Extended Data Fig. 2: Functional superiority of endogenous antigen-responsive CD8⁺ T cells residing in lymph nodes.

(a, b) P14 T cells expressing IRF4-tdTomato were analysed using flow cytometry on d25 p.i. with chronic LCMV. The expression of IRF4 in T_PEX and T_EX subsets in the spleen and LN was quantified. (a) Flow cytometry plots and (b) quantification showing the expression of IRF4 in exhausted T cell subsets (n = 8). (c–h) Naïve wildtype mice were infected with LCMV-Docile. On d26 p.i., CD8⁺ T cells from spleen and pooled inguinal, brachial, axillary, cervical and mandibular lymph nodes (LN) were analysed using flow cytometry. (c, d) Flow cytometry plots and quantification showing the frequencies of (c) CX3CR1⁺ (n = 21) and (d) CD101⁺ cells (n = 15) among endogenous T_EX cells (gated as TCF1⁻). (e, f) Flow cytometry plots and quantification showing expression of (e) CXCR6 (n = 9) and (f) Ly108 (n = 9) among endogenous CX3CR1⁺ T_EX cells in the spleen and LN. (g) Flow cytometry plots and corresponding quantification showing the frequencies of Ki67⁺ among Ly108^intCX3CR1⁺ and Ly108^loCX3CR1⁺ T_EX cells in spleen and LN (n = 14). (h) Flow cytometry plots and corresponding quantification showing the frequencies of Ki67⁺CX3CR1⁺ cells among endogenous PD-1⁺CD8⁺ T cells in spleen and LN (n = 8). (i–n) Naïve congenically marked (CD45.1⁺) P14 CD8⁺ T cells were adoptively transferred into naïve wildtype (CD45.2⁺) mice, which were subsequently infected with LCMV-cl13. On d32 p.i., P14 T cells in the spleen and pooled LN were analysed using flow cytometry. (i, j) Flow cytometry plots and quantification showing the frequencies of (i) T_PEX cells among P14 cells (n = 9) and (j) CD62L⁺ cells among T_PEX cells (n = 9). (k) Flow cytometry plots and quantification showing the frequencies of CD101⁺ cells among P14 T_EX cells (n = 12). (l) Flow cytometry plots and quantification showing the frequencies of Ki67⁺CX3CR1⁺ cells among P14 cells in spleen and LN (n = 10). (m, n) Flow cytometry plots and quantification showing the frequencies of Ly108^intCX3CR1⁺ cells among (m) P14 cells (n = 23) and (n) endogenous antigen-responsive CD8⁺PD-1⁺ cells (n = 9). Flow cytometry plots are representative. Dots in graphs (b–n) represent individual mice; bars represent median. Quantification and statistics derive from unpaired two-tailed t tests (c–f, h–n) and one-way ANOVA (a, g) and based on all data points across at two (f, h, i–l, n) or three (b–e, g, m) independent experiments.