Mapping Of Infiltrating Innate Lymphoid Cells In Hepatocellular Carcinoma

Innate lymphoid cells(ILCs)are heterogeneous cells derived from bone marrow lymphocyte progenitors which do not express TCR or BCR and do not undergo antigen-specific receptor gene rearrangement.ILCs approved to be divided into five groups by the International Union of Immunological Societies,including NK cells,ILC1,ILC2,ILC3 and LTi cells.Most ILCs are tissue-resident cells that play an important role in immune regulation in the body’s microenvironment.ILCs have been reported to accumulate in the tumor microenvironment,but their exact function remains unclear.In addition,under the influence of specific cytokines in the tissue microenvironment,each subset of ILCs will undergo mutual transformation,thus making rapid response to changes in the local environment.Liver is an important organ for the study of innate immune cells.In addition,due to the immune tolerance characteristics of liver,the incidence of liver cancer is high,so what role does liver ILCs play in it?The advent of single-cell RNA sequencing provides us with a good means to study the heterogeneity of scarce cells,which has been used to discover previously neglected cell subsets.However,there are few studies on the functional status of human liver ILCs in normal liver or in the liver tumors.Therefore,we hope to find a new way to treat hepatocellular carcinoma by studying the transcriptional map of ILCs in normal liver,hepatocellular carcinoma tumor and tumor-adjacent tissue.The results are as follows:1.Human liver NK cells differ from spleen and peripheral blood NK cellsA comparative analysis of the single-cell transcription map of NK cells in human liver,spleen and peripheral blood showed that the transcription map of NK cells in spleen and peripheral blood were similar,while the NK cells in liver had a significant difference from them,indicating the organ specificity of NK cells in human liver.2.Seven subsets of ILCs in human liver identified by using unsupervised clusteringSeven ILCs subsets(L1～L7)were found in human normal liver by single-cell RNA sequencing data integration analysis.In addition to the known subsets,we also found a liver resident NK cell subset(L4-LrNK-FCGR3A)expressing CD 16 and a NK cell subset(L3-NK-HLA)expressing both a moderate level of CD56bright NK cell genes and a moderate level of CD56dim NK cell genes.And these two newly discovered subsets play a positive role in immune regulation.3.Six subsets of ILCs in liver cancer identified by using unsupervised clusteringIntegration analysis of single-cell RNA sequencing data revealed that there were six ILCs subsets(T1～T6)in human hepatocellular carcinoma,and two NK cell subsets with positive immunoregulation were missing compared with normal liver.At the same time,the large concentration of ILC2 in liver tumors suggested that ILC2 may play a role in promoting tumor development.4.The proportion of ILC2 in hepatocellular carcinoma was significantly higher than that in the tumor-adjacent tissueComparison of single-cell RNA sequencing data of ILCs from tumor and tumoradjacent tissue in a hepatocellular carcinoma patient further confirmed that the proportion of ILC2 in human hepatocellular carcinoma was significantly higher than that in the tumor-adjacent tissue.5.Four prognosis-related genes were elevated simultaneously in tumor and peripheral blood of hepatocellular carcinoma patientsTo find indicators that are convenient for clinical use,we explored genes that were elevated in both peripheral blood and tumors.Through TCGA database analysis,we finally determined four prognosis-related genes for hepatocellular carcinoma patients.Among them,patients with high expression of RHOB or HLA-DPA1 had better survival prognosis,while patients with high expression of TKT or S100A11 had worse survival prognosis.6.SLAMF1 is a potential immune checkpoint moleculeWe further verified that abundant ILC2 were infiltrated into hepatocellular carcinoma tumors by experiments.Through cell cycle analysis and RNA dynamics analysis,we found that ILCl in tumors had a tendency to transform into ILC3 and eventually into ILC2.At the same time,we also identified that ILC2 in tumor expressed higher level of SLAMF1 by flow cytometry.Combined with the clinical data and survival analysis of patients,we found that hepatocellular carcinoma patients with high expression of SLAMF1 had a poor survival prognosis.And this phenomenon existed in many types of tumors,including kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma.Finally,we further confirmed that SLAMF1+ILC2 played an immunosuppressive role in human hepatocellular carcinoma by bulk RNA sequencing.In conclusion,positive regulatory NK subsets were absent in hepatocellular carcinoma tumors compared to healthy liver tissues.While inhibitory ILC2 accumulated in hepatocellular carcinoma tumors,which may be caused by the conversion of other types of ILCs to ILC2.High expression of SLAMF1 in tumorinfiltrating ILC2 was associated with poor survival outcomes in hepatocellular carcinoma and other tumor types.It can be said that SLAMF1 is a potential immune checkpoint molecule.Our study identified four genes associated with survival prognostic that are elevated in both hepatocellular carcinoma tumor tissues and peripheral blood,and detection of these four genes in patients’ blood may provide a simple way to predict the prognosis of hepatocellular carcinoma patients.At the same time,we found a new immune checkpoint molecule SLAMF1,which may be used for immunotherapy of hepatocellular carcinoma and other tumors.