Extended Data Fig. 4: Preventive anti-PD1 treatment drives hepatocarcinogenesis in a CD8-dependent manner in NASH.
From: NASH limits anti-tumour surveillance in immunotherapy-treated HCC
a, Histological staining of hepatic tissue with H&E, Sirius Red and PD1 from mice fed for 12 months with ND or CD-HFD and treated for 8 weeks with IgG, anti-CD8 or anti-PD1 antibodies (H&E: ND n = 24 mice; CD-HFD n = 40 mice; CD-HFD + anti-CD8 n = 29 mice; CD-HFD + anti-PD1 n = 36 mice; Sirius Red: ND n = 19 mice; CD-HFD n = 53 mice; CD-HFD + anti-CD8 n = 24 mice; CD-HFD + anti-PD1 n = 33 mice; PD1: ND n = 5 mice; CD-HFD n = 5 mice; CD-HFD + anti-CD8 n = 5 mice; CD-HFD + anti-PD1 n = 7 mice). Arrowheads, PD1+ cells. Scale bars, 50 μm. b–d, NAS evaluation by H&E (b), ALT levels (c) and histological staining of hepatic tissue by H&E and Sirius Red (d) of mice fed for 12 months with ND or CD-HFD, and untreated or treated for 8 weeks with anti-CD8 or anti-CD8 + anti-NK1.1 antibodies (fibrosis ND n = 19 mice; CD-HFD n = 53 mice; CD-HFD + anti-CD8 n = 27 mice; CD-HFD + anti-CD8/NK1.1 n = 6 mice; NAS: ND n = 24 mice; CD-HFD n = 40 mice; CD-HFD + anti-CD8 n = 29 mice; CD-HFD + anti-CD8/NK1.1 n = 6; ALT: ND n = 22 mice; CD-HFD n = 42 mice; CD-HFD + anti-CD8 n = 31 mice; CD-HFD + anti-CD8/NK1.1 n = 6). Scale bar, 100 μm. e, f, Flow cytometry plots of hepatic cells from mice fed for 12 months with ND or CD-HFD and treated for 8 weeks with anti-CD8 (e) or anti-CD8 + anti-NK1.1 (f) antibodies. g, Quantification by immunohistochemistry of PD1+ cells in hepatic tissue from mice fed for 12 months with ND or CD-HFD and untreated or treated for 8 weeks treatment with anti-CD8 or anti-PD1 antibodies (ND n = 5 mice; CD-HFD n = 5 mice; CD-HFD + anti-CD8 n = 5 mice; CD-HFD + anti-PD1 n = 7 mice). h, Assessment of metabolic tolerance by intraperitoneal glucose tolerance test of mice as in g (CD-HFD n = 8 mice; CD-HFD + anti-CD8 n = 10 mice; CD-HFD + anti-PD1 n = 9 mice). i, Relative quantification of hepatic leukocytes of mice as in g (CD3, NK T: CD-HFD n = 9 mice; CD-HFD + anti-CD8 n = 14 mice; CD-HFD + anti-PD1 n = 8 mice; CD4, CD8, CD19, NK, CD11b+, mDC: CD-HFD n = 9 mice; CD-HFD + anti-CD8 n = 17 mice; CD-HFD + anti-PD1 n = 8 mice; pDC: CD-HFD n = 9 mice; CD-HFD + anti-CD8 n = 13 mice; CD-HFD + anti-PD1 n = 8 mice; Kupffer cells (KC): CD-HFD n = 9 mice; CD-HFD + anti-CD8 n = 12 mice; CD-HFD + anti-PD1 n = 8 mice). More MHCII+ myeloid cells were found in the respective sub-populations. j, Flow cytometry analysis for polarization of hepatic CD4+ T cells from mice as in g (CD-HFD n = 12 mice; CD-HFD + anti-CD8 n = 17 mice; CD-HFD + anti-PD1 n = 17 mice). k, Flow cytometric analysis for polarization of hepatic myeloid cells of mice fed for 12 months with CD-HFD and untreated or treated for 8 weeks with anti-PD1 antibodies (CD-HFD n = 8 mice; anti-PD1 + CD-HFD n = 12 mice). l, Flow cytometric analysis for polarization of hepatic CD8+ T cells from mice as in k (CD-HFD n = 10 mice; anti-PD1 + CD-HFD n = 14 mice). m, Confocal analyses revealed clusters of CD8+ T cells with adjacent cleaved caspase 3+ hepatocytes that were strongly increased by anti-PD1-related immunotherapy in liver tissue from mice fed for 12 months with ND or CD-HFD and treated for 8 weeks with IgG or anti-PD1 antibodies, suggesting increased necro-inflammation in the vicinity of CD8+ T cells (n = 27 FOV in 3 mice per group). Scale bars, 30 μm. n, GSEA of RNA-seq data for hepatic tissue from mice fed for 12 months with CD-HFD and treated for 8 weeks with anti-CD8, anti-CD8 + anti-NK1.1 or anti-PD1 antibodies (n = 5 mice per group) revealed enrichment for TNF signalling via NF-κB and inflammatory responses. Deletion of NK1.1+ cells altered the cholesterol homeostasis-related signature, suggesting a link between NK T cells and aberrant cholesterol metabolism. Moreover, tissue from mice treated with anti-PD1 antibodies revealed positive enrichment of apoptosis, inflammatory responses and epithelial–mesenchymal transition, indicating a pro-inflammatory, pro-carcinogenic liver environment upon anti-PD1 treatment. o, Livers from mice fed for 12 months with CD-HFD and treated for 8 weeks with IgG or anti-PD1 antibodies. Arrowheads, tumours or lesions. Scale bar, 10 mm. All data are shown as mean ± s.e.m. b, c, One-way ANOVA and Fisher’s LSD test. h, Two-way ANOVA and Sidak’s multiple comparison test. i–l, Two-tailed Student’s t-test. m, Two-tailed Mann–Whitney test.
