Il 12

Agonistic mouse anti-CD40 (clone FGK4.5/FGK45, BioXCell, Lebanon, NH, USA), anti-CD154/CD40L (MR-1, BioXCell), anti-NK1.1 (PK136, BioXCell), anti-CD19 (1D3, BioXCell), anti-IL12 (R1-5D9, BioXCell), and anti-IFNγ (XMG1.2, BioXCell) antibodies were diluted to 200 µg in PBS and administered 24 h prior to viral challenge. Anti-CD4 (YTS 177, BioXCell) and anti-CD8 (2.43, BioXCell) antibodies were diluted to 200 µg/100 µL in PBS, pooled to a total of 400 µg (200 µL), and administered 24 h prior to downstream use. Isotype IgG controls (BioXCell) were used at equivalent doses in all experiments. All antibodies were administered in a final volume of 100–200 µL via intraperitoneal (i.p.) injection. Cytokines used in this study include IL-12 (StemCell Technologies, Vancouver, CA, USA, catalog #78028.1) and IFN-γ (StemCell Technologies, catalog #78020.1), which were given at 5 µg in 100 µL of PBS via i.p. injection 24 h prior to viral challenge. For the disruption of IL-12 production, the inhibitor apilimod (MedChemExpress, Monmouth Junction, NJ, USA, catalog #HY-14644) was administered via i.p. injection at a concentration of 2.5 mg/kg. The drug was administered on day −1, day 0, and day 1 post-infection with rVSV-EBOV GP.

Human PBMC (2 × 10⁵ cells/well) were stimulated with 1 ng/ml of IL-12 (R&D Systems) and 1 ng/ml of LPS in the presence of an IL-1R antagonist at 100 nM (anakinra, Amgen/Biovitrium), an IL-18 antagonist at 33 nM (IL-18BPa-Fc, R&D Systems) or a control IgG1 at 33 nM (MedImmune, UK). Alternatively, NK cells were isolated from PBMC by negative selection (Stem Cell Technologies) and plated (1 × 10⁴ cells/well) in medium containing 1 ng/ml IL-12. Supernatants taken from HRV-activated NHBE cells or LPS-activated monocytes were mixed with antagonists indicated above and then added to NK cells at a dilution of 1:4. For transwell and co-culture experiments, 5 × 10⁴ NK cells were seeded either in the well along with 5 × 10⁴ monocytes (co-culture) or in an insert (transwell). Supernatants were collected after 24 h of incubation to measure IFNγ production.