MpDCs induce the generation of CD25⁺Foxp3⁺ natural Tregs from positively selected CD69^hiTCR-αβ^hi DP autologous thymocytes. (A) CD69 versus TCR-αβ phenotype of primary large-sized DP thymocytes enriched in CD69^hi cells by magnetic sorting (L-DP CD69^hi) and unselected small-sized DP thymocytes mostly composing CD69^lo cells (S-DP CD69^lo) (left histograms, day 0). Numbers indicate percentages of gated cells. Flow cytometric analysis of Foxp3 versus CD25 expression on L-DP CD69^hi and S-DP CD69^lo thymocytes cultured onto OP9 stromal cells with rhIL-2 plus rhIL-7 for 7 days either in the presence or absence of autologous MpDCs is shown on the right (day 7). Numbers in quadrants indicate percentages of CD25⁺Foxp3⁺ cells. Results are representative of 1 of 3 independent experiments. (B) Relative numbers of CD25⁺Foxp3⁺ nTregs generated from L-DP CD69^hi and S-DP CD69^lo thymocytes cultured as in panel A. Data are mean ± SEM (n = 3). *P < .05. (C) Flow cytometric analysis of CD4 (left) or CD8 (right) versus CD25 expression on L-DP CD69^hi thymocytes cultured as in panel A (top panels). Foxp3 expression on electronically gated CD8^hiCD25⁻ (R1), CD8^intCD25⁺ (R2), and CD8⁻CD25⁺ (R3) populations is shown (bottom panels). Numbers indicate MFI values. Results are representative of 1 of 3 independent experiments.