Cd3 apc af750

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.

The cryopreserved PBMCs were thawed and cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO). Then the cells were stimulated with co-stimulatory antibodies (CD28, CD49d - 1μg/mL) (BD Bioscience, San Diego, CA) and test peptides/antigens such as ESAT-6/CFP-10 peptides, PPD, MTB300 peptide pool, Candida antigen, and anti-CD3 with specified concentrations as described above. Cells cultured under similar conditions without any stimulation served as a negative (nil) control. The culture plate was incubated for 40 hours at 37°C with 5% CO_2, and the cells were stained with specific surface markers of anti-human monoclonal antibodies such as CD4 BV650 (BD Bioscience, San Diego, CA), and CD3 APC-AF750, CD8 krome Orange, CD25 APC-AF700, CD134 APC (Beckman Coulter, Brea, CA). Finally, cells were washed and fixed with 0.5% paraformaldehyde and at least 250,000 cells were acquired by BD LSRFortessa (BD Bioscience, San Diego, CA). Files were exported in FCS 3.0 format and the percentage of phenotypic markers was analyzed by Kaluza software (Beckman Coulter, Brea, CA). The background (nil) response was subtracted from the tested antigen stimulations. Considering the cell count, number of tested antigens, reagent cost and that this is a small pilot study, we performed the flow cytometry in a single well manner for each antigenic condition.