Weidanz J A, Card K F, Edwards A, Perlstein E, Wong H C
Sunol Molecular, Miramar, FL 33025, USA.
J Immunol Methods. 1998 Dec 1;221(1-2):59-76. doi: 10.1016/s0022-1759(98)00153-7.
The ability to display functional T-cell receptors (TCR) on the surface of bacteriophage could have numerous applications. For instance, TCR phage-display could be used to develop new strategies for isolating TCRs with unique specificity or it could be used to carry out mutagenesis studies on TCR molecules for analyzing their structure-function. We initially selected a TCR from the murine T-cell hybridoma, DO11.10, as our model system, and genetically engineered a three domain single-chain TCR (scTCR) linked to the gene p8 protein of the Escherichia coli bacteriophage fd. Immunoblotting studies revealed that (1) E. coli produced a soluble scTCR/p8 fusion protein and (2) the fusion protein was packaged by the phage. Cellular competition assays were performed to evaluate the functionality of the TCR and showed the DO11.10 TCR-bearing phage could significantly inhibit stimulation of DO11.10 T hybridoma cells by competing for binding to immobilized MHC/peptide IA(d)/OVA(323-339). Flow cytometric analysis was carried out to evaluate direct binding of DO11.10 TCR-bearing phage onto the surface of cells displaying either IAd containing irrelevant peptide or OVA peptide. The results revealed binding of DO11.10 TCR-bearing phage only on cells expressing IA(d) loaded with OVA peptide showing TCR fine specificity for peptide. To illustrate the generality of TCR phage-display, we also cloned and displayed on phage a second TCR which recognizes a peptide fragment from human tumor suppressor protein p53 restricted by HLA-A2. These findings demonstrate functional TCR can be displayed on bacteriophage potentially leading to the development of novel applications involving TCR phage-display.
在噬菌体表面展示功能性T细胞受体（TCR）的能力可能有众多应用。例如，TCR噬菌体展示可用于开发新策略以分离具有独特特异性的TCR，或者可用于对TCR分子进行诱变研究以分析其结构功能。我们最初从鼠T细胞杂交瘤DO11.10中选择了一种TCR作为我们的模型系统，并对与大肠杆菌噬菌体fd的基因p8蛋白相连的三结构域单链TCR（scTCR）进行基因工程改造。免疫印迹研究表明：（1）大肠杆菌产生了可溶性scTCR/p8融合蛋白；（2）该融合蛋白被噬菌体包装。进行细胞竞争试验以评估TCR的功能，结果显示携带DO11.10 TCR的噬菌体可通过竞争结合固定化的MHC/肽IA(d)/OVA(323 - 339) 来显著抑制DO11.10 T杂交瘤细胞的刺激。进行流式细胞术分析以评估携带DO11.10 TCR的噬菌体与展示含有无关肽或OVA肽的IAd的细胞表面的直接结合。结果显示携带DO11.10 TCR的噬菌体仅与表达负载OVA肽的IA(d)的细胞结合，表明TCR对肽具有精细特异性。为了说明TCR噬菌体展示的普遍性，我们还克隆了第二种TCR并在噬菌体上展示，该TCR识别受HLA - A2限制的人肿瘤抑制蛋白p53的肽片段。这些发现表明功能性TCR可展示在噬菌体上，这可能会导致涉及TCR噬菌体展示的新应用的开发。
