The large drop in the proportion of CD25int mTreg with a demethylated TSDR post expansion, regardless of the exposure to TAC, identifies a considerable risk to adoptively transferring these cells into patients to tolerize allograft recipients. Although the CD25int mTreg population showed superior IL-10 production and stability to suppress when expanded in the presence of TAC, the lack of a demethylated TSDR is known to identify cells with an unstable Treg phenotype that are susceptible to loosing suppressive function.22 Indeed, we observed that CD25int mTreg produce substantially greater amounts of IL-17A and IFN-γ compared with naive Treg. Regulatory T cells with an unstable regulatory phenotype have been shown to have the potential to develop into pathogenic cells in mice23 and similar could be true for human Treg cells. It is possible that CD25int mTreg cells, possibly containing a large proportion of cells derived from Teff cell contaminants on day 0 that expand faster than conventional Treg, will lose suppressive function after adoptive transfer to patients and damage the allograft.