Cd15 af700

The following antibodies were used: CD45-PB, CD14-APC (both from Beckman Coulter, Brea, CA, USA, clones J33 and RMO52, respectively), CD15-AF700 and anti-ASC-PE (both from Biolegend, San Diego, CA, USA, clones W6D3 and HASC-71, respectively). Following reagents were used: phosphate-buffered saline (PBS) (Eurobio Scientific, Luxembourg, Luxembourg), Ficoll (Dutscher, Bruxelles, Belgium), RPMI HEPES (Eurobio, Les Ulis, France), penicilline/streptomycine (Clinisciences, Nanterre, France), fungizone (Cheplapharm Arzneimittel, Mesekenhagen, Germany), glutamine (Ozyme, Saint-Cyr-l’École, France), versalyse (Beckman Coulter), lipopolysaccharide (LPS) (Sigma-Aldrich, single mix of references L3137, L2637 and L3012, Saint-Louis, MO, USA), nigericin (InvivoGen, San Diego, CA, USA), water for injection (Aguettant, Lyon, France), Cytofix/Cytoperm, Fixation/Permeabilization Kit, stain buffer (both from BD Bioscience, Franklin Lakes, NJ, USA) and human AB serum (SAB) (“Etablissement Français du Sang”, Lyon, France).

At each time point, in addition to leukocyte count, we assessed immature neutrophils (CD16^low) and LOX-1⁺ MDSC (CD15⁺, CD45^dim, LOX-1⁺ polymorphonuclear cells) percentages as described by Coudereau et al. (2022) (link) and immune checkpoint inhibitor (PD-1 and TIM-3) expression on CD3, CD4 and CD8 T lymphocytes. Cell staining was performed on fresh whole blood sample within 4 h after sampling. We used the following antibodies: CD45-PB, CD3-APC-AF750, CD4-FITC, CD8-Kro, CD14-PB, CD16-APC from BeckmanCoulter (Brea, CA) and: PD1-APC, TIM-3-PE-Dazzle, CD15-AF700, LOX1-PE from BioLegend (San Diego, CA). Isotype control antibodies (BioLegend) were used to determine the percentages of positive cells for PD-1, TIM-3 and LOX-1. Samples were run on Navios flow cytometer (Beckman Coulter). T lymphocytes subsets’ absolute quantification was performed on Aquios flow cytometer (Beckman Coulter). Detailed protocols are presented in supplementary methods. Results were expressed as absolute counts for neutrophil subsets and T lymphocyte subsets (i.e., cells/mm3). Results were expressed as absolute cell counts for immature neutrophils and LOX-1⁺ MDSC. Immune checkpoint inhibitor expressions on T lymphocyte subsets were expressed as percentages of positive cells based on isotype controls.

Fresh tumor tissues were processed immediately after collection. The tissues were mechanically minced and subjected to enzymatic digestion using 1 mg/mL type IV collagenase (Worthington Biochemical, NJ, USA) with 150 μg/mL DNase I (Worthington Biochemical, NJ, USA) for 60 min in a 37°C rotating incubator. The dissociated samples were filtered using a 70 μm strainer, washed and resuspended with fluorescence activated cell sorting (FACS) buffer. Cells were incubated with 5 μL TruStain FcX block antibody (BioLegend, CA, USA) in 100 µL FACS buffer for 10 min on ice and then stained with fluorochrome-conjugated antibodies and Fixable Viability Dye eFluor 780 (Invitrogen, CA, USA) for 30 min on ice. Antibodies used were: anti-human CD45–FITC(HI30), CD3–BV650(OKT3), CD19–PE/Dazzle(HIB19), CD15–AF700(HI98), CD14–BV605(M5E2), HLA-DR–BV785(L243), CD11c–BV510(3.9), CD53–PE(HI29), all from BioLegend, and Paired immunoglobulin-like receptor α (PILRα)–AF647(2175D) from R&D (MN, USA). Stained cells were fixed with 1× Intracellular (IC) fixation buffer (Invitrogen, CA, USA) for 30 min on ice. Samples were acquired on a LSRFortessa flow cytometer (BD Biosciences, CA, USA) and data were analyzed with FlowJo software version 10.4.0 (BD Biosciences, CA, USA).