Human CD4^high Treg induced from naive CD4⁺CD25⁻ T cells by CD40-activated allogeneic B cells are alloantigen-specific Treg. (A) Characteristics of CD4^high Treg induced from naive CD4⁺CD25⁻ T cells. Freshly purified naive CD4⁺CD25⁻ T cells were labeled with CFSE and cocultured with CD40-activated allogeneic B cells for 7 days. Representative data of CD4 and CD25 expression (left panel), CFSE dilution (top right panel), and Foxp3 expression (bottom right panel) from 6 independent experiments. Open histograms show the CFSE fluorescence intensity (top right panel) and Foxp3 expression (bottom right panel) of CD4^mediumCD25⁻ cells. Filled histograms represent the CFSE fluorescence intensity (top right panel) and Foxp3 expression (bottom right panel) of CD4^highCD25⁺ cells. (B) CD4^highCD25⁺ Treg generated from naive CD4⁺CD25⁻ T cells potently suppressed MLR in an alloantigen-specific manner, and unsorted CD4⁺ T cells generated from naive CD4⁺CD25⁻ T cells had similar suppressor capacities in MLR. Freshly purified CD4⁺CD45RA⁺CD25⁻ T cells were cocultured with CD40-activated allogeneic B cells for 7 days. The sorted CD4^highCD25⁺ (■) and CD4^mediumCD25⁻ (□), and unsorted CD4⁺ T cells (×) were added into MLR culture system as described in “Mixed lymphocyte reaction assays.” Proliferation (y-axis) was shown for day 3 of MLR. The results shown are representative of 8 independent experiments.