Extended Data Fig. 4: Transcriptional characterization of individual genes and pathways in EnhDel cells early and late in chronic infection.
From: Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control
(a) Heatmap of the pseudobulk normalized expression per gene, averaged across sample replicates (n = 2 per genotype). Genes ordered by gene weight in axis 1 from the PCA model (PC2 loading). A curated set of genes is highlighted. (b) Violin plot of Car2 (left) and Cd7 (right) gene expression across cells grouped by cluster. The terminal cluster was used as reference; significance calculated using a Wilcoxon rank sum test. (c) Violin plot of Tox (left) and Ikzf2 (right) gene expression, cells grouped by cluster and genotype. Genotypes within a cluster were compared; significance calculated using a Wilcoxon rank sum test. (d) Pre-ranked GSEA of an apoptosis signature (HAMAI_APOPTOSIS_VIA_TRAIL_UP) in EnhDel versus WT (top) and EnhDel versus Pdcd1-KO (bottom) differentially expressed genes, analyzed separately by cluster (left: Prog., center-left: Eff.-like, center-right: Trans., right: Term.). Rank of genes at the bottom. (e) Violin plots of genes containing potential CRISPR off-target editing sites. Gene name indicated at column header. Cells grouped by genotype. Significance calculated using a Wilcoxon rank sum test. (f) Venn diagrams of overlap between D9 and D30 significant gene sets. Significant gene sets identified by pre-ranked GSEA analysis comparing EnhDel and WT cells. Top: Prog., bottom: Term. Number of gene sets in each Venn area. Significance of D9 vs. D30 overlap (Fisher’s exact test) listed. (g) Pre-ranked GSEA of an oxidative phosphorylation signature (HALLMARK_OXIDATIVE_PHOSPHORYLATION) in D9 EnhDel versus WT differentially expressed genes, analyzed separately by cluster (left: Prog., right: Term.). Rank of genes at the bottom. (h) Pre-ranked GSEA of an effector-versus-exhausted CD8+ T cell signature (GSE9650_EFFECTOR_VS_EXHAUSTED_CD8_TCELL_UP) in D9 EnhDel versus WT differentially expressed genes, analyzed separately by cluster (left: Prog., right: Term.). Rank of genes at the bottom. Asterisks used to indicate significance correspond to the following: ns, not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.
