Identification of tumor antigens by the use of human γδ TCR tetramers

在本项目中，进行了以下研究。(1) γδ TCR的高分辨率x射线晶体学分析(2)基于x射线晶体学分析的γδ TCR四聚体设计。(3) γδ TCR四聚体的制备。(4)利用γδ TCR四聚体鉴定肿瘤抗原。对于(1)，在x射线初步分析中，截断的γ链205和δ链232给出了高分辨率的衍射图。利用peg4000为基础的缓冲液，可获得重复性好的单晶，其空间群是同步胞浆分析的理想选择。在SPring 8上进行分析，得到了分辨率为1.9A的衍射图，足以进行高分辨率的晶体学分析。经过分子建模和细化，在高达2.4A的分辨率下，晶体结构没有矛盾。在(2)中，δ205的下游引入了一个生物素化序列，这是最有效的再折叠速率。在(3)中，原γ-链和生物素化的δ-链的包涵体混合并重新折叠，生物素附着于其上。(4)利用新制备的四聚体对肿瘤细胞进行广谱分析时，发现RPMI8226能够表达该四聚体的配体。基于这些发现，γδ T细胞明显识别肿瘤细胞上表达的某些抗原。此外，IRP60被鉴定为可能参与γδ T细胞对非肽抗原识别的分子之一。通过对多种肿瘤细胞系的分析，发现该分子的四聚体可识别U937。综上所述，我们为阐明γδ T细胞对非肽抗原的识别机制奠定了坚实的基础。

In the present project, the following studies were performed. (1) High resolution X-ray crystallographic analysis of γδ TCR (2) Design of γδ TCR tetramers based on the X-ray crystallographic analysis. (3) Preparation of γδ TCR tetramers. (4) Identification of tumor antigens by using γδ TCR tetramers. Regarding (1), the truncated γ-chain at 205, and δ-chain at 232 gave diffraction patterns at high resolution on preliminary X-ray analysis. By employing PEG 4000-based buffer, single crystals were obtained reproducibly, whose space group was ideal for synchrocydotron analysis. On analysis at SPring 8, diffraction patterns were obtained at the resolution limit of 1.9A, which was enough for high resolution crystallographic analysis. After molecular modeling and refinement, no contradiction was obtained for crystal structure at the resolution of up to 2.4A. In (2), a biotinylation sequence was introduced immediate downstream of δ205, which gave the most efficient refolding rate. As for (3), inclusion bodies of the original γ-chain and the biotinylated δ-chain were mixed and refolded, to which biotin was attached. Finally the biotinlated TCR was tetramerized using PE-conjugated strep〓, (4) When analyzing a wide spectrum of tumor cells by using the newly prepared tetramers, RPMI8226 was found to express ligands for the tetramers. Based on these findings, γδ T cells apparently recognize certain antigens expressed on tumor cells. In addition, IRP60 was identified as one of the molecules, which may be involved in the γδ T cell recognition of nontpeptide antigens. On analysis of various kinds of tumor cell lines, the tetramer of this molecule was found to recognize U937. In conclusion, we could take a firm stand to elucidate the mechanism for γδ T cell recognition of nonpeptide antigens.