María F. Quiroga

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.

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.

Signaling Lymphocytic Activation Molecule Expression and Regulation in Human Intracellular Infection Correlate with Th1 Cytokine Patterns

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-γ production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-γ production from tuberculoid patients and slightly increased the levels of IFN-γ in lepromatous patients. In addition, IFN-γ augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-γ treatment of Ag-stimulated cells enhanced IFN-γ production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-γ by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-γ production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-γ in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.

Activation of Signaling Lymphocytic Activation Molecule Triggers a Signaling Cascade That Enhances Th1 Responses in Human Intracellular Infection

T cell production of IFN-γ contributes to host defense against infection by intracellular pathogens, including mycobacteria. Lepromatous leprosy, the disseminated form of infection caused by Mycobacterium leprae, is characterized by loss of cellular response against the pathogen and diminished Th1 cytokine production. Relieving bacterial burden in Ag-unresponsive patients might be achieved through alternative receptors that stimulate IFN-γ production. We have previously shown that ligation of signaling lymphocytic activation molecule (SLAM) enhances IFN-γ in mycobacterial infection; therefore, we investigated molecular pathways leading from SLAM activation to IFN-γ production in human leprosy. The expression of the SLAM-associated protein (an inhibitory factor for IFN-γ induction) on M. leprae-stimulated cells from leprosy patients was inversely correlated to IFN-γ production. However, SLAM ligation or exposure of cells from lepromatous patients to a proinflammatory microenvironment down-regulated SLAM-associated protein expression. Moreover, SLAM activation induced a sequence of signaling proteins, including activation of the NF-κB complex, phosphorylation of Stat1, and induction of T-bet expression, resulting in the promotion of IFN-γ production, a pathway that remains quiescent in response to Ag in lepromatous patients. Therefore, our findings reveal a cascade of molecular events during signaling through SLAM in leprosy that cooperate to induce IFN-γ production and strongly suggest that SLAM might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.

Modulation of the phenotype and function of Mycobacterium tuberculosis-stimulated dendritic cells by adrenal steroids

Cell-mediated immunity, cytokines induced during the specific immune response and T-cell populations are crucial factors for containing Mycobacterium tuberculosis infection. Recent reports suggest a cross-regulation between adrenal steroids (glucocorticoids and dehydroepiandrosterone, DHEA) and the function of antigen-presenting cells (APCs). Therefore, we investigated the role of adrenal hormones on the functional capacity of M. tuberculosis-induced dendritic cells (DCs). Cortisol significantly inhibited the functions of M. tuberculosis-induced DCs. Interestingly, the presence of DHEA enhanced the M. tuberculosis-induced expression of MHC I, MHC II and CD86 and also increased ERK1/2 phosphorylation. Moreover, DHEA improved the production of IL-12 in response to M. tuberculosis stimulation, diminished IL-10 secretion and could not modify TNF-α synthesis. Importantly, we observed that DHEA enhanced the antigen-specific T-cell proliferation and IFN-γ production induced by M. tuberculosis-stimulated DC. These data show for the first time the relevance of the adrenal axis (especially of DHEA) in the modulation of DC function in the context of tuberculosis, a disease where the induction of a Th1 environment by APCs is crucial for the development of an effective immune response to the mycobacteria.

Cross-Talk between CD31 and the Signaling Lymphocytic Activation Molecule—Associated Protein during Interferon-γ 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.

Dynamics of Adrenal Steroids Are Related to Variations in Th1 and Treg Populations during Mycobacterium tuberculosis Infection in HIV Positive Persons

Tuberculosis (TB) remains the most frequent cause of illness and death from an infectious agent, and its interaction with HIV has devastating effects. We determined plasma levels of dehydroepiandrosterone (DHEA), its circulating form DHEA-suphate (DHEA-s) and cortisol in different stages of M. tuberculosis infection, and explored their role on the Th1 and Treg populations during different scenarios of HIV-TB coinfection, including the immune reconstitution inflammatory syndrome (IRIS), a condition related to antiretroviral treatment. DHEA levels were diminished in HIV-TB and HIV-TB IRIS patients compared to healthy donors (HD), HIV+ individuals and HIV+ individuals with latent TB (HIV-LTB), whereas dehydroepiandrosterone sulfate (DHEA-s) levels were markedly diminished in HIV-TB IRIS individuals. HIV-TB and IRIS patients presented a cortisol/DHEA ratio significantly higher than HIV+, HIV-LTB and HD individuals. A positive correlation was observed between DHEA-s and CD4 count among HIV-TB individuals. Conversely, cortisol plasma level inversely correlated with CD4 count within HIV-TB individuals. M. tuberculosis-specific Th1 lymphocyte count was increased after culturing PBMC from HIV-TB individuals in presence of DHEA. We observed an inverse correlation between DHEA-s plasma level and Treg frequency in co-infected individuals, and CD4+FoxP3+ Treg frequency was increased in HIV-TB and IRIS patients compared to other groups. Strikingly, we observed a prominent CD4+CD25-FoxP3+ population across HIV-TB and HIV-TB IRIS patients, which frequency correlated with DHEA plasma level. Finally, DHEA treatment negatively regulated FoxP3 expression without altering Treg frequency in co-infected patients. These data suggest an enhancing role for DHEA in the immune response against M. tuberculosis during HIV-TB coinfection and IRIS.