基于高通量测序技术分析多发性硬化症患者TCR图谱特征

doi:10.3969/j.issn.1673-8640.2025.05.002

摘要/Abstract

摘要：

目的分析多发性硬化症（MS）患者T细胞受体（TCR）β链互补决定区3（CDR3）的图谱特征。方法从ImmuneAccess数据库下载11例女性MS患者（MS组）和12名健康女性（正常对照组）的T细胞免疫组库测序数据。系统分析所有研究对象的TCR免疫图谱，包括CDR3频率分布、CDR3长度分布、TRβ V、TRβ D和TRβ J使用频率、V-J组合频率和Vdels、Jdels、D5dels、D3dels、n1ins、n2ins位点的插入缺失长度分布。基于D50指数、Shannon指数、Simpson指数和Inverse simpson指数计算TCR图谱的多样性。结果 MS组TCRβ链CDR3的多样性显著大于正常对照组（P<0.01）。与正常对照组比较，MS组TCRβ链CDR3核苷酸长度在57、60、63、66 nt的频率显著增加（P<0.05），TCRβ链CDR3氨基酸序列中CASSLRETQYF序列显著降低（P<0.05）。MS组和正常对照组 In和Out序列的插入缺失长度分布、TCRβ链CDR3第6位和第7位氨基酸中的20种氨基酸利用率，以及亲水性氨基酸、中性氨基酸和疏水性氨基酸的使用频率差异均无统计学意义（P>0.05）。MS组和正常对照组的TRβ V、TRβ D和TRβ J基因亚家族的使用频率差异有统计学意义（P<0.05）。结论 MS组TCR图谱存在明显异常，可为MS患者的诊断和治疗提供参考。

关键词: T细胞受体, 互补决定区3, 多发性硬化症, 图谱

Abstract:

Objective To investigate the profile characteristics of complementary determining region 3 （CDR3） of T cell receptor （TCR） β chain in patients with multiple sclerosis （MS）. Methods The T cell immune repertoire sequencing data of 11 female MS patients （MS group） and 12 healthy females （healthy control group） were downloaded from the ImmuneAccess database. The TCR profiles of all research subjects were analyzed systematically，which included CDR3 frequency distribution，CDR3 length distribution，TRβ V，TRβ D and TRβ J usage frequencies，V-J combination frequency and insertion/deletion length distribution of Vdels，Jdels，D5dels，D3dels，n1ins and n2ins sites. The diversity of TCR profiles based on D50 index，Shannon index，Simpson index and Inverse simpson index was calculated. Results The diversity of TCRβ chain CDR3 in MS group was higher than that in healthy control group （P<0.01）. Compared with healthy control group，the frequency of TCRβ chain CDR3 nucleotide lengths at 57，60，63 and 66 nt in MS group was increased （P<0.05），while the CASSLRETQYF sequence in TCRβ chain CDR3 amino acid sequence was decreased （P<0.05）. There was no statistical significance in the length distribution of insertion deletion in the In and Out sequences，the utilization frequency of 20 amino acids in the 6th and 7th amino acids of TCRβ chain CDR3 and the utilization frequency of hydrophilic，neutral and hydrophobic amino acids between MS group and healthy control group（P>0.05）. There was statistical significance in the utilization frequency of TRβ V，TRβ D and TRβ J gene subfamilies between MS group and healthy control group（P<0.05）. Conclusions The TCR profile of MS group is different from that of healthy control group，which provides a reference for the diagnosis and treatment of MS patients.

Key words: T cell receptor, Complementary determining region 3, Multiple sclerosis, Profile

中图分类号:

R446.62

陈晓艳, 莫倩儿, 黄方滢, 胡子鹏, 杨珊珊, 农菁菁, 杨雪丽. 基于高通量测序技术分析多发性硬化症患者TCR图谱特征[J]. 检验医学, 2025, 40(5): 421-427.

CHEN Xiaoyan, MO Qianer, HUANG Fangying, HU Zipeng, YANG Shanshan, NONG Jingjing, YANG Xueli. Characteristics of TCR profile in multiple sclerosis patients based on high-throughput sequencing technology[J]. Laboratory Medicine, 2025, 40(5): 421-427.

图/表 6

图1 MS组和正常对照组TCRβ链CDR3多样性分析注（a）D50指数；（b）Inverse simpson指数；（c）Shannon指数；（d）Simpson指数。

图2 MS组和正常对照组TCRβ链CDR3氨基酸和核苷酸长度分布注：（a）氨基酸长度分布；正常对照组、MS组；（b）功能性TCRβ链CDR3序列（in frame）长度分布；（c）非功能性TCRβ链CDR3序列（out of frame）长度分布。

图3 MS组、正常对照组PBMC中单个TCRβ CDR3核苷酸序列长度分布频率和In、Out序列的InDel长度分布频率比较注：（a）PBMC中单个TCRβ链CDR3核苷酸序列的长度分布频率；正常对照组、MS组；与正常对照组比较，*P<0.05；（b）In序列的插入长度分布；（c）In序列的缺失长度分布；（d）Out序列的插入长度分布；（e）Out序列的缺失长度分布。

图4 MS组、正常对照组TCRβ链CDR3氨基酸序列第6位20种氨基酸和亲水性氨基酸、中性氨基酸、疏水性氨基酸的使用频率注：（a）MS组和正常对照组TCRβ链CDR3氨基酸序列第6位20种氨基酸的使用频率（占总CDR3的百分比）；（b）MS组和正常对照组TCRβ链CDR3氨基酸序列第6位亲水性氨基酸、中性氨基酸和疏水性氨基酸的使用频率（占总CDR3的百分比）；亲水性氨基酸、中性氨基酸、疏水性氨基酸。

图5 MS组、正常对照组TCRβ链CDR3氨基酸序列第7位20种氨基酸和亲水性氨基酸、中性氨基酸、疏水性氨基酸的使用频率注：（a）MS组和正常对照组TCRβ链CDR3氨基酸序列第7位20种氨基酸的使用频率（占总CDR3的百分比）；（b）MS组和正常对照组TCRβ链CDR3氨基酸序列第7位亲水性氨基酸、中性氨基酸和疏水性氨基酸的使用频率（占总CDR3的百分比）；亲水性氨基酸、中性氨基酸、疏水性氨基酸。

图6 MS组、正常对照组TCRβ链CDR3氨基酸频率和TRβ V、TRβ D、TRβ J基因亚家族的表达分析注：（a）TCRβ链CDR3氨基酸频率比较；正常对照组；MS组；与正常对照组比较，*P<0.05；（b）TRβ V基因亚类的表达；（c）TRβ D基因亚类的表达；（d）TRβ J基因亚类的表达；蓝色表示低表达，红色表示高表达。

参考文献 30

[1]	刘锦仪, 黄伟雄. 多发性硬化34例临床分析[J]. 中国医药指南, 2013, 11(16):120-121.
[2]	巫嘉陵, 王跃. 多发性硬化50例临床分析[J]. 中国实验诊断学, 2002(4):259-260.
[3]	王佳颖, 阮邹荣, 江波. 治疗多发性硬化症药物临床试验现状及展望[J]. 中国现代应用药学, 2022, 39(18):2405-2411.
[4]	周文斌, 崔玉真, 肖波. 多发性硬化的流行病学研究(综述)[J]. 中国神经免疫学和神经病学杂志, 2005(6):373-375.
[5]	RAMIEN C, YUSKO E C, ENGLER J B, et al. T cell repertoire dynamics during pregnancy in multiple sclerosis[J]. Cell Rep, 2019, 29(4):810-815. DOI PMID
[6]	THOMPSON A J, BANWELL B L, BARKHOF F, et al. Diagnosis of multiple sclerosis:2017 revisions of the McDonald criteria[J]. Lancet Neurol, 2018, 17(2):162-173.
[7]	ROBINS H, DESMARAIS C, MATTHIS J, et al. Ultra-sensitive detection of rare T cell clones[J]. J Immunol Methods, 2012, 375(1-2):14-19. DOI PMID
[8]	CARLSON C S, EMERSON R O, SHERWOOD A M, et al. Using synthetic templates to design an unbiased multiplex PCR assay[J]. Nat Commun, 2013, 4:2680.
[9]	ELHANATI Y, SETHNA Z, CALLAN C G Jr, et al. Predicting the spectrum of TCR repertoire sharing with a data-driven model of recombination[J]. Immunol Rev, 2018, 284(1):167-179. DOI PMID
[10]	POGORELYY M V, MINERVINA A A, CHUDAKOV D M, et al. Method for identification of condition-associated public antigen receptor sequences[J]. Elife, 2018, 7:e33050.
[11]	MURUGAN A, MORA T, WALCZAK A M, et al. Statistical inference of the generation probability of T-cell receptors from sequence repertoires[J]. Proc Natl Acad Sci U S A, 2012, 109(40):16161-16166.
[12]	GOMEZ-TOURINO I, KAMRA Y, BAPTISTA R, et al. T cell receptor β-chains display abnormal shortening and repertoire sharing in type 1 diabetes[J]. Nat Commun, 2017, 8(1):1792.
[13]	HOU X, YANG Y, CHEN J, et al. TCRβ repertoire of memory T cell reveals potential role for Escherichia coli in the pathogenesis of primary biliary cholangitis[J]. Liver Int, 2019, 39(5):956-966.
[14]	HUANG C, LI X, WU J, et al. The landscape and diagnostic potential of T and B cell repertoire in Immunoglobulin A Nephropathy[J]. J Autoimmun, 2019, 97:100-107.
[15]	HOU X, ZENG P, CHEN J, et al. No difference in TCRβ repertoire of CD4+ naive T cell between patients with primary biliary cholangitis and healthy control subjects[J]. Mol Immunol, 2019, 116:167-173.
[16]	STEWART J J, LEE C Y, IBRAHIM S, et al. A Shannon entropy analysis of immunoglobulin and T cell receptor[J]. Mol Immunol, 1997, 34(15):1067-1082. DOI PMID
[17]	VENTURI V, KEDZIERSKA K, TURNER S J, et al. Methods for comparing the diversity of samples of the T cell receptor repertoire[J]. J Immunol Methods, 2007, 321(1-2):182-195. DOI PMID
[18]	林念童, 石尚金. T细胞受体疫苗与多发性硬化[J]. 中华神经科杂志, 1999, 32(6):382.
[19]	BIEGLER B W, YAN S X, ORTEGA S B, et al. Clonal composition of neuroantigen-specific CD8+ and CD4+ T-cells in multiple sclerosis[J]. J Neuroimmunol, 2011, 234(1-2):131-140. DOI PMID
[20]	SALOU M, GARCIA A, MICHEL L, et al. Expanded CD8 T-cell sharing between periphery and CNS in multiple sclerosis[J]. Ann Clin Transl Neurol, 2015, 2(6):609-622.
[21]	LUTTER L, SPIERINGS J, VAN RHIJN-BROUWER F C C, et al. Resetting the T cell compartment in autoimmune diseases with autologous hematopoietic stem cell transplantation:an update[J]. Front Immunol, 2018, 9:767.
[22]	AMORIELLO R, CHERNIGOVSKAYA M, GREIFF V, et al. TCR repertoire diversity in multiple sclerosis:high-dimensional bioinformatics analysis of sequences from brain,cerebrospinal fluid and peripheral blood[J]. EbioMedicine, 2021, 68:103429.
[23]	PLANAS R, METZ I, MARTIN R, et al. Detailed characterization of T cell receptor repertoires in multiple sclerosis brain lesions[J]. Front Immunol, 2018, 9:509.
[24]	YAMOUT B I, ALROUGHANI R. Multiple sclerosis[J]. Semin Neurol, 2018, 38(2):212-225. DOI PMID
[25]	HOU X L, WANG L, DING Y L, et al. Current status and recent advances of next generation sequencing techniques in immunological repertoire[J]. Genes Immun, 2016, 17(3):153-164. DOI PMID
[26]	NOROOZI KARIMABAD M, KHANAMANI FALAHATI-POUR S, HASSANSHAHI G. Significant role(s)of CXCL12 and the SDF-1 3'A genetic variant in the pathogenesis of multiple sclerosis[J]. Neuroimmunomodulation, 2016, 23(4):197-208.
[27]	LENG Q, NIE Y, ZOU Y, et al. Elevated CXCL12 expression in the bone marrow of NOD mice is associated with altered T cell and stem cell trafficking and diabetes development[J]. BMC Immunol, 2008, 9:51.
[28]	ROBEY I F, PETERSON M, HORWITZ M S, et al. Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice[J]. J Immunol, 2004, 172(7):4624-4629. PMID
[29]	HOU X, WANG M, LU C, et al. Analysis of the repertoire features of TCR beta chain CDR3 in human by high-throughput sequencing[J] Cell Physiol Biochem, 2016, 39(2):651-667. DOI PMID
[30]	GLANVILLE J, HUANG H, NAU A, et al. Identifying specificity groups in the T cell receptor repertoire[J]. Nature, 2017, 547(7661):94-98.