Extended Data Fig. 4: Isolation of CD8⁺ TILs for scRNA-seq and phenotypic characterisation of CD8⁺ TIL populations.

Ptger2^−/−Ptger4^fl/fl, Cd4^crePtger2^−/−Ptger4^fl/fl or Gzmb^crePtger2^−/−Ptger4^fl/fl mice were transplanted with 2 × 10⁶ control BRAF^V600E melanoma cells. After 11 days, CD8⁺ TIL populations were sorted from n = 4 tumours per group and analysed by scRNA-seq (b,d-h) or flow cytometry (c,i-k). a, Flow cytometry plots showing the sorting strategy. b, UMAP plots showing transcript expression of Cd44, Tox and Pdcd1 (encoding PD-1) as determined by scRNA-seq. c, Flow cytometric analysis of CD44, TOX and PD-1 protein expression in CD8⁺ TILs. d, Heatmap showing the expression of cluster signature transcripts (top 50). e,f, Correlation of TCF1⁺CD8⁺ TIL cluster gene expression with gene signatures of e naive CD8⁺ T cells, memory stem cell CD8⁺ T cells (TSCM) and central memory CD8⁺ T cells (TCM) or f naive CD8⁺ T cells, tumour antigen-specific CD8⁺ T cells in tdLNs and tumour-infiltrating stem-like CD8⁺ T cells. g, UMAP visualisation of transcript expression of indicated immune genes as determined by scRNA-seq. h, Expression levels of selected immune genes across CD8⁺ TIL clusters. i, Flow cytometric analysis of GZMB and CD62L expression among TCF1⁺ and TIM-3⁺ CD8⁺ TIL populations from a WT mouse. j, Flow cytometric analysis of TIM-3 and CXCR6 protein expression in CD8⁺ TILs from a WT mouse. k, Analysis of GZMB expression in activated (CD44⁺) CD8⁺ T cells isolated from tumours, tdLNs and spleen. Numbers indicate percentage of GZMB⁺ cells compared to isotype control. Plots in a,c,i-k show data for one tumour representative for n = 6 tumours from one (a,b), two (c,i,k), or three (j) independent experiments or pooled data from n = 4 biological replicates from one experiment (d-h). P values in e,f are from pairwise comparisons using Wilcoxon rank sum test and Bonferroni correction for multiple testing. P ≥ 0.05, not significant (NS).