Extended Data Fig. 4: The development of CX3CR1⁺ T_EX cells is defined by KLF2 expression.

(a) Diffusion maps showing all annotated cell clusters (as per Fig. 3a) and cluster distribution according to origin, spleen and LN. (b) UMAP representations (as per Fig. 3a) showing enrichment of transcriptional signatures for Trajectory 1 (Cx3cr1⁺ T_EX cells, top 50 genes) and Trajectory 2 (Cd101⁺ T_EX cells, top 50 genes), with reciprocal enrichments of these signatures in T_PEX cells bordered. (c) Box plots showing enrichment scores of Trajectory 1 and Trajectory 2 signatures among Sell⁺ and Sell⁻ T_PEX cell clusters. (d) Diffusion maps (as per Fig. 3e) showing the expressions of selected Traj. 2 genes that were also expressed in some T_PEX cells. (e–g) UMAP representation after correction using muVI single cell trajectory analysis using Palantir and CellRank of P14 cells scRNA-seq data showing (e) cell differentiation originating from Tcf7⁺ T_PEX cells and (f) progressing into either Cx3cr1⁺ (Traj. 1; upper) or Cd101⁺ (Traj. 2; lower) T_EX cells. (g) Box plot showing fate probabilities towards Traj. 1 among Sell⁺ and Sell⁻ T_PEX cell clusters. (h–o) 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 or LCMV-cl13. On d28 p.i. CD8⁺ T cells from spleen and pooled inguinal, brachial, axillary, cervical and mandibular lymph nodes (LN) were analysed using flow cytometry. (h, i) Flow cytometry plots showing the expression of (h) KLF2 and CD69, and (i) CD69 and CD62L expression among KLF2⁺ and KLF2⁻ T_PEX cells in the spleen and LN. (j) Quantification showing the frequency of CD69-expressing T_PEX cells stratified by KLF2 expression (n = 9). (k, l) Flow cytometry plots showing the expression of (k) KLF2 and CD69, and (l) CD69 and CD101 among KLF2⁺ and KLF2⁻ T_EX cells in the spleen and LN. (m) Quantification showing the frequencies of CD69-expressing T_EX cells stratified by KLF2 expression (n = 9). (n, o) Flow cytometry plots and corresponding quantification showing the frequencies KLF2⁺ cells among (n) T_PEX (n = 6) and (o) T_EX cell subsets (n = 6) in the spleen and LN. Dots in graphs (j, m–o) represent individual mice; bars represent median. Flow cytometry plots are representative. Box plots in (c, g) define ranges (maxima and minima), interquartile ranges and medians of the datasets. Quantification and statistics derive from unpaired two-tailed t tests (c, g) and one-way ANOVA (j, m–o) and based on all analysed mice across at least two independent experiments.