应用CD4＋Vα9-J27／Vβ29-D1-J2四聚体检测结核分枝杆菌感染的初步研究

The preliminary study of CD4＋Vα9-J27/Vβ29-D1-J2 tetramers in detecting Mycobacterium tubercu-losis infections

目的初步探讨CD4＋Vα9-J27／V1329-D1-J2TCR四聚体对结核分枝杆菌感染检测的特异性。方法应用果蝇s2恒定细胞株表达、纯化获得生物素化TCR复合物单体制备四聚体。流式检测PE-四聚体与细胞株膜结核抗原肽／HLA-DR复合物以及肺结核患者外周血的单核细胞的结合百分率，并设立病例对照组。激光共聚焦倒置显微镜观察肺结核病理组织及对照组织中四聚体与结核抗原双染阳性的细胞及四聚体与CD14双染阳性细胞。结果四聚体对表达C14／HLA—DRB1＊1504的细胞株结合能力最强。四聚体与结核患者外周血单核细胞结合率显著高于各对照组。结核组织标本可观察到四聚体与CD14双染阳性及四聚体与结核抗原双染阳性的细胞；而对照组织均为阴性。结论CD4＋TCR四聚体对结核检测有一定的特异性，为TCR四聚体应用于结核的诊断和免疫机制研究提供了一定的实验资料。

Objective To investigate the specificity of CD4＋Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis（MTB） infections. Methods The above TCR tetramer by using bioti- nylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell（S2 cell） lines was prepared. The PE-labled TCR tetramer was used to costain with S2 cell lines expressing MTB prptide/ HLA-DR complexes on the cell membrane, and also was used to detect tetramer-bound CD14＋ monocytes and macrophages in the peripheral blood mononuclear cells （PBMC） of pulmonary tuberculosis （PTB） patients and three control groups by flow cytometric analysis. And the FITC-labled tetramer was used to examine tet- ramer-bound CD14＋ monocytes and macrophages, and MTB antigen-specific and tetramer-bound cells by in situ staining. Results The TCR tetramer was well binding with S2 cell lines expressing C14/HLA-DR ＊ 1504 on the cell membrane. By flow cytometric analysis, the percentage of tetramer-bound CD14＋ monocytes and macrophages in PTB patients group was higher than the other three control groups（ P〈0.001 ）. By in situ staining, tetramer-bound CD14＋ monocytes and macrophages, and MTB antigen-specific and tetramer-bound cells were positive in PTB tissue and negative in control pneumonia tissue. Conclusion The spcificity of TCR tetramer in monitoring MTB infections by flow cytometric analysis and in situ staining could be seen, which laid a laboratory foundation in the diagnosis and immune mechanism research of TB by using TCR tet-ramer.