Hunting for Endogenous Ligands for Invariant Natural Killer T Cells

描述（由申请人提供）：本研究项目旨在寻找iNKT细胞发育的真正内源性配体。免疫系统的常规CD4+和CD8+ T细胞分别识别与主要组织相容性复合体（MHC） II类或MHC I类分子结合的特异性肽抗原。相反，T细胞的一个特殊亚群称为不变自然杀伤T （iNKT）细胞识别由MHC i类分子CD1d呈递的糖脂抗原。经刺激后，iNKT细胞快速爆发产生大量的辅助性T 1 （Th1）、Th2细胞因子和趋化因子，具有广泛的免疫影响，包括对自身免疫性疾病的保护、宿主对寄生虫和细菌的反应以及抗肿瘤反应。虽然众所周知的a-GalCer可以显著刺激iNKT细胞，但它是从海绵中分离出来的外源性抗原。在胸腺iNKT细胞从T细胞前体发育的过程中，一种内源性抗原是由CD1d向随机生成的TCR呈递的过程所必需的。据报道，iGb3可能是小鼠和人体内的内源性抗原。然而，来自独立实验室的研究挑战了iGb3是小鼠iNKT细胞发育的主要配体的假设。特别地，NP-HPLC法在小鼠和人胸腺中均未检测到iGb3。iGb3合成酶敲除（iGb3S-/-）小鼠胸腺、脾脏和肝脏的iNKT细胞数量也正常。进一步的实验表明，通过对iGb3S mRNA的分析，没有在任何人体组织中发现iGb3S，并且含有人iGb3S催化结构域的嵌合分子由于至少一个氨基酸取代而没有显示出合成iGb3的任何功能活性。尽管在小鼠和人胸腺样本或其他组织中是否存在iGb3存在争议，但iGb3在刺激iNKT细胞中的活性是公认的。这为我们在胸腺内寻找iNKT细胞发育的真正的iNKT细胞配体提供了重要线索。因此，本研究基于糖脂在小鼠和人体内的生物合成和降解途径，基于CD1d/1-GalCer/TCR和CD1d/iGb3复合物的晶体结构，以及iGb3的三维结构，假设一种糖脂作为潜在的一级配体。可能的抗原是三种血型糖基神经酰胺，它们具有与iGb3相似的结构，并且在小鼠和人体内都有相应的合成途径。所有这些配体在半乳糖的C2位置上都有一个聚焦片段。血液B型糖基神经酰胺的计算对接表明，该化合物可以被容纳在TCR和CD1d之间的空腔中。尽管迄今为止对这些血型糖基神经酰胺的研究要少得多，但其中两种已在猪组织中被发现，这意味着它们也可能存在于小鼠和人体组织中，如胸腺和脾脏。本项目提出了三个具体目标：1。制备三种不同血型的糖神经酰胺，包括Fuc1(1,2)Gal2(1,4)Glc2（1,1）神经酰胺（O型血）、GalNAc1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2（1,1）神经酰胺（A型血）和Gal1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2（1,1）神经酰胺（B型血）。并制备了以硫原子为连接键的代谢稳定类似物。2. 用不同的模型评价这些血型糖基神经酰胺及其代谢类似物的生物活性。评估血型糖基神经酰胺对iNKT细胞发育的影响。3. 鉴定小鼠组织中潜在的血型糖基神经酰胺抗原。iNKT细胞是连接先天免疫系统和适应性免疫系统的桥梁。鉴定iNKT细胞的内源性配体有助于了解iNKT细胞发育和增殖的机制，以及iNKT细胞与其他免疫细胞（如T细胞、B细胞、巨噬细胞）的关系。这也将有助于研究人员开发将该原理应用于临床的方法。公共卫生相关性：iNKT细胞是一种新发现的T细胞亚群。在糖脂刺激下，iNKT细胞迅速释放大量细胞因子，在炎症反应和抑制肿瘤增殖中起着重要作用。iNKT细胞是连接先天免疫系统和适应性免疫系统的桥梁。鉴定iNKT细胞的内源性配体有助于了解iNKT细胞发育和增殖的机制，以及iNKT细胞与其他免疫细胞（如T细胞、B细胞、巨噬细胞）的关系。这也将有助于研究人员开发将该原理应用于临床的方法。

DESCRIPTION (provided by applicant): This research program targets at the search for the true endogenous ligands for the iNKT cells development. Conventional CD4+ and CD8+ T cells of the immune system recognize specific peptide antigens bound to major histocompatibility complex (MHC) class II or MHC class I molecules, respectively. In contrast, a specialized subpopulation of T cells called invariant natural killer T (iNKT) cells recognizes glycolipid antigens presented by the MHC class I-like molecule, CD1d. Upon stimulation, iNKT cells produce large amounts of T helper 1 (Th1), Th2 cytokines and chemokines in a rapid burst which has a broad immunological influence, including protection against autoimmune diseases, host responses to parasites and bacteria, and anti-tumor responses. Although the well-known a-GalCer can dramatically stimulate the iNKT cells, it's an exogenous antigen isolated from the marine sponge. During the development of iNKT cells in the thymus from the T cell precursor, an endogenous antigen is necessary for the process of being presented by CD1d to the randomly generated TCR. iGb3 was reported as a possible endogenous antigen in both mice and human. However, studies from independent laboratories have challenged the hypothesis that iGb3 is the primary ligand responsible for iNKT cell development in mice. Specially, no iGb3 was detected in either the mice or human thymus by NP-HPLC analysis. The iGb3 synthase knockout (iGb3S-/-) mice furthermore showed normal iNKT cells numbers in the thymus, spleen, and liver. Further experiments showed that there is no iGb3S identified in any human tissue by analysis of the iGb3S mRNA, and the chimeric molecules containing the catalytic domain of human iGb3S did not show any functional activity in synthesis of iGb3 due to at least one amino acid substitution. Despite the dispute of the existence of iGb3 in mice and human thymus samples or other tissues, the activity of iGb3 in the stimulation of iNKT cells is well accepted. This provides us with important clues in the search for the genuine iNKT cell ligands inside the thymus for iNKT cell development. Accordingly, in this proposal, we hypothesize one type of glycolipids as the potential primary ligands based on the biosynthesis and degradation pathway of glycolipids in mouse and human, on the crystal structures of CD1d/1-GalCer/TCR and CD1d/iGb3 complexes, as well as on the three dimensional structure of iGb3. The possible antigens are three blood type glycosylceramides which possess structures similar to that of iGb3 and have the corresponding synthetic pathway in both mice and humans. All of these ligands possess a fucose moiety on the C2 position of a galactose. The computational docking of the blood B type glycosylceramide shows that this compound can be accommodated in the cavity between TCR and CD1d. Although these blood type glycosylceramides are much less studied so far, two of them have been identified in pig tissues, which implies that they may also exist in mouse and human tissues, such as thymus and spleen. Three specific aims are proposed in this program: 1. Preparation of three different blood type glycoceramides which include Fuc1(1,2)Gal2(1,4)Glc2(1,1)ceramide (blood O type), GalNAc1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2(1,1)ceramide (blood A type) and Gal1(1,3)[Fuc1(1,2)]Gal2(1,4)Glc2(1,1)ceramide (blood B type). The metabolic stable analogs with thio-atom as linkage between the saccharides will also be prepared. 2. Evaluate the biological activity of these blood type glycosylceramides and metabolic analogs with different models. Evaluate the effects of blood type glycosylceramides on iNKT cells development. 3. Identify the potential blood type glycosylceramide antigens in mice tissues. iNKT cells serve as the bridge between innate and adaptive immune system. Identification of endogenous ligand of iNKT cells will benefit the understanding of the mechanism of iNKT cells developing and proliferation, and its relationship with other immune cells, such as T cells, B cells, and macrophage cells. This will also help researchers to develop methods to apply the principle into clinic application. PUBLIC HEALTH RELEVANCE: iNKT cells are a newly discovered sub-population of T cells. Upon stimulation with glycolipids, iNKT cells rapidly release a large amount of cytokines which is very important in the inflammatory response and suppressing the tumor proliferation. iNKT cells serve as the bridge between innate and adaptive immune system. Identification of endogenous ligand of iNKT cells will benefit the understanding of the mechanism of iNKT cells developing and proliferation, and its relationship with other immune cells, such as T cells, B cells, and macrophage cells. This will also help researchers to develop methods to apply the principle into clinic application.