Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-γ production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-γ production from these individuals. Moreover, M. tuberculosis-induced IFN-γ participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-γ-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-γ responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.

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.

Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon gamma (IFN-gamma), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PD-L1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-gamma expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-gamma production of NK cells against M. tuberculosis. Furthermore, PD-1(+) NK cells displayed a diminished IFN-gamma mean fluorescence intensity, denoting the relevance of PD-1 on IFN-gamma regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.

Interferon-γ (IFN–γ) is crucial for protection against Mycobacterium tuberculosis, and the transcription factor cAMP response element binding protein (CREB) increases IFN-γ transcription. We determined whether the transmembrane receptor signaling lymphocyte activation molecule (SLAM) and interleukin-17 (IL-17) affect CREB phosphorylation and IFN-γ production in persons with tuberculosis. When T cells from patients with tuberculosis were activated with M. tuberculosis, 80% of SLAM+ T cells expressed phosphorylated CREB, and SLAM activation increased CREB phosphorylation and IFN-γ production. In contrast, IL-17 down-regulated SLAM expression, CREB phosphorylation, and IFN-γ production. Therefore, IL-17 and SLAM have opposing effects on IFN-γ production through CREB activation in persons with tuberculosis.