Fig. 4

Azacytidine increases Treg frequency and induces demethylation of Foxp3 enhancer and IL-2 promoter. Balb/cJ mice were injected i.v. with 10.10⁶ bone marrow cells and 70.10⁶ splenocytes from B10.D2 donor mice after lethal irradiation. Mice were then given (or not) azacytidine (AZA, 0.5 mg/kg or 2 mg/kg) administered subcutaneously every 48 h from day +10 to day +30. a, b FACS analyses showing the Tregs frequency in two cohorts of mice combined (a) and in a third cohort of mice (b). c FACS analysis of activated CD103⁺ cells among Tregs in the spleens of AZA on day +52 (data from the third cohort). d, e Methylation status of the Foxp3 enhancer assessed by methylation-sensitive restriction enzyme and qPCR performed on genomic DNA of Tregs (n = 3) and Tconvs (n = 9) sorted from the spleen of three unmanipulated B10.D2 mice (d) or of the spleens of control (n = 4) or AZA-treated (n = 5) recipient Balb/cJ mice at day +52 post transplantation (e). f FACS comparison of phosphorylated-STAT5 mean fluorescence intensity (MFI) between Tregs in spleen of control (n = 8) and AZA-treated (n = 9) mice. g Comparison of ratio, for each mouse treated or not with AZA, of pSTAT5 MFI of Tregs versus pSTAT5 MFI of Tconvs. h Methylation status of the IL-2 promoter assessed by MSRE-qPCR performed on genomic DNA of spleens from control (n = 4) and AZA-treated (n = 5) recipient Balb/cJ mice at day +52 post transplantation. Results are expressed as median, 25th and 75th percentiles of the distribution (boxes), and whiskers extending to the 5th and 95th percentiles. *P < 0.05; **P < 0.01