Characterization of Human Cellular Immune Responses to Novel Mycobacterium tuberculosis Antigens Encoded by Genomic Regions Absent in Mycobacterium bovis BCG

Patients.Heparinized venous blood was collected from newly diagnosed and culture-confirmed pulmonary TB patients (n = 48) attending the Chest Diseases Hospital, Kuwait. Buffy coats were obtained from M. bovis BCG-vaccinated and PPD-positive healthy subjects (n = 45) donating blood at the Central Blood Bank, Kuwait. The groups of healthy donors and TB patients were serologically negative for human immunodeficiency virus infection and included Kuwaiti, as well as non-Kuwaiti, citizens. Informed consent was obtained from all the subjects, and the study was approved by the Ethical Committee of the Faculty of Medicine, Kuwait University, Kuwait.

Complex mycobacterial antigens and synthetic peptides of M. tuberculosis.The complex mycobacterial antigens used in this study were whole-cell killed M. tuberculosis H37Rv and M. bovis BCG (1, 39) and M. tuberculosis culture filtrate (MT-CF) and cell walls (MT-CW) provided by P. J. Brennan (Colorado State University, Fort Collins) through the repository of TB research materials at the National Institute of Allergy and Infectious Diseases (NIH contract no. AI-25147).

A total of 1,648 peptides (25-mers overlapping neighboring peptides by 10 amino acids) spanning the sequence of putative proteins encoded by genes in the RD1, RD4 to RD7, RD9 to RD13, and RD15 genomic regions were designed based on the amino acid sequence deduced from the nucleotide sequences of the respective genes (Table 1). These peptides were commercially synthesized by Thermo Hybaid GmBH (Ulm, Germany) using fluonerylmethoxycarbonyl chemistry, as described previously (38, 39). The stock concentrations (5 mg/ml) of the peptides were prepared in normal saline (0.9%) by vigorous pipetting, and the working concentrations were prepared by further dilution in tissue culture medium RPMI 1640, as previously described (38, 39).

Isolation of PBMC and stimulation for antigen-induced proliferation and cytokine secretion.PBMC were isolated from the blood of TB patients and healthy subjects by flotation on Lymphoprep gradients using standard procedures (2, 30). The cells were finally suspended in complete tissue culture medium (RPMI 1640 plus 10% human AB serum, penicillin [100 U/ml], streptomycin [100 μg/ml], gentamicin [40 μg/ml], and fungizone [2.5 μg/ml]) and counted in a Coulter counter (Coulter Electronics Ltd., Luton, Bedfordshire, United Kingdom).

Antigenic stimulation of PBMC for proliferation and cytokine secretion was performed according to standard procedures (2, 35). In brief, 2 × 10⁵ PBMC suspended in 50 μl were seeded into the wells of 96-well tissue culture plates (Nunc, Denmark). Antigens in 50 μl complete medium were added at optimal concentrations to the wells in triplicate. Whole bacilli were used at 10 μg/ml (wet weight) and MT-CW at 1 μg/ml. All other antigens and peptides were used at an optimal concentration of 5 μg/ml. The cells in the control wells did not receive any mycobacterial antigen/peptide. The final volume of the culture in each well was adjusted to 200 μl. The plates were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. On day 6, the culture supernatants (100 μl) were collected from each well and frozen at −20°C until they were used to determine cytokine concentrations. The remaining cultures were pulsed with 1 μCi [³H]thymidine (Amersham Life Science, Amersham, United Kingdom) and harvested (Skatron Instruments As, Norway) according to standard procedures (36, 37).

Interpretation of proliferation results.The radioactivity incorporated was obtained as cpm. The average cpm was calculated from triplicate cultures stimulated with each antigen or peptide pool, as well as from triplicate wells of negative control cultures lacking antigen. The cell proliferation results were presented as the stimulation index (SI), which is defined as follows: SI = cpm in antigen or peptide-stimulated cultures/cpm in cultures lacking antigen or peptide. A patient was considered a responder to a given antigen if the PBMC yielded an SI of ≥3 (2). Based on the median SI and percentages of responders in antigen-induced proliferation assays, the antigens were considered strong (median SI, >10, with >70% responders), moderate (median SI, >3.0 but <10, with 50 to 70% responders), or weak (median SI, <3.0, with <50% responders) stimulators of PBMC.

Immunoassays for the quantitation of IFN-γ by enzyme-linked immunosorbent assay (ELISA).The concentrations of IFN-γ in the supernatants of PBMC from TB patients (n = 48) and healthy subjects (n = 45) were measured using Predicta immunoassay kits (Coulter/Immunotech, S.A., Marseille, France) as specified by the manufacturer. The detection limit of the IFN-γ assay kit was 0.08 U/ml. Secretion of IFN-γ in response to a given antigen was considered positive when delta IFN-γ (the IFN-γ concentration in cultures stimulated with antigen minus the IFN-γ concentration in cultures without antigen) was ≥3 U/ml (3). Based on the median delta IFN-γ and percentages of responders in IFN-γ assays, the antigens were considered strong (delta IFN-γ, >10, with >70% responders), moderate (delta IFN-γ, >3.0 but <10, with 50 to 70% responders), or weak (delta IFN-γ, <3.0, with <50% responders) stimulators of PBMC.

Immunoassays for the quantitation of cytokines by FlowCytomix kits.The supernatants, collected from the cultures of PBMC of TB patients (n = 17) before the [³H]thymidine pulse, were assayed to determine the concentrations of secreted cytokines by using FlowCytomix kits (Bender Medsystems Inc., Austria) according to the manufacturer's instructions. These kits allow simultaneous quantification of 10 cytokines, including Th1 cytokines, IFN-γ, IL-2, and TNF-β; Th2 cytokines, IL-10, IL-4, and IL-5; and the proinflammatory cytokines, TNF-α, IL-6, IL-8, and IL-1β. In brief, FlowCytomix technology is based on spectrally discrete microspheres that are used as the solid phase in an immunoassay. The beads are internally dyed with Starfire Red, a far-red (685- to 690-nm)-emitting fluorochrome that is excited by UV, argon, or HeNe lasers. The test samples were analyzed by flow cytometry using Coulter Epics FC500 (Beckman Coulter Inc.). For each analysis, up to 10,000 events were acquired. The mean concentration of each cytokine was expressed as pg/ml. By using the above-mentioned kits, the minimum detectable concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, TNF-β, and IFN-γ were 4.5 pg/ml, 8.9 pg/ml, 6.4 pg/ml, 5.3 pg/ml, 4.7 pg/ml, 6.4 pg/ml, 6.9 pg/ml, 7.9 pg/ml, 3.2 pg/ml, and 7.0 pg/ml, respectively. In response to antigenic stimuli, the values of E/C that were ≥2 were considered positive responses (4). E/C is defined as follows: E/C = cytokine concentration in antigen-stimulated cultures/cytokine concentration in cultures lacking antigen.

In the experiments where the concentrations of cytokines in control cultures lacking antigens were not detectable, the E/C values were determined by dividing the concentration of a given cytokine in antigen-stimulated cultures with the minimum detectable concentration of the same cytokine.

Statistical analysis.The antigen-induced proliferation and IFN-γ secretion results with antigens and peptide pools showing strong to moderate responses were statistically analyzed for significant differences between TB patients and healthy subjects using a nonparametric Mann-Whitney U test for two independent samples. P values of <0.05 were considered significant.

Antigen-induced proliferation of PBMC in response to complex mycobacterial antigens and RD peptides.In antigen-induced proliferation assays, PBMC from all TB patients and healthy subjects responded to one or more complex mycobacterial antigens (data not shown). In addition, all complex mycobacterial antigens were strong stimulators of PBMC from TB patients and healthy subjects, as indicated by high median SI values (14.3 to 25.3 and 19 to 22, respectively) and percentages of responders (77 to 90% and 96 to 98%, respectively) (Fig. 1A and B). Furthermore, the proliferation of PBMC from TB patients was strong in response to the RD1 peptide pool (median SI values, 11, and 75% responders); moderate in response to peptide pools of RD4, RD6, RD7, and RD9 (median SI values, 3.0 to 3.7, and 50 to 57% responders); and weak to peptide pools of RD5, RD10, RD11, RD12, RD13, and RD15 (median SI values, <3.0, and <50% responders) (Fig. 1C). In healthy subjects, only peptide pools of RD1 and RD7 induced moderate responses (SI values, 4.4 and 3.1, and responders, 62 and 51%, respectively), whereas all other peptide pools were weak stimulators of PBMC (median SI values, <3.0, and <50% responders) (Fig. 1D). The statistical analysis of antigen-induced proliferation responses showed that none of the complex mycobacterial antigens induced responses that were significantly different in TB patients than in healthy subjects (P > 0.05), whereas among the peptide pools inducing strong to moderate responses, only RD1 and RD4 induced significantly better responses (P < 0.05) in TB patients than in healthy subjects (Fig. 1A, B, C, and D).

• Open in new tab
• Download powerpoint

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.

Estimation of antigen-induced IFN-γ secretion by PBMC in response to complex mycobacterial antigens and RD peptides by ELISA.Antigen-induced IFN-γ was secreted by PBMC from all TB patients and healthy subjects in response to one or more complex mycobacterial antigens (data not shown). Furthermore, all complex mycobacterial antigens were strong stimulators of IFN-γ secretion by PBMC of TB patients (median delta IFN-γ, 12 to 18 U/ml, and responders, 73 to 89%) (Fig. 2A) and healthy subjects (median delta IFN-γ, 42 to 50 U/ml, and responders, 98 to 100%) (Fig. 2B). The statistical analysis of data showed that healthy subjects secreted significantly more IFN-γ than TB patients in response to all complex mycobacterial antigens (P < 0.05). In response to peptide pools, only RD1 peptides induced moderate responses in both TB patients (median delta IFN-γ, 4.0 U/ml, and 56% responders) and healthy subjects (median delta IFN-γ, 7.0 U/ml, and 62% responders), with no statistical difference between the two groups (P > 0.05). The peptide pools of all other RDs were weak stimulators of IFN-γ secretion in both TB patients and healthy subjects (median delta IFN-γ, <3.0 U/ml, and <50% responders) (Fig. 2C and D, respectively).

• Open in new tab
• Download powerpoint

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.

Estimation of antigen-induced secretion of cytokines by PBMC in response to complex mycobacterial antigens and RD peptides by FlowCytomix assays.Antigen-induced secretion of the proinflammatory Th1 and Th2 cytokines was studied with PBMC in 17 randomly selected TB patients from the group described above in response to the complex mycobacterial antigens and peptide pools of RDs. The secretion of the proinflammatory cytokine IL-6 by PBMC was observed in response to all of the complex mycobacterial antigens and peptide pools of RDs (data not shown), whereas IL-1β and TNF-α were secreted only in the presence of the complex mycobacterial antigens (data not shown), and IL-8 secretion was not enhanced by stimulation of PBMC with any antigen (data not shown).

Among the Th1 and Th2 cytokines, the secretion of TNF-β was induced by complex mycobacterial antigens and peptide pools of RD1, RD6, and RD13 (data not shown), whereas IL-2, IL-4, and IL-5 secretion by PBMC could not be detected in response to any of the antigens and RD peptides (data not shown). However, strong antigen-induced IFN-γ secretion was observed in response to all complex mycobacterial antigens, with the percentages of responders ranging from 76 to 88%. The peptide pools of RD1, RD4, RD5, RD6, RD7, RD9, and RD10 induced secretion of IFN-γ with 53 to 31% positive responders, whereas RD11, RD12, RD13, and RD15 were weak inducers of IFN-γ secretion, as indicated by 19 to 25% positive responders. Strong antigen-induced secretion of IL-10 was observed with all the complex mycobacterial antigens, with 76 to 100% positive responders. Among the peptide pools, RD6, RD12, RD13, and RD15 showed antigen-induced secretion of IL-10, with 38 to 47% positive responders, whereas RD1, RD7, RD9, RD10, and RD11 induced IL-10 secretion from PBMC of less than 25% of patients.

The determination of relative concentrations of Th1 and Th2 cytokines by calculating IFN-γ/IL-10 ratios indicated distinct Th1 biases in response to MT-CF and peptide pools of RD1, RD5, RD7, RD9, and RD10 (ratio of IFN-γ to IL-10 = 32 to 5.0), whereas in response to other complex mycobacterial antigens and peptide pools of RDs, no Th2 biases were observed (ratio of IFN-γ to IL-10 = 1.1 to 0.4).

Antigen-induced IL-10 secretion and effects of RD12 and RD13 peptides on antigen-induced proliferation and IFN-γ secretion by PBMC of healthy subjects.To further evaluate IL-10 secretion in response to complex mycobacterial antigens and peptide pools in healthy subjects, randomly selected culture supernatants from PBMC of 18 M. bovis BCG-vaccinated healthy subjects were tested for IL-10 concentrations using ELISA. The analysis of the results showed that PBMC of healthy subjects secreted IL-10 in response to all complex mycobacterial antigens (median concentrations, 284 to 846 pg/ml), but in response to RD peptides, only RD12 and RD13 showed antigen-induced IL-10 secretion (median concentrations, 215 and 140 pg/ml, respectively) (Fig. 3A and B).

• Open in new tab
• Download powerpoint

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.

To determine if secretion of IL-10 in response to RD12 and RD13 peptides could have any effect on the response of PBMC to Th1 cell-stimulating antigens, experiments were performed with PBMC from healthy subjects (n = 10) in the presence of four different concentrations (0.2, 1.0, 5.0, and 25 μg/ml) of these RD peptides, either alone or in combination with RD1. The results of antigen-induced proliferation and IFN-γ secretion assays showed that when tested alone, RD12 and RD13 failed to induce proliferation or IFN-γ secretion at all concentrations, whereas RD1 peptides showed optimal responses in both assays at 5 μg/ml (Fig. 4A; data are shown for proliferation only). Further experiments with PBMC from the same donors at the optimal (5 μg/ml) (Fig. 4B) and suboptimal (1 μg/ml) (Fig. 4C) concentrations showed that addition of either RD12 or RD13 peptides to the PBMC cultures, at concentrations equal to those of RD1 peptides, inhibited RD1-induced proliferation and IFN-γ secretion at both concentrations (Fig. 4B and C; data are shown for proliferation only). These inhibitory effects were not due to a toxic effect of RD12 and RD13 on PMBC, because the cells were >98% viable (as determined by the trypan blue exclusion technique) at the end of the culture period in cultures with and without RD12 and RD13.

• Open in new tab
• Download powerpoint

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.