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

描述（由申请人提供）：据报道，cd1限制性T细胞（或“iNKT细胞”）调节极其多样化的免疫反应和疾病。这些T细胞的功能障碍与自身免疫，特别是自身免疫性糖尿病的发展明显相关。尽管cd1限制性T细胞在这种疾病中的重要性，但这些T细胞如何正常发挥作用以及疾病相关缺陷的确切性质尚不清楚。在这方面，预计对1型糖尿病有重大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激活诱导的Th1和Th2细胞因子的分泌。在小鼠模型中，iNKT细胞分泌Th2细胞因子与保护自身免疫性糖尿病有关。相反，从1型糖尿病患者身上克隆的iNKT细胞，除了其他缺陷外，还存在极端的Th1细胞因子偏倚。在正常人类志愿者中，CD4+亚群在体内负责Th2细胞因子的产生，而CD4-（或“DN”）亚群则强烈倾向于Th1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的DN亚群显著增加。因此，CD4+ 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CD4细胞与DN iNKT细胞的比例向DN iNKT细胞倾斜。此外，对促炎DN iNKT细胞的偏向和CD4+ iNKT细胞上调Foxp3和其他潜在免疫调节基因的失败可能导致1型糖尿病的进展。为了确定iNKT细胞亚群如何使用相同的高度进化保守的TCR/CD1d抗原呈递系统进行差异信号传递，我们建议开发蛋白质组学方法，以定量确定位点特异性磷酸化信号事件，目的如下：分析Foxp3在CD4+ iNKT细胞中优先激活的条件，确定CD4+ iNKT细胞发挥免疫调节功能的机制。1 b。测定CD4+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。在美国，每300人中就有1人患有这种自身免疫性疾病。尽管在疾病管理和使用的胰岛素制剂方面取得了进展，但30岁以下的1型糖尿病患者的寿命缩短了10-15年。30-50%的1型糖尿病患者出现微血管和大血管并发症，2005年仅糖尿病的卫生保健支出估计就超过了1 200亿美元。与2型糖尿病相比，1型糖尿病在此类支出中所占的份额不成比例。这种疾病被认为是失明（视网膜病变）、心脏病、周围血管疾病、肾功能衰竭和阳痿的主要原因。因此，一种涉及疾病活动早期检测的方法，以及一种防止胰腺细胞破坏的方法，如iNKT细胞的激活，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.