Intracellular Galectin-9 Enhances Proximal TCR Signaling and Potentiates Autoimmune Diseases

This information is current as Heng-Yi Chen, Yen-Fei Wu, Feng-Cheng Chou, Yu-Hsuan of September 29, 2021. Wu, Li-Tzu Yeh, Kuo-I Lin, Fu-Tong Liu and Huey-Kang Sytwu J Immunol published online 22 January 2020 http://www.jimmunol.org/content/early/2020/01/21/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 22, 2020, doi:10.4049/jimmunol.1901114 The Journal of Immunology

Intracellular Galectin-9 Enhances Proximal TCR Signaling and Potentiates Autoimmune Diseases

Heng-Yi Chen,*,† Yen-Fei Wu,‡ Feng-Cheng Chou,x,{ Yu-Hsuan Wu,‖ Li-Tzu Yeh,x Kuo-I Lin,‡,1 Fu-Tong Liu,#,1 and Huey-Kang Sytwu*,†,x,1

Galectin-9 is a risk gene in inflammatory bowel disease. By transcriptomic analyses of ileal biopsies and PBMCs from inflam- matory bowel disease patients, we identified a positive correlation between galectin-9 expression and colitis severity. We observed that galectin-9–deficient T cells were less able to induce –mediated colitis. However, several mouse-based studies reported that galectin-9 treatment induces T cell and ameliorates autoimmune diseases in an exogenously modulated manner, indicating a complicated regulation of galectin-9 in T cells. We found that galectin-9 is expressed mainly inside T cells, and its secreted form is barely detected under physiological conditions. Endogenous galectin-9 was recruited to immune synapses upon

T cell activation. Moreover, proximal TCR signaling was impaired in galectin-9–deficient T cells, and proliferation of these cells Downloaded from was decreased through an intracellularly modulated manner. Th17 cell differentiation was downregulated in galectin-9–deficient T cells, and this impairment can be rescued by strong TCR signaling. Taken together, these findings suggest that intracellular galectin-9 is a positive regulator of T cell activation and modulates the pathogenesis of autoimmune diseases. The Journal of Immunology, 2020, 204: 000–000.

nflammatory bowel disease (IBD) is a chronic relapsing Th1/Th17-mediated inflammatory process, affects any part of the http://www.jimmunol.org/ disorder of the gastrointestinal (GI) tract with pathological GI tract from the mouth to the anus, whereas UC seems to be a Th2- I characterization of intestinal inflammation and epithelial mediated disease characterized with inflammation confined to the injury. Patients with IBD experience severe diarrhea, abdominal top layers of colon (1–3). The current therapeutic strategies for IBD pain, fatigue, and weight loss. IBD is categorized into two major are limited by low responsive rate, moderate effectiveness, high types, Crohn disease (CD) and ulcerative colitis (UC). CD, a costs, and/or side effects, underlining the importance of identifying novel molecular markers and/or targets for optimized therapies. Jostins et al. (4) conducted an imputation-based association *Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, † analysis by using autosomal genotype-level data from 15 genome-

Taiwan; National Institute of Infectious Diseases and Vaccinology, National Health by guest on September 29, 2021 Research Institutes, Miaoli 350, Taiwan; ‡Genomics Research Center, Academia wide association studies of IBD and identified various IBD loci Sinica, Taipei 115, Taiwan; xDepartment and Graduate Institute of Microbiology markedly enriched in genes for immune regulation (ADA, CD40, { and Immunology, National Defense Medical Center, Taipei 114, Taiwan; Laboratory IFNGR2, IL12B, IRF8, STAT1, STAT3, TAP1, TAP2, and TYK2). of Translational Medicine Office, Development Center for Biotechnology, Taipei 115, Taiwan; ‖School of Medicine, National Defense Medical Center, Taipei 114, Interestingly, LGALS9 was identified as an IBD gene for a total Taiwan; and #Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan 163 IBD loci, suggesting its putative role in modulating autoim- 1K.-IL., F.-T.L., and H.-K.S. share senior authorship. mune diseases (4). ORCIDs: 0000-0001-9283-6144 (H.-Y.C.); 0000-0003-4350-4068 (Y.-F.W.); 0000- Expressed by many organisms, including and 0003-0868-258X (Y.-H.W.); 0000-0003-4477-0798 (K.-IL.). (5), galectins are b-galactoside–binding with conserved Received for publication September 16, 2019. Accepted for publication December carbohydrate-recognition domains (CRDs) that bind to cell surface 18, 2019. (6). Currently, 15 galectin members have been identified in This work was supported by the Ministry of Science and Technology, Taiwan (MOST mammals with broad tissue distribution (7–9). Galectin-9 is highly 106-2320-B-400-032-MY3, MOST 106-2321-B-400-014, MOST 107-2321-B-400-016, and MOST 108-2321-B-400-018) and the Tri-Service General Hospital (TSGH-C107- expressed in lymphoid organs and plays unique roles in immune 008-S02, TSGH-C108-007-008-S02, VTA107-T-1-1, and VTA108-T-1-2). regulation (10, 11). Galectin-9 is expressed in thymic epithelial cells H.-Y.C. carried out the majority of experiments and analyzed data. Y.-F.W. and K.-IL. and modulates the process of negative selection during T cell de- conducted the –related analysis. F.-C.C., Y.-H.W., and L.-T.Y. contributed to velopment (11). Galectin-9 is also an apoptosis-inducing factor that individual experiments. K.-IL., F.-T.L., and H.-K.S. gave advice. H.-Y.C. and H.-K.S. designed experiments and wrote the manuscript. binds to T cell Ig domain 3 (Tim-3) on Th1 cells (12). Address correspondence and reprint requests to Prof. Huey-Kang Sytwu, National Galectin-9 can bind to 4-1BB on T cells and promote their effector Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, functions (13). Current studies show that galectin-9 interacts with Miaoli 350, Taiwan. E-mail address: [email protected] CD44 or DR3, which further increases the stability and function of The online version of this article contains supplemental material. adaptive regulatory T cells (Tregs) (14, 15). Overall, these previous Abbreviations used in this article: BMDC, bone marrow–derived ; CD, reports provide strong evidence of the diverse roles of galectin-9 in Crohn disease; CLN, cervical lymph node; CT, cholera toxin; CTR, CellTrace Far T cell development and function. However, these data have been Red; CTV, CellTrace Violet; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; GC, germinal center; GEO, Gene Expression Omnibus; GI, gastro- obtained mainly using an exogenous galectin-9–based approach in intestinal; IBD, inflammatory bowel disease; iTreg, induced Treg, inducible Treg; KLH, which the physiological role of endogenous galectin-9 in the mod- keyhole limpet hemocyanin; MLN, mesenteric lymph node; MOG, myelin oligoden- drocyte ; MS, multiple sclerosis; NP, (4-hydroxy-3-nitrophenyl) acetyl; ulation of T cell responses may have been missed and/or masked. PLCg1, phospholipase Cg1; qPCR, quantitative real-time PCR; SRBC, sheep RBC; Interestingly, galectins are also localized in the and Tg, transgenic; Tim-3, T cell Ig mucin domain 3; Treg, regulatory T cell; UC, ulcerative nucleus, where they interact with other intracellular regulators colitis; WT, wild-type. to modulate a variety of cellular functions in a -dependent Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 or glycan-independent manner (16). For example, galectin-1 and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901114 2 INTRACELLULAR GALECTIN-9 IN T CELLS galectin-3 can serve as splicing factors by directly interacting to eliminate erythrocytes, washed, and resuspended in RPMI 1640 with Gemin4 during the process of formation (17). supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, Moreover, galectin-3 positively regulates macrophage phagocy- 0.1 mg/ml streptomycin, and 10 mM HEPES (Life Technologies, Waltham, MA). tosis through an intracellular mechanism (18). Other studies have + – demonstrated that galectin-3 and galectin-8 modulate antibacterial Isolation of CD4 CD25 T cells in opposite ways by binding to different host glycans Mouse CD4+ T cells were isolated by negative selection using a Mouse exposed on damaged phagosomes (19, 20). In T cells, galectin-3 CD4 T Lymphocyte Enrichment Set (BD Pharmingen, San Jose, CA). To is induced in an activation-dependent manner and is expressed deplete the Tregs, biotin-conjugated anti-mouse CD25 was added in the Ab . mainly in the cytosol but is barely detectable on the surface of mixtures. The purity of isolated cells was 90%. activated T cells (21). Another report has indicated that endogenous Isolation of lamina propria mononuclear cells galectin-3 interacts with Alix via a glycan-independent manner to Lamina propria mononuclear cells were isolated from mouse colons tissues trigger TCR internalization and to downregulate proximal TCR followed by previous report (27). Briefly, the colon was removed and cut signaling (22). One study has reported that galectin-9 can be into pieces, and the colon pieces were incubated with EDTA and DTT in expressed in the cytosol of Jurkat human leukemia cells (23), which HBSS, the suspension including epithelial cells, villus cells, subepithelial suggests an intracellular role of galectin-9 in T lymphocytes. cells, and intraepithelial lymphocytes were removed. The remaining lamina However, the physiological functions of intracellular galectin-9 in propria was digested by collagenase type VIII (Sigma-Aldrich, St Louis, MO), and the suspension was subjected to Percoll-gradient separation T cells are not completely understood. (GE Healthcare Bio-Sciences, Uppsala, Sweden). The collected cells were Although administration of exogenous galectin-9 or over- stained with Abs and analyze by flow cytometry. expression of galectin-9 plasmids in mice can suppress autoim- Downloaded from Immunofluorescence staining mune diseases by engaging different cell surface or + – (12, 14, 24–26), the precise effects on these diverse CD4 CD25 T cells were attached to poly-L-lysine–coated coverslips for receptor-bearing cells are difficult to identify. Moreover, the 15 min at 37˚C, then stained with anti-mouse CD4–Alexa Fluor 488 for 1 h at 4˚C, and the coverslips were fixed with 2% paraformaldehyde to stop the potential mechanisms through which endogenous galectin-9 reaction for 1 h. The cells were then stained with anti-mouse galectin-9– mediates T cell responses may have been overlooked in previ- Alexa Fluor 594 (137906; BioLegend) at 4˚C overnight and stained with ous studies that induce autoimmune diseases in conventional DAPI (422801; BioLegend) before microscope analysis. For the stimula- http://www.jimmunol.org/ + – Lgals92/2 mice. To examine the modulatory potential of galectin-9 tion, CD4 CD25 T cells stimulated with anti-CD3 (1 mg/ml, 145-2C11; m in T cells, we analyzed the expression kinetics and localization of BD Pharmingen) and anti-CD28 (1 g/ml, 37.51; BioLegend, San Diego, CA) for 30 min and then cross-linked with mouse anti-hamster IgG mix- endogenous galectin-9 in T cells and investigated the modula- ture (25 mg/ml, G94-56 and G70-204; BD Pharmingen) for 1 h. After tory role of endogenous galectin-9 in T cell–mediated autoim- stimulation, cells were attached to poly-L-lysine–coated coverslips for mune diseases in Lgals92/2 mice. We found that galectin-9 was 15 min, and the coverslips were fixed with 2% paraformaldehyde to stop recruited to immune synapses through T cell activation and pro- the reaction for 1 h. The cells were then stained with anti-mouse Zap70– Alexa Fluor 594 (693505; BioLegend) and anti-mouse galectin-9–Alexa moted T cell proliferation and Th17 cell differentiation. These Fluor 594 (137906; BioLegend) at 4˚C overnight and stained with DAPI actions may be involved as positive regulators of the pathogen- (422801; BioLegend) before microscope analysis. Images were ac- esis of T cell–mediated colitis and experimental autoimmune quired at room temperature with Zen 2 (black edition) version 2.0 by guest on September 29, 2021 encephalomyelitis (EAE). (ZEISS, Oberkochen, Germany) using an LSM880 confocal microscope (ZEISS) equipped with Plan-Apochromat 1003 with 1.4 numerical aper- ture oil objectives (ZEISS). Data were analyzed by Zen 2 (black edition) Materials and Methods version 2.0. Analyses of microarray and RNA sequence datasets T cell proliferation assay Publicly available microarray and RNA sequence datasets were down- To measure cell proliferation, CD4+CD25– T cells were cultured in trip- loaded from Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih. licate wells of 96-well, flat-bottom plates (4–5 3 105 cells/200 ml/well) gov/geo/) along with appropriate chip annotation data. All analyses were with the indicated amounts of plate-bound anti-CD3/CD28. After 48 h, carried out using Excel. GEO GSE57945 and GEO GSE3365 datasets were the cultured cells were pulsed with 1 mCi [methyl-3H] thymidine used for human IBD analysis, GEO GSE27302 dataset for analysis of (PerkinElmer, Shelton, CT) per well and harvested after 16–18 h. The mouse colitis, and GEO GSE78244 for human multiple sclerosis (MS) plates were harvested into a UniFilter-96 GF/C microplate (PerkinElmer), analysis. To analyze behaviors of specific genes in different clusters, and the incorporated [methyl-3H] thymidine was detected with a Packard fraction of cells expressing the gene of interest and average expression TopCount Microplate Scintillation Counter (PerkinElmer). To measure cell values among expressing cells were calculated (z score). The value of division, CD4+CD25– T cells were labeled with CFSE, CellTrace Far Red expression above the average expression values were showed in red color, (CTR), or CellTrace Violet (CTV) Cell Proliferation Kit (all from Life and the value of expression below the average expression values were Technologies) by incubating 1 3 107 cells in 1 ml of PBS with 2.5 mM showed in blue color. Correlation analysis was performed on normalized CFSE and 5 mM CTR or 10 mM CTV for 10 min at 37˚C. The reaction was expression values of interest genes in each classification. quenched by adding 10 ml of cold complete RPMI 1640 medium, and the Mice cells were washed twice. The labeled cells were cultured in 96-well, flat- bottom plates (5 3 105 cells/200 ml/well) and stimulated with plate-bound 2 2 C57BL/6 Lgals9 / mice were a gift from J. C. Paulson (The Scripps anti-CD3 (0.5 mg/ml) and anti-CD28 (0.5 mg/ml) for 3 d. Data were col- Research Institute, San Diego, CA) and were kindly provided by F.-T. Liu lected on an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), and For bone marrow–derived dendritic cell (BMDC)–T cell coculture, purified 2 2 C57BL/6 2D2 transgenic (Tg) and Rag1 / mice were purchased from wild-type (WT) or Lgals92/2 CTV–labeled CD4+CD25– T cells (2.5 3 The Jackson Laboratory (Bar Harbor, ME) and subsequently bred at the 105 cells/200 ml/well) were cocultured with BMDCs from C57BL/6 mice Animal Center of the National Defense Medical Center in Taipei, Taiwan, (5 3 104 cells/200 ml/well) in the presence of 50 mg of myelin oli- under specific pathogen-free conditions. Mice were treated in accordance godendrocyte glycoprotein (MOG)35–55 for 3 d. Cells were collected, with the Institutional Animal Care and Use Committee of the National and cell division was analyzed with an FACSVerse flow cytometer Defense Medical Center guidelines for experiments and approved by a (BD Biosciences). committee in the same office. BMDC cell isolation and culture Isolation of splenocytes and lymph node cells Bone marrow cells from tibias and femurs of C57BL/6 mice were cultured The cells were isolated from mouse spleen, mesenteric lymph node (MLN), with 10% FBS (Life Technologies), 5 3 1025 M2-ME(LifeTechnol- and cervical lymph node (CLN) by mash tissues between two frosted ogies), 5 mg/ml L-glutamine (Sigma-Aldrich), 100 U/ml penicillin, microscope slides. The cells were further treated with RBC lysis buffer 100 mg/ml streptomycin (Life Technologies), and 20 ng/ml GM-CSF The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. LGALS9 expression positively correlates with IBD severity. (A–C) Analyses of human galectin and T cell–related gene transcripts in ilea from healthy controls, UC, colon-only CD (cCD), ileal CD without deep ulcers (iCD-noDU), and ileal CD with deep ulcers (iCD-DU) patients. Data were derived from GEO dataset GSE57945. (A) The average expression of each individual. (B) The value of the T cell–related genes and LGALS9 in human UC and CD ileal biopsies compared with controls. (C) The correlation analyses between T cell–related genes and LGALS9 in human (Figure legend continues) 4 INTRACELLULAR GALECTIN-9 IN T CELLS

(Sigma-Aldrich). Myeloid progenitor cells were cultured in a 10-cm dish NP-specific Ab titers were measured in WT or Lgals92/2 mice as previ- at 4 3 106 cells. On day 3 of culture, 10 ml of fresh medium containing ously described (28). Briefly, the wells of Nunc-Immuno ELISA micro- GM-CSF was added to the culture. On day 6, half of the medium was plates (Sigma-Aldrich) were coated with 5 mg of either NP32–BSA (to replaced by fresh medium containing 20 ng/ml GM-CSF. On day 8, the measure the total amount of IgG) or NP4–BSA (to measure the amount of final nonadherent population was BMDCs. high-affinity IgG) (N-5050; Biosearch Technologies) for 2 h at 37˚C. The plates were washed five times with TBST, blocked with 1% BSA for Analysis of TCR signaling after cell stimulation 30 min, and then washed three times with TBST. Serum samples collected from WT or Lgals92/2 mice were diluted and added to the plates, and the + – m CD4 CD25 T cells were stimulated at 37˚C with anti-CD3 (1 g/ml, 145- plates were incubated for 90 min. The plates were washed five times with m 2C11; BD Pharmingen) and anti-CD28 (1 g/ml, 37.51; BioLegend) and TBST and then incubated with secondary Ab, HRP-conjugated anti-mouse m then cross-linked with mouse anti-hamster IgG mixture (25 g/ml, G94-56 IgG (1:20,000; Sigma-Aldrich). Finally, the plates were washed five times and G70-204; BD Pharmingen) for the indicated time periods. After with TBST, and 3,39,5,59-tetramethylbenzidine substrate was added (BD stimulation, cells were immediately resuspended in 23 lysis buffer Pharmagen). The absorbance was measured at 450 nm with an ELISA (50 mM Tris [pH 7.4], 10% glycerol, and 150 mM NaCl) containing plate reader (SpectraMax M2; Molecular Devices, Sunnyvale, CA). To protease inhibitor mixture (Sigma-Aldrich) and phosphatase inhibitor analyze Ig isotypes, mouse serum was collected and analyzed using a mixture (Roche, Mannheim, Germany). Cell lysates were separated by bead-based multiplex assay and the MILLIPLEX MAP Mouse Ig Isotyping 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and Magnetic Bead Panel (MilliporeSigma, Billerica, MA). probed with Abs to Zap70 (2705), phospho-Zap70 Tyr319 (2701), Lck (2752), phospho-Lck Tyr394 (2101), phospholipase Cg1 (PLC-g1) (2822), In vitro stimulation and differentiation of B cells phospho–PLC-g1 Tyr394 (2821) ( Technology, Beverly, MA), and b-actin (AC-15; Sigma-Aldrich). Mouse splenic B cells were purified by positive selection using B220 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purified 6 T cell differentiation B cells (1–2 3 10 /ml) were stimulated with goat anti-mouse F(ab9)2 Downloaded from + (10 mg/ml; Jackson ImmunoResearch Laboratories), anti-CD40 (1 mg/ml; Naive CD4 T cells were stimulated for 3 d with the indicated amounts of BD Biosciences), and IL-21 (200 ng/ml; Life Technologies). The frequency plate-coated anti-CD3 (0.05–10 mg/ml) and soluble anti-CD28 (1 mg/ml) of plasma cells was determined 5 d later. To assess B cell activation, isolated mAbs under conditions that stimulate Th1 cells (10 ng/ml IL-12, 5 ng/ml + B220 cells were stimulated with goat anti-mouse F(ab9)2 (10 mg/ml; IL-2, and 5 mg/ml anti–IL-4); Th2 cells (30 ng/ml IL-4, 5 ng/ml IL-2, and Jackson ImmunoResearch Laboratories) and harvested 24 h later. 5 mg/ml anti–IFN-g); Th17 cells (50 ng/ml IL-6, 5 ng/ml TGF-b, 5 ng/ml IL-2, anti–IL-4, and 5 mg/ml anti–IFN-g); or induced Tregs (iTregs) RNA extraction, reverse transcription, and quantitative (5 ng/ml TGF-b; 5 ng/ml IL-2). For intracellular staining, real-time PCR http://www.jimmunol.org/ T cells were stimulated with PMA and ionomycin in the presence of monensin (all from Sigma-Aldrich) for 3.5 h. The cells were analyzed by Cells were lysed with TRIzol Reagent (Invitrogen, Carlsbad, CA). Nu- gatingonCD4+ T cells. Data were collected on an FACSVerse flow cleoprotein complexes were then separated through bromochloropropane cytometer (BD Biosciences). (Sigma-Aldrich), and the samples were centrifuged at 12,000 rpm at 4˚C for 15 min. RNA was collected and precipitated with an equal volume of T cell apoptosis assay isopropanol. The samples were incubated at 25˚C for 10 min and centri- fuged at 12,000 rpm at 4˚C for 15 min. The RNA pellets were collected + – m The purified CD4 CD25 T cells were stimulated with anti-CD3 (0.5 g/ml) and washed with ethanol by vigorous mixing. RNA was obtained by m plus anti-CD28 (0.5 g/ml) for various lengths of time (days). For centrifugation at 7500 rpm at 4˚C for 10 min. After air-drying, the RNA recombinant galectin-9 treatment, purified CD4+CD25– T cells from WT 2 2 pellets were dissolved in RNase-free double-distilled water. Reverse or Lgals9 / mice were labeled with CTV and stimulated with anti-CD3 transcription was performed using a Reverse Transcription Kit (Applied by guest on September 29, 2021 and anti-CD28. After 72 h, the cells were purified with Histopaque (Sigma- Biosystems, Carlsbad, CA) to synthesize cDNA. Quantitative real-time Aldrich) and then cultured for 18 h at a density of 2 3 105 cells per well in 3 5 PCR (qPCR) was performed using Power SYBR Green Master Mix the presence of recombinant galectin-9 or PBS. The cells (1 10 cells/ (Life Technologies), and cDNA was amplified using a StepOnePlus Real- m 100 l/tube) were collected and stained with annexin V–FITC and Time PCR System (Applied Biosystems). Mouse b-actin mRNA was 7-aminoactinomycin D in annexin V binding buffer (all from BD Phar- used for internal normalization in all experiments. The fold change for mingen) for 15 min at room temperature in the dark. After staining, the each gene was calculated as the reciprocal of each mRNA normalized cells were analyzed within 1 h on an FACSCalibur or FACSVerse flow to the internal control. The SYBR green primer sequences were Lgals9 cytometer. forward, 59-CTTTCTACACCCCCATTCCA-39 and Lgals9 reverse, Immunization of mice and in vivo BrdU incorporation 59-CTCGTAGCATCTGGCAAG-39 and Actb forward, 59-CATTGCTG- ACAGGATGCAGAAGG-39 and Actb reverse, 59-TGCTGGAAGGTGG- Lgals92/2 or WT mice were i.p. immunized with 100 mg of (4-hydroxy-3- ACAGTGAGG-39. nitrophenyl) acetyl (NP)32–keyhole limpet hemocyanin (KLH) (Biosearch Technologies, Petaluma, CA). Lgals92/2 or WT mice were immunized Flow cytometry for B cell analysis m m with 10 l of sheep RBCs (SRBCs) in 500 l of saline by i.p. injection, CellswereresuspendedinPBSsupplemented with 0.5% BSA and 1 mM and serum was collected on the indicated days or at the time of sacrifice on EDTA (FACS buffer) and stained with the Abs listed below at 4˚C for day 7. The frequency of apoptotic germinal center (GC) B cells was de- 30 min. Mouse cell suspensions were preincubated with anti-mouse termined using an Annexin V Apoptosis Detection Kit (BD Pharmingen). CD16/32 (Fc Block, clone 93; eBioscience) for 5 min before the pri- To label cells with BrdU in vivo, immunized mice were injected i.p. with mary Abs were added. The anti-mouse Abs used for flow cytometric 1 mg of BrdU (Sigma-Aldrich) in PBS 24 h before sacrifice, The splenocytes analysis were the following: anti-CD45R/B220–allophycocyanin/allo- were isolated, and a BrdU Flow Kit (BD Pharmingen) was used to analyze phycocyanin–Cy7 (RA3-6B2), anti–GL-7–FITC (GL-7), anti-CD95–PE– the frequency of BrdU-incorporated GC B cells. Cy7 (Jo2), annexin V–allophycocyanin (550474), anti-CD86–PE (GL1), ELISA and Ig isotyping analysis anti-CD4–Alexa Fluor 700/FITC (GK1.5/RM4-5), anti-CD62L–PE (MEL-14), anti-CD44–allophycocyanin (IM7), anti-IgM–PerCP–Cy5.5 Culture supernatant fractions from T cell cultures were analyzed for levels (R6-60.2), anti–BP-1–PE (BP-1), anti-CD43–allophycocyanin (S7), and of IFN-g, IL-17, TNF-⍺, galectin-1, galectin-3, and galectin-9. The pro- anti-CD5–FITC (53-7.3) (all from BD Pharmingen); anti-CD38–PE–Cy7 cedures followed the standard protocols provided by R&D Systems (90), anti-BrdU–allophycocyanin (Bu20a), anti-CD69–allophycocyanin (Minneapolis, MN) and LifeSpan BioSciences systems (Seattle, WA). (H1.2F3), anti-CD138–allophycocyanin (281-2), anti-IgM–PE (RMM-1),

UC and CD ileal biopsies compared with controls. (D–F) Analyses of human galectin and T cell–related gene transcripts in PBMCs isolated from healthy controls, UC and CD. Data were derived from GEO GSE3365. (D) The average expression of each individual. (E) The correlation analyses between CD4 and LGALS9 in human UC and CD compared with controls. (F) The value of the IL17 and IFNG in human UC and CD compared with controls. (G and H) Analyses of mouse galectin and T cell–related gene transcripts in colon from T cell–transferred recipients. Data were derived from GEO GSE27302. (G) The average expression of each individual. (H) The correlation analyses between T cell–related genes and LGALS9 in colon from recipients. All bar graphs show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Galectin-9 contributes to the pathogenesis of T cell–mediated colitis. (A) CD4+CD45RBhi T cells from WT or Lgals92/2 mice were sorted using an FACSAria Fusion flow cytometer and then transferred into immunodeficient Rag12/2 recipients. (B) Body weight of Rag12/2 mice transferred with WT or Lgals92/2 CD4+CD45RBhi T cells (n = 4). Data are representative of five independent experiments. (C–F) At 35 d after transfer, Rag12/2 recipients were sacrificed, and colon morphology (original magnification 32) (C), weight (D), length (E), and ratio (F) were measured (n = 11). (G and H) Colon sections were collected (original magnification 3100), and a colitis histology score was calculated (n = 6; each colon were segmented for four sections). (I and J) The number of MLNs (I) and lamina propria cells (J) were calculated (n = 11). (K and L) Isolated lymphocytes from MLNs from Rag12/2 recipients were stained with (K) Ki67 (n =4)or(L) stimulated with PMA and ionomycin and analyzed for IL-17A and IFN-g production (n = 11). All bar graphs show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 6 INTRACELLULAR GALECTIN-9 IN T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Galectin-9 is recruited to immune synapses in activated T cells and participates in T cell homeostasis. (A–C) Flow cytometric analyses of the percentages of naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD4+ T cells in the spleen (SP), MLN, and CLN of 6-wk (n = 5)– and 12-wk (n = 6)–old WT and Lgals92/2 mice. (D–H) Purified CD4+CD25– T cells from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 for the number of days indicated (n = 3). Data are representative of three independent experiments. (D) Lgals9 expression was determined by (Figure legend continues) The Journal of Immunology 7 anti-CD21/35–allophycocyanin (7E9), and anti-CD23–PE–Cy7 (B3B4) patients (32)and revealed that the expression of LGALS9 was (from BioLegend). moderately increased in PBMCs from IBD patients (Fig. 1D), and T cell–mediated colitis model its expression was positively correlated with the expression of CD4 (Fig. 1E). However, the expression levels of major cytokine The purified CD4+CD25– T cells from 6- to 8-wk-old WT or Lgals92/2 + hi hi genes (IL17A and IFNG) were indistinguishable between healthy mice were further sorted into CD4 CD45RB CD62L T cells using an FACSAria Fusion flow cytometer (BD Biosciences) and then transferred control and IBD patients (Fig. 1D, 1F). These results could be into 6–8-wk-old immunodeficient Rag12/2 recipients. The body weight of explained by the immunosuppressive treatment in those patients the recipients was monitored twice a week. At 35 d after transfer, the (33–35). To further investigate whether an increased Lgals9 ex- recipients were sacrificed, and the colon length and weight were measured. pression is also positively correlated with disease severity in The lymphocytes were isolated from the MLNs and lamina propria, and the percentages of IFN-g+ and IL-17+ T cells were measured. mouse model, we analyzed the published transcriptomic data of colon from T cell–transferred recipients (36). The expressions T cell–mediated EAE model of Lgals9 and T cell–related markers (Cd5, Cd3d, Cd44, Cd3g,

Mouse 2D2 T cell clone can specifically recognize MOG35–55 presented by Cd69, Cd4, Il21r, Il2rg, Il17ra, Il2rb, Tnf, Ifng, Il1b, Il2, Il17a, MHC class II molecule H2-IAb and orchestrate immune responses to de- Il1a, and Il6) were also positively correlated with disease severity stroy myelin sheath. Therefore, CD4 T cells harvested from 2D2 Tg mice (Fig. 1G, 1H). Overall, only galectin-9 is significantly increased in can be considered as autoreactive T cells to induce EAE (29, 30). To in- intestines and/or PBMCs among all galectins in various tran- vestigate the potential role of galectin-9 in T cell and its subsequent effect in the pathogenesis of EAE, we generated Lgals92/2 2D2 Tg mice. After scriptomic databases, suggesting a modulatory role of this mole- modified from previous report (29, 30), purified CD4+CD25– T cells from cule in the pathogenesis of colitis. 2/2 6- to 8-wk-old WT 2D2 Tg mice or Lgals9 2D2 Tg mice were trans- Downloaded from ferred into 6–8-wk-old immunodeficient Rag12/2 recipients, and the re- Galectin-9 contributes to the pathogenesis of cipients were injected i.p. with pertussis toxin twice at days 1 and 2. EAE T cell–mediated colitis clinical manifestations were evaluated by daily assignment of scores from 0 to 5 as follows: 0, no clinical manifestation; 0.5, partial weakness of limb To evaluate the potential contribution of galectin-9 on the de- tail; 1, complete paralysis of the tail; 1.5, paralysis of the tail and waddling velopment of T cell–mediated colitis, we transferred WT or 2 2 2 2 gait; 2, paralysis of one hind limb; 2.5, paralysis of one hind limb and Lgals9 / CD4+CD45RBhi T cells into Rag1 / recipients partial paralysis of the other hind limb; 3, paralysis of both hind limbs;

(Fig. 2A). We found that intestinal inflammation, as shown by http://www.jimmunol.org/ 3.5, forelimb weakness; 4, forelimb paralysis; and 5, moribund or dead. weight loss, colon length, colon weight, and histology, was less At 28 d after transfer, the recipients were sacrificed, and the lymphocytes 2/2 were isolated from the CNS for analysis of the percentage of IFN-g+ and severe in recipients of Lgals9 T cell transfer (Fig. 2B–H). IL-17+ Tcells. Although the numbers of MLN cells were similar in WT and Lgals92/2 recipients, the Lgals92/2 group showed lower fre- Statistics quencies and numbers of T cells in the lamina propria (Fig. 2I, 2J). Prism v8.00 software (GraphPad Software, San Diego, CA) was used to Moreover, the percentages of proliferating cells (Ki67+) and Th17 generate graphs and for statistical analysis. Student unpaired t test was used cells were lower in MLNs (Fig. 2K, 2L). These results demon- for statistical analysis of the experiments in this study. All figures are presented as mean 6 SEM. A p value ,0.05 was defined as significant strate a positive role of galectin-9 in the development of colitis. (*p , 0.05, **p , 0.01, ***p , 0.001, and ****p , 0.0001). However, previous reports have indicated that recombinant by guest on September 29, 2021 galectin-9 ameliorates autoimmune diseases by engaging dif- Results ferent cell surface glycoproteins, including Tim-3, DR3, CD44, and 4-1BB (12, 14, 24–26). This discrepancy may be due to the Galectin-9 expression positively correlates with IBD severity differential modulatory roles of exogenous and endogenous Previous report has demonstrated that LGALS9 is a risk gene of galectin-9 in T cells or indirect effects by other galectin-9–acted IBD (4). However, the modulatory role of LGALS9 in IBD path- cells such as dendritic cells (DCs), intestinal epithelial cells, or ogenesis is still unknown. We first sought to interrogate whether B cells. LGALS9 expression is increased within the GI tract during intes- tinal inflammation. Through analyses of published transcriptomic Endogenous galectin-9 positively modulates T cell homeostasis data of ileal biopsies from a pediatric cohort of newly diagnosed Previous report has demonstrated that administration of galectin-9 IBD patients (31), we found an enrichment of LGALS9 transcripts in mice promotes thymocyte negative selection and T cell apoptosis within inflamed intestine and a positive correlation between its (9). However, these exogenous and high-dose approaches may not expression level and disease severity (Fig. 1A, 1B). Interestingly, elaborate the physiological roles of galectin-9 in T cell develop- the expression of genes related to T cell activation and ment and homeostasis. To address this issue, we compared thy- (CD4, CD44, IL17A, IL17F, IL6, IL1A, IL1B, TNF, and IFNG) mocyte maturation and T cell development between WT and were also increased in IBD patients, and the patterns were similar Lgals92/2 mice. The size and cellularity of thymi during different to LGALS9 (Fig. 1A, 1B), suggesting that T cells of IBD patients stages did not differ between Lgals92/2 mice and WT mice express higher level of LGALS9. To address this issue, we ana- (Supplemental Fig. 1A–C), which indicated that galectin-9 is dis- lyzed the correlation between LGALS9 and CD4, IL1R1, and IL6. pensable for thymocyte maturation. Moreover, the cell numbers in In our results, LGALS9 expression level was positively correlated spleen and lymph nodes and percentages of different lymphocyte with these T cell–related markers (Fig. 1C). We further analyzed populations (CD4+, CD8+, and CD19+) were indistinguishable other published transcriptomic data of PBMCs from adult IBD between Lgals92/2 and WT mice (Supplemental Fig. 1D, 1E).

qPCR. (E–G) The expression of surface and total galectin-9 was determined by flow cytometry with galectin-9 Ab (clone 108A2). (H) The culture su- pernatants were collected, and the galectin-1, galectin-3, and galectin-9 concentration was measured by ELISA. The galectin concentrations (picograms per milliliter) were normalized with viable cell numbers in each time point (picograms per 1 3 106 cells) (I and J) Purified CD4+CD25– T cells from WT mice were stimulated with anti-CD3 and anti-CD28 for 1 h and stained with DAPI, anti-CD4, anti-Zap70, and anti–galectin-9. The cells were further analyzed by confocal microscopy. Data are representative of three independent experiments (original magnification 31000). All bar graphs show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 8 INTRACELLULAR GALECTIN-9 IN T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Lgals92/2 T cells have impaired proximal TCR signaling. (A–C) Purified CD4+CD25– T cells from WT or Lgals92/2 mice were cultured with anti-CD3 and anti-CD28 for the number of days indicated, and the supernatant was collected for ELISA to quantify the concentrations of IFN-g, IL-17, and TNF-a. Data are representative of four independent experiments. (D and E) Purified CD4+CD25– T cells from WT or Lgals92/2 mice were cultured with the indicated amounts of (D) anti-CD3 and anti-CD28. The cells were cultured for 3 d, and T cell proliferation was measured as the in- corporation of [methyl-3H] thymidine. To measure cell division, (E) CFSE-labeled CD4+CD25– T cells were analyzed by flow (Figure legend continues) The Journal of Immunology 9

Although previous reports have indicated that galectin-9 in- analyzed the cytokine production by CD4+ T cells from WT and creases the population of adaptive Tregs (14, 15), the percentages Lgals92/2 mice. CD3/CD28-stimulated Lgals92/2 T cells secreted of -derived and peripherally derived Tregs in spleen and less IFN-g, IL-17, and TNF-a than did WT T cells (Fig. 4A–C). lymph nodes (Supplemental Fig. 1F) were similar between Cell proliferation was significantly lower for Lgals92/2 T cells than Lgals92/2 and their control littermates. These results suggest that for WT T cells, as determined by 3H-thymidine incorporation assay endogenous galectin-9 is not critical during the development of (Fig. 4D) and CFSE-labeling cell-division assay (Fig. 4E). thymocytes and peripheral lymphocytes. To investigate further whether galectin-9 mediates T cell pro- To investigate further whether endogenous galectin-9 modu- liferation in an Ag-specific manner, WT or Lgals92/2 2D2 T cells lates T cell homeostasis in an age-dependent manner, we ana- were cocultured with BMDCs in the presence of MOG35–55. lyzed the frequencies of naive (CD44loCD62Lhi) and memory-like Consistent with the results from anti-CD3/CD28 stimulation, Ag- (CD44hiCD62Llo) T cells in WT and Lgals92/2 mice at different specific T cell proliferation was diminished in Lgals92/2 2D2 ages. The age-dependent increase in the memory-like phenotype T cells (Fig. 4F). Interestingly, Lgals92/2 T cells proliferated was attenuated in Lgals92/2 Tcells(Fig.3A–C),whichdem- similarly to WT T cells when stimulated with PMA and ion- onstrates that galectin-9 positively regulates the homeostasis of omycin (Fig. 4G), which suggests that galectin-9 modulates memory T cells in peripheral lymphoid organs. T cells in the proximal signaling stage. To examine this issue further, we analyzed the phosphorylation status of proximal Intracellular galectin-9 is recruited to immune synapses upon signaling-related molecules from stimulated T cells. The phos- T cell activation phorylation of TCR-mediated Lck, Zap70, and PLCg1 was at- 2 2 Galectins usually colocalize with glycosylated surface molecules tenuated in Lgals9 / T cells (Fig. 4H, Supplemental Fig. 2), Downloaded from but are also detected in the cytosol and nucleus, where they in- and this subsequently impaired T cell proliferation. teract with various regulators to modulate a variety of cell func- Although we found that endogenous galectin-9 facilitates T cell tions (16, 22, 37). However, the expression kinetics and pattern of activation, it was unclear whether the modulatory effect of galectin- galectin-9 in T cells are unclear. We analyzed the kinetics of 9 in T cells occurs through an intrinsic or extrinsic manner. To galectin-9 RNA expression in resting and activated T cells. The examine this issue, we assessed cell division by coculturing 2/2 + expression of Lgals9 decreased significantly after anti-CD3/CD28 CTR-labeled WT and CFSE-labeled Lgals9 CD4 T cells. The http://www.jimmunol.org/ stimulation (Fig. 3D). proliferative impairment of Lgals92/2 T cells could not be rescued To investigate further whether galectin-9 is expressed inside or in cocultured WT T cells, which suggests that galectin-9 modu- on the surface of T cells or is secreted by T cells, we used flow lates T cell proliferation in an intracellular manner (Fig. 4I). cytometric analysis to identify its surface and intracellular ex- Considering that WT T cells secrete a very low amount of pression and ELISA to quantify its secreted form. Galectin-9 was galectin-9 (Fig. 3H), we next added recombinant galectin-9 in this barely detected on the T cell surface but was abundantly expressed assay. An addition of high level of exogenous galectin-9 increased in the cytosol of resting T cells. Similar to the RNA kinetics, the apoptosis in T cells (Supplemental Fig. 3B), which is correlated expression level of galectin-9 decreased at different time points with previous reports (12, 38, 39). Interestingly, the proliferation after T cell activation (Fig. 3E–G). We also detected the amounts defect in Lgals92/2 T cells could not be recovered by recombinant by guest on September 29, 2021 of galectin-1 and galectin-3 in the supernatants, because these two galectin-9 (Fig. 4J). This finding supports an intracellular role of galectins have been reported to be expressed in T cells. In our galectin-9 in T cell activation and proliferation. results, galectin-1 and galectin-3 were abundantly detected in the Intracellular galectin-9 plays a limited role in T cell apoptosis supernatants. Again, galectin-9 was barely detected in the super- natants (Fig. 3H). These results suggest that endogenously pro- Previous reports have demonstrated that galectin-9 can interact duced galectin-9 can physiologically modulate T cell functions with Tim-3 to trigger cell death (12, 38, 39). We found that Tim-3 in an intracellular manner. was induced after TCR stimulation and that the Tim-3 levels were 2/2 To evaluate galectin-9 localization in T cells further, we per- indistinguishable between WT and Lgals9 T cells (Supplemental formed confocal microscopic analysis. Galectin-9 expression Fig. 3A). We also observed that the addition of exogenous galectin- 2/2 exhibited a disperse distribution within the cytosol of resting T cells 9 increased apoptosis in both WT and Lgals9 Tcells (Fig. 3I, 3J). Interestingly, galectin-9 was recruited to immune (Supplemental Fig. 3B). However, it was unclear whether en- synapses after anti-CD3/CD28 stimulation (Fig. 3I, 3J). These dogenous galectin-9 also participates in T cell apoptosis. To ex- results suggest that galectin-9 may play a role in T cell activation. amine this issue, we analyzed activation-induced cell death without the addition of exogenous galectin-9 and found similar Intracellular galectin-9 potentiates T cell activation percentages of apoptotic cells in WT and Lgals92/2 T cells and proliferation (Supplemental Fig. 3C, 3D). These findings suggest that exoge- Our confocal microscopic data led us to hypothesize that galectin-9 nous galectin-9 can trigger Tim-3–dependent, but endogenous modulates TCR signaling and influences T cell functions. We next galectin-9–independent, cell death. Endogenous galectin-9 appears

2/2 cytometry. (F) Purified CTV-labeled WT or Lgals9 2D2 T cells were cultured with BMDCs in the presence of MOG35–55 for 72 h, and T cell division was analyzed by flow cytometry. Data are representative of four independent experiments. (G) Purified CD4+CD25– T cells from WT or Lgals92/2 mice were cultured with the indicated amounts of PMA and ionomycin. The cells were cultured for 3 d, and T cell proliferation was measured as the incor- poration of [methyl-3H] thymidine. (H) Purified CD4+CD25– T cells from WT or Lgals92/2 mice were stimulated at 37˚C with anti-CD3/CD28 and then cross-linked with mouse anti-hamster IgG for the indicated times. After stimulation, the cells were immediately resuspended in lysis buffer and subjected to immunoblotting analysis. Data are representative of three independent experiments. (I) CTR-labeled WT and CFSE-labeled Lgals92/2 CD4+CD25– T cells were cultured separately or together and stimulated with anti-CD3 and anti-CD28 for 3 d, and the data were collected using flow cytometry. Data are representative of three independent experiments. (J) Purified CD4+CD25– T cells from WT or Lgals92/2 mice were labeled with CTV and stimulated with anti-CD3 and anti-CD28. After 60–72 h, the cells were purified with Histopaque and plated for 18 h at a density of 2 3 105 cells per well in the presence of recombinant galectin-9 or PBS. Analysis of CTV-labeled CD4+CD25– T cells. Data were collected by flow cytometry. Data are representative of three independent experiments. All bar graphs show mean 6 SEM. **p , 0.01, ***p , 0.001, ****p , 0.0001. 10 INTRACELLULAR GALECTIN-9 IN T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Galectin-9 contributes to T cell–dependent Ab responses and positively regulates Th17 differentiation. (A) qPCR showing the mRNA levels of galectin-9 in GC B and naive B cells isolated from mice immunized with NP32–KLH on day 14 (n = 3). (B and C) ELISA showing the NP-specific IgG 2/2 titers against NP32–BSA (B)orNP4–BSA (C) in the serum of Lgals9 and littermate control WT mice on the indicated days after i.p. immunization with NP32–KLH [WT (n = 4) and KO (n =7)in(B); WT (n = 6) and KO (n =8)in(C)]. (D) The ratio of OD obtained (Figure legend continues) The Journal of Immunology 11 to be directly involved in T cell activation and proliferation but Endogenous galectin-9 promotes Th17 cell differentiation may be dispensable for cell death. TCR activation and strength differentially modulate Th1, Th2, and Galectin-9 positively regulates B cell functions in a Th17 cell differentiation (42–45). To investigate whether galectin- T cell–dependent manner 9 regulates the differentiation of Th cells, we polarized Lgals92/2 In addition to the regulatory role of galectin-9 in T cells, recent T cells into different Th cells. Strikingly, Th17 polarization was 2/2 reports have also indicated that addition of galectin-9 suppresses significantly impaired in Lgals9 T cells, compared with WT B cell signaling by binding to N-glycans of the BCR (40, 41). control cells (Fig. 5L), which suggested a positive role of galectin- However, the role of endogenous galectin-9 in B cell development 9 in Th17 differentiation. To investigate further whether the defect is unclear. The cellularity of B cells in bone marrows or spleens in Th17 differentiation could be rescued by strong TCR signaling, 2/2 during the different developmental stages was similar in WT and we stimulated Lgals9 T cells with higher anti-CD3 concen- Lgals92/2 mice (Supplemental Fig. 4A). Interestingly, the IgA trations than those used normally in Th17 polarization. The in- 2/2 and IgG3 levels were significantly lower in Lgals92/2 than in WT creased TCR signaling rescued the defect of Lgals9 Th17 mice (Supplemental Fig. 4B), which suggests an endogenous role differentiation (Fig. 5M). This observation supports the idea that of galectin-9 in B cell function. Th17 differentiation can be positively sustained by galectin-9 in To investigate whether impairment of Ab production is affected the presence of TCR signaling below a certain range. However, by galectin-9 expression, we analyzed the galectin-9 RNA ex- the percentages of Th1, Th2, and iTreg cells were similar between 2/2 pression in naive and GC B cells. The expression of Lgals9 was Lgals9 and WT T cells, which suggests that galectin-9 is dispensable in the differentiation of Th cells with a lower TCR downregulated in GC B cells compared with naive B cells Downloaded from (Fig. 5A). This activation-dependent decrease in galectin-9 ex- signaling requirement. pression in B cells is consistent with previous observation in Galectin-9 contributes to the pathogenesis of T cells (Fig. 3D). To examine whether galectin-9 modulates the T cell–mediated EAE affinity maturation of Abs, we analyzed the production of high- affinity NP-specific IgG in NP-KLH–immunized Lgals92/2 mice. Our data showed that galectin-9 positively modulates T cell ac- The amounts of total and high-affinity NP-specific IgG in the tivation and Th17 differentiation, suggesting that it may also in- http://www.jimmunol.org/ serum were lower in Lgals92/2 mice than in WT controls volve in the pathogenesis of Th17-mediated autoimmune diseases (Fig. 5B, 5C). Interestingly, the ratio of high-affinity/total NP- such as MS. We analyzed the published transcriptomic data of + specific IgG was lower in Lgals92/2 mice than in WT controls, CD4 T cells isolated from PBMCs of MS patients. Our results especially on day 14 (Fig. 5D). Moreover, the number of GC revealed that LGALS9 was highly expressed in patients, compared B cells was lower in NP–KLH-immunized Lgals92/2 mice than in with healthy controls. Moreover, expression of T cell activation controls (Fig. 5E). Taken together, these findings suggest a posi- markers (CD44, CD5, and CD69), cytokine receptors (IFNGR1, tive role of galectin-9 in affinity maturation. IL13RA1, and IL17RA), and cytokine (IL2) were also increased in We also evaluated B cell proliferation and apoptosis after im- patients when compared with healthy controls (Fig. 6A, 6B). Overall, these results support a positive role of galectin-9 in the munization with SRBCs. B cell proliferation decreased after im- by guest on September 29, 2021 munization in Lgals92/2 mice (Fig. 5F). However, the percentage development of MS. To validate further whether the pathogenesis of apoptotic cells was similar between WT and Lgals92/2 B cells of EAE is mediated by endogenous galectin-9 in T cells, we + 2/2 (Fig. 5G). These results suggest that endogenous galectin-9 pref- transferred naive CD4 T cells isolated from WT or Lgals9 2/2 erentially affects B cell proliferation, but not apoptosis, which 2D2 TCR Tg mice into Rag1 mice and then injected the mice was similar to our findings in T cells (Fig. 4D, Supplemental Fig. with pertussis toxin (Fig. 6C). We found that the disease was 2/2 3C, 3D). We next investigated whether impairment of B cell ameliorated in recipients of Lgals9 T cells (Fig. 6D, 6E). On functions in Lgals92/2 mice is B cell autonomous. Anti-IgM– day 27 after transfer, the mice were sacrificed for cell analysis. stimulated B cell activation (Fig. 5H) and plasmablast populations Consistent with our data for T cell–mediated colitis, the total T + (Fig. 5I) were similar between isolated WT and Lgals92/2 B cells, and IL-17 T cell numbers in the inflamed CNS were significantly 2/2 which suggests that intrinsic galectin-9 may be dispensable in lower in Lgals9 -transferred mice (Fig. 6F–H). These results modulating B cell functions. Therefore, the functional impairment indicate that endogenous galectin-9 potentiates T cell activation of B cells, in terms of cell division, GC B cell population, Ab and differentiation and further involves in the pathogenesis of production, and affinity maturation observed in vivo may relate autoimmune diseases, such as colitis and EAE. to the defects in Lgals92/2 T cells. This assumption is supported by the finding that the frequencies of CD4+ Tcellsandmemory Discussion T cells decreased after NP–KLH immunization in Lgals92/2 Our finding demonstrates an intracellular role of galectin-9 in T cell mice (Fig. 5J, 5K). activation and proliferation. Moreover, the proliferation defect in

from (C) versus (B). (E) Flow cytometric analysis showing the frequency of GC B cells in splenic B cells of WT and Lgals92/2 mice on day 14 after + immunization with NP32–KLH (WT [n = 5] and KO [n = 6]). (F and G) Flow cytometric analysis showing the frequency of (F) BrdU GC B cells and (G) annexin V+ GC B cells in the spleens of WT and Lgals92/2 mice on day 7 after immunization with SRBCs (WT [n = 8] and KO [n = 6]). (H) Flow cytometric analysis showing the expression of CD86 and CD69 in splenic B cells isolated from WT and Lgals92/2 mice 24 h after anti-IgM stimulation. Data are representative of two independent experiments. (I) Flow cytometric analysis showing the frequency of CD138+B220lo plasmablasts among WT or Lgals92/2 splenic B cells stimulated with IL-21, anti-CD40, and anti-IgM for 5 d. Data are representative of two independent experiments. (J) Flow + 2/2 cytometric analysis showing the frequency of splenic CD4 T cells on day 7 after NP32–KLH immunization in WT and Lgals9 mice (WT [n = 4] and KO [n = 6]). (K) Flow cytometric analysis showing the frequency of central memory T cells (CD62L+CD44+) in spleens of WT and Lgals92/2 mice on day + – 2/2 7 after NP32–KLH immunization (n = 5). (L) Purified CD4 CD25 T cells from WT or Lgals9 mice were stimulated with anti-CD3 and anti-CD28 in the presence of Th0-, Th1-, Th17-, and iTreg-polarizing conditions for 3 d (n = 4). (M)WTorLgals92/2 CD4+CD25– T cells were differentiated in the presence of varying amounts of anti-CD3 (plus anti-CD28) and in the presence of Th17 polarization and analyzed by flow cytometry. Data are repre- sentative of three independent experiments. All bar graphs show mean 6 SEM. *p , 0.05, **p , 0.01. 12 INTRACELLULAR GALECTIN-9 IN T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Galectin-9 contributes to the pathogenesis of T cell–mediated EAE. (A and B) Analyses of human galectin and T cell–related gene transcripts in healthy controls and MS CD4+ T cells. Data were derived from GEO GSE78244. (B) The value of the T cell–related genes (CD44 and IFNGR1) and LGALS9 in human UC and CD ileal biopsies compared with controls. (C) CD4+CD25– T cells from WT or Lgals92/2 mice were isolated and then transferred into immunodeficient Rag12/2 recipients, and pertussis toxin was injected i.p. twice on days 1 and 2. (D) EAE (Figure legend continues) The Journal of Immunology 13

Lgals92/2 T cells could not be recovered by recombinant Previous reports have indicated a divergent role of galectin-9 in galectin-9 (Fig. 4J). However, a previous report has demonstrated Lgals92/2 EAE models (13, 26). Oomizu et al. (26) reported that that exogenous galectin-9 promotes Lck phosphorylation in Jurkat the disease was more severe in Lgals92/2 mice. By contrast, cells (46). There are two possible explanations for this discrep- Madireddi et al. (13) reported that Lgals92/2 mice developed ancy in TCR signaling: 1) the Jurkat human leukemia cell has an EAE with severity similarly to WT mice. They also found that the intrinsic mitogenic effect that may potentiate galectin-9–mediated action of agonistic anti–4-1BB in suppressing EAE was galectin-9 activation, or 2) the potential effect of exogenous galectin-9 dependent, because anti–4-1BB treatment was unable to suppress in primary T cells may be masked by strong anti-CD3/CD28 the disease in Lgals92/2 mice. These controversial results may stimulation. highlight a divergent role of galectin-9 in regulating various cell A previous report indicated that the BCR signaling was increased functions through different pathways. For example, our prelimi- in anti-IgM–stimulated Lgals92/2 B cells (40), which is different nary data revealed that Lgals92/2 DCs have increased ability to from our finding demonstrated that intrinsic galectin-9 may be promote T cell responses (data not shown). Therefore, the en- dispensable in modulating B cell functions (Fig. 5H). This dis- dogenous role of galectin-9 in T cells may be missed or masked in crepancy implies that intrinsic galectin-9 may be involved in BCR conventional Lgals92/2 disease models. signaling during early phase stimulation (40) and that stronger Our findings have demonstrated that IFN-g+IL-17+, but not signaling may override the potential defect in Lgals92/2 B cell IFN-g–IL-17+, T cells in the inflamed CNS were significantly activation (Fig. 5H). lower in Lgals92/2-transferred mice. Although Th17 cells play Previous reports indicated that recombinant galectin-9 major pathogenic role in EAE, several reports have demon- 2 2 2 2 suppresses Th17 differentiation (26, 47). However, this ex- strated that Il17a / or Il17f / mice were only partially re- Downloaded from ogenously provided dose may not represent the physiological sistant to EAE pathogenesis (49, 50). Moreover, Th17 cells condition and could mask the potential role of endogenous were plastic, which have been reported to acquire IFN-g ex- galectin-9 in Th17 differentiation. Recently, Liang et al. (48) pression, and were expanded in the CNS during EAE that used Lgals92/2 mice to dissect the role of endogenous further induced CNS autoimmunity (51–53). Our results also galectin-9 in mucosal immunity. They found that cholera toxin indicated that these IFN-g+IL-17+ T cells were accumulated 2/2 (CT)–immunized Lgals9 mice developed severe diarrhea in CNS, which actively participate the pathogenesis of EAE. http://www.jimmunol.org/ and that this could be modulated by impaired mucosal CT- Several reports have demonstrated that a transition from specific IgA responses through a non–B cell–autonomous the Th17 cells into IFN-g–producing cells were found in au- pathway. Moreover, the difference in mucosal IgA responses toimmune disease, and these cells can promote disease path- between immunized WT and Lgals92/2 mice disappeared ogenicity (51, 54), suggesting that IFN-g+IL-17+ T cells after in vivo IL-17 blockade, which suggests that galectin-9– may further transit to Th1-like cells and promote the EAE mediated mucosal immunity is Th17 dependent (48). Our severity. finding that galectin-9 modulates B cell responses through Most studies have demonstrated that galectin-9 regulates cell a T cell–autonomous manner (Fig. 5J, 5K) is consistent with responses through the CRDs and that treatment can di- the findings of Liang et al. (48). However, galectin-9 is minish these galectin-9–mediated effects (8, 55). A recent report by guest on September 29, 2021 expressed in various cell types, including T cells, B cells, indicated that galectin-9 binds to dectin-1 on macrophages DCs, fibroblasts, and epithelial cells. Whether the decrease through a glycan-independent pathway (56); this observation in Th17 numbers in CT-immunized Lgals92/2 mice is mod- provides a striking insight into a divergent role of galectins. ulated by endogenous galectin-9 in T cells and/or the subse- Similarly, Chen et al. (22) reported that intracellular galectin-3 quent effects by other galectin-9-bearing cells remain to be interacts with Alix in T cells through short proline-rich motifs. elucidated. In our results, intracellular galectin-9 was recruited to the im- Previous studies have reported that galectin-9 is highly expressed mune synapse during T cell activation. This finding suggests that by iTregs, and it increases their functions and stability (14, 47). galectin-9 interacts with some activation-related molecules; Both studies indicated that the impaired suppressive function of however, the precise mechanism of action of endogenous galectin- Lgals92/2 iTregs could be rescued by exogenous galectin-9 9 in T cells is not clear. Nevertheless, our results demonstrate that treatment, which suggests an extracellular role of galectin-9 in the expression of galectin-9 in CD4+ T cells isolated from PBMCs iTreg. We found that galectin-9 was expressed mainly in the cy- is increased in patients and positively correlated with disease se- toplasm of T cells and that its was very limited, which verity, providing a critical insight into the development of galec- provides evidence of its endogenous role in T cell function. tin-9–based biomarker for autoimmune diseases. In conclusion, However, a deficiency of galectin-9 did not affect iTreg differ- we found that galectin-9 promotes proximal TCR signaling in entiation (Fig. 5L), which supports the idea that galectin-9 is T cells and that this affects Th cell differentiation and B cell dispensable for the induction of Tregs. Interestingly, we observed responses. Endogenous galectin-9 in T cells appears to modulate that suboptimal Th17 polarization by low-TCR signaling pro- positively the pathogenesis of some autoimmune diseases. moted iTreg differentiation in Lgals92/2 T cells. This finding Nevertheless, the possibility of its extracellular functions cannot implies a dual role of galectin-9 in sustaining Th17 and sup- be excluded. Further studies using conditional Lgals92/2 mice pressing iTreg differentiation in a low-TCR signaling–dependent are warranted to dissect the roles of galectin-9 in various cell manner. types.

clinical score in Rag12/2 mice transferred with WT or Lgals92/2 CD4+CD25– T cells. At 27 d after transfer, Rag12/2 recipients were sacrificed (n = 11). Data are representative of two independent experiments. (E) The brain and spinal cord sections were collected and stained with H&E. Data are repre- sentative of two independent experiments. (F and G) The cell number (F) and percentage (G) of CD4+ T cells in the CNS were calculated (n = 7). Data are representative of two independent experiments. (H) Isolated lymphocytes from the CNS from Rag12/2 recipients were stimulated with PMA and ion- omycin and analyzed for IL-17A and IFN-g production (WT [n = 5] and KO [n = 6]). Data are representative of two independent experiments. All bar graphs show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 14 INTRACELLULAR GALECTIN-9 IN T CELLS

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AB C 100 1.5 108 WT 80 KO WT KO 80 p=0.07 60 p=0.1248 8 1.0 10 60 40 40 5.0 107 Cell number

% of thymocytes 20

20 % of thymocytes

0.0 0 0 WT KO DN DP CD4 SP CD8 SP DN1 DN2 DN3 DN4 DE 8 2.0 10 WT 80 WT

KO s KO 1.5 108 60

1.0 108 40 lymphocyte f Cell number

5.0 107 o 20 %

0.0 0 SP MLN CLN ILN CD4 F 20 Foxp3+Nrp1- Foxp3+Nrp1+

15 T cells + 10

5 % of CD4

0 SP MLN PLN ILN CLN SP MLN PLN ILN CLN Supplementary Figure 1 (S1): Galectin-9 deficiency does not affect T-cell development. (A) Mice were sacrificed at 6–8 wk, and the cell number was counted in the thymus (n=6). (B) Flow cytometric analysis of the percentages of double-negative (CD4–CD8–), double-positive (CD4+CD8+), CD4 single positive (CD4+CD8–), and CD8 (CD4–CD8+) cells (n=6). Data are representative of two independent experiments. (C) The different stages of double-negative cells were analyzed by flow cytometry (n=6). Data are representative of two independent experiments. (D) The mice were sacrificed at 6–8 wk, and the cell number of spleen and LNs (n=4). Data are representative of four independent experiments. (E) Flow cytometric analysis of the percentages of CD4, CD8, and B cells (n=4). Data are representative of four independent experiments. (F) Flow cytometric analysis of the percentage of Tregs. (n=4). Data are representative of four independent experiments. All bar graphs show mean ± SEM.

1 Supplementary Figure 2 (S2): Lgals9–/– T cells have impaired proximal TCR signaling. Purified CD4+CD25– T cells from WT or Lgals9–/– mice were stimulated at 37°C with anti-CD3/CD28 and then cross-linked with mouse anti-hamster IgG for the indicated times. After stimulation, the cells were immediately resuspended in lysis buffer and subjected to immunoblotting analysis. Data are representative of three independent experiments.

2

Supplementary Figure 3 (S3): Galectin-9 deficiency does not affect the T-cell apoptosis. (A) Purified CTV-labeled CD4+CD25– T cells from WT or Lgals9–/– mice were stimulated with anti-CD3 and anti-CD28 in the presence of Th1-polarizing conditions for 3 d. The expression of Tim-3 was analyzed by flow cytometry with anti-Tim-3. Data are representative of four independent experiments. (B,C) Purified CD4+CD25– T cells from WT or Lgals9–/– mice were stimulated with anti-CD3 and anti-CD28 for the number of days indicated and then labeled with Annexin V–FITC and 7-AAD. Data were collected by flow cytometry (n=3). Data are representative of three independent experiments. (D) Purified CD4+CD25– T cells from WT or Lgals9–/– mice were labeled with CTV and stimulated with anti-CD3 and anti-CD28. After 60–72 h, cells were purified on Histopaque and then plated for 18 h at a density of 2 ´ 105 cells/well in the presence of recombinant galectin-9 or PBS. Cell death was analyzed by staining with anti-Annexin V and 7-AAD. Data are representative of three independent experiments. All bar graphs show mean ± SEM.

3 A

100 90 WT 80 KO 70 60 50 40 30 20 10 8 % of total cells total of % 6 4 2 0

tB T1 T2 Pro-B Pre-B B1 B B2 B Mature B Mature B Immature B ProB+PreB Follicular B

Marginal zone B

Bone marrow Spleen

B IgM IgG3 IgG2b ** 15000 2000 15000

1500 10000 10000 1000 μ g/mL μ g/mL μ g/mL 5000 5000 500

0 0 0 WT KO WT KO WT KO

IgG2a IgG1 IgA * 100 20000 6000 80 15000 4000 60 10000 μ g/mL μ g/mL 40 μ g/mL 2000 20 5000

0 0 0 WT KO WT KO WT KO Supplementary Figure 4 (S4): Normal B-cell development in mice lacking galectin-9. (A) To determine the frequency of each B-cell subset, bone marrow and splenic B cells were harvested for flow cytometric analysis with antibodies against CD43, BP-1, IgM, B220, CD5, CD21, and CD23 (n=3). (B) Sera of 12-wk-old mice were harvested and subjected to Ig subtype analysis using a multiplexed isotyping assay (n=10). All bar graphs show mean ± SEM. *, p < 0.05 ; **, p < 0.01.

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