Immunological Mechanisms Underlying T1dm Pathogenesis

1型糖尿病（T1 DM）是一种自身免疫性疾病，由T细胞介导的胰腺中产生胰岛素的β细胞破坏引起。目前的治疗包括通过注射替代胰岛素、频繁的血糖监测和饮食/锻炼纪律，可以预防因激素不足而死亡，但不能治愈，也不能完全预防长期并发症，包括神经损伤和对大小血管的血管损伤。血管损伤反过来又会导致各种器官的损伤，包括心脏、大脑、肾脏和眼睛。与没有T1 DM的个体相比，预期寿命估计缩短了三分之一。这项工作于20世纪90年代中期在海军医学研究所开始，并在德国的乌尔姆大学进一步发展，随后在过去的2年中在NIH进行，利用大鼠胰岛素启动子（RIP）-CD 80转基因小鼠（RIP-CD 80小鼠）表征了独特的自身免疫性糖尿病鼠模型。这些动物基于其共刺激分子CD 80的β细胞特异性表达而易受免疫介导的β细胞破坏。RIP-CD 80小鼠很少发生自发性糖尿病，（发病率6.7%，相比之下野生型小鼠无发病率），我们已经观察到并初步确定了这些小鼠在用β细胞自身抗原免疫后表现出的进行性胰岛浸润和最终胰岛素依赖性糖尿病（IDDM）的高度易感性（即由这些小鼠的胰腺β细胞特异性表达的蛋白质分子）。例如，与非转基因同窝出生的动物不同，这些动物在用胰岛素前体（强烈怀疑参与人T1 DM发病机制的β细胞自身抗原）免疫后一致地发生胰岛细胞破坏和胰岛素依赖性糖尿病（IDDM）。我们已经表明，至少有一个相关的自身抗原性CTL表位（H-2b）必须存在于成熟的胰岛素分子，这与目前的基于算法的表位预测模型。此外，到目前为止，我们的模型比非肥胖糖尿病（NOD）小鼠（一种最广泛用于研究自身免疫性糖尿病的模型）更能预测对治疗药物的免疫反应。我们使用RIP-CD 80小鼠模型的目的可以概括如下：（1）鉴定作为发展自身免疫应答的初始靶标的重要自身抗原（Ag）和CTL表位。我们将探索几种途径与候选自身抗原免疫，包括肽免疫，DNA疫苗，逆转录病毒转导的树突状细胞（DC），他们的能力，引起MHC I类限制性T细胞反应，胰岛炎，糖尿病。(2)研究过继转移模型中的β细胞特异性T细胞应答，其中原发性自身反应性T细胞致敏将在体外或通过体内免疫发生，如上文（1）中所提出的。这些研究将集中在发展中的β细胞破坏性免疫反应的机制，并将试图确定新的治疗方法来干扰正在进行的免疫反应。(3)继续探索我们的初步数据，表明非专业抗原呈递细胞（APC），如胰腺β细胞或成纤维细胞，可以在体内引发幼稚前体CTL，并刺激慢性自身免疫反应。由非专职APC诱导的免疫应答与由专职APC（即源自造血谱系的那些，如树突状细胞）产生的免疫应答形成对比，因为专职APC诱导均匀和暴发性胰岛破坏和糖尿病。重要的是要认识到，在人类T1 DM中观察到的胰岛破坏通常不是暴发性的，而是与我们观察到的慢性过程更一致。(4)在小鼠模型中测试正在考虑用于新发T1 DM患者试验的潜在免疫抑制剂预防疾病的能力。RIP-CD 80模型中的糖尿病诱导可以偏向于几乎完全依赖于CD 8 + T细胞的糖尿病诱导（抗原脉冲的成纤维细胞免疫）或使用不同的免疫策略（例如DNA疫苗接种）偏向于依赖于CD 4+和CD 8 + T细胞的糖尿病诱导。(5)我们已经将RIP-CD 80模型调整为能够测试被认为代表β细胞祖细胞的细胞产生功能性胰岛素产生细胞的能力的体内系统。

Type 1 diabetes mellitus (T1DM) is an autoimmune disease resulting from the T cell mediated destruction of insulin-producing beta cells located in the pancreas. Current treatment, which includes insulin replacement by injection, frequent blood glucose monitoring, and dietary/exercise discipline, can prevent death from hormonal insufficiency, but is not curative and does not completely prevent the long-term complications including nerve damage, and vascular damage to both large and small blood vessels. The blood vessel damage in turn results in injury to various organs, including the heart, brain, kidneys, and eyes. Life expectancy is shortened by an estimated one-third compared to individuals without T1DM. This work, initiated in the mid 1990's at the Naval Medical Research Institute and further developed at the University of Ulm in Germany and subsequently for the past 2 years at the NIH has characterized a unique murine model of autoimmune diabetes using rat-insulin promoter (RIP)-CD80 transgenic mice (RIP-CD80 mice). These animals are predisposed to immune mediated beta cell destruction based on their beta cell-specific expression of the costimulatory molecule CD80. While RIP-CD80 mice rarely develop spontaneous diabetes (incidence 6.7%, compared to none of wild type mice), we've observed and have initially characterized the profound susceptibility these mice display to develop progressive islet infiltration and eventually insulin dependent diabetes mellitus (IDDM) following immunization with beta cell autoantigens (i.e. protein molecules that are expressed specifically by the pancreatic beta cells of these mice). For instance, these animals, unlike non-transgenic littermates, uniformly develop islet cell destruction and insulin dependent diabetes mellitus (IDDM) upon immunization with an insulin precursor, a beta cell autoantigen strongly suspected to be involved in the pathogenesis of human T1DM. We have shown that at least one relevant autoantigenic CTL epitope (H-2b) must be present in the mature insulin molecule, which contrasts with current algorhythm-based epitope prediction models. Moreover, our model has so far been more predictive of immune responses to therapeutic agents than the non-obese diabetic (NOD) mouse, a model most widely used to study autoimmune diabetes. Our objectives using the RIP-CD80 mouse model can be summarized as follows: (1) Identify important self-antigens (Ag) and CTL-epitopes serving as the initial targets for the developing autoimmune response. We will explore several routes to immunize with candidate auto-antigens, including peptide immunizations, DNA-vaccines, and retrovirally transduced dendritic cells (DC), for their ability to elicit MHC class I restricted T cell responses, insulitis, and diabetes. (2) Study beta cell specific T cell responses in an adoptive transfer model where the primary autoreactive T cell sensitization will take place either in vitro, or by immunizing in vivo, as proposed above in (1). These studies will focus on the mechanisms of the developing beta cell destructive immune responses, and will attempt to identify novel therapeutic approaches to interfere with the ongoing immune responses. (3) Continue to explore our preliminary data suggesting that non-professional antigen-presenting cells (APC), such pancreatic beta cells or fibroblastoid cells, can prime naive precursor CTL in vivo, and stimulate a chronic autoimmune response. The immune response induced by the non-professional APCs contrasts with that produced by professional APC (i.e. those derived from the hematopoetic lineage, such as dendritic cells) in that professional APCs induce a uniform and fulminant islet destruction and diabetes. It is important to recognized that the islet destruction observed in human T1DM is typically not fulminant but is more consistent with the chronic process we've observed. (4) Test potential immunotherapeutic agents being considered for trials in patients with new onset T1DM for their ability to prevent disease in the mouse model. Diabetes induction in the RIP-CD80 model can be biased toward one dependent almost exclusively on CD8+ T cells (antigen pulsed fibroblast immunization) or using different immunization strategies (e.g. DNA vaccination) toward one dependent upon both CD4+ and CD8+ T cells. (5) We have adapted the RIP-CD80 model as an in vivo system capable of testing cells felt to represent progenitors for beta cells for their capacity to generate functional insulin producing cells.