Leukocyte activation kit

Whole blood samples from peripheral venipuncture or indwelling port were collected in sterile tubes containing lithium heparin, on cycle 1 day 1 (C1D1, baseline) and again on cycle 2 day 1 (C2D1). Total PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ). 3 × 10⁶ PBMCs were labeled in PBS-1% FCS-EDTA using anti-CD3, -CD4, -CD11c, -CD25, (BD Biosciences, San Jose, CA), -CD8 (Invitrogen, Carlsbad, CA), -CD11b, -CD14, -CD45RO, -CD45RA, -CD62L (BioLegend, San Diego, CA), -CD127, -HLA-DR, -FoxP3, -PD-1, -IL-17A, or -IFN-γ (eBioscience, San Diego, CA) monoclonal antibodies, For cytokine detection, 3 × 10⁶ PBMCs were stimulated for 5 hours with Leukocyte Activation kit (BD bioscience), stained for surface markers, permeabilized using BD Fix/Perm protocol and stained intracellular cytokines. Cells were acquired using a BD LSRII flow cytometer (BD Biosciences), flow data analyzed using FlowJo (Ashland, OR) and statistics done with GraphPad Prism software (La Jolla, CA).

Fresh tumor tissues were processed into single-cell suspensions using a gentleMACS™ Dissociator with a Human Tumor Dissociation Kit (Miltenyi Biotec). Two flow cytometry panels focused on T cells and myeloid cells were applied to single-cell suspensions of blood and tumors. All cell suspensions were initially incubated with a Leukocyte Activation Kit (BD Pharmingen) at 37 °C for 5 h and then divided into two tubes (1 × 10⁶ cells/tube). Each tube was stained for viability and blocked with the Fc-block reagent (BD Pharmingen). The cells were then stained for surface markers (Additional file 1: Table S1-2). After fixation and permeabilization, cells were stained for intracellular markers (Additional file 1: Table S1-2). The limits for quadrant markers were always set based on negative populations. Cells were acquired using a Cytek Aurora cytometer and analyzed using FlowJo 10.8.0 software.