Vit C promotes Foxp3 expression and stability in CD8⁺iTregs. (A) Allogeneic CD8⁺ iTregs were generated by coculturing CD8⁺ T cells isolated from C57BL/6 mice with allogeneic dendritic cells isolated from BALB/c mice in the presence of IL-2 (5 ng/mL), TGF-β (5 ng/mL), retinoic acid (40 nM), and Vit C (0 µg/mL to 20 µg/mL). After 5 days, the expression of Foxp3⁺ on CD8⁺ cells was analyzed by flow cytometry (n = 3 per group). (B) CD8⁺ iTregs (Vit C 10 μg/mL) were enriched from the bulk culture using positive selection with CD25⁺ MicroBeads. FACS plots show Foxp3⁺ expression after generation and CD25⁺ enrichment. (C) In vitro stability of CD8⁺ iTregs under IL-2-alone conditions and under IL-2+IL-12 conditions (n = 3 per group) were evaluated. Foxp3⁺ expression retained after 3 and 6 days is shown. In vitro suppressive function of CD8⁺ iTregs was assessed by carboxyfluorescein diacetate succinimidyl ester dilution of CD4⁺ (D) and CD8⁺ (E) responder T cells (n = 3 per group). (F) Demethylation status in the CNS2 region of the Foxp3 gene (⃝, demethylated CpGs; ●, methylated CpGs) (n = 21 per group). (G) Bar graph showing the percentage of demethylation in control (green) and Vit C–treated (red) CD8⁺ iTregs. Data are representative of 2 (A,C-G) or >10 (B) independent experiments. *P ≤ .05, **P ≤ .01, ****P ≤ .0001, 2-tailed Student t test.