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Gammadelta+ and CD4+ alphabeta+ human T cell subset responses upon stimulation with various Mycobacterium
1Dipartimento di Biomedicina Sperimentale, Infettiva e Pubblica, Università degli Studi di Pisa, Italy.
Insights
Preparation methods for Mycobacterium tuberculosis extracts significantly impact T cell responses. Autoclaving releases components that stimulate gammadelta T cells, while sonication primarily activates these cells, suggesting differential antigen presentation.
Area of Science:
- Immunology
- Microbiology
Background:
- Soluble extracts from Mycobacterium tuberculosis (M. tuberculosis) and M. avium stimulate human gammadelta+ T cells.
- The preparation method of M. tuberculosis extracts influences their T cell stimulatory capacity.
Purpose of the Study:
- To investigate how different preparation protocols for M. tuberculosis extracts affect T cell proliferation.
- To characterize the T cell subsets stimulated by M. tuberculosis extracts prepared via sonication versus autoclaving.
Main Methods:
- Flow cytometry to identify proliferating T cells.
- Preparation of M. tuberculosis extracts using heat (85°C sonication vs. 121°C autoclaving).
- Protease digestion and Fast Performance Liquid Chromatography (FPLC) for component analysis.
Main Results:
- Sonication-prepared extracts (TBe) strongly stimulated gammadelta+ T cells.
- Autoclaving released soluble components that predominantly stimulated CD4+ alphabeta+ T cells.
- Autoclaving-released antigens were protease-sensitive, while TBe's activity was only partially reduced by proteases.
- FPLC fractionation showed gammadelta+ T cell activity in low molecular weight fractions.
Conclusions:
- Bacterial preparation methods critically determine the type of T cell response elicited by M. tuberculosis extracts.
- Autoclaving releases distinct antigens that modulate T cell responses differently than sonication.
- Low molecular weight components are key stimulators for gammadelta+ T cells.
- Potential regulatory role of gammadelta+ T cells on CD4+ alphabeta+ T cells suggested.
Abstract:
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 gammadelta+ 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 gammadelta+ 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 gammadelta+ 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+ alphabeta+ T cells was achieved in the responding population. The stimulatory activity for gammadelta+ 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 gammadelta+ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on gammadelta+ 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 gammadelta+ 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+ alphabeta+ T cells only. The gammadelta+ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of gammadelta+ T cells on CD4+ alphabeta+ T cells.