IL-21 stimulation does not affect survival and does not alter cytokine production and secretion in pDCs upon TLR activation. (A) Freshly isolated human thymic pDCs were cultured with and without TLR7 agonist R848 (10 μg/mL) in the presence or absence of IL-21 (25 ng/mL) for 4 days and subsequently stained with an annexinV-conjugated APC antibody and 7-AAD. The percentages of annexinV⁺7-AAD^−/+ early and late apoptotic cells were analyzed by flow cytometry. Numbers represent percentages of cells in the indicated gates. (B) Freshly isolated pDCs were cultured overnight with and without CpG–A (10 μg/mL) in the presence or absence of IL-21, as indicated. Cells were incubated with GolgiPlug for the last 4 hours and analyzed by flow cytometry after intracellular staining using a PE-conjugated antibody directed against IFN-α[2b] protein. CD45RA expression was analyzed to confirm the presence of pDCs. Numbers represent percentages of cells in the indicated gates, which were set on the basis of an IgG-PE isotype control antibody. (C,D) Freshly isolated pDCs were cultured in the presence of CpG-B (10 μg/mL) (C) or R848 (10 μg/mL) (D) with IL-21 (black bars) or without IL-21 (white bars) for 20 hours. Culture supernatants were analyzed for the presence of IL-6 and TNF-α by CBA using flow cytometry. One representative experiment out of 2 is depicted.