An increased frequency of Foxp3⁺Helios⁺ T cells is seen in the presence of the CpG ODN during expansion of distinct populations of Tregs. (A) Sorting strategy used for isolation of CD127^hiCD25⁺, CD127^loCD25^int, and CD127^lo25^hi T cells. Expression of Foxp3 and Helios was analyzed in the different populations after sorting. (B) CpG ODN-mediated enhancement of the frequency of Foxp3⁺Helios⁺ cells in the sorted populations. (C) Sorting strategy for the isolation of CD25⁺CD45RA⁺ and CD25⁺CD45RA⁻ Treg subpopulations. Expression of Foxp3 and Helios in the cells was performed after sorting. (D) CpG ODN-mediated enhancement of the frequency of Foxp3⁺Helios⁺ cells in the sorted CD25⁺CD45RA⁺ and CD25⁺CD45RA⁻ Treg subpopulations after activation and subsequent expansion. (E) Enhancement of Foxp3⁺Helios⁺ cells in 2 week-expanded CD25⁺CD45RA⁺ Tregs (n = 5).