Flow cytometry gating strategy.

Gating strategy (A and B) and representative examples of SARS-CoV-2 spike-specific IFN-γ, IL-2 and TNF-α production in memory CD4+ and CD8+ T cells (C). (D) Representative examples of SARS-CoV-2 spike-specific IFN-γ or IL-2 production in CD4+ and CD8+ T cells on which memory phenotyping were gated. (E) Representative plots showing spike-specific T cell immune phenotyping. ED: early differentiated, LD: late differentiated, Eff: effector, Inter: intermediate. Polyfunctional profile of SARS-CoV-2-specific CD4+ (F) and CD8+ T cells (G) at the 1M (red bar), 6M (green bar) and post boost (blue bar) time-points. PBMCs from patients with either no prior infection (black), infected with D614G (blue), or infected with Beta (red) are represented by closed circles and BTIs are represented as white squares. The x-axis illustrates each combination which is indicated with a black circle for the presence of IFN-g, IL-2 and TNF-a. The medians and interquartile range are shown. Each response pattern (any possible combination of IFN-g, IL-2 and TNF-a production) is color coded and summarized in the pie charts, with each pie slice representing the median contribution of each combination to the total SARS-CoV-2 responses. The permutation test was used to compare the statistical differences between the pie charts and the Mann Whitney Sum Test to compare response patterns between the time-points; p values (TIFF)