RAPA inhibited Treg expansion less than Tconv and preserved suppressive activity and Foxp3 expression. (A) In vitro expansion of luciferase transgenic CD4⁺CD25^high or CD4⁺CD25⁻ T cells (C57BL/6) in the presence of CD3/CD28 stimulation, IL-2 (100 IU/mL), for 48 hours in complete media (cRPMI) and the indicated RAPA (R) doses. Percentages represent the relative reduction compared with the absence of RAPA (100%). One representative experiment of 3 independent experiments is shown. (B) Mean fluorescence intensity (MFI) for Foxp3 surface expression of CD4⁺CD25^high cells at different timepoints during stimulation as described in panel A is quantified in the presence or absence of RAPA (*P < .05). (C) A representative histogram on day 6 of culture for the 3 different culture conditions described in panel A is shown. (D) Suppressive activity of Tregs recovered from the respective cultures. Thymidine incorporation of conventional T cells is significantly reduced when Tregs are included in the culture. Tregs derived from RAPA-containing cultures are more suppressive in vitro (*P < .05). One representative proliferation analysis of 3 independent experiments is shown.