CFSE-based proliferation analysis. (A) In vivo expansion of CFSE-labeled C57B/6 Treg (H-2k^b, Thy-1.1) in Balb/c recipients (H-2k^d) on day 6 after BMT is depicted. The congenic markers Thy-1.1 and Thy-1.2 were used to distinguish between Treg and T_CONV cells, respectively. All Balb/c recipients (H-2k^d) were given 5 × 10⁶ C57B6 TCD-BM cells (H-2k^b) after lethal irradiation with 800 cGy. Additionally 5 × 10⁵ Treg (day 0) plus 1 × 10⁶ CD4⁺/CD8⁺ T cells (day 2) were given (both H-2k^b). Histograms show the FACS profile of CFSE⁺ Treg cells (H-2k^bThy1.1). Numbers of events in each cell division (n) are indicated below the respective peak. Percentages of cells having undergone either 0 or 1 cell division are indicated in each histogram. Proliferation of Treg cells (first column from left) is slightly reduced in the presence of CSA (10 mg/kg; second column from left) and to a lesser extent by MMF (90 mg/kg; fourth column from left). Addition of RAPA (0.5 mg/kg; third column from left) has no impact on Treg expansion. Cells are gated on the donor marker H-2k^b and the congenic marker Thy 1.1. (B) Intracellular expression of Foxp3 in donor-derived Thy1.1⁺H-2k^b+CD4⁺CD25^high+ T cells derived from Balb/c recipients having received T_CONV (Thy-1.2) and Treg (Thy-1.1) cells alone (first column from left) in conjunction with CSA (second column from left), RAPA (third column from left), or MMF (fourth column from left). Cells were sorted, fixed onto glass slides, and analyzed by immunofluorescence. FoxP3 (Alexa 546), red; DNA (DAPI), blue; double positive, purple; magnification × 200. Percentage of FoxP3⁺ cells within the donor CD4⁺CD25⁺ population (average of 3 independent experiments): 95% ± 3% no immunosuppressant, 57% ± 3.2% CSA (second column from left), 94% ± 3.5% RAPA (third column from left), 89% ± 1.7% MMF (fourth column from left).