Low or absent CD127 cell surface expression in combination with CD25 expression is an important and long-established marker to aid the distinction of Treg from T effector (Teff) cells that express higher levels of CD127 in humans. Forkhead box protein (FOX)3 expression inversely correlates with CD127 expression and has been shown to directly regulate CD127 expression through binding the CD127 promoter.8,9 Human CD25+CD127−/lo Treg cells have been demonstrated to be highly suppressive in vivo in a humanized mouse model.10,11 Live human CD25+CD127−/lo Treg cells may be purified and subsequently validated as bona fide Treg by ensuring a high enrichment of FOXP3+ cells and demethylation of the Treg cell-specific demethylated region (TSDR) of the FOXP3 promoter.12 The latter finding is important because downregulation of CD127 and upregulation CD25 and FOXP3 expression by human activated conventional CD4+ T cells are possible.12 Distinct populations within total CD4+ T cells have also been identified using CD25 and CD45RA cell surface markers, each with differential FOXP3 expression and suppressive capacity.13,14 Here we demonstrate that CD4+CD25+CD127−/lo Treg sorted according to differential CD45RA expression distinguishes Treg subpopulations that, after in vitro expansion with a physiological concentration of TAC, have a differential TSDR demethylated phenotype and suppressive function. These observations will inform the design of protocols to deliver Treg cellular therapy to transplant patients receiving CNIs, including TAC.