Regulation of Phosphoprotein Signalling in CD4+ and DN iNKT Cell Subsets

描述（由申请人提供）：据报道，CD 1d限制性T细胞（或“iNKT细胞”）可调节极其多样化的免疫应答和疾病。这些T细胞的功能障碍显然与自身免疫的发展相关，特别是自身免疫性糖尿病。尽管CD 1d限制性T细胞在这种疾病中的重要性，但这些T细胞如何正常发挥功能以及疾病相关缺陷的确切性质仍不清楚。在这方面，预测对1型糖尿病具有显著影响的潜在调节功能包括最近描述的iNKT细胞与树突状细胞的关键相互作用以及活化诱导的Th 1和Th 2细胞因子分泌。在鼠模型中，iNKT细胞分泌的Th 2细胞因子与自身免疫性糖尿病的保护相关。相反，从1型糖尿病患者克隆的iNKT细胞被发现，除其他缺陷外，具有极端的Th 1细胞因子偏好。在正常人志愿者中，CD 4+亚群负责体内Th 2细胞因子的产生，而CD 4-（或“DN”）亚群强烈偏向Th 1细胞因子的分泌和细胞毒性功能。有趣的是，有1型糖尿病风险的个体在DN亚群中显著增加。因此，CD 4 + iNKT细胞可能有助于预防糖尿病的进展，而DN iNKT细胞可能促进促炎反应。该提议的一个主要假设是CD 4与DN iNKT细胞的比率偏向DN iNKT细胞。此外，倾向于促炎性DN iNKT细胞和CD 4 + iNKT细胞未能上调Foxp 3和潜在的其他免疫调节基因可能有助于进展为1型糖尿病。为了确定iNKT细胞亚群如何使用相同的高度进化保守的TCR/CD 1d抗原呈递系统差异地发出信号，我们建议开发蛋白质组学方法，该方法将允许定量测定位点特异性磷酸化信号传导事件，目的如下：分析CD 4 + iNKT细胞中Foxp 3优先激活的条件，并确定CD 4 + iNKT细胞发挥免疫调节功能的机制。1b.测定CD 4+和DN iNKT细胞的磷酸化和细胞因子分泌模式。1型糖尿病是一种自身免疫性疾病，导致产生胰岛素的胰腺细胞被破坏。这种自身免疫性疾病在美国影响多达1/300的人。尽管在疾病管理和使用的胰岛素制剂方面取得了进展，但在30岁以下的1型糖尿病患者中，寿命缩短了10-15年。30-50%的1型糖尿病患者发生微血管和大血管并发症，2005年仅糖尿病的医疗保健支出估计就超过1200亿美元。1型糖尿病占此类支出的比例不成比例（与2型糖尿病相比）。这种疾病被认为是失明（视网膜病）、心脏病、外周血管疾病、肾衰竭和阳痿的主要原因。因此，一种涉及疾病活动的早期检测的方法，以及一种防止胰腺细胞破坏如iNKT细胞活化的方法是有吸引力的。

DESCRIPTION (provided by applicant): CD1d-restricted T cells (or "iNKT cells") have been reported to regulate an extremely diverse set of immunologic responses and diseases. Dysfunction of these T cells is clearly correlated with the development of autoimmunity, and in particular autoimmune diabetes. Despite the importance of CD1d-restricted T cells in this disease, how these T cells function normally and the exact nature of the disease-associated defects remains unclear. In this regard, potential regulatory functions that would be predicted to have significant impact on type 1 diabetes include recently described critical interactions of iNKT cells with dendritic cells and the activation-induced secretion of Th1 and Th2 cytokines. Th2 cytokine secretion by iNKT cells has been associated with protection from autoimmune diabetes in murine models. Conversely, iNKT cells cloned from patients with type 1 diabetes were found, amongst other defects, to have an extreme Th1 cytokine bias. In normal human volunteers, the CD4+ subset is responsible for Th2 cytokine production in vivo, whereas the CD4- (or "DN") subset was strongly biased towards the secretion of Th1 cytokines and cytotoxic function. Interestingly, individuals at risk for type 1 diabetes have a significant increase in the DN subset. Hence, CD4+ iNKT cells might serve to prevent progression to diabetes whilst DN iNKT cells might promote pro-inflammatory responses. A major hypothesis of the proposal is that the ratio of CD4 to DN iNKT cells is skewed toward DN iNKT cells. In addition, a bias towards proinflammatory DN iNKT cells and the a failure of CD4+ iNKT cells to up regulate Foxp3 and potentially other immunoregulatory genes might contribute to the progression to type 1 diabetes. To determine how iNKT cell subsets differentially signal using the same highly evolutionarily conserved TCR/CD1d antigen presentation system, we propose to develop proteomic methodology that will allow for the quantitative determination of site-specific phosphosignaling events in the following aim: Specific Aims: 1a. Analyze the requirements for the preferential activation of Foxp3 in CD4+ iNKT cells and determine the mechanism by which CD4+ iNKT cells exert immunoregulatory function. 1b. Determine phosphorylation and cytokine secretion patterns of CD4+ and DN iNKT cells. Type 1 diabetes is an autoimmune disease that results in destruction of pancreatic ¿ cells that produce insulin. This autoimmune disease affects as many as 1 in 300 persons in the United States. Despite advances in disease management and in the insulin preparations used, life span is shortened by 10-15 years in those developing type 1 diabetes under the age of 30. Microvascular and macrovascular complications occur in 30-50% of type 1 diabetes patients, and health care expenditures for diabetes alone in 2005 were estimated to have been in excess of $120 billion. Type 1 diabetes accounts for a disproportional share of such expenditures (in comparison to type 2 diabetes). The disease is recognized as a leading cause of blindness (retinopathy), heart disease, peripheral vascular disease, renal failure, and impotence. Therefore, an approach that involves early detection of disease activity, as well as a method of preventing pancreatic ¿ cell destruction such as activation of iNKT cells is appealing.