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.

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. f. The bar chart showing the proportion of 12 main cell types in each sample.

An overview of the 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 (n=3) and CON (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 (n=3) and CON (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 CASE and CON. BP, biological processes; CC, cellular components; MF, molecular function. e. The scatter showing the incoming and outgoing interaction strength of all cell subtypes in CASE and CON.

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 of macrophages between the 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 of Kupffer cells between the two groups by gene scoring. c. The violin plots displaying maturation, phagocytosis, chemotaxis, chemokine activity and Type interferon signaling pathway scores of neutrophils between the two groups by gene scoring. d. The heatmap showing the relative expression level of genes of the 3 subtypes of DC cells between the two groups. The color represents the relative expression level. e. The heatmap showing the relative expression level of genes of B cells and plasma cells between the two group. The color represents the relative expression level. f. The dot plot showing biological process of GO enrichment on differentially expressed genes (DEGs) in monocytes between the two group. Upper, upregulation; down, downregulation. g. The violin plots displaying cytotoxicity and exhaustion scores of the 6 cell subtypes of CD8 T cells between the two groups by gene scoring. h. The violin plots displaying Treg scores of the 3 cell subtypes of CD4 T cells between the 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 two group. ***, P<0.001; Wilcox test. b. Representative immunofluorescence staining for NK cells marked by CD56 and expression of KLRC1 in liver tissues of WD and control. The nucleus is stained by DAPI. Scale bars, 100 μm for 40X and 50 μm for 100X. KLRC1+ NK cells are highlighted by arrows. The bar chart showing the proportion of KLRC1+ NK cells. Quantifications were performed by assessing three random fields per slide. Data are mean± S.D. Unpaired two-tailed t-test. c. Gating strategy applied in flow cytometry. Upper: NK cells were determined by CD3-CD56+. Down: NK cells with positive indicators (KLRC1, TIGIT, and IFNγ) were recorded. d. The bar charts showing the percentage of KLRC1+ (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.