Cd200r pe

All experiments and assays were performed on freshly isolated samples. Isolated PBMCs were incubated with directly conjugated fluorescent antibodies for 30 min at 4 °C. The cells were washed before flow cytometry analysis. Monocytes were separated from other cells by gating on CD3/15/19⁻ cells combined with forward scatter (FSC)/ side scatter (SSC) characteristics and CD45 expression. The gating strategy used is shown in Fig. S1. Antibodies used included anti-human CD160-Alexa Fluor 488, CD4-APC-Fire750, CD8-BV510, HLA-DR-Alexa Fluor 700, CD14-APC, PD-1-PE, 2B4-PE-CF594, CD16-BV711, TIM-3-BV650, CD200R-PE, BTLA-BV650, CD45-BV786 (BD Biosciences, San Diego, CA, USA), CX3CR1-BV421, CD3-PerCP-Cy5.5, CD15-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD29-Alexa Fluor 488, CD62L-BV650, CD11b-BV605, CCR2-PE (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, and LAG-3-APC (eBioscience, San Diego, CA, USA), along with the corresponding isotype controls. BD Trucount Tubes (BD Biosciences), combined with specific antibodies (CD45/3/4/8 cocktail; BD Biosciences), were used to determine the absolute counts of leukocytes in the blood with flow cytometry according to the manufacturer’s instructions. The absolute numbers (cells per microliter) of leukocytes and T cells were determined by comparing cellular and bead events.

For the differentiation and polarization of isolated human monocytes into M1 and M2 macrophages, cells were cultured for 7 days at 37 °C in a 5% CO₂ atmosphere in complete medium composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution in the presence or absence of different cytokines. Monocytes were seeded in 96- or 6-well culture plates at 2 × 10⁵ cells/well or 1 × 10⁶ cells/well, respectively, and the following cytokines were added for 7 days: 50 ng/mL GM-CSF (for M1 polarization) or 50 ng/mL M-CSF (for M2 polarization). At day 6, 100 ng/mL LPS was added to M1 polarized macrophages and 20 ng/mL IL-4 was added to M2 polarized macrophages. Seven days later, cells were detached from plates and stained with a cocktail of conjugated antibodies as follows: 10 µL CD14-FITC, 5 µL CD16-APC-H7, 5 µL CD80-PE-Cy5, 5 µL CD163-APC, and 5 µL CD200R-PE from BD Biosciences. Data acquisition was performed on 10,000 cells with constant PMT values on an FACS Canto II cytometer (BD Biosciences). Data analysis was carried out using Diva software (BD FACSDiva v9.0software, BD).