Sulfatide-reactive type II NKT cells: repertoire and function

描述（由申请人提供）：自然杀伤T（NKT）细胞识别由CD 1d分子呈递的脂质抗原，并可根据其分别使用半不变TCR或不同TCR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细胞在小鼠中占主导地位。自身糖脂反应性CD 1d限制性II型NKT细胞的生物学和功能知之甚少。我们已经确定了一个主要的子集的II型NKT细胞，识别髓鞘衍生的糖脂，硫苷脂。实验性自身免疫性脑脊髓炎（EAE）期间，炎症组织中富含硫酸脂酶反应性II型NKT细胞，而非I型NKT细胞。重要的是，硫苷脂介导的II型NKT细胞的活化诱导一种新的调节途径，伴随着I型NKT细胞中的无反应性诱导和对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基因库的抗原精细特异性，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，涉及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（pDC），骨髓源性抑制细胞（MDSC），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基因的光谱分析来确定硫苷脂/CD 1d-四聚体分选细胞的TCR库。在使用逆转录病毒载体介导的显性TCR 1和2链基因转染到TCR缺陷细胞系中确定抗原特异性后，大多数亲和TCR将与加载有硫苷脂的CD 1d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基磺基转移酶CST-/-或saposin B-/-小鼠中研究过量内源性硫苷脂的存在或不存在对TCR谱系的影响。将使用B细胞缺陷型5 MT小鼠及其骨髓嵌合体研究B细胞在II型NKT细胞活化中的作用及其调节能力。由于几种自身免疫性疾病与抗硫苷脂抗体应答相关，因此将在CD 1d-/-、J118-/-和II型NKT耗竭小鼠以及硫苷脂/CD 1d-四聚体+细胞接受者中检查II型NKT细胞为抗体应答提供帮助的能力。将使用CFSE标记的CD 1d-/-受体中的CD 1d-四聚体+细胞研究pDC与mDC在II型NKT细胞活化和调节中的作用。II型NKT细胞在MDSC积累中的作用及其抑制致脑炎性CD 4 + T细胞的能力将在过继转移试验中以及在用全反式视黄酸处理的小鼠中的损伤后进行检查。这些研究不仅对于理解II型NKT细胞的生物学和免疫调节机制很重要，而且由于CD 1d分子在物种间的高度保守性，它们将形成操纵人类自身免疫性疾病的基础，并将对增强抗肿瘤免疫应答产生影响。 公共卫生相关性：我们已经确定了一个独特的群体的白色血液淋巴细胞，成为激活的自我糖脂，并存在于小鼠和人类。这些细胞被激活后，能够预防和治疗模拟人类多发性硬化症（MS）的实验性自身免疫性疾病。该提案旨在了解这些淋巴细胞控制致病细胞的机制，以努力开发治疗此类疾病的新治疗方法。

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. PUBLIC HEALTH RELEVANCE: We have identified a distinct population of white blood lymphocytes that becomes activated by a self-glycolipid and is present in both mice and in humans. Upon their activation these cells are able to prevent and treat experimental autoimmune diseases mimicking multiple sclerosis (MS) in humans. This proposal seeks to understand the mechanism by which these lymphocytes control disease-causing cells in an effort to develop novel therapeutic approaches for the treatment of such diseases.