Invariant natural killer T (iNKT) cell exhaustion in sarcoidosis.
ABSTRACT: Invariant natural killer T (iNKT) cells are integral components of immune responses during many chronic diseases, yet their surface phenotypes, subset distribution, and polyfunctional capacity in this environment are largely unknown. Therefore, using flow cytometry, we determined iNKT cell phenotypic and functional characteristics in subjects with chronic inflammatory disease sarcoidosis and matched controls. We found that sarcoidosis subjects displayed lower iNKT-cell frequencies, which correlated with lung fibrosis, C-reactive protein levels, and other measures of clinical disease. The CD4(-) CD8(-) (double negative, DN) iNKT-cell population was selectively lower in diseased individuals and the remaining DN iNKT cells exhibited higher frequencies of the activation markers CD69 and CD56. Functionally, both total IFN-?(+) and the dual-functional IFN-?(+) TNF-?(+) iNKT cells were decreased in sarcoidosis subjects and these functional defects correlated with total iNKT-cell circulating frequencies. As the loss of polyfunctionality can reflect functional exhaustion, we measured the surface antigens programmed death-1 receptor and CD57 and found that levels inversely correlated with dual-functional iNKT-cell percentages. These findings reveal that, similar to traditional T cells, iNKT cells may also undergo functional exhaustion, and that circulating iNKT-cell frequencies reflect these defects. Programmed death-1 receptor antagonists may therefore be attractive therapeutic candidates for sarcoidosis and other iNKT-cell-mediated chronic diseases.
Project description:Investigation of the Th1 immune response in sarcoidosis CD4+ T cells has revealed reduced proliferative capacity and cytokine expression upon TCR stimulation. In other disease models, such cellular dysfunction has been associated with a step-wise, progressive loss of T cell function that results from chronic antigenic stimulation. T cell exhaustion is defined by decreased cytokine production upon TCR activation, decreased proliferation, increased expression of inhibitory cell surface receptors, and increased susceptibility to apoptosis. We characterized sarcoidosis CD4+ T cell immune function in systemic and local environments among subjects undergoing disease progression compared to those experiencing disease resolution. Spontaneous and TCR-stimulated Th1 cytokine expression and proliferation assays were performed in 53 sarcoidosis subjects and 30 healthy controls. PD-1 expression and apoptosis were assessed by flow cytometry. Compared to healthy controls, sarcoidosis CD4+ T cells demonstrated reductions in Th1 cytokine expression, proliferative capacity (p < 0.05), enhanced apoptosis (p < 0.01), and increased PD-1 expression (p < 0.001). BAL-derived CD4+ T cells also demonstrated multiple facets of T cell exhaustion (p < 0.05). Reversal of CD4+ T cell exhaustion was observed in subjects undergoing spontaneous resolution (p < 0.05). Sarcoidosis CD4+ T cells exhibit loss of cellular function during progressive disease that follows the archetype of T cell exhaustion.
Project description:Based on the phenotypic and functional characteristics unconventional T-lymphocytes such as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells link the innate and adaptive immune responses. Up to now data are scarce about their involvement in pulmonary disorders including chronic obstructive pulmonary disease (COPD). This study explores simultaneously the frequencies of iNKT and MAIT cells in the peripheral blood and sputum of stable and exacerbating COPD patients.By means of multicolor flow cytometry frequencies of total iNKT and MAIT cells and their subsets were enumerated in peripheral blood and sputum samples of healthy controls, and COPD patients. In addition, gene expression of TCR for iNKT, MAIT cells, and CD1d, MR1 were assessed by qPCR in the study cohorts.Percentages of total iNKT and MAIT cells were dramatically dropped in blood, and reduced numbers of iNKT cells were observed in the sputum of COPD patients. Furthermore decreased DN and increased CD4+ iNKT subsets, while increased DN and decreased CD8+ MAIT subpopulations were measured in the blood of COPD patients. Reduced invariant TCR mRNA levels in COPD patients had confirmed these previous findings. The mRNA expression of CD1d and MR1 were increased in stable and exacerbating COPD patients; however both molecules were decreased upon antibiotic and systemic steroid treatments.Our results support the notion that both invariant T-cell populations are affected in COPD. Further detailed analysis of invariant T cells could shed more light into the complex interactions of these lymphocyte groups in COPD pathogenesis.
Project description:Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.
Project description:RATIONALE:Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. OBJECTIVES:To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. METHODS:Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. MEASUREMENTS AND MAIN RESULTS:Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. CONCLUSIONS:Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.
Project description:Invariant natural killer T (iNKT) cells develop in the thymus and branch off from the maturation pathway of conventional T cell at the DP stage. While different stages of iNKT cellular development have been defined, the actual time that iNKT cell precursors spend at each stage is still unknown.Here we report on maturation dynamics of post-selection DN iNKT cells by injecting wild-type DP(dim) thymocytes into the thymus of TCR?(-/-) mice and using the V?14-J?18 rearrangements as a molecular marker to follow the maturation dynamics of these cells.This study shows that the developmental dynamics of DN iNKT cells in DP(dim) are very rapid and that it takes less than 1 day to down-regulate CD4 and CD8 and become DN. These DN cells are precursors of peripheral DN iNKT cells and appear in the spleen in 1-2 days. Thymic DN iNKT residents are predominantly derived from cells that quickly return from the periphery. The expansion of a very small subset of DN iNKT precursors could also play a small role in this process. These data are an example of measuring T cell maturation in the thymus and show that the maturation dynamics of selected DN iNKT cells fall within the same general time frame as conventional ?? T cells.
Project description:In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells.We compared iNKT cell expansion and cytokine production in C57BL/6J (B6) and apolipoprotein E-deficient (apoE(-/-)) mice. In response to in vivo stimulation with alpha-galactosylceramide, a prototypic iNKT cell glycolipid antigen, apoE(-/-) mice showed significantly decreased splenic iNKT cell expansion at 3 days after injection, a profile associated with iNKT cell anergy due to chronic stimulation. This decrease in expansion and cytokine production was accompanied by a 2-fold increase in percentage of iNKT cells expressing the inhibitory marker programmed death-1 in apoE(-/-) mice compared with controls. However, in vivo and in vitro blockade of programmed death-1 using monoclonal antibody was not able to restore functions of iNKT cells from apoE(-/-) mice to B6 levels. iNKT cells from apoE(-/-) mice also had increased intracellular T cell receptor and Ly49 expression, a phenotype associated with previous activation. Changes in iNKT cell functions were cell autonomous, because dendritic cells from apoE(-/-) mice were able to activate B6 iNKT cells, but iNKT cells from apoE(-/-) mice were not able to respond to B6 dendritic cells.These data suggest that chronic dyslipidemia induces an iNKT cell phenotype that is unresponsive to further simulation by exogenous glycolipid and that sustained unresponsiveness is iNKT cell intrinsic.
Project description:Chronic inappropriate immune activation is the central defect-driving loss of CD4(+) T helper cells and progression to AIDS in persons with HIV-1 infection, but the mechanisms remain controversial. We examined key regulatory invariant receptor natural killer T (iNKT) cells in the gut, the largest reservoir of lymphocytes and a key arena of HIV-1 pathogenesis. In healthy control persons, the anti-inflammatory CD4(+) iNKT-cell subset predominated over the pro-inflammatory CD4(-) iNKT-cell subset in the gut, but not in the blood, compartment. HIV-1 infection resulted in a preferential loss of this anti-inflammatory CD4(+) iNKT-cell subset within the gut. The degree of loss of the CD4(+) iNKT-cell subset in the gut, but not in the blood, correlated to the systemic immune activation and exhaustion that have been linked to disease progression. These results suggest a potentially important contribution of gut iNKT-cell imbalance in determining the systemic immune activation that is the hallmark of HIV-1 pathogenesis.
Project description:Systemic low-grade chronic inflammation has been intensively investigated in obese subjects. Recently, various immune cell types, such as macrophages, granulocytes, helper T cells, cytotoxic T cells, and B cells, have been implicated in the pathogenesis of adipose tissue inflammation. However, the roles of invariant natural killer T cells (iNKT cells) and the regulation of iNKT cell activity in adipose tissue are not thoroughly understood. Here, we demonstrated that iNKT cells were decreased in number in the adipose tissue of obese subjects. Interestingly, CD1d, a molecule involved in lipid antigen presentation to iNKT cells, was highly expressed in adipocytes, and CD1d-expressing adipocytes stimulated iNKT cell activity through physical interaction. iNKT cell population and CD1d expression were reduced in the adipose tissue of obese mice and humans compared to those of lean subjects. Moreover, iNKT cell-deficient J?18 knockout mice became more obese and exhibited increased adipose tissue inflammation at the early stage of obesity. These data suggest that adipocytes regulate iNKT cell activity via CD1d and that the interaction between adipocytes and iNKT cells may modulate adipose tissue inflammation in obesity.
Project description:Increasing drug resistance and the lack of an effective vaccine are the main factors contributing to <i>Mycobacterium tuberculosis</i> (Mtb) being a major cause of death globally. Despite intensive research efforts, it is not well understood why some individuals control Mtb infection and some others develop active disease. HIV-1 infection is associated with an increased incidence of active tuberculosis, even in virally suppressed individuals. Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are innate T cells that can recognize Mtb-infected cells. Contradicting results regarding the frequency of MAIT cells in latent Mtb infection have been reported. In this confirmatory study, we investigated the frequency, phenotype, and IFN? production of MAIT and iNKT cells in subjects with latent or active Mtb infection. We found that the frequency of both cell types was increased in subjects with latent Mtb infection compared with uninfected individuals or subjects with active infection. We found no change in the expression of HLA-DR, PD-1, and CCR6, as well as the production of IFN? by MAIT and iNKT cells, among subjects with latent Mtb infection or uninfected controls. The proportion of CD4- CD8+ MAIT cells in individuals with latent Mtb infection was, however, increased. HIV-1 infection was associated with a loss of MAIT and iNKT cells, and the residual cells had elevated expression of the exhaustion marker PD-1. Altogether, the results suggest a role for MAIT and iNKT cells in immunity against Mtb and show a deleterious impact of HIV-1 infection on those cells.
Project description:Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.