A fast and robust method to clone and functionally validate T-cell receptors
A fast and robust method to clone and functionally validate T-cell receptors
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DOI：
10.1016/j.jim.2009.05.001
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发表时间：
2009-07-31
影响因子：
2.2
通讯作者：
Schaft, Niels
中科院分区：
文献类型：
--
作者：
Birkholz, Katrin;Hofmann, Christian;Schaft, Niels
免疫治疗和免疫学研究中通常需要对来自T细胞克隆的T细胞受体（TCR）α链和β链进行测序、克隆和功能测试。然而，通过简单的PCR测定TCR链是不可能的，因为与3'恒定结构域和非翻译区（UTR）相反，5'区中不存在保守序列。此外，克隆TCR的后续功能测试需要费力的细胞培养实验，通常涉及原代人材料和耗时的病毒转导策略。在这里，我们提出了一个通用的基于PCR的协议，改编自帽开关技术，允许扩增的TCR α和β链mRNA的TCR可变结构域亚型的知识，通过附加一个设计的序列到mRNA的51端。以这种方式克隆了两种不同的MelanA/HLA-A2特异性TCR和一种HIVgag/HLA-A2特异性TCR，并通过新开发的简单，快速和低成本的方法进行了功能测试：我们同时用TCR编码RNA和NFAT报告基因构建体电穿孔Jurkat T细胞，并测量细胞在特异性刺激后的激活状态。该测定的结果与电穿孔的大量T细胞的细胞因子释放、功能亲合力、增殖活性和识别MelanA/HLA-A2呈递肿瘤细胞的能力相关。编码相同TCR的RNA。这两种方案共同代表了用于T细胞克隆的TCR的鉴定和功能测试的快速且低成本的工具，其然后可以应用于免疫疗法或免疫学研究。(c)2009 Elsevier B. V.保留所有权利。
Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell-culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 51 end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with. RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research. (c) 2009 Elsevier B.V. All rights reserved.