pDCs and cDCs from human thymus mature in response to different stimuli. (A) CD123 versus CD13 flow cytometry analysis of thymocyte suspensions depleted of T cells and monocytes/macrophages shows the presence of CD123⁺CD13⁻ (R1), CD123^loCD13⁺ (R2), and CD123^loCD13⁻ (R3) cells (left histogram). Isotyped-matched control Abs were used to define background staining. Isolation of pDCs and cDCs was performed by magnetic sorting based on reciprocal CD123 and CD13 expression. Postsorting phenotype of one of 10 experiments is shown (right histogram). (B) Flow cytometry analysis of BDCA2, CD11c, CD86, CD80, HLADR, CD45RA, and CD45RO expression in the populations electronically gated as defined in panel A (shaded histograms). Background staining was defined with isotype-matched Abs (empty histograms). Results of 1 of 4 experiments are shown. (C) Cell recoveries of either pDCs cultured for 24 hours with TSLP or with CD40L plus IL-3 or cDCs cultured with TSLP. Absolute DC numbers were normalized to 10⁵ input cells. Data (mean ± SD) correspond to 3 independent experiments. *P < .05. (D) pDCs do not mature in response to TSLP but mature in the presence of CD40L plus IL-3. Flow cytometry analysis of CD80 and CD86 expression (shaded histograms) of pDCs and cDCs cultured as described in panel C. (E) Bright-field microscopy of thymic pDCs cultured with CD40L plus IL-3 for 24 hours (top, original magnification ×40), showing an amplification of the lower right inset (bottom).