Self-glycolipid-reactive T cells: repertoire & function

描述(申请人提供)：一些T细胞识别由I类MHC样CD1分子呈递的脂类抗原。尽管激活它们的自体脂类抗原尚未被确定，但大多数NK T细胞被认为是自身反应的，并可以被替代的糖脂配体α-半乳糖基神经酰胺或α-GalCer迅速激活。为了表征自身糖脂反应性T细胞、其表型和生理功能，我们重点研究了一种主要的髓磷脂来源的糖脂硫脂(3‘-硫酸基β-半乳糖神经酰胺)的识别。我们已经产生了硫脂-CD1d-四聚体，并在幼龄C57BL/6小鼠的肝脏、胸腺和脾中发现了硫脂反应性T细胞群。我们还产生了硫脂反应性T细胞杂交瘤。在抗原诱导的实验性自身免疫性脑脊髓炎(EAE)过程中，中枢神经系统内硫脂-CD1d-四聚体+T细胞选择性增加数倍。硫脂治疗可以预防和逆转野生型EAE，但不能预防CD1d缺陷小鼠。基于我们的发现，我们认为硫脂是一种自身糖脂配体，可被CD1d限制性T细胞识别，可被激活以调节自身免疫反应。我们将使用硫脂-和a-GalCer-CD1d-四聚体和针对NK T细胞上各种细胞表面标志的抗体来表征硫脂反应性T细胞的表型。硫脂反应性T细胞杂交瘤将用于分析抗原的良好特异性和TCRV基因库。利用糖脂四聚体和ELISPOT分析，我们将确定它们的激活动力学，并研究不同脂质反应性T细胞群体之间的相互作用。我们将测定野生型和CD1d缺陷小鼠注射硫脂后NK细胞、B细胞和髓鞘蛋白反应性致病CD4T细胞的数量、表型和命运。在过继转移实验中，我们将直接检测硫脂-CD LD-四聚体+T细胞的调节潜力。中和抗体和缺乏1型和2型细胞因子的小鼠将被用来确定它们在硫脂调节EAE中的作用。我们将优化硫脂对正在进行的疾病的治疗，以探索其治疗潜力。这些研究不仅对于理解自然产生的自身糖脂反应性T细胞群体的生物学意义重大，而且因为CD1d分子跨物种的高度保守性，它们将形成操纵人类自身免疫性脱髓鞘疾病的基础，如MS。

DESCRIPTION (provided by applicant): Some T cells recognize lipid antigens presented by class I MHC-like CD1 molecules. Although self-lipid antigens activating them have not yet been identified, most NK T cells are thought to be autoreactive and can be rapidly activated by a surrogate glycolipid ligand alpha-galactosyl ceramide or alpha-GalCer. To characterize self-glycolipid-reactive T cells, their phenotype and their physiological function, we have focused on the recognition of a major myelin-derived glycolipid, sulfatide, (3'-sulfated beta-galactosyl ceramide). We have generated sulfatide-CD1d-tetramers and have identified sulfatide-reactive T cell populations in the liver, thymus and spleen from naive C57BL/6 mice. We have also generated sulfatide-reactive T cell hybridomas. During the course of antigen-induced experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) in humans, sulfatide-CD1d-tetramer+ T cells are selectively increased several-fold within the central nervous system. Treatment with sulfatide can both prevent and reverse ongoing EAE in wild type but not in CD1d-deficient mice. Based on our findings we propose that sulfatide is a self-glycolipid ligand recognized by a distinct population of CD1d-restricted T cells that can be activated to modulate autoimmune responses. We will use sulfatide- and a-GalCer-CD1d-tetramers and antibodies against various cell surface markers on NK T cells to characterize the phenotype of sulfatide-reactive T cells. Sulfatide-reactive T cell hybridomas will be used to analyze the antigen fine specificity and the TCR V-gene repertoire. Using glycolipid tetramers and Elispot analysis we will determine the dynamics of their activation and investigate mutual interactions among different lipid-reactive T cell populations. We will determine the number, phenotype and the fate of NK cells, B cells and myelin protein-reactive pathogenic CD4 T cells following sulfatide injection of wild type and CD1d-deficient mice. In adoptive transfer experiments we will directly examine the regulatory potential of sulfatide-CD ld-tetramer+ T cells. Neutralizing antibodies and mice genetically deficient for type 1 and 2 cytokines will be used to determine their role in the modulation of EAE by sulfatide. We will optimize treatment of ongoing disease with sulfatide in order to explore its therapeutic potential. These studies are important not only for understanding the biology of a naturally occurring self-glycolipid-reactive T cell population, but also because of the highly conserved nature of CD1d molecules across species they will form the basis for manipulation of human autoimmune demyelinating diseases, such as MS.