Verónica E. García

Programmed Death (PD)-1:PD-Ligand 1/PD-Ligand 2 Pathway Inhibits T Cell Effector Functions during Human Tuberculosis

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.

MANNOSE-CAPPED LIPOARABINOMANNAN- AND PROSTAGLANDIN E2-DEPENDENT EXPANSION OF REGULATORY T CELLS IN HUMAN Mycobacterium tuberculosis INFECTION

We evaluated the role of regulatory T cells (CD4+ CD25+ Foxp3+ cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin‐negative individuals. The M. tuberculosis mannose‐capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19‐kDa protein and heat shock protein 65 had no effect. Anti‐IL‐10 and anti‐TGF‐β alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme‐2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat‐killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF‐β and IL‐10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis‐responsive CD4+ IFN‐γ cells. Culturing M. tuberculosis‐expanded Tregs with autologous CD8+ cells decreased the frequency of IFN‐γ+cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.

IL-17 and IFN-γ expression in lymphocytes from patients with active tuberculosis correlates with the severity of the disease

Th1 lymphocytes are crucial in the immune response against Mycobacterium tuberculosis. Nevertheless, IFN‐γ 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‐γ 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‐γ and IL‐17, but in comparison with BCG‐vaccinated healthy donors, IFN‐γ results were reduced significantly, and IL‐17 was markedly augmented. Moreover, the main source of IL‐17 was represented by CD4+IFN‐γ+IL‐17+ lymphocytes, a Th1/Th17 subset regulated by IFN‐γ. Interestingly, the ratio of antigen‐expanded CD4+IFN‐γ+IL‐17+ 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+IFN‐γ+IL‐17+ cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4+IFN‐γ+IL‐17+ T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.

Role Played by the Programmed Death-1-Programmed Death Ligand Pathway during Innate Immunity against Mycobacterium tuberculosis

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.

IL‐10 down‐regulates costimulatory molecules on Mycobacterium tuberculosis‐pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients

Activation of T cells requires both TCR‐specific ligation and costimulation through accessory molecules during T cell priming. IFNγ is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL‐10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNγ and IL‐10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). γ‐irradiated‐Mtb (i‐Mtb) induced IL‐10 production from CD14+ cells from TB patients. Moreover, CD3+ T cells of patients with advanced disease also produced IL‐10 after i‐Mtb stimulation. In healthy donors, IL‐10 decreased the lytic activity of CD4+ and CD8+ T cells whereas it increased γδ‐mediated cytotoxicity. Furthermore, we found that the presence of IL‐10 induced a loss of the alternative processing pathways of antigen presentation along with a down‐regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL‐10 or addition of IFNγ to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8+ CTL together with an up‐regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i‐Mtb presentation. Taken together, our results demonstrate that the presence of IL‐10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC‐restricted cytotoxicity against infected macrophages.

Secretory Leukocyte Protease Inhibitor : A Secreted Pattern Recognition Receptor for Mycobacteria

RATIONALE Human secretory leukocyte protease inhibitor (SLPI) displays bactericidal activity against pathogens such as Escherichia coli and Streptococcus. Furthermore, it has been reported that murine SLPI shows potent antimycobacterial activity. OBJECTIVES The aim of the present study was to investigate whether human recombinant SLPI not only kills mycobacteria but also acts as a pattern recognition receptor for the host immune system. METHODS For the in vivo experiment, BALB/c mice were infected by intranasal instillation with Mycobacterium bovis BCG and viable BCG load in lung homogenates was later determined. For the in vitro experiments, SLPI was incubated overnight with a suspension of M. bovis BCG or the virulent strain Mycobacterium tuberculosis H37Rv, and the percentage survival as well as the binding of SLPI to mycobacteria was determined. Furthermore, bacteria phagocytosis was also determined by flow cytometry. MEASUREMENTS AND MAIN RESULTS Intranasal SLPI treatment decreased the number of colony-forming units recovered from lung homogenates, indicating that SLPI interfered with M. bovis BCG infection. Moreover, SLPI decreased the viability of both M. bovis BCG and H37Rv. We demonstrated that SLPI attached to the surface of the mycobacteria by binding to pathogen-associated molecular pattern mannan-capped lipoarabinomannans and phosphatidylinositol mannoside. Furthermore, we found that in the sputum of patients with tuberculosis, mycobacteria were coated with endogenous SLPI. Finally, we showed that phagocytosis of SLPI-coated mycobacteria was faster than that of uncoated bacteria. CONCLUSIONS The present results demonstrate for the first time that human SLPI kills mycobacteria and is a new pattern recognition receptor for them.

Inducible Costimulator: A Modulator of IFN-γ Production in Human Tuberculosis

Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-γ production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-γ production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-γ from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-γ production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-γ secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-γ released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-γ secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens.

IFN-γ Production during Active Tuberculosis Is Regulated by Mechanisms That Involve IL-17, SLAM, and CREB

Interferon-gamma (IFN-gamma) is crucial for protection against Mycobacterium tuberculosis, and the transcription factor cAMP response element binding protein (CREB) increases IFN-gamma transcription. We determined whether the transmembrane receptor signaling lymphocyte activation molecule (SLAM) and interleukin-17 (IL-17) affect CREB phosphorylation and IFN-gamma 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-gamma production. In contrast, IL-17 down-regulated SLAM expression, CREB phosphorylation, and IFN-gamma production. Therefore, IL-17 and SLAM have opposing effects on IFN-gamma production through CREB activation in persons with tuberculosis.

Expression of Signaling Lymphocytic Activation Molecule- Associated Protein Interrupts IFN-γ Production in Human Tuberculosis

Production of the Th1 cytokine IFN-γ by T cells is considered crucial for immunity against Mycobacterium tuberculosis infection. We evaluated IFN-γ production in tuberculosis in the context of signaling molecules known to regulate Th1 cytokines. Two populations of patients who have active tuberculosis were identified, based on their T cell responses to the bacterium. High responder tuberculosis patients displayed significant M. tuberculosis-dependent T cell proliferation and IFN-γ production, whereas low responder tuberculosis patients displayed weak or no T cell responses to M. tuberculosis. The expression of the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) on cells from tuberculosis patients was inversely correlated with IFN-γ production in those individuals. Moreover, patients with a nonfunctional SAP gene displayed immune responses to M. tuberculosis similar to those of high responder tuberculosis patients. In contrast to SAP, T cell expression of SLAM was directly correlated with responsiveness to M. tuberculosis Ag. Our data suggest that expression of SAP interferes with Th1 responses whereas SLAM expression contributes to Th1 cytokine responses in tuberculosis. The study further suggests that SAP and SLAM might be focal points for therapeutic modulation of T cell cytokine responses in tuberculosis.

Induction of cell-mediated immunity to Staphylococcus aureus in the mouse mammary gland by local immunization with a live attenuated mutant.

ABSTRACT The efficacy of intramammary (Ima) immunization with a live attenuated (la) Staphylococcus aureus mutant to protect the mouse mammary gland from infection has previously been established. The present study was aimed at evaluating whether Ima immunization with la-S. aureus can induce cell-mediated immune responses to the pathogen within the mammary gland. Mice were immunized by Ima route with la-S. aureus, and regional lymph node mononuclear cells were obtained thereafter. A higher expression of the interleukin-2 receptor was found on B and T cells from immunized mice when they were compared with control mice. Immunization with la-S. aureus induced strong proliferative responses to S. aureus. Moreover, significantly increased levels of gamma interferon (IFN-γ) were produced by CD4+ T cells when lymphocytes from immunized mice, but not from control mice, were cultured in the presence of staphylococcal antigens. Moreover, a significant increase in the percentage of IFN-γ-producing CD4+ and CD8+ T cells was observed after S. aureus Ima challenge in immunized mice compared to challenged control mice. Our results demonstrated that Ima immunization with la-S. aureus induced primed lymphocyte populations capable of responding against staphylococcal antigens during in vitro stimulation, as well as during in vivo infection by S. aureus. CD4+ and CD8+ T cells appear to be the main lymphocyte subpopulations involved in this response. It is suggested that IFN-γ production induced by Ima immunization may play a pivotal role in the eradication of intracellular staphylococci.