(A) Spike-specific CD154+CD137+ Th cells from all groups were concatenated and subjected to unsupervised analysis using tSNE; highlighted (z dimension) are areas with IFN-γ+, TNF-α+, or IL-2+ cells. Spike- or CEF-specific CD154+CD137+ Th cells were further examined after manual gating for expression of (B) IFN-γ (spike/CEF: ANOVA), (C) TNF-α (spike: Kruskal-Wallis test; CEF: ANOVA), (D) IL-2 (spike/CEF: Kruskal-Wallis test) with n as in Figure 2B, respectively, or (E) IL-4 (spike: ANOVA; CEF: Kruskal-Wallis test; HC: spike, n = 35; CEF: n = 31; KTx: spike, n = 11; CEF, n = 12; HD: spike, n = 24; CEF: n = 22). (F) Portions (left) of spike-specific T cells expressing 3, 2, 1, or 0 cytokines at a time based on the respective mean values of each group or (right) frequencies of spike- or CEF-specific Th cells staining triple positive for IFN-γ, TNF-α, and IL-2 with n as in Figure 2B and Kruskal-Wallis testing, respectively. IL-4 was excluded from polyfunctionality analyses due to the limited sample size in the KTx group. Graphs show mean ± SD.