Tnfα pe

Testing of T cell responses was performed via stimulation of peripheral blood mononuclear cells with peptide libraries encompassing SARS-CoV-2 structural proteins as previously described [12 (link)]. Cells were then cultured for 10 days in 96-well plates with IL-4 (400 IU/mL) and IL-7 (10 ng/mL). On day 10, expanded viral specific T cells (VSTs) were harvested. 2 × 10⁵ VSTs were plated in a 96-well plate and re-stimulated with SARS-CoV-2 structural proteins pooled pepmixes or actin (negative control) with CD28/CD49d (BD Biosciences) and anti-CD107a- Pe-Cy7 antibody. After 1 h of stimulation, brefeldin A (Golgiplug; BD Biosciences, San Jose, CA, USA) and monensin (GolgiStop; BD Biosciences, San Jose, CA, USA) were added. Cells were then incubated for an additional 4 h. Cell viability was assessed using Live-Dead Aqua. Cells were surface stained with fluorophore-conjugated antibodies against CD3-BV785, CD4-BV605, CD8- BV421, TCRαβ-PerCP Cy5.5, TCRγδ- APC-Fire750, CCR7-FITC, CD45-RO-PE Dazzle, HLA-DR-Alexaflour700, and CD56-BV650 (Miltenyi Biotec; BioLegend). Cells were fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and subsequently stained with IFN-γ-APC and TNF-α-PE (Miltenyi Biotec). All samples were acquired on a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA). The gating strategy for analysis is presented in Supplemental Fig. 2 .