Improving TCR affinity on 293T cells
Improving TCR affinity on 293T cells
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DOI：
10.1016/j.jim.2018.11.010
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发表时间：
2019-03-01
影响因子：
2.2
通讯作者：
Kuzushima, Kiyotaka
中科院分区：
文献类型：
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作者：
Ohta, Rieko;Demachi-Okamura, Ayako;Kuzushima, Kiyotaka
本研究提供了一种提高TCR亲和力的有效方法，包括1)CDR引导的TCR基因的饱和突变，2)通过简单的质粒法在表达CD3的HEK293T(CD3-293T)细胞上显示TCR，3)用HLA四聚体染色，以及4)在流式细胞仪上对CD8非依赖四聚体结合的细胞进行多轮分选。使用这些程序，我们成功地从人类端粒酶逆转录酶(HTERT)特异的人类端粒酶逆转录酶(HTERT)限制性TCR中鉴定出结合增强的突变TCR。分离到两个这样的克隆，2A7A和2D162，分别在TCRβ的CDR1和CDR2上存在突变，两者都显示出顺序的4个氨基酸替换。当与野生型TCRα一起在CD3-293T细胞上表达时，这些突变体的TCR分子及其组合突变与人类白细胞抗原-A24/hTERT-四聚体的结合比野生型TCR更强，而不与对照四聚体结合。此外，为了便于TCR的功能研究，我们建立了一个人工T细胞系，命名为CD8I-J2，通过修饰Jurkat来源的J.RT3-T3.5细胞来表达人CD8和干扰素-γ产生盒。分析表达野生型或亲和力增强的hTERT特异性TCRs的CD8I-J2细胞对HLAA*24：02转导的T2细胞上序列稀释的同源肽的识别能力。表达每个突变型TCR的CD8I-J2细胞识别hTERT多肽的浓度低于野生型TCR。这些突变的TCR与CD3-293T细胞上的四聚体结合是一致的，并且到目前为止，这些突变的TCR都没有与据报道存在于人类白细胞抗原-A*24：02分子上的无关多肽发生交叉反应。因此，这些方法对于从其他感兴趣的TCR获得高亲和力突变体可能是有用的。
This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCR beta, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCR alpha, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers.Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-gamma producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.