使用特异性TCR寡核苷酸探针在体外检测多发性硬化症患者及对照中髓鞘碱性蛋白反应性T细胞。

Ex vivo detection of myelin basic protein-reactive T cells in multiple sclerosis and controls using specific TCR oligonucleotide probes.

作者信息

Hong Jian, Zang Ying C Q, Li Sufang, Rivera Victor M, Zhang Jingwu Z

机构信息

Department of Neurology and Baylor-Methodist Multiple Sclerosis Center, Baylor College of Medicine, Houston, USA.

Joint Immunology Laboratory of Health Science Center and Shanghai Institute of Immunology, Shanghai Institutes of Biological Sciences - Shanghai Second Medical University, Shanghai, China.

出版信息

Eur J Immunol. 2004 Mar;34(3):870-881. doi: 10.1002/eji.200324790.

DOI:10.1002/eji.200324790

PMID:14991617

Abstract

T cell reactivity to candidate myelin autoantigens, such as myelin basic protein (MBP), may play an important role in the pathogenesis of multiple sclerosis (MS). Although MBP-reactive T cells have been found to undergo in vivo activation in patients with MS, their true precursor frequency in MS is unknown as current frequency analysis is commonly based on the T cell functional responses to MBP. In this study, we developed a TCR sequence-based ex vivo detection system using colony hybridization with oligonucleotide probes specific for CDR3 of selected T cell clones for the analysis of true T cell precursor frequency in PBMC. The results revealed that the precursor frequency of five independent T cell clones recognizing the immunodominant MBP(83-99) region was found to be in the range of 1.6 x 10(-4) in total T cells in three HLA-DR2 patients with MS compared to that of 0.25 x 10(-4) in HLA-DR2 healthy individuals. The observed frequency of MBP(83-99)-reactive T cells in MS patients was considerably higher than those measured in parallel by cell culture-based analysis (2.3 x 10(-6)) or by enzyme-linked immunospot assay (3.9 x 10(-5)) in the same peripheral blood mononuclear cell specimens. Furthermore, the study showed that MBP(83-99)-reactive T cells detected ex vivo belonged to CD45RA+, CD25+ and CD95- T cell subsets as evidenced by preferential expression of specific TCR transcripts in these cell fractions.

摘要

T细胞对候选髓鞘自身抗原（如髓鞘碱性蛋白，MBP）的反应性可能在多发性硬化症（MS）的发病机制中起重要作用。尽管已发现MBP反应性T细胞在MS患者体内会发生激活，但由于目前的频率分析通常基于T细胞对MBP的功能反应，其在MS中的真实前体频率尚不清楚。在本研究中，我们开发了一种基于TCR序列的体外检测系统，使用与选定T细胞克隆的CDR3特异性寡核苷酸探针进行菌落杂交，以分析外周血单个核细胞（PBMC）中真实的T细胞前体频率。结果显示，在三名HLA - DR2型MS患者中，识别免疫显性MBP（83 - 99）区域的五个独立T细胞克隆的前体频率在总T细胞中为1.6×10⁻⁴，而在HLA - DR2型健康个体中为0.25×10⁻⁴。在MS患者中观察到的MBP（83 - 99）反应性T细胞频率显著高于在相同外周血单个核细胞标本中通过基于细胞培养的分析（2.3×10⁻⁶）或酶联免疫斑点测定（3.9×10⁻⁵）并行测量的频率。此外，该研究表明，体外检测到的MBP（83 - 99）反应性T细胞属于CD45RA⁺、CD25⁺和CD95⁻T细胞亚群，这些细胞组分中特异性TCR转录本的优先表达证明了这一点。