Characterization of a 10- to 14-kilodalton protease-sensitive Mycobacterium tuberculosis H37Ra antigen that stimulates human gamma delta T cells.
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.
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4984.
W H
WH
Boom
K N
KN
Balaji
R
R
Nayak
K
K
Tsukaguchi
K A
KA
Chervenak
D000942:Antigens, Bacterial / Q000276:immunology*; D003600:Cytosol / Q000276:immunology; D005434:Flow Cytometry; D006358:Hot Temperature; D006801:Humans; D007526:Isoelectric Point; D008213:Lymphocyte Activation / Q000276:immunology*; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000276:immunology*; D011402:Pronase / Q000378:metabolism; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D000942:Antigens, Bacterial; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011402:Pronase