肺结核患者治疗期外周血及病理切片多肽特异性CD4~+T细胞的检测

目的应用四聚体技术研究肺结核患者治疗前后各时间点外周血中E7多肽特异性CD4+T细胞的变化及结核患者肺组织切片中特异性CD4+T细胞的分布。方法培养能稳定分泌表达E7/HLA-DRB1*0404和E7/HLA-DRB1*1602的果蝇S2细胞株,表达纯化获得2种蛋白质单体,并制备成PE标记的相应四聚体,流式细胞分析检测肺结核患者外周血中特异性CD4+T细胞。制备FITC标记的四聚体,对肺结核患者的肺组织切片进行原位染色,观察特异性CD4+T细胞的分布。结果肺结核患者治疗前组及治疗1～60 d组分别与治疗61 d后各组及脐带血组之间的四聚体阳性多肽特异性CD4+T细胞差异均有统计学意义(P<0.05),但治疗前组与治疗1～60 d组之间、治疗61 d后各组之间及分别与脐带血之间、2个四聚体检测组之间差异均无统计学意义(P>0.05)。在肺结核患者的肺组织切片中可观察到特异性CD4+T细胞。结论制备的2种E7/HLA-DR四聚体对于结核病的检测具有一定的意义,可以用于检测研究肺结核患者治疗前后外周血E7多肽特异性CD4+T细胞的变化及肺结核患者局部病理组织中特异性CD4+T细胞的分布。

Detection of tetramer-bound/peptide-specific peripheral blood CD4~+ T cells and pathological analysis of tissue sections from patients with pulmonary tuberculosis

Objective To evaluate the percentage of tetramer-bound and peptide E7-specific CD4 + T cells in peripheral bloods(PBLs) during the treatment,and analyze the in situ staining of specific CD4 + T cells in the lung tissue sections from the patients with pulmonary tuberculosis(PTB).Methods E7 / HLA-DR(E7 / HLA-DRB1 * 0404 and E7 / HLA-DRB1 * 1602) tetramers were prepared from stable Drosophila Schneider 2 cell(S2 cell) lines and then labeled with PE.Specific CD4 + T cells from the PBL of PTB patients were analyzed with flow cytometric method.Tetramers labeled with FITC were used in the in situ staining of lung tissue slices for the examination of specific CD4 + T cells.Results Both the pre-treatment and PTB patients receiving 1 to 60 days treatment have statistical differences with other PTB patients groups(P<0.05) in the two tetramer tests.Meanwhile,both pre-treatment and 1～60 days′ PTB patients groups have statistical differences with control umbilical cord bloods group.In contrast,there were no statistical differences between the PBLs of pre-treatment PTB patients group and the PBLs of 1～60 days′ PTB patients group in the two tetramer tests(P>0.05).Antigen-specific CD4 + T cells were observed in the lung tissue slices of PTB patient by in situ tetramer staining.Conclusion Two types of E7 / HLA-DR tetramers were used for PTB detection.The tetramers could be used to track the change of the peptide-specific CD4 + T cells in the PBL of PTB patients during the treatment.It could also be used in the in situ staining of peptide-specific CD4 + T cells in the tissues of PTB patients.