichi Oomizu,2* Keiichi Kontani,‡ Atsuya Nobumoto,*¶ Hiroaki Tateno,ʈ Kota Watanabe,¶ Toshiro Niki,¶ Shigeki Katoh,§ Minoru Miyake,† Syun-Ichiro Nagahata,† Jun Hirabayashi,ʈ Vijay K. Kuchroo,# Akira Yamauchi,§ and Mitsuomi Hirashima3*

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of -mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-␥-producing Tim-3؉ CD8؉ T cells with enhanced granzyme B and perforin expression, although it induced CD4؉ T cell . It simultaneously increased the number of Tim-3؉CD86؉ mature dendritic ␥-cells (DCs) in vivo and in vitro. Coculture of CD8؉ T cells with DCs from Gal-9-treated mice increased the number of IFN producing cells and IFN-␥ production. Depletion of Tim-3؉ DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-␥-producing CD8؉ T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8؉ T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8؉ T cells. The Journal of Immunology, 2008, 181: 7660–7669.

alectin-9 (Gal-9),4 a ␤-galactoside-binding , was cule”), which resulted in the suppression of experimental autoim- first identified as an chemoattractant and ac- mune encephalitis (8). More recently, we showed that Gal-9 ame- G tivation factor (1–4). Subsequent studies in our labora- liorated another representative autoimmune model, collagen- tory revealed that Gal-9, similar to other galectins, modulates a induced arthritis, by inducing the apoptosis of synoviocytes (9), variety of biological functions such as cell aggregation and adhe- suppressing the generation of Th17 cells, and up-regulating the sion, apoptosis of tumor cells, and others (5, 6). We showed that induction of regulatory T cells (Tregs) (10). These results suggest Gal-9 induced the apoptosis of activated but not resting human that Gal-9 induces immunotolerance in animals with exaggerated CD4ϩ T cells (7). We also reported that Gal-9 induced apoptosis immune responses, including autoimmune diseases. of Th1 cells that expressed Tim-3 via Gal-9-Tim-3 interactions Nevertheless, the fact that Gal-9 can be a prognostic factor with (where Tim is “T cell Ig- and domain-containing mole- antimetastatic potential in melanoma and breast led us to postulate that Gal-9 exhibits antitumor activity in tumor-bearing hosts (11, 12). We recently found that Gal-9 induces the matura- *Department of Immunology and Immunopathology, †Department of Oral and Max- tion of human dendritic cells (DCs) from immature DCs (iDCs) illofacial Surgery, ‡Department of Respiratory, Breast, and Endocrine Surgery, and §Cell Regulation, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa, Japan; (13) and that it also stimulates innate immune cells, such as mono- ¶Research Center, Galpharma Company, Takamatsu, Kagawa, Japan; ʈResearch Cen- cytes and DCs, to secrete low levels of TNF-␣ via Gal-9-Tim-3 ter for Medical Glycoscience, National Institute of Advanced Industrial Science and interactions in both mouse and human model systems (14). These Technology, Tsukuba, Ibaraki, Japan; #Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical results led us to further hypothesize that Gal-9 ameliorates immune School, Boston, MA 02115 suppression in tumor-bearing hosts by promoting innate and adap- Received for publication June 19, 2008. Accepted for publication October 3, 2008. tive immunity, specifically via DC maturation. The costs of publication of this article were defrayed in part by the payment of page The purpose of the present study is to test the hypothesis that charges. This article must therefore be hereby marked advertisement in accordance Gal-9 potentiates immune responses in the context of tumor-in- with 18 U.S.C. Section 1734 solely to indicate this fact. ϩ duced immune suppression by maturing Tim-3 DCs and Tim- 1 This study was supported in part by a grant from the Japanese Ministry of Educa- ϩ ϩ ␥ tion, Culture, Sports, Science, and Technology. 3 CD8 T cells to produce IFN- via Gal-9-Tim-3 interactions between DCs and CD8 Tϩ cells. 2 K.N., T.A., and S.O. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Mitsuomi Hirashima, Depart- ment of Immunology and Immunopathology, Faculty of Medicine, Kagawa Univer- Materials and Methods sity, 1750- 1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0791, Japan. E-mail address: Animals [email protected] 4 Female BALB/c and BALB/c nude mice, 6- to 8-wk old, were purchased Abbreviations used in this paper: Gal-9, Galectin-9; CRD, carbohydrate recognition from Japan SLC and CLEA Japan, respectively. All animals were kept domain; DC, ; iDC, immature DC; mDC, mature dendritic cell; mIL-2, murine IL-2; MSC, myeloid-derived suppressor cell; PEC, peritoneal exudate cell; PI, under standard conditions in a 12-h day/night rhythm with free access to propidium iodide; poly-LN, polylactosamine; Tim, T cell Ig- and mucin domain- food and water ad libitum and received humane care in accordance with containing molecule; Treg, regulatory T cell. international guidelines and national law. Study protocols were reviewed and approved by the Animal Care and Use Committee of Kagawa Univer- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 sity (Kita-gun, Kagawa, Japan). www.jimmunol.org The Journal of Immunology 7661

Culture medium Granzyme B and perforin in PECs and spleen cells The medium used for cell culture experiments was RPMI 1640 (Sigma- PECs and spleen cells were harvested from BALB/c mice 7 days after Aldrich) supplemented with 10% FBS (JRH Biosciences), antibiotic solu- peritoneal Meth-A cell inoculation. Spleen cells were cultured in culture tion (Sigma-Aldrich), and 2-ME (Invitrogen). medium containing murine IL-2 (mIL-2) (1 ng/ml; Peprotech) for 5 days and incubated with monensin (2 ␮M; Biolegend) for 14 h. Spleen cells and PECs were fixed/permeabilized with Cytofix/Cytoperm solution (BD Bio- Inoculation of Meth-A cells into mice sciences). The following Abs were used: granzyme B-FITC (16G6; eBio- science) and perforin-PE (eBio MAK-D; eBioscience). The cells were an- Meth-A cells (a methylcholanthrene-induced sarcoma of BALB/c ori- alyzed by flow cytometry. gin; 5 ϫ 105) were passaged in the peritoneal cavities of mice (15). Meth-A cells were then cultured for 1 wk in culture medium at 37°C in Flow cytometric analysis of intracellular phosphorylated STAT 5% CO2 to avoid the contamination of immune cells. Expression and purification of recombinant human stable Gal-9 have been previously Spleen cells were harvested from BALB/c mice 7 days after peritoneal described (16), and stable Gal-9 was used instead of wild-type Gal-9 Meth-A cell inoculation. Spleen cells from PBS- or Gal-9-treated tumor- throughout the present experiments. Meth-A cells (5 ϫ 105) were in- bearing mice were pipetted into 96-well plates at 1 ϫ 106 cells/well for 6 h. oculated into the peritoneal cavities of BALB/c mice, and 10, 30, or 100 Spleen cells were fixed/permeabilized with Cytofix/Cytoperm solution (BD ␮g of Gal-9 per mouse was administered daily from day 0 to day 14. In Biosciences). The following Abs were used: anti-Stat4-PE (pY693; BD some experiments, we started the daily treatment of tumor-bearing mice Biosciences), anti-Stat6 Alexa Fluor 488 (pY641; BD Biosciences), and with Gal-9 (100 ␮g/mouse) from days 0, 3, 7, and 10 and stopped it on anti-Stat3-Alexa Fluor 647 (pY705; BD Biosciences). The cells were an- day 14. Meth-A cells were inoculated into the peritoneal cavities of alyzed by flow cytometry. BALB/c nude mice identically as in the method described above for wild-type BALB/c mice. Further, CD4ϩ and CD8ϩ T cells (1.5 ϫ 107) Apoptosis assay from naive BALB/c mice were transferred i.p. to BALB/c nude mice 1 ϩ ϩ CD4 and CD8 T cells were purified from naive BALB/c mice by mag- day before the i.p. inoculation of Meth-A cells. Gal-9 (100 ␮g/mouse) ϩ ϩ netic cell sorting using CD4 and CD8 T cell isolation kits (Miltenyi was administrated daily from day 0 to day 14 postchallenge, and the ϩ Biotech) according to the manufacturer’s instructions. The purity of CD4 survival rates of mice were monitored. ϩ ϩ and CD8 T cells was Ͼ90% as assessed by flow cytometry. CD4 or CD8ϩ T cells were cultured in anti-CD3 Ab-coated plates (2 ␮g/ml) at 3 ϫ 5 Cytotoxicity assay 10 cells/well for 24 h, followed by stimulation with Gal-9 at the indicated concentrations for 6 h. The proportion of apoptotic cells was determined by Peritoneal exudate cells (PECs) were obtained from Gal-9- or PBS- staining with propidium iodide (PI) and annexin V. treated tumor-bearing mice. NK cells were depleted by positive immu- noselection using MACS anti-DX5 beads (Miltenyi Biotech) according Lectin microarray analysis to the manufacturer’s instructions, followed by purifying mononuclear ϩ ϩ Purified CD4 and CD8 T cells from spleens of naive BALB/c mice were cells by density gradient centrifugation using Percoll (GE Healthcare). 51 also used for lectin microarrays as previously described (17). Briefly, Cr-labeled Meth A cells (target cells) were added at varying E:T ϩ ϩ CMRA-labeled CD4 and CD8 T cells (2.5 ϫ 106 cells/well) suspended ratios. After a 6-h culture, supernatants were harvested and transferred in PBS/BSA were added to each well of a glass slide (100 ␮l/well) and to Luma plates (PerkinElmer). Released 51Cr was counted using a incubated at 4°C for 1 h. After unbound cells were separated from the wells gamma counter (PerkinElmer). Spontaneous 51Cr release was deter- by gravity in cold PBS, cells bound to immobilized on a glass slide mined by incubating the radiolabeled target cells in the absence of were detected with an evanescent-field fluorescence scanner, SC-Profiler effector cells. Maximal 51Cr release was determined by incubating tar- (Moritex), under Cy3 mode. get cells in 2% Triton X-100. Cytotoxicity was calculated as follows: ϭ Ϫ lysis (%) ([cpm of test sample cpm of spontaneous release]/[cpm Analysis of DC subset of maximal release Ϫ cpm of spontaneous release]) ϫ 100. Spleen cells from tumor-bearing mice treated with PBS or Gal-9-for 7 days

were washed in PBS with 0.5% FCS and 0.1% NaN3, and incubated with ELISPOT assay fluorochrome-labeled Abs as follows: anti-mouse Tim-3-PE (8B.2C12; eBioscience), CD11c-allophycocyanin (N418; eBioscience), I-A/I-E-PE Ag-specific responses were enumerated by an IFN-␥ ELISPOT assay (BD (M5/114.15.2; eBioscience), and CD86-FITC (GL1; eBioscience). The Biosciences) according to the manufacturer’s instructions. Spleen cells cells were analyzed by flow cytometry. from PBS- or Gal-9-treated tumor-bearing mice were cocultured with ␮ Meth-A cells treated with 50 g/ml mitomycin C (MMC) in a MultiScreen DC generation from bone marrow-derived cells 96-well plate (Millipore) precoated with anti-IFN-␥ Abs for 18 h at 37°C

in 5% CO2. After culture, the membranes were thoroughly washed with iDCs were prepared from bone marrow precursors as previously described distilled water and incubated with biotinylated anti-mouse IFN-␥ for 1.5 h (18, 19). Briefly, bone marrow-derived cells obtained from femurs and at 37°C. The IFN-␥ spots were developed by a 3-amino-ethylcarbazone tibias of BALB/c mice were cultured in medium supplemented with 20 (AEC) substrate reagent (BD Biosciences) and counted with a dissecting ng/ml mouse GM-CSF (Peprotech). On days 2 and 4, nonadherent cells microscope. were removed, and adherent cells were cultured further. On day 6, nonad- herent and loosely adherent iDCs were harvested and cultured in medium with varying concentrations of Gal-9 for another 24 or 48 h to assess the Quantification of production effects of Gal-9 on DC maturation from iDCs to mature DCs (mDCs). The cells were analyzed by flow cytometry. For quantification of cytokine production, spleen cells from PBS- or Gal-9-treated tumor-bearing mice were plated in anti-CD3 Ab-coated (2 In vitro generation of cytotoxic CD8ϩ T cells ␮g/ml) plate at 3 ϫ 105 cells/well for 3 days. The amounts of IL-4 and IFN-␥ in the supernatants of cultured cells were measured with ELISA T cells were depleted from spleen cells harvested from tumor-bearing kits (R&D Systems) according to the manufacturer’s instructions. To mice treated with PBS or Gal-9 for 7 days by magnetic cell sorting evaluate the number of cytokine-producing cells, intracellular cytokine using CD90 microbeads (Miltenyi Biotec) according to the manufac- staining was performed. Spleen cells were cultured using the above turer’s instructions. T cell-depleted splenocytes were used as APCs. conditions for 3 days and incubated with PMA (50 ng/ml; Sigma-Al- After T cell depletion, CD3ϩ cells were Ͻ3% and I-A/I-Eϩ cells were drich), ionomycin (1 ␮g/ml; Sigma-Aldrich), and monensin (2 ␮M; Ͼ80%. In some experiments, Tim-3ϩ cells were depleted from isolated BioLegend) during the last 14 h. The cells were fixed and permeabilized APCs by complement-dependent lysis using anti-Tim-3 mAbs as de- with Cytofix/Cytoperm solution (BD Biosciences). The following Abs scribed previously (20). CD8ϩ T cells were also purified from spleens were used for staining: CD4-FITC (L3T4; eBioscience), CD8-FITC of naive BALB/c mice by magnetic cell sorting as described above. The (Ly-2; eBioscience), IFN-␥-allophycocyanin (XMG1.2; eBioscience), generation of CTLs was performed in a 96-well plate in culture medium IL-4-allophycocyanin (clone 11B11; eBioscience), Tim-1-PE (RMT1–4; Bio- supplemented with anti-CD3 Ab (0.5 ␮g/ml) and mIL-2 (1 ng/ml). To Legend), and Tim-3-PE (8B.2C12; eBioscience). Stained cells were analyzed inhibit Gal-9 activity, (30 mM) was added to the culture me- using a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo soft- dium. Sucrose (30 mM) was used as a control. Tim-3-Ig (100 ng/ml) ware (Tree Star). was added to the culture medium to suppress the effects of Tim-3. Both 7662 GALECTIN-9-TIM-3 INTERACTION PROMOTES ANTITUMOR IMMUNITY

APCs and CD8ϩ T cells were plated at 1.5 ϫ 105 cells per well, cul- tured for 5 days, and incubated with PMA (50 ng/ml), ionomycin (1 ␮g/ml), and monensin (2 ␮M) for an additional 14 h. The cells were harvested and stained with anti-CD8 Ab and anti-IFN-␥ Ab as described above. The levels of IFN-␥ in the supernatant of the cultured cells were measured with ELISA kits (R&D Systems) according to the manufac- turer’s instructions.

Statistical analysis For statistical comparisons, nonparametric two-tailed Mann-Whitney U test, log-rank test, and one-way ANOVA were used. Comparisons be- tween two groups were done by Mann-Whitney U test. All statistical analyses were done with Prizm 4 software (GraphPad Software).

Results Gal-9 prolongs the survival of tumor-bearing mice in a T cell-dependent manner

We first designed experiments to determine whether Gal-9 exerts FIGURE 1. Antitumor effect of Gal-9 in tumor-bearing mice is de- cell death-inducing activity in vitro against Meth-A cells (a murine pendent upon T cells. BALB/c or BALB/c nude mice were inoculated fibrosarcoma cell line). Gal-9 apparently induced the cell death of with 5 ϫ 105 Meth-A cells i.p., and the impact of Gal-9 treatment on Meth-A cells in a dose-dependent manner. Cell death was induced tumor rejection was evaluated by monitoring the survival outcomes. in almost all Meth-A cells by treatment with 10 ␮g/ml Gal-9 Day 0 represents the day of tumor inoculation. A, Tumor-bearing mice (supplemental figure 1).5 Such cell death was suppressed by were treated daily starting on day 0 with varying concentrations of ␮ ϭ lactose but not sucrose (data not shown). Thus, our next exper- Gal-9 (10, 30, and 100 g/mouse; n 10, each). B, Gal-9 treatment was started on various days after tumor inoculation (days 0, 3, 7, and 10; iments were designed to clarify whether Gal-9 exhibits antitu- n ϭ 10, each). C, Tumor-bearing nude mice were treated with 100 ␮g mor activity in vivo. Meth-A cells were inoculated into the per mouse of Gal-9 after tumor inoculation (PBS, n ϭ 7; Gal-9, n ϭ peritoneal cavities of BALB/c mice. Intraperitoneal Gal-9 treat- 10). D, BALB/c nude mice were adoptively transferred with CD4ϩ and ment was started at 1 h after the inoculation and continued CD8ϩ T cells from naive BALB/c mice i.p. 1 day before tumor inoc- every day until day 14. All of the control Meth-A-inoculated ulation, followed by daily treatment with either PBS or 100 ␮g/mouse ,p Ͻ 0.001; NS ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(mice died by 16 days postchallenge, whereas Gal-9 treatment of Gal-9 (n ϭ 10, each dramatically prolonged the survival of Meth-A-bearing mice in not significant. a dose-dependent manner (Fig. 1A). Complete tumor rejection was observed in eight of 10 mice treated with 100 ␮g of Gal-9. Gal-9 treatment at 30 ␮g also resulted in significant survival Gal-9 enhances cytotoxic T cell activity and granzyme B and prolongation, and complete rejection was observed in three of perforin expression in tumor-bearing mice 10 mice (Fig. 1A). We next designed experiments to determine whether Gal-9 en- To assess the therapeutic effects of Gal-9, daily treatment hances the cytotoxic activity of spleen cells in tumor-bearing mice with Gal-9 was started on day 3, 7, or 10 postchallenge and on day 7 after treatment. We harvested PECs from tumor-bearing stopped on day 18. Two of 10 mice treated with Gal-9 starting mice treated with Gal-9 and found a significant increase in the 3 days after Meth-A inoculation survived longer than 30 days number of activated CD8ϩ T cells (CD8ϩCD44ϩCD62LϪ)inthe (Fig. 1B). Further, even when the treatment was started 10 days peritoneums of Gal-9-treated tumor-bearing mice compared with after Meth-A inoculation, Gal-9 treatment significantly pro- CD8ϩ T cells in PBS-treated tumor-bearing mice on day 7 (Fig. longed the survival of Meth-A-bearing mice, although the dif- 2A). Granzyme B and perforin in PEC CD8ϩ T cells were mea- ference was modest (Fig. 1B). These results suggest that Gal-9 sured as surrogate markers of cytotoxic activity (22, 23). The lev- exhibits antitumor activity against Meth-A in vivo in dose- and els of granzyme B and perforin in PEC CD8ϩ T cells from Gal- time-dependent manners. 9-treated tumor-bearing mice were significantly higher than those Because T cell-mediated immune responses are required for suf- of PBS-treated tumor-bearing mice on day 7 posttreatment (Fig. ficient antitumor activity (21), we did experiments in Meth-A- 2B and supplemental figure 2A). Indeed, NK cell-depleted PECs bearing BALB/c nude mice to test the contribution of T cells or to from Gal-9-treated tumor-bearing mice exhibited higher cytolytic see whether Gal-9 directly kills tumor cells. Gal-9 treatment did activity against Meth-A cells than those from PBS-treated mice, not prolong the survival of Meth-A-bearing nude mice compared suggesting that Gal-9 treatment results in enhanced cytotoxic T with PBS-treated mice (Fig. 1C), in contrast to our results in wild- cell activity (Fig. 2C). 7 ϩ type BALB/c mice (Fig. 1A). When either 1.5 ϫ 10 CD4 or Next, we asked whether spleen CD8ϩ T cells also express gran- ϩ CD8 cells from naive spleen cells of normal mice were trans- zyme B and perforin. Spleen cells were harvested on day 7 after ferred into nude mice 1 day before Meth-A inoculation, Gal-9 Gal-9 treatment and cultured with mIL-2 for 5 days ex vivo. treatment significantly prolonged the survival of those mice Spleen CD8ϩ T cells from Gal-9-treated mice expressed signifi- ϩ ϩ (CD4 , p Ͻ 0.0001; CD8 , p ϭ 0.0045) (Fig. 1D). These results cantly higher levels of granzyme B and perforin compared with suggest that although Gal-9 did not directly induce sufficient cell PBS-treated mice (Fig. 2D and supplemental figure 2B), indicating ϩ death in Meth-A cells in vivo, the involvement of both CD4 and that Gal-9 treatment may potentiate the expression of granzyme B ϩ CD8 T cell-mediated immune responses is critical for Gal-9- and perforin in CD8ϩ T cells of tumor-bearing hosts to promote induced antitumor activity. antitumor immunity. To assess whether Gal-9-mediated cytotoxicity is associated with the activation of a STAT-signaling pathway, we compared the 5 The online version of this article contains supplemental material. phosphorylation of STAT-3, STAT-4, and STAT-6 in spleen The Journal of Immunology 7663

FIGURE 2. Gal-9 enhances cytotoxic T cell activity and granzyme B and perforin expression in tumor-bearing mice. PECs and spleens obtained from PBS- or Gal-9-treated (i.p., 100 ␮g/mouse) tumor-bearing mice were compared for the presence of infiltrating cytotoxic CD8ϩ T cells 7 days after challenge. A, Number of CD8ϩCD44ϩCD62LϪ T cells in the peritoneal cavities of each group. Results are the mean Ϯ SEM of five animals for each group. B, Percentage of granzyme Bϩ or perforinϩ cells in the peritoneal cavities of each group (gated on CD8ϩ T cells). Results are the mean Ϯ SEM of five animals for each group. C, Specific cytotoxicity was measured by 51Cr release assay. NK cell-depleted PECs were incubated for 6 h with Meth-A cells labeled with 51Cr. Percentage of specific lysis was as shown in Material and Methods. D, Spleen cells from PBS or Gal-9-treated tumor-bearing mice were cultured with 1 ng/ml mIL-2 for 5 days, and then the percentages of granzyme Bϩ and perforinϩ CD8ϩ T cells were evaluated by flow cytometry. Results are the mean Ϯ SEM of five animals for each group. E, Spleen cells from PBS- or Gal-9-treated tumor-bearing mice were cultured for 6 h and analyzed for intracellular expression of the indicated molecules. Histograms for the indicated ,ء .molecules (solid lines) and isotype-matched controls (filled histograms) are shown. Numbers are mean fluorescence intensity for each molecule .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05

CD8ϩ T cells of Gal-9-treated tumor-bearing mice with those from in tumor-bearing mice. We stimulated spleen cells with mitomycin PBS-treated tumor-bearing mice by flow cytometric analysis after C-treated Meth-A cells as a specific Ag and observed that the incubation for 6 h. It was evident that STAT-4 in CD8ϩ T cells number of IFN-␥-secreting cells in the Meth-A-stimulated spleen from Gal-9-treated tumor-bearing mice was activated (mean fluo- cells from PBS-treated tumor-bearing mice was very low, suggest- rescence intensity ϭ 11.1 Ϯ 0.37), whereas no change was de- ing that immunosuppression occurred in the tumor-bearing mice. tected in CD8ϩ T cells from PBS-treated mice (mean fluorescence In contrast, Meth-A stimulation significantly increased the number intensity ϭ 4.14 Ϯ 0.12) ( p ϭ 0.0286) (Fig. 2E). In contrast, the of IFN-␥-secreting cells in Gal-9-treated tumor-bearing mice (n ϭ phosphorylation of STAT-3 and STAT-6 remained unchanged in 5) compared with PBS-treated tumor-bearing mice (n ϭ 3, p ϭ the two groups (STAT-3: PBS ϭ 2.12 Ϯ 0.01, Gal-9 ϭ 2.09 Ϯ 0.0357) (Fig. 3A), suggesting an immunopotentiating role of Gal-9 0.01; STAT-6: PBS ϭ 1.47 Ϯ 0.02, Gal-9 ϭ 1.63 Ϯ 0.05) (Fig. in tumor-bearing mice. In the case of IL-4, the cytokine-secreting 2E). This suggested that Gal-9 treatment preferentially activated cells were not detectable even after Meth-A-stimulation (data not STAT-4, which is closely correlated with the generation of gran- shown). zyme B and perforin (24), as well as IFN-␥ (25). We next used anti-CD3 stimulation for 3 days to further assess the enhanced T cell cytokine production due to the effects of Gal-9 ␥ Gal-9 enhances IFN- and IL-4 production in tumor-bearing treatment. ELISA analyses revealed that the spleen cells of Gal- mice 9-treated tumor-bearing mice produced significantly higher levels CD8ϩ T cell-mediated cytotoxic activity and T cell-derived cyto- of both IFN-␥ and IL-4 compared with those of PBS-treated tu- kines, such as IFN-␥ and IL-4, may play critical roles in T cell- mor-bearing mice (n ϭ 5, each) (Fig. 3B), indicating that Gal-9 mediated antitumor immunity (26). Therefore, experiments were may exhibit antitumor activity by enhancing both IFN-␥ and IL-4 done to assess the effects of Gal-9 on IFN-␥ and IL-4 production production. 7664 GALECTIN-9-TIM-3 INTERACTION PROMOTES ANTITUMOR IMMUNITY

FIGURE 3. Gal-9 up-regulates the Ag-specific responses against tu- mor cells. Analysis of IFN-␥ and IL-4 production by spleen cells ob- tained from PBS- and Gal-9-treated tumor-bearing mice. A, Spleen cells obtained from PBS- or Gal-9-treated tumor-bearing mice on day 14 were stimulated with Meth-A cells treated with mitomycin C and ana- lyzed by ELISPOT assay as described in Materials and Methods. Re- sults are the mean Ϯ SEM (PBS: n ϭ 3; Gal-9: n ϭ 5). B, Spleen cells from PBS- or Gal-9-treated tumor-bearing mice on day 7 were stimu- lated on anti-CD3 Ab-coated plates for 72 h. IFN-␥ and IL-4 concen- trations in the culture supernatant were determined by ELISA. Results ,p Ͻ 0.001; NS ,ءء ;p Ͻ 0.05 ,ء .(are the mean Ϯ SEM (n ϭ 5, each not significant.

Additional experiments were done to identify the cellular source of IFN-␥ and IL-4. Spleen cells from PBS- or Gal-9- treated tumor-bearing mice were stimulated with anti-CD3 fol- lowed by incubation with PMA, ionomycin, and monensin for FIGURE 4. The source of IFN-␥ in tumor-bearing mice. Spleen cells an additional 14 h. The proportions of intracellular IFN-␥- and from PBS- or Gal-9-treated tumor-bearing mice were harvested on day ϩ ϩ IL-4-positive CD4 and CD8 T cells were assessed by flow 7, stimulated in anti-CD3 Ab-coated plates for 5 days, and cytokine cytometry. This analysis revealed that the up-regulation of production was assessed by flow cytometry. Harvested spleen cells IFN-␥ occurred for CD8ϩ but not CD4ϩ T cells (Fig. 4A). were restimulated with PMA and ionomycin in medium containing mo- ϩ nensin for an additional 14 h as described in Materials and Methods. A, IFN-␥ production in CD4 T cells was slightly decreased by ϩ ϩ ϩ ϩ Percentages of IFN-␥-producing cells gated on CD8 or CD4 cells. Gal-9 treatment (Fig. 4A), possibly because the Tim-3 CD4 T Numbers shown are the percentages of IFN-␥-positive cells (upper) and ␥ ϩ ϩ cells that are normally responsible for IFN- production were negative cells (lower)inCD8 or CD4 T cells. B, Percentages of induced to apoptosis by Gal-9 treatment (8, 10). In con- IL-4-positive CD8ϩ or CD4ϩ T cells. Numbers are the percentages of trast to IFN-␥, increases of IL-4-producing cells were de- IL-4-positive cells (upper) and negative cells (lower)inCD8ϩ or CD4ϩ ,p Ͻ 0.05, NS ,ء .(tected in CD4ϩ but not in CD8ϩ T cells, although the increase T cells. Results are the mean Ϯ SEM (n ϭ 4, each was not significant ( p Ͼ 0.05) (Fig. 4B). These results led us to not significant. assume that there is little or no involvement of type 2 cytotoxic, IL-4-producing CD8ϩ T cells in Gal-9-mediated antitumor activity. Gal-9 increases the number of Tim-3ϩCD8ϩ T cells in We found that Gal-9 preferentially induces cell death, includ- tumor-bearing mice ϩ ing apoptosis, in CD3-stimulated CD4 T cells and to a lesser ϩ ϩ We found that Tim-3 expression in CD3-stiumlated CD8 T extent in CD8ϩ T cells in a dose-dependent manner (Fig. 5, A cells from Gal-9-treated tumor-bearing mice was significantly up- and B). It thus became intriguing to clarify the reason for such ϩ ϩ ϩ regulated compared with that in CD8 T cells from PBS-treated divergent effects of Gal-9 on CD4 and CD8 T cells. We ␥ ϩ ϩ mice (Fig. 6A). Furthermore, the percentage of IFN- -producing hypothesized that CD4 and CD8 T cells express different sets of cells for Tim-3ϩCD8ϩ T cells in Gal-9-treated tumor-bearing mice N-orO-. Surface glycome profiling experiments using a was significantly higher than that for Tim-3ϪCD8ϩ T cells (Fig. ϩ lectin microarray revealed that CD8 T cells were more evidently 6B). These results suggest that Gal-9 increases the number of Tim- bound to lectins having affinities for O-glycans and polylac- 3ϩCD8ϩ T cells and that the source of IFN-␥ is naturally the ϩ tosamines (poly-LN) than CD4 T cells (Fig. 5C), thus indicating Tim-3ϩCD8ϩ T cell. ϩ that CD8 T cells evidently express more O-glycans and poly-LN IL-4 is normally produced by Tim-1ϩCD4ϩ T cells (Th2) in on their surfaces than do CD4ϩ T cells. allergic conditions (27) and also by type 2 cytotoxic CD8ϩ T cells The Journal of Immunology 7665

FIGURE 6. Gal-9 up-regulates Tim-3 expression on CD8ϩ T cells in vitro. Spleen cells from PBS or Gal-9-treated tumor-bearing mice were harvested on day 7 and then stimulated in anti-CD3 Ab-coated plates for 5 days. Tim-3 expression was evaluated by flow cytometry. A, Tim-3 expression was significantly induced in CD8ϩ T cells obtained from Gal-9-treated-mice. B, The frequency of IFN-␥-producing cells for Tim-3ϩCD8ϩ T cells was significantly higher than those for Tim- .p Ͻ 0.05 ,ء .(3ϪCD8ϩ T cells. Results are the mean Ϯ SEM (n ϭ 4

FIGURE 5. Divergent effects of Gal-9 treatment on CD4ϩ and CD8ϩ and Gal-9-treated tumor-bearing mice (supplemental figure 4A). T cells. CD4ϩ and CD8ϩ T cells from naive BALB/c mice cells were Thus, it is unlikely that Gal-9 treatment ameliorates tumor-in- cultured in anti-CD3 Ab-coated plates for 24 h, followed by stimulation duced immunosuppression by reducing Treg numbers. We also with Gal-9 at the indicated concentrations for 6 h. A, Annexin V and PI ϩ ϩ addressed whether MSCs are involved in the Gal-9-mediated staining in PBS or Gal-9-treated CD4 and CD8 T cells. One set of antitumor activity and found that Gal-9 decreased the number of representative data from five independent experiments is shown. B, Per- ϩ Ϫ ϩ ϩ MSCs weakly but significantly (supplemental figure 4B). This centages of annexin V PI cells in Gal-9-treated CD4 and CD8 T cells. (Annexin VϩPIϪ cells represent early apoptotic cells.) Results are suggested that the decreased number of MSCs by Gal-9 treat- p Ͻ 0.001 C, Differential lectin binding ment was at least partly involved in Gal-9-mediated antitumor ,ءءء .(the mean Ϯ SEM (n ϭ 5 profiling of CD4ϩ and CD8ϩ T cells. Freshly isolated CD4ϩ and CD8ϩ activity. T cells (2.5 ϫ 106 cells) were labeled with CMRA and allowed to bind to a lectin microarray. Bound cells were detected by an evanescent-field Gal-9 promotes DC maturation in tumor-bearing mice fluorescent scanner. Red rectangles indicate O- binding lectins We previously reported that Gal-9 induces human DC maturation and yellow rectangles indicate poly-LN binding lectins. M; Marker. in vitro (13) and also activates human and mouse DCs to release low levels of TNF-␣ (14). Together, these results raise the possi- bility that Gal-9 induces DC maturation in tumor-bearing mice. (28). Although we found that spleen cells from Gal-9-treated tu- We therefore assessed the effects of Gal-9 on DC maturation in mor-bearing mice produce an increased level of IL-4 (Fig. 3B), tumor-bearing mice in vivo. When tumor-bearing mice were Gal-9 did not increase the percentage of IL-4-producing CD4ϩ T treated daily with Gal-9 for 7 days, the percentage of I-A/I- cells (Fig. 4B). Moreover, we also found that the frequency of EϩCD86ϩ cells in the spleens of Gal-9-treated tumor-bearing mice Tim-1ϩCD4ϩ T cells was not enhanced by Gal-9 treatment (sup- (n ϭ 5) was higher than that of PBS-treated tumor-bearing mice plemental figure 3A) and confirmed that the source of IL-4 is, as (n ϭ 5) (Fig. 7A). expected, Tim-1ϩCD4ϩ T cells in Gal-9-treated tumor-bearing Furthermore, the percentage of CD86ϩ cells for CD11cϩ mice (supplemental figure 3B). We thus suggest that although cells from the spleens of Gal-9-treated tumor-bearing mice (n ϭ Gal-9 may not increase the number of IL-4-producing cells, it does 5) was significantly higher than that of PBS-treated tumor-bear- enhance the amount of IL-4 produced by Tim-1ϩCD4ϩ T cells. ing mice (n ϭ 5) (Fig. 7B). We did additional experiments to However, additional studies are required to clarify the exact investigate whether a different level of Tim-3 expression is ob- mechanisms. served between CD86ϪCD11cϩ (iDCs) and CD86ϩCD11cϩ From these results, it is quite likely that Gal-9 contributes to T (mDCs), as we have described that Gal-9-Tim-3 interactions are cell-mediated antitumor immunity. There are at least two expla- involved in LPS-induced DC activation. We found that Tim-3 nations for the Gal-9-mediated up-regulation of antitumor immu- expression for mDCs was greater than for iDCs (Fig. 7C). nity; one is that Gal-9 decreases the number or function of reg- Moreover, Gal-9 treatment significantly increased the number ulatory T cells (Tregs) and myeloid-derived suppressor cells of Tim-3ϩ mDCs in the spleens of tumor-bearing mice (n ϭ 5) (MSCs) capable of down-regulating proinflammatory T cell im- compared with those in PBS-treated mice (n ϭ 5) (Fig. 7D), mune responses, and another is that Gal-9 up-regulates DC mat- indicating that Gal-9 may promote maturation of DCs with uration resulting in T cell activation. To address the first pos- Tim-3 expression. sibility, we next designed experiments to determine whether We also did in vitro experiments using iDCs generated from Gal-9 treatment affects Tregs. However, we found there was no bone marrow-derived cells to assess the effects of Gal-9 on DC significant difference in the percentages of Tregs between PBS maturation. GM-CSF-induced iDCs were harvested on day 6 7666 GALECTIN-9-TIM-3 INTERACTION PROMOTES ANTITUMOR IMMUNITY

FIGURE 8. Gal-9 induces DC maturation in vitro. GM-CSF-induced iDCs were cultured for 24 or 48 h in varying concentrations of Gal-9 (1, 3, and 10 ␮g/ml). A, Percentages of CD86ϩ in CD11cϩ cells at 24 h. B, Percentage of Tim-3ϩ cells in CD11cϩ cells after 24 (upper) and 48 h ,ء .(lower) of culture in Gal-9. Results are the mean Ϯ SEM (n ϭ 4, each) FIGURE 7. Gal-9 induces DC maturation associated with the expres- p Ͻ 0.05. sion of Tim-3. Spleens obtained from PBS-treated or Gal-9-treated tu- mor-bearing mice were harvested on day 7 and the frequency of mDCs was assessed by flow cytometry. A, Percentages of I-A/I-Eϩ CD86ϩ cells from PBS- or Gal-9-treated tumor-bearing mice. B, CD86ϩcells in CD11cϩ cells from PBS- or Gal-9-treated tumor-bearing mice. Results used as a source of DCs. Coculture of DCs from Gal-9-treated are the mean Ϯ SEM (n ϭ 5, each). C, Comparison of Tim-3 expression tumor-bearing mice significantly enhanced IFN-␥ production by between iDC and mDCs in Gal-9-treated tumor-bearing mice. Histo- CD8ϩ T cells compared with DCs from PBS-treated mice (Fig. grams of Tim-3 (solid lines) and isotype-matched controls (filled his- 9A) and also increased the percentage of IFN-␥-producing CD8ϩ tograms) are shown. D, Comparisons of the number of T cells during coculture (Fig. 9B). These results suggest that DCs CD11cϩCD86ϩTim-3ϩ cells in spleens in PBS- and Gal-9-treated tu- from Gal-9-treated mice may exhibit a higher potential to fully ϩ ,ء .(mor-bearing mice. Data represent the mean Ϯ SEM (n ϭ 5, each .p Ͻ 0.01. prime naive CD8 T cells ,ءء ,p Ͻ 0.05 Because lactose, a representative ␤-galactoside, can interrupt binding between Gal-9 and its ligand, we cultured CD8ϩ T cells and shown to be Ͼ80% CD11cϩ. After washing, iDCs were with DCs from Gal-9-treated tumor-bearing mice in the presence further cultured with Gal-9 for an additional 24 or 48 h. Gal-9 of lactose. Lactose significantly suppressed IFN-␥-producing cells, induced the maturation of iDCs to mDCs in a dose-dependent although sucrose, a control disaccharide, did not (Fig. 9C). This manner as measured by CD86 expression of CD11cϩ cells after suggests that the lectin nature of Gal-9 is involved in the activation 24 h culture (Fig. 8A). However, Gal-9 failed to induce the of IFN-␥ production by CD8ϩ T cells. We also deleted Tim-3ϩ up-regulation of Tim-3ϩ in mDCs after 24 h of stimulation (Fig. DCs to assess the effects of Tim-3 expression on DCs with regard 8B). When iDCs were cultured for an additional 48 h with to DC function, as up-regulation of Tim-3 expression is observed Gal-9, the number of Tim-3ϩ mDCs was significantly enhanced not only in DCs but also in CD8ϩ T cells. Removal of Tim-3ϩ (Fig. 8B), suggesting that CD86 expression is followed by DCs resulted in a decreased percentage of IFN-␥-producing CD8ϩ Tim-3 expression. Taken together, our results suggest that T cells (Fig. 9D), suggesting a critical role for Tim-3ϩ DCs in Gal-9 enhances T cell-mediated antitumor immunity by increas- eliciting IFN-␥ production by CD8ϩ T cells. Therefore, additional ϩ ϩ ϩ ing not only Tm-3 CD8 T cells but Tim-3 mDCs as well. experiments were done to clarify the involvement of Gal-9-Tim-3 interactions in the activation of CD8ϩ T cells during coculture Gal-9 enhances IFN-␥ production from CD8ϩ T cells via ϩ with DCs. Gal-9-Tim-3 interactions between DCs and CD8 T cells To confirm the above possibility, we assessed the effects of a To determine whether DCs from Gal-9-treated tumor-bearing mice fusion , Tim-3-Ig, which inhibits the binding of Gal-9 with have an increased potential to activate CD8ϩ T cells compared Tim-3 (8, 14). As expected, the percentage of IFN-␥-producing with those from PBS-treated tumor-bearing mice, we compared the cells was significantly suppressed in the presence of Tim-3-Ig (Fig. effects of DCs from PBS- and Gal-9-treated tumor-bearing mice on 9E). Collectively, the present results suggest that Gal-9-Tim-3 me- IFN-␥ production in coculture experiments with naive CD8ϩ T diated interactions between DCs and CD8ϩ T cells play a critical cells stimulated with anti-CD3. T cell-depleted spleen cells were role for potentiating IFN-␥ production by Tim-3ϩCD8ϩ T cells. The Journal of Immunology 7667

FIGURE 9. Critical role for Tim-3- expressing DCs on CD8ϩ T cell acti- vation. Naive CD8ϩ T cells were cocultured with DCs from PBS- or Gal-9-treated tumor-bearing mice and stimulated with CD3 and mIL-2. A, The levels of IFN-␥ in culture super- natants of CD3-stimulated CD8ϩ T cells cocultured with DCs from PBS- or Gal-9-treated tumor-bearing mice. B, Percentages of IFN-␥-producing CD8ϩ T cells cocultured with DCs from PBS or Gal-9-treated tumor-bearing mice as mea- sured by flow cytometry. C, Suppressive effect of lactose on IFN-␥ production by CD8ϩ T cells. Naive CD8ϩ T cells and DCs from Gal-9-treated tumor-bearing mice were stimulated with CD3 and mIL-2 in the presence of 30 mM lactose (Lac). Sucrose (Suc) was used as a con- trol. D, Percentages of IFN-␥-produc- ing CD8ϩ T cells cocultured with DCs from Gal-9-treated tumor-bearing mice before and after Tim-3ϩ cell depletion. E, The effects of Tim-3-Ig on IFN-␥- production by CD8ϩ T cells cocultured with DCs from Gal-9-treated tumor- bearing mice. Cells were cultured in the presence or absence of 100 ng/ml Tim- 3-Ig. Percentage of IFN-␥-producing CD8ϩ T cells was assessed by flow cy- tometry. Results are the mean Ϯ SEM .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(n ϭ 5)

Discussion exosomes is a useful therapy for some cancer patients (37, 38). We In the present experiments, we have reported that Gal-9 treatment have shown that Gal-9 induces maturation of human immature induces antitumor activity in a T cell-dependent manner, suggest- DCs (13), as well as activating human and mouse Tim-3-express- ing a possible immunopotentiating activity for Gal-9 in the context ing DCs to produce low levels of TNF-␣ (14). In the present ex- of tumor immunity. This model is inconsistent with our recent periments we have shown that Gal-9 increases the number of Tim- ϩ ϩ findings that Gal-9 suppresses hyperimmune conditions in auto- 3 CD86 mDCs in vivo and in vitro. However, the mechanisms immune animal models by inducing the cell death of Tim-3ϩ Th1 by which Gal-9 can affect the maturation, number, and Tim-3 ex- and Th17 cells and increasing Treg generation (10), and with more pression of DCs are still unclear, although it is imperative to elu- ϩ ϩ recent findings by Wang et al. that the accumulation of Tim- cidate them. Moreover, we have found that mature Tim-3 CD86 ϩ ϩ ϩ 3ϩCD8ϩ T cells in a skin graft model was suppressed by Gal-9 DCs activate Tim-3 CD8 T cells to produce IFN-␥ and CD4 T treatment (29). Thus, it is quite important to elucidate which mech- cells to produce IL-4. Although IFN-␥ usually contributes to an- anisms are involved in the Gal-9-mediated enhancement of anti- titumor activity for many tumors (39, 40), IL-4 has been found to tumor immunity in tumor-bearing hosts. contribute antitumor activity for certain cell types such as Meth-A Tumor cells can escape the attack of the host immune system (41). Thus, it is conceivable that Gal-9 exhibits antitumor activity through various mechanisms, including immune evasion, immu- in Meth-A-bearing mice because Gal-9 enhances both IFN-␥ and nosuppression, or others (30). Progressive tumor growth seems to IL-4 production. be at least partly ascribed to tumor cell-induced down-regulation The fact that Gal-9 treatment also increases the number of Tim- ϩ ϩ of T cell-mediated immune responses (31). Indeed, an increased 3 CD8 T cells in tumor-bearing mice is consistent with the find- ϩ ϩ number or enhanced activity of Tregs (32, 33) and MSCs (34, 35) ings that the increases of Tim-3 CD8 T cells and Tim-3 ligand is observed in tumor-bearing mice and/or patients with cancer, and are observed in a mouse acute graft-vs-host disease model, al- it has been thought to be the major cause of immunosuppression in though there is no direct evidence that the Tim-3 ligand in these tumor hosts. It is, however, unlikely that Gal-9 exhibits antitumor experiments is Gal-9 (42). The exact mechanisms of Gal-9-in- activity by decreasing Tregs, because Gal-9 treatment does not duced up-regulation of Tim-3ϩ CD8ϩ T cells remain unclear, and induce a decrease in Treg numbers. In contrast, a decreased func- future research should focus on this area. tioning of MSCs may, at least in part, contribute to Gal-9-mediated Gal-9 induces the apoptosis of not only Tim-3-expressing Th1 antitumor activity, because Gal-9 decreased the frequency of Gr- cells (8) but also CD8 T cells (29) through Gal-9-Tim-3 interac- 1ϩCD11bϩ cells, probably MSCs, in the spleens of Gal-9-treated tions. In the present experiments we have, however, shown that tumor-bearing mice. Gal-9 does not induce cell death in activated CD8ϩ T cells, al- One strategy for combating tumors is to increase the number and though the apoptosis of activated CD4ϩ T cells is evidently in- activity of cytotoxic CD8ϩ T cells against tumor cells (36). Indeed, duced by Gal-9. These divergent effects of Gal-9 on CD4ϩ and there is increasing evidence that inoculation of DCs or DC-derived CD8ϩ T cells may be explained by the findings that CD4ϩ and 7668 GALECTIN-9-TIM-3 INTERACTION PROMOTES ANTITUMOR IMMUNITY

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