Foxp3⁺Helios⁺ Tregs have uniformly demethylated TSDR and do not produce inflammatory cytokines. (A) CD4⁺CD25^hi cells were isolated from the buffy coat of a male donors (n = 7), fixed-permeabilized, and stained for intracellular expression of Foxp3 and Helios. Stained cells were sorted into the 4 different fractions. Upper plot shows the staining and gating condition before sorting. For DNA methylation analysis of the TSDR, bisulfite modification of genomic DNA was performed after extraction from the sorted fractions. Methylation of CpG was read and analyzed by the pyrosequencing method. Results from 7 donors are shown in the lower plot. (B) CD4⁺CD25⁺ cells were sorted from the buffy coat and were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 6 days. The cells were then expanded for an additional 5 days with anti-CD3/anti-CD28 antibody-coated magnetic beads (CD3/CD28-beads). Intracellular staining for IFNγ (n = 4), IL17A (n = 5), and IL2 (n = 4) was performed after restimulation with PMA and ionomycin. Top dot plot is an example to show the gating of each subset separated by Foxp3 and Helios expression in the expanded population.