Il 2 bv421

Approximately 1.5 × 10⁶ cells were stained with antibodies and antibody application was followed by the recommendation. Mouse lymphocytes were stimulated with the peptide pool of SARS-CoV-2 RBD and incubated with monensin [BioLegend (Cat. No. 420701)] for 9 hours. Then, the cells were harvested. For surface staining, cells were stained with fluorescence-labeled mAbs of CD3-FITC (BioLegend, USA), CD4-APC-Cy7 (BioLegend, USA) and CD8-AF700 (BioLegend, USA). The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences, USA) and intracellularly stained with fluorescence-labeled mAbs of IFN-γ-BV605 (BioLegend, USA), IL-2-BV421 (BioLegend, USA) and IL-4-PE (BioLegend, USA). All stained cells were detected on BD LSRFortessa™ X-20.

Splenocytes were isolated from the vaccinated mice and seeded into 24‐well plates to quantify the percentages of antigen‐specific T cells; ≈10⁶ cells were added per well. Subsequently, stimulated the cells with mosaic peptides pool (GenScript) at a final concentration of 2 g mL⁻¹ and co‐stimulated with 2 µg mL⁻¹ anti‐CD28 (1000× Biolegend 102116) at 37 °C with 5% CO₂ for 1 h. DMSO was used as a negative control, and PMA/ionomycin was used as a positive control. Splenocytes were then blocked with 5 µg mL⁻¹ brefeldin A (TargetMOL T6062/20350156) and 2 µM monensin (TargetMOL T1033/22373780) for 5 h to prevent cytokines from passing through the cell membrane. Then the cells were extracellularly stained with fluorochrome‐conjugated monoclonal antibodies for 20 min within PBS containing 0.5% BSA on ice. The following antibodies were used: FVD (Invitrogen 65‐0865‐14), CD3‐ PerCP‐Cy5.5 (Biolegend 100328), CD4‐PE‐Cy7 (Biolegend 566939), CD44‐FITC (Biolegend 103021), followed by treatment of 200 µL IC Fixation buffer (BD Biosciences) overnight at 4 °C. The next day, cells were performed with the 1 mL Permeabilization buffer (Invitrogen 00‐8333‐56). Then the antibodies IL‐2‐BV421(Biolegend 503820), TNF‐α‐BV510 (Biolegend 506339), IFN‐γ‐APC (Biolegend 505810), IL‐4‐PE (Biolegend 504104) were used for the intracellular staining and the flow cytometry analysis.

T2 cells (1 × 10⁵ cells per condition) expressing HLA-A3 and/or HLA-A2 were pulsed overnight with 10 µg/mL of the indicated myelin peptide or no antigen. Myelin tetramer-positive CD8⁺ T cells (5 × 10⁴ to 2 × 10⁵) were added to the pulsed T2 cells (total volume, 100 µL) and stimulated for 6 h in the presence of 1:500 GolgiStop (BD), 1:500 GolgiPlug (BD), and 1:200 CD28/CD49d (FastImmune; BD). Cells were washed with FACS buffer and stained with the cell surface antibodies as above (anti-CD8, dump channel antibody mixture, and live/dead dye). Cells were washed, fixed, and stained with antibodies for intracellular cytokines in permeabilization buffer. Intracellular antibodies included IFN-γ Alexa 647 (BioLegend; 4S.B3), TNFα Alexa 488 (BioLegend; Mab11), IL-2 BV421 (BioLegend; MQ1-17H12), GM-CSF PE (BioLegend; BVD2-21C11), CD107a APC/Cy7. Cells were then washed and collected on an LSRFortessa.