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TCR mispairing in genetically modified T cells was detected by fluorescence resonance energy transfer

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Abstract

Adoptive transfer of T lymphocytes genetically modified with antigen-specific T cell receptor (TCR) constitutes a promising approach for the treatment of malignant tumors and virus infections. One of the challenges in this field of TCR gene therapy is TCR mispairing defining the incorrect pairing between an introduced TCR α or β chain and an endogenous TCR β or α chain, which results in diluted surface expression of the therapeutic TCR αβ. Although there is currently no clinical evidence for TCR mispairing-induced autoreactivity, the generation of autoreactive TCRs upon TCR mispairing cannot be excluded. So it is important to detect TCR mispairing to evaluate the efficiency of TCR gene therapy. Currently there is no available quantitative assay for the measurement of TCR mispairing. Fluorescence resonance energy transfer (FRET) is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. In this study, we described the method of FRET for the measurement of TCR mispairing. It was found that the average FRET efficiency was 12.2 ± 7.5% in HeLa cells and 8.4 ± 3.3% in Jurkat cells (P = 0.026605). The reduction of FRET efficiency in lymphocytes reflected the presence of mispaired TCRs, indicating there were ~30% TCR mispairing in lymphocytes. This study provides a quantitative intracellular assay that can be used to detect TCR mispairing in genetically modified T lymphocytes.

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Acknowledgements

We thank Drs. Tao Deng and Jian Liu for their helpful reviews and suggestions. This work is supported by a grant from National Scientific and Technological Major Special Project for “Significant Creation of New Drugs” (No. 2009ZX09103-708), a grant from Nurturing Project for the Excellent and Creative Youth Scholars of Colleges and Universities of Guangdong (No. 2008342), and a grant from Faculty Development and Research Funds of GDPU.

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  1. School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, People’s Republic of China

    Hongwei Shao, Wenfeng Zhang, Qinglian Hu, Fenglin Wu, Han Shen & Shulin Huang

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  1. Hongwei Shao
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  2. Wenfeng Zhang
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  3. Qinglian Hu
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Correspondence to Shulin Huang.

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Shao, H., Zhang, W., Hu, Q. et al. TCR mispairing in genetically modified T cells was detected by fluorescence resonance energy transfer. Mol Biol Rep 37, 3951–3956 (2010). https://doi.org/10.1007/s11033-010-0053-y

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