Extended Data Figure 6: IgA ablation inhibits, while CD8 deficiency accelerates HCC development.

a, b, Total DNA was extracted from HCC nodules of 11-month-old mice (n = 16) of the indicated genotypes with or without anti-PD-L1 treatment, and subjected to exome sequencing. Shown are the number of point mutations identified per sample (a) and mutational signatures (b). Horizontal axis shows the 96-substitution patterns with substitution subtypes on top, and vertical axis indicates the probability of each pattern in b. c, Top 20 hallmark gene sets sorted by normalized enrichment score (NES) are shown to depict HCC progression in MUP-uPA and MUP-uPA/Iga^−/− mice by gene set enrichment analysis. Immune-related gene sets are coloured blue. The gene sets previously described^20,57 for human HCC are marked with a red asterisk. d, Total RNA was extracted from livers of 6-month-old mice and from HCC nodules of 11-month-old mice of the indicated genotypes and subjected to RNA-seq analysis. Hierarchical clustering of gene expression profiles comparing HCC and non-HCC mouse samples according to the RNA-seq. The top three enriched pathways/functional categories from Metascape are reported for major clusters of genes. e, Representative images of liver histology at different time points and indicated strains are shown with detailed n values. Scale bars, 100 μm (3 months), 250 μm (6, 11 months). f, Total tumour numbers in 3-month-old MUP-uPA mice (n = 11, 7, 8). g, Heat map depicting differential expression of 17 liver-specific genes and 33 HCC-related genes in the indicated strains, illustrating the upregulation of some HCC-related genes in MUP-uPA/Cd8a^−/− livers at 6 months of age (total mice number 29; n = 3 or 4 per group). h–j, BL6 mice of the indicated phenotype were subjected to the STAM protocol and their tumour volumes (n = 14, 6, 6, 4, 3, 9, 3) (h) and histopathology (i, j) were evaluated at 25 weeks of age. The data were validated at least in two or three experiments. Paraffin-embedded and frozen liver sections from these mice were stained with haematoxylin and eosin, Sirius Red, or Oil Red O, as indicated. Shown are typical images of tumour-containing and tumour-free areas, the borders between which are marked by the black lines. Scale bars: haematoxylin and eosin, 100 μm; Sirius Red, 100, 250, or 500 μm; Oil Red O, 50 μm. Oil Red O-positive areas were quantitated and are shown on the right. The Sirius Red-stained areas for each mouse were calculated by image analysis of the whole-tissue scan and normalized to the haematoxylin and eosin stain (n = 3 or 4 per group). k, Tumour volumes are shown for STAM-WT (n = 10), STAM-Iga^−/− (n = 13), and STAM-Iga^−/− after CD8 depletion (n = 3). l, MUP-uPA/Iga^−/−-HFD mice were injected weekly with anti-CD8 for 6 weeks and tumour multiplicity was determined (n = 3). k, l, CD8 depletion experiments were repeated using two different HCC models (MUP and STAM). m, Heat map depicting the differential expression of 59 genes involved in allograft rejection, IFNγ response, and inflammation (total mouse number 41; 6 months: n = 3 and 11 months: 3 or 4 per group). n, o, Paraffin-embedded and frozen liver sections from 11-month-old mice (n) and adoptively transferred mice (o) were stained with haematoxylin and eosin, Sirius Red, or Oil Red O as indicated and analysed (Sirius Red: n = 9, 16, 14, 6; Oil Red O: n = 8, 5, 5, 8 for n). Shown are typical images of tumour-containing and tumour-free areas, the borders between which are marked by the black lines. p, Liver cells from MUP-uPA/Rag1^−/− mice 1 week after being adoptively transferred with CFSE-labelled T cells with or without B cells as indicated (n = 3 in each group) were stained and analysed by flow cytometry. Shown are the percentage of CD8⁺ T cells among CD45⁺ cells (left), and histogram of proliferating CFSE-labelled T cells with the corresponding mean fluorescence intensity (right). q, Liver sections for MUP-uPA/Rag1^−/− and the corresponding adoptive lymphocyte transfer mice (4 weeks after adoptive transfer (AT)) were stained with alpha-SMA, IgA, CD3, and CD8 antibodies, counterstained with DAPI and examined by fluorescent microscopy (scale bars, 50 μm). For CD3/CD8 staining, images with higher magnification are shown (scale bars, 20 μm). Single-cell suspensions were prepared from the corresponding liver, stained with antibodies to CD45, IgA, CD19, B220, CD8, and CD4, and analysed by flow cytometry. Shown are representative scatter plots. MUP;Rag1^−/− mouse livers have been used for validation of CD4, CD8, IgA, and CD19, both for flow cytometry and for immunofluorescence analyses. The data were validated in at least two experiments. r, STAM-BL6 mice of the indicated phenotypes were analysed for IgA serum amounts by ELISA (n = 11, 4, 4, 4, 4). s, Absolute IgA⁺ cell number in livers of indicated STAM-BL6 mice (n = 13, 4, 4, 9, 8). Each dot represents a mouse. Two-sided t-test (means ± s.e.m.; f, j, l, n, r) and Mann–Whitney test (median; h, k, n, s) were used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001. N values for each group are shown either in individual panels or in legends for each group from left to right accordingly.