Deaf1 isoforms control changes in PTA expression in the NOD PLN during T1D pathog

描述（由申请人提供）：1型糖尿病（T1D）由自身反应性T细胞对胰腺β细胞的自身免疫破坏发展而来。在胸腺中逃避消除的潜在自反应性T细胞随后可能在周围被删除或耐受。最近，有报道称，淋巴结基质细胞通过诱导各种外周组织抗原（PTAs）的异位表达，并以诱导耐受（缺失或调节）的方式呈递给T细胞，从而控制自身耐受。目前，T1D中PTA在淋巴结表达的转录调控和外周耐受的失调尚不清楚。我们的实验室最近发现Deaf1是外周淋巴结中PTA基因表达的转录调节因子。我们的研究结果表明，在12周龄的非肥胖糖尿病（NOD）小鼠的胰腺淋巴结（PLN）中，包括胰岛素和其他β细胞抗原在内的PTAs基因表达下调。这种表达的变化与自身反应性T细胞破坏β细胞的开始时间一致。初步研究表明，Deaf1调控PLN中多个PTA基因的表达。到目前为止，在12周龄NOD小鼠的PLN中发现了两种形式的Deaf1，一种全长功能形式和一种非功能变体（Deaf1- var）。在这个年龄段，与NOD相比，NOD的PLN中Deaf1- var的表达明显升高，而Deaf1的表达明显降低。B10老鼠。Deaf1- var在细胞质中表达，自身的转录活性很小，通过与Deaf1异源二聚化并将其保留在细胞质中来抑制其转录活性。值得注意的是，我们在T1D患者的PLN中发现了一个等效的选择性剪接的Deaf1亚型。这种人类变异在T1D患者的PLN中比对照组多20倍，其表现与小鼠Deaf1-VAR相似。该变体的高表达也与T1D患者PLN中缺乏胰岛素基因表达相关。基于这些数据，我们假设Deaf1控制PLN中某些胰腺β细胞抗原（包括胰岛素）的表达并维持其外周耐受性，并且Deaf1剪接的改变有助于T1D的发病。为了验证这一假设，提出的研究解决以下问题：1)PLN中的哪种细胞类型表达Deaf1， Deaf1- var和PTAs？2) Deaf1如何在分子水平上介导PTA基因转录？3) PLN的炎症或各种“遗传因素”是否调节了Deaf1的剪接？4)敲除pta表达细胞中的Deaf1表达是否会加剧NOD疾病，如果是这样，这是由于缺乏自身反应性T细胞缺失还是缺乏调节性T细胞发育？最后，自身免疫性葡萄膜视网膜炎的小鼠模型或人类APS 1患者是否存在与疾病相关的Deaf1变化？这些研究的结果将提供对Deaf1及其异构体如何维持外周耐受性的全面理解，从而了解Deaf1剪接的改变或Deaf1的突变如何促进自身免疫性疾病的发展。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by self-reactive T cells. Potentially self-reactive T cells escaping elimination in the thymus may subsequently be deleted or tolerized in the periphery. Recently, stromal cells of lymph nodes were reported to control self-tolerance by inducing the ectopic expression of various peripheral tissue antigens (PTAs) and presenting them to T cells in a manner that induces tolerance (deletion or regulation). Currently, both the transcriptional control of PTA expression in lymph nodes and the dysregulation of peripheral tolerance in T1D are unclear. Our laboratory recently identified Deaf1 as a transcriptional regulator of PTA gene expression in peripheral lymph nodes. Our findings show that the gene expression of PTAs, including insulin and other beta cell antigens, are down- regulated in the pancreatic lymph nodes (PLN) of non-obese diabetic (NOD) mice at 12 weeks of age. This change in expression coincides in time with the onset of beta cell destruction by autoreactive T cells. Preliminary studies show that Deaf1 regulates the expression of multiple PTA genes in the PLN. Thus far, two forms of Deaf1, a full-length functional form and a non-functional variant (Deaf1-VAR) have been identified in the PLN of 12 week-old NOD mice. At this age, Deaf1-VAR expression is significantly higher and Deaf1 expression is significantly lower in the PLN of NOD compared to NOD.B10 mice. Deaf1-VAR is expressed in the cytoplasm, has little transcriptional activity of its own, and inhibits the transcriptional activity of Deaf1 by heterodimerizing with it and retaining it in the cytoplasm. Remarkably, we identified an equivalent alternatively spliced Deaf1 isoform in the PLN of T1D patients. This human variant which was ~20-fold more abundant in the PLN of T1D patients than controls, behaves like the mouse Deaf1-VAR. The high expression of this variant also correlated with the absence of insulin gene expression in the PLN of T1D patients. Based on these data, it is hypothesized that Deaf1 controls the expression of and maintains peripheral tolerance to certain pancreatic beta-cell antigens, including insulin, in the PLN, and that a change in Deaf1 splicing contributes to the pathogenesis of T1D. To examine this hypothesis, proposed studies address the following: 1) What cell type in the PLN expresses Deaf1, Deaf1-VAR and PTAs? 2) How does Deaf1 mediate PTA gene transcription on a molecular level? 3) Does inflammation of the PLN or various "genetic factors" regulate the splicing of Deaf1? 4) Will knock-out of Deaf1 expression in PTA-expressing cells exacerbate NOD disease, and if so, is this due to a lack of autoreactive T cell deletion or a lack of regulatory T cell development? Finally, do either mouse models of autoimmune uveoretinitis or human APS 1 patients have changes in Deaf1 that can be correlated with disease? The results of these studies will provide an overall understanding of how Deaf1 and its isoforms maintain peripheral tolerance and thus, how a change in the splicing of Deaf1 or a mutation in DEAF1 may contribute to the development of autoimmune disease. PUBLIC HEALTH RELEVANCE: Type 1 diabetes can result from a break in peripheral tolerance that is, in part, mediated by the expression of peripheral tissue antigens (PTAs) on lymph node cells. We have identified a transcriptional regulator, Deaf1, that regulates the expression of PTAs in the pancreatic lymph node (PLN), and have data to suggest that the alternative splicing of this gene leads to reduced PTA expression in the non-obese diabetic mouse model and in human type 1 diabetic patients. The studies proposed here will examine the role of Deaf1 in maintaining peripheral tolerance and help us understand how a change in Deaf1 splicing may contribute to the pathogenesis of T1D.