Lox 1 pe

Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).

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.