Cytokine release

Peripheral blood mononuclear cells (PBMC) collected from 5 to 8 donors were added to tissue culture wells (200,000/well) precoated with SRK-181, positive control (anti-CD3/anti-CD28 antibody cocktail), or vehicle (IgG) control at concentrations of 0.8 to 100 μg/mL. Cells were then incubated at 37 °C for 48 hours prior to supernatant collection. Supernatant was measured in triplicate by Luminex multiplex assay (Luminex) for interferon γ (IFNγ), interleukin 2 (IL-2), interleukin 1β (IL-1β), tumor necrosis factor α (TNFα), C-C Motif Chemokine Ligand 2 (CCL2), and interleukin 6 (IL-6). Cell culture supernatant was diluted 1:1 in Luminex Assay Buffer (Luminex). Logistically fit standard curves were used to calculate the concentration of each cytokine per well and a minimum of 5 donors were analyzed for each analyte. If variability across triplicates was greater than 10-fold that data point was flagged, and if an analyte had more than 2 flagged data points for a particular donor, then it was removed from analysis for that analyte.