Multiple molecules can be used synergistically with rapamycin to decrease conventional CD4⁺ T cells and induce Treg. Percentage of CD4⁺ T cells (A) or CD25⁺FoxP3⁺ Treg/CD4⁺ T cells (B) in spleens of DO11.10-tg x Rag-2^−/− BALB/c mice IP injected 3 times per week for 4 weeks with 100 μg of OVA_323-339 plus rapamycin (4 mg/kg) and either Flt3L (80 μg/kg), Fc-GITR-L (8 mg/kg), a combination of Flt3L/Fc-GITR-L, IL-2 (50 ng/kg), or IL-10 (50 ng/kg). Untreated naïve animals serve as controls (n = 4-5 per group). Data are average ± standard deviation (SD). Statistically significant differences were determined by 2-way analysis of variance (ANOVA). (C) Examples of Treg induction with Flt3L/OVA_323-339/rapamycin compared with OVA_323-339/rapamycin and untreated control mouse.