Mmha 2

Purified NK cells were plated in a 96-well round-bottom plate (2 × 10⁶ cells/mL) and in vitro differentiated- or adult pDC were added in a NK:pDC ratio of 10:1. pDC were stimulated by adding a TLR-9 ligand (CpG-A ODN2216, 10 µg/mL, InvivoGen, San Diego, CA, USA) or a TLR-7 ligand (Imiquimod, 0.8 µg/mL, Sigma), and NK/pDC co-cultures were incubated overnight at 37 °C and 5% CO₂ atmosphere. Similarly, NK cells were co-cultured with mo-DC, peripheral blood or in vitro generated mDC. mDC and mo-DC were stimulated by adding a TLR-3 ligand (poly-IC, 10 µg/mL, Sigma). Unstimulated NK cells or NK cells cultured with unstimulated pDC were used as negative controls. NK cells stimulated with IFN-α (1000 IU/mL) were used as a positive control for each experiment. Increasing doses of IL-2 (200–20,000 IU/mL; Novartis Pharmaceuticals Canada, Dorval, Quebec, Canada) were also used to stimulate NK cells. IFN-α signaling neutralization assays were performed by incubating NK cells and pDC with neutralizing antibodies (anti-IFN-α/β receptor chain2 and anti-IFN-α) (20 µg/mL, MMHAR-2 and MMHA-2, respectively, PBL Assay Science, Piscataway, NJ, USA) for 30 min prior to the addition of TLR ligands or IFN-α. Blocking antibodies were kept in culture medium overnight.

NK cells were plated in a 96-well round-bottom plate (2×10⁶ cells/mL) and incubated overnight without stimulation or with human IFN-α (1000 UI/ml) or co-cultured with pDCs (Lin⁻HLA-DR⁺CD123⁺) (ratio NK:pDC of 10:1) and CpG-A ODN2216 (10μg/ml) (Invivogen, San Diego, CA) at 37°C in a 5% CO₂ atmosphere. Of note, pDCs and NK cells were not prepared from the same donor. In some experiments, 24-well microplates equipped with a transwell insert (0.4μm, Greiner bio-one) were used to prevent direct contact between NK cells and pDCs. For IFN-α signaling neutralization assays, NK cells were incubated for 30 minutes with neutralizing anti-IFN-α/β receptor Chain2 and anti-IFN-α antibodies (20μg/ml each) (MMHAR-2 and MMHA-2 respectively; PBL Assay Science, Piscataway, NJ), prior to the addition of IFN-α or pDCs and overnight incubation.

Autocrine and paracrine effects of the IFN-I response were neutralized (Fig 2) by treating mDCs with cocktails of neutralizing antibodies. The anti-IFN cocktail contained antibodies neutralizing IFNAR (MMHAR-2, 5 μg/mL), IFNα (MMHA-2, 2.5 μg/mL), and IFNβ (MMHB-3, 2.5 μg/mL), all from PBL Assay Science. In the control condition, the cocktail contained corresponding control isotype antibodies, IgG1 (5 μg/mL) and IgG2a (5 μg/mL), both from ThermoFisher Scientific. One dose of antibody cocktail was administrated to the cells at the time of infection. Half a dose was added to the cell culture 1 and 3 dpi. Cells were infected at a MOI = 0.1 by wild type or mCherry-expressing MOPV or LASV. For MOPV_WT- and LASV_WT-infected mDCs, small volumes of supernatant were harvested at various times post-infection and titrated. For mCherry-expressing MOPV and LASV, mCherry fluorescence was measured by fluorescence microscopy with Leica DMIRB.