Thymic mature pDCs and cDCs can prime autologous CD69^hiTCR-αβ^hi DP thymocytes to differentiate into nTregs with distinct cytokine expression profiles. (A) Absolute (top panel) and relative numbers (bottom panel) of CD25⁺Foxp3⁺ cells derived from CD69^hiTCR-αβ^hi DP thymocytes cocultured with either MpDCs or McDCs as described in Figure 3A. Data are mean ± SEM (n = 4). *P < .05. **P < .01. (B) Flow cytometry of CD25 and Foxp3 expression on cells generated from McDC- or MpDC-primed L-DP CD69^hi thymocytes as described in panel A (top panels) and on the CD25^hi-enriched population isolated from them by magnetic sorting (bottom panels). (C) Suppressive function of CD25^hi cells isolated as in panel B was assessed by [³H]-thymidine incorporation as described in Figure 2D at the indicated CD25^hi/T-cell ratios Data are mean ± SD (cpm) of triplicate cultures from a representative experiment. **P < .01. (D) IL-10 and TGF-β production by L-DP CD69^hi thymocytes cultured either in the absence or presence of McDCs or MpDCs as in panel A was assessed by ELISA. Data are mean ± SD (pg/mL) of triplicates for a representative experiment of 3. *P < .05. **P < .01. (E). Suppressive function of CD25^hi cells generated from L-DP CD69^hi thymocytes on MpDC or McDC priming and isolated as in panel B was assessed by [³H]-thymidine incorporation at a 1:2 CD25^hi/T-cell ratio either in the absence or presence of anti-IL-10 plus anti-IL-10R blocking Abs or TGF-βRI inhibitor. Isotyped-matched Abs and dimethyl sulfoxide carrier were used as controls. Data represent fold inhibition of PBCD4 T-cell proliferation normalized to cpm values obtained in cultures performed in the absence of blocking reagents. Data are mean ± SEM values of triplicate cultures from a representative experiment. **P < .01.