Ril 2

Approximately 1.5 × 10⁶ PBMCs were stimulated with A*01/S₈₆₅, A*02/S₂₆₉ or A*03/S₃₇₈-specific peptides (5 μM) and anti-CD28 monoclonal antibody (0.5 μg ml⁻¹, BD) and expanded for 14 days in complete RPMI culture medium containing rIL-2 (20 IU ml⁻¹, StemCell Technologies). Intracellular cytokine production and degranulation was assessed with spike-specific peptides (15 μM) in the presence of anti-CD107a (H4A3, 1:100) (BD Bioscience) for 1 h at 37 °C. Afterwards, brefeldin A (GolgiPlug, 0.5 μl ml⁻¹) and monensin (GolgiStop, 0.5 μl ml⁻¹) (all BD Biosciences) were added for additional 5 h, followed by surface and intracellular staining. The expansion capacity was calculated based on peptide-loaded HLA class I tetramer staining as previously described^{24 (link)}.

1.5 × 10⁶ PBMCs were stimulated with the spike protein-derived peptides A*01/S₈₆₅ or A*02/S₂₆₉ and anti-CD28 monoclonal antibody (0.5 µg/mL) for 14 days in RPMI cell culture medium supplemented with rIL-2 (20 IU/ml, StemCell Technologies). At day 4, 7 and 11, 50% of the culture medium was exchanged with freshly prepared medium containing 20 IU/mL rIL-2. After 14 days, PBMCs were stimulated with peptides again, and stained for CD107a for 1 h at 37 °C to analyze degranulation. Subsequently, brefeldin A (GolgiPlug, 0.5 μl/mL) and monensin (GolgiStop, 0.5 μl/mL) (all BD Biosciences) were added and incubation continued for four more hours, followed by surface and intracellular staining with anti-IFNy, anti-TNF and anti-IL-2-specific antibodies. For calculation of the expansion capacity and to assess the cytotoxic capacity of the expanded cells, peptide-loaded HLA class I tetramer staining was performed together with intracellular staining of Granzyme B, Granzyme K, Perforin and Granulysin.