Production of animal models to define how CTLA-4 impacts to T1D susceptibility

描述(由申请人提供)：制作新的动物模型以确定CTLA-4如何促进T1D易感性。该应用程序解决了挑战领域15：翻译科学和特定挑战主题15-DK-102：开发改进的NIDDK疾病动物模型。T1D的遗传基础已被深入研究。在啮齿动物和人类中分别定位了20多个和5个T1D易感基因，其中MHC基因多态为这两个物种的风险提供了最好的预测指标。CTLA4是人类和小鼠共有的唯一其他T1D易感基因。它编码一种属于T细胞共刺激家族的蛋白，该蛋白的表达是在T细胞激活时诱导的，并具有抑制T细胞激活的功能。CTLA-4也在调节性FOXP3+CD4+T(Treg)细胞亚群上有结构性表达。Treg细胞对小鼠预防T1D至关重要，CTLA-4和Treg细胞之间在控制T1D方面存在功能联系，特别是考虑到缺乏CTLA-4或FOXP3的小鼠会因无节制的自我反应性T细胞扩张和渗透到组织中导致4周前死亡而导致多器官衰竭。最近，我们和其他人利用T细胞亚群受限表达CTLA-4的新动物模型明确地证明了CTLA-4对于正常Treg细胞功能是必不可少的。有了这一见解，并重新调整了对CTLA-4功能的研究重点，这项应用的目标是生成更合理设计的动物模型，以了解CTLA-4与其他疾病易感性参数如何影响T1D进展。何时以及在多大程度上，CTLA-4功能的异常可以特异性地导致T细胞介导的胰岛细胞的破坏是尚未解决的问题。建立分析CTLA-4功能的动物模型的困难一直是研究进展的主要障碍。在体内分别研究CTLA-4在常规T细胞和Treg细胞中的功能尤其具有挑战性，因为操纵后者会导致T细胞动态平衡的严重破坏和早期死亡。因此，目前还没有明确定义的涉及CTLA-4功能改变的T1D进展的动物模型。通过简单地将CTLA-4的表达限制在特定的T细胞亚群，我们现在已经在糖尿病抵抗的C57BL/6(B6)背景下产生了T1D的小鼠模型，该模型发展为100%外显性的自发性岛炎，但不会进展为完全的糖尿病。这些小鼠被称为CT4act，是一系列针对不同T细胞亚群表达CTLA-4的小鼠模型之一。使用这些模型，我们已经证明Treg细胞以CTLA-4依赖的方式维持T细胞的动态平衡。CT4激活的小鼠，CTLA-4只在激活的T细胞中表达，由于缺乏CTLA-4的功能受损的Treg细胞，导致淋巴增殖。然而，它们可以活到8-12个月大，而不是CTLA4-/-小鼠的3-4周。这种相对的寿命可以归因于CT4作用的小鼠非淋巴组织中活化的T细胞很少，这表明异常激活的T细胞需要CTLA-4来防止致病T细胞向非淋巴组织的渗透。CTLA-4为组织完整性提供的保护的一个例外是胰腺的胰岛细胞。这些结果强烈地表明，Treg细胞通过CTLA-4依赖的方式预防胰腺炎。用CT4充当小鼠 在主要平台上，我们正在生成一系列T1D的B6小鼠模型，这些模型可以完全概括疾病的发展过程，以确定是什么过程控制了从胰岛素炎到糖尿病的转变。为此，我们已经建立了几个小鼠模型，将用于评估Treg细胞的CTLA-4非依赖功能与MHC等位基因的相对贡献，以及胰腺自身抗原对Treg和致病T细胞的TCR谱系。一旦建立了控制糖尿病诱导的关键参数，我们将利用小鼠模型，其中CTLA-4可以以时间调节和细胞类型特定的方式被消融，以确定CTLA-4如何影响T1D检查点参数。 公共卫生相关性：CTLA-4功能受损被强烈认为是人类和啮齿动物T1D易感性的主要因素。这项应用将产生新的小鼠模型，以确定CTLA-4和其他调节T1D进展的因素之间的关系，从而可以制定出更多针对CTLA-4治疗T1D的合理疗法。

DESCRIPTION (provided by applicant): Production of new animal models to determine how CTLA-4 contributes to T1D susceptibility. This application addresses Challenge Area 15: Translational Sciences and specific Challenge Topic 15-DK-102: Develop improved animal models of NIDDK diseases. The genetic basis of T1D has been intensely investigated. More than 20 and 5 T1D-susceptibility genes have been mapped in rodents and humans respectively, with MHC polymorphisms providing the best predictive measure of risk in both species. Ctla4 is the only other T1D susceptibility gene common to humans and mice. It encodes for a protein belonging to the T cell costimulatory family whose expression is induced upon T cell activation and which functions to inhibit T cell activation. CTLA-4 is also expressed constitutively on the regulatory FOXP3+ CD4+ T (Treg) cell subset. Treg cells are critical for preventing T1D in mice and a functional link between CTLA-4 and Treg cells in controlling T1D has been suggested, especially given that mice lacking CTLA-4 or FOXP3 succumb to multi-organ failures caused by unchecked self-reactive T cell expansion and infiltration into tissues leading to death before 4 weeks of age. Recently, we and others have definitively shown that CTLA-4 is essential for normal Treg cell function using new animal models with T cell subset-restricted expression of CTLA-4. With this insight, and a realigned research focus on where CTLA-4 functions, the goal of this application is to generate more rationally designed animal models to understand how CTLA-4, in conjunction with other disease susceptibility parameters, impact T1D progression. When, and to what extent, aberrations in CTLA-4 function can specifically lead to T cell mediated destruction of ¿-islet cells are unresolved issues. The difficulty in generating animal models to dissect CTLA-4 function has been a major impediment to research progress. It has been particularly challenging to study CTLA-4 function separately in conventional T cells and Treg cells in vivo, since manipulations of the latter subset result in profound deregulation of T cell homeostasis and early mortality. As a result, there are no well-defined animal models of T1D progression that involves alterations in CTLA-4 function. By simply restricting CTLA-4 expression to particular T cell subsets, we have now generated a mouse model of T1D in diabetes resistant C57BL/6 (B6) background, which develops spontaneous insulitis with 100% penetrance, but does not progress to full-blown diabetes. These mice, referred to as CT4act, are one of a series of mouse models that target CTLA-4 expression to distinct T cell subsets. Using these models, we have shown that Treg cells maintain T cell homeostasis in a CTLA-4-dependent manner. CT4act mice, where CTLA-4 is expressed only in activated T cells, are afflicted with lymphoproliferation due to functionally impaired Treg cells that lack CTLA-4. However, they live to 8-12 months of age instead of 3-4 weeks for Ctla4-/- mice. This relative longevity can be attributed to a paucity of activated T cells in non-lymphoid tissues in CT4act mice, suggesting that CTLA-4 is required in aberrantly activated T cells to prevent the infiltration of pathogenic T cells into non-lymphoid tissues. One exception to this protection afforded by CTLA-4 for tissue integrity is the ¿-islet cells of the pancreas. These results strongly suggest that Treg cells prevent insulitis in a CTLA-4-dependent manner. Using CT4act mice as the main platform, we are generating a series of B6 mouse models of T1D that can fully recapitulate disease progression to determine what processes control insulitis to diabetes transition. Towards this purpose, we have already generated several mouse models that will be used to assess the relative contributions of CTLA-4-independent function of Treg cells in conjunction with MHC alleles, and TCR repertoire for pancreatic self-antigens on Treg and pathogenic T cells. Once the critical parameter controlling diabetes induction is established, we will utilize mouse models in which CTLA-4 can be ablated in a temporally-regulated and cell type-specific manner to determine how CTLA-4 impacts T1D checkpoint parameters. PUBLIC HEALTH RELEVANCE: Impaired CTLA-4 function has been strongly implicated as a major factor in T1D susceptibility in humans and rodents. This application will generate new mouse models to determine the relationship between CTLA-4 and other factors that regulate T1D progression so that more rationale therapies designed to target CTLA-4 to treat T1D can be formulated.