Combination of RAPA + IL-2 has additive effects on the expansion of CD4⁺Foxp3⁺ and CD4⁺CD25⁺ cells after infusion of donor Tregs and CD25 depleted Tcons. BALB/c mice (Thy1.2 and H-2k^d) were injected with 5 × 10⁶ TCD-BM cells from GFP transgenic C57BL/6 mice (Thy1.1 and H-2k^b), 5 × 10⁵ Treg from wild-type C57BL/6 mice (Thy1.2 and H-2k^b) and 5 × 10⁵ CD25⁻ Tcons (CD4⁺CD8⁺CD25⁻ T cells) from congenic Thy1.1 C57BL/c mice (Thy1.1 and H-2k^b) after lethal irradiation with 800 cGy on day 0. Mice received either PBS alone, RAPA, IL-2 or a combination of RAPA + IL-2 from day 0 to day 7 after BMT (5 mice per group). Cells were reisolated from mLNs, pLNs, and spleen and analyzed by flow cytometry after staining with CD4, CD25, and intracellular Foxp3 on day 7 after BMT. Cells from each lymphoid organ were stained with (A) anti-CD4 and anti-Foxp3 intracellular staining, and (B) anti-CD4 and anti-CD25; data shown are gated on H-2k^b+ lymphocytes. These data are representative of 3 separate experiments. The combination of RAPA + IL-2 increased the expansion of donor-type CD4⁺Foxp3⁺ T cells and CD4⁺CD25⁺ T cells, and reduced donor-type CD4⁺CD25⁻ T cells from donor-type Tregs and CD25 depleted Tcons. The graphs compare the proportion of (C) CD4⁺Foxp3⁺ cells, (D) CD4⁺CD25⁺ T cells, and (E) CD4⁺CD25⁻ T cells from peripheral LNs in each group (PBS alone, ●; IL-2, ■; RAPA, ▴; RAPA + IL-2, ▾). Absolute cell number of CD4⁺Foxp3⁺ T cells and CD4⁺CD25⁻ T cells in pLNs, mLNs cells and splenocytes from each treatment group are shown (F-H). Data are derived from 3 separate experiments. Results are displayed as the mean ± SD. *P < .05.