Effects of IFN-α on pDC development and function. (A) FACS purification strategy of DC progenitor cells (lin⁻ CD11c⁻ B220⁻ Flt3⁺ cells) and pDCs (CD11c⁺ CD11b⁻ B220⁺ cells) from BM, as well as cDCs (CD11c⁺ CD11b⁺ B220⁻ cells) from spleen. (B) Proportion of pDCs (top left quadrant) and cDCs (bottom right quadrant) after 4 days of culture of DC progenitors in Flt3L (FL, 50 ng/mL), GM-CSF (GM, 25 ng/mL), IFN-α (IFN, 300 U/mL), or the indicated combinations. Results were gated on the CD11c⁺ population (not shown); B220 and CD11b analysis is shown. Data represent 3 independent experiments. (C) pDCs were generated from total BM cells cultured in Flt3L, Flt3L + IFN-α, GM-CSF + IFN-α, or Flt3L + GM-CSF + IFN-α as indicated in panel B for 4 days. pDCs were then purified by FACS and stimulated with or without 5 μM CpGA overnight, as indicated. Culture supernatants were assayed by ELISA. Error bars represent SEM of values obtained from triplicate wells. (D) pDCs were purified from total BM and treated with or without IFN-α for 24 hours in the presence of Flt3L. pDCs were then stimulated with CpGA and analyzed for IFN-α secretion as indicated in panel C. (E) Representative surface staining of pDCs purified from Flt3L alone (dashed line) or Flt3L + IFN-α cultures (solid line) with antibodies to PDCA-1, Siglec-H, or CCR9. Shaded area represents isotype control staining. Data represent 3 independent experiments. (F) Gene expression in DC progenitors stimulated with Flt3L, Flt3L + IFN-α, GM-CSF, or GM-CSF + IFN-α for 4 days, as indicated, or immediately after purification (NT). Data are the mean ± SEM of 3-5 independent experiments. (G) Annexin V and 7-amino-actinomycin D (7-AAD) staining of DC progenitors cultured 4 days with Flt3L (dashed line) or Flt3L + IFN-α (solid line). Shaded area represents isotype control staining. Data represent 3 independent experiments. (H) Phenotype of FACS-purified BM pDCs and splenic cDCs. After purification, pDCs and cDCs were cultured 4 days in the presence of Flt3L + IFN-α and analyzed by flow cytometry (middle panels) or analyzed immediately (left panels); pDC and cDC frequencies within the CD11c⁺ population are shown. (Right panels) Purified DCs were stained with CFSE, cultured 4 days in Flt3L + IFN-α, and analyzed by flow cytometry.