Delfia readout

3–5 × 10⁵ PBMCs were seeded in 96-well plates and stimulated with MEDI9197 (in DMSO), CpG (ODN 2395 VacciGrade™ in H₂O, Invivogen), STING agonist (2′3’-c-di-AM (PS)2 (Rp,Rp) VacciGrade™ in H₂O, Invivogen), Resiquimod (in DMSO, Sigma-Aldrich), or Imiquimod (in DMSO, R&D Systems) at the indicated concentrations for 24 h at 37 °C. Flow cytometry was performed by standard procedures with intracellular fixation buffer from eBioscience. See Additional file 1 for a list of Flow cytometry antibodies. IFN-gamma was analysed by ELISA (Standard: human recombinant IFNγ (R & D). Capture antibody: Anti-human IFNγ (Clone NIB42, Pharmingen). Detection antibody: Biotinylated anti-human IFNγ (Clone 4S.B3, Pharmingen) or MSD (Mesoscale Diagnostics). IFN-alpha was analysed by ELISA (MABTECH) and IL-12p70 by MSD (Mesoscale Diagnostics). For ELISAs, DELFIA readout (Perkin Elmer) was used.

2 × 10⁵ human PBMCs were seeded in 96-well plates and treated with MEDI9197 or DMSO for 2.5 h, then incubated with 10 μg/mL PHA-L (Roche) for 45.5 h at 37 °C. IL-5 was assessed in supernatants using human IL-5 ELISA Set (BD OptEIA,) with DELFIA readout (Perkin Elmer).