Tlrl 2216

pDC-Gen2.2 cells were cultured at 1–2 10^6 cells/ml in complete RPMI 1640 on the MS-5 feeder cells. Before the experiment, the non-adherent fraction of the culture was harvested, pelleted at 1500 rpm and re-suspended in a fresh medium. 50,000 cells were plated in 96-well round bottom plates and stimulated with 0.25μM CpGA (Invivogen, tlrl-2216), 0.25μM CpGB (Invivogen, tlrl-2006), 3.75μg/ml R848 (Invivogen, tlrl-r848), 0.5μg/ml LPS (Invivogen, tlrl-b5lps) or were left unstimulated (Mock) in total volume of 150μl. The stimulation assay was performed with and without 5μM RBV (1221 ng/ml) (Sigma, R9644). At time points (indicated in figures), the culture supernatants were analyzed for IFNα by ELISA and cells were harvested for RNA isolation and qRT-PCR analysis of gene expression. Where total PBMCs were used, 50,000 cells were stimulated with 0.25μM CpGA +/- RBV and analyzed as above.

Buffy coats from healthy donors were used for isolation of peripheral blood mononuclear cells by density-gradient centrifugation (650g, 22°C, 30 minutes) (catalog MST00T41004; Lymphosep). pDCs were obtained with the CD304 (BDCA-4/Neuropilin-1) MicroBead Kit, human, isolation kit (catalog 130-090-532; Miltenyi Biotec) at a purity greater than 95%. pDCs were cultured in RPMI-1640/l-glutamine supplemented with 10% v/v heat-inactivated FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 mm HEPES (Gibco) for up to 18 hours. Prior to stimulation with NETs (25% v/v NET-containing supernatants or cleaved IL-33–containing supernatants) and CpG-A (0.1 μM; catalog tlrl-2216; Invivogen), pDCs were pretreated with aST2L (3 μg/mL; catalog AF523; R&D) and FcR blocking reagent (catalog 130-090532; Miltenyi Biotec) to minimize any IC carryover effect and nonspecific binding of aST2L. IC SLE NET-containing supernatants were pretreated with goat anti–human IL-33 antibody (4 μg/mL; catalog AF3625; R&D) or left untreated at 37°C for 45 minutes before their administration to pDC cultures. pDCs were also pretreated with cytochalasin D (5 μg/mL; catalog C2618; Sigma-Aldrich) or chloroquine (4 μM; Plaquenil, ATC code 8P01BA02; Sanofi Aventis) for 30 minutes to block endocytosis and TLR trafficking, respectively.