Ifn α2b

MUTZ-3 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ], Braunschweig, Germany) were maintained by seeding 4 × 10⁵ cells every three days in MEM-α medium (Gibco, Life Technologies Co., Carlsbad, CA, USA), supplemented with 20% FBS (Gibco), 5 ng/mL human recombinant GM-CSF (Peprotech, Rotterdam, The Netherlands). MUTZ-3-derived IL-4- and IFN-DCs were induced by culturing MUTZ-3 cells (3 × 10⁵ cells/mL) in AIM-V medium (serum-free medium, Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 250 ng/mL GM-CSF (Peprotech, The Netherlands), 12 ng/mL TNF-α (Peprotech), 2nM Mitoxantrone (Sigma-Aldrich, Zwijndrecht, The Netherlands), and either 10 ng/mL IL-4 (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) or 5 ng/mL IFN-α2b (Peprotech) for the induction of immature MUTZ-3-derived IL-4- and IFN-DCs. After 3 days, the M-imIL4-DC or M-imIFN-DC were harvested, counted, and maturated by seeding 3 × 10⁵ cells/mL M-imIL4-DC or M-imIFN-DC in AIM-V medium, supplemented with 120 ng/mL TNF-α (Peprotech), 0.75 ng/mL IL-1β (Peprotech), and 1 μg/mL PGE2 (Kyowa Pharma Chemical Co., Ltd., Toyama, Japan). Either M-mIL4-DC or M-mIFN-DC were harvested after 24 h and used for subsequent experiments. Immature and mature MUTZ-3-derived IL-4- and IFN-DCs are referred to as “M-imIL-4-DC”, “M-imIFN-DC”, “M-mIL-4-DC”, and “M-mIFN-DC”, respectively.

PBMCs were cultured in 5 mL round-bottom polystyrene tubes at a concentration of 2 × 10⁶ cells/mL in 10% FBS RPMI at 37 °C with 5% CO₂. 1 mL of cell culture was used per sample. For analysis of purified pDC, 100,000 pDC in 100 µL of 10% FBS RPMI per sample were cultured in a round-bottom 96-well plate. PBMC were stimulated with a multiplicity of infection (MOI) of 1 of HSV-1 and IAV and purified pDCs were stimulated with an MOI of 10 (which yields a similar concentration of virus volume:volume as in the PBMCs). PBMCs were stimulated with 500 ng p24 equivalents/mL of HIV-MN. 200 ng/mL of ultrapurified LPS (Invivogen, San Diego, CA, USA) and 5 ug/mL CpGA (ODN 2336) and CpGB (ODN 2006) (Invivogen) were used to stimulate both PBMCs and enriched pDCs. 10 μM R848 (Resiquimod) was used to stimulate PBMCs (Invivogen). Supernatants were collected from PBMC cultures stimulated with LPS and kept at −20 °C until use. pDCs and PBMCs stimulated with supernatant were cultured in media from autologous PBMCs. For inhibition of LPS binding by polymyxin B, supernatants were pre-treated with 1 μg/mL of polymyxin B (Invivogen) for a half hour before use. For cytokine stimulation, PBMCs were cultured with 10, 100, 1000, 5000, or 10,000 IU IFNα2b or 0.2, 1, 10 ng/mL of IL-10 or TNFα (PeproTech, Rocky Hill, NJ, USA).