Abstract
The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRα- and β-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-γ secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRα- and β-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.
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Acknowledgments
We thank S. Shalapour for helpful advice on how to design and analyze real-time PCR assays and K. Hummel and J. Hauchwitz for excellent technical assistance. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich TR36 and SPP1230) and the Leukaemia Research Fund.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s00109-008-0361-z
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Leisegang, M., Engels, B., Meyerhuber, P. et al. Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J Mol Med 86, 573–583 (2008). https://doi.org/10.1007/s00109-008-0317-3
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DOI: https://doi.org/10.1007/s00109-008-0317-3