Intracellular cytokine staining

Spleens were collected from mice 7 days after vaccination, homogenized, passed through a 70-μm nylon mesh, and mixed with ACK lysis buffer (Gibco) to lyse red blood cells. Splenocytes were then resuspended in RPMI media with 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin (Fisher Scientific). 2E–6 cells were added to a non-treated U-bottom 96-well plate and co-incubated with either 500 nM of PepTivator SARS-CoV-2 Prot S1 pool (Miltenyi Biotec), 500 nM of PepTivator SARS-CoV-2 Prot N pool (Miltenyi Biotec), 5 μM each of VACV F2/E3 peptides (SPGAAGYDL/VGPSNSPTF; Genscript),⁸³ or 0.2% DMSO. After 4 h of incubation at 37°C, Brefeldin A (Invitrogen) was added to block cytokine release from the Golgi apparatus, and the splenocytes were co-incubated with the peptides for an additional 16 h at 37°C. CD16/CD32 antibodies (553142; BD) were then added to block Fc receptors followed by staining with Fixable Viability stain 510 (BD). Cells were then stained with antibodies against extracellular receptors (detailed in Table 1) in PBS with 0.5% BSA. Cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (Cat No. 554714) and then stained with antibodies against IFNγ and TNFα (detailed in Table 1). Since TNFα was undetected in the samples, it was not discussed throughout the results. After staining, cells were resuspended in 1% PFA in PBS and analyzed at the University of Ottawa’s Flow Cytometry Core Facility or Ottawa Hospital Research Institute Core facility using the BD LSR Fortessa (BD) Flow Cytometers. Analysis of flow data was conducted using FlowJo v10 (FlowJo, Ashland, OR). The gating protocol is shown in Figure S5.