Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette
Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette
复制标题
DOI：
10.1007/s00109-008-0317-3
复制
发表时间：
2008-05-01
影响因子：
4.7
通讯作者：
Uckert, Wolfgang
中科院分区：
文献类型：
--
作者：
Leisegang, Matthias;Engels, Boris;Uckert, Wolfgang
T细胞受体（TCR）基因的转移允许赋予T细胞新的抗原特异性。对于TCR重定向的T细胞的临床应用，转基因TCR的有效功能表达是关键的先决条件。在这里，我们比较了转基因盒对鼠TCR P14（识别LCMV gp 33表位）和人TCR WT-1（识别肿瘤相关抗原WT-1的表位）的表达和功能的影响。我们构建了不同的载体，其中TCR α链和β链基因（a）通过内部核糖体进入位点（IRES）连接，（B）通过2A肽组合，或（c）引入两个单独的逆转录病毒构建体。虽然在TCR缺陷型T细胞系中，TCR P14被所有构建体同样良好地表达，但我们发现，在携带TCR的细胞系和原代鼠T细胞中，使用IRES（而不是2A）的TCR表达受到阻碍，其中转基因TCR必须与内源性TCR链竞争。类似地，如通过四聚体结合和肽特异性IFN-γ分泌所测量的，2A连接的TCR WT-1基因产生最高的表达和功能。如实时PCR所示，表达差异与拷贝数整合无关。因此，通过2A肽连接TCR α链和β链基因对于TCR表达和T细胞功能上级IRES。
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 TCR alpha- and beta-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-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCR alpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.