DC_regs induce donor-derived alloreactive CD4⁺CD25⁺Foxp3⁺ T_R cells from CD4⁺CD25⁻Foxp3⁻ T cells in the mice that received transplants. The irradiated recipient BALB/c mice received T-cell-depleted BM cells and CD4⁺CD25⁻ T cells obtained from B10.D2 mice, and were then treated with or without the recipient-type mDCs, DC_regs after the transplantation as described in Figure 3A,B. Subsequently, donor-derived CD4⁺ T cells were collected from each transplant recipient 30 days after the transplantation. (A) Donor-derived CD4⁺ T cells (2 × 10⁵) were cultured with the irradiated mDCs (2 × 10⁴) obtained from BALB/c mice in the presence or absence of IL-2 (10³ U/mL) or mAbs to CD3 and CD28 (each 10 μg/mL) for 3 days, and the proliferative response was measured. The error bars indicate SD. *P < .01 compared with normal mice by Student paired t test. Three replicate experiments with similar results were pooled. (B,C) The expression of CD25 and Foxp3 on CD4⁺ T cells was analyzed by flow cytometry, and data are represented by a dot plot (B) and are expressed as percentage positive cells (C). The error bars indicates SD. *P < .01 compared with normal mice by Student paired t test. Three replicate experiments with similar results were pooled. (D) CD4⁺CD25⁻ T cells obtained from B10.D2 mice (5 × 10⁴) were cultured with the irradiated allogeneic mDCs (5 × 10³) obtained from BALB/c mice in the presence of CD4⁺CD25⁺ T cells (6.25 × 10³-5 × 10⁴) obtained from B10.D2 mice or from each group of the transplanted mice for 3 days, and the proliferative response was measured. The error bars are SD. *P < .01 compared with CD4⁺CD25⁺ nT_R cells by Student paired t test. Results of three replicated experiments were pooled. (E) The production of IL-2, IL-4, IL-10, and IFN-γ by freshly isolated CD4⁺CD25⁺ T cells from B10.D2 mice and the DC_reg-treated recipient mice was analyzed by flow cytometry, and data are represented by a dot plot. Results of 3 replicated experiments were pooled.