Sulfatide-reactive type II NKT cells: repertoire and function

描述(申请人提供)：自然杀伤T(NKT)细胞识别由CD1d分子呈递的脂类抗原，并可根据其对半不变TCR或不同TCR的使用而分为I型或II型。II型NKT细胞在人类中占优势，而I型NKT细胞在小鼠中占优势。自身糖脂反应性CD1d限制性的II型NKT细胞的生物学和功能还知之甚少。我们已经确定了II型NKT细胞的一个主要亚群，它识别一种髓磷脂衍生的糖脂，硫脂。在实验性自身免疫性脑脊髓炎(EAE)的炎症组织中，硫脂反应型II型NKT细胞而不是I型NKT细胞丰富。重要的是，硫脂介导的II型NKT细胞的激活诱导了一种新的调节途径，伴随着I型NKT细胞的无能诱导和对自身免疫性疾病的保护。在这里，我们建议从抗原的精细特异性、组织分布和细胞因子分泌模式等方面详细描述II型NKT细胞的TCRV基因谱系。此外，我们还将研究它们的激活和免疫调节机制，涉及NKT细胞与其他天然类细胞的相互作用，包括浆细胞样树突状细胞(PDCs)和髓系来源的抑制细胞(MDSCs)，后者反过来控制获得性免疫。硫脂/CD1d-四聚体分选细胞的TCR谱系将通过流式细胞术、DNA测序以及TCRV1和V2基因的谱型来确定。在通过逆转录病毒载体将主要的TCR1和TCR2链基因导入TCR缺陷的细胞系进行抗原特异性测定后，大多数仿制的TCRs将与负载硫脂的CD1d分子折叠，用于三分子复合体的结构研究。缺乏或存在过量的内源性硫脂对TCR谱系的影响将分别在神经酰胺半乳糖磺基转移酶CST-/-或皂苷B-/-小鼠中进行研究。将利用B细胞缺陷的5MT小鼠及其骨髓嵌合体来研究B细胞在II型NKT细胞激活中的作用及其调节能力。由于几种自身免疫条件与抗硫脂抗体反应有关，将在CD1d-/-、J118-/-和II型NKT耗竭小鼠以及硫脂/CD1d-四聚体+细胞的接受者中检测II型NKT细胞为抗体反应提供帮助的能力。CD1d-/-受体的CD1d-四聚体+细胞将通过CFSE标记的CD1d-四聚体+细胞来研究pDC和mDCs在II型NKT细胞激活和调节中的作用。II型NKT细胞在MDSCs积累中的作用以及它们抑制脑源性CD4+T细胞的能力将在过继转移试验中以及在接受全反式维甲酸治疗的小鼠的损伤后进行检测。这些研究不仅对了解II型NKT细胞的生物学和免疫调节机制具有重要意义，而且由于CD1d分子跨物种的高度保守性，它们将形成人类自身免疫性疾病的操作基础，并将对增强抗肿瘤免疫反应具有重要意义。

DESCRIPTION (provided by applicant): Natural killer T (NKT) cells recognize lipid antigens presented by CD1d molecules and can be classified into type I or type II based upon their usage of a semi-invariant TCR or diverse TCRs, respectively. Type II NKT cells are predominant in humans, whereas type I NKT cells are prevalent in the mouse. The biology and function of self-glycolipid-reactive CD1d-restricted type II NKT cells is poorly understood. We have identified a major subset of type II NKT cells that recognizes a myelin-derived glycolipid, sulfatide. Sulfatide-reactive type II but not type I NKT cells are enriched in the inflamed tissues during experimental autoimmune encephalomyelitis (EAE). Importantly, sulfatide-mediated activation of type II NKT cells induce a novel regulatory pathway accompanied by anergy induction in type I NKT cells and protection from autoimmune diseases. Here we propose to characterize in details the TCR V-gene repertoire of type II NKT cells with respect to the antigen fine specificity, tissue distribution and cytokine secretion pattern. Also we will study their activation and mechanism of immune regulation that involves interactions of NKT cells with other innate-like cells, including plasmacytoid dendritic cells (pDCs), myeloid-derived suppressor cells (MDSCs) which in turn control adaptive immunity. The TCR repertoire of sulfatide/CD1d-tetramer-sorted cells will be determined using flow cytometry, DNA sequencing and spectratyping of TCR V1 and V2 genes. Following determination of antigen specificity using retroviral vector-mediated transfection of dominant TCR 1 and 2 chain genes into a TCR-deficient cell line, most affine TCRs will be folded with sulfatide-loaded CD1d molecules for structural studies of the tri-molecular complex. The impact of the absence or presence of an excess amount of endogenous sulfatide on the TCR repertoire will be studied in ceramide galactosyl sulfotransferase CST-/- or saposin B-/- mice, respectively. The role of B cells in the activation of type II NKT cells and their regulatory ability will be investigated using B cell-deficient 5MT mice and their bone marrow chimeras. Since several autoimmune conditions are associated with the anti-sulfatide antibody response, the ability of type II NKT cells to provide help for the antibody response will be examined in CD1d-/-, J118-/- and type II NKT-depleted mice as well as in recipients of sulfatide/CD1d-tetramer+ cells. The role of pDCs vs. mDCs in activation of type II NKT cells and in regulation will be studied using CFSE-labeled CD1d-tetramer+ cells in CD1d-/- recipients. The role of type II NKT cells in the accumulation of MDSCs and their ability to suppress the encephalitogenic CD4+ T cells will be examined in adoptive transfer assays and following their impairment in mice treated with all-trans-retinoic acid. These studies are important not only for understanding the biology of type II NKT cells and the mechanisms of immune regulation, but also because of the highly conserved nature of CD1d molecules across species they will form the basis for manipulation of autoimmune diseases in humans and will have implications for augmenting anti-tumor immune responses.