Stability, plasticity, and functionality of in vitro expanded Treg-A and Treg-B. (A) Representative methylation status of TSDR in HD-expanded Treg-A, HD-expanded Treg-B, AA-expanded Treg-A, and AA-expanded Treg-B compared with non-Treg. (B) Bar chart shows the percentage of methylation in HD-expanded Treg-A (n = 2), HD-expanded Treg-B (n = 2), AA-expanded Treg-A (n = 3), AA-expanded Treg-B (n = 3), and non-Treg (n = 2). (C) Representative plots show the percentage of IL-17A⁺ population in expanded Treg-A and Treg-B cultured with anti-CD3/CD28 beads (Treg:bead ratio = 6.25:1), 10 ng/mL IL-1b, 25 ng/mL IL-6, and 10 IU/mL IL-2 for 5 days and stimulated with Leukocyte Activation Cocktail, with BD GolgiPlug. The x-axis indicates log fluorescence intensity of IL-17A APC. Th17, T-helper 17 cell. (D) Chart shows the mean fluorescence intensity (MFI) quantification of IL-17A staining. Error bars represent mean ± SD. *P ≤ .05. (E) The percentage of proliferation of CFSE-stained T_con cells when cocultured with autologous HD and AA-expanded total Tregs in 8:1, 4:1, 2:1, and 1:1 T_con:Treg ratios for 5 days in the presence of anti-CD3/CD28 beads (Treg:beads = 20:1). (F) The proliferation index of CFSE-stained T_con cells when cocultured with autologous AA-expanded Treg-A or Treg-B in 8:1, 4:1, 2:1, and 1:1 T_con:, Treg ratios for 5 days in the presence of anti-CD3/CD28 beads (Treg:, beads = 20:1). Error bars represent mean ± SD. **P ≤ .01; ***P ≤ .001. (G) Representative proliferation plots of CFSE-stained T_con cells when cocultured with autologous AA-expanded Treg-A and Treg-B in different T_con:Treg ratios (8:1, 4:1, 2:1, and 1:1).