Ifn α2b intron a

EBV–B cell lines were cultured in RPMI (Invitrogen), and SV40-fibroblasts and 293T HEK cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Saimiri T cells were cultured in a 1:1 mixture (by volume) of RPMI and Panserin 401 (PAN-Biotech GmbH), supplemented with 10% FBS, 350 µg/ml glutamine, 100 µg/ml gentamycin, and 10 U/ml hIL-2 (Roche). PBMCs were isolated from blood by Ficoll-Hypaque density gradient centrifugation (GE Healthcare) and cultured at a density of 10⁶ cells/ml in RPMI supplemented with 10% FBS. Stimulations were performed, at the doses stated and for the indicated times, with IL-12 (R&D Systems), BCG (donated by C. Nathan, Weill Cornell Medical College, New York, NY), IL-18 (R&D Systems), PMA/ionomycin (Sigma-Aldrich), IL-2 (BD), IL-23 (R&D Systems), IFN-γ (Imukin; Boehringer Ingelheim), IL-27 (R&D Systems), IFNα-2b (Intron A; Schering Plough), IFNβ-1b (PeproTech), LPS (Sigma-Aldrich), IL-21 (R&D Systems), IL-29 (R&D Systems), IL-28B (R&D Systems), IL-10 (R&D Systems), IL-6 (R&D Systems), hyper–IL-6 (hIL-6; donated by S. Rose-John), and LIF (EMD Millipore). hIL-6 was prepared as described elsewhere (Fischer et al., 1997 (link)).

IAV (A/H1N1/CA/2009) was provided by C. Basler (Georgia State University, Atlanta, GA). For IAV infection, 4 × 10⁴ SV40-fibroblasts were plated in poly-L-lysine–coated individual wells of 48-well plates 1 d before infection. We used IAV at a MOI of 1, 5, or 10 to infect cells in PBS supplemented with 0.3% BSA, 1 mM CaCl₂, 1 mM MgCl₂, and penicillin/streptomycin. After 1 h, the cells were washed twice with Dulbecco's PBS and transferred to 200 µl of DMEM supplemented with 0.3% BSA, 0.1% FBS, penicillin/streptomycin, and 1 µg/ml of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)–treated trypsin (Sigma-Aldrich), in which they were maintained until harvesting at 1, 8, 12, 24, or 36 h. Where indicated, cells were pretreated with IFN-α2b (Intron A; Schering-Plough) at a concentration of 10⁴ IU/ml for 18 h before infection. Viral replication was assessed by determining viral titers in plaque assays on Madin-Darby canine kidney cells. 10-fold serial dilutions of viral suspensions were allowed to adsorb onto the cells for 1 h at room temperature. The cells were washed twice with DPBS, and incubated in agar overlay containing MEM, 0.5% dextran, 2.5% NaHCO₃, 1 µg/ml TPCK-treated trypsin, and 0.8% agar.

For infection with viruses, 5 × 10⁴ SV40-fibroblasts were plated in 48-well plates 1 d before infection. We used EV30 and EV71 at a MOI of 0.1 and HSV-1 at a MOI of 0.001 to infect cells in 500 µl DMEM supplemented with 10% FBS. After 2 h, the cells were washed once with Dulbecco’s PBS and transferred to 500 µl DMEM supplemented with 2% FBS, in which they were maintained until harvesting at the various time points indicated. Where indicated, cells were treated with IFN-α2b (Intron A; Schering-Plough) at a concentration of 10⁴ IU/ml before or after infection at various time points. Viral replication was assessed by determining viral titers on Vero cells, according to the Reed and Muench calculation (Zhang et al., 2007 (link)).