The Effects of Low-Dose IL-2 Therapy on Beta Cell Dysfunction in Type 1 Diabetes

项目摘要 1型糖尿病(T1D)是一种破坏性的自身免疫性疾病，目前影响着超过125万美国人 无法治愈。针对T1D患者自身免疫的治疗已从广泛的免疫抑制转向 环孢素等药物，现在的重点是增强免疫耐受性。小剂量白介素2 已经在临床前和临床试验中表明可以诱导调节性T细胞(Tregs)的增殖 促进免疫耐受，防止自身免疫破坏产生胰岛素的β细胞。最近，它 临床前模型显示，IL-2/CD25融合蛋白可诱导Tregs和Tregs更大的增殖 具有比传统重组IL-2更长的半衰期。对IL-2及其类似物的研究主要集中在 免疫耐受的机制以及它如何防止β细胞团的损失。近期临床发现 在确诊前至少五年证明胰岛素分泌受损，而β细胞团仍然存在 这表明，除了β细胞质量的丧失外，β细胞功能障碍对 T1D的发病机制。目前尚不清楚IL-2/CD25治疗如何影响β细胞功能障碍。这 代表着必须解决的知识方面的严重差距。初步数据显示，IL-2/CD25诱导 局部免疫渗入的变化，增加Tregs比例，降低Tregs比例 胰岛的效应性T细胞(T细胞)。体外研究表明，促炎细胞因子通过以下途径释放 通过对内质网(ER)的应激，渗入Teffs并导致β细胞功能障碍。因此，我 假设IL-2/CD25通过阻止细胞因子介导的β细胞而延缓T1D进展 功能障碍，而不仅仅是通过避免细胞死亡。我计划在以下具体目标中检验这一假设：1) 确定IL-2/CD25治疗如何保护胰岛功能和葡萄糖稳态，2)确定IL-2/CD25如何 2/CD25处理可维持胰岛组织的动态平衡。在第一个目标下，我将使用一种新的方法来使用生活 胰腺切片分析IL-2/CD25后胰岛素分泌和细胞内钙动态的变化 治疗。在第二个目标下，我将确定恢复胰岛稳态所涉及的具体机制 在IL-2/CD25治疗后，通过免疫染色和单细胞RNA测序寻找应激标志物， 增殖和去分化。我提议的研究具有重要意义，因为它将产生关于 IL-2/CD25介导的局部免疫渗透变化对胰岛生物学的影响。这一贡献是巨大的 因为它将提供基本的知识，将完善和修改关于免疫疗法的模型 T1D。这项研究支持国家卫生研究院的战略计划，包括推进 通过投资基础科学和开发治疗/治疗方法获得生物医学研究的机会 疾病。

Project Summary Type 1 diabetes (T1D) is a devastating autoimmune disease affecting over 1.25 million Americans that currently has no cure. Therapies which target autoimmunity in T1D have shifted away from broadly immunosuppressive agents such as cyclosporine, and now focus on enhancing immune tolerance. Low-doses of interleukin-2 (IL-2) have been shown in pre-clinical and clinical trials to induce proliferation of regulatory Tcells (Tregs) which promote immune tolerance and prevent autoimmune destruction of the insulin producing beta cell. Recently, it has been shown in preclinical models that an IL-2/CD25 fusion protein induces greater proliferation of Tregs and has a longer half-life than traditional recombinant IL-2. Studies on IL-2 and its analogues have focused on mechanisms of immune tolerance and how it prevents loss of beta cell mass. Recent clinical findings demonstrate that insulin secretion is impaired at least five years before diagnosis, while beta cell mass is still preserved, suggesting that beta cell dysfunction, in addition to loss of beta cell mass, is critically important to the pathogenesis of T1D. It is currently unknown how IL-2/CD25 therapy affects beta cell dysfunction. This represents a critical gap in knowledge that must be addressed. Preliminary data shows that IL-2/CD25 induces changes in the local immune infiltrate, increasing the proportion of Tregs and decreasing the proportion of effector T cells (Teffs) at the islets. In vitro studies have shown that pro-inflammatory cytokines are released by infiltrating Teffs and cause beta cell dysfunction via stress on the endoplasmic reticulum (ER). Therefore, I hypothesize that IL-2/CD25 works to delay T1D progression by preventing cytokine-mediated beta cell dysfunction, not just by averting cell death. I plan to test this hypothesis in the following specific aims: 1) Determine how IL-2/CD25 treatment preserves islet function and glucose homeostasis, 2) Determine how IL- 2/CD25 treatment preserves islet tissue homeostasis. Under the first aim, I will use a novel approach using living pancreas slices to analyze changes in insulin secretion and intracellular Ca2+ dynamics after IL-2/CD25 treatment. Under the second aim, I will identify the specific mechanisms involved in restoring islet homeostasis after IL-2/CD25 therapy by using immunostaining and single-cell RNA sequencing for markers of stress, proliferation, and dedifferentiation. My proposed study is significant because it will yield new information about how changes in local immune infiltration mediated by IL-2/CD25 affect islet biology. This contribution is significant because it will provide fundamental knowledge that will complete and revise models about immunotherapies for T1D. This study supports the strategic plans of the National Institutes of Health which include advancing opportunities in biomedical research by investing in fundamental science and developing treatments/cures for disease.