Naturally occurring CD49b⁺CD200R3⁺ DC_regs induce donor-derived alloreactive CD4⁺CD25⁺Foxp3⁺ T_regs in the transplanted recipient mice. (A-D) The transplanted recipient BALB/c mice were prepared as described in Figure 5A and B. Subsequently, serum and donor-derived CD4⁺ T cells were collected from each transplanted recipient mouse on day 30 after transplantation. (A) Serum production of TNF-α, IL-12p70, and IFN-γ was measured by enzyme-linked immunosorbent assay. *P < .01 compared with the untreated group. (B) Donor-derived CD4⁺ T cells (2 × 10⁵) were cultured with the irradiated CD11c⁺ DCs (2 × 10⁴) obtained from BALB/c or C57BL/6 mice for 3 days, and the proliferative response was measured. *P < .01 compared with normal mice. (C) The expression of CD25 and Foxp3 on CD4⁺ T cells was analyzed by flow cytometry, and data are percentage positive cells. *P < .01 compared with the untreated recipient mice. (D) CD4⁺CD25⁻ T cells obtained from B10.D2 mice (5 × 10⁴) were cultured with CD11c⁺ DCs from BALB/c mice (5 × 10³) in the presence of a graded dose (6.25 × 10³-5 × 10⁴) of CD4⁺CD25⁺ T cells obtained from normal B10.D2 mice (normal mice) or the recipient mice treated with CD49b⁺CD200R3⁺ cells for 3 days, and the proliferative response was measured. *P < .01 compared with normal mice. Data are expressed as mean plus or minus SEM, and the results are combined from 3 replicate experiments (A-D). (E,F) The transplanted recipient BALB/c mice were prepared as described in Figure 5C and D. (E) The expression of CD200R3 among gated CD49b⁺ cells was analyzed on day 0 by flow cytometry, and data are represented by a histogram. Data are representative of 3 replicate experiments. (F) Donor-derived CD4⁺ T cells were collected from each transplanted recipient mouse on day 30 after transplantation. The expression of CD25 and Foxp3 on CD4⁺ T cells was analyzed by flow cytometry, and data are percentage positive cells. *P < .01 compared with the untreated recipient mice. Data are mean ± SEM, and the results are combined from 3 replicate experiments.