Molecular profiling defines a signature of memory differentiation shared by natural T_CM, T_(TN), and T_(TCM) lymphocytes. (A) Comparison of the expression pattern of natural T_N and T_CM identifies a human memory phenotype signature defined by a set of 65 genes up- and down-regulated during the differentiation of naive CD8⁺ T cells into memory lymphocytes. Hierarchical clustering of T_(TN) and T_(TCM) within the conserved CD8⁺ T-cell memory signature classifies T_(TN) lymphocytes as a separate subgroup of the memory branch. (B) Enrichment profile of genes of the CD8⁺ T-cell memory signature (up-regulated in the T_CM gene set) in T_N versus T_(TN) cells. GSEA evidences that the T_CM gene set is significantly overrepresented in T_(TN) cells (FDR q value = 0) compared with T_N. (C) All genes differentially expressed (2-fold cutoff and q value ≤ 0.05) between T_(TN) and memory lymphocytes (T_CM and T_(TCM); supplemental Table 2) are functionally classified under broad categories based on information of the Gene Ontology (pie chart) and of the Molecular Signature Database (enrichment analysis using GSEA). The normalized enrichment score (NES) indicates that all 11 pathways statistically enriched (at FDR q value ≤ 25%) are inversely correlated to the T_(TN) phenotype. (D) Representative FACS plots of CD95 expression on T_N, T_(TN), T_CM, and T_(TCM). Cells are gated on CD8. Gray histograms represent fluorochrome-matched isotype controls. (E) Quantification of CD95⁺ cells among the different subsets.