Mechanisms of Fas Ligand Control of Insulitis in Autoimmune Diabetes

描述（由申请人提供）：尽管胰岛素输送得到改善，但在许多1型糖尿病（T1D）患者中，保持严格控制葡萄糖稳态仍然是一个挑战，导致严重的低血糖和高血糖发作以及严重的长期并发症。因此，开发一种针对这种疾病的免疫疗法仍然是一个主要目标。然而，要实现这一目标，需要对糖尿病发生过程的各个方面有深入的了解，这通常被认为是由致病和调节机制之间的不平衡引发的，这种不平衡允许致糖尿病T细胞浸润胰岛并破坏产生胰岛素的细胞。因此，在易感个体和动物模型中，识别和理解各种分子和细胞类型在免疫致病途径中的作用，对于开发有效的免疫治疗非常重要。当TNF家族的凋亡诱导成员Fas配体（FasL）在遗传或药理学上失活时，该研究探讨了强有力地控制ss细胞特异性自身反应性T细胞的机制。此前，由于缺乏合适的模型和有效的FasL阻断单抗，严重阻碍了此类研究。在这项应用中，我们将使用FasL单倍缺乏的NOD小鼠（NOD- gold /+小鼠）和FasL中和单抗（MFL4克隆）来研究使用MFL4单抗阻断FasL的潜在机制和治疗意义。nod - gold /+小鼠完全免受T1D的侵害，免疫功能正常，免疫稳态正常。此外，MFL4 mAb在不改变免疫稳态的情况下保护NOD-wt小鼠免受糖尿病的侵害，更重要的是，我们的初步数据显示，它在逆转新发病例的高血糖方面具有良好的功效。基于我们使用这些模型系统产生的初步数据，我们假设产生il -10的调节性B细胞亚群抑制糖尿病自身反应性T细胞受到FasL的负调控。在NOD-wt小鼠中，fasl介导的凋亡消除了产生IL- 10的调节性B细胞，从而消除了对自身反应性t细胞的抑制（Aim 1）。我们假设MFL4单抗可用于逆转新发糖尿病（在Aim 2中进行了测试）。在NOD小鼠中，FasL （gold /+）的单倍不足或FasL的单抗阻断可阻止产生il -10的B细胞的消除，从而控制致糖尿病T细胞和抑制胰岛素（Aim试验）

DESCRIPTION (provided by applicant): Despite improvement in insulin delivery, maintaining tight control of glucose homeostasis continues to be a challenge that results in bouts of severe hypo and hyperglycemia and serious long-term complications in many type 1 diabetes (T1D) patients. Therefore, developing an immunotherapy for the disease remains a major goal. Reaching this goal, however, requires deep knowledge of all facets of the diabetogenic process - which is generally believed to be initiated by an imbalance between pathogenic and regulatory mechanisms that allows diabetogenic T cells to infiltrate pancreatic islets and destroy insulin-producing ss-cells. Therefore, identifying and understanding roles of various molecules and cell types that tip the balance towards the immunopathogenic pathways in susceptible individuals and animal models is important for developing effective immunotherapy. This proposal investigates mechanisms that powerfully control ss-cell specific autoreactive T cells when Fas ligand (FasL), an apoptosis-inducing member of TNF family, is genetically or pharmacologically inactivated. Previously, the lack of appropriate models and efficacious FasL blocking mAb has severely hampered such investigation. In this application, we will use NOD mice that are haploinsufficient for FasL (NOD-gld/+ mouse) and a FasL- neutralizing mAb (MFL4 clone) to investigate the underlying mechanisms and therapeutic significance of FasL blockade using the MFL4 mAb. NOD-gld/+ mice are completely protected from T1D, immunocompetent, and have normal immune homeostasis. In addition, MFL4 mAb protects NOD-wt mice from diabetes without altering immune homeostasis and, more importantly, our preliminary data show it has promising efficacy in reversing hyperglycemia in new-onset cases. Based on our preliminary data generated using these model systems, we hypothesize that an IL-10-producing regulatory B cell subset that suppresses diabetogenic autoreactive T cells are negatively regulated by FasL. In NOD-wt mice, FasL-mediated apoptosis eliminates IL- 10-producing regulatory B cells thereby removing the brakes on autoreactive T-cells (tested Aim 1). We hypothesize that the MFL4 mAb can be used to reverse new-onset diabetes (tested in Aim 2). In NOD mice, haploinsufficiency for FasL (gld/+) or mAb blockade of FasL prevents IL-10-producing B cell elimination, leading to control of diabetogenic T cells and suppression of insulitis (tested in Aim 3). In this revised application, we will also assess relevance of our preclinical data to the human disease in samples from newly diagnosed patients at Hopkins and tissues provided by the JDRF sponsored nPOD project (Aim 3). Because the role of FasL in normal immune response and ss-cell death are dispensable, understanding how FasL modulates the diabetogenic process can lead to new mechanistic insights into the disease pathogenesis that could have important therapeutic implications.