The Foxp3⁺Helios⁺ T cells expanded in the presence of the CpG ODN are functional Tregs. (A) The TSDR of CD4⁺CD25⁺ T cells expanded in the presence of the CpG ODN is fully demethylated. FACS-sorted CD4⁺CD25⁺ cells were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of CpG ODN (2.5μM) for 5 days. The cells were then expanded 14 to 16 days in IL-2 containing media without CpG ODN and methylation analysis was performed as in Figure 1A. (B) Two-week expanded, CpG ODN-treated Tregs were treated as in Figure 1B with PMA/ionomycin and analyzed for intracellular cytokine expression. (C) The population of cells expanded in the presence of the CpG ODN exhibits greater suppressive activity than cells expanded in the absence of the ODN. Tregs were expanded as in panel A and cultured for 3 days at different ratios with freshly isolated CD4⁺CD25⁻ cells (5 × 10⁴ cells/well) in the presence of γ-irradiated PBMCs (5 × 10⁴) and soluble anti-CD3 (2 μg/mL). ³H-TdR incorporation was measured during the last 18 hours of culture.