Long-term maintenance of SARS-CoV-2 spike-reactive T cells after homologous and heterologous vaccination. (a)-(b) Longitudinal characterization of spike-specific T cells, quantified by IFN-γ spot forming units (SFU) after stimulation with SARS-CoV-2 spike peptide pools S1 and S2. (a) Vaccinees of Munich study center quantified by IFN-γ Fluorospot. “early after #1”: 55 to 137 days after initial ChAd vaccination, n = 26. “early after #2”: 12 to 36 days after second BNT vaccination, n = 29. “late after #2”: 91 to 153 days after second BNT vaccination, n = 22. (b) Vaccinees of Erlangen study center quantified by IFN-γ ELISPOT. “before”: pre-vaccination, n = 16. “early after #1”: 9-12 days after first BNT vaccination, n = 21. “early after #2”: 9-11 days after second BNT vaccination, n = 17. “late after #2”: 148–210 days after second BNT vaccination, n = 12. The time points “before”, “early after #1”, and “early after #2” refer to a separate cohort of vaccinees that was included for contextualization. (c) Cohort comparison of spike-specific T cells after stimulation with SARS-CoV-2 spike peptide pools S1 and S2, in dilution of solvent (Neg. ctrl.), or with PMA/ionomycin (Pos. ctrl.). Vaccinees of Erlangen study center 141–210 days after second vaccination (late after #2). Representative data (left) and quantification of IFN-γ SFU for all donors of indicated vaccination cohorts (right) are displayed. (d)-(f) Flow cytometric analyses of polyfunctional spike-specific T cells after stimulation with SARS-CoV-2 spike peptide pools S1 and S2, in dilution of solvent (Neg. ctrl.), or with PMA/ionomycin (Pos. ctrl.). Vaccinees of Erlangen study center 141–210 days after second vaccination (late after #2). (d) Representative flow cytometry data. Shown gates are pre-gated for CD4+ living lymphocytes. Quantification of IL-2 and IFN-γ double-positive CD4 (e) and CD8 (f) T cells for all donors of indicated vaccination cohorts. Dots represent individual vaccinees. Numbers indicate vaccinees with a positive response defined by a detectable T-cell response above background. Non-responsive vaccinees are represented as not detected (n.d.). For inter-group statistics concerning one time point Kruskal-Wallis test was performed followed by Dunn's multiple comparisons test ((c), (e), (f)). Over-time comparison within one group was done by Mann-Whitney test ((a), (b)). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s. indicates not significant.