Identification of Islet Proteins Stimulatory for T Cells from Diabetes Patients

描述（由申请人提供）：鉴定胰岛蛋白刺激的T细胞来自糖尿病患者。摘要。1型糖尿病（T1DM）是一种细胞介导的针对β细胞的自身免疫性疾病，而2型糖尿病（T2DM）不是自身免疫性疾病。近年来，一组自身免疫性表型T2DM患者的研究表明，T细胞介导的自身免疫可能与T1DM和T2DM中β细胞衰竭的发病和进展有关。在两次NIH赞助的研讨会上，我们的细胞免疫印迹T细胞测定，使用分离的人类胰岛，已被证实可以区分T1DM患者和对照组，具有出色的特异性和敏感性。有趣的是，来自T1DM患者的T细胞对胰岛蛋白的选择分子量区域（初步数据）有反应，这是通过细胞免疫印迹法确定的。因此，我们假设个体胰岛蛋白负责自身免疫性糖尿病患者中观察到的T细胞反应性，并且识别这些蛋白将提供对自身免疫性糖尿病中T细胞反应性的深入了解。以前，用于T细胞检测的胰岛蛋白是基于自身抗体反应性而不是直接的T细胞反应性来鉴定的。目前需要的是鉴定直接刺激自身免疫性糖尿病患者T细胞的胰岛蛋白。与蛋白质组学专家合作，我们建议使用以下三种方法从自身免疫性糖尿病患者中分离和鉴定刺激T细胞的胰岛蛋白：a)。从感兴趣的分子量区分离T细胞刺激蛋白，通过细胞免疫印迹鉴定，使用电洗脱的一和二维SDS-PAGE，然后使用串联质谱（MS/MS）鉴定蛋白质。b)。顺序生化方法分离蛋白质，使其接近均匀性，然后进行质谱/质谱鉴定。c)。一种新的正交分离策略来识别激活T细胞的蛋白质。一旦T细胞刺激胰岛蛋白被分离和鉴定，我们将继续通过分析自身免疫性糖尿病患者的T细胞反应性来验证分离的胰岛蛋白。鉴定直接刺激糖尿病患者T细胞的单个抗原胰岛细胞蛋白可能导致新的基于抗原的检测和干预策略的发展，并为跟踪和评估新的治疗试验提供创新的方法。

DESCRIPTION (provided by applicant): Identification of Islet Proteins Stimulatory for T cells from Diabetes Patients. Abstract. Type 1 Diabetes Mellitus (T1DM) is a cell-mediated autoimmune disease directed against the beta cells whereas Type 2 diabetes (T2DM) is not autoimmune. In recent years, a group of autoimmune phenotypic T2DM patients have been described suggesting that T cell mediated autoimmunity may be associated with the pathogenesis and progression of beta cell failure in both T1DM and T2DM. In two masked NIH sponsored workshops, our cellular immunoblotting T cell assay, which uses isolated human islets, has been validated to distinguish T1DM patients from controls with excellent specificity and sensitivity. Interestingly, T cells from T1DM patients respond to select molecular weight regions of islet proteins (preliminary data) as identified using cellular immunoblotting. Therefore, we hypothesize that individual islet proteins are responsible for the T cell reactivity observed in autoimmune diabetes patients and that identifying these proteins will provide insight into T cell reactivity in autoimmune diabetes. Previously, islet proteins utilized in T cell assays were identified based on autoantibody reactivity and not direct T cell reactivity. What is currently needed is the identification of islet proteins that directly stimulate T cells from autoimmune diabetes patients. In collaboration with experts in proteomics, we propose to isolate and identify islet proteins stimulatory to T cells from autoimmune diabetes patients using the following three approaches: a). Isolation of T cell stimulatory proteins from molecular weight regions of interest, identified by cellular immunoblotting, using electro-elution of one and two-dimensional SDS-PAGE followed by tandem mass spectrometric (MS/MS) identification of proteins. b). Sequential biochemical approaches to isolate proteins to near homogeneity followed by MS/MS identification of the purified proteins. c). A novel orthogonal separation strategy to identify proteins that activate T cells. Once the T cell stimulatory islet proteins are isolated and identified, we will proceed to verify the isolated islet proteins by analysis of T cell reactivity in autoimmune diabetes patients. Identification of individual antigenic islet cell proteins that directly stimulate T cells from diabetes patients could lead to the development of new antigen based assays and intervention strategies, and provide innovative methods to follow and assess new therapeutic trials.