Foxp3⁺ regulatory T cells (Tregs) maintain self-tolerance and adoptive therapy, and using Foxp3⁺ Tregs has been proposed as treatment for autoimmune diseases. The clinical use of Tregs will require large numbers of cells and methods for in vitro expansion of Tregs are being developed. Foxp3⁺ Tregs can be divided into 2 subpopulations based on expression of the transcription factor, Helios. Foxp3⁺Helios⁺ Tregs (70%) are thymic-derived, whereas Foxp3⁺Helios⁻ Tregs (30%) are induced in the periphery. Foxp3⁺Helios⁺ Tregs differ from Foxp3⁺Helios⁻ Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression on days to weeks of expansion in vitro. Addition of a 25 mer DNA oligonucleotide of random composition for a short period during the expansion of Foxp3⁺ Tregs in vitro results in prolonged stabilization of the Foxp3⁺Helios⁺ subpopulation and yields an optimal population for use in cellular biotherapy.