Ag presentation by pDCs induces T reg cell development. (A) CD4⁺ T cells were purified from the LNs of 2D2 mice, labeled with CFSE, and transferred into WT:WT and pIII+IV^−/−:WT chimeras that had been immunized 1 d previously with MOG_35–55+CFA. Nonimmunized WT recipients were used as controls. 3 d after transfer, 2D2 T cells in the LNs were analyzed by flow cytometry for dilution of CFSE labeling (left and right) and the frequency of Foxp3⁺ cells (right). Percentages of cells in the peaks corresponding to divisions 0–7 (D0–D7) are indicated on the left. Percentages of Foxp3⁺ cells are indicated on the top and right. The results are representative of two independent experiments. (B) CD4⁺ T cells were purified from the LNs of 2D2 mice, labeled with CFSE, and transferred into WT:WT, pIII+IV^−/−:WT, µMT:WT, and µMTxpIII+IV^−/−:WT chimeras that had been immunized 1 d previously with MOG_35–55+CFA. 3 d after transfer, 2D2 T cells in the dLNs were analyzed by flow cytometry to determine the frequency of Foxp3⁺ T reg cells. The results are representative of two independent experiments. The means and SEM were derived from three chimeras per group. ns, not significant; *, P < 0.05; **, P < 0.01. (C) CD4⁺ T cells were purified from LNs of 2D2 mice, labeled with CFSE, and transferred into WT:WT and pIII+IV^−/−:WT chimeras that had been immunized 1 d previously with MOG_35–55+CFA. 3 d after transfer, dLN sections were stained using antibodies against PDCA-1 and Foxp3. Representative images derived from two independent experiments are shown. Arrowheads indicate CFSE⁺ Foxp3⁺ 2D2 T reg cells; circles indicate CFSE⁺ Foxp3⁺ 2D2 T reg cells that interact with pDCs. (D) The histogram shows the percentages of CFSE⁺ 2D2 T cells that interact with pDCs. The ratios above the bars represent the number of interacting 2D2 T cells over the total number of 2D2 T cells. (E) The histograms show the percentages of Foxp3⁺ T reg cells among CFSE⁺ 2D2 T cells found in close proximity to pDCs. The ratios above the bars represent the number of Foxp3⁺ 2D2 T cells over the number of 2D2 T cells interacting with pDCs. (D and E) The number of confocal images analyzed (N) is indicated at the bottom of each bar. Results were pooled from two independent experiments with two mice per group. Histograms show the means and SEM derived from two independent experiments with two mice per group. ***, P < 0.001.