IL-3–differentiated CCR6⁺ CCR10⁺ blood pDCs preserve their ability to produce high levels of IFN-α, TNF-α, and CXCL10 in response to flu virus. (A) Freshly purified blood pDCs were cultured in IL-3 for different time periods before flu virus and CpG-A ODN2336 were added for another 24 hours. IFN-α was measured in the supernatants by ELISA. Data are mean ± SD of duplicate wells of culture and are representative of 3 independent experiments. (B) Intracellular production of IFN-α, TNF-α, and CXCL10 assessed by flow cytometry. pDCs were cultured in IL-3 for 96 hours and then activated with flu virus for an additional 6 hours. Percentages of each population are indicated in dot plots, and data are representative of 3 independent experiments.