Extended Data Fig. 4: Phenotypic, functional, transcriptional and epigenetic characterization of TCR_TAG and TCR_OT1 cells in liver tumours.

a, Approximately 3 × 10⁴ TCR_TAG (TAG, red; Thy1.1⁺) and TCR_OT1 (OT1, black; Ly5.1⁺) T cells were transferred into wild-type B6 mice or liver tumour-bearing AST×Alb-Cre mice and immunized with 5 × 10⁶ CFU of Listeria Lm_TAG-I-OVA. Three to four weeks after immunization, livers from AST×Alb-Cre mice and spleens from B6 mice were analysed for the presence of donor TAG and OT1 T cells by FACS; the percentages of CD8 T cells are shown. Expression of CD62L, CD44, CD69 and Ki67 of TAG and OT1 T cells. Naive T cells are shown in grey as a control. CD107 expression after 4-h TAG or OVA peptide stimulation of TAG and OT1 TILs isolated 3–4 weeks after transfer. Flow plots are gated on CD8⁺Thy1.1⁺ and CD8⁺Ly5.1⁺ cells. Data are representative of three independent experiments. b, Heat map of RNA-seq-normalized expression values (log₂(counts per million)) across all samples (colour corresponds to z-scores) for genes differentially expressed between TAG and OT1 T cells (FDR < 0.05). c, GSEA of RNA-seq data generated from TAG and OT1 T cells isolated from AST×Cre liver lesions 3 weeks after adoptive transfer and Listeria infection. Gene sets used: T cell exhaustion during chronic viral infection²⁰ (GEO accession GSE30962) and mutant/constitutively-active form of NFAT1-overexpressing CD8 T cells²¹. NES, normalized enrichment score. d, Venn diagrams showing the numbers and percentage of significantly opening (left) and closing (right) peaks between TAG and OT1 T cells (FDR < 0.05, log₂-transformed fold change > 2). e, Genome browser view of ATAC-seq signal intensities of TAG and OT1 T cells at Pdcd1, Entpd1, Cd38 and Cd244 loci. Red or blue boxes indicate peaks that become significantly more accessible or inaccessible in TAG versus OT1 T cells, respectively. ATAC-seq peaks from naive TAG T cells are shown in grey as a control. f, Chromatin accessibility heat map for TAG and OT1 T cells. Each row represents one peak (differentially accessible between TAG and OT1 T cells; FDR < 0.05) displayed over a 2-kb window centred on the peak summit; regions were clustered using k-means clustering. Genes associated with individual clusters are highlighted.