Pe clone h4a3

Degranulation of T cells was determined by extracellular staining of CD107a as previously described in more detail¹⁹ using an anti-CD107a antibody conjugated with PE (clone H4A3, BD biosciences). For this assay, 0.5 × 10⁵ target cells were blocked with human serum (10% final concentration) of an HCMV-seronegative individual for 10 minutes at 4 °C and 0.5 × 10⁵ effector cells were added (effector:target (E:T) ratio of 1:1) followed by addition of an anti-CD107a antibody. After 1 h of incubation at 37 °C, 5 µM Monensin was added to prevent internalization and the incubation was continued for 4 h. Afterwards, the cells were transferred to FACS staining tubes, washed and stained with antibodies against CD3, CD4 and CD8 as described above. Round-bottom 96-well plates were used for this assay to enhance cell-to-cell contact during the incubation period.

SARS-CoV-2 spike protein in carbonate/bicarbonate solution (2.5 µg/ml) was added to 96-well Nunc MaxiSorp ELISA plates and incubated for 16 h at 4 °C. Plates were washed six times with PBS and blocked with 5% BSA in PBS for one hour at 37 °C. Plasma samples were added neat and in duplicate, and plates were incubated for two hours at 37 °C. Following another wash step, 10⁵ natural killer NK-92 cells expressing human CD16 (PTA-8836 cell line, American Type Culture Collection) described by^{69 (link)} were added to each well with brefeldin A (10 µg/mL; Sigma Aldrich), GolgiStop (BD Biosciences) and CD107a (1:20 dilution; PE, clone H4A3, BD Biosciences). Plated cells were incubated for five hours at 37 °C and then transferred to V-bottom plates, incubated with fixable LIVE/DEAD staining (1:500 dilution; R780, BD Biosciences) and fixed. Data was acquired using a BD Fortessa and percentages of CD107a expressing NK cells relative to control wells with spike protein and blocking buffer only were determined using FlowJo Software (version 10.7.1). To assess inter-assay variation, both a pre-pandemic pool of three donors and a pool of six hospitalised SARS-CoV-2-infected individuals were plated in triplicate on each plate.