Cd4 apc fire750

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.

Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells were then incubated with an antibody at 4 °C for 30 min in the dark in PBS with 2% normal FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to standard techniques. To characterize splenic cells, the monoclonal antibodies used spleen cell suspensions prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells’ suspensions were incubated for 20 min with the following antibodies. All antibodies were obtained from Biolegend (San Diego, CA, USA) and used at a dilution of 1:100 unless otherwise mentioned: CD3 FITC, CD4 APC Fire 750 (BD Biosciences, San Jose, CA, USA), CD8 BV 605, CD69 PEDazzle594, CD40 PercPCy5.5, B220 APC, CD44 BV650, MHC II eFluor450 (DAPI) (eBioscience, San Diego, CA, USA), F4/80 BV711 (dilution 1:200), CD11b PercP Cy5.5, CD80 BV421, CD86 FITC, CD206 Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA. Dilution 1:200), Ly6C PECy7.

PBMCs were stimulated with anti-CD3/CD28 (5 µg/mL, Ebioscience) for 5 h in the presence of anti-CD107a BV421 (BioLegend) and Golgiplug (BD Biosciences). CD107a expression was measured as a marker of degranulation on CD8⁺ T cells after stimulation. The cells were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with TNF-α-BV711, IL-2-BV650 (BioLegend), or IFN-γ-AF700 (Ebioscience) antibodies. For Ki67, perforin, Granzyme B, T-bet, or Eomes staining, PBMCs were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with Granzyme B-AF700, T-bet-BV421 (BD Biosciences), Ki67-BV711, perforin-APC (BioLegend), or Eomes-PE-CY7 (Ebioscience) antibodies. A fixable viability dye eFluor^® 506 (Ebioscience) was used to label dead cells.