Antigenic peptide-pulsed DC_regs induce Ag-specific peripheral generation of CD4⁺CD25⁺ Foxp3⁺ T_R cells from CD4⁺CD25⁻Foxp3⁻ T cells. (A) Rag2^−/−KJ1–26⁺ T cells (5 × 10⁴) were cultured with the irradiated syngeneic DCs (5 × 10³) for 3 days, and the proliferative response was measured. The error bars indicate SD. *P < .01 compared with mDCs by Student paired t test. Results of 4 replicated experiments were pooled. (B-D) Rag2^−/−KJ1–26⁺ T cells were adoptively transferred with or without OVA_323–339 peptide/mDCs or OVA_323-339 peptide/DC_regs into BALB/c mice. On day 8 (B,C) or the indicated days (D) after the adoptive transfer, KJ1–26⁺ T cells were isolated from the recipient mice. (B) KJ1–26⁺ T cells (10⁵) obtained from each group of the mice that received an adoptive transfer were cultured with or without the irradiated syngeneic APCs (10⁵) in the presence of OVA_323-339 peptide (1 μM), IL-2 (10³ U/mL), and/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 KJ1–26⁺ T cells by Student paired t test. Results of 3 replicated experiments were pooled. (C,D) The expression of CD25 and Foxp3 on KJ1–26⁺ T cells was analyzed by flow cytometry, and data are represented by a dot plot (C) and expressed as percentage of positive cells (D) of the OVA_323-339 peptide/mDC-adoptive transfer group (left panel) and OVA_323-339 peptide/DC_regs-adoptive transfer group (right panel). The error bars indicate SD. Results of 3 replicated experiments were pooled.