T-cell responses to the Mycobacterium 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-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma 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-gamma 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-gamma 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-gamma 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.

FIG. 1.

Evaluation of IFN-γ production in response to rESAT-6 antigen in vitro. (A) PBMCs obtained from Brazilian TB patients with either untreated (n = 26) or treated (n = 24) pulmonary TB and controls (n = 20) were cultured in the presence of ESAT-6 (5 μg/ml), and after 5 days, supernatants were harvested and assayed by ELISA. (B) Cells obtained from pleural TB patients (n = 10) and from pulmonary TB patients with (n = 18) and without (n = 32) lung cavity versus controls (n = 20) were also assayed in vitro in response to ESAT as described above. (C) The responses of healthy controls (HC) who presented a positive (>10 mm induration, n = 6) or negative (≤4 mm induration, n = 10) TST and the responses of leprosy patients (n = 14) were evaluated in vitro after PBMC stimulation with ESAT-6 and compared to those of TB patients. Results are mean picograms of protein per milliliter ± standard error for each group of individuals with positive responses (IFN-γ, >100pg/ml) to the antigen. PBMC responses to recombinant protein controls (vector controls) were mostly below 30 pg/ml. ∗, significant difference compared to controls or to leprosy patients; ∗∗, significant difference compared to TST-negative healthy controls. Differences in response between TB patients and TST-negative healthy controls were marginally significant (P = 0.05).

FIG. 2.

T-cell responses of TB patients and controls to mycobacterial antigens. (A) Secretion of IFN-γ by PBMCs was assessed after in vitro stimulation with PPD, when after 5 days, supernatants were harvested and assayed by ELISA. The values shown are mean IFN-γ level ± standard error for all groups studied: pleural TB (n = 10), pulmonary TB (n = 50), TST-positive healthy controls (HC; n = 6), and TST-negative healthy controls (n = 10). No major differences were found in the IFN-γ levels among the groups. (B) The PBMC response to the 85B antigen (2.5 μg/ml) was also evaluated in TB patients (treated TB, n = 18; untreated TB, n = 14) and in the control group (healthy TST-negative donors and non-TB patients; n = 17). Horizontal bars represent mean IFN-γ values that were not significantly different among the groups; (C) The in vitro T-cell responses to mycobacterial antigens of the TST-positive healthy controls and the TST-negative donors were compared. Following in vitro stimulation of PBMCs with either ESAT-6 (5 μg/ml), PPD (5 μg/ml), or Ag85B (2.5 μg/ml), culture supernatants were assayed by ELISA. A total of 6 TST-positive healthy controls and 10 TST-negative healthy controls were evaluated. The values shown represent the mean level of IFN-γ ± standard error and were significantly higher in the former group for all the antigens tested (∗, P < 0.05). All results are expressed as already subtracted from the values obtained in the unstimulated cultures. PBMC responses to recombinant protein controls were mostly below 30 pg/ml.

FIG. 3.

Intracellular flow cytometric detection of IFN-γ production in CD4⁺ and CD8⁺ T cells. PBMCs obtained from TB patients were stimulated with ESAT-6 (5 μg/ml) and anti-CD28 antibody as described in Materials and Methods. After the culture period, cells were harvested and evaluated by flow cytometry. Cells were acquired in the gated lymphocyte population, and the quadrants were set up with an isotype-matched control. The numbers in the figure represent the percentage of positive cells in each quadrant. The results are presented as already subtracted from the values obtained in nonstimulated control cultures with anti-CD28 and brefeldin A treatments. Data from one representative experiment are shown.