Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging
Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging
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DOI：
10.1016/s0022-1759(97)00197-x
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发表时间：
1997-12-29
影响因子：
2.2
通讯作者：
Boehm, O
中科院分区：
文献类型：
--
作者：
Manfras, BJ;Rudert, WA;Boehm, O
在各种生理和病理条件下的人T细胞受体（TCR）库的表征已经成为研究免疫应答的重要工具。因此，已经描述了用于TCR库的半定量分析的许多基于PCR的策略。TCR cDNA的家族特异性扩增已被用于许多研究中，结果往往相互矛盾。我们已经开发了一种策略，利用外源性标准品与同源引物结合位点的α/β T细胞受体库的定量分析。该系统允许基于定量PCR（Q-PCR）和竞争性PCR（C-PCR）检测T细胞群体中的甚至微小差异。本文呈现的结果表明，当超过总库的1%时，可以容易地监测由可变基因区段的特异性定义的T细胞亚群的扩增。此外，所提出的方法揭示了CDR 3大小异质性的直接信息，并且可以用于估计T细胞库复杂性和监测克隆扩增。我们讨论的变量，如细胞数量和实验条件影响分析的准确性和再现性。我们已经使用了这个协议的基础上，非放射性技术的特征的T细胞库在外周和器官浸润T淋巴细胞的精细的特异性。这些分析揭示了关于在各种刺激（例如T细胞亚群的超抗原刺激以及自身免疫性和感染性疾病中α/β T细胞库的抗原驱动的成形）后体内和体外T细胞的多克隆或克隆扩增的信息。(C)1997年Elsevier Science B.V.
The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the alpha/beta T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire, In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions, We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the alpha/beta T-cell repertoire in autoimmune and infectious diseases. (C) 1997 Elsevier Science B.V.