Human alloreactive CD4^high cells induced by CD40-activated B cells are Treg. (A) CD4 expression in CD4⁺CD25⁻ T cells stimulated with allogeneic B cells for 5 days (top panels), and its relationship with cell proliferation based on the loss of CFSE label and CD45RA expression. Top panels represented the T cells gated on CD4. The percentage of CD4⁺ T cells in each gate is indicated. For the bottom panel, open histograms indicate the CFSE fluorescence intensities of the unstimulated control T cells, and the filled histograms represent the CFSE fluorescence intensities of the allostimulated T cells. The numbers in each histogram represent the percentage of cells that have undergone mitosis from each cell subset. (B) CD4^high cells express both CD25 and Foxp3. The dot plot on the left shows CD25 expression after 5 days of allostimulation. Open histograms on the right show the Foxp3 expression, and filled histograms indicate the isotype controls. The results shown are representative of 4 different experiments. (C) CD4^highCD25⁺ Treg generated from CD4⁺CD25⁻ T cells potently suppressed MLR in an antigen-nonspecific manner. Freshly purified CD4⁺CD25⁻ T cells were cocultured with CD40-activated allogeneic B cells for 7 days. The sorted CD4^highCD25⁺ (■) and CD4^mediumCD25⁻ (□) cells were added into MLR culture system as described in “Mixed lymphocyte reaction assays.” Proliferation (y-axis) is shown for 3 days of MLR. The results shown are representative of 5 different experiments.