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（T1 D）由自身反应性T细胞对胰腺β细胞的自身免疫性破坏发展而来。在胸腺中逃避消除的潜在自身反应性T细胞随后可能在外周中被删除或耐受。最近，据报道，淋巴结的基质细胞通过诱导各种外周组织抗原（PTA）的异位表达并以诱导耐受（缺失或调节）的方式将其呈递给T细胞来控制自身耐受。目前，淋巴结中PTA表达的转录控制和T1 D外周耐受的失调都不清楚。我们的实验室最近确定Deaf 1作为外周淋巴结PTA基因表达的转录调节因子。我们的研究结果显示，在12周龄的非肥胖糖尿病（NOD）小鼠的胰腺淋巴结（PLN）中，PTA（包括胰岛素和其他β细胞抗原）的基因表达下调。这种表达的变化与自身反应性T细胞对β细胞的破坏在时间上一致。初步研究表明，Deaf 1调节PLN中多个PTA基因的表达。到目前为止，已经在12周龄NOD小鼠的PLN中鉴定了两种形式的Deaf 1，全长功能形式和非功能变体（Deaf 1-VAR）。在这个年龄，与NOD.B10小鼠相比，NOD的PLN中Deaf 1-VAR表达显著更高，Deaf 1表达显著更低。Deaf 1-VAR在细胞质中表达，自身几乎没有转录活性，并且通过与Deaf 1异源二聚化并将其保留在细胞质中来抑制Deaf 1的转录活性。值得注意的是，我们在T1 D患者的PLN中鉴定了一种等效的选择性剪接的Deaf 1亚型。这种人类变体在T1 D患者的PLN中的丰度比对照组高约20倍，其行为类似于小鼠Deaf 1-VAR。该变体的高表达也与T1 D患者PLN中胰岛素基因表达的缺乏相关。基于这些数据，假设Deaf 1控制PLN中某些胰腺β细胞抗原（包括胰岛素）的表达并维持外周耐受性，并且Deaf 1剪接的变化有助于T1 D的发病机制。为了检验这一假设，提出的研究解决了以下问题：1）PLN中的哪种细胞类型表达Deaf 1，Deaf 1-VAR和PTA？2)Deaf 1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Deaf 1的剪接？4)敲除PTA表达细胞中Deaf 1的表达是否会加重NOD疾病，如果是的话，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患者是否具有与疾病相关的Deaf 1变化？这些研究的结果将提供一个全面的了解如何Deaf 1及其亚型维持外周耐受性，因此，如何在Deaf 1的剪接或突变的Deaf 1的变化可能有助于自身免疫性疾病的发展。 公共卫生相关性：1型糖尿病可由外周耐受性的破坏引起，外周耐受性的破坏部分地由淋巴结细胞上的外周组织抗原（PTA）的表达介导。我们已经确定了一个转录调节因子，Deaf 1，调节PTA在胰腺淋巴结（PLN）的表达，并有数据表明，该基因的选择性剪接导致减少PTA在非肥胖糖尿病小鼠模型和人类1型糖尿病患者的表达。本文提出的研究将研究Deaf 1在维持外周耐受性中的作用，并帮助我们了解Deaf 1剪接的变化如何有助于T1 D的发病机制。

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.