Characterization of a 10- to 14-kilodalton protease-sensitive Mycobacterium tuberculosis H37Ra antigen that stimulates human γδ T cells

Abstract

γδ T-cell receptor-bearing T cells (γδ T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for γδ 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 γδ T cells. γδ T-cell activation was measured by determining the increase in percentage of γδ T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of γδ 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 γδ 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 γδ 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, γδ T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. γδ 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 γδ T cells.