Th1 lymphocytes are crucial in the immune response against Mycobacterium tuberculosis. Nevertheless, IFN-␥ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL-17-producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN-␥ and IL-17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine-producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN-␥ and IL-17, but in comparison with BCG-vaccinated healthy donors, IFN-␥ results were reduced significantly, and IL-17 was markedly augmented. Moreover, the main source of IL-17 was represented by CD4 ϩ IFN-␥ ϩ IL-17 ϩ lymphocytes, a Th1/Th17 subset regulated by IFN-␥. Interestingly, the ratio of antigen-expanded CD4 ϩ IFN-␥ ϩ IL-17 ϩ lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4 ϩ IFN-␥ ϩ IL-17 ϩ cells was detected in Low Responder TB patients, individuals displaying se-vere pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4 ϩ IFN-␥ ϩ IL-17 ϩ T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.

Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)-producing CD4(+) lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis-induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ-producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis.

IFN-γ release assays (IGRAs) are better indicators of Mycobacterium tuberculosis infection than the tuberculin skin test (TST) in Bacillus Calmette–Guérin (BCG)-vaccinated populations. However, IGRAs do not discriminate active and latent infections (LTBI) and no gold standard for LTBI diagnosis is available. Thus, since improved tests to diagnose M. tuberculosis infection are required, we assessed the efficacy of several M. tuberculosis latency antigens. BCG-vaccinated healthy donors (HD) and tuberculosis (TB) patients were recruited. QuantiFERON-TB Gold In-Tube, TST and clinical data were used to differentiate LTBI. IFN-γ production against CFP-10, ESAT-6, Rv2624c, Rv2626c and Rv2628 antigens was tested in peripheral blood mononuclear cells. LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD. Additionally, Rv2626c peptide pools to which only LTBI responded were identified, and their cumulative IFN-γ response improved LTBI discrimination. Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients. ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients. Therefore, since only LTBI recognizes specific epitopes from Rv2626c, this antigen could improve LTBI diagnosis, even in BCG-vaccinated people.

Production of IFN-γ contributes to host defense against Mycobacterium tuberculosis (Mtb) infection. We previously demonstrated that Signaling lymphocytic activation molecule-associated protein (SAP) expression on cells from tuberculosis (TB) patients was inversely correlated with IFN-γ production. Here we first investigated the role of NK, T and B cell antigen (NTB-A)/SAP pathway in the regulation of Th1 response against Mtb. Upon antigen stimulation, NTB-A phosphorylation rapidly increases and afterwards modulates IFN-γ and IL-17 secretion. To sustain a healthy immune system, controlled expansion and contraction of lymphocytes, both during and after an adaptive immune response, is essential. Besides, restimulation-induced cell death (RICD) results in an essential homeostatic mechanism for precluding excess T-cell accumulation and associated immunopathology during the course of certain infections. Accordingly, we found that the NTB-A/SAP pathway was required for RICD during active tuberculosis. In low responder (LR) TB patients, impaired RICD was associated with diminished FASL levels, IL-2 production and CD25high expression after cell-restimulation. Interestingly, we next observed that SAP mediated the recruitment of the Src-related kinase FYNT, only in T cells from LR TB patients that were resistant to RICD. Together, we showed that the NTB-A/SAP pathway regulates T cell activation and RICD during human TB. Moreover, the NTB-A/SAP/FYNT axis promotes polarization to an unfavorable Th2-phenotype.

IFNγ-secreting cells are critical in the defense against M. tuberculosis (Mtb) infection, but other cells might be required to eradicate the pathogen. IL-9-producing cells were reported to be involved in inflammation and immune diseases, but their role during tuberculosis remains to be clarified. Here we analyzed the involvement of IL-9-secreting cells in the immunity of patients with active disease. We demonstrated that Mtb induced a marked expansion of CD4 IL-9+ cells in peripheral blood from tuberculosis patients. After antigen stimulation, patients with robust Th1 responses to Mtb (high responders) produced higher amounts of IL-9 compared to those with weak Th1 responses (low responders) and a direct significant correlation between IL-9 and IFNγ production against Mtb was detected. To investigate the role of the IL-9 secreted by those lymphocytes, we inhibited IL-9 expression with siRNA, which significantly diminished the production of IFNγ against Mtb. Addition of IL-9 markedly increased the secretion of IFNγ against Mtb, even in low responders. Furthermore, treatment with IL-9 inhibited Mtb-induced apoptosis of IFNγ+ cells, enhancing the survival of the cells. Finally, by studying the effect of neutralizing IL-9 antibodies on polarized Th9 cells, we confirmed that IL-9 participates in the up-regulation of IFNγ production against Mtb. Thus, our results indicate that IL-9 enhances Th1 responses during tuberculosis promoting the cell-mediated immunity to the pathogen.