Rapamycin does not block AICD and proliferation of murine CD4⁺ T cells. (A) DO11.10 tg CD4⁺ T cells were cultured with APCs plus OVA (medium) or APCs plus OVA plus rapamycin (rapamycin) and AICD was monitored by FACS after 24 and 72 hours of culture. Percentage of propidium iodide-positive (PI⁺)-annexin V⁺ cells is indicated in each dot plot. One representative experiment of 2 is presented. (B) Fold expansion of DO11.10 tg CD4⁺ T cells 1, 2, and 3 weeks after culture in the presence of APCs plus OVA (medium, □) or APCs plus OVA plus rapamycin (rapamycin, ▪) was evaluated by direct cell counts. One representative experiment of 6 is presented. (C) After 3 rounds of stimulation with APCs plus OVA (medium) or APCs plus OVA plus rapamycin (rapamycin), DO11.10 tg CD4⁺ T cells were stained with CFSE and restimulated with APCs plus OVA in the absence of the compound and of exogenous IL-2. CFSE dilution was monitored 5 days after activation. One representative experiment of 3 is presented. (D) After 3 rounds of stimulation with APCs plus OVA (medium) or APCs plus OVA plus rapamycin (rapamycin), DO11.10 tg CD4⁺ T cells were left resting for one additional week with no further stimulation in the presence of IL-2 (50 U/mL). At the end of the 7 days cell size was analyzed by FACS by plotting forward scatter (FSC) versus side scatter (SSC) parameters. Small (solid line) and big (dotted line) cells are circled. Naive CD4⁺ T cells from a DO11.10 tg mouse were used as control. One representative experiment of 6 is presented.