Peripheral blood immune correlatives
SEPARATION
Peripheral blood was collected at baseline (pre-treatment), C2D1, and post-cycle 3 (C4D1 or C6D1) for flow cytometry analysis. Peripheral blood was obtained using heparin collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 6 h using Ficoll-Paque Separation according to manufacturer’s instructions (GE Healthcare). PBMCs were cryopreserved in 10% DMSO, 90% FBS and thawed rapidly for flow cytometry analysis. Single-cell suspensions were prepared on ice in 2% FBS in PBS. Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences). Samples were acquired using a Cytek Aurora spectral cytometer with user settings established by Spectroflo QC beads. Unmixing and compensation were performed in Spectroflo software using a mix of reference controls from either single stained PBMCs or single stained OneComp compensation beads (eBioscience). Samples were stained with fluorescently tagged antibodies (Supplemental Table 4). Antibodies were titrated for optimal signal-to-noise ratio prior to use. Flow cytometry analysis was performed using Flowjo vX, and the CATALYST R package was used for FlowSOM analysis and UMAP projections [