Background
Tuberculosis (TB), an infectious disease caused by the bacillus
Mycobacterium tuberculosis (MTB), is a global health problem. Overall, a relatively small proportion (5–15%) of the estimated 2–3 billion people infected with
MTB will develop TB disease during their lifetimes [
1]. However, the probability of developing TB is much higher among people infected with human immunodeficiency virus (HIV), indicating that the adaptive immune response plays a vital role in controlling
MTB pathogenesis. The components of this protective immune response have been investigated with an emphasis on the role of T cells secreting type-1 cytokines, specifically TNF-α and IFN-γ, in the formation of granulomas in the lung [
2‐
6].
However, a failing immune response in the lung may be involved in the formation of pulmonary cavities. With regard to the TNF-α and IFN-γ responses of cavitary TB, several investigators have produced conflicting results. Casarini noted only that IFN-γ but not TNF-α was reduced in cavities compared with areas of infiltrates as measured by ELISA [
4], and Condos demonstrated similar results [
5]. However, Mazzarella pointed to a reduction in type-1 cytokines from both cavities and areas of infiltrates [
6]. Fan et al. also recently demonstrated that the
MTB antigen-specific Th1 response was decreased when pulmonary TB (PTB) lesions developed in severe cavities [
7].
T cells specific for multiple immunogenic antigens of
MTB have recently been identified, including the Ag85 complex, culture filtrate protein 10 (CFP-10), early secretory antigenic target 6 (ESAT-6), heat shock protein 65, and TB10.4 [
8‐
11]. In particular, ESAT-6 and CFP-10 have been utilized as antigens for the immune diagnosis of
MTB infection based on the presence of IFN-secreting cells; both have been shown to be more specific and sensitive than tuberculin skin tests [
12].
Flow cytometry (FCM) is a powerful technology for characterizing each cell in terms of both the intracellular cytokines, and also activation markers. In addition, FCM is better than enzyme-linked immunosorbent assay (ELISA) to detect cytokines in peripheral blood mononuclear cells (PBMCs) from patients with TB, because ELISA cannot reveal which specific cells are producing and secreting the measured cytokines.
To clarify MTB-specific lymphocyte responses in TB patients with pulmonary cavities, we utilized ESAT-6 and CFP-10 as immunogenic antigens and FCM to determine the blood lymphocyte responses of CD4+ and CD8+ T lymphocytes producing and secreting IFN-γ and TNF-α in order to study the local immune responses of subjects with cavitary disease as compared to individuals having pulmonary infiltrates.
Methods
Study subjects and ethics statement
Our study included 46 newly diagnosed patients with PTB and 23 household contacts from the physical examination center of our hospital with both TST and T-SPOT negative. All information on the patients was recorded in the clinical database of the Infectious Disease Hospital of Wuxi, China. PBMCs were isolated before treatment from blood samples by means of centrifugation with Ficoll-Hypaque (Sigma). PTB was diagnosed when subjects with clinical and/or imaging features compatible with TB met at least one of the following criteria: positive sputum smear for acid-fast bacilli; positive culture for MTB, biopsy suggestive of TB, and/or full response to anti-TB treatment. A radiologist reviewed the posteroanterior chest radiographs (CXRs) of all 48 newly diagnosed PTB patients for the presence or absence of cavities. A respiratory clinician reviewed all CXRs at the same time, with both agreeing on the results.
Synthetic peptides
Peptides (16- to 18-mers overlapping by 10 aa) corresponding to the sequence of the
MTB antigens CFP-10 and ESAT-6 were synthesized on an automated peptide synthesizer. For initial screening purposes, peptides were arranged into pools of 9 peptides each in matrix fashion, such that each peptide was uniquely represented in 2 pools [
13].
Intracellular cytokine staining and immunophenotyping
PBMCs were isolated from heparin anticoagulant whole blood and incubated with stimulated antigens using 200 μL of per stimulation in a polypropylene tube. Each patient had four different stimulation setups: negative (medium alone), positive [Phorbol 12-myristate 13-acetate (PMA), each at 5 mg/mL], CFP-10 (10 mg/mL), and ESAT-6 (10 mg/mL). Tubes were vortexed, covered, and kept overnight (16 h) at 37 °C in a 5% CO2 incubator. In addition, 10 μg of Brefeldin A (Sigma) was added after 2 h of incubation. Following overnight stimulation, the cells were washed and stained with the following surface antibodies at room temperature for 15 min: energy-coupled dye (ECD)-conjugated anti-CD3 and fluorescein isothiocyanate (FITC)-conjugated anti-CD8. The cells were washed, fixed, and permeabilized (with Caltag reagents A and B) (Caltag Laboratories). The following intracellular antibodies were then added: phycoerythrin–Cy5.5 (PE-Cy5.5)-conjugated anti-IFN-γ and phycoerythrin (PE)-conjugated TNF-α. All antibodies were purchased from Becton–Dickinson.
The cells were incubated for 15 min and then washed and placed on an FC500 (Beckman Coulter) flow cytometer. Negative controls consisting of PBMCs incubated with medium alone were included in each assay. Responses ≥0.03% above the background value were considered to be positive. For phenotypic analysis of CD8 cells, peptide-stimulated cells were gated on CD3+/CD8+ cells and analyzed for expression of TNF-α and IFN-γ. For phenotypic analysis of CD4 cells, peptide-stimulated cells were gated on CD3+/CD8− cells and analyzed for expression of TNF-α and IFN-γ.
Statistical methods
Statistical analyses were conducted using GraphPad Prism software version 4.0. The unpaired, nonparametric t test (Mann–Whitney test), Chi square test, and Spearman rank correlation analysis were performed. Values of P < 0.05 were considered to be statistically significant for all analyses.
Discussion
Type-1 immune dominance during TB was shown by the predominance of CD4+ and CD8+ T cells responsive to
MTB antigens by rapid IFN-γ and TNF-α synthesis. These cells were found in both radiologically involved and uninvolved pulmonary sites but were conspicuously reduced in areas of cavity formation [
14]. Our results indicate that proportions of TNF-α- and IFN-γ-producing cells within both the CD4+ and CD8+ subsets were significantly reduced in TB patients with cavities compared with those with radiological infiltrates after stimulation of ESAT-6 or CFP-10, respectively, although the activities of IFN-γ and TNF-α were broad and included both beneficial and detrimental effects [
15]. IFN-γ and TNF-α may help to recruit cells to the site of infection and promote the antimicrobial activity of macrophages [
16,
17]. In addition, TNF-α can lead to TNF-mediated apoptosis of infected macrophages, thus helping to eliminate the pathogen [
14]. Therefore, type-1 cytokines, IFN-γ and TNF-α, may generate protective granulomas and enhance the killing of
MTB within macrophages.
However, the IFN-γ and TNF-α responses of both CD4+ and CD8+ T lymphocytes from TB patients were remarkably impaired in those individuals with pulmonary cavities. The pulmonary cavity has been the classic hallmark of TB and is the site of a very high
MTB burden.
MTB infection triggers the recruitment of leukocytes and the activation of intercellular networks, which then results in tissue destruction [
18]. Successful immune responses lead to the formation of granulomas and curtailment of the disease process, whereas cavitation indicates a failing immune response [
19]. Histological examination also demonstrates a predominance of acid-fast bacilli only at the internal surfaces of the cavities, at which site there are few CD4 and CD8 lymphocytes [
19]. The pulmonary cavities are the sites of high
MTB burdens.
Cavitation has also been reported to be associated with local neutrophilia and relative lymphopenia [
14]. Our results show that TB patients with cavities had higher absolute numbers of neutrophils than did patients with infiltrates; moreover, further analysis indicated an inverse correlation between the proportions of
MTB-specific IFN-γ-secreting T cells and neutrophil counts. The accumulation of neutrophils together with these impaired
MTB-specific lymphocyte responses probably plays an important role in the pathogenesis of cavitary TB.
Our study also has limitations. The eligible patients represented a fraction of the patients diagnosed with active TB, raising a concern for a selection bias. The antigen of CD4 was endocytosed with the stimulation of PMA and also impaired by the process of permeabilization. Therefore for the phenotypic analysis of CD4 cells, peptide-stimulated cells were gated on CD3+ CD8− cells. Except for these limitations, we conclude that MTB-specific lymphocyte responses are impaired in TB patients with pulmonary cavities, which may explain the high MTB burden at the cavity site and the severity of cavitary.
Authors’ contributions
HP and JW conceived and designed the experiments. JW, JL and YD performed the experiments. JW analyzed the data. All authors read and approved the final manuscript.
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