Spleen and draining lymph nodes were harvested 2 weeks after the final vaccination. Harvested spleens were digested with gentle MACSTM Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) and lymph nodes were manually ground to single-cell suspension, which was then RBC-lysed with RBC lysis buffer (Biolegend, San Diego, CA, USA) for 5 min at 4 °C. Single-cell suspension of lymph nodes was stained with immune-cell-specific antibodies for flow cytometry (BD Accuri C6, BD Bioscience, Franklin Lakes, NJ, USA) as follows: PERCP anti-CD45.2 antibody, APC anti-CD3 antibody, FITC anti-CD4 antibody for CD4 T cell; PERCP anti-CD45.2 antibody, APC anti-CD3 antibody, FITC anti-CD8 antibody for CD8 T cell; and PERCP anti-CD45.2 antibody, APC anti- antibody, FITC anti- antibody, PE anti-B220 antibody for B cell.
Single-cell suspension of spleen was seeded into a 96-well plate and challenged with SARS-CoV-2-spike-protein–peptide-pool (Miltenyi peptide pool; 130-127-958, 130-128-482). After incubating for 5 h, the supernatant was collected for TNFα and IFNγ ELISA (R&D systems, Minneapolis, MN, USA), which were performed according to the manufacturer’s protocol. The cells were centrifuged to stain with PE-conjugated anti-IL-4 antibody (Biolegend).
Single-cell suspension of spleen was seeded into a 96-well plate and challenged with SARS-CoV-2-spike-protein–peptide-pool (Miltenyi peptide pool; 130-127-958, 130-128-482). After incubating for 5 h, the supernatant was collected for TNFα and IFNγ ELISA (R&D systems, Minneapolis, MN, USA), which were performed according to the manufacturer’s protocol. The cells were centrifuged to stain with PE-conjugated anti-IL-4 antibody (Biolegend).