Milliplex analytes

The levels of 41 cytokine molecules were measured using Luminex based bead array, the MILLIPLEX^® MAP Human Cytokine/Chemokine panel on LuminexxMAP^® platform using a magnetic bead format (MILLIPLEX^® Analytes, Millipore, MA, USA,) for the following biomarkers: EGF, FGF, Eotaxin, TGF-α, G-CSF, Flt, GM-CSF, Fractalkine, IFN-α2, IFN-γ, GRO, IL-10, MCP, IL-12P40, MDC, IL-12P70, IL-13, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, TNF-β, VEGF, PDGF-AA, PDGF-BB, and RANTES. In brief, 25 µL plasma samples were tested for the levels of the cytokines, using the multiplex immunoassay containing fluorescent labeled beads conjugated with specific monoclonal antibody for the target molecule, according to the manufacturer’s recommendations [14 (link),20 ]. Appropriate standards including 41 beads and quality control (provided in the kit) were run with the test sample plate. The software determines and interprets the data and concentrations of analytes in samples determined from the standards run in the test. Samples were considered for analysis using the standards that were run in each of the plates. Each run contained appropriate quality controls run in duplicates and results were interpreted from the standard using Bioplex 200 system (Bio-Rad, Hercules, CA, USA).

The concentrations of 13 ICPs were measured on MAGPIX (MERCK, Darmstadt, Germany) with a Luminex-based bead array, the MILLIPLEX^® MAP Human Immuno-Oncology Checkpoint Protein Panel 1 (MERCK, Darmstadt, Germany), and the MILLI-PLEX^® MAP Human Immuno-Oncology Checkpoint Protein Panel 2 (MERCK, Darmstadt, Germany) on the Luminex xMAP^® platform using a magnetic bead format (MILLIPLEX^® Analytes, Millipore, MA, USA) for the following biomarkers: sTIM-3, sCTLA-4, sCD80, sCD86, sCD28, sPD-1, sPD-L1, sPD-L2, sHVEM, sCD112, sCD155, sLAG-3, and sVISTA (B7-H5). For each sample, 25 µL of serum was used to assess the concentration of soluble ICPs and their ligands. All procedures were performed according to the manufacturer’s recommendations. Quality assurance was maintained through the inclusion of appropriate standards and quality controls provided in the kits. Each run incorporated relevant quality controls, and results were calculated using the xPONENT system (MERCK, Darmstadt, Germany). For specific data regarding the upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ), please refer to Supplementary Materials, Table S1.