Cd28 clone 28.2

Blood was collected in sodium heparin tubes and processed within 4-6 hours 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 ^{81 (link),82 (link)}. Briefly, 500 μl of blood was stimulated for 24 hours 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 hours, blood was treated with EDTA (2 mM) for 15 minutes followed by red blood cell lysis and white cell fixation using FACS lysing solution (BD Biosciences) for 10 minutes. Cells were then cryopreserved in freezing media (90% fetal bovine serum (FBS) and 10% DMSO) and stored at −80°C until batched analysis.

The assessment of the percentage of CD4 and CD8 T cells was performed on cryopreserved fixed whole blood as described⁵⁵ . Briefly bood was collected in sodium heparin tubes and processed within 3 h of collection. We adapted this assay to detect SARS-CoV-2 specific T cells using synthetic SARS-CoV-2 PepTivator peptides (Miltenyi Biotec, Surrey, UK), consisting of 15-mer sequences with 11 amino acid overlap covering the immunodominant parts of the spike (S) protein, and the complete sequence of the nucleocapsid (N) and membrane (M) proteins⁵⁵ . All peptides were combined in a single pool and used at a final concentration of 1 µg/ml. Briefly, 400 µl whole blood was stimulated with the SARS-CoV-2 S, N and M protein peptide pool at 37 °C for 5 h in the presence of co-stimulatory antibodies against CD28 (clone 28.2) and CD49d (clone L25) (1 µg/ml each; BD Biosciences, San Jose, CA, USA) and Brefeldin-A (10 µg/ml, Sigma-Aldrich, St Louis, MO, USA). Unstimulated blood was incubated with co-stimulatory antibodies, Brefeldin-A and an equimolar amount of DMSO. Red blood cell lysis and white cell fixation was performed in a single step using a Transcription Factor Fixation buffer (eBioscience, San Diego, CA, USA) for 20 min. Cells were then cryopreserved in freezing media (50% fetal bovine serum, 40% RPMI and 10% dimethyl sulfoxide) and stored in liquid nitrogen until batched analysis.

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.