FIGURE 2.

Absence of SIRT3 in T cells reduced nonspecific TCR as well as allogeneic responses in vitro. (A) Naive CD90.2⁺ T cells were isolated from B6 WT spleen with MACS and stimulated with anti-CD3 (1–10 μg/ml)/CD28 (1 μg/ml) Abs. The expression of SIRT3 at various time points after stimulation was evaluated with real-time quantitative PCR. Data presented are representative of one of three independent experiments. (B) Isolated splenic CD90.2⁺ T cells from either B6 WT or SIRT3^−/− mice were incubated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) Abs for 48 h and analyzed for proliferation following [³H]thymidine incorporation during the last 6 h of incubation. (C) In vitro MLR. Isolated splenic CD90.2⁺ T cells from either B6 WT or SIRT3^−/− mice were cultured with BMDCs from syngeneic B6 or allogeneic BALB/c mice for 96 h and analyzed for proliferation following [³H-]thymidine incorporation during the last 16 h of incubation. (D and E) IFN-γ (D) and IL-2 (E) production from allogeneic-stimulated T cells from (C) was measured by ELISA. (F) T_reg suppression assay. BMDCs from BALB/c mice were used as stimulators, cocultured with effector T cells (CD4⁺CD25⁻) and T_reg cells (CD4⁺CD25⁺) from either B6-WT or SIRT3^−/− mice at different ratios in an MLR, and analyzed for T cell proliferation following [³H]thymidine incorporation during the last 16 h of incubation. Error bars show the mean ± SEM. Three independent experiments were performed for (B–F). *p < 0.05, **p < 0.01, ***p < 0.001.