Alterations of leukocyte integrin signaling leading to diabetes and autoimmunity

项目摘要/摘要 对1型糖尿病(T1D)遗传基础的研究从罕见的检查中受益匪浅 可能患有该疾病单基因形式的个人。与GWAS相结合，在 免疫调节基因已被定义为导致T1D遗传风险的基因。使用完整的外显子 在单基因糖尿病登记处登记的患有T1D的个体的测序，我们确定了一名患有T1D的个体 SKAP2基因以及其他几个具有潜在致病性的T1D患者的功能获得突变 其他白细胞整合素信号基因的变异。这些患者往往有许多自身免疫性疾病 除T1D外的其他表现，表明免疫耐受的关键途径存在缺陷。多个GWA 研究发现SKAP2基因多态与T1D之间存在很强的遗传关联(频率为 ~20%)，但SKAP2基因改变导致自身免疫性T1D的机制尚不清楚。 SKAP2主要在髓系细胞中表达，在髓系细胞中作为整合素信号转导的适配蛋白发挥作用 将细胞表面整合素连接到WASP和发生在白细胞后的肌动蛋白重排的途径 粘附力。我们患者的SKAP2 G153R突变导致SKAP2与WASP的结构性关联 导致患者或工程巨噬细胞培养的巨噬细胞出现高黏附表型 以包含Skap G153R替换。为了了解白细胞整合素信号的激活如何 对T1D的贡献，我们已经产生了包含小鼠Skap2中G153R替代的敲入(Ki)小鼠， 关于诺德的遗传背景。雌性NOD.SKAP2KI小鼠的发病率更高，发病更早 雄性NOD.SKAP2也发生T1D(发病率~50%)，而雄性NOD.WT 不要出现坦率的高血糖。对这些小鼠的初步分析揭示了持续炎症的证据 在生命的早期，伴随着广泛的自身反应抗体的发展。来源的树突状细胞 NOD.SKAP2Ki小鼠对胰岛特异性转基因T细胞的抗原提呈活性增强，而 这些小鼠的中性粒细胞显示整合素信号增强的证据。这些观察结果表明 NOD.SKAP2Ki小鼠适当地模拟了在我们患者中观察到的自身免疫性T1D疾病。这个 项目建议完成对这些小鼠的分析，假设增加细胞黏附在 树突状细胞导致DC-T细胞相互作用时间延长，这推动了对自身反应性T细胞克隆的选择 导致与广谱自身免疫相关的T1D的发生。我们将在以下方面测试这一假设 各种过继细胞转移实验，通过条件敲入小鼠的生成和通过对 炎症胰岛中DC-T细胞的相互作用。将对其他候选白细胞进行类似的研究 在单基因T1D注册表中发现的整合素信号突变。这项研究将讨论是否 白细胞整合素信号的失调可能是T1D的一个未知的遗传风险因素。 为这些患者提供了潜在的替代治疗方法。

Project Summary/Abstract The study of the genetic basis for type 1 diabetes (T1D) has benefited tremendously from examination of rare individuals with likely monogenic forms of the disease. Combined with GWAS, a number of polymorphisms in immune regulatory genes have been defined that contribute to genetic risk for T1D. Using whole exome sequencing of individuals with T1D enrolled in a monogenic diabetes registry, we identified an individual with a gain-of-function mutation in the SKAP2 gene, as well as several other T1D patients with potentially pathogenic variants in other leukocyte integrin signaling genes. These patients tend to have a number of autoimmune manifestations in addition to T1D, indicating defects in critical pathways of immune tolerance. Multiple GWAS studies have identified a strong genetic linkage between SKAP2 polymorphisms and T1D (at a frequency of ~20%), however the mechanisms by which alteration of SKAP2 could lead to autoimmune T1D are unknown. SKAP2 is expressed primarily in myeloid cells, where it functions as an adapter protein in the integrin signaling pathway, linking cell surface integrins to WASP and actin rearrangements that occur following leukocyte adhesion. The SKAP2 G153R mutation in our patient resulted in constitutive association of SKAP2 with WASP leading to a hyperadhesive phenotype in macrophages cultured from the patient or macrophages engineered to contain the SKAP G153R substitution. To understand how activation of leukocyte integrin signaling may contribute to T1D, we have generated knock-in (KI) mice containing the G153R substitution in murine Skap2, on the NOD genetic background. Female NOD.SKAP2 KI mice have a higher incidence and earlier onset of T1D than do NOD.WT animals; male NOD.SKAP2 also develop T1D (incidence ~50%) while male NOD.WT do not develop frank hyperglycemia. Initial analysis of these mice reveals evidence of ongoing inflammation early in life with development of a broad spectrum of auto-reactive antibodies. Dendritic cells from NOD.SKAP2 KI mice have increased antigen presenting activity to islet-specific transgenic T-cells while neutrophils from these mice show evidence of increased integrin signaling. These observations demonstrate that the NOD.SKAP2 KI mice appropriately model the autoimmune T1D disease observed in our patient. The project proposes to complete the analysis of these mice, under the hypothesis that increased cell adhesion in dendritic cells leads to prolonged DC-T cell interactions, which drives selection of auto-reactive T-cell clones leading to development of T1D, associated with broad spectrum autoimmunity. We will test this hypothesis in a variety of adoptive cell transfer experiments, by generation of conditional knock-in mice and by imaging of DC-T cell interactions in the inflamed islets. Similar studies will be performed for other candidate leukocyte integrin signaling mutations identified in the monogenic T1D registry. This study will address whether dysregulation of leukocyte integrin signaling may constitute an unrecognized genetic risk factor for T1D, suggesting potential alterative therapeutic approaches for these patients.