Addition of CpG ODN increases the frequency of Foxp3⁺Helios⁺ cells during in vitro expansion. (A) FACS-sorted CD4⁺CD25⁺ cells (left plot, square gate) were stimulated with plate-bound anti-CD3 and anti-CD28 in the presence of TGFβ (5 ng/mL) and CpG ODN (ODN2395, 2.5μM) for 5 days. The cells were then washed and incubated for 2 additional days in IL-2 containing media. Foxp3 and Helios staining was performed on gated viable CD4⁺ cells. (B) Summary of CpG ODN mediated enhancement of Foxp3⁺Helios⁺ cells during expansion. Culture conditions for stimulation and expansion are same as in panel A. Donors (n = 15) ranged in age from 19 to 75 and were all male. (C) The CpG ODN enhances the frequency of Foxp3⁺Helios⁺ cells in both freshly isolated and preactivated T cells. Cells were isolated and stimulated for 5 days as in panel A. The CpG ODN was added at the time points indicated in the figure.