Anti human il 17a

Enzyme-linked immunosorbent spot (ELISPOT) assays were used to identify PBMCs secreting IFN-γ and IL-17. Multiscreen HTS HA Opaque 96-well filtration plates (Millipore) were coated with mouse anti-human IFN-γ antibody (BD Biosciences, 2 μg/ml) or IL-17A antibody (eBioscience, 5 μg/ml) and blocked with RPMI/10% FBS. Cells were not stimulated or were stimulated with 1 μg/ml pooled hemagglutinin (H) or N overlapping peptides, 5.8 μg/ml MeV-infected Vero cell lysate (Advanced Biotechnologies) or 5 μg/ml concanavalin A. Freshly isolated PBMCs (10⁵) were added to wells stimulated with ConA, H or N peptides and 5 × 10⁵ PBMCs were added to non-stimulated and MeV lysate wells and incubated at 37 °C/5%CO₂ for 40–42 h. Biotinylated anti-human IFN-γ (Mabtech, 7-B6-1; 1 μg/ml) or anti-human IL-17A (eBioscience, 64DEC17; 2 μg/ml) antibody was added for 2 h. Plates were developed with avidin-horseradish peroxidase (BD Biosciences; 1:2000) and diaminobenzidine substrate. Plates were read and analyzed using an ImmunoSpot plate reader and ImmunoSpot 5.0 software (C.T.L.). Data are presented as total spot-forming cells (SFCs)/10⁶ PBMCs. MeV-specific SFCs stimulated to secrete cytokines ex vivo were determined by subtracting the in vivo activated (no in vitro stimulation) SFCs from the total SFCs at each time point. All assays were done in duplicate.

Neutralizing antibodies against proinflammatory cytokines (anti-human IFN-γ (eBioscience, 14-7318-85) and anti-human IL-17A (eBioscience, 16-7178-85)) were used during allogeneic antigen stimulation. Anti-HLA-ABC (BioLegend, 311412), anti-HLA-DR (BioLegend, 307612), and anti-HLA-DQ (BioLegend, 361502) antibodies were used to block ADSC surface antigens. All of these antibodies were added at a concentration of 1 μg/ml every 7 days when exchanging the medium.