Parameters T-Cell Receptor avidity during thymic selection in autoimmune diabetes

参数 自身免疫性糖尿病胸腺选择过程中 T 细胞受体亲合力

• 批准号: 8900917
• 负责人: Matthew Bettini
• 金额: $ 10.73万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8900917
• 关键词: Address; Affinity; Agonist; Antibodies; Antigens; Autoimmune Diabetes; Autoimmune Diseases; Autoimmune Process; Autoimmune Responses; Avidity; B-insulin; Beta Cell; Biological Markers; Bypass; Cells; Cleaved cell; Development; Diabetes prevention; Dose; Frequencies; Gene Transfer; Generations; Health; Inbred NOD Mice; Injection of therapeutic agent; Insulin; Insulin-Dependent Diabetes Mellitus; Islets of Langerhans; Knowledge; Lead; Link; Lymphocyte; Modeling; Mus; Mutate; Non obese; Peptide antibodies; Peptides; Peripheral; Receptor Signaling; Reporter; Retroviral Vector; Role; Signal Pathway; Signal Transduction; Stem cells; T Cell Receptor Signaling Pathway; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Testing; Thymocyte Development; Thymus Gland; Transgenic Mice; Transgenic Model; autoreactive T cell; base; central tolerance; diabetic; early onset; human disease; insight; islet; mutant; novel; novel strategies; peripheral tolerance; pressure; promoter; retroviral-mediated; thymocyte; vector

DESCRIPTION (provided by applicant): Type I Diabetes (T1D) or juvenile diabetes, is an early onset autoimmune disease that culminates in the targeting and destruction of β cells found within the pancreatic islets of Langerhans. Little is known about how autoreactive lymphocytes escape negative selection in the thymus, resulting in the breakdown in central tolerance. Understanding the mechanisms of autoimmune T cell selection and development is crucial for identifying the underlying issues in the breakdown in central tolerance and may be applicable in establishing peripheral tolerance to islet antigen specific autoimmune T cells. Due to the low clonal frequency of autoreactive T cells in the thymus and periphery, studies into underlying mechanisms in self-reactive T cell selection and survival have been challenging. To bypass this issue, T cell receptor (TCR) transgenic models have been utilized, however only a limited number of islet antigen specific TCR transgenic models have been described [1-4]. Therefore, there has been limited study into the mechanisms of autoreactive TCR selection in the thymus, and most of the knowledge we have about the role of negative selection in limiting self-reactivity is based on studies done with model antigens, not with TCRs obtained during an autoimmune response. To address this issue, we have composed a panel of ten insulin specific TCRs with variable reactivity to insulin, the majority of which have been isolated from the islets of spontaneously diabetic non-obese diabetic (NOD) mice. Insulin has been shown to be a major antigen in development of autoimmune diabetes on the NOD background, and antibodies to insulin are used as predictive biomarkers in human disease. In this proposal we plan to study the development of these T cell clones in the thymus through the use of a new approach for the rapid generation of TCR transgenic mice, called retrogenic mice, using retroviral-mediated stem cells gene transfer and novel 'self-cleaving' 2A peptide-linked multicistronic retroviral vectors that express both TCR chains in a single vector [5-14]. This will allow us to quickly generate a large number of TCR retrogenic mice with ten different TCR's to study the fate of a single TCR during positive and negative selection. We will be able to elucidate the role of thymically expressed insulin in central tolerance by utilizing mice with reduced thymic expression of insulin and mice with mutated insulin TCR contact residue. In addition, we want to establish whether insulin reactive TCR's can be deleted in the thymus using DEC-205 insulin and insulin mimetope fusion peptide antibodies. Finally, we propose to express insulin in an inducible manner, under the Aire promoter, in an effort to establish central tolerance. We hypothesize that our unique panel of insulin reactive TCRs can be used to define the threshold for positive and negative selection of autoreactive insulin specific thymocytes.

描述（由申请人提供）：I型糖尿病（T1D）或青少年糖尿病，是一种早发性自身免疫性疾病，最终以朗格汉斯胰岛内发现的β细胞的靶向和破坏为高潮。对于自身反应性淋巴细胞如何逃避胸腺的负选择，从而导致中枢耐受性的破坏，我们知之甚少。了解自身免疫T细胞选择和发育的机制对于确定中枢耐受性破坏的潜在问题至关重要，并且可能适用于建立对胰岛抗原特异性自身免疫T细胞的外周耐受性。由于自身反应性T细胞在胸腺和外周的克隆频率较低，对自身反应性T细胞选择和存活的潜在机制的研究一直具有挑战性。为了绕过这一问题，人们利用了T细胞受体（TCR）转基因模型，但目前只报道了数量有限的胰岛抗原特异性TCR转基因模型[1-4]。因此，对胸腺自身反应性TCR选择机制的研究有限，而且我们对阴性选择在限制自身反应性中的作用的大部分知识都是基于对模型抗原的研究，而不是对自身免疫反应中获得的TCR的研究。为了解决这个问题，我们组成了一个由10个胰岛素特异性tcr组成的小组，这些tcr对胰岛素具有不同的反应性，其中大多数是从自发性糖尿病非肥胖糖尿病（NOD）小鼠的胰岛中分离出来的。胰岛素已被证明是NOD背景下自身免疫性糖尿病发展的主要抗原，胰岛素抗体被用作人类疾病的预测性生物标志物。在本提案中，我们计划通过使用一种新的方法来研究这些T细胞克隆在胸腺中的发展，该方法使用逆转录病毒介导的干细胞基因转移和新的“自裂”2A肽连接的多顺反转录病毒载体，在单个载体中表达两个TCR链，从而快速生成TCR转基因小鼠，称为逆转录小鼠[5-14]。这将使我们能够快速生成大量具有10种不同TCR的TCR逆转录小鼠，以研究单个TCR在阳性和阴性选择中的命运。我们将能够通过使用胸腺胰岛素表达降低的小鼠和TCR接触残留突变的小鼠来阐明胸腺表达胰岛素在中枢耐受中的作用。此外，我们想要确定是否可以使用DEC-205胰岛素和胰岛素米托基融合肽抗体在胸腺中删除胰岛素反应性TCR。最后，我们建议在Aire启动子下以诱导方式表达胰岛素，以努力建立中心耐受性。我们假设我们独特的胰岛素反应性tcr面板可以用来定义阳性和阴性选择自身反应性胰岛素特异性胸腺细胞的阈值。