Preferential Involvement of Tim-3 in the Regulation of Hepatic CD8⁺ T Cells in Murine Acute Graft-versus-Host Disease

Coexpression of Tim-3 and Tim-3L on T cells in aGVHD mice. Splenocytes and HMC from intact and aGVHD mice were obtained as described in Fig. 1. Cells were stained with FITC-conjugated anti-Tim-3 mAb, purified Tim-3/B6-Ig, followed by biotinylated anti-mouse IgG2a mAb and PE-streptavidin, PerCP-conjugated anti-CD3 mAb, and allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb or with appropriate fluorochrome-conjugated control Igs. Samples were analyzed by flow cytometry. If needed, an electronic gate was set on either CD3⁺ (T), CD4⁺CD3⁺ (CD4 T) or CD8⁺CD3⁺ (CD8 T) lymphocytes; the expressions of the indicated Ags are shown as dot plots (four-decade log scale). Representative FACS profiles of ten mice in each group are shown. The quadrant markers have been positioned to include >98% of control Ig-stained cells (data not shown) in the lower left. The underlined values of the right corner show Tim-3L positive percentages within CD3⁺, CD4⁺CD3⁺, or CD8⁺CD3⁺ T cells. The values of each quadrant in the double staining with Tim-3 and Tim-3L are positive percentages within CD4⁺ CD3⁺ or CD8⁺CD3⁺ T cells.

Expression of Tim-3 and Tim-3L on DCs and macrophages. Splenocytes and HMC from intact and aGVHD mice were stained with FITC-conjugated anti-CD11c or anti-F4/80 mAb, and either biotinylated anti-Tim-3 mAb, followed by PE-streptavidin, or unlabeled Tim-3Ig, followed by biotinylated anti-mouse IgG2a mAb and PE-streptavidin, or with appropriate fluorochrome-conjugated control Igs. Samples were analyzed by flow cytometry. Representative FACS profiles in each group of ten mice are displayed. The profiles of control Ig staining are shown in the left of each panel. The markers have been positioned to include >98% of control Ig-stained cells. A, the expressions of CD11c and either Tim-3 or Tim-3L within lymphocyte gate were analyzed. The underlined values of the right corner show Tim-3 or Tim-3L positive percentages within CD11c⁺ DCs. B, In the GVHD-induced mice, the F4/80⁺ cells did not contain a major population of forward scatter (FSC)^high/side scatter (SSC)^high characteristic macrophages. Therefore, an electronic gate was set on FSC^high/SSC^high cells, and then Tim-3 or Tim-3L expression was analyzed. The data are displayed as dot plots with Tim-3 or Tim-3L expression and FSC. Splenocytes and HMC within the macrophage gate contained 79% and 80% F4/80⁺ cells in an intact mouse and 58% and 30% F4/80⁺ cells in a GVHD mouse, respectively. The values are positive percentages in the FSC^high/SSC^high cells.

Effects of anti-Tim-3 mAb treatments on manifestation of acute GVHD. The BDF₁ recipient mice transferred 5 × 10⁷ B6 splenocytes were treated with either RMT3-23 (•), RMT3-8 (▪), or control rat IgG (○). Body weight was measured on the indicated days. The change in mean body weight (A), total cell numbers of splenocytes and HMC at 14 days after transfer (B) are shown. The values are the mean ± SD from three experiments with each group of five mice. ∗, Statistically different from control IgG-treated group (p < 0.05) (A). ∗, Statistically different (p < 0.05) (B). C, H&E staining of the livers from aGVHD mice. Representative staining from each group of three mice are shown. Scale bars = 25 μm.

Enhanced IFN-γ expression in T cells by the anti-Tim-3 mAb treatment. Splenocytes and HMC from aGVHD mice treated with either RMT3-23, RMT3-8, or control IgG were cultured with PMA and ionomycin in the presence of brefeldin A for 4 h. A, Cells were cell surface stained with FITC-conjugated CD8 mAb and PerCP-conjugated anti-CD3 mAb, or with appropriated fluorochrome-conjugated control Igs. After fixation and permeabilization, the cells were stained intracellularly with PE-conjugated anti-IFN-γ mAb or control Ig. B, Cells were cell surface stained with FITC-conjugated Tim-3 mAb and PerCP-conjugated anti-CD3 mAb, and either allophycocyanin-conjugated anti-CD4 or anti-CD8 mAb, or with appropriated fluorochrome-conjugated control Igs, and then stained intracellularly with PE-conjugated anti-IFN-γ mAb or control Ig. Samples were analyzed by flow cytometry. An electronic gate was set on either the CD3⁺ (A), CD3⁺CD4⁺ (CD4 T), or CD3⁺CD8⁺ (CD8 T) (B) cells; the expressions of the indicated Ags are shown as dot plots. Representative FACS profiles are shown. The quadrant markers have been positioned to include >98% of control Ig-stained cells (data not shown) in the lower left. Values are the mean ± SD for each group of ten mice.