Anti mouse cd4 bb700

ICS assay was performed according to our previous method [30 (link)]. Briefly, mouse splenic lymphocytes were seeded in the 96-well plates at 2 × 10⁶ per well and stimulated with the SARS-CoV-2 N peptide pool (2 μg/mL for each peptide) at 37 °C for 1.5 h. Then, brefeldin A (BD, Franklin Lakes, NJ, USA) was added and incubated for 16 h at 37 °C. The cells were harvested and stained with anti-mouse CD3-FITC, anti-mouse CD4-BB700, and anti-mouse CD8-PE cy7 (BD, Franklin Lakes, NJ, USA) for 30 min, and protected from light at room temperature. Then, the cells were fixed and permeabilized with cytofix/cytoperm (BD, Franklin Lakes, NJ, USA) for 20 min, and protected from light at 4 °C. After being washed with perm/wash (BD, Franklin Lakes, NJ, USA), the cells were stained with anti-mouse IFN-γ-APC, anti-mouse IL-2-BV605, and anti-mouse TNF-α-PE (BD, Franklin Lakes, NJ, USA) for 1 h, and protected from light at 4 °C. Finally, the cells were washed with FACS washing buffer (PBS supplement with 2% heat-inactivated FBS) three times and resuspended in PBS. The data were obtained with Beckman CytExpert software and analyzed using FlowJo software (version 10.8.1).

Groups of mice were mock-vaccinated (PBS) and vaccinated with WNPRBD and WNPRBD-R266 as indicated. BALF and lung were harvested at 7 days post-second vaccination. Cells derived from BALF and lung-disassociated cells were used for the T cell activation assay. Cells were counted and activated by the peptide pool (PepMix SARS-CoV-2 S-RBD; JPT Peptide Technologies, Berlin, Germany) overnight. Cells were then stained with antibodies including anti-mouse CD8a-APC-Fire750 (Rat IgG2ak; 0.2 mg/mL; Biolegend 100766) 1:400; anti-mouse CD3-SB780 (Rat IgG2bk; 0.2 mg/mL; eBioscience 78-0032-82) 1:100; anti-mouse IL2-PE (Rat IgG2bk; 0.2 mg/mL; Biolegend 503808) 1:100; anti-mouse TNFα-BV421 (Rat IgG1k; 0.2 mg/mL; Biolegend 506328) 1:200; anti-mouse IFNγ-APC (Rat IgG1k; 0.2 mg/mL; Biolegend 505809) 1:100; anti-mouse CD4-BB700 (Rat IgG2ak; 0.2 mg/mL; BD 566408) 1:400. Data were collected by LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo version 10 (BD Biosciences).

Mouse splenic lymphocytes were resuspended at 1 × 10⁶/mL in 0.1% FBS/PBS and incubated at 37 °C for 10 min with 0.2 μM CFSE (ThermoFisher, Waltham, MA, USA). An equal volume of ice-cold RPMI 1640 was added and an ice bath was run for 5 min to terminate the staining. After the addition of serum and washes with RPMI 1640, cells were resuspended at 1 × 10⁶ cells/mL and plated into 48-well U-bottom plates at 500 μL volumes and simulated with a SARS-CoV-2 N peptide pool (2 μg/mL for each peptide). After being simulated for five days, cells were harvested and washed with FACS washing buffer and stained with anti-mouse CD3-PE, anti-mouse CD4-BB700, anti-mouse CD8-PE-Cy7, and fixable viability stain 780 (BD, Franklin Lakes, NJ, USA). Cells were washed and a flow cytometric analysis was conducted using Beckman CytExpert. The data were analyzed using FlowJo software (version 10.8.1).