Effector functions of heparin-binding hemagglutinin-specific CD8+ T lymphocytes in latent human tuberculosis

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS: HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION: During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.     

Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.     

分枝杆菌肝素结合血凝素研究进展

结核病是由分枝杆菌感染引起的世界范围内流行的人畜共患病。肝素结合血凝素(heparin-binding adhesin,HBHA)是分枝杆菌表达的一种蛋白质,致病性分枝杆菌的HBHA蛋白具有黏附上皮细胞、扩散感染范围等功能。其发挥黏附功能的C端结构域能够刺激机体产生有效的Th1细胞免疫反应;HBHA特异的黏膜免疫反应能够提供一定的保护效果。这提示HBHA具有制作为疫苗的潜力。而且,HBHA蛋白在活动性结核病患者和潜伏感染者中引发的免疫反应存在差异,这使其可以成为新的诊断抗原。此外,在细胞水平上,HBHA可以利用补体系统进入巨噬细胞,促使巨噬细胞向M1极化,同时能够逃避自噬并诱发线粒体损伤和内质网应激介导功能,这为结核病的致病机制提供了更多的解释。HBHA与宿主之间相互作用认识的积累,可以为HBHA的应用性研究提供依据。     

Regulatory T cells depress immune responses to protective antigens in active tuberculosis

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.     

Protective immunity against Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen with which over a billion people have been infected and 3 million people die annually. The bacterium induces vigorous immune responses, yet evades host immunity, persisting within phagosomes of the infected macrophages. Thus, it is necessary to delineate that the virulence-related intracellular survival mechanism and the host immune responses to eradicate M. tuberculosis on the molecular basis. In this regard, recent findings clearly indicated that Toll-like receptors (TLRs) play an essential role in the recognition of MTB components by macrophages and dendritic cells, resulting in not only activation of innate immunity but also development of antigen-specific adaptive immunity. It has been also reported that induction of early death of the infected cells may be one of the strategy of host defense against MTB because macrophages go into apoptosis upon infection with MTB, resulting in suppression of the intracellular replication. Furthermore, recent report has shown that autophagy is induced by IFN-gamma and suppress intracellular survival of mycobacteria, suggesting that activation of autophagy pathway is required to overcome phagosome maturation arrest induced by MTB. In addition, it is known that IFN-gamma plays an important role in protection. The cytokine that is produced from NK cells and dendritic cells at the early period of infection strongly induces not only macrophage activation but also development of antigen-specific IFN-gamma-producing CD4+T cells. Since antigen-specific CD8+ T cells and CD1-restricted T cells are also reported to contribute to the protective immunity, cooperation of these T cells is essential for the host resistance. In this paper, I am going to summarize the recent progress of the understanding of protective immunity against MTB.     

Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis

Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M. tuberculosis. CCR2(-/-) mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2(+/+) mice. CCR2(-/-) mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung. CCR2(-/-) mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar in CCR2(-/-) and CCR2(+/+) mice. However, T cell priming was delayed in the MLNs of the CCR2(-/-) mice, and fewer CD4(+) and CD8(+) T cells primed to produce IFN-gamma accumulated in the lungs of the CCR2(-/-) mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M. tuberculosis.     

Lack of alpha-1 integrin alters lesion morphology during pulmonary Mycobacterium tuberculosis infection

The hallmark of Mycobacterium tuberculosis infection is the granuloma, a highly dynamic immune structure that contains the bacilli during chronic infection. Here, we examined if alpha1beta1 integrin is required in the development and maintenance of the granulomatous structure during pulmonary infection using the alpha1 integrin knockout (alpha1-null) mouse. The alpha1beta1 integrin is expressed on activated macrophages and T cells, and interacts with collagen molecules in the extracellular matrix (ECM), and thus may play a role in the granulomatous process. Following pulmonary infection with virulent M. tuberculosis, lungs of alpha1-null infected mice had striking differences in granuloma structure, as well as distinct and markedly thickened alveolar septae. By day 180, there were regions of cell death within granulomatous lesions, characterized by cellular debris in these mice. To determine if this molecule was necessary for T cell trafficking within the lungs, the expression of CD4, CD44 and CD62L was monitored. The number of activated and IFN-gamma-producing CD4(+) T cells increased in the lungs of alpha1-null mice during the chronic phase of infection, although they had decreased concentrations of TNF-alpha and MMP-9. These results suggest that while alpha1beta1 integrin is not required for trafficking or maintenance of T cells in M. tuberculosis infected lungs, it does play a role in granuloma structure and integrity during the chronic phase of infection.     

Compartmentalized bronchoalveolar IFN-gamma and IL-12 response in human pulmonary tuberculosis

Human tuberculosis (TB) principally involves the lungs, where local immunity impacts on the load of Mycobacterium tuberculosis (M.tb). Because concomitants of local Th1 immunity are still under-explored in humans, we characterized immune responses in bronchoalveolar cells (BACs) and systemically in peripheral blood mononuclear cells (PBMCs) in persons with active pulmonary TB and in healthy community controls. PPD- and live M.tb-induced IFN-gamma-production were observed in CD4(+), CD8(+), gammadeltaTCR(+), and CD56(+) alveolar T cell subpopulations and NK cells (CD3(-)CD56(+)). IFN-gamma-producing CD4(+) T cells (mostly CD45RO(+)) were more abundant (p<0.05). M.tb-induced IL-12p70, but interestingly also IL-4, was increased (p<0.05) in BACs from TB patients. Constitutive expression of IL-12Rbeta1 and IL-12Rbeta2 mRNA in BACs and PBMCs and IFN-gammaR1 in BACs was similar in both study groups. Data were normalized to account for differences in proportions of alveolar T cells and macrophages in the study groups. IFN-gamma-production and its induction by IL-12R engagement occur virtually unimpaired in the bronchoalveolar spaces of patients with pulmonary TB. The reasons for the apparent failure to control M. tuberculosis growth during active pulmonary TB disease is unknown but could be the expression of locally acting immunosuppressive mechanisms that subvert the antimycobacterial effects of IFN-gamma.     

Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.     

Human immunity to M. tuberculosis: T cell subsets and antigen processing

A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control infection through acquired immunity, in which antigen specific T cells and macrophages arrest growth of M. tuberculosis bacilli and maintain control over persistent bacilli. In addition to CD4+ T cells, other T cell subsets such as, gammadelta, CD8+ and CD1-restricted T cells have roles in the immune response to M. tuberculosis. A diverse T cell response allows the host to recognize a wider range of mycobacterial antigens presented by different families of antigen-presenting molecules, and thus greater ability to detect the pathogen. Macrophages are key antigen presenting cells for T cells, and M. tuberculosis survives and persists in this central immune cell. This is likely an important factor in generating this T cell diversity. Furthermore, the slow growth and chronic nature of M. tuberculosis infection results in prolonged exposure to antigens, and hence further T cell sensitization. The effector mechanisms used by T cells to control M. tuberculosis are poorly understood. To survive in macrophages, M. tuberculosis has evolved mechanisms to block immune responses. These include modulation of phagosomes, neutralization of macrophage effector molecules, stimulating the secretion of inhibitory cytokines, and interfering with processing of antigens for T cells. The relative importance of these blocking mechanisms likely depends on the stage of M. tuberculosis infection: primary infection, persistence, reactivation or active tuberculosis. The balance of the host-pathogen interaction in M. tuberculosis infection is determined by the interaction of T cells and infected macrophages. The outcome of this interaction results either in control of M. tuberculosis infection or active disease. A better understanding of this interaction will result in improved approaches to treatment and prevention of tuberculosis.     

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-gamma-secreting CD4(+) and CD8(+) T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8(+) T cells in infected tissues of challenged mice. In contrast, specific IFN-gamma production by CD4(+) T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-gamma secretion by CD4(+) T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.     

Half-truths and selective memory: Interferon gamma, CD4(+) T cells and protective memory against tuberculosis

Efforts to develop a new, more effective vaccine for tuberculosis have been hampered by a lack of understanding of what constitutes a protective memory immune response. While interferon gamma production by CD4(+) T cells after vaccination is commonly used as a surrogate of protective memory immunity, its use in this regard appears to have little predictive value. We argue that this is due to the different requirements for interferon gamma-mediated protection in the primary response versus the memory recall response. In this review, we present evidence that suggests memory CD4(+) T cells can protect against tuberculosis in the absence of interferon gamma, and discuss potential mechanisms that may be involved such as IL-17 and regulatory T cells. A comprehensive understanding of the requirements for protective memory immunity to tuberculosis is essential for the development of an effective vaccine.     

Increased frequency of CD4(+)CD25(high) Treg cells inhibit BCG-specific induction of IFN-gamma by CD4(+) T cells from TB patients

Cell-mediated immunity plays a considerable role in the protection against Mycobacterium tuberculosis infection. The immune response to tuberculosis (TB) was dominated by both CD4(+) T cells with the T helper 1 type cytokines and CD8(+) T cells. Recent studies have suggested that the circumstances in which protective or tissue-damaging T cell responses to microbes are affected by the activity of Treg (CD4(+)CD25(high)) cells. In the present study, we demonstrated that the frequencies of CD4(+)CD25(+) and CD4(+)CD25(high) T cells in TB patients were significantly higher compared to normal individuals. These Treg cells expressed CTLA-4 and Foxp3 at protein level and displayed activation and memory phenotypes as assessed by flow cytometric analysis. The frequencies of CD4(+)CD25(high)CTLA-4(+) and CD4(+)CD25(high)Foxp3(+) T cells within the total CD4(+) T cell population were significantly increased in the blood of TB patients compared to healthy donors. Moreover, the expression of GITR on Treg cells was higher in TB patients than in normal donors. The phenotypic analysis demonstrated that CD4(+)CD25(high) Treg expressed higher levels of CD45RO and HLA-DR, and lower levels of CD45RA compared to CD4(+)CD25(low) and CD4(+)CD25(-) T cells. The addition of CD4(+)CD25(high) T cells back to cultures could significantly suppress the antigen-specific production of IFN-gamma induced by BCG-stimulated CD4(+)CD25(-) T cells, suggesting that Treg might play a key role in the control of cellular immune responses in TB infection.     

结核分枝杆菌HBHA的研究进展

HBHA是结核分枝杆菌的一种表面蛋白,它与细菌的黏附作用、诱导MФ细胞凋亡以及肺外播散型结核的发生相关,在抗肿瘤免疫中也有一定的作用;也可以作为新一代的血清学诊断标识,鉴别诊断活动性与非活动性结核病,并在开发结核新疫苗及抗结核病新药中显示出潜在的的应用价值。     

Preexposure to live Brugia malayi microfilariae alters the innate response of human dendritic cells to Mycobacterium tuberculosis

BACKGROUND: Mycobacterium tuberculosis and helminth coinfection is highly prevalent, and the presence of helminths may modulate the Th1 response necessary for M. tuberculosis control. METHODS: Elutriated human monocytes, differentiated into dendritic cells (DCs) and macrophages, were exposed in vitro to live microfilariae (mf). The influence that mf had on M. tuberculosis infectivity, expression of cell surface molecules, and production of cytokines was determined. RESULTS: Compared with mf-unexposed, M. tuberculosis-infected cells, mf-exposed, M. tuberculosis-infected DCs had decreased expression of CD14, CD54, and human leukocyte antigen-DR, and mf-exposed, M. tuberculosis-infected macrophages had decreased expression of CD40. DCs that were mf exposed and M. tuberculosis infected produced more interleukin (IL)-1 beta than did mf-unexposed, M. tuberculosis-infected DCs. Also, mf-exposed, M. tuberculosis-infected DCs and macrophages expressed less IL-10 and interferon (IFN)- alpha than did mf-unexposed, M. tuberculosis-infected cells. When they were cultured with autologous CD4+ T cells, mf-exposed, M. tuberculosis-infected DCs were less capable of stimulating the production of IFN- gamma than were other DCs. Exposure of DCs to mf decreased the surface expression of DC-specific intercellular adhesion molecule-3 grabbing nonintegrin, a receptor required by M. tuberculosis for entry into DCs. CONCLUSIONS: Exposure to mf reduces a key receptor on the DC surface, which perhaps renders these cells less susceptible to infection with M. tuberculosis. Exposure to mf changes the surface expression of adhesion and costimulatory molecules on DCs and macrophages and alters their expression of cytokines and chemokines in a way that renders them less capable of immunologic responses.     

Cross-talk between CD31 and the signaling lymphocytic activation molecule-associated protein during interferon- gamma production against Mycobacterium tuberculosis

Effective host defense against tuberculosis requires Th1 cytokine responses. We studied the regulation of interferon (IFN)- gamma production during tuberculosis by investigating the role of CD31, a receptor that attenuates T cell receptor signals. After antigen stimulation, CD3(+)CD31(+) blood lymphocytes decreased in healthy donors and in tuberculosis patients with robust Th1 responses to Mycobacterium tuberculosis and IFN- gamma was secreted only by CD31(-) T cells. In contrast, in patients with weak Th1 cytokine responses to M. tuberculosis, the level of CD3(+)CD31(+) lymphocytes was increased and IFN- gamma production was low. Furthermore, the inverse relationship between CD31 expression and IFN- gamma production was in contrast to signaling lymphocytic activation molecule (SLAM) expression, an IFN- gamma inducer in tuberculosis. Interestingly, CD31 bound to SLAM-associated protein (SAP), an IFN- gamma inhibitor in tuberculosis, and when CD31 and SAP were coexpressed in lymphocytes, their association inhibited the IFN- gamma response to M. tuberculosis. Thus, CD31, when binding to SAP, interferes with Th1 responses, suggesting that CD31 has a key regulatory role in the signaling pathway(s) leading to the IFN- gamma response to M. tuberculosis.     

Shifts of T4/T8 T lymphocytes from BAL fluid and peripheral blood by clinical grade in patients with pulmonary tuberculosis

OBJECTIVES: We investigated the shifts of T4/T8 lymphocytes from BAL fluid (BALF) and peripheral blood by the clinical grade of pulmonary tuberculosis (TB), which is determined by factors such as extent of pulmonary involvement, fever, and loss of body weight. MATERIALS AND METHODS: In order to explore these questions, BALF was collected from 45 patients presenting with active pulmonary TB and 14 healthy control subjects. The percentages for T-lymphocyte subpopulations, including CD4(+), CD8(+), and CD3(+) T cells, were measured using two-color flow cytometry. RESULTS: A higher percentage of CD3(+)CD4(+) T lymphocytes, with a relatively lower percentage of CD3(+)CD8(+) T lymphocytes, was revealed for the patients with a higher grade of pulmonary TB, compared to patients with a lower grade of pulmonary TB, resulting in an increased BALF C4(+)/CD8(+) ratio. By contrast, a higher percentage of CD3(+)CD8(+) T lymphocytes with a relatively low percentage of CD3(+)CD4(+) T lymphocytes was demonstrated for these patients with a higher grade of pulmonary TB, resulting in a decreased peripheral blood CD4(+)/CD8(+) ratio. CONCLUSIONS: Our findings suggest that compartmentalization of the CD4(+) T lymphocytes in the infected lungs may occur for patients with higher grades of pulmonary TB.     

A model on the influence of age on immunity to infection with Mycobacterium tuberculosis

Increasing susceptibility of the elderly to many infectious diseases is highly associated with the loss or delay in the generation of antigen-specific CD4(+) T cells. For Mycobacterium tuberculosis infection, where antigen-specific CD4(+) T cell derived IFN-gamma is essential, such a loss can lead to a significant failure to control infection. The present paper develops a mathematical model of infection with M. tuberculosis in old mice. The model includes an early resistance to infection which is mediated by CD8(+) T cells. A subsequent reversal of this phenotype results from the slow generation of CD4(+) T cell mediated immunity in old age. The model simulations corroborate experimental data and hence, the model was used to test whether immunity to infection could be improved in old mice, if CD4(+) T cell responses were enhanced. Our simulations indicate that boosting antigen presentation and T cell proliferation can decrease the M. tuberculosis burden in the lung.