Extended Data Fig. 1: Lymph nodes preserve stemness, proliferative and metabolic fitness, and polyfunctionality of CD8⁺ T cells.

(a–e) Naïve congenically marked (CD45.1⁺) P14 CD8⁺ T cells were adoptively transferred into naïve wildtype (CD45.2⁺) mice, which were subsequently infected with LCMV-Docile. On d25 post-infection (p.i.) CD8⁺ T cells from spleens and pooled inguinal, brachial, axillary, cervical and mandibular lymph nodes (LN) were analysed using flow cytometry. (a) Quantification showing IL-2 production by P14 T_PEX cells (n = 7). (b) Flow cytometry plots and quantification showing IFN-γ and TNF production in CD62L⁺ and CD62L⁻ T_PEX cells from spleen and LN after incubation with gp33 peptide (n = 11). (c) Flow cytometry plots and quantification of Ki67⁺ cells among the P14 T_EX cells (n = 21). (d) Quantification showing ex vivo puromycin uptake in splenic and LN T_EX cells. (e) Flow cytometry plots and quantification showing IFN-γ and TNF production in P14 T_EX cells from spleen and LN after incubation with gp33 peptide (n = 10). (f–k) Naïve wildtype mice were infected with LCMV-Docile. On d25 p.i. CD8⁺ T cells from spleen and pooled LN were analysed using flow cytometry. (f) Flow cytometry plots and quantification showing the expression of PD-1 and TOX in endogenous polyclonal antigen-responsive CD8⁺ T cells in spleen and LN in comparison to naïve CD8⁺ T cells (n = 10). (g) Flow cytometry plots and quantification of T_PEX cells among endogenous PD-1⁺CD8⁺ T cells in spleen and LN (n = 10). (h) Flow cytometry plots and quantification of CD62L⁺ cells among endogenous T_PEX cells in spleen and LN (n = 20). (i) Flow cytometry plots and quantification showing the frequencies of Ki67⁺ cells among endogenous T_PEX and T_EX cells (n = 12). (j) Quantification showing ex vivo puromycin uptake of spleen and LN-derived endogenous antigen-responsive CD8⁺ T cells (n = 8). (k) Flow cytometry plots and quantification showing IFN-γ and TNF production in endogenous PD-1⁺CD8⁺ T cells from spleen and LN after stimulation in the presence or absence of gp33 peptide (n = 26). Flow cytometry plots are representative. Dots in graphs (a–k) represent individual mice; bars represent median. Quantification and statistics derive from unpaired two-tailed t tests (a, c, e, g–i, k) and one-way ANOVA (b, d, f) and are based on all data points across at least two independent experiments.