aPC induces epigenetic and metabolic alterations in mouse CD4⁺CD25⁻ cells, resulting in a Treg-like phenotype. (A) 5mC (mouse, n = 4) analysis showing methylation alterations in CD4⁺CD25⁻ cells without (T) or with aPC (T + aPC) preincubation followed by stimulation with αCD3 and αCD28 for 48 hours. A representative flow cytometry histogram (left) and a bar graph with a dot plot (right) summarizing data as the MFI. (B-C) Exemplary gel image of Foxp3 promoter methylation (methylation-specific PCR, mouse; B, bottom, n = 4) and dot plot summarizing the results (B, top) of promoter methylation in 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. Heat map (C, left) and bar graph with dot plot (C, right) summarizing results of pyrosequencing of 5 CpG motifs in the Foxp3 promoter in murine CD4⁺CD25⁻ cells as described in panel B. Color code indicates the degree of methylation at each CpG motif, n = 3. (D) Bar graph with dot plot summarizing Foxp3 promoter activity determined in EL4 murine T cells expressing Foxp3 promoter–driven luciferase 24 hours after stimulation with αCD3 and αCD28 without (T) or with aPC (T + aPC) preincubation (n = 3). (E) T cells isolated from mice 2 weeks after induction of GVHD (E, top). T cells were preincubated with aPC (20 nM, 1 hour) before transplantation. Representative line graph showing (E, bottom) summarizing the results (E, n = 5). (F) Representative flow cytometry plot (top) and bar graph with dot plot (bottom) showing FOXP3 expression in T cells isolated from DEREG-GFP mice and stimulated with αCD3 and αCD28 (96 hours) (n = 5). Control CD4⁺ GFP⁻ cells (T) compared with CD4⁺ GFP⁻ cells preincubated with aPC (T + aPC, 1 hour, 20 nM) or with supplementation of α-KG (48 hours, 3.5 mM, T + aPC + α-KG) or glutamine (48 hours, 4 mM, T + aPC + Q). The data are shown as the mean ± SEM, and statistical significance was determined by 2-tailed Student t test for panels A-D and 1-way ANOVA for panel F: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005; ns, nonsignificant.