Endogenous glycosphingolipid antigens for NKT cells

描述(申请人提供)：自然杀伤T细胞(NKT)是一种NK细胞和T细胞的杂交细胞类型，但只在抗原刺激下被激活。NKT细胞通过识别来自微生物和自身来源的脂质配体，连接天然免疫和获得性免疫。内源性NKT配体是自身脂质的代谢产物，参与感染性疾病、自身免疫性疾病和癌症的免疫病理过程。然而，由于技术障碍，这些内源性配体的分子鉴定一直很困难。最近，我们发现抑制葡萄糖神经酰胺合成酶的药物NB-DGJ完全阻断了NKT细胞的发育。更重要的是，我们确定了NKT细胞的第一个已知的天然配体--异寡糖三己糖神经酰胺，这是NB-DGJ抑制的目标糖鞘糖脂之一。然而，iGb3合成酶基因敲除小鼠在NKT细胞的发育和功能方面没有缺陷，表明存在其他GSL和/或非GSL配体。糖脂组学分析的不断发展，在离子俘获质谱学技术的辅助下，增强了我们进一步了解这些机制和识别新配体的能力。在这个项目中，我们将检验这样的假设，即NKT细胞中至少存在一个内源性配体，而不是iGb3，并且与NKT细胞的发育和激活有关。识别一个或多个内源性配体可能会为调节NKT细胞在癌症和自身免疫性疾病等疾病环境中的功能提供新的方法。该项目的具体目标是：1)通过生化分离和分析确定刺激性GSL和/或非GSL抗原的身份；2)利用遗传途径识别潜在的内源性NKT配体。NKT细胞天然配体的分子鉴定不仅是天然免疫领域的一项紧迫任务，也是一项具有挑战性的任务。尽管寻找这些配体的过程已经持续了十多年，但它们仍然难以捉摸。因此，我们选择了生化分离/鉴定和关键酶的基因敲除相结合的方法来帮助实现这一探索。人类基因组计划的完成和质谱学技术的进步为脂类抗原领域的发展提供了框架，并有可能为一些最复杂的免疫相关疾病的新的治疗方法的确定提供了可能。更重要的是，该项目将提供对细胞脂质组学研究的早期见解，特别关注糖脂的重要免疫功能。人类基因组中超过10%的基因用于脂类代谢；因此，该项目获得的知识和经验可能对功能脂组学的研究产生深远的影响，与蛋白质组学和基因组学相比，功能脂组学是一个重要但鲜为人知的领域。公共卫生相关性：NKT淋巴细胞是天然免疫的重要组成部分，可以启动获得性免疫反应并调节免疫结果。了解NKT细胞天然配体的特性是研究NKT细胞在病理生理条件下的调控的前提。我们的长期目标是阐明NKT细胞天然配体生物合成途径的分子机制，并确定可药物调控的靶点。该项目的重要临床益处是1)脂代谢干扰药物在NKT细胞免疫调节中的新的治疗应用；2)鉴定新的天然NKT配体作为自身免疫性疾病和癌症免疫治疗的靶点。

DESCRIPTION (provided by applicant): Natural Killer T cells (NKT) are a hybrid cell type of NK cells and T cells, but are activated only by antigenic stimulation. NKT cells bridge innate and adaptive immunity through recognition of lipid ligands from both microbial and self origins. Endogenous NKT ligands, which are metabolism products of self lipids, are involved in immuno-pathology of infectious diseases, auto-immune diseases and cancer. However, molecular identification of these endogenous ligands has been difficult due to technical barriers. Recently, we found that NB-DGJ, a drug which inhibits glucosylceramide synthase, completely abolishes the development of NKT cells. More importantly, we identified isoglobotrihexosylceramide, one of the target glycosphingolipids being inhibited by NB-DGJ, as the first known natural ligand for NKT cells. However, iGb3 synthase knockout mice showed no defect in NKT cell development and function, indicating that other GSL and/or non-GSL ligands exist. Continued development of glycolipidomics assays, assisted by ion trap mass spectrometry technology, enhances our ability to further understand these mechanisms and identify new ligands. In this project, we will test the hypothesis that at least one endogenous ligand of NKT cells, other than iGb3, exists and is responsible for the development and activation of NKT cells. Identifying one or more endogenous ligands may permit new approaches to regulating the function of NKT cells in disease settings such as cancer and autoimmune diseases. The specific aims of this project are: 1) Determine the identities of stimulatory GSL and/or non-GSL antigens by biochemical fractionation and analysis; 2) Utilize the genetic pathways to identify potential endogenous NKT ligands. The molecular identification of natural ligands for NKT cells is not only an urgent, but also a challenging task in the field of innate immunity. Although the search for these ligands has lasted more than a decade, they remain elusive. We therefore have chosen a combined approach of biochemical fractionation/characterization and genetic knockdown of key enzymes to help bring this search to fruition. The completion of human genome project and the advancement of mass spectrometry technology provide the framework to move the field of lipid antigens forward and potentially permit the identification of new therapeutic approaches to some of the most complex immune-related diseases. More important, this project will provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycolipids. More than 10% of the genes in the human genome are used for lipid metabolism; thus, knowledge and experience gained in this project may have profound implications on studies of functional lipidomics, an important but little-understood area compared with proteomics and genomics. PUBLIC HEALTH RELEVANCE: NKT lymphocytes represent an essential component of innate immunity, that can jump start the adaptive immune responses and regulate the immune outcome. Understanding the identities of the natural ligands for NKT cells is a prerequisite to study the regulation of NKT cells in patho-physiological conditions. Our long-term goal is to elucidate the molecular mechanisms of the biosynthesis pathways of natural ligands for NKT cells, and to identify druggable targets of regulation. The important clinical benefit of this project is 1) novel therapeutic application of lipid-metabolism-interfering drugs in immuno-regulation of NKT cells; 2) identification of new natural NKT ligands as targets for immuno-therapy of autoimmune diseases and cancer.