Extended Data Fig. 9: Co-culture experiment of cell line-PBN-CD8⁺ T cells and analyses of two IFIT⁺ neutrophil subsets.

a, Experimental workflow. b, Gating strategy separating neutrophils from CD8⁺ T cells in the bottom chamber of co-culture system in a. c, FACS analysis showing the expression of CD25 (n = 3), CD69 (n = 3), and IFNγ (n = 4) in PBNs co-cultured with different cell lines. Student’s t-test, one-sided. Data are presented as mean values ± SEM. d, FACS analysis showing the expression of IFNγ in CD8⁺ T cells when anti-PD-L1 or the IgG control is added to the co-culture system. e, FACS analysis showing the expression of CD25, IFNγ, GZMB, and PRF1 in CD8⁺ T cells co-cultured with matched TAN or non-TAN isolated from patients with liver cancer. f, Volcano plot showing differentially expressed genes between Neu_03_ISG15 and Neu_09_IFIT1. Benjamini-Hochberg adjusted Wilcoxon rank-sum test, two-sided. g, Heatmap showing the predicted ligand activity by NicheNet on genes highly expressed in Neu_09_IFIT1. Pearson correlation indicates the ability of each ligand to predict the target genes, and better predictive ligands are thus ranked higher. h, Dot heatmap showing the selected ligand-receptor pairs between different cell populations and Neu_09_IFIT1. Benjamini-Hochberg adjusted permutation test. i, Boxplots showing the proportion of two IFNG⁺ populations between patients with or without Neu_09_IFIT1. Wilcoxon rank-sum test, two-sided. (Yes, n = 33 cases, No, n = 78 cases). For boxplots, centre line shows median, box limits indicate upper and lower quartiles, and whiskers extend 1.5 times the interquartile range, while data beyond the end of the whiskers are outlying points that are plotted individually. j, Pearson correlation between the expression of CD274 and IFNG in TIME cells in this study (left) or in the collected bulk RNA-seq datasets (right). In c and i, n denotes biologically independent samples.

Source data