桥本甲状腺炎患者甲状腺与外周血T细胞的单细胞转录组分析

Single-cell transcriptome analysis of T cells in thyroid and peripheral blood of patients with Hashimoto′s thyroiditis

目的构建桥本甲状腺炎(Hashimoto′s thyroiditis,HT)患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)和甲状腺组织T细胞的单细胞转录图谱,分析在HT疾病状态下,T细胞各个亚群在比例和功能上的改变。方法对5例HT患者的PBMCs和甲状腺组织进行单细胞转录组测序,并从公共数据库中获取5名健康对照者PBMCs的单细胞转录组测序数据,初步聚类后,将表达CD3E的细胞聚类提取并再次聚类,根据已知的细胞标志物和高表达的基因确定每个聚类细胞亚群的细胞类型,统计每个细胞亚群的比例,并对不同样本的差异表达基因进行分析。结果将质量控制后得到的71533个T细胞分成了19个细胞亚群。其中,在HT患者甲状腺组织T细胞中,C1_CD4^(+)初始T细胞亚群、C3_CD4^(+)Treg细胞亚群、C7_CD8^(+)初始T细胞亚群、C8_GNLY-CD8^(+)T细胞亚群、C10_RORC^(+)CD8^(+)T细胞亚群、C11_GZMK^(+)CD8^(+)T细胞亚群、C12_CCL4^(+)CD8^(+)T细胞亚群和C18_PTGDS^(+)NK细胞亚群的比例与功能与其在健康对照者的PBMCs和HT患者的PBMCs T细胞中的比例和功能有着显著差异。结论HT患者甲状腺组织中多种T细胞在比例上与其在PBMCs中有着明显差异,其中3种高表达细胞杀伤相关基因的CD8^(+)T细胞亚群在HT患者甲状腺组织T细胞中的占比高于其在PBMCs T细胞中的占比。此外,HT患者甲状腺组织中多种T细胞的功能也与其在PBMCs中有着明显差异,与HT患者PBMCs中的细胞相比,在HT患者甲状腺组织中,一类GZMK^(+)CD8^(+)T细胞PD1通路相关的基因表达明显降低,且一类CCL4^(+)CD8^(+)T细胞中,IL-12相关信号通路基因的表达明显降低。

Objective To construct single-cell transcription landscape of T cell in peripheral blood mononuclear cells(PBMCs)and thyroid tissue of patients with Hashimoto′s thyroiditis(HT),and to analyze the changes in the proportion and functionality of T cell clusters in HT disease state.Methods Single cell RNA sequencing was performed on PBMCs and thyroid tissue from 5 HT patients.Single cell RNA sequencing data of PBMCs from 5 healthy individuals were retrieved from public databases.After preliminary clustering,the clusters expressing CD3E were extracted and clustering again,and the names of each cluster were determined according to the known cell markers.The proportion of each cell subtype was compared,and the differentially expressed genes in different samples were analyzed.Results After quality control,the 71533 T cells were classified into 19 cell clusters.Among them,the proportion and function of C1_CD4^(+)Naive T cell clusters,C3_CD4^(+)Treg cell clusters,C7_CD8^(+)Naive T cell clusters,C8_GNLY-CD8^(+)T cell clusters,C10_RORC^(+)CD8^(+)T cell clusters,C11_GZMK^(+)CD8^(+)T cell clusters,C12_CCL4^(+)CD8^(+)T cell clusters,and C18_PTGDS^(+)NK cell clusters in thyroid tissue of HT patients were significantly different from those in PBMCs of healthy controls and HT patients.Conclusion The proportion of multiple T cells in thyroid tissue of HT patients were significantly different from those in PBMCs.Among them,the proportion of three of CD8^(+)T cell subsets with high expression of cell killing-related genes in thyroid tissue T cells of HT patients is higher than that in PBMCs T cells,and it is statistically significant.In addition,the functionality of various T cells in the thyroid tissue of HT patients are also significantly different from those in PBMCs.A cluster of GZMK^(+)CD8^(+)T cells showes significantly lower expression of genes related to PD1 pathway in thyroid tissues of HT patients compared with cells in PBMCs of HT patients,also a cluster of CCL4^(+)CD8^(+)T cells showes significantly lower expression of genes related to IL-12 pathway.