人胸腺基质发育及退化的单细胞转录组分析

doi:10.3969/j.issn.1674-8115.2021.07.004

摘要/Abstract

摘要：

目的·探索人类胸腺基质细胞在不同发育阶段的转录动态。方法·利用人类胸腺单细胞转录组数据E-MATB-8581，借助蛋白质相互作用数据库分析不同类型胸腺基质细胞在胚胎期、婴儿期和成人期的分群特点，并计算基因表达差异，结合富集分析推测它们功能变化。结果·相比于树突状细胞，胸腺上皮细胞（thymic epithelial cell，TEC）、内皮细胞（endothelial cell，Endo）和成纤维细胞（fibroblast，Fb）由不同发育阶段造成的基因表达差异更为显著。Ⅰ型髓质TEC（medullary TEC，mTEC）细胞在成年期的抗原提呈功能下降，主要组织相容性复合物（major histocompatibility complex，MHC）Ⅱ类分子表达下调。在出生后的胸腺中，Fb逐渐富集参与T细胞抗原识别、分化和激活的功能。在胚胎期和婴儿期，Endo上调表达的基因主要富集与细胞外基质等相关的通路，而出生后则富集抗原提呈相关通路，并上调2类MHC分子的表达。结论·生物信息学方法揭示了mTEC、Fb和Endo是易受胸腺发育和退化影响的细胞类型。人类胸腺生理性退化可能主要影响T细胞分化中的阴性选择。

关键词: 胸腺基质细胞, 发育, 阴性选择, 单细胞RNA测序, 基因表达

Abstract:

Objective·To explore the transcriptomic dynamics of human thymic stroma cells across different developmental stages.

Methods·The single-cell transcriptome datasets of human thymus were downloaded from ArrayExpress (E-MATB-8581). The cellular maps of thymic stroma cells across different developmental stages were analyzed based on protein-protein interaction. The differentially expressed genes of fetal, infant and adult stages were calculated. The cell state alternation across different developmental stages was predicted based on functional enrichment analysis.

Results·The transcriptomic dynamics of thymic epithelial cells (TEC), endothelial cells (Endo) and fibroblasts (Fb) were more remarkable compared with dendritic cells. In adult thymus, there was decline of antigen presenting function and down-regulation of major histocompatibility complex (MHC) class Ⅱ genes in medullary TEC type Ⅰ cells. The fibroblasts enriched functions related to antigen recognition, differentiation and activation of T cells in postnatal thymus. The up-regulated genes of thymic Endo enriched functions such as extracellular matrix during fetal and infant stages, while up-regulated MHC genes and enriched pathways associated with antigen presenting in adult thymus.

Conclusion·Bioinformatics analysis revealed that mTEC, Fb and Endo were the susceptible stroma cell types in response to thymic development and senescence. The involution of human thymus may affect the negative selection of T cell differentiation mostly.

Key words: thymic stroma, development, negative selection, single-cell RNA-sequencing, gene expression

中图分类号:

R714.51

李雨晨, 白丽莲, 黄荷凤, 刘欣梅. 人胸腺基质发育及退化的单细胞转录组分析[J]. 上海交通大学学报(医学版), 2021, 41(7): 865-875.

Yu-chen LI, Li-lian BAI, He-feng HUANG, Xin-mei LIU. Single-cell transcriptomic analysis of development and involution of human thymic stroma[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 865-875.

图/表 8

图1 人类胸腺基质细胞的UMAP分群图Note： A. Cells were colored according to cell populations (top-left), developmental time points (top-right), and developmental stages (bottom-left). B. Visualization of cell-type marker gene expression with UMAP. PCW—post-conception weeks; M—months; Y—years.

Fig 1 UMAP plots of human thymic stroma cells

图2 人类cTECs差异基因的KEGG通路分析Note： A. Up-regulated genes. B. Down-regulated genes.

Fig 2 The KEGG pathway analysis of differentially expressed genes of human cTECs

图3 人类mTECs差异基因的KEGG通路分析Note： A. mTEC(Ⅰ). B. mTEC(Ⅱ).

Fig 3 KEGG pathway analysis of differentially expressed genes of human mTECs

图4 人类胸腺DCs差异基因的KEGG通路分析Note： A. DC1. B. DC2.

Fig 4 KEGG pathway analysis of differentially expressed genes of human thymic DCs

图5 人类胸腺APCs中HLA基因的表达差异Note： A. cTEC, mTEC(Ⅰ) and mTEC(Ⅱ). B. DC1 and DC2.

Fig 5 Differential expression of HLA genes in human thymic APCs

图6 人类胸腺Fb1和Fb2在不同发育阶段的差异表达基因Note： A. Differentially expressed genes. B. Differentially expressed collagen genes. C. Expression of CCL19 and CCL21 in human mTEC (Ⅰ).

Fig 6 Differentially expressed genes of human thymic Fb1 and Fb2 across development stages

图7 人类胸腺Fb 1型和Fb 2型差异基因的KEGG通路分析Note： A. Fb1. B. Fb2.

Fig 7 KEGG pathway analysis of differentially expressed genes of human thymic Fb1 and Fb2

图8 人类胸腺Endo在不同发育阶段的差异表达基因Note： A. Differentially expressed genes. B. Expression of HLA genes. C. KEGG pathway analysis of differentially expressed genes of human thymic Endo.

Fig 8 Differentially expressed genes of human thymic Endo across development stages

参考文献 31

1	Han J, Zúñiga-Pflücker JC. A 2020 view of thymus stromal cells in T cell development[J]. J Immunol, 2021, 206(2): 249-256.
2	Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells[J]. Annu Rev Immunol, 2003, 21: 139-176.
3	Farley AM, Morris LX, Vroegindeweij E, et al. Dynamics of thymus organogenesis and colonization in early human development[J]. Development, 2013, 140(9): 2015-2026.
4	Nishino M, Ashiku SK, Kocher ON, et al. The thymus: a comprehensive review[J]. Radiographics, 2006, 26(2): 335-348.
5	Palmer S, Albergante L, Blackburn CC, et al. Thymic involution and rising disease incidence with age[J]. Proc Natl Acad Sci U S A, 2018, 115(8): 1883-1888.
6	Dik WA, Pike-Overzet K, Weerkamp F, et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[J]. J Exp Med, 2005, 201(11): 1715-1723.
7	Mingueneau M, Kreslavsky T, Gray D, et al. The transcriptional landscape of αβ T cell differentiation[J]. Nat Immunol, 2013, 14(6): 619-632.
8	Tang F, Barbacioru C, Wang Y, et al. mRNA-Seq whole-transcriptome analysis of a single cell[J]. Nat Methods, 2009, 6(5): 377-382.
9	Macosko EZ, Basu A, Satija R, et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets[J]. Cell, 2015, 161(5): 1202-1214.
10	Schier AF. Single-cell biology: beyond the sum of its parts[J]. Nat Methods, 2020, 17(1): 17-20.
11	Zeng Y, Liu C, Gong Y, et al. Single-cell RNA sequencing resolves spatiotemporal development of pre-thymic lymphoid progenitors and thymus organogenesis in human embryos[J]. Immunity, 2019, 51(5): 930-948.e6.
12	Park JE, Botting RA, Domínguez Conde C, et al. A cell atlas of human thymic development defines T cell repertoire formation[J]. Science, 2020, 367(6480): eaay3224.
13	Stuart T, Butler A, Hoffman P, et al. Comprehensive integration of single-cell data[J]. Cell, 2019, 177(7): 1888-1902.e21.
14	Dong J, Zhou P, Wu Y, et al. Enhancing single-cell cellular state inference by incorporating molecular network features[EB/OL]. (2019-10-15)[2021-03-20]. .
15	Oughtred R, Rust J, Chang C, et al. The BioGRID database: a comprehensive biomedical resource of curated protein, genetic, and chemical interactions[J]. Protein Sci, 2021, 30(1): 187-200.
16	Becht E, McInnes L, Healy J, et al. Dimensionality reduction for visualizing single-cell data using UMAP[J]. Nat Biotechnol, 2018: 38-44.
17	Yu G, Wang LG, Han Y, et al. clusterProfiler: an R package for comparing biological themes among gene clusters[J]. OMICS, 2012, 16(5): 284-287.
18	Jardine L, Haniffa M. Reconstructing human DC, monocyte and macrophage development in utero using single cell technologies[J]. Mol Immunol, 2020, 123: 1-6.
19	De Val S, Black BL. Transcriptional control of endothelial cell development[J]. Dev Cell, 2009, 16(2): 180-195.
20	Thiele BJ, Doller A, Kähne T, et al. RNA-binding proteins heterogeneous nuclear ribonucleoprotein A1, E1, and K are involved in post-transcriptional control of collagen Ⅰ and Ⅲ synthesis[J]. Circ Res, 2004, 95(11): 1058-1066.
21	van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition[J]. Annu Rev Immunol, 2003, 21: 659-684.
22	Thomas R, Wang W, Su DM. Contributions of age-related thymic involution to immunosenescence and inflammaging[J]. Immun Ageing, 2020, 17: 2.
23	Fletcher AL, Seach N, Reiseger JJ, et al. Reduced thymic aire expression and abnormal NF-κB2 signaling in a model of systemic autoimmunity[J]. J Immunol, 2009, 182(5): 2690-2699.
24	Oh J, Wang W, Thomas R, et al. Capacity of tTreg generation is not impaired in the atrophied thymus[J]. PLoS Biol, 2017, 15(11): e2003352.
25	Cavadini P, Vermi W, Facchetti F, et al. AIRE deficiency in thymus of 2 patients with omenn syndrome[J]. J Clin Invest, 2005, 115(3): 728-732.
26	Passos GA, Speck-Hernandez CA, Assis AF, et al. Update on aire and thymic negative selection[J]. Immunology, 2018, 153(1): 10-20.
27	Kecha O, Brilot F, Martens H, et al. Involvement of insulin-like growth factors in early T cell development: a study using fetal thymic organ cultures[J]. Endocrinology, 2000, 141(3): 1209-1217.
28	Kozai M, Kubo Y, Katakai T, et al. Essential role of CCL21 in establishment of central self-tolerance in T cells[J]. J Exp Med, 2017, 214(7): 1925-1935.
29	Halkias J, Melichar HJ, Taylor KT, et al. Tracking migration during human T cell development[J]. Cell Mol Life Sci, 2014, 71(16): 3101-3117.
30	Wieczorek M, Abualrous ET, Sticht J, et al. Major histocompatibility complex (MHC) class Ⅰ and MHC class Ⅱ proteins: conformational plasticity in antigen presentation[J]. Front Immunol, 2017, 8: 292.
31	Pober JS, Merola J, Liu R, et al. Antigen presentation by vascular cells[J]. Front Immunol, 2017, 8: 1907.

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