Expansion and characterization of allo-specific Tregs. Tregs were seeded at 1 × 10⁵/well in a 48-well plate with irradiated allogeneic DCs (1 × 10⁴), rapamycin (100 ng/mL), IL-2 (10 U/mL), and IL-15 (10 ng/mL). Cells were cultured for 12 days. (A) The total Treg numbers at each time point were calculated by flow cytometry of timed events. The numbers of starting Tregs were normalized to 1 million at time 0. (B) The diagram outlines the numbers of allo-specific Tregs before and after in vitro expansion, normalized to 10 000 Tregs at time 0. The frequency of allo-specific Tregs at time 0 was calculated by LDA. The frequencies of allo-specific Tregs at subsequent times were calculated by flow microfluorometry of CFSE-labeled Tregs (C). (D-F) Flow cytometry plots showed intracellular expression of Foxp3 and cell-surface expression of CD45RA, CCR7, and CD62L on gated CD4⁺, CD25⁺, and CD127⁻ Tregs at the end of culture. Dot plot shows the analysis of CFSE dilution versus Ki-67 nuclear protein (G) or Foxp3 (H) staining in expanded Tregs at the end of culture. We observed a dual level of Foxp3 expression with the BD Biosciences kit (panel H), but not with the eBioscience kit (panel D).