Tim3–Gal9 interaction stimulates antimycobacterial activity. (A) H37Rv-infected macrophages (MP) were cultured alone or in the presence of increasing amounts of Tim3-Ig fusion protein or with 30 µg/ml of HuIgG as a control. Data are representative of three independent experiments. Error bars indicate mean ± SEM from three to six replicate cultures. (B) Mtb-infected WT or Tim3−/− macrophages were cultured alone or in the presence of 10 µg/ml of sTim3-Ig or HuIgG (control). (C) Mtb-infected macrophages were cultured alone or treated with 10 µg/ml Tim3-Ig in the presence of increasing amounts of lactose. Data are representative of three independent experiments. Error bars indicate mean ± SEM from three replicate cultures. (D) Mtb-infected WT or Gal9−/− macrophages were cultured alone or in the presence of 10 µg/ml Tim3-Ig or HuIgG (control). Data are representative of three independent experiments. Bars indicate mean ± SEM from three replicate cultures. (E) Mtb-infected macrophages cultured alone or with splenocytes from uninfected WT (WT Spl) or Tim3tg (Tg Spl) mice, or with Tim3+CD4+ T cells from uninfected Tim3tg mice. In all experiments (unless otherwise noted), day 1 (d1) is the CFU in infected macrophages alone 24 h after infection, before the addition of cells or Tim3-Ig or HuIgG and represents initial inoculum, whereas day 4 (d4) is the CFU recovered from macrophages 4 d after infection in the absence of any treatment. Data are representative of 3–11 independent experiments. Error bars indicate mean ± SEM from three to six replicate cultures. **, P < 0.01; ***, P < 0.001, one-way ANOVA compared with day 4 macrophages alone. (F) Pulmonary and splenic bacterial burden 7 and 14 d after intravenous transfer of H37Rv-infected WT or Gal9−/− macrophages into naive Tim3tg mice. Bacteria in lungs and spleen on day 1 after adoptive transfer were 60 and 1088, respectively. Data are from a single experiment with two time points (n = 5 mice per group per time point; error bars represent the SEM). **, P < 0.01; ***, P < 0.001, unpaired two-tailed Student’s t test.