In vitro–generated pDCs produce IFN-α upon stimulation with either stimulatory CpG or virus. CD34⁺CD1a^– thymic progenitors were cultured on OP9-Jag1 cells. (A) After 6 days of coculture, cells were stimulated with CpG (2216) (10 μg/mL) for 16 hours or left unstimulated. Flow cytometric analysis was performed after intracellular staining of IFN-α and surface staining of CD123. (B-C) After 5 days of coculture, cells were stimulated for 72 hours with (B) CpG (2216) (1 μg/mL) or (C) supernatant of HIV-1_LAI–infected PM1 cells (2 ng or 10 ng p24 per milliliter) in the presence or absence of neutralizing antibodies against type I IFNs (10 μL/mL). Control CpG (2243) and supernatant of uninfected PM1 cells served as controls. Flow cytometry was performed after intracellular staining of MxA. (B) Left histogram: MxA protein expression of cultures treated with CpG (2216) (MFI, 114), exogenously added IFN-α (1000 U/mL; MFI, 148) (positive control), control CpG (2243) (MFI, 11), or unstimulated cells (MFI, 12) (negative controls). Right histogram: MxA levels after preincubation with neutralizing antibody to type I IFNs. (C) MxA protein expression in cultures stimulated with 10 μL or 2 μL of control virus supernatant (MFI, 10 and 10, respectively) or HIV-1_LAI supernatant in the absence (MFI, 146 and 169, respectively) or presence of neutralizing antibodies (MFI, 85 and 75, respectively). Results are representative of 3 independent experiments.