SIRT inhibition increases Treg functionality. Isolated primary T cells were cultured in the presence of IL-2, anti-CD3, and anti-CD28. Cells were treated with the SIRT inhibitor NAM or carrier as control. (A) Foxp3 levels per cells of different T-cell sources were analyzed by FACS, and data represent the mean fluorescence intensity of the Foxp3⁺ population. (B) Cells from a human T-cell clone were stimulated and treated with NAM or control. Foxp3 transcription was determined with quantitative PCR, and results were corrected for the housekeeping gene β₂M and Foxp3⁺ cell numbers. (C) CD4 T cells isolated from mouse splenocytes were stimulated using IL-2, anti-CD3, and anti-CD28 beads and treated with NAM or control. CD25⁺ cells were sorted, and the function of these induced Tregs was analyzed using a standard suppression assay. A total of 10 000 effector T cells were cocultured with 2000 sorted CD25⁺ cells for 4 days (without NAM). Proliferation of effector T cells on day 4 was measured by CFSE dilution within the CD4⁺ effector T-cell population. Data shown are representative of at least 3 independent experiments. **P < .01.