Generation of CD4⁺CD25⁺Foxp3⁺ iT_regs from CD4⁺CD25⁻Foxp3⁻ T cells by naturally occurring CD49b⁺CD200R3⁺ DC_regs. (A-C) Rag2^−/−KJ1-26⁺ T cells (10⁷/mouse) were transferred with or without OVAp/CD11c⁺ DCs, OVAp/CD49b⁺CD200R3⁻ cells, or OVAp/CD49b⁺CD200R3⁺ cells (10⁶/mouse) into mice. On day 8 after the adoptive transfer, Rag2^−/−KJ1-26⁺ T cells were isolated from the recipient mice. (A,B) The expression of CD25 and Foxp3 among gated KJ1-26⁺ T cells was analyzed by flow cytometry, and data are represented by a dot plot (A) and are percentage positive cells (B). *P < .01 compared with Rag2^−/−KJ1-26⁺ T cells and OVAp/CD11c⁺ DCs. Data are representative of 4 replicate experiments (A). Data are mean ± SEM, and the results are combined from 4 replicate experiments (B). (C) Rag2^−/−KJ1-26⁺ T cells (2 × 10⁴) were cultured with splenocytes (2 × 10⁴) plus anti-CD3ϵ mAb (1 μg/mL) in the presence of a graded dose (2.5 × 10³-2 × 10⁴) of Rag2^+/+KJ1-26⁺CD25⁺ T cells or Rag2^−/−KJ1-26⁺CD25⁺ T cells obtained from the adoptively transferred mice for 3 days, and the proliferative response was measured. *P < .01 compared with Rag2^−/−KJ1-26⁺ T cells. Data are mean ± SEM, and the results are combined from 4 replicate experiments. (D) Rag2^−/−KJ1-26⁺ T cells (2 × 10⁶) were cultured with OVAp/CD11c⁺ DCs, OVAp/CD49b⁺CD200R3⁻ cells, or OVAp/CD49b⁺CD200R3⁺ cells (2 × 10⁵) in the presence or absence of TGF-β1 (0.1 ng/mL), anti–TGF-β mAb, anti-CD200R3 mAb, or control Ig (each 5 μg/mL) for 6 days, and the expression of CD25 and Foxp3 among gated KJ1-26⁺ T cells was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the percentage of CD25⁺Foxp3⁺ cells among gated KJ1-26⁺ T cells. Data are representative of 4 replicate experiments.