DNA Immunizations with GAD65 to Induce Tolerance

1型糖尿病是一种自身免疫性疾病，其特征是T细胞介导的朗格汉斯胰岛胰岛素生成细胞的破坏。胰岛素依赖型糖尿病（IDDM）的小鼠模型，即非肥胖型糖尿病（NOD）小鼠，由于该疾病的许多特征是相同的，因此已成为人类疾病的有用模型。在小鼠和人类糖尿病的发展之前，大量的淋巴样细胞浸润胰岛，包括CD4+、CD8+ T细胞、树突状细胞和单核细胞。最近发现，T细胞对神经元酶谷氨酸脱羧酶65 （GAD65）的反应是最早在年轻NOD小鼠的脾脏中检测到的，反应早在4周时就被检测到。此外，研究发现，通过胸腺内或静脉注射使NOD对GAD65产生耐受性，可以预防糖尿病。我们最近的研究表明，用单一GAD65肽脉冲的树突状细胞治疗NOD小鼠可以预防糖尿病，这是通过启动GAD65特异性Th2反应发生的。根据免疫途径的不同，使用质粒DNA进行遗传免疫可导致Th1或Th2应答。我们在非糖尿病模型中有初步证据表明，转染蛋白的细胞定位也深刻影响免疫反应的性质。当蛋白处于细胞质中时，可以获得强烈的Th1反应（CTL），而当蛋白在卵白蛋白（OVA） cDNA基因枪免疫后分泌时，可以看到Th2主导的反应。因此，基因免疫可以定制，以诱导所需的反应。在NOD小鼠的糖尿病病例中，研究表明，诱导Th2对特定GAD65肽的反应可以导致破坏性胰岛素炎的停止和糖尿病的预防。1)构建GAD65 cDNA质粒，使GAD65能够作为分泌蛋白和跨膜蛋白在细胞质中表达；2)研究三种GAD65构建体对NOD和非糖尿病易感菌株免疫后的免疫应答；3)确定DNA免疫GAD65构建体是否能预防糖尿病和胰岛素的发生。

Type 1 diabetes is an autoimmune disease characterized by T cell- mediated destruction of the insulin-producing cells of the islets of Langerhans. The murine model of insulin dependent diabetes mellitus (IDDM), the non-obese diabetic (NOD) mouse has served as a useful model of the human disease since many of the features of the disease are shared. In both mouse and human the development of diabetes is preceded by heavy islet infiltration with lymphoid cells, including CD4+, CD8+ T cells, dendritic cells and monocytes. Recently it was found that T cell responses to the neuronal enzyme glutamic acid decarboxylase 65 (GAD65) were among the first to be detected in the spleen of young NOD mice, with responses being detected as early as 4 weeks. In addition it was found that if NOD were made tolerant to GAD65, by intra-thymic or intravenous injection, diabetes was prevented. We have recently shown that treatment of NOD mice with dendritic cells pulsed with a single GAD65 peptide can prevent diabetes and that this occurs through the initiation of a GAD65-specific Th2 response. Genetic immunization using either plasmid DNA, depending on the route of immunization, can lead to either a Th1 or Th2 response. We have preliminary evidence in a non-diabetes model that the cellular localization of the transfected protein also profoundly influences the nature of the immune response. Strong Th1 responses, with CTL, are obtained when the protein is cytoplasmic, whereas a Th2 dominated response is seen when the protein is secreted following gene gun immunization of ovalbumin (OVA) cDNA. Genetic immunization can thus be tailored to induce the desired response. In the case of diabetes in the NOD mouse, it has been shown that the induction of Th2 responses to specific GAD65 peptides can lead to a halt in the destructive insulitis and the prevention of diabetes. In this proposal are: 1) To construct GAD65 cDNA plasmids which will allow expression of GAD65 in the cytoplasm, as a secreted protein and as a transmembrane protein; 2) To characterize the immune response initiated following immunization of NOD and non diabetes-prone strains with the three GAD65 constructs; 3) To determine whether DNA immunization GAD65 constructs prevent the initiation of diabetes and insulitis.