gamma-delta+ and CD4+alpha-beta+ human T cell subset responses upon stimulation with various Mycobacterium tuberculosis soluble extracts

By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for human gamma delta(+) T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their gamma delta(+) T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85 degrees C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of gamma delta(+) T cells after 6-7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121 degrees C, 25 min) and then washed by centrifugation, a predominant proportion of CD4(+) alpha beta(+) T cells was achieved in the responding population. The stimulatory activity for gamma delta(+) T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving of M. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human gamma delta(+) T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on gamma delta(+) T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of gamma delta(+) T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4(+) alpha beta(+) T cells only. The gamma delta(+) T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of gamma delta(+) T cells on CD4(+) alpha beta(+) T cells