Extended Data Fig. 3: CX3CR1⁺ T_EX cells develop in lymph nodes.

(a–d) 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 d26 p.i., mice were subjected to daily FTY720 treatment for 4 days. On d30 p.i., P14 T cells from the spleen and lymph nodes (LN) were analysed using flow cytometry. (a, b) Quantification showing the (a) frequencies and (b) numbers of P14 T_PEX and T_EX cells in the presence and absence of FTY720 in the spleen and LN (n = 10). (c) Representative flow cytometry plots and corresponding quantification showing the frequencies of Ly108^intCX3CR1⁺ cells among endogenous antigen-responsive PD-1⁺CD8⁺ T cells (n = 10). (d) Quantification showing the absolute numbers of endogenous T_PEX and T_EX cells in the presence and absence of FTY720 in the spleen and LN (n = 10). (e–i) 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. P14 cells in the spleen and LN were harvested on d30 p.i. and subjected to scRNA-seq. (e) Normalised gene expression of key identity genes of exhausted T cell populations projected onto the UMAPs. (f) Heatmap showing the expression of cluster signature genes for each cluster from across all cell populations as annotated in Fig. 3a. (g) Annotated UMAP plot of single P14 T cells from spleen and LN of LCMV-Docile infected mice showing expression of the stem-like T_PEX cell transcriptional signature²². (h) Box plot showing transcriptional diversity scores (CytoTRACE)³³ among splenic and LN-derived T_PEX (Spleen: n = 2175; LN: n = 995) and T_EX cells (Spleen: n = 2266; LN: n = 1346). (i) Annotated volcano plots showing differentially expressed (DE) genes between splenic and LN total Cx3cr1⁺ T_EX cells (left) and between Cx3cr1⁺S1pr5⁺ T_EX and Cx3cr1⁺ T_EX cells independent of tissue of origin (right). Dots in graphs (b–d) represent individual mice; bars represent median or mean. Box plot in (h) defines range (maxima and minima), interquartile range and median of the dataset. Quantification and statistics derive from two-tailed t tests (h) and one-way ANOVA (a–d) and based on all data points across at least two independent experiments (a–d).