Extended Data Fig. 1: T cell activation and hepatic abundance correlate with NASH pathology.
From: NASH limits anti-tumour surveillance in immunotherapy-treated HCC
a–c, Time kinetics of haematoxylin and eosin (H&E) staining of liver tissue (a), ALT (b), and NAS (c) in mice fed ND, CD-HFD, or WD-HTF (n ≥ 5 mice per group). Scale bar, 100 μm. H&E 3 months: ND n = 5 mice; CD-HFD n = 5 mice; WD-HTF n = 3 mice; 6 months: ND n = 16 mice; CD-HFD n = 8 mice; WD-HTF n = 8 mice; 12 months: ND n = 9 mice; CD-HFD n = 12 mice; WD-HTF n = 6 mice; ALT 3 months: ND n = 15 mice; CD-HFD n = 46 mice; WD-HTF n = 23 mice; 6 months: ND n = 46 mice; CD-HFD n = 59 mice; WD-HTF n = 21 mice; 12 months: ND n = 25 mice; CD-HFD n = 69 mice; WD-HTF n = 5 mice; NAS 3 months: ND n = 5 mice; CD-HFD n = 5 mice; WD-HTF n = 3 mice; 6 months: ND n = 16 mice; CD-HFD n = 8 mice; WD-HTF n = 8 mice; 12 months: ND n = 9 mice; CD-HFD n = 12 mice; WD-HTF n = 6 mice. d, e, H&E staining (d) with NAS evaluation by H&E (e, left) and ALT (e, right) of mice fed with ND, HFD or CD-HFD for 3 months. NAS: ND n = 7 mice; CD-HFD n = 7 mice; HFD n = 5 mice; ALT: ND n = 8 mice; CD-HFD n = 8 mice; HFD n = 7 mice. Scale bar, 50 μm. f, g, Representative flow cytometry plots (f) and PD1 expression (g) of hepatic T cells from mice fed for 3 months with ND, HFD or CD-HFD (n = 4 mice per group). h, Heat map showing the median marker expression of the defined CD45+ subsets displayed in i by flow cytometry of cells from mice fed for 12 months with ND or CD-HFD (ND n = 4 mice; CD-HFD n = 8 mice). i, UMAP representation of FlowSOM-guided clustering and quantification of hepatic immune cell composition of mice fed for 12 months with ND or CD-HFD (ND n = 4 mice; CD-HFD n = 8 mice). j, k, Abundance (j), flow cytometry plots (k, left) and PD1 expression (k, right) of hepatic CD8+ T cells from mice fed for 6 or 12 months with ND or CD-HFD (abundance of CD8 6 months: ND n = 17 mice; CD-HFD n = 10 mice; WD-HTF n = 7 mice; 12 months: ND n = 11 mice; CD-HFD n = 6 mice; WD-HTF n = 5 mice; PD1 expression in CD8+ T cells 6 months: ND n = 15 mice; CD-HFD n = 14 mice; WD-HTF n = 7 mice; 12 months: ND n = 10 mice; CD-HFD n = 6 mice; WD-HTF n = 5 mice). l, m, Abundance (l), flow cytometry plots (m, left) and PD1 expression (m, right) of hepatic CD4+ T cells from mice fed for 6 or 12 months with ND or CD-HFD (abundance of CD4 6 months: ND n = 17 mice; CD-HFD n = 10 mice; WD-HTF n = 7 mice; 12 months: ND n = 11 mice; CD-HFD n = 6 mice; WD-HTF n = 5 mice; PD1 expression in CD4+ T cells 6 months: ND n = 15 mice; CD-HFD n = 14 mice; WD-HTF n = 7 mice; 12 months: ND n = 10 mice; CD-HFD n = 6 mice; WD-HTF n = 5 mice). n, H&E, CD8 and PD1 hepatic staining (top), and quantification of CD8+ cells and PD1+ cells by immunohistochemistry (bottom) from 32-week-old hURI-tetOFFhep and non-transgenic littermate control mice (n = 6 mice/group). Arrowheads, specific positive-staining cells. Scale bar, 100 μm. o, Hepatic abundance of TCRγδ T cells from mice fed for 6 or 12 months with ND or CD-HFD (6 months ND n = 8 mice; CD-HFD n = 6 mice; 12 months ND n = 8 mice; CD-HFD n = 6 mice). p, Left, quantification of hepatic Cd274+ expression by mRNA in situ hybridization of mice fed for 6 or 12 months with ND or CD-HFD (6 months: ND n = 6 mice; CD-HFD n = 6 mice; 12 months: ND n = 3 mice; CD-HFD n = 3 mice). Middle, quantification of hepatic PDL1+ expression by immunohistochemistry of mice fed for 12 months with ND or CD-HFD (ND n = 8 mice; CD-HFD n = 6 mice). Right, mRNA in situ hybridization (top) and PD1-stained micrographs (bottom). Scale bars, 100 μm. q, RNA velocity indicating transcriptional activity, gene expression, and the trajectory of CD8+ cells by scRNA-seq from 12 months ND or CD-HFD-fed mice. Root cells: yellow; blue cells: farthest away from root. End points: yellow indicates end point; blue cells: farthest away from defined end point. Latent time: pseudo-time by RNA velocity, dark color: start of velocity, yellow: end point of latent time. RNA velocity flow: Blue cluster: start point; orange cluster: intermediate; green: end point. Arrows: cell trajectory (n=3 mice/group). All data are shown as mean ± s.e.m. a, b, j–m, o, p, Two-tailed Student’s t-test. d–g, One-way ANOVA and Fisher’s LSD test. i, n, q, Two-tailed Mann–Whitney test.
