CHARACTERIZATION OF A 10-KILODALTON 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 Mycobac terium tuberculosis. The bacterial antigens responsible for gamma delt a T-cell activation remain poorly characterized. We have found that he at treatment of live M. tuberculosis bacilli released into the superna tant an antigen which stimulated human gamma delta T cells, gamma delt a T-cell activation was measured by determining the increase in percen tage of gamma delta T cells by flow cytometry in peripheral blood mono nuclear cells stimulated with antigen and by proliferation of gamma de lta T-cell lines with monocytes as antigen-presenting cells. Supernata nt from heat-treated M. tuberculosis was fractionated by fast-performa nce liquid chromatography (FPLC) on a Superose 12 column. Maximal gamm a delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing dem onstrated peak activity at a pi of <4.0. On two-dimensional gel electr ophoresis, the 10- to 14-kDa FPLC fraction contained at least seven di stinct molecules, of which two had a pi of <4.5. Protease treatment re duced the bioactivity of the 10- to 14-kDa FPLC fraction for both rest ing and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay w ith 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-stimulati ng antigen was not found in culture filtrate of M. tuberculosis but wa s associated,vith the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kD a, cell-associated, heat-stable, low-pI protein antigen of M. tubercul osis which is a major stimulus for human gamma delta T cells.