TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning
TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning
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DOI：
10.1002/ijc.10317
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发表时间：
2002-05-01
影响因子：
6.4
通讯作者：
Schrier, PI
中科院分区：
文献类型：
--
作者：
Aarnoudse, CA;Krüse, M;Schrier, PI
新肿瘤抗原的鉴定对于有效的癌症免疫治疗是极其重要的。该领域的主要障碍是肿瘤特异性细胞毒性T淋巴细胞（CTL）在体外的有限寿命。因此，我们寻找分离这些CTL的肿瘤特异性的方法，即，它们的T细胞受体（TCR）并将其转移到永生化的T细胞系。为此目的，TCR阴性Jurkat T细胞系配备有活化T细胞核因子（NFAT）-荧光素酶报告基因构建体以允许测量TCR介导的活化。为了建立这种肿瘤特异性TCR转导的可行性，我们将对肿瘤抗原CAMEL（黑色素瘤上的CTL识别抗原）特异性的已知T细胞克隆的TCR基因克隆到逆转录病毒构建体中。测试用该构建体Jrt-TCR α 3 β 5转导的Jurkat报告细胞对表达CAMEL的黑素瘤细胞、负载肽的T2细胞和CAMEL转染的COS-1细胞的反应性。黑色素瘤细胞系识别不佳，但肽脉冲和转染的细胞有效地刺激NFAT信号传导。TCR+ Jurkat报告细胞的活化显示依赖于靶细胞上的抗原密度和Jurkat细胞上辅助受体CD 8的表达水平。为了验证这种TCR重建方法对于鉴定新抗原的益处，将最初克隆CAMEL的cDNA文库的库转染到COS-1细胞中并用Jrt-TCR α 3 β 5筛选。发现这些细胞和CAMEL特异性CTL克隆的相同cDNA池呈阳性。我们的研究结果表明，TCR重建的Jurkat报告细胞是一个有用的工具，在识别新的肿瘤抗原的cDNA表达克隆。(C)2002 Wiley-Liss，Inc.
The identification of novel tumor antigens is of extreme importance for effective immunotherapy against cancer. A major obstacle in this field is the limited life span of tumor-specific cytotoxic T lymphocytes (CTLs) in vitro. Therefore we searched for a method to isolate the tumor specificity of these CTLs, i.e., their T-cell receptors (TCRs) and transfer it to an immortalized T-cell line. For this purpose, a TCR-negative Jurkat T-cell line was equipped with a nuclear factor of activated T cells (NFAT)-luciferase reporter construct to allow measurement of TCR-mediated activation. To establish the feasibility of this tumor-specific TCR transduction, we cloned the TCR genes of a known T-cell clone specific for the tumor antigen CAMEL (CTL-recognized antigen on melanoma) into a retroviral construct. Jurkat reporter cells transduced with this construct, Jrt-TCRalpha3beta5, were tested for their reactivity against CAMEL-expressing melanoma cells, peptide-loaded T2 cells and CAMEL-transfected COS-1 cells. The melanoma cell lines were poorly recognized, but peptide-pulsed and -transfected cells effectively stimulated NFAT signaling. The activation of TCR+ Jurkat reporter cells was shown to be dependent on the antigen density on the target cells and the expression level of the coreceptor CD8 on the Jurkat cells. To verify the benefit of this TCR reconstitution method for identification of novel antigens, pools of the cDNA library from which CAMEL was originally cloned were transfected in COS-1 cells and screened with Jrt-TCRalpha3beta5. Identical cDNA pools were found that were positive with these cells and with the CAMEL-specific CTL clone. Our results illustrate that TCR-reconstituted Jurkat reporter cells are a useful tool in the identification of novel tumor antigens by cDNA expression cloning. (C) 2002 Wiley-Liss, Inc.