Hunting for Endogenous Ligands for Invariant Natural Killer T Cells

寻找恒定自然杀伤 T 细胞的内源配体

• 批准号: 7699675
• 负责人: Peng George Wang
• 金额: $ 18.54万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7699675
• 关键词: Amino Acid Substitution; Anabolism; Antigens; Autoimmune Diseases; B-Lymphocytes; Bacteria; Biological; Biological Assay; Blood; Blood typing procedure; CD8B1 gene; Catalytic Domain; Cell Count; Cells; Ceramides; Clinic; Complex; Degradation Pathway; Development; Disputes; Docking; Family suidae; Fucose; Galactose; Glycolipids; Histocompatibility Antigens Class II; Human; Immune; Immune response; Immune system; Inflammatory Response; Investigation; Knock-out; Laboratories; Ligands; Liver; Major Histocompatibility Complex; Marines; Messenger RNA; Metabolic; Methods; Modeling; Mus; Parasites; Pathway interactions; Peptides; Population; Porifera; Positioning Attribute; Preparation; Process; Reporting; Research; Research Personnel; Sampling; Series; Spleen; Structure; T-Cell Development; T-Lymphocyte; Thymus Gland; Tissues; UDP-galactose beta-D-galactosyl-1,4-glycosylceramide alpha-1,3-galactosyltransferase; analog; antigen binding; base; chemokine; cytokine; human tissue; killer T cell; macrophage; programs; public health relevance; research study; response; three dimensional structure; tumor

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.

描述（由申请方提供）：本研究项目旨在寻找用于iNKT细胞开发的真正内源性配体。免疫系统的常规CD 4+和CD 8 + 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。相比之下，一种称为不变自然杀伤T（iNKT）细胞的特化T细胞亚群识别由MHC I类分子CD 1d呈递的糖脂抗原。在刺激后，iNKT细胞以快速爆发的方式产生大量的T辅助细胞1（Th 1）、Th 2细胞因子和趋化因子，其具有广泛的免疫影响，包括针对自身免疫性疾病的保护、对寄生虫和细菌的宿主应答以及抗肿瘤应答。虽然众所周知的α-GalCer可以显著刺激iNKT细胞，但它是从海绵中分离的外源性抗原。在胸腺中从T细胞前体发育成iNKT细胞的过程中，内源性抗原对于由CD 1d呈递给随机产生的TCR的过程是必需的。据报道，iGb 3在小鼠和人中均为可能的内源性抗原。然而，来自独立实验室的研究挑战了iGb 3是负责小鼠中iNKT细胞发育的主要配体的假设。特别地，通过NP-HPLC分析，在小鼠或人胸腺中均未检测到iGb 3。此外，iGb 3合酶敲除（iGb 3S-/-）小鼠在胸腺、脾脏和肝脏中显示正常的iNKT细胞数量。进一步的实验表明，通过分析iGb 3S mRNA，在任何人体组织中均未鉴定出iGb 3S，并且由于至少一个氨基酸取代，含有人iGb 3S催化结构域的嵌合分子在iGb 3合成中未显示任何功能活性。尽管iGb 3在小鼠和人胸腺样品或其他组织中的存在存在争议，但iGb 3在刺激iNKT细胞中的活性是公认的。这为我们在胸腺内寻找用于iNKT细胞发育的真正iNKT细胞配体提供了重要线索。因此，在本建议中，我们假设一种类型的糖脂作为潜在的主要配体的基础上的糖脂在小鼠和人类的生物合成和降解途径，在CD 1d/1-GalCer/TCR和CD 1d/iGb 3复合物的晶体结构，以及在iGb 3的三维结构。可能的抗原是三种血型糖基神经酰胺，它们具有与iGb 3类似的结构，并且在小鼠和人中都具有相应的合成途径。所有这些配体在半乳糖的C2位置上具有岩藻糖部分。血液B型糖基神经酰胺的计算对接表明，该化合物可以容纳在TCR和CD 1d之间的空腔中。虽然这些血型糖基神经酰胺的研究少得多，迄今为止，其中两个已被确定在猪组织，这意味着它们也可能存在于小鼠和人体组织，如胸腺和脾脏。该计划提出了三个具体目标：1。制备三种不同血型的糖神经酰胺，其包括Fuc 1（1，2）Gal 2（1，4）Glc 2（1，1）神经酰胺（血O型）、GalNAc 1（1，3）[Fuc 1（1，2）] Gal 2（1，4）Glc 2（1，1）神经酰胺（血A型）和Gal 1（1，3）[Fuc 1（1，2）] Gal 2（1，4）Glc 2（1，1）神经酰胺（血B型）。还将制备具有硫原子作为连接基团之间的键的代谢稳定的类似物。2.用不同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.确定小鼠组织中潜在的血型糖基神经酰胺抗原。iNKT细胞是先天性免疫系统和适应性免疫系统之间的桥梁。iNKT细胞内源性配体的鉴定将有助于了解iNKT细胞的发育、增殖机制及其与T细胞、B细胞、巨噬细胞等免疫细胞的关系。这也将有助于研究人员开发将该原理应用于临床应用的方法。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，这在炎症反应和抑制肿瘤增殖中非常重要。iNKT细胞是先天性免疫系统和适应性免疫系统之间的桥梁。iNKT细胞内源性配体的鉴定将有助于了解iNKT细胞发育和增殖的机制及其与T细胞、B细胞和巨噬细胞等其他免疫细胞的关系。这也将有助于研究人员开发将该原理应用于临床应用的方法。