Fig. 1: Transcriptional signature of human CD8⁺ T cell subsets.

a) Representative image of CD8⁺ T cells gated on CD27⁺CD45RA⁺ T_N cell, CD27⁺CD45RA⁻ T_CM cells, CD27⁻CD45RA⁻T_EM cells, and CD27⁻CD45RA⁺T_EMRA cells isolated from the PBMCs from 6 healthy donors. Numbers in gates represent percentages of cells in each subset from a representative donor. Similar results were obtained in other experiments. b) Heat map of gene expression of Affymetrix U133 plus 2 microarrays of sorted T_N and T_EMRA CD8⁺ T cell subsets, showing downregulated (in yellow) and upregulated genes (in blue). c) The relative fold-change (log10) of differentially expressed genes of interest in T_CM, T_EM and T_EMRA CD8⁺ T cell subsets compared to T_N CD8⁺ T cells. The list of genes of interest is shown in Supplementary table 2 and the complete list of differentially expressed genes from the whole-transcriptome analysis (≥2-fold change, p<0.05, FDR<0.05%) is available in Supplementary table 1. d) NKR expression T_N, T_CM, T_EM, and T_EMRA CD8⁺ T cells assessed by flow cytometry on PBMCs from 22 healthy donors (median age = 52, range 25–83). e) Representative immunoblots of proximal TCR components Lck, PLCγ1, LAT and Zap70 on CD27⁺CD28⁺, CD27⁺CD28⁻ and CD27⁻CD28⁻ CD8⁺ T cells freshly isolated from PBMCs using magnetic activated cell. Similar data were obtained in 4 independent experiments. Summary data (n = 4) of Lck, Zap70, PLCγ1 and LAT expression normalized to the loading control (GAPDH) and presented relative to the basal expression in CD27⁺28⁺CD8⁺ cells set to 1. d) Two-way ANOVA with Dunnett’s post-test correction and e) one-way ANOVA with Tukey’s correction (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).