Mmhb 3

Adherent CD14+ monocytes were infected with mock, UV irradiated or TB40/E strain as described above in the presence of excess neutralizing anti-Human Interferon α [3µg; Clone: MMHA-6; EC50 20ng/ml (40 (link))], anti-Human Interferon β [4µg/ml; Clone : MMHB-3 (41 (link))] (both PBL Assay Science, USA) and Ultra-leaf anti-human Interferon γ [10µg/ml; Clone: B27 (42 (link))] (BioLegend, London, UK) antibodies or Mouse IgG1 isotype control (Clone: 11711) (R & D Systems). The secretomes generated were harvested after 10 days, clarified and then analyzed by ELISA for CXCL10 and CCL8 cytokines.

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.