Origin of CD4⁺Foxp3⁺cells. 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 H2-k^b), 5 × 10⁵ Treg from wild-type C57BL/6 mice (Thy1.2 and H-2k^b) and 5 × 10⁵ CD25-depleted Tcons (CD4⁺CD8⁺CD25⁻ T cells) from congenic Thy1.1 C57BL/c mice (Thy1.1 and H2-k^b) after lethal irradiation with 800 cGy on day 0. Mice of each group received PBS alone, RAPA, IL-2, or a combination of RAPA + IL-2 from day 0 to day 7 after BMT. Cells were reisolated from mLNs, pLNs, and spleen on day 7 after BMT and analyzed by flow cytometry. The combination of RAPA + IL-2 had a synergistic effect resulting in an increase in the frequency and absolute number of adoptively transferred donor-type Tregs in each lymphoid organ. The absolute cell number of CD4⁺Foxp3⁺ cells by expansion of adoptively transferred donor CD4⁺CD25^highFoxP3⁺ Tregs (A) and by increased conversion from CD25⁻ Tcons (B) in each treatment group. Results were shown as mean ± SD using horizontal column bar graphs. Data are derived from 3 independent experiments. *P < .05.