HUMAN T CELL REACTIVITY TO GAD AND ITS ROLE IN IDDM

越来越多的人热衷于针对早期 对临床前IDDM患者进行干预，以停止或减缓 自身免疫性疾病发作时β细胞功能下降。 这样的试验产生了对两种表型标记的需求。第一、 需要一种能够预测谁将发展为 在胰岛素依赖型糖尿病发病率较低的普通人群中， 这种性质的可靠的、特异性的标记物将允许 需要进行干预试验，以证明拟议的 治疗要减少。第二，在预处理的临床干预试验中， 对于目前所考虑的糖尿病患者，治疗效果主要是 通过患者在3-5年内是否变成糖尿病来确定， 个或多个.对替代标记物有额外的迫切需求。 这将比这更快地反映治疗效果。在这 项目，我们假设细胞免疫谷氨酸 脱羧酶（GMD）可能是进展为IDDM的重要预测因子。 将使用有限稀释法测定T 外周血淋巴细胞系列样品中对GMD反应的细胞 来自糖尿病前期患者（基于多个标志物阳性定义）和HLA 匹配的控制。将测试GMD T细胞前体的频率， 肽特异性和细胞因子（IL-2 vs. IL-4）分泌 纵向。将通过以下方法克隆对GMD表位反应的T细胞： 几种方法，包括一种新的分子方法。最后，TCR的使用 将确定这些克隆中的DNA含量，并将进行原位PCR/FACS测定。 用于测量GMD反应性TCR携带T细胞的频率， 来自糖尿病前期患者和对照组的连续PBL样本。这些目标 研究开发和验证对GMD的细胞免疫测定， 胰岛素依赖型糖尿病进展的新预测工具。 这是一个跨学科糖尿病研究计划的项目#2。作为 它受益于，并有助于，一些合作和 胰岛素依赖型糖尿病发病机制中早期免疫学事件的相互作用研究。为 例如，该项目将严重依赖临床核心（A）， 为研究提供充分表征的患者材料，并将取决于 在其他项目中，用于鉴定GMD肽， DQ3.2（项目#1），肽优先加工， 由不同类型的抗原呈递细胞呈递（项目#3和 #4）。反过来，该项目将提供重组人GMD和GMD 反应性T细胞的其他项目，并作为一个试验场， 这些数据的预测和治疗效用 项目

There is increasing enthusiasm for clinical trials aimed at early intervention in the pre-clinical IDDM patient in order to halt or slow the decline in beta cell function that occurs with autoimmune attack. Such trials generate a need for two kinds of phenotypic markers. First, there is a need for markers that allow prediction of who will develop disease in the general population where the incidence of IDDM is low. Reliable, specific markers of this nature would allow the size of intervention trials necessary to demonstrate the efficacy of proposed therapies to be reduced. Second, in clinical intervention trials of pre- diabetics as currently contemplated, therapeutic efficacy is mainly determined by whether or not the patient becomes diabetic over 3-5 years, or more. There is an additional critical need for surrogate markers. which will reflect therapeutic efficacy sooner than that. In this project, we hypothesize that cellular immunity to glutamic acid decarboxylase (GMD) may be an important predictor of progression to IDDM. A limiting dilution assay will be used to measure the frequencies of T cells reactive to GMD in serial samples of peripheral blood lymphocytes from prediabetics (defined based on multiple marker positively) and HLA matched controls. GMD T cell precursors will tested for their frequency, peptide specificity, and cytokine (IL-2 vs. IL-4) secretion longitudinally. T cells reactive to GMD epitopes will be cloned by several methods including a novel molecular approach. Finally, TCR usage in these clones will be determined and an in situ PCR/FACS assay will be used to measure the frequencies of GMD reactive TCR bearing T cells in the serial PBL samples from prediabetics and controls. The goal of these studies to develop and validate assays for cellular immunity to GMD as new predictive tools for IDDM progression. This is Project #2 of an interdisciplinary diabetes research program. As such it benefits from, and contributes to, a number of collaborative and interactive studies of early immunologic events in IDDM pathogenesis. For example, this project will rely heavily on the clinical core (A) to provide well characterized patient material for study, and will depend on the other projects for the identification of GMD peptides that bind to DQ3.2 (project #1), and peptides preferentially processed and presented by different types of antigen presenting cells (projects #3 and #4). In turn, this project will provide recombinant human GMD and GMD reactive T cells to the other projects and serve as a testing ground for the predictive and therapeutic utility of data derived from those projects.