Extended Data Fig. 6: The KLF2-instructed differentiation program involves molecules associated with lymph node trafficking, including CCR7 and S1PR1, which contribute to CX3CR1⁺ T_EX cell differentiation.

(a) Enrichment of a transcriptional signature for stem-like Sell⁺ T_PEX cells (ref. ²²) in control versus Klf2^Δ T_PEX cells. (b) Annotated UMAP plot showing single P14 T cells in spleen and LN. (c, d) Enrichment of transcriptional signatures projected onto a UMAP plot among genes (c) upregulated and (d) down-regulated comparing control and Klf2^Δ T_PEX cells, showing enrichment in Cx3cr1⁺ T_EX cells and Cd101⁺ T_EX cells, respectively. (e) Enrichment of transcriptional signatures among single P14 T cells derived from spleen and LN based on upregulated and down-regulated genes between control and Klf2^Δ T_PEX cells, showing reciprocal enrichment in splenic and LN P14 cells, respectively (Spleen T_PEX: n = 2175; LN T_PEX: n = 995; Spleen T_EX: n = 2266; LN T_EX: n = 1346). (f–h) Naïve congenically marked control and CCR7-deficient (Ccr7^Δ) P14 CD8⁺ T cells were generated using CRISPR-Cas9-mediated editing. Control and Ccr7^Δ P14 cells were cultured in the presence of IL7 for 2 days in vitro and surface expression of CCR7 were analysed using flow cytometry. (f) Schematic of in vitro experimental setup. (g) Flow cytometry plot and quantification of CCR7 expression in control and Ccr7^Δ P14 cells (n = 10). (h) Quantification showing the absolute numbers of control and Ccr7^Δ T_EX cell subsets in the spleen and LN on d26 p.i. (n = 10). (i–l) Naïve congenically marked control and S1pr1^Δ P14 CD8⁺ T cells co-transferred at a 1:1 ratio into naïve wildtype mice, which were subsequently infected with chronic LCMV-cl13 and analysed on d7 and d26 p.i. (i) Flow cytometry plots and quantification showing the expression of CD69 among control and S1pr1^Δ P14 cells in the blood at the specified time points (n = 9). (j) Flow cytometry plots and quantification showing the expression of CD69 among control and S1pr1^Δ P14 cells in the spleen and LN on d26 p.i. (n = 9). (k) Quantification showing the relative accumulation of control and S1pr1^Δ T_PEX and T_EX cells in the spleen and LN on d7 (n = 8) and d26 p.i. (n = 13). (l) Flow cytometry plots and corresponding quantification showing the frequencies of Ki67⁺CX3CR1⁺ cells among control and S1pr1^Δ P14 cells in spleen and LN (n = 9). Results in (g) represent four independent experiments. Flow cytometry plots are representative. Dots in graphs (h–l) represent individual mice; bars represent median. Box plots in (d, e) define ranges (maxima and minima), interquartile ranges and medians of the datasets. Quantification and statistics derive from two-tailed t tests (e, g) and one-way ANOVA (d, h–l) and based on all data points across at least two independent experiments (h–l).