Response of CD4⁺CD25^- T cells activated in the presence of rapamycin. (A) The same experiment described in Figure 3A (left panel) with cells from DO11.10 tg mice was performed using CD4⁺CD25^- T cells cultured for 3 weeks with APCs plus OVA (medium cells) or APCs plus OVA plus rapamycin (rapamycin cells). The cultured cells were added in equal number to naive KJ1-26⁺CFSE⁺ cells (10⁵:10⁵) and proliferation was monitored by CFSE dilution. One representative experiment of 3 is presented. FACS profile of the cells used before culture (starting population) is shown on top. (B) Relative levels of mRNA FoxP3 were determined by real-time quantitative RT-PCR in CD4⁺ (top) or CD4⁺CD25^- (bottom) T cells repetitively activated in vitro with or without rapamycin. The amounts of FoxP3 mRNA are expressed relative to that in splenocytes depleted of CD4⁺CD25⁺ T cells (which was given an arbitrary value of 1). Relative levels of mRNA FoxP3 in the cells before culture (▪) are also indicated. One representative experiment of 3 is presented.