aPC reduces FOXP3 promoter methylation. (A-C) Frequencies of CD4⁺FOXP3⁺ (A), CD4⁺T-bet⁺ (B), and CD4⁺IFNγ⁺ (C) cells without (T) or with aPC (T + aPC) preincubation (1 hour, 20 nM) as determined by flow cytometry 96 hours after activation of human CD4⁺CD25⁻ cells with αCD3 and αCD28 (A-B, n = 5; C, n = 3). Bar graphs with dot plots summarizing data. (D) Frequency of CD4⁺FOXP3⁺ cells without (T_n) or with aPC (T_n + aPC) preincubation (1 hour, 20 nM) under Treg polarization condition as assessed by flow cytometry 5 days after activation of human näive T cells (T_n) with αCD3 and αCD28 (n = 5). Bar graphs with dot plots summarizing data. (E-F) Global methylation changes, as reflected by 5-methylcytosine (5mC) (E, n = 4, human), and H3K27Me3 (F, n = 4, human) in CD4⁺CD25⁻ cells without (T) or with aPC (T + aPC) preincubation followed by stimulation with αCD3 and αCD28 for 48 hours. Representative histogram (E, left) and bar graph with dot plot (E, right; F) summarizing the results from flow cytometry as the mean fluorescence intensity (MFI). (G-H) Schematic representation of human FOXP3 promoter showing CpG sites in different regions and amplicons used for methylation-specific polymerase chain reaction (PCR) (MSP) (G). FOXP3 promoter methylation of different regions (methylation-specific PCR, n = 4 each group, H) and dot plot summarizing results of MSP in the FOXP3 promoter in human CD4⁺CD25⁻ cells without (T) or with aPC (T + aPC) preincubation (1 hour, 20 nM) followed by 48 hours of stimulation with αCD3 and αCD28. The data are shown as the mean ± standard error of the mean (SEM); statistical significance was determined by 2-tailed Student t test (A-H): ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005.