Identification of intermediate CLP-derived pDC precursors. (A) The transient pDC precursor. CLPs were cultured with FL for 1-2 days. CLP-derived cells were analyzed for expression of the DC markers BST-2 and CD11c. (B) pDC products of this precursor. CD11c⁻BST-2⁺ cells were sorted after 2 days of culture, recultured with FL, and analyzed after 2 days for the presence of CD11c⁺BST-2⁺ pDCs. (C) B-cell products of this precursor. CD11c⁻BST-2⁺ precursors were sorted and cultured on OP9 stromal cells with IL-7 and FL. Progeny were analyzed after 7 days for the presence of CD19⁺ B cells. (D) An equivalent precursor population in BM. BM from C57Bl/6 was depleted for a selection of lineage antigens (CD3, CD11b, CD19, TER119, and Ly6G) and then BST-2⁺CD11c⁻IL-7Rα⁺ cells were sorted from the lin⁻ BM. (E) In vivo products of this BM precursor. The sorted cells were adoptively transferred into lethally irradiated CD45.1 recipients. Six to 14 days after transfer, recipient spleens were analyzed for the presence of CD45.2⁺ donor-derived cells and within the donor-derived cells for the presence of Siglec H⁺CD11c^int pDCs, Siglec H⁻CD11c^high cDCs, CD24⁺CD19⁺ B cells, CD49b⁺CD161c⁺ NK cells, and CD3⁺ T cells. Results represent the peak responses at day 6 for DCs and at day 14 for lymphoid cells. Results are representative of 5 (A), 3 (B), or 2 (C-E) independent experiments.