Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG

Abstract

Comparative genomics has identified several regions of differences (RDs) between the infectious Mycobacterium tuberculosis and the vaccine strains of Mycobacterium bovis BCG. We aimed to evaluate the cellular immune responses induced by antigens encoded by genes predicted in 11 RDs. Synthetic peptides covering the sequences of RD1, RD4 to RD7, RD9 to RD13, and RD15 were tested for antigen-induced proliferation and secretion of Th1 cytokine, gamma interferon (IFN-gamma), by peripheral blood mononuclear cells (PBMC) obtained from culture-proven pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects. Among the peptide pools, RD1 induced the best responses in both donor groups and in both assays. In addition, testing of TB patients' PBMC for secretion of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 6 [IL-6], IL-8, and IL-1beta), Th1 cytokines (IFN-gamma, IL-2, and TNF-beta), and Th2 cytokines (IL-4, IL-5, and IL-10) showed differential effects of RD peptides in the secretion of IFN-gamma and IL-10, with high IFN-gamma/IL-10 ratios (32 to 5.0) in response to RD1, RD5, RD7, RD9, and RD10 and low IFN-gamma/IL-10 ratios (<1.0) in response to RD12, RD13, and RD15. Peptide-mixing experiments with PBMC from healthy subjects showed that secretion of large quantities of IL-10 in response to RD12 and RD13 correlated with inhibition of Th1 responses induced by RD1 peptides. In conclusion, our results suggest that M. tuberculosis RDs can be divided into two major groups--one group that activates PBMC to preferentially secrete IFN-gamma and another group that activates preferential secretion of IL-10--and that these two groups of RDs may have roles in protection against and pathogenesis of TB, respectively.

FIG. 1.

Antigen-induced proliferation of PBMC of TB patients and healthy subjects in response to complex mycobacterial antigens (A and B, respectively) and RD peptides (C and D, respectively). PBMC obtained from pulmonary TB patients (n = 48) and M. bovis BCG-vaccinated healthy subjects (n = 45) were cultured in the presence of complex mycobacterial antigens and pools of RD peptides. Each point represents the SI by PBMC from an individual donor in response to a given antigen. The median SIs are represented by horizontal bars, and the percentages of positive responders to each antigen are presented. The dashed lines indicate cutoff SI values of ≥3.

FIG. 2.

IFN-γ secretion by PBMC of TB patients and healthy subjects in response to complex mycobacterial antigens (A and B, respectively) and RD peptides (C and D, respectively). PBMC obtained from pulmonary TB patients (n = 48) and M. bovis BCG-vaccinated healthy subjects (n = 45) were cultured in the presence of complex mycobacterial antigens and pools of RD peptides, and the supernatants collected on day 6 were tested for concentrations of secreted IFN-γ (U/ml). Each point represents the concentration of the cytokine secreted by PBMC from an individual donor in response to a given antigen. The median concentrations of IFN-γ are represented by horizontal bars, and the percentages of positive responders in response to each antigen are presented.

FIG. 3.

IL-10 secretion by PBMC of M. bovis BCG-vaccinated healthy subjects in response to complex mycobacterial antigens (A) and RD peptides (B). PBMC obtained from M. bovis BCG-vaccinated healthy subjects (n = 18) were cultured in the presence of complex mycobacterial antigens and pools of RD peptides. The culture supernatants were collected on day 6 and tested for concentrations of secreted IL-10 (pg/ml). Each point represents the concentration of IL-10 secreted by PBMC from an individual donor in response to a given antigen. The median concentration of each cytokine is represented by a horizontal bar.

FIG. 4.

Antigen-induced proliferation of PBMC at various concentrations of RD1, RD12, and RD13 peptides (A) and the effects of adding RD12 or RD13 peptides on the proliferation of PBMC induced by RD1 peptides at suboptimal (1-μg/ml) (B) and optimal (5-μg/ml) (C) concentrations. (A) PBMC obtained from healthy subjects (n = 10) were cultured for antigen-induced proliferation in the presence of RD1, RD12, and RD13 peptides at 0.2, 1, 5, and 25 μg/ml. The results are expressed in terms of the SI, and the mean values at each concentration ± standard errors of the mean are presented. (B and C) The effects of RD12 and RD13 peptides on the proliferation induced by RD1 peptides were studied by stimulating PBMC at suboptimal (1-μg/ml) (B) and optimal (5-μg/ml) (C) concentrations of RD1 peptides and adding the peptides of RD12 or RD13 to the cultures at concentrations equal to those of RD1 peptides. The proliferation results are expressed as the SI, and the mean values, along with the standard errors of the mean, are represented by bars and vertical lines, respectively. The percentage inhibition (SI in the presence of RD1 peptide and RD12 or RD13 peptide × 100/SI in the presence of RD1 peptide alone) values are given above the corresponding bars and are statistically significant (P < 0.05; Mann-Whitney U test) at suboptimal (B) and optimal (C) peptide concentrations.