Extended Data Fig. 4: RNA velocity analysis of a divergent clone and regulatory programs of exhaustion.

(a) Heat maps depict the gene expression program of a gp33–reactive divergent clone, differentiating from the Tex^int stage into either Tex^term or Tex^KLR fates (left). Pseudotime order (direction of differentiation) was determined by RNA velocity analysis and is presented on a UMAP (right). (b) UMAPs of gp33⁺ CD8⁺ T cells from Arm infection at D8 and D21 and gp33⁻ at D21. Color gradient (RNA velocity pseudotime order) indicates directions of T cell differentiation fates determined by RNA velocity analysis. (c) UMAP of scATAC-seq results of D8 and D21 gp33⁺ and gp33⁻T cells from Cl13 infection. UMAP is colored by the annotated T cell subsets. Small UMAPs (right) show T cells that originate from the indicated gp33 fractions and timepoints. (d) Heat map of Peak score values at the unique open chromatin regions (OCRs) of the T cell subsets determined by scATAC-seq with a list of annotated putative target genes based on proximity (left, log₂ FC > 1, FDR < 0.05, p-values determined by two-sided Wilcoxon Rank Sum test and adjusted using the Benjamini & Hochberg procedure to obtain FDRs). Heat map of motif enrichment results at the unique OCR sets of the annotated T cell subsets (right, p-values determined by hypergeometric enrichment and adjusted using the Bonferroni correction method). (e) Upset plot of differentially accessible OCRs relative to T^naive at the Tox gene locus and their overlap among the different Tex subsets (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). Violin plot shows the gene expression level of Tox in the identified Tex subsets. Box center line, median; box limits, upper and lower quartiles; box whiskers, 1.5× interquartile range.