Differentiation of nTregs from L-DP CD69^hi thymocytes primed with MpDCs or McDCs involves CD80 and CD86 coreceptors. (A) Flow cytometric analysis of ICOS versus CD25 expression on nTregs derived from L-DP CD69^hi thymocytes primed with McDCs or MpDCs as in Figure 4. (B) Flow cytometry analysis of ICOS and CD28 expression on L-DP CD69^hi and L-DP CD69^lo primary thymocytes. Numbers indicate percentages of positive cells. MFI values of CD28 expression are indicated. (C) ICOSL expression analyzed by flow cytometry on thymic pDCs and cDCs either ex vivo-isolated or after maturation in vitro. Numbers indicate percentages of positive cells. Results of one of 3 experiments are shown. (D-E) Effect of blocking anti-ICOSL, anti-CD80, anti-CD86, or both anti-CD80 and anti-CD86 mAbs in the generation of Tregs from MpDC- or McDC-primed L-DP CD69^hi thymocytes. Generation of CD25⁺Foxp3⁺ nTregs was analyzed by flow cytometry at day 7. (D) Data are mean ± SEM of relative numbers of CD25⁺Foxp3⁺ cells normalized to numbers in control cultures with isotype-matched control antibodies (CAb) (n = 3). *P < .05. **P < .01. (E) Flow cytometry analysis of CD25⁺Foxp3⁺ cell generation of 1 representative experiment of 3 in panel D.