Rapamycin-exposed T cells suppress proliferation of syngeneic naive CD4⁺ T cells. (A) Naive KJ1-26⁺ CD4⁺ tg T cells isolated from spleens of DO11.10 tg mice were stained with CFSE and were activated with APCs alone or APCs plus OVA. DO11.10 CD4⁺ T cells activated for 3 weeks with APCs plus OVA (medium cells) or APCs plus OVA plus rapamycin (rapamycin cells) were added in equal number to naive CFSE⁺ cells (10⁵:10⁵) as shown in the left panel. Alternatively, naive CD4⁺ cells isolated from spleens of Balb/c mice were labeled with CFSE and cultured alone (unstimulated) or with αCD3 mAb. Balb/c CD4⁺ T cells activated for 3 weeks with αCD3 plus αCD28 mAbs (medium cells) or αCD3+αCD28 mAbs plus rapamycin (rapamycin cells) were added in equal number to naive CFSE⁺ cells (10⁵:10⁵) as shown in the right panel. After 5 days of culture, cell division was monitored by levels of CFSE dilution. Histograms show the FACS profile of CD4⁺CFSE⁺ T cells. Number of events in each cell division (n) are indicated on top of each peak. The amount of CD4⁺CFSE⁺ cells proliferating in the absence or presence of cultured T cells was calculated as described in “Materials and methods” and percentages of undivided cells in each culture condition is indicated. Percentages of suppression in comparison to proliferation of naive control cells is indicated. One representative experiment of 9 (for DO11.10) and 1 of 7 (for Balb/c) is presented. (B) Using the same cells described in panel A (right), the experiment was performed in a transwell system in which responder naive CD4⁺ T cells were activated with αCD3 mAb at the bottom of the transwell while medium or rapamycin cells were preactivated with αCD3 mAb for 6 hours and then added on top of the transwell (right). Data obtained in the transwell system were compared to data obtained in the coculture system (left). One representative experiment of 3 is presented.