Single-cell Sequencing Reveals That The Interaction Between HLA-C And KIR2DL Inhibits The Cytotoxicity Of NK Cells In Patients With Hepatoblastoma

Objective: This study aimed to explore the changes in immune cells in patients with hepatoblastoma(HB)using single-cell transcriptome sequencing technology.We focused on immune-targeted therapy and explored potential immunotherapeutic targets by analyzing the expression changes of immune-related genes in HB patients.Methods: This study selected two children with HB(experimental group S1 and S2)and one healthy child(control group S3)as subjects and collected peripheral blood as samples for comparative analysis.Peripheral blood mononuclear cells(PBMCs)were isolated from the samples using density gradient centrifugation,and sequencing libraries were constructed for high-throughput sequencing.The raw data were processed using Cell Ranger software,and further quality control was performed using the R programming language to obtain PBMC gene expression profile information.The R programming language was used for principal component analysis,cell clustering,marker gene selection,and cell annotation to obtain the characteristics of changes in immune cell numbers.KEGG,GO,and GSEA analyses were conducted on NK cell genes,key genes were identified,and differences in gene expression among different groups were described.KEGG pathway analysis was conducted on the signaling pathways and biological processes of the key genes.Results: After quality control,transcriptome information of 9014,8178,and 8615 cells from experimental group(S1,S2)and control group(S3)were obtained.Using cell clustering,S1,S2,and S3 were clustered into 13,17,and 12 cell clusters,respectively.After merging the three samples,20 cell clusters were obtained.Based on the marker genes of these cell clusters,cell annotation was performed,and 11 cell types were identified.Natural killer(NK)cells were the cell type with the most significant change in number.Further differential gene expression analysis was performed on NK cells,revealing 310 differentially expressed genes,including 122 upregulated genes and 188 downregulated genes.GO and KEGG analysis showed that KIR2DL4,a gene upregulated in NK cells,was closely related to the cytotoxicity of NK cells.Further GSEA analysis of the NK cell gene set identified 96 genes associated with the cytotoxicity of NK cells,ofwhich 34 core genes(including KIR2DL1,KIR2DL3,and KIR2DL4)were highly expressed in HB.The average expression levels of KIR2DL1,KIR2DL3,and KIR2DL4 genes in different groups were further described,and significantly upregulated expression levels were observed in the experimental group.Conclusion: Compared to healthy children,this study found that the number of NK cells increased significantly in children with HB,and KIR2 DL genes were highly expressed in NK cells of children with HB.Analysis of the NK cell cytotoxicity signaling pathway(KEGG pathway: map04650)revealed that the interaction between the HLA-C ligand on the surface of tumor cells and the inhibitory receptor KIR2 DL on the surface of NK cells could suppress NK cell cytotoxicity,leading to immune evasion of tumors.KIR2 DL may become a potential therapeutic target to block the interaction between HLA-C and KIR2 DL,enhance NK cell cytotoxicity,improve anti-tumor effects,and provide a new strategy for the treatment of HB.