首页|基于单细胞转录组测序的细粒棘球蚴感染小鼠肝T细胞异质性分析

基于单细胞转录组测序的细粒棘球蚴感染小鼠肝T细胞异质性分析

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目的 从单细胞水平探究细粒棘球蚴感染小鼠不同时期肝组织微环境细胞中T细胞亚型组成及其转录谱特征.方法 从课题组前期细粒棘球蚴感染后1个月(1只)、3个月(1只)和6个月(2只)的BALB/c小鼠和健康小鼠(1只,对照组)肝组织的单细胞转录组测序数据集(中国国家生物信息中心组学原始数据归档库:CRA008416)(https://ngdc.cncb.ac.cn/gsa)中提取测序数据进行质控.采用统一流形逼近与投影(UMAP)算法对单细胞群聚类进行可视化作图,采用共享最近邻相似度(SNN)聚类算法分析最优细胞分群.采用SingleR软件包基于immgen参考数据集对细胞亚群进行细胞类型注释.使用Seurat软件包的FindMarkers函数分析不同时期感染小鼠和对照组小鼠的调节性T细胞(Treg)和CD8+T细胞的差异表达基因(DEG).利用基因本体论(GO)和京都基因与基因组百科全书(KEGG)分别对DEG进行功能富集分析和通路富集分析.结果 质控后获得37 760个细胞,人工优化后分为8种类型细胞.对T细胞重聚类后获得12个细胞群.注释后鉴定获得7种T细胞,包括CD4+初始T细胞、CD4+效应T细胞、Treg、CD8+初始T细胞、CD8+T细胞、增殖性T细胞、γ8 T细胞.在细粒棘球蚴感染1个月后T细胞各亚群占比无明显变化,感染后3个月增殖性T细胞(11.91%,56/470)和Treg(13.40%,63/470)占比均高于对照组(3.51%,38/1 082;4.34%,47/1 082),感染后6个月CD8+T细胞占比(30.20%,1 145/3 791)高于对照组(15.43%,167/1 082).Treg在感染后3个月高表达肿瘤坏死因子-α诱导蛋白8(Tnfaip8)、Maf、伊卡洛斯家族锌指3(Ikzf3)等维持Treg的基因;CD8+T细胞在感染后6个月高表达白细胞表面分化抗原40配体(Cd40lg)、类几丁质酶3(Chil3)、分泌型磷蛋白1(Spp1)等耗竭性基因.GO分析结果显示,感染后3个月Treg的DEG主要富集于转化生长因子β受体复合物组装、正调节T细胞活化、环磷酸腺苷介导的信号传导等通路;感染后6个月CD8+T细胞的DEG主要富集调节血管内皮生长因子受体、色氨酸分解代谢过程、细胞外基质细胞信号等通路.KEGG分析结果显示,感染后3个月Treg的DEG主要参与原发性免疫缺陷和Ras信号传导途径等通路;感染后6个月CD8+T细胞DEG主要参与脂肪酸代谢、谷胱甘肽代谢、叶酸代谢等通路.结论 细粒棘球蚴感染后3个月、6个月小鼠的肝组织T细胞亚型存在差异,感染后3个月Treg占比增加,感染后6个月CD8+T细胞占比增加,Treg、CD8+T细胞的DEG及其主要富集的通路存在差异.
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.

Echinococcus granulosusSingle-cell RNA sequencingT cellsImmune microenvironment

江楠、苏雅馨、蒋小凤、沈玉娟、曹建平

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中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),传染病溯源预警与智能决策全国重点实验室,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,科技部国家级热带病国际研究中心,上海 200025

上海交通大学医学院-国家热带病研究中心全球健康学院,上海 200025

细粒棘球绦虫 单细胞转录组测序 T细胞 免疫微环境

国家自然科学基金国家自然科学基金

8207230782272369

2024

中国寄生虫学与寄生虫病杂志
中华预防医学会,中国疾病预防控制中心寄生虫病预防控制所

中国寄生虫学与寄生虫病杂志

CSTPCD北大核心
影响因子:1.155
ISSN:1000-7423
年,卷(期):2024.42(3)