pDCs produce type-I and type-III interferon, but not TNF-α, upon stimulation of cGAS-STING pathway. (A)Purified pDCs were stimulated for 3, 6, 9, 12, and 15 hr. with cyclic GMP-AMP (cGAMP), HSV and the TLR9 agonist CpG-A and the expression of IFN-α and IFN-λ were tested via flow cytometry. BFA was added 2 hrs prior to each time point. Cells were stained for pDC markers, fixed, permeabilized, stained for intracellular cytokines and acquired by flow cytometer. Data are from a representative experiment from two individual donors. (B) Purified pDCs (0.25 × 106 cells/ml) were stimulated with HSV, CpG-A or lipofected with cGAMP (10 and 50 μg/ml) and supernatant was collected at each time point to perform ELISA for IFN-α and IFN-λ. Data are shown as mean ± 1 SD (n = 3 independent experiments). (C) pDCs were lipofected with ISD for 18 hours. Supernatants were collected and production of IFN-α and IFN-λ was measured by ELISA. Data are shown as mean ± 1 SD (n = 3 independent experiments). (C) pDCs were lipofected with ISD for 18 hours. Supernatants were collected and production of IFN-α was measured by ELISA. Data are shown as mean ± 1 SD (n = 3 independent experiments). (D, E) pDCs were stimulated with HSV, CpG-A or lipofected with cGAMP (50 μg/ml) or ISD (5 μg/ml) for 18 hours. Supernatants were collected and production of (D) TNF-α and (E) IFN-β was measured by ELISA. Data are shown as mean ± 1 SD (n = 3 independent experiments).