CD25int mTreg maintained similar FOXP3 protein expression before and after expansion (median, 83.7% and 74.9%, respectively) in DMSO control media, whereas the proportion of naive Treg that expressed FOXP3 increased (median, 78.7% to 95.8% after expansion) (Figure 3B). As the extensive loss of TSDR demethylation status in the CD25int mTreg population is not reflected by reduced FOXP3 expression, it appears that the CD25int expanded population contains a greater proportion of activated Teff cell or induced Treg (iTreg) cell that may transiently express FOXP3 without having a demethylated TSDR. A reduced level of TSDR demethylation alongside a sustained FOXP3 expression altered the ratio of FOXP3 expressing cells compared with TSDR demethylated cells after expansion in the CD25int mTreg population (Figure 3D).