DC_regs generate alloreactive CD4⁺CD25⁺Foxp3⁺ T_R cells from CD4⁺CD25⁻ Foxp3⁻ T cells in vitro. (A) CD4⁺CD25⁻ T cells (2 × 10⁵) obtained from B10.D2 mice were cultured with or without the irradiated DCs (2 × 10⁴) obtained from BALB/c mice for 3 days, and the proliferative response was measured. The error bars indicate SD. *P < .01 compared with mDCs. Results of 4 replicated experiments were pooled. (B-D) CD4⁺CD25⁻ T cells (5 × 10⁶) obtained from B10.D2 mice were cultured with the irradiated DCs (5 × 10⁵) obtained from BALB/c mice for 7 days, and CD4⁺ T cells were collected. (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% positive cells (C). The error bars indicate SD. *P < .01 compared with the CD4⁺ T cells primed with allogeneic mDCs by Student paired t test. Results of 4 replicated experiments were pooled. (D) CD4⁺CD25⁻ T cells obtained from B10.D2 mice (5 × 10⁴) were cultured with the irradiated 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 each culture for 3 days, and the proliferative response was measured. The error bars indicate SD. *P < .01 compared with CD4⁺CD25⁺ nT_R cells by Student paired t test. Results of 4 replicated experiments were pooled. (E) CD4⁺CD25⁻ T cells (10⁶) obtained from B10.D2 mice and the irradiated DC_regs (10⁵) obtained from BALB/c mice were cocultured or placed separately in the presence or absence of anti–IL-10 mAb, anti–TGF-β mAb or control Ig for 7 days, and CD4⁺ T cells were collected. The expression of CD25 and Foxp3 on CD4⁺ T cells was analyzed by flow cytometry, and data are expressed as percentage of cells positive for both CD25 and Foxp3. The error bars indicate SD. *P < .01 compared with the primed CD4⁺ T cells primed with allogeneic DC_regs by Student paired t test. Results of 3 replicated experiments were pooled.