Effect of TIM-3 blockade on liver inflammation and CD4⁺ T cell immune response.

<p>Isotype or TIM-3 mAb (100 µg per mouse) was administrated i.v. to Balb/c mice (n = 8 per group) 30 min before Con A injection (20 mg kg^–1). (A) TIM-3 expression of CD4⁺ T cell in spleen was detected 24 h following Con A injection. (B) The effect of anti-TIM-3 on the binding of galectin-9 to mouse Th1 cells. CD4⁺ T cells were purified from splenocytes of normal mice by negative selection with magnetic beads. Cells (1×10⁶ cells/ml) were cultured for 5 d with phytohemagglutinin (1 mg/ml) and IL-2 (8 ng/ml) in polarizing conditions: IL-12 (2 ng/ml) plus antibody to IL-4 (anti-IL-4; 100 ng/ml; MP4-25D2); Cells (5×10⁵ cells/ml) were collected and incubated for 1 h at 4°C with biotinylated galectin-9 in the presence or absence of increasing anti-TIM-3 (2 ug/ml or 10 ug/ml). Cells were then incubated for 45 min at 4°C with fluorescein isothiocyanate–conjugated streptavidin, were washed and were analyzed by FACS. Serum ALT (C) and AST (D) levels were measured 24 h after Con A injection. The results were presented as the mean ± SD of three separate experiments. **, p<0.01; *, p<0.05. (E) The livers were removed 24 h later. Paraffin sections were stained with hematoxylin and eosin (H&E) staining. Representative liver sections were shown for each group, original magnification: ×200. (F) Percentages and phenotype (surface TIM-3) of CD4⁺ T cells in spleen of mice are shown. Normal, normal mice; Control, PBS treatment in Con A-treated mice; anti-TIM-3, anti-TIM-3 mAb pretreatment in Con A-treated mice.</p

Effect of galectin-9 treatment on inflammatory cytokines secretion <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Time course of IFN-γ, TNF-α and IL-6 release into serum after Con A injection, with or without galectin-9 (100 µg per mouse) administered i.v. for 30 minutes before Con A injection. Serum cytokine levels were determined using ELISA-assay kits. Data points represent the mean ± SD for 8 animals killed at each point. **, p<0.01; *, p<0.05 vs galectin-9 pretreated mice. (B) Spleen macrophages (1×10⁶ cells/mL) were incubated for 24 hours with 100 ng/mL LPS, with or without galectin-9 treatment (10 µg/ml). Aliquots of supernatant were then collected and stored at −80°C until assayed. IFN-γ, TNF-α and IL-6 concentrations were measured by using specific ELISA kits. The results were presented as the mean ± SD of three separate experiments. ***, p<0.001; **, p<0.01.</p

Effect of galectin-9 treatment on cellular infiltration in spleen of Con A treated mice.

<p>Galectin-9 (100 µg per mouse) or PBS was administrated i.v. to Balb/c mice (n = 8 per group) 30 min before Con A injection (20 mg kg^–1). The splenocytes were isolated 24 h later. Percentages and phenotype (surface TIM-3) of CD4⁺ T cells in spleen of mice are shown in (A). (B) The frequencies of Th1, Th17 and Treg subsets were detected by FACS. Statistically significant differences were indicated in (C). The results (A, C) were presented as the mean ± SD of three separate experiments. **, p<0.01; *, p<0.05 vs PBS treatment.</p

T cells and Con A-induced hepatitis.

<p>Isotype, CD4 and/or CD8 mAbs (100 µg per mouse) was administrated i.p. to Balb/c mice (n = 8 per group) 24h before Con A injection (20 mg kg^–1). The effect of anti-CD4 orCD-8 antibodies administration on depleting these cells in the liver of mice prior injecting Con A was determined by FACS. Representative results were shown in (A). Sera were collected 24 h after Con A injection. Serum ALT (B) and AST (C) levels were measured. The results were presented as the mean ± SD of three separate experiments. ***, p<0.001 vs control. (D) The livers were removed 24 h later. Paraffin sections were stained with hematoxylin and eosin (H&E) staining. Representative liver sections were shown for each group, original magnification: ×200.</p