The Mechanism Of CXCR6 Regulating Invariant Natural Killer T Cells In Liver Anti-tumor Immunity

ObjectivesGlobally,hepatocellular carcinoma ranks second in the world in terms of incidence and mortality,and hepatocellular carcinoma is the most common liver cancer,accounting for approximately 90%of all liver cancer incidence.An increasing number of studies have shown that invariant natural killer T(iNKT)cells have an important regulatory role in the hepatic immune system and are closely associated with the development of liver diseases.iNKT cells may be involved in regulating the progression of hepatocellular carcinoma and become a new immunotherapeutic target,but their specific mechanisms of targeting tumor cells are not fully understood.The aim of this study was to investigate the specific mechanisms of iNKT cells involved in anti-tumor immunity during the formation of hepatocellular carcinoma.MethodsWe constructed Hepa1-6 cell line with stable high expression mCherry fluorescent protein,and after counting 2×105 cells,the cells were injected in situ into the left lobe of CXCR6GFP/+mouse liver to construct a mouse in situ hepatocellular carcinoma model.Changes in the proportion and distribution of hepatic iNKT cells were observed by flow cytometry and confocal microscopy in vivo imaging of mice at the early stage of hepatocellular carcinoma.Flow cytometry was used to verify whether the distribution of iNKT cells in the liver were altered after CXCR6 knockdown.While primary hepatocytes were extracted,immune cells were sorted by magnetic beads,we mixed hepatocytes and immune cells in proportion and performed single-cell sequencing.We analyzed the single-cell sequencing results to found the specific mechanism by which CXCR6 drives the altered distribution of iNKT cells.We assessed the activation status of liver iNKT cells by observing their motility trajectory by confocal microscopy,and their activation markers and tumor cell killing markers were detected by flow cytometry.The expression of CXCR6 ligand CXCL16 protein were analyzed by Western-blot technique.We analyzed the results of single cell sequencing to find the cells which secreted CXCL16 to induce altered distribution of iNKT cells in the liver,then we verified it by flow cytometry.The interaction of iNKT cells with tumor cells was observed by confocal microscopy.To further observe the effect of blocked hepatic sinusoidal transmigration to the liver parenchyma on tumor formation,liver tumorigenesis was observed in CXCR6-/-mice at 2 weeks of in situ injection of hepatocellular carcinoma cells.The expression levels of tumor cell killing molecules such as IFN-γ and IL-4 in CXCR6+/-,CXCR6-/-mice liver iNKT cells and the ratio of CD4+T cells,CD8+T cells in the liver were detected by flow cytometry.Results1 Construction of in situ hepatocelluar carcinoma modelHepa1-6 cells stably expressing mCherry fluorescent protein were constructed and then injected into the liver of CXCR6GFP+mice at an amount of 2×105 per mouse in situ to visualize iNKT cells and Hepa1-6 cells.2 Confirming iNKT cells transmigrated from hepatic sinusoidal to parenchymal at the early stage of tumor formationFlow cytometry and confocal microscopy results showed that iNKT cells were significantly recruited in the liver within 8 h of in situ injection of Hepa1-6 cells.We use PEC AM-1 label hepatic sinusoids and the confocal microscopy results showed significant recruitment of iNKT cells in hepatic parenchyma during in situ injection of Hepa1-6 cells for 8 h;flow cytometry results by CD45-APC labeling of iNKT cells in hepatic sinusoids further verified that the proportion of iNKT cells in hepatic blood sinusoids was significantly reduced during in situ injection of Hepa1-6 cells for 8 h,and the proportion of hepatic parenchyma iNKT cell was significantly increased.3 CXCR6 mediates iNKT cells transmigrate from hepatic sinusoidal to parenchymal Flow cytometry results showed that iNKT cells transmigrate from hepatic sinusoidal to parenchymal was regulated by CXCR6.Single-cell sequencing results showed that immune function-related pathways expressed by hepatic iNKT cells were significantly downregulated in CXCR6-/-mice compared to CXCR6+/-mice upon in situ injection of Hepa1-6 cells at 8 h.4 Hepatic parenchyma iNKT cells activated molecules and tumor cell killing molecules were enhancedFlow cytometry results showed that in CXCR6+/-mice,iNKT cells entering the liver parenchyma expressed significantly higher activated markers and tumor cell killing markers;and most iNKT cells in the liver were quiescent under confocal microscopy.In contrast,in CXCR6-/-mice,the expression levels of activated molecules and tumor cell killing markers of iNKT cells in the liver parenchyma were not altered,and the iNKT cells in the liver still appeared more active under confocal microscopy.5 Hepatic sinusoidal endothelial cells secreted CXCL16 to recruit iNKT cells into liver parenchymaWestern-blot results showed that the expression level of CXCL16 protein,the ligand of CXCR6,was significantly higher in the mice,which was injected Hepa1-6 cell for 8 hours.Single-cell sequencing and flow cytometry results showed that hepatic sinusoidal endothelial cells can secret CXCL16 to recruit iNKT cells form hepatic sinusoid into hepatic parenchyma.6 Recorded the interaction between iNKT cells and tumor cellsThe whole process of tumor cell killing by iNKT cells was recorded by confocal microcopy.During tumor formation,the tumor cells gathered into clusters and iNKT cells were wrapped in the tumor cell clusters,while CXCR6 blocked iNKT cells hepatic sinusoidal-solid transition,there was no iNKT cells were wrapped in the tumor cell clusters.7 iNKT cells exacerbate tumorigenesis and attenuate CD4+T cell and CD8+T cell recruitment after blocked iNKT cells transmigrate to hepatic parenchymalHepatic tumorigenesis was exacerbated in CXCR6-/-mice after in situ injection of hepatocellular carcinoma cells for 2 weeks.The tumor cell killing molecules expression of liver iNKT cells were detected by flow cytometry,it showed that the expression of IFN-γ and IL-4 increased significantly with time,while the expression of IFN-γ and IL4 did not change in CXCR6-/-mice.Both liver CD4+T cells and CD8+T cells were significantly recruited at 2 weeks of in situ injection of hepatocellular carcinoma cells,whereas the ratio of liver CD4+T cells and CD8+T cells was not altered in CXCR6-/mice.ConclusionIn the early stage of hepatocellular carcinoma formation,hepatic iNKT cells were significantly aggregated and CXCR6 driven iNKT cells transmigrate from hepatic sinusoidal to parenchymal,and the activation and tumor cell killing function molecules of iNKT cells entering the hepatic parenchyma were significantly enhanced.During iNKT cells transmigrate from hepatic sinusoidal to parenchymal,CXCL16,the ligand of CXCR6,protein expression level increased significantly.CXCL16 was secreted by hepatic sinusoidal endothelial cells to recruit hepatic sinusoidal iNKT cells into hepatic parenchyma.The iNKT cells entering the liver parenchyma interacted with tumor cells and exerted anti-tumor immune functions.At advanced stages of hepatocellular carcinoma formation,liver tumorigenesis was intensified in mice after CXCR6 blocked iNKT cells transmigrate from hepatic sinusoidal to parenchymal,tumor cell killing molecules expressed by hepatic iNKT cells were not altered,and the recruitment of liver CD4+T cells and CD8+T cells was significantly blocked.