RAPA allows for polyclonal Treg expansion in vivo. (A) Expansion of luc⁺ Tconv (CD4⁺CD25⁻) or luc⁺ Tregs as shown for 5 representative animals of each group in the presence or absence of RAPA (1.5 mg/kg) on days 4 and 16 after BMT (C57BL/6 → BALB/c). Addition of RAPA reduces the expansion of luc⁺ Tconv (first vs second column of 5 animals from left), whereas expansion of luc⁺ Tregs is not significantly affected (third vs fourth column of 5 animals from left). (B) Expansion of luciferase-labeled T cells as quantified in emitted photons over total body area at serial timepoints after BMT. BLI signal intensity of mice receiving TCD-BM (△, n = 10), with luc⁺ T cells with PBS (□, n = 10) or RAPA 1.5 mg/kg (■, n = 10), luc⁺ Treg with PBS (○, n = 10) or RAPA 1.5 mg/kg (●, n = 10). Signal intensity is significantly higher in animals receiving T cells + PBS compared with T cells + RAPA (□ vs ■, * P < .05). (C) Frequency of splenic CD4⁺FoxP⁺ T cells on day 5 after transplantation is not different when PBS compared with RAPA is given in vivo. One representative FACS analysis of 3 independent experiments is shown. (D) BALB/c mice were given 5 × 10⁶ TCD-BM together with 5 × 10⁵ Tregs (C57BL/6) after lethal irradiation with 800 cGy and were injected with PBS or RAPA (1.5 mg/kg) daily. Expression of the indicated markers on splenic Thy1.1⁺CD4⁺CD25⁺ on day 5 after transplantation is shown. Black histogram, recipient received Treg + PBS. Light gray histogram, Treg + RAPA. Dark gray histogram, Isotype control Ab. (E) TCR Vβ usage of Thy1.1⁺CD4⁺CD25⁺ on day 30 after transplantation in BALB/c recipients is shown.