Mechanism of hypomethylating agent-induced generation of Tregs. (A) Perforin (Prf1) is partially required for the suppressor function of dcT. DcTs from both WT and granzyme B (GzmB)–deficient mice were equally suppressive while those from Prf1-deficient mice were significantly less suppressive. A pool of 2 independent experiments. (B-C) The impact of dcTs (B) and azacTs (C) from WT, Foxp3 heterozygote (Foxp3⁺/Y) and Foxp3 deficient (Foxp3⁻/Y) mice on Teffs were compared in proliferation assays. They are equally suppressive regardless of their origins. Neg: negative control, CFSE-labeled Teffs alone; pos: CFSE-labeled Teffs with stimulators, anti-CD3/CD28 antibody coated beads or allogeneic APC; all others contain both CFSE-labeled Teffs and stimulators plus indicated cells. (D) GA-Mantel³³  analysis was run 100 times. Forty-nine probes appeared in at least 30 of 100 solutions (Mantel correlation = 0.97). Foxp3 is the most frequently selected probe. Heat map shows these 49 probes that separate the 3 Treg groups from the pbsT group. nnTreg: naive nTregs (n = 2), anTreg: activated nTregs (n = 3), dcT (n = 3), pbsT (n = 3). (E) AzaC treatment prolongs survival of Foxp3 deficient mice. A treatment of 2 mg/kg AzaC was given every other day (4 doses) starting at 16 to 26 days of age. A pool of 4 independent experiments. (F) Shown are 41-day-old Foxp3 KO mice treated with PBS (left) and AzaC (right); 25% (n = 8) had WT-looking ears.