Effector function of natural killer T (NKT) cells in sarcoidosis

描述(申请人提供)：结节病是一种病因不明的全身性炎症性疾病，无法治愈。它被认为是由对自身或非自身抗原的异常免疫反应引起的。结节病的特征是以干扰素-γ为主要细胞因子的Th1免疫反应。尽管有这些知识，但对这种异常免疫反应的调节因素知之甚少，而且目前的治疗方法(如全身皮质类固醇和甲氨蝶呤)是非特异性的，具有显著的毒性。自然杀伤T细胞(NKT)是一种免疫调节性T细胞，可根据局部刺激促进或抑制免疫反应。在小鼠身上，NKT细胞被发现在几种自身免疫性疾病中具有保护作用。在人类中，两项独立的临床研究发现，肺结节病患者的外周血和肺NKT细胞数量明显低于对照组。因此，NKT细胞在结节病中被异常调节。然而，关于NKT细胞在该病中的细胞亚群和效应功能方面的知识存在很大差距。明确的目标。由于NKT细胞的CD4亚群缺陷可能使免疫反应偏向Th1反应(结节病的一个特征)，我们假设结节病的整体NKT细胞缺陷是由于CD4NKT细胞亚群的选择性丢失导致Th2细胞因子的缺乏和细胞因子平衡的倾斜，有利于Th1细胞因子产生的CD4-NKT细胞。因此，我们将1)在横断面研究中确定CD1d配体刺激后的CD1d细胞在结节病中的分布及其细胞因子反应；2)在纵向研究中确定NKT细胞效应器功能如何与结节病患者的疾病严重程度、进展和表型相关，以及免疫抑制对NKT细胞亚群和细胞因子反应的影响。实验方法：采集正常志愿者和结节病患者外周血单个核细胞。我们将使用新的体外刺激方法和多色流式细胞仪分析，直接在体外测定CD4和CD4-NKT细胞的数量以及它们对几种NKT细胞配体的细胞因子反应。这些反应将与匹配的健康志愿者的反应进行比较。我们还将每隔6个月纵向分析结节病患者的NKT细胞亚群和细胞因子反应，并将这些测量与疾病严重程度相关联。此外，我们还将确定全身免疫抑制剂对NKT细胞亚群和细胞因子反应的影响。最后，我们将在5-10名肺结节病患者的初步研究中比较血液和肺NKT细胞效应功能。结果的意义。由于NKT细胞可能在结节病的发病机制中发挥作用，这项研究的发现可以确定一种新的细胞类型，可能成为使用CD1d配体的新治疗策略的靶点，这被最近的NHLBI研讨会确定为结节病研究的主要优先领域。公共卫生相关性：我们正在寻求NIH支持的临床项目的科学目标是全面分析最近发现的免疫调节细胞--自然杀伤T细胞--在结节病患者中的功能。该项目的首要目标之一是促进对结节病免疫学的了解，以便将其转化为更具体的治疗方法，以减少患者的痛苦，从而促进肺部健康和改善患者的生活。此外，通过更好地了解结节病的免疫学，我们可能能够预防这种肺部疾病。

DESCRIPTION (provided by applicant): Sarcoidosis is a systemic inflammatory disease of unknown etiology and has no cure. It is thought to be caused by an abnormal immune response to either a self or non-self antigen. Sarcoidosis is characterized by a Th1 immune response with interferon-gamma being the predominant cytokine. Despite this knowledge, little is known about the regulators of this aberrant immune response, and current treatment approaches (e.g. systemic corticosteroids and methotrexate) are non-specific and have significant toxicities. Natural killer T (NKT) cells are immunoregulatory T cells that can either promote or suppress immune responses depending on local stimuli. In mice, NKT cells have been found to be protective in several autoimmune diseases. In humans, two independent clinical studies found that the numbers of peripheral blood and lung NKT cells were significantly lower in patients with pulmonary sarcoidosis compared to controls. Thus, NKT cells are abnormally regulated in sarcoidosis. However, large gaps in knowledge exist about the cellular subsets and effector function of NKT cells in this disease. Specific Aims. Because a deficiency of the CD4+ subset of NKT cells may skew immune responses towards Th1 responses (a characteristic of sarcoidosis), we hypothesize that the overall NKT cell deficiency in sarcoidosis is due to a selective loss of the CD4+ NKT cell subset leading to a paucity of Th2 cytokines and a skewing of the cytokine balance in favor of Th1 cytokine-producing CD4- NKT cells. Therefore, we will 1) determine the distribution of CD4+ vs. CD4- NKT cells in sarcoidosis and their cytokine responses after stimulation with CD1d ligands in a cross-sectional study and 2) determine how NKT cell effector function relates to disease severity, progression and phenotype in sarcoidosis subjects and the effect of immune- suppression on NKT cell subsets and cytokine responses in a longitudinal study. Experimental Approach: We will collect peripheral blood mononuclear cells from normal volunteers and subjects with sarcoidosis. Directly ex-vivo we will determine the number of CD4+ and CD4- NKT cells and their cytokine responses to several NKT cell ligands using novel in vitro stimulation methods and multi-color flow cytometric analysis. These responses will be compared to those from matched healthy volunteers. We will also analyze NKT cell subsets and cytokine responses within sarcoidosis subjects longitudinally at ~6 month intervals and correlate these measurements to disease severity. Additionally, we will determine the effect of systemic immunosuppressants on NKT cell subsets and cytokine responses. Finally, we will compare blood and lung NKT cell effector function in a pilot study of 5-10 pulmonary sarcoidosis subjects. Significance of the results. Since NKT cells may play a role in the pathogenesis of sarcoidosis, findings from this study could identify a new cell type that may be targeted in novel treatment strategies using CD1d ligands, which was identified as a major priority area of research in sarcoidosis by a recent NHLBI workshop. PUBLIC HEALTH RELEVANCE: The scientific aims of the clinical project for which we are seeking support from the NIH is to comprehensively analyze the function of a recently identified immunoregulatory cell, the natural killer T cell, in patients with sarcoidosis. One of the overriding goals of this project is to advance the knowledge of the immunology of sarcoidosis so that this may be translated into more specific therapies to reduce suffering of patients and thereby promote lung health and improve patients' lives. In addition, by achieving a better understanding of the immunology of sarcoidosis, we may be able to prevent this lung disease.