Cytokine and Degranulation Assay for T Cells

Following identification of CMV positive and negative donors, 2 × 10⁶ cells were incubated at 37°C for 5 h with either PMA and ionomycin, CMV peptide mix (10 (link)) or EBV peptide mix at 10 μg/ml (Supplementary Table 6), along with protein cocktail inhibitor mix (eBiosciences). Live/dead red dye (Invitrogen), anti-CD3 APC-Cy7 (Biolegend) anti-CD8 Amcyan (BD Biosciences), were then applied before fixation and permeabilisation and IFN-γ AF700 (Biolegend) and TNF-α PE-Cy7 staining (eBioscience). Example flow plots can be found in supplementary (Figure S1). For assessing immune cell activation and cytotoxic degranulation, 2 × 10⁶ cells were stimulated with either the above peptide mixes overnight at 37°C or a cell stimulation cocktail (Invitrogen) for 5 h. At the time of stimulation, CD107a FITC (Biolegend), along with brefeldin A and monensin was incorporated into the stimulation panel (example staining of CD107a can be found in supplementary).