Simple SummaryNonalcoholic fatty liver disease (NAFLD) is rapidly becoming a major cause of cirrhosis and hepatocellular carcinoma (HCC). The risk of liver-related mortality, including HCC, significantly increases with the progression of liver fibrosis in NAFLD patients. This study aimed to characterize the molecular and cellular changes occurring during liver fibrosis progression in NAFLD patients. The identified gene and immune cell signatures may lead to novel surveillance and prevention strategies.AbstractThe expression of immune- and cancer-related genes was measured in liver biopsies from 107 NAFLD patients. The strongest difference in overall gene expression was between liver fibrosis stages F3 and F4, with 162 cirrhosis-associated genes identified. Strong correlations with fibrosis progression from F1 to F4 were observed for 91 genes, including CCL21, CCL2, CXCL6, and CCL19. In addition, the expression of 21 genes was associated with fast progression to F3/F4 in an independent group of eight NAFLD patients. These included the four chemokines, SPP1, HAMP, CXCL2, and IL-8. A six-gene signature including SOX9, THY-1, and CD3D had the highest performance detecting the progressors among F1/F2 NAFLD patients. We also characterized immune cell changes using multiplex immunofluorescence platforms. Fibrotic areas were strongly enriched in CD3+T cells compared to CD68+macrophages. While the number of CD68+macrophages increased with fibrosis severity, the increase in CD3+T-cell density was more substantial and progressive from F1 to F4. The strongest correlation with fibrosis progression was observed for CD3+CD45R0+memory T cells, while the most significant increase in density between F1/F2 and F3/F4 was for CD3+CD45RO+FOXP3+CD8−and CD3+CD45RO−FOXP3+CD8−regulatory T cells. A specific increase in the density of CD68+CD11b+Kupffer cells with liver fibrosis progression was also observed.