Sulfatide-reactive type II NKT cells: repertoire and function

描述（由申请人提供）：自然杀伤T （NKT）细胞识别CD1d分子呈递的脂质抗原，根据其使用的半不变TCR或不同TCR，可分为I型或II型。II型NKT细胞在人类中占主导地位，而I型NKT细胞在小鼠中普遍存在。自糖脂反应性cd1 -限制性II型NKT细胞的生物学和功能尚不清楚。我们已经确定了II型NKT细胞的一个主要亚群，它可以识别髓磷脂衍生的糖脂，即硫脂。实验性自身免疫性脑脊髓炎（EAE）期间，炎症组织中富含硫脂反应型ⅱ型而非ⅰ型NKT细胞。重要的是，硫脂介导的II型NKT细胞激活诱导了一种新的调节途径，伴随着I型NKT细胞的能量诱导和自身免疫性疾病的保护。在这里，我们建议详细描述II型NKT细胞的TCR v基因库，包括抗原精细特异性、组织分布和细胞因子分泌模式。此外，我们将研究它们的激活和免疫调节机制，包括NKT细胞与其他先天样细胞的相互作用，包括浆细胞样树突状细胞（pDCs），髓源性抑制细胞（MDSCs），这些细胞反过来控制适应性免疫。将使用流式细胞术、DNA测序和TCR V1和V2基因的谱分型来确定硫脂/ cd1 -四聚体分类细胞的TCR库。利用逆转录病毒载体介导的TCR 1和2链显性基因转染TCR缺陷细胞系，确定抗原特异性后，大多数仿射TCR将与装载硫脂的CD1d分子折叠，用于三分子复合物的结构研究。将分别在神经酰胺半乳糖磺酸转移酶CST-/-或皂苷B-/-小鼠中研究缺乏或过量内源性硫脂对TCR库的影响。B细胞在II型NKT细胞激活中的作用及其调控能力将在B细胞缺陷5MT小鼠及其骨髓嵌合体中进行研究。由于几种自身免疫性疾病与抗硫脂抗体反应相关，因此将在CD1d-/-， J118-/-和II型NKT耗尽小鼠以及硫脂/ cd1 -四聚体+细胞受体中检测II型NKT细胞为抗体反应提供帮助的能力。pDCs与mDCs在II型NKT细胞激活和调控中的作用将在CD1d-/-受体中使用cfse标记的CD1d-四聚体+细胞进行研究。II型NKT细胞在MDSCs积累中的作用及其抑制致脑性CD4+ T细胞的能力将在过继性转移试验中进行检测，并在全反式维甲酸处理的小鼠中进行损伤。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很重要，而且由于CD1d分子在物种间的高度保守性，它们将形成人类自身免疫性疾病的基础，并将对增强抗肿瘤免疫反应产生影响。

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.