IFN-α–producing plasmacytoid cells differentiate into DCs after culture in IL-3.

Cytospins of sorted CD11c⁺B220⁺ cells were stained using antibodies to MHC class II, a biotinylated goat anti–rat IgG antibody, and then by streptavidin-alkaline phosphatase and were developed using Fast Red substrate. These data demonstrate that (A) directly after sorting, cells are round and lack DC attributes and that (B) after 2 days in culture with rmIL-3 and activating anti-CD40 antibodies, many cells develop DC morphology with long protrusions and strong MHC class II expression. (C) Cytospins of CD11c⁺B220⁺ DCs were infected for 24 hours with HSV (10 PFU/cell) and then stained with isotype control or (D) rat anti–mouse IFN-α₂ antibodies and were developed using Fast Red substrate. (E) Transmission electron microscopy of FACS-sorted CD11c⁺B220⁺ DCs. White bar represents 2 μm. Original magnification, × 2500. (F) Irradiated CD11c⁺CD11b⁺ myeloid DCs (filled triangles) CD11c⁺B220⁺ pDC (filled circles) and CD11c⁺CD8α⁺ lymphoid DCs (open circles) were cultured with total allogeneic CD4⁺ T cells (200 000/well) for 6 days in triplicate in 96-well, round-bottomed plates, and³H-thymidine was added for the last 18 hours of culture. Cells were harvested, and proliferation was measured in a scintillation counter. Data are representative of 3 experiments yielding comparable data.