Rapamycin is required for Treg induction. Flt3L alone or with antigen fails to induce Treg. (A) Induction of OVA-specific CD4⁺CD25⁺FoxP3⁺ Treg cells in DO11.10-tg Rag-2^−/− BALB/c mice treated with Flt3L/OVA_323-339/rapamycin combination. Mice treated with OVA_323-339, Flt3L, Flt3L/OVA_323-339, or rapamycin only failed to generate Treg (n = 3-5 per group). Statistical differences were determined by 1-way ANOVA with Bonferonni’s multiple comparison posttest analysis. All groups tested were significantly different from the Flt3L/OVA_323-339/Rapa treatment group (P < .0001). (B) Representative examples of stains for CD25 and FoxP3 (gated off CD4⁺ cells) for naïve controls, Flt3L-treated, rapamycin-treated, and Flt3L/OVA_323-339/rapamycin-treated mice. (C) Expression of CTLA-4, Helios, CD62L, and GITR molecules in the induced Treg. Data are average ± SD (n = 5 per group).