Sirt-1 deacetylates Foxp3 and decreases iTregs stability. (A) Allogeneic CD4 iTregs were generated in vitro by coculturing naive CD4 T cells with allogeneic dendritic cells in the presence of IL-2, TGF-β, and retinoic acid for 5 days. CD4 iTregs were enriched for CD25^hi cells. (B) Enriched CD25⁺ CD4iTregs were analyzed by western blot analysis for detection of global acetylation. (C) Five-day in vitro-generated CD4 iTregs were restimulated with IL-2 and analyzed for pSTAT5 expression. (D) Enriched CD4 iTregs were cocultured with allogeneic APCs in the presence of IL-2 or IL-2+IL-12 for 3 days. Percentage Foxp3 retention was analyzed on day 3 (n = 4). (E) Experimental scheme: lethally irradiated BALB/c mice were adoptively transferred with 5 × 10⁶ Rag1^−/− BM and 0.5-1 × 10⁶ CD4 iTregs (Ly5.2). On day 3, 0.5-2 × 10⁶ CD25-depleted T-cells from B6Ly5.1 congenic mice were injected to induce GVHD in recipients. On day 7 or 14 after allo-BMT, spleen was harvested and analyzed. (F) Percentages of Foxp3 and CD25 expressions on CD4iTregs analyzed on day 7 (gated on Ly5.2⁺CD4⁺) are shown. (G) Percentages of Foxp3 and IFN-γ expressions of transferred CD4 iTregs are shown (n = 4-5 mice per group). Data represent the mean ± standard error of the mean. Student t-test was used for statistical analysis. *P < .05; **P < .01; ***P < .001; ****P < .0001.