Sirt-1 enhances T-cell alloreactivity through p53 deacetylation. (A-B) Purified WT or Sirt-1^−/− T cells were labeled with CFSE and cocultured with T-cell-depleted splenocytes as APCs from BALB/c mice for 5 days in the presence of either dimethyl sulfoxide or Ex-527 (10 µg/mL). Cells were restimulated with phorbol 12-myristate 13-acetate and ionomycin for cytokine measurement. Average percentages of CFSE-diluted and IFN-γ⁺ gated on donor CD4 or CD8 T cells are shown. (C) CD4⁺ T cells isolated from WT or Sirt-1^−/− or WT T cells treated with Ex-527 were polarized into Th1 cells in the presence of syngeneic APCs with 1 μg/mL anti-mouse CD3ε (clone 145-2C11); 10 ng/mL mIL-12, and 1 ng/mL mIFN-γ were used for Th1 polarization. For western blot analysis, polarized cells were pretreated with 2 μM Trichostatin A (Sigma Aldrich) for 45 minutes to induce basal protein acetylation for detection of global acetylation. (D) Purified CD4 T cells from WT or Sirt-1^−/− or Ex-527-treated cells were polarized under Th1 condition for 3 days and analyzed by western blot analysis for α-acetylated p53 and total p53. (E-F) T cells isolated from WT were labeled with CFSE and cocultured with allogeneic APCs for 5 days in the presence of Ex-527 or Pifithrin-μ, p53 inhibitor (10 μg/mL each) or in combinations. The frequency of CFSE diluted and IFN-γ gated on donor CD4 or CD8 T cells were analyzed. Data represent mean ± standard error of the mean. Ordinary 1-way analysis of variance with Sidak multiple comparisons test was used for statistical analysis (n = 6 per group). *P < .05; **P < .01; ***P < .001; ****P < .0001.