Extended Data Fig. 1: Age-related changes in A2/M1₅₈⁺CD8⁺ T cell frequencies and phenotypes.

a, Representative FACS panels indicate the gating strategy used to characterize the total CD8⁺ T cell and the A2/M1₅₈⁺CD8⁺ T cell populations. The second line indicates unenriched, enriched and flowthrough fractions of the TAME assay. The unenriched fraction was used to define the frequency and phenotype of the total CD8⁺ T cell population (gray gate and cell populations), whereas the A2/M1₅₈ tetramer-positive CD8⁺ T cells of the enriched fraction were used to define the frequency and phenotype of the total A2/M1₅₈⁺CD8⁺ T cell population (red gate and cell populations). Naïve/memory T cell subsets were identified as T_cm (CD27⁺CD45RA⁻) cells, T_em (CD27⁻CD45RA⁻), T_emra (CD27⁻CD45RA⁺), T_naïve (CD27⁺CD45RA⁺CD95⁻) and T_scm (CD27⁺CD45RA⁺CD95⁺) cells. Boolean gating was used to identify frequencies of individual and combined expression of CD57 and PD-1 and combined expression of CD38, HLA-DR and CD71. b, number of A2/M1₅₈⁺phenotype⁺CD8⁺ T cells per 10⁶ CD8⁺ T cells across all age groups. Co-expression of CD57 and PD-1 in total CD8⁺ T cells (c) and A2/M1₅₈⁺CD8⁺ T cells (d-top panel) and number of A2/M1₅₈⁺CD57⁺CD8⁺ T cells (d-middle panel) or A2/M1₅₈⁺PD-1⁺CD8⁺ T cells (d-bottom panel) per 10⁶ CD8⁺ T cells across all age groups. Co-expression of CD38 and HLA-DR in total CD8⁺ T cells (e) and A2/M1₅₈⁺CD8⁺ T cells (f). Numbers of A2/M1₅₈⁺phenotype⁺CD8⁺ T cells per 10⁶ CD8⁺ T cell data were right shifted by 0.001 (that is absence of A2/M1₅₈ events in a specific phenotypic population are displayed as 0.001) (b,d-middle and bottom panel). Only samples with 10 or more total A2/M1₅₈⁺ events were included for phenotype analysis (b-f). Horizontal bars indicate the median, dots represent individual donors, with n = 10 newborns, n = 12 children, n = 20 adults and n = 16 older adults in b, d and f and n = 11 newborns, n = 12 children, n = 20 adults and n = 18 older adults in c and e. Technical replicates were not performed due to limited samples. Statistical analysis was performed using a two-sided Kruskal-Wallis with Dunn’s correction for multiple tests (b, d-middle and bottom panel) or a two-sided Tukey’s multiple comparisons test (c, d-top panel, e, f). Exact significant p-values are indicated above the graphs. N, newborns; C, children; A, adults; OA, older Adults.

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