Luminex
PBMCs from patients were thawed and rested overnight at 37°C in complete RPMI (cRPMI, table S7). 96-well flat bottom plates were coated with 1 μg/mL of anti-CD3 (UCHT1, #BE0231, BioXell) in PBS at 4°C overnight. The next day, cells were collected and plated at 1 × 105/well in 100 μl in duplicate. 2 μg/mL of anti-human CD28/CD49d was added to the wells containing plate-bound anti-CD3 (Clone L293, 347690, BD). PBMCs were stimulated or left unstimulated for 16 hours, spun down (1200 rpm, 10 min) and 85 μL/well of supernatant was collected. Plasma from matched subjects was thawed on ice, spun (3000 rpm, 1 min) to remove debris, and 85 μl collected in duplicate. Luminex assay was run according to manufacturer’s instructions, using a custom human cytokine 31-plex panel (EMD Millipore Corporation, SPRCUS707). The panel included: EGF, FGF-2, Eotaxin, sIL-2Ra, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12P40, IL-12P70, IL-13, IL-15, IL-17A, IL-1Ra, HGF, IL-1β, CXCL9/MIG, IL-2, IL-4, IL-5, IL-6, IL-7, CXCL8/IL-8, CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, RANTES, TNF-α, and VEGF. Assay plates were measured using a Luminex FlexMAP 3D instrument (Thermofisher, Cat#APX1342).
Data acquisition and analysis were done using xPONENT software www.luminexcorp.com/xponent/). Data quality was examined based on the following criteria: The standard curve for each analyte has a 5P R2 value > 0.95 with or without minor fitting using xPONENT software. To pass assay technical quality control, the results for two controls in the kit needed to be within the 95% of CI (confidence interval) provided by the vendor for >25 of the tested analytes. No further tests were done on samples with results out of range low (<OOR). Samples with results that were out of range high (>OOR) or greater than the standard curve maximum value (SC max) were not tested at higher dilutions without further request.