Whole Blood Assay for SARS-CoV-2 T Cell Detection

Blood was collected in sodium heparin tubes and processed within 4–6 h of collection. The whole blood assay sample processing used for this study was adapted from a whole blood intracellular cytokine detection assay designed to detect SARS-CoV-2 specific T cells in adults.⁸⁸ (link)^,89 For this study, 500 μL of blood was stimulated for 24 h at 37°C with a combined pool of SARS-CoV-2 peptides including S, N and M, all at 1 μg/mL in the presence of costimulatory antibodies against CD28 (clone 28.2) and CD49d (clone L25) (1 μg/mL each; BD Biosciences) and Brefeldin A (10 μg/mL, Sigma-Aldrich). Unstimulated blood was incubated with costimulatory antibodies, Brefeldin A and an equimolar amount of DMSO as a background control. After 24 h, blood was treated with EDTA (2 mM) for 15 min followed by red blood cell lysis and white cell fixation using FACS lysing solution (BD Biosciences) for 10 min. Cells were then cryopreserved in freezing media (90% fetal bovine serum (FBS) and 10% DMSO) and stored at −80°C until batched analysis.