Marisol Hernández-Garay1, Patricia Méndez-Samperio. 1. Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, ENCB, Instituto Politécnico Nacional, IPN, Mexico City, Mexico.
Abstract
BACKGROUND: A comprehensive understanding of the immune response induced by Mycobacterium bovis Bacille Calmette-Guérin in activation of protective T cells against tuberculosis is important to develop effective therapies to combat this disease. In this study, our experiments were designed to determine effects of transforming growth factor (TGF)-beta on M. bovis-induced T-cell activation and survival. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used for detection of apo-ptotic cells by three different methods: 1). scattered light change during early phase of apoptosis; 2). detection of hypodiploid DNA, or 3). terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) technique. Quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: TGF-beta added at initiation of culture did not alter percentage of viable cells. By contrast, TGF-beta added 72 h post-activation decreased percentage of viable cells. This effect was statistically significant (p <0.05). Furthermore, addition of anti-TGF-beta MoAb together with TGF-beta abolished the ability of this cytokine to decrease survival in post-activated human T cells. Role of TGF-beta on post-activated human T cells was further confirmed by staining apoptotic nuclei with propidium iodide, which detects late events of apoptosis, and by DNA fragmentation determined using TUNEL assay. Interestingly, TGF-beta did not promote Fas-mediated killing. Finally, TGF-beta increased apoptosis of CD4(+) T cells after mycobacterial stimulation. CONCLUSIONS: This study indicated an important role for TGF-beta in suppression of protective immune response against M. bovis by promoting elimination of post-activated T cells. Furthermore, results showed that TGF-beta had no direct effect on M. bovis-induced up-regulation of Fas (CD95).
BACKGROUND: A comprehensive understanding of the immune response induced by Mycobacterium bovis Bacille Calmette-Guérin in activation of protective T cells against tuberculosis is important to develop effective therapies to combat this disease. In this study, our experiments were designed to determine effects of transforming growth factor (TGF)-beta on M. bovis-induced T-cell activation and survival. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used for detection of apo-ptotic cells by three different methods: 1). scattered light change during early phase of apoptosis; 2). detection of hypodiploid DNA, or 3). terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) technique. Quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS:TGF-beta added at initiation of culture did not alter percentage of viable cells. By contrast, TGF-beta added 72 h post-activation decreased percentage of viable cells. This effect was statistically significant (p <0.05). Furthermore, addition of anti-TGF-beta MoAb together with TGF-beta abolished the ability of this cytokine to decrease survival in post-activated human T cells. Role of TGF-beta on post-activated human T cells was further confirmed by staining apoptotic nuclei with propidium iodide, which detects late events of apoptosis, and by DNA fragmentation determined using TUNEL assay. Interestingly, TGF-beta did not promote Fas-mediated killing. Finally, TGF-beta increased apoptosis of CD4(+) T cells after mycobacterial stimulation. CONCLUSIONS: This study indicated an important role for TGF-beta in suppression of protective immune response against M. bovis by promoting elimination of post-activated T cells. Furthermore, results showed that TGF-beta had no direct effect on M. bovis-induced up-regulation of Fas (CD95).
Authors: Adrian G Rosas-Taraco; David M Higgins; Joaquín Sánchez-Campillo; Eric J Lee; Ian M Orme; Mercedes González-Juarrero Journal: Tuberculosis (Edinb) Date: 2010-12-31 Impact factor: 3.131