Role of the B7 family in antigen-specific de novo generation of CD4⁺Foxp3⁺ iT_regs from CD4⁺Foxp3^– T cells in Sp and MLNs. (A-C) Sp KJ1-26⁺Foxp3^EGFP− T cells were transferred into WT mice and B7^−/− mice (5 per group), and the animals were subsequently fed PBS (none) or OVA protein the day after the adoptive transfer. Expression of Foxp3^EGFP among gated KJ1-26⁺ T cells in Sp (A-B) and MLNs (A,C) on day 11 after the adoptive transfer was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of Foxp3^EGFP+ cells among gated KJ1-26⁺ T cells in each quadrant (A) and are the percentage of positive cells (B-C). *P < .01 compared with WT mice. Data are the mean ± SD, and the results are representative of 4 independent experiments. (D-F) CFSE-labeled Sp Rag2^−/−KJ1-26⁺ T cells (3 × 10⁶/mouse) were transferred into WT mice (5 per group) with or without Sp CD4⁺CD25⁺ T cells (10⁶/mouse) obtained from Foxp3^EGFPDO11.10 mice (D,F), WT mice (E,F), and B7^−/− mice (E-F) that had been fed PBS (none) or OVA protein, and the animals were subsequently injected with OVA protein the day after the adoptive transfer. CFSE dilution among gated KJ1-26⁺ T cells on day 3 after the injection with OVA protein was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of CFSE dilution among gated KJ1-26⁺ T cells in each quadrant (D-E) and are the percentage of dividing cells (F). *P < .01 compared with Rag2^−/−KJ1-26⁺ T cells plus OVA. Data are the mean ± SD, and the results are representative of 4 independent experiments.