Clinical manifestation of WD patients and WD causes poor prognosis of cholecystitis. a.

DNA sequencing result of 20 WD patients was summarized in the graph. b. The schematic diagram illustrates copper transport disturbances caused by ATP7B mutations, along with structural and functional impairments in mitochondria. Created with BioRender.com. c. Masson-trichrome staining of liver tissues of patients with WD and control. d. The picture of Kayser-Fleischer (KF) ring in the cornea of a patient with WD, captured during clinical examination. e. The table of clinical statistics of patients in 3 cohorts. Data are mean± S.D. Unpaired two-tailed t-test and chi-square test.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Single-cell profiling of non-parenchymal cells in the liver of WD patients. a.

The workflow of scRNA-seq. Created with BioRender.com. b. UMAP visualization of 26 principal components of all the single cells. c. UMAP visualization of 26 principal components by samples. d. UMAP visualization of 12 main cell types. e. The dot plot showing the expression of top 3 marker genes in 12 main cell types.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

The altered immune microenvironment in WD patients. a.

UMAP visualization of MPs colored and labeled by cell subtypes (left). The bar chart showing the average percentage of different subtypes in case group (n=3) and control group (n=3) (right). Data are mean ± S.D. b. UMAP visualization of T and NK cells colored and labeled by cell subtypes (left). The bar chart showing the average percentage of different subtypes in case group (n=3) and control group (n=3) (right). Data are mean± S.D. c. The heatmap showing the top 20 differently expressed genes (DEGs) for all immune cells. d. The dot plot showing GO enrichment on DEGs of all immune cells between the case and control group. BP, biological processes; CC, cellular components; MF, molecular function. e. The scatter showing the strength of all cell subtypes in signal incoming and outgoing. Upper, the case group; down, the control group.

The dysfunction of main immune cells in WD patients. a.

The violin plots displaying M1 polarization, M2 polarization, pro-inflammatory signature, and interferon responsed signature of the 4 subtypes within macrophages between two groups by gene scoring. b. The violin plots displaying M1 polarization, M2 polarization, pro-inflammatory signature, and interferon responsed signature of the 3 subtypes within Kupffer cells between two groups by gene scoring. c. The violin plots displaying maturation, phagocytosis, chemotaxis, chemokine activity and Type Ⅰ interferon signaling pathway scores within neutrophils between two groups. d. The heatmap showing the relative expression level of genes in each cell subtype between the case and control group. e. The dot plot showing biological process of GO enrichment on differentially expressed genes (DEGs) in monocytes between the case and control group. Upper, upregulation; down, downregulation. f. The violin plots displaying cytotoxicity and exhaustion scores of the 6 cell subtypes within CD8 T cells between two groups by gene scoring. g. The violin plots displaying Treg scores of the 3 cell subtypes within CD4 T cells between two groups by gene scoring.

Identification of NK cell exhaustion in WD patients. a.

The violin plots showing the relative expression level of genes in NK cells between the case and control group. ***, P<0.001; Wilcox test. b. Trajectories of NK cells in the case and control group. c. Representative immunofluorescence staining for NK cells (CD56) and expression of NKG2A in liver tissues of WD and control. Scale bars, 100 μm for 40X and 50 μm for 100X. NKG2A+ NK cells are highlighted by arrows. Quantifications were performed by assessing three random fields per slide. Data are mean± S.D. Unpaired two-tailed t-test. d. Gating strategy applied in flow cytometry. e. The bar charts showing the percentage of NKG2A+ (WD, n=5; Control, n=5), TIGIT+ (WD, n=3; Control, n=3), and IFNγ+ (WD, n=4; Control, n=4) NK cells. Data are mean± S.D. Unpaired two-tailed t-test.

NK cell exhaustion predicts poor prognosis of inflammatory diseases. a.-d.

Kaplan-Meier survival curves for cholecystitis, pancreatitis, lung adenocarcinoma, and hepatocellular carcinoma according to the markers of NK cell exhaustion in the GSE166915, GSE91035, GSE75037, and GSE41804 datasets.