Before expansion, the majority of naive Treg and CD25int mTreg expressed FOXP3 and possessed a demethylated TSDR (Figure 1). Naive Treg and CD25int mTreg cells showed differential stability of this Treg cell phenotype during expansion in DMSO control media. A significantly higher proportion of CD25int mTreg cells did not have a demethylated TSDR phenotype (median, 14.2% remaining demethylated) compared to the naive Treg cell subpopulation (median, 78.7% remaining demethylated) (Figure 3A, P < 0.05) by day 14. This finding correlated with a substantial reduction in the population that expressed CD27, a marker known to be associated with highly suppressive Treg,18 by CD25int mTreg compared with naive Treg expanded in DMSO control media (Figure 3C).