Fig. 4. T_EMRA subpopulations with stem-like signatures are enriched in Omicron-reactive CD8⁺ T cells.

(A) Representative plots show gating on CD8⁺ T_EMRA population as well as expression of CD27, CD127, and CX3CR1. (B) Boolean gating of CD8⁺ T_EMRA cells based on CD27, CD127, and CX3CR1 expression. Quantification shows frequencies of three different T_EMRA subpopulations between bulk and Omicron-reactive after booster doses. Each dot represents one donor, and lines depict the median. Wilcoxon matched-pairs signed rank test with Holm-Šidák posttest. (C) UMAP of bulk memory CD8⁺ T cells upon stimulation with Omicron full spike peptide pool (n=5 healthy donors, activation-markers excluded from UMAP calculation). (D) UMAP from (C) with an overlay of Omicron-reactive CD8⁺ T_EMRA subsets. (E) Heatmap of key differentiation markers from data shown in (C, D) (n=11 healthy donors). (F) Representative plots showing Cell Trace Violet (CTV) dilution after five days of CD3/CD28 stimulation of sorted CD8⁺ T_EMRA subsets and T_CM as control. (G) Proliferative capacity shown as division and proliferation index calculated with data measured in (F). Each dot represents one donor, and lines depict the mean ± SD. Mann-Whitney test between indicated groups. (B, G) ****p <0.0001, ^***p <0.001, **p <0.01, *p <0.05.