Résumé
Objective To evaluate the utility of CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14+ monocytes in the blood of tuberculosis (TB) patients .Methods CD4+ TCR tetramers were used to detect tetramer‐positive CD14+ monocytes in the peripheral blood (PBL ) samples of inpatients with advanced pulmonary TB (PTB) by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14+ monocytes and Mycobacterium tuberculosis (MTB) antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of tetramer‐bound CD14+ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4+ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4+ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14+ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14+ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect M TB antigen‐specific CD14+ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .
Résumé
ObjectiveTo investigate the specificity of CD4+ Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis(MTB) infections.MethodsThe above TCR tetramer by using biotinylated 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 tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells by in situ staining.ResultsThe 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.ConclusionThe 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 tetramers.