Genetic Regulation of Human Beta Cell Destruction

 描述（由申请人提供）：1型糖尿病（T1 D）的发展依赖于免疫系统细胞之间复杂的相互关系[例如，DC、CD 8 + T细胞]和赋予对疾病的易感性或抗性的基因，这些基因是产生胰岛素的胰腺β细胞的自身免疫破坏的基础。虽然有大量证据支持这一观点（我们自己也相信这是真的），但促进自身免疫性β细胞破坏的确切机制仍不清楚。此外，每个方面的相对贡献（即，细胞、基因）在这一过程中发挥的作用在很大程度上仍然是未知的。T1 D相关易感等位基因的机制研究因多基因遗传而复杂化，因此没有两个个体是真正相似的。因此，研究受到人群缺乏力量的严重阻碍，并且无法分离出一个人的功能影响。 一种特殊的细胞类型。在这里，我们提出了一个解决方案，专注于个别等位基因使用创新的等基因模式，利用尖端技术的优势。我们已经创建了一个实验平台来研究特定的遗传风险变体如何加速免疫失调，导致细胞毒性CD 8 + T淋巴细胞（CTL）活化和β细胞破坏。我们假设PTPN 22中的遗传调节缺陷促进：i）免疫原性DC，ii）TH 1应答，iii）胰腺血管炎症和CTL归巢，以及iv）针对β细胞的致病性CTL活性，以及减少的活化诱导的CTL死亡：这些租户中的每一个形成了该资助的目的。此外，我们认为，当免疫细胞和内皮细胞对刺激炎症的内源性或外源性因子的激活过度敏感时，缺陷达到了全部潜力，从而将T1 D中的环境和免疫遗传学联系起来。 在这里，我们将利用一种新的实验管道，其中PTPN 22 R（T1 D抗性）、PTPN 22 W（T1 D易感）或PTPN 22缺失（PTPN 22-/-）等位基因由从诱导多能干细胞[iPSC]工程化的同基因人类免疫和内皮细胞携带。iPSC系统允许精确控制T1 D疾病等位基因，其中易感等位基因可以被抗性等位基因取代（反之亦然），提供恒定的遗传背景，在此基础上可以研究单一风险同种异型的影响，而不会使上位效应复杂化， 以类似于转基因小鼠研究的方式。本文提出的这个系统将提供前所未有的能力来询问在等基因条件下的分子和细胞相互作用，以提供对PTPN 22等位基因如何调节T1 D发病机制的各个步骤以及这些步骤如何相互关联以引起T1 D发作的机制理解。重要的是，这项研究也将为未来使用这种创新策略研究单个或多个T1 D易感基因奠定基础。

 DESCRIPTION (provided by applicant): The development of Type 1 Diabetes (T1D) relies on complex interrelationships between cells of the immune system [e.g., DC, CD8+ T cells] and genes imparting susceptibility or resistance to the disease that underlie the autoimmune destruction of insulin producing pancreatic β cells. While a broad body of evidence certainly exists to support this notion (and we ourselves believe it true), the exact mechanism by which autoimmune β cell destruction is facilitated remains unclear. In addition, the relative contributions of each facet (i.e., cells, genes) play in the process remain, to a large extent, unknown. Mechanistic studies of T1D-associated susceptibility alleles are complicated by polygenic inheritance such that no two individuals are truly alike. Hence, studies are severely hampered by a lack of power in populations, and the inability to isolate the functional impact of a variant to a specific cell type. Here we present a solution that focuses on individual alleles usin an innovative isogenic mode that takes advantage of cutting edge technologies. We have created an experimental platform to study how specific genetic risk variants precipitate immune dysregulation leading to cytotoxic CD8+ T lymphocyte (CTL) activation and β cell destruction. We hypothesize that genetically regulated defects in PTPN22 promote; i) immunogenic DC, ii) TH1 responses, iii) pancreatic vascular inflammation and CTL homing, and iv) pathogenic CTL activity towards β cells coupled with reduced activation induced CTL death: each of these tenants form an aim of this grant. Further, we posit that defects reach full potential when immune cells and endothelial cells are excessively sensitive to activation by endogenous or exogenous factors that stimulate inflammation, thus linking environment and immunogenetics in T1D. Here we will utilize a novel experimental pipeline where PTPN22R (T1D resistant), PTPN22W (T1D susceptible) or PTPN22 deletion (PTPN22-/-) alleles are carried by isogenic human immune and endothelial cells engineered from induced pluripotent stem cells [iPSC]. The iPSC system allows exquisite control of T1D disease alleles, where the susceptible allele can be replaced by the resistant allele (and vice versa) providing a constant genetic background upon which effects of a single risk allotype can be studied without complicating epistatic effects, in a manner analogous to studies in genetically modified mice. This system proposed here will provide an unprecedented capacity to interrogate molecular and cellular interactions under isogenic conditions to provide mechanistic understanding of how PTPN22 alleles regulate individual steps of T1D pathogenesis and how those steps interrelate to bring upon T1D onset. Importantly, this study will also lay the groundwork for future investigations of single or multipl T1D susceptibility genes using this innovative strategy.