(A and B) CD25⁺ percentages from CB and AB naive CD4⁺ T cells after 96 hours of anti-CD3/CD28 stimulation under No Cytokine or IL-2 + TGF-β conditions. (C–E) Marker expression on CD25⁺ fraction by flow cytometry. (F and G) ELISA of cytokines in supernatant. (H) Allogeneic MLR measuring suppressive capacity of CB and AB No Cytokine and IL-2 + TGF-β-stimulated cells. (I) Allogeneic MLR assay for CD25⁺ versus CD25^neg iTreg-stimulated cells. (J–L) FOXP3 CNS2 methylation was assessed by bisulfide sequencing in CD4⁺CD25⁺CD127^lo cells from iTreg cultures and endogenous blood Tregs (CD4⁺FOXP3⁺CD25⁺CD127^lo). Two separate experiments were analyzed together with adjustment for batch effects. (J) Heatmap, (K) violin plot, and (L) graphs of CpG island methylation ratios. (M) FOXP3 stability assessed through 2 rounds of stimulation and rest. CD25⁺ and FOXP3 MFI on CD25⁺ cells after second rest. Data points represent individual donors. (H and I) N = 5/group. (A–E) One of 3 representative experiments. (H) One of 2 representative experiments. (F, G, I, and M) Performed once. (J–L) Combined data from 2 experiments. (B–G and M) Two-way ANOVA with multiple comparisons used. (I) One-way ANOVA with multiple comparisons used. (L) Linear mixed model (included batch correction). *P ≤ 0.05, **P ≤ 0.02, ***P ≤ 0.001, ****P < 0.0001. Horizontal bars and column heights in panels B–G and M depict mean values. In H and I, mean value plus standard deviation is shown.