Extended Data Fig. 5: Molecular programs of early effector- and progenitor-exhausted T cells and fate mapping experiments.

(a) UMAPs of scATAC-seq (left) and scRNA-seq (right) results from the infection models. Small UMAPs are colored by sample of origin (bottom). (b) Stacked bar plot depicts the phenotypic distribution of gp33⁺ CD8 T cells from scRNA-seq. (c) Volcano plot of differentially expressed genes (DEGs) between memory precursor T cells (T^mp) from Arm and the precursor exhausted T cells (Tex^prec) from Cl13 infections (top left). Differential gene expression analyses were performed as follows: log₂ FC > 0.25, Bonferroni adjusted p-value < 0.05, p-values determined by two-sided Wilcoxon Rank Sum test. Ingenuity pathway analyses of the T^mp andTex^prec specific gene sets. Volcano plot of the differential open chromatin regions (OCRs) of the T^mp and Tex^prec populations (top right). Differential OCR analyses were performed as follows: log₂ FC > 1, FDR < 0.1, p-values determined by two-sided Wilcoxon Rank Sum test and adjusted using the Benjamini & Hochberg procedure to obtain FDRs. Enriched transcription factor (TF) motifs in specific OCRs of T^mp andTex^prec subsets are shown (p-values determined by hypergeometric enrichment and adjusted using the Bonferroni correction method). (d) DEGs between the D8 effector T cells (T^eff) from Arm and early effector Tex cells (Tex^eeff) from Cl13 infections (top left). Same statistical approach was used as in (c). Ingenuity pathway analyses of the T^eff and Tex^eeff specific gene sets (bottom left). Volcano plot depicts the differentially accessible OCRs of T^eff and Tex^eeff populations (right). Same statistical approach was used as in (c). Enriched TF motifs in T^eff and Tex^eeff specific OCR sets (p-values determined by hypergeometric enrichment and adjusted using the Bonferroni correction method). (e) Expression of the indicated genes profiled by scRNA-seq. Box center line, mean; limits, upper and lower quartiles; whiskers, minimum and maximum values. (f) Schematic of the Tex^eeff adoptive transfer experiment with pre-transfer sorting strategy (top). Representative flow cytometry plots show the analysis of the phenotypic content of recovered T cells. Stacked bar plots show the phenotypic distribution from 3 biologically independent animals (mean % of each subset is shown with SDs). (g) Heat map of differentially expressed TFs in Tex subsets. (h) Upset plot of differentially accessible OCRs of Tex^KLR and Tex^term relative to Tex^int (log₂ FC > 1, FDR < 0.01, p-values determined by two-sided Wilcoxon Rank Sum test and adjusted using the Benjamini & Hochberg procedure to obtain FDRs) and their overlap. (i) Schematic of lineage tracing experiment (left). Gating strategy to analyze tdTomato⁺ fractions of Tex subsets. Boxplot of the % of tdTomato⁺ Tex subsets are shown. Box center line, mean; limits, upper and lower quartiles; whiskers, minimum and maximum values (n = 4 biologically independent animals).

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