Heterogeneity analysis of T cells in liver of mice infected with Echinococcus granulosus based on single-cell RNA sequencing
Objective To explore the composition and transcriptional profile characteristics of T cell subtypes in liver tissue microenvironment cells of mice infected with Echinococcus granulosus at different time points at the single-cell level.Methods Data were extracted from the single-cell RNA sequencing dataset(genome sequence ar-chive:CRA008416)of BALB/c mouse liver tissue at 1 month(1 mouse),3 months(1 mouse)and 6 months(2 mice)after E.granulosus infection and healthy mouse(1 mouse,control group)in the previous study of the research group and quality control was conducted.The uniform manifold approximation and projection(UMAP)method was used to vi-sualize the single cell clusters,and the clustering algorithm adopted shared nearest neighbour(SNN)to obtain the opti-mal cell clusters.SingleR software package was used for cell type annotation of cell subsets based on the immgen ref-erence dataset.FindMarkers function from Seurat software package was used to analyze differentially expressed genes(DEGs)of regulatory T cells(Tregs)and CD8+T cells in mice infected at different time points and control group mice.Functional enrichment and pathway enrichment of DEGs were analyzed using gene ontology(GO)and Kyoto En-cyclopedia of Genes and Genomes(KEGG),respectively.Results After quality control,37 760 cells were obtained,which were divided into 8 types after manual optimization.After re-clustering the T cells,12 cell groups were obtained.Seven T cell subtypes were annotated and identified,including CD4+naive T cells,CD4+effector T cells,Tregs,CD8+naive T cells,CD8+T cells,proliferative T cells,γδ T cells.The proportion of each T cell subtype did not change significantly at 1 month after E.granulosus infection.The proportion of proliferative T cells(11.91%,56/470)and Tregs(13.40%,63/470)were significantly higher than those in control group(3.51%,38/1 082;4.34%,47/1 082)at 3 months after infec-tion.The proportion of CD8+T cells(30.20%,1 145/3 791)was significantly higher than that of the control group(15.43%,167/1 082)at 6 months after infection.Tregs showed high expression of tumor necrosis factor-α-induced pro-tein 8(Tnfaip8),Maf,IKAROS family zinc finger 3(Ikzf3)and other Treg-maintaining genes at 3 months after infec-tion,while CD8+T cells showed high expression of depletion genes such as CD40 ligand(Cd40lg),chitinase-like 3(Chil3),secreted phosphoprotein 1(Sppl)at 6 months after infection.GO analysis showed that DEGs of Tregs were mainly concentrated in transforming growth factor beta receptor complex assembly,positive regulation of T cell activation,cyclic adenosine monophosphate(cAMP)mediated signalling pathway at 3 months after infection;while the DEGs of CD8+T cells were mainly concentrated in the regulation of vascular endothelial growth factor receptor,tryptophan catabolic pro-cess,extracellular matrix-cell signalling pathways at 6 months after infection.KEGG analysis showed that DEGs of Tregs were mainly involved in primary immune deficiency and Ras signalling pathway at 3 months after infection;while the DEGs of CD8+T cells were mainly involved in fatty acid metabolism,glutathione metabolism,folate metabolism and other pathways at 6 months after infection.Conclusion There are differences in T cell subtypes in liver of mice at 3 months and 6 months after E.granulosus infection;the proportion of Tregs increased at 3 months,and CD8+T cells increased at 6 months after infection.There were differences in DEGs and their main enrichment pathways of Tregs and CD8+T cells.