Sorted CD4⁺CD25⁺ Tr cells exposed to rapamycin suppress proliferation of syngeneic naive CD4⁺ T cells and preserve FoxP3 expression. (A) The same experiment described in Figure 3A (right) with cells from Balb/c mice was performed using CD4⁺CD25⁺ T cells cultured for 3 weeks with αCD3 plus αCD28 plus 1000 U/mL IL-2 (medium cells) or αCD3 plus αCD28 plus 1000 U/mL IL-2 plus rapamycin (rapamycin cells). The cultured cells were added in equal number to naive CD4⁺ T cells isolated from Balb/c mice (10⁵:10⁵) and proliferation was monitored by CFSE dilution. (B) Relative levels of mRNA FoxP3 were determined by real-time quantitative RT-PCR in Balb/c CD4⁺CD25⁺ T cells repetitively activated in vitro with or without rapamycin. The amounts of FoxP3 mRNA are expressed as relative to amounts 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 (start) are also indicated.