Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

Heparin-Binding Hemagglutinin Adhesin (HBHA) Is Involved in Intracytosolic Lipid Inclusions Formation in Mycobacteria.

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Contribution of a heparin-binding haemagglutinin interferon-gamma release assay to the detection of Mycobacterium tuberculosis infection in HIV-infected patients: comparison with the tuberculin skin test and the QuantiFERON-TB Gold In-tube.

BACKGROUND: The screening and treatment of latent tuberculosis (TB) infection reduces the risk of progression to active disease and is currently recommended for HIV-infected patients. The aim of this study is to evaluate, in a low TB incidence setting, the potential contribution of an interferon-gamma release assay in response to the mycobacterial latency antigen Heparin-Binding Haemagglutinin (HBHA-IGRA), to the detection of Mycobacterium tuberculosis infection in HIV-infected patients. METHODS: Treatment-naïve HIV-infected adults were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the HBHA-IGRA in parallel to a classical method consisting of medical history, chest X-ray, tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT). Prospective clinical and biological follow-up ensued, with repeated testing with HBHA-IGRA. A group of HIV-infected patients with clinical suspicion of active TB was also recruited and tested with the HBHA-IGRA. Multiplex analysis was performed on the culture supernatants of this in-house assay to identify test read-outs alternative to interferon-gamma that could increase the sensitivity of the test. RESULTS: Among 48 candidates enrolled for screening, 9 were identified with latent TB by TST and/or QFT-GIT results. Four of these 9 patients and an additional 3 screened positive with the HBHA-IGRA. This in-house assay identified all the patients that were positive for the TST and showed the best concordance with the presence of a M. tuberculosis exposure risk. During follow-up (median 14 months) no case of active TB was reported and HBHA-IGRA results remained globally constant. Fourteen HIV-infected patients with clinical suspicion of active TB were recruited. Active TB was confirmed for 6 of them among which 3 were HBHA-IGRA positive, each with very high interferon-gamma concentrations. All patients for whom active TB was finally excluded, including 2 non-tubercular mycobacterial infections, had negative HBHA-IGRA results. Multiplex analysis confirmed interferon-gamma as the best read-out. CONCLUSIONS: The HBHA-IGRA appears complementary to the QuantiFERON-TB Gold In-Tube for the screening of latent TB in HIV-infected patients. Large-scale studies are necessary to determine whether this combination offers sufficient sensitivity to dismiss TST, as suggested by our results. Furthermore, HBHA-IGRA may help in the diagnosis work-up of clinical suspicions of active TB.

Key role of effector memory CD4+ T lymphocytes in a short-incubation heparin-binding hemagglutinin gamma interferon release assay for the detection of latent tuberculosis.

The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4(+) T lymphocytes, a finding suggestive of continuous HBHA presentation during latency.

Heterogeneity of expression of IgA receptors by human, mouse, and rat eosinophils.

IgA is the most abundant class of Abs at mucosal surfaces where eosinophils carry out many of their effector functions. Most of the known IgA-mediated functions require interactions with IgA receptors, six of which have been identified in humans. These include the IgA FcR FcalphaRI/CD89 and the receptor for the secretory component, already identified on human eosinophils, the polymeric IgR, the Fcalpha/muR, asialoglycoprotein (ASGP)-R, and transferrin (Tf)R/CD71. In rodents, the existence of IgA receptors on mouse and rat eosinophils remains unclear. We have compared the expression and function of IgA receptors by human, rat, and mouse eosinophils. Our results show that human eosinophils express functional polymeric IgR, ASGP-R, and TfR, in addition to CD89 and the receptor for the secretory component, and that IgA receptors are expressed by rodent eosinophils. Indeed, mouse eosinophils expressed only TfR, whereas rat eosinophils expressed ASGP-R and CD89 mRNA. These results provide a molecular basis for the differences observed between human, rat, and mouse regarding IgA-mediated immunity.

Peroxisome proliferator-activated receptors alpha and gamma down-regulate allergic inflammation and eosinophil activation.

Allergic asthma is characterized by airway hyperresponsiveness, eosinophilia, and mucus accumulation and is associated with increased IgE concentrations. We demonstrate here that peroxisome proliferator-activated receptors (PPARs), PPAR-alpha and PPAR-gamma, which have been shown recently to be involved in the regulation of various cell types within the immune system, decrease antigen-induced airway hyperresponsiveness, lung inflammation, eosinophilia, cytokine production, and GATA-3 expression as well as serum levels of antigen-specific IgE in a murine model of human asthma. In addition, we demonstrate that PPAR-alpha and -gamma are expressed in eosinophils and their activation inhibits in vitro chemotaxis and antibody-dependent cellular cytotoxicity. Thus, PPAR-alpha and -gamma (co)agonists might be of therapeutic interest for the regulation of allergic or inflammatory reactions by targeting both regulatory and effector cells involved in the immune response.