CpG ODN acts directly on Foxp⁺Helios⁺ cells. The enhancement of the frequency of Foxp3⁺Helios⁺ Tregs by CpG ODN is not secondary to differential effects of the ODN on the proliferation of Foxp3⁺Helios⁺, Foxp3⁺Helios⁻, or Foxp3⁻Helios⁻ subpopulations. A mixture containing 50% CD4⁺CD25⁺ and 50% CD4⁺CD25⁻ T cells was created after sorting. After labeling with CFSE, the mixture was stimulated for 5 days with plate-bound anti-CD3/CD28 in the presence or absence of the CpG ODN (2.5μM). (A) The extent of CFSE dilution was analyzed by gating on the 3 subsets (Foxp3⁺Helios⁺, Foxp3⁺Helios⁻, and Foxp3⁻Helios⁻). (B) The proliferation of the Foxp3⁺ and Foxp3⁻ T cells is identical in the presence and absence of the ODN. (C) The optimal size ODN do not inhibit Treg expansion. CD4⁺CD127^loCD25⁺ cells (1 × 10⁵) were isolated by FACS sorting, stimulated for 5 days in the presence of different length ODNs (10 mer, 10-bp long ODN; 25 mer, 25-bp long ODN; 50 mer, 50-bp long ODN; 100 mer, 100-bp long ODN). Stimulated cells were washed and expanded for an additional 7 days in the absence of the ODN. Cell yield and percent Foxp3⁺ and Helios⁺ cells were quantitated by FACS. The graph indicates the absolute number (mean ± SD) of Foxp3⁺Helios⁺ cells (open box) and non-Foxp3⁺Helios⁺ cells (closed box) recovered on day 12. Data shown is the 1 of 4 independent experiments from different donors.