To investigate whether TAC differently affected the expansion of Treg subpopulations, we analyzed the effect of TAC on cell expansion in the final 7 days of a 14-day culture, comparing cell numbers at day 7 with day 14, when cells were exposed to TAC (8 ng/mL) or DMSO vehicle control. CD25hi mTreg were highly anergic and could not be expanded sufficiently to perform experiments. As expected, TAC significantly reduced expansion of all Treg populations. CD25int mTreg expanded a median of 25- and 13-fold; naive Treg by a median of 21- and 6-fold; and total Treg cells expanded a median of 16- and 6-fold in the presence of DMSO or TAC, respectively (Figure 2A). Analysis of the ratio of proliferation between Treg incubated with DMSO and TAC demonstrated that TAC did not inhibit proliferation of any 1 Treg cell subpopulation more than any other (Figure 2B).