T-Cell Responses to theMycobacterium tuberculosis-Specific Antigen ESAT-6 in Brazilian Tuberculosis Patients

Abstract

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-␥), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-␥ secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-␥ in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-␥ in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-␥ production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.Tuberculosis (TB) remains a major public health problem in the 21st century. A third of the world's population is infected with Mycobacterium tuberculosis, and 5 to 10% of the infected population will develop the disease during their lifetime. TB is responsible for more than 2 million deaths per year worldwide (9). The situation is exacerbated by coinfections with human immunodeficiency virus (HIV) and the emergence of multidrug-resistant strains of M. tuberculosis. The Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine available against TB, and yet its efficacy is controversial. BCG has shown extremely variable levels of protection in different populations (12), ranging from 0% (38) in the study of Chingleput, South India, to 80% in The British MRC BCG Trial, Great Britain (37). A cell-mediated immune (CMI) response of the Th1 type, characterized by elevated production of gamma interferon (IFN-␥) and interleukin-12 (IL-12), is essential to mount a protective immunity against M. tuberculosis (8,13,26). Besides, a basic principle for selecting novel antigen candidates for subunit vaccine design is their capacity to induce a protective Th1 response. The definition of new antigens to be applied in an immune-based diagnostic assay for early detection of TB also has a high priority. Purified protein derivative (PPD) from M. tuberculosis is a mixture of complex antigens that has been long used as a skin...

Flow cytometric measurements of TB‐specific T cells comparing with QuantiFERON‐TB gold

“…It has been reported that BCG vaccination of human newborns induces a specific IFN-cþ CD8þ T cell response to BCG in vitro, although it is quantitatively smaller than the CD4þ T cell response (27). Regarding the non-Target cells (IFN-cþ CD4À T cells) positive case in the no TB group of this study, the significance of increased MTB antigen-stimulated IFN-c-producing CD8þ T cells should be elucidated in further studies (6,27,28).…”

“…The ICCFC has the advantage of identifying the composition of the secreting cells to reference populations of peripheral blood (2,6,(9)(10)(11)(12)(13)(14), bronchoalveolar lavage fluid (9,15), or induced sputum (16) using polychromatic immunophenotyping. Despite the obvious advantage of diagnostic accuracy, studies comparing ICCFC with two other methodologies have not yet been performed (7), with the exception of two recent reports, one with QFT (14), and the other with ELISpot (16).…”

“…The commercial kit for the latter is Quanti-FERON-TB Gold (QFT) (Cellestis Ltd, VIC, Australia). In addition, intracellular cytokine flow cytometry (ICCFC) assay can be performed according to a protocol developed for diagnosis using commercially available reagents (6)(7)(8).…”

Evaluation of T-Cell Responses to Novel RD1- and RD2-EncodedMycobacterium tuberculosisGene Products for Specific Detection of Human Tuberculosis Infection

“…Two of the low-molecular-mass potent T-cell antigens identified by Andersen and colleagues, early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP10), are encoded in RD1 (2, 10) and have been intensively investigated in animal models and humans over the last few years (4,7,15,16,26,30,32,33,36,40). Studies using several different assays, including lymphoproliferation, gamma interferon (IFN-␥) enzyme-linked immunosorbent assay (ELISA), and IFN-␥ enzyme-linked immunospot (ELISPOT) assay, have shown that they are strong targets of the cellular immune response in animal models, TB patients, and contacts (4,11,24,28,30,35,(37)(38)(39).…”

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

“…Pulmonary tuberculosis and leprosy were diagnosed according to the criteria established by the World Health Organization (WHO) and the Brazilian Ministry of Health. The detailed methodology for the screening and diagnosis of the tuberculosis and leprosy patients has been described elsewhere (15,16). The patients were treated according to the recommendations of the Ministry of Health.…”