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

在本项目中，开展了以下研究。(1)γδ四聚体的高分辨X射线晶体分析(2)基于X射线结晶学分析的γδ四聚体的设计(3)γδ-TCR四聚体的制备。(4)用γδ-TCR四聚体鉴定肿瘤抗原。关于(1)，在205点截断的γ链和在232点的δ链在初步的X射线分析中给出了高分辨率的衍射图。采用基于PEG4000的缓冲液，可重复性地获得单晶，其空间群是同步加速器分析的理想空间群。在第8春的分析中，获得了1.9A的分辨率极限的衍射图，这足以进行高分辨率的结晶学分析。经过分子模拟和细化，在2.4A的分辨率下没有发现晶体结构上的矛盾。在(2)中，在δ205的下游引入了生物素化序列，从而获得了最有效的复性速度。至于(3)，原来的γ链和生物素标记的δ链的包涵体混合复性，生物素连接到包涵体上。(4)用新制备的四聚体对肿瘤细胞进行广谱分析时，发现〓表达四聚体的配体。根据这些发现，γδT细胞显然识别肿瘤细胞上表达的某些抗原。此外，IRP60可能参与了γδT细胞对非多肽抗原的识别。通过对多种肿瘤细胞系的分析，发现该分子的四聚体能够识别U937。综上所述，我们可以采取坚定的立场来阐明γδT细胞识别非肽抗原的机制。

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.