Fig. 3. Functional properties of T_STEM and T_PEX cells.

a, Representative flow cytometric analysis of FACS-purified T_STEM, T_PEX, and T_EM cells showing the expression of CD25 and CD69 before (us, unstimulated) and after stimulation with SEB for 24 h. Similar data were obtained from other donors (n = 4). Numbers colored to match each subset indicate percent marker⁺ cells with the corresponding median fluorescence intensity (MFI) in brackets. b, Dot plot showing the expression of T-bet among CD25⁺ CD69⁺ T_STEM, T_PEX, and T_EM cells before (us) and after stimulation as in a. Data are shown in terms of MFI. Each dot represents one donor (n = 5 from two independent experiments). Bars indicate mean ± SEM. *P < 0.05 (two-tailed paired t-test). c, Representative flow cytometric analysis showing the expression of CD107a, IFN-γ, IL-2, and TNF among T_STEM, T_PEX, and T_EM cells stimulated with anti-CD3 plus CD28 for 12 h. Numbers indicate percentages in the drawn gates. d, Dot plot summarizing the data obtained as in c. Each dot represents one donor (n = 7 from four independent experiments). Bars indicate mean ± SEM. **P < 0.01 (two-tailed paired t-test). e, Dot plot showing the function⁺ populations identified in d in terms of MFI. Each dot represents one donor (n = 3 from four independent experiments for CD107a, n = 7 from four independent experiments for IFN-γ, IL-2, and TNF). Bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed paired t-test). f, Representative flow cytometric analysis showing the expression of CD107a, IFN-γ, IL-2, and TNF among T_STEM, T_PEX, and T_EM cells after stimulation of magnetically-enriched CD8⁺ T cells with PMA and ionomycin for 3 h. Numbers indicate percentages in the drawn gates. Subsets were gated as CCR7⁺ GZMK⁻ (T_STEM), CCR7⁺ GZMK⁺ (T_PEX), or CCR7⁻ CD45RO⁺ CD95⁺ (T_EM). g, Dot plot summarizing the data obtained as in f. Each dot represents one donor (n = 6 from three independent experiments). Bars indicate mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed paired t-test). h, Schematic layout of the serial transfer experiments. i, Line chart showing the absolute numbers of CD8⁺ T cells in PB on days 11, 18, and 28 after transfer of T_STEM or T_PEX cells into secondary NSG recipients. Data were pooled from two independent experiments (total n = 8 mice). Bars indicate mean ± SEM. *P = 0.0117 (two-way ANOVA). j, Dot plot showing the absolute numbers of CD8⁺ T cells in spleen on day 28 after transfer of T_STEM or T_PEX cells into secondary NSG recipients. Each dot represents one mouse (n = 8 from two independent experiments). Bars indicate mean ± SEM. *P = 0.0148 (two-tailed Mann-Whitney U test).