a, Flow cytometry analysis of sorted human CD45RO⁻CD62L⁺ naïve CD8⁺ T cells prior to and 14 days after stimulation with α-CD3/CD2/CD28-coated beads and IL-2 in the presence or absence of 5µM TWS119. Numbers indicate the percentage of cells in the CD45RO⁻CD62L⁺ gate. b, Flow cytometry analysis of TWS119-generated CD45RO⁻CD62L⁺ naïve-like CD8⁺ T cells overlaid with CD45RO⁻CD62L⁺ naïve and memory (non-CD45RO⁻ CD62L⁺) cells from a healthy donor (HD). c, Flow cytometry analysis of PBMC from a healthy donor. Dot plots show the gating strategy to identify CD95⁺, IL2Rβ⁺ T_SCM cells. d, Percentages of circulating CD8⁺ T-cell subsets in 29 healthy donors. e, Flow cytometry analysis of PBMC from a representative healthy donor. Overlaid histogram plots show expression levels of a given molecule in different CD8⁺ T-cell subsets. CD8⁺ T-cell subsets were defined as follows: T_N cells, CD3⁺CD8⁺CD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL7Rα⁺CD95⁻; T_SCM cells, CD3⁺CD8⁺CD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL7Rα⁺CD95⁺; T_CM cells, CD3⁺CD8⁺CD45RO⁺CD45RA⁻ CCR7⁺CD62L⁺; T_EM cells, CD3⁺CD8⁺CD45RO⁺CD45RA⁻CCR7⁻CD62L⁻.

a, TCR excision circle (TREC) copy number in sorted CD8⁺ T-cell subsets relative to T_N cells. Data are represented as mean ± s.e.m. of 4 donors. b, Intracellular cytokine staining of PBMC from a representative healthy donor after stimulation with SEB. Graphs show naïve-like (NL) gated T cells. NL, CD45RO⁻CCR7⁺CD45RA⁺CD27⁺CD28⁺. Numbers represent the percentage of CD95⁺ (T_SCM) and CD95⁻ (T_N) cells producing a single cytokine. c, Percentages of CD8⁺ T-cell subsets producing IFN-γ, IL-2 and TNF-α in 6 healthy donors (obtained as described in panel b). d, Pie charts depicting the quality of the cytokine response in CD8⁺ T-cell subsets in 6 healthy donors as determined by the Boolean combination of gates identifying IFN-β⁺, IL-2⁺ and TNF-α⁺ cells. e, CFSE dilution in sorted CD8⁺ T-cell subsets after stimulation with 25 ng ml⁻¹ of IL-15 for 10 days. Data are shown after gating on CD8⁺ cells. PD: percentage divided; PI: proliferation index. f, Percentage divided cells and g, Proliferation index of different CD8⁺ T-cell subsets after stimulation as in panel e. Data are represented as means ± s.e.m of 9 donors. h, Flow cytometry analysis of PBMC from HLA-A2⁺ donors. Graphs show tetramer-binding cells vs. CD95 expression in the NL (CD45RO⁻CCR7⁺CD45RA⁺CD27⁺IL7Rα⁺) gate. i, Percentage of tetramer-binding cells expressing CD95 in the NL gate determined as in panel h. Data represent the donors tested for tetramer specificity. HD: healthy donor; MP: melanoma patient. j, Frequency of two immunodominant CMV-specific TCRβ clonotypes relative to all CMV-specific TCRβ clonotypes in pp65 -specific T-cell subsets isolated over a period of 22 months from a representative donor. The figure legend shows the CDR3β amino acid sequences. Changes in the frequencies of immunodominat clonotypes are depicted as the thickness of the bars, with the magnitude scale shown on the right. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; ns= not significant (t test, c,f,g,I and χ² permutation test, d).

a, Heat map of differentially expressed genes (P < 0.01 One-Way Repeated Measures ANOVA, FDR < 5% Benjamini-Hochberg's method) among CD8⁺ T-cell subsets. Red and blue colors indicate increased and decreased expression respectively. b, Robust Multichip Analysis (RMA)-normalized intensity of selected display of genes progressively downregulated (naïve associated genes) or upregulated (effector associated genes) from T_N cells →T_SCM cells →T_CM cells →T_EM cells. Data are represented as means ± s.e.m of 3 donors. c) Multidimensional scaling (MDS) analysis of differentially expressed genes (P < 0.01, FDR < 5%). Numbers represent the differentially regulated genes among each CD8⁺ T-cell subset (P < 0.01 (t test) and > 2 fold change in expression level). d, Heat map of differentially-expressed genes among T_SCM and T_CM cells (P < 0.01 (t test) and > 2 fold change in expression level). Red and blue colors indicate increased and decreased expression, respectively. Full gene names are listed in the Supplementary Information.

a, Percentage of CD8⁺ T cells expressing CCR7 and CD62L (right panel) and CD45RA (left panel) relative to cell division after exposure to 25ng ml⁻¹ of IL-15 for 10 days. Slopes (S) were compared using a Wilcoxon rank test, *= P = 0.0391. where g = generation number and p = percentage of CD62L⁺CCR7⁺ cells (n = 8). b, Percentage of CFSE-diluted CD8⁺ T cells that retained the parental phenotype following stimulation with 25ng ml⁻¹ of IL-15 for 10 days. c, Percentage of CD8⁺ T cells expressing CCR7 and CD62L (right panel) and CD45RA (left panel) relative to cell division after stimulation with α-CD3/CD2/CD28-coated beads for 6 days. d, Percentage of CFSE-diluted CD8⁺ T cells with a given phenotype following stimulation with α-CD3/CD2/CD28-coated beads for 6 days. e, Stemness index of CD8⁺ memory T-cell subsets. Stemness index was calculated by multiplying self-renewal (SI) and multipotency (MI) indexes. SI was calculated as follows: SI= 2^PIP_RP, PI=Proliferation index, P_RP= Percentage of cells retaining the input phenotype and MI was calculated as the net entropy of the progeny T-cell subsets where p = percentage of a given T-cell subset generated following α-CD3/CD2/CD28 stimulation. Data are represented as mean ± s.e.m of 4 donors; * = P < 0.05 (t test) (n = 4).

a, ³H-thymidine incorporation by sorted CD8⁺ T-cell subsets after stimulation with α-CD3/CD2/CD28-coated beads. Data are represented as means ± s.e.m of 10 donors. Results are normalized to the number of seeded cells, as different cell numbers were obtained from different sorts. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 (t test) b, Flow cytometry analysis of human T cells in the spleen, lymph nodes (LN) and liver of a representative NSG mouse at 4 weeks following adoptive transfer of CD4⁺ T cells (5 × 10⁶) with or without sorted CD8⁺ T-cell subsets (10⁶). Graphs show T cells after gating on human CD45⁺ cells. Numbers indicate the percentage of cells in the CD4⁺CD8⁻ or CD4⁻CD8⁺ gates. c, Total human CD8⁺ T-cell recovery in the spleens, LN and livers from 6 NSG mice 4 weeks following adoptive transfer of CD4⁺ T cells with or without sorted CD8⁺ T-cell subsets. A total of 6 mice per T-cell subset from two independent experiments (3 replicate mice per T-cell subset per experiment) are shown. Horizontal bars indicate median values. * = P < 0.05; ** = P < 0.01 (t test) d–f, In vivo bioluminescent imaging (d), percentage change of body weight (e), and survival of NSG mice (f) bearing M108-luciferase mesothelioma after adoptive transfer of CD4⁺ T cells (10⁶) with or without sorted CD8⁺ T-cell subsets (3 × 10⁶) expressing a mesothelin-specific chimeric antigen receptor. *** = P < 0.001 One-Way Repeated Measures ANOVA (e) and Log-rank (Mantel-Cox) Test (f).