RAPA/Tregs protected from aGVHD by reducing alloreactive T-cell expansion in vivo. (A) Single timepoints showing the expansion of luciferase transgenic (luc⁺) donor T cells in 3 representative BALB/c mice (of 10 animals per group) receiving T cells in combination with Tregs at different ratios (1:4 and 1:8) combined with RAPA as indicated. T-cell expansion is significantly reduced when Tregs are combined with RAPA (column 3 vs 4 and 5 vs 6 from the 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 T cells (△, n = 15), T cells and RAPA 0.5 mg/kg (*, n = 10), Treg:T cells (1:4) + PBS (□, n = 12) Treg:T cells (1:4) + RAPA (○, n = 12). Signal intensity is significantly higher in animals receiving Treg:T cells (1:4) + PBS compared with Treg:T cells (1:4) + RAPA (□ vs ○, P < .05). (C) BLI signal intensity of mice receiving TCD-BM (●, n = 10), with T cells (△, n = 15), T cells and RAPA 0.5 mg/kg (*, n = 10), Treg:T cells (1:8) + PBS (□, n = 12) Treg:T cells (1:9) + RAPA (○, n = 12). Signal intensity was significantly higher in animals receiving Treg:T cells (1:8) + PBS compared with Treg:T cells (1:8) + RAPA (□ vs ○, P < .05).