Both CD25int mTreg and naive Treg cell subpopulations had a similar high percentage of cells with a demethylated TSDR and FOXP3 expression before expansion. Loss of TSDR demethylation during expansion was restricted to CD25int mTreg cells. It is possible that the CD25int mTreg cell population is contaminated with memory Teff cell that expand faster than CD25int mTreg cell to become the predominant constituent of cultured CD127−/loCD25intCD45RA− sorted cells after 14 days of polyclonal stimulation. Regulatory T cell TSDR demethylation status is described to be highly stable when cells are subjected to extended periods of in vitro expansion.12 It is therefore less likely that CD127−/loCD25+CD45RA− cells that are TSDR demethylated on day 0 undergo epigenetic alteration to possess a methylated TSDR during expansion. Both CD25int mTreg and naive Treg cells express high levels of FOXP3 after expansion, yet the former may be accounted for by CD25int Teff cell contaminants expressing FOXP3 after activation. The combined observations of a greater level of IL-10 production, a lack of a TSDR demethylated status, and expression of FOXP3 by CD25int mTreg expanded cells strongly indicates that a large proportion of this population is comprised of de novo produced iTreg cells that may derive from Teff cells that contaminate the sorted population of day 0 and expand to form the main population constituent. Contaminating CD4+ Teff cells may have been converted to iTreg cells in TGF-β–rich culture conditions,20 derived from conventional Treg cells21 sorted alongside. The contaminating non-Treg population may have comprised a small constituent of the sorted CD25int mTreg cell population on day 0 because the proportion of CD25int mTreg and naive Treg with demethylated TSDR is similar at day 0. The dominant method by which naive Treg and CD25int mTreg exert suppression may differ, given the differential level of IL-10 production and susceptibility of interference of suppression by TAC.