Boom W H, Balaji K N, Nayak R, Tsukaguchi K, Chervenak K A
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4984.
Infect Immun. 1994 Dec;62(12):5511-8. doi: 10.1128/iai.62.12.5511-5518.1994.
gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells. gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pI of < 4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pI of < 4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated with the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.
携带γδ T细胞受体的T细胞（γδ T细胞）很容易被细胞内细菌病原体如结核分枝杆菌激活。负责γδ T细胞激活的细菌抗原仍未得到充分表征。我们发现，对活的结核分枝杆菌进行热处理后，会释放出一种能刺激人γδ T细胞的抗原到上清液中。通过流式细胞术测定外周血单核细胞中γδ T细胞百分比的增加以及以单核细胞作为抗原呈递细胞时γδ T细胞系的增殖来测量γδ T细胞的激活。将热处理的结核分枝杆菌的上清液在Superose 12柱上通过快速高效液相色谱（FPLC）进行分离。对于10至14 kDa的级分，测量到最大的γδ T细胞激活。通过制备性等电聚焦分离上清液显示在pI < 4.0时具有峰值活性。在二维凝胶电泳上，10至14 kDa的FPLC级分包含至少七个不同的分子，其中两个的pI < 4.5。蛋白酶处理降低了10至14 kDa的FPLC级分对静息和活化的γδ T细胞的生物活性。针对10至14 kDa级分产生的鼠抗体通过酶联免疫吸附测定法与结核分枝杆菌裂解物中10至14 kDa的抗原发生反应。此外，γδ T细胞对结核分枝杆菌裂解物中存在的10至14 kDa的抗原产生增殖反应。在结核分枝杆菌的培养滤液中未发现γδ T细胞刺激抗原，但与结核分枝杆菌的细菌沉淀和裂解物相关。这些结果提供了对结核分枝杆菌一种10至14 kDa、细胞相关、热稳定、低pI蛋白抗原的初步表征，该抗原是人类γδ T细胞的主要刺激物。
