This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, China, 1998). The protocol was approved by the Medical Laboratory Animal Care and Use Committee of Anhui Province (Permit no. 2009–0132) as well as the Ethical Committee of Yijishan Hospital of Wannan Medical College (Permit no. 20090015).

Rabbit anti-Smad2/3, p-Smad2 and p-Smad3 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse anti-p-ERK1/2, mouse anti-Smad4, mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (MAB374) and rabbit anti-hemagglutinin (anti-HA) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The CD4⁺CD25⁺ T cell isolation kit was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).

Spleens were isolated from 8- to 10-week-old Balb/c mice. CD4⁺CD25⁻ naive T cells were isolated from splenocytes using a CD4⁺CD25⁺ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of CD4⁺CD25⁻ T cells was >90%.

The isolated naive CD4⁺ T cells in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) with 10% heat-inactivated fetal bovine serum, IL-2 (20 ng/ml, R&D Systems, Minneapolis, MN, USA) and anti-CD28 (2 μg/ml, Becton-Dickinson, Bedford, MA, USA) were distributed into anti-CD3 coated 96-well plates at 2×10⁵ cells/0.1 ml/well in the presence or absence of human TGF-β1 (5 ng/ml, R&D Systems) and human galectin-9 (0.3, 1 and 3 μg/ml, ProSpec-Tany TechnoGene, Ltd., Israel), and maintained for 4 days at 37°C in an atmosphere containing 5% CO₂. Then the percentage of CD4⁺CD25⁺Foxp3⁺ cells was detected by fluorescence-activated cell sorting (FACS). For generation of Tr1 cells, FACS-sorted naive CD4⁺CD25⁻ T cells and irradiated splenic APCs were cultured 1×10⁶ cells/ml/well in 24-well plates in the presence of suboptimal IL-10 (10 ng/ml) or IL-10 and galectin-9 (1 μg/ml). The cultured T cells were then restimulated with freshly prepared irradiated splenic APCs in the presence of IL-10 at intervals of 10–14 days. The stimulation under the same conditions was repeated weekly for 3 consecutive weeks. The percentage of Tr1 was then detected by FACS.

For the analysis of suppressor function, various numbers of CD4⁺CD25⁺ cells or the whole cell suspension obtained from the conversion assay were added to 1×10⁵ CD4⁺CD25⁻ carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled wild-type T cells. Cells were stimulated with CD3mAb and CD28mAb in 96-well flat-bottomed plates for 96 h in serum-free medium. Proliferation was analyzed by FACS assessing the CFSE dilution.

Total RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA. PCR was performed to evaluate the relative expression of Foxp3. The forward and reverse primers for Foxp3 (5′-GCTCAGGCTCCGTTTCAC-3′ and 5′-AGGATCGTC TAAGTCCAG-3′) and the GAPDH house-keeping gene (5′-GAAGGTGAAGGTCGGAGTCA-3′ and 5′-GAAGAT GGTGATGGGATTTC-3′) were used. The expression of Foxp3 was normalized using GAPDH expression.

Purified CD4⁺CD25⁻ T cells from Balb/c mice were cultured with IL-2 (20 ng/ml, R&D Systems), TGF-β1 (5 ng/ml, R&D Systems) and/or galectin-9 (1 μg/ml), as indicated, under activating conditions for 4 days. 5-Bromo-2-deoxyuridine (BrdU; Upstate, Lake Placid, NY, USA) was added to the cultured cells at a final concentration of 50 μM, 16 h prior to the end of the 4-day incubation period. The cells were stained with conjugated anti-mouse CD4, CD25 antibody (eBioscience, Inc., San Diego, CA, USA), fixed in IC fixation buffer (eBioscience, Inc.) for 30 min and permeabilized in permeabilization buffer (eBioscience, Inc.) for 20 min. Cells were then incubated at room temperature for 30 min in 0.15 M NaCl, 4.2 mM MgCl₂ and 10 mM HCl in the presence of 2 units DNaseI (Invitrogen), followed by staining with conjugated anti-BrdU antibody (BD Biosciences) for 30 min, and were finally analyzed by FACS.

Cells were lysed in RIPA extraction solution [(15 mM Tris, pH 7.5, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 2 mM EDTA, 0.1 mM dithiothreitol, 0.5% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich)]. Lysates were resolved by SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed sequentially with specific polyclonal antibodies against p-Smad2, p-Smad3 and Smad2/3 and monoclonal antibodies against Smad4 and GAPDH. Membranes were then incubated with secondary goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated antibodies (1:10,000; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) and developed using an ECL kit (Amersham Biosciences, Uppsala, Sweden).

The isolated naive T cells in RPMI-1640 (Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum, IL-2 (20 ng/ml, R&D Systems) and anti-CD28 (2 μg/ml, Becton-Dickinson) were distributed into anti-CD3 coated 96-well plates at 2×10⁵ cells/0.1 ml/well. They were then serum-starved and treated with TGF-β1 (5 ng/ml, R&D Systems) or TGF-β1 and galectin-9 (1 μg/ml) for 30 min. Cells were then scraped into ice-cold PBS and lysed with lysis buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% nonidet P-40, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol and protease inhibitors (Sigma-Aldrich). After being spun at top speed in a microcentrifuge to remove cellular debris, the supernatant was transferred to a clean test tube and precleared for 1 h with 50 μl of protein A. Following preclearing, rabbit anti-Smad2/3 antibody and control rabbit anti-HA antibody, preincubated with 30 μl of protein A, were added to the lysate. Immune complexes were precipitated overnight at 4°C. The immunoprecipitated material was washed three times with 1 ml of washing buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin and 0.1% NP-40. The pellet was then resuspended in SDS sample buffer and analyzed by western blotting.

Data are shown as the means ± SEM. Statistical analysis of the data was performed with the two-tailed independent Student’s t-test and ANOVA analysis using SPSS, version 12.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

TGF-β1 is essential for the conversion of CD4⁺CD25⁻ T cells into iTregs (5,6). As this may represent an important physiological process to regulate and restrict inflammation, we aimed to investigate the roles for galectin-9 regarding the conversion of naive T cells into iTregs. We stimulated CD4⁺CD25⁻ T cells with plate-bound CD3mAb and soluble CD28mAb and cultured the cells for 4 days in the presence of TGF-β1 and/or galectin-9. We found that galectin-9 increased the conversion to Foxp3⁺ cells induced by a concentration (5 ng/ml) of TGF-β1 (15.80 vs. 23.25%; Fig. 1), but not in a dose-dependent manner. However, in the absence of TGF-β1, galectin-9 (3 μg/ml) could not induce Foxp3 expression (Fig. 1B). In addition, we explored the role of galectin-9 in the differentiation of Tr1 regulatory T cells (Tr1) in vitro. We found that galectin-9 could not increase the conversion to Tr1 in the presence of suboptimal IL-10 (10 ng/ml) in vitro. These data show that galectin-9 potently enhances TGF-β1-dependent Treg induction.

The suppressive activity of Tregs induced by galectin-9 and TGF-β1 was measured relative to that of TGF-β1-induced Tregs and freshly harvested natural Tregs (nTregs) in a suppressor assay in vitro. The data show that Tregs induced by galectin-9 and TGF-β1 are potent suppressors of effector T cells in vitro (Fig. 2).

The observation that Treg cells can be induced by galectin-9 in the presence of TGF-β1 led us to question whether this occurs by galectin-9 functioning to interrupt T cell proliferation. To visualize this, purified CD4⁺CD25⁻ T cells were stimulated in the presence of TGF-β1 (5 ng/ml) alone or with galectin-9 (1 μg/ml), with T cell proliferation determined by BrdU incorporation assay. The addition of TGF-β1 to these cultures generated Tregs at expected frequencies (data not shown), with no impediment in cell proliferation observed due to the presence of galectin-9 (Fig. 3A). Notably, galectin-9 enhances TGF-β1-mediated Foxp3 expression (Fig. 3B). Collectively, these data show that galectin-9 enhances Foxp3 induction through a mechanism independent of dampening T cell proliferation.

TGF-β1-induced transcriptional regulation is mediated mainly through phosphorylation of Smad2/3 proteins, followed by their translocation into the cell nucleus to further activate specific genes (28). Accordingly, it was speculated whether the increase of TGF-β1-induced gene expression mediated by galectin-9 involves alterations in the status of Smad2/3 following treatment. To test this possibility, CD4⁺CD25⁻ naive T cells were isolated from splenocytes. The isolated naive T cells in RPMI-1640 with 10% heat-inactivated fetal bovine serum, IL-2 and anti-CD28 were distributed into anti-CD3-coated 96-well plates at 2×10⁵ cells/0.1 ml/well. They were then serum-starved and treated with TGF-β1 or TGF-β1 and galectin-9 for 30 min and a western blot analysis was performed using antibodies against the phosphorylated form of Smad2 and Smad3. As demonstrated in Fig. 4A, a significant induction in Smad3 phosphorylation was already observed 30 min after treatment with TGF-β1. In contrast to TGF-β1, galectin-9 and TGF-β1-treated cells exhibited a significant increase in the phosphorylation of Smad3. By analyzing the status of the Smad2 protein, we noticed that similar to the case for Smad3, galectin-9 enhanced the phosphorylation of Smad2 following treatment of the cells with TGF-β1 for 30 min (Fig. 4A). It was already demonstrated that in addition to the Smad-dependent signaling pathway, TGF-β1 also has the ability to induce MAPK or phosphatidylinositol 3-kinase signaling pathways (31). Accordingly, we examined whether, as with Smad2/3, galectin-9 also promotes other TGF-β1-induced signaling pathways. To that end, ERK1/2 phosphorylation was analyzed by using specific antibodies against the phosphorylated form of the proteins. As depicted in Fig. 4B, treating cells with galectin-9 and TGF-β1 for 30 min significantly induced the phosphorylation of ERK1/2 proteins compared with TGF-β1 treatment alone. We also examined the involvement of galectin-9 in the formation of a complex between Smad2 and Smad4 upon TGF-β1 and galectin-9 treatment. As shown in Fig. 4C, TGF-β1-treated cells induced an association between Smad2 and Smad4, as detected by coimmunoprecipitation followed by western blot analysis. By contrast, treating cells with galectin-9 and TGF-β1 yielded more of the complex. These findings suggest that galectin-9, which promotes TGF-β1-induced activation of Smad2/3, eventually enhances the association between Smad2 and Smad4 following TGF-β1 treatment.