Role of Tim-3:Bat-3 pathway in inducing tolerogenic DCs and peripheral tolerance

项目总结 我们克隆了TIM3作为一种在产生干扰素-g的T细胞上差异表达的分子，并且已经成为一种主要的 终止效应器T细胞反应所必需的抑制分子。TIM 3的表达增加 人类慢性病毒感染和癌症中的效应T细胞，使其功能障碍。相比之下，在人类中 自身免疫性疾病中，效应器T细胞上TIM3表达缺失，使其高度支持 炎症性和致病性的。由于其在T细胞衰竭中的作用，TIM 3正成为多个临床的靶点 癌症试验。TIM-3在树突状细胞(DC)上也有组成性表达，但其作用和功能 从临床试验的角度来看，TIM 3对DC的作用还不是很清楚，理解这一点尤其重要 正在用抗TIM 3抗体进行研究。 与在T细胞中一样，TIM 3与其适配蛋白BAT-3在DC中共表达，其中BAT-3起到分子“门-门”的作用。 Keeper“，这限制了TIM 3的信号和功能。为了了解TIM 3在DC中的功能，我们有 在DC中产生TIM 3和BAT-3的条件性“敲除”小鼠。初步研究表明，不受限制 在没有BAT-3的情况下，TIM 3的信号转导使BAT-3缺陷的DC具有耐受性，从而使它们不能有效地 诱导炎性T细胞反应，小鼠对自身免疫的发展具有抵抗力。基于我们的 初步数据显示，我们假设树突状细胞中TIM-3信号的未减弱促进了耐受树突状细胞的产生。 为了解决这一假设，我们提出了两个具体目标： 1.确定TIM 3/BAT-3相互作用如何调节致耐受树突状细胞的发育。我们观察到 TIM 3通过删除BAT-3，特别是DC中的非相反信号抑制多发性硬化的发展 包括实验性自身免疫性脑脊髓炎(EAE)在内的自身免疫性疾病是重点 这项提议。在DC中使用TIM 3和BAT-3的条件性“敲除”小鼠，我们建议 确定BAT-3 CKO小鼠对自身免疫的抵抗力是否部分或完全恢复 从同一组DC中删除TIM 3。此外，这也将使我们能够确定损失如何 BAT-3调节DC的表型和功能。 2.确定TIM-3与Smad/TGF-b途径相互作用的分子机制 促进产生耐受树突状细胞。使用无偏蛋白质组筛选来鉴定分子 在没有BAT-3的情况下，我们鉴定了Smad-2，它是转化生长因子-b途径的转导分子， 作为一种TIM-3相互作用蛋白。这一新颖的观察结果使我们能够研究TIM 3 介导其抑制功能，特别是我们将能够确定时间的分子基础 3/Smad/TGF-b途径促进耐受树突状细胞的发育。使用高密度时间 TIM 3和BAT-3缺陷DC的转录分析，我们建议发展转录 TIM3：BAT-3通路在DC中介导其抑制功能的网络。 拟议的研究将确定TIM 3：BAT-3途径如何使DC产生耐受，提供关键的 可以被利用来造福于多种人类疾病的信息。在抑制TIM 3功能的同时， 增强慢性病毒感染和癌症的免疫反应，增强TIM 3信号可能会抑制 自身免疫性疾病和促进抗原特异性耐受。

PROJECT SUMMARY We cloned TIM 3 as a molecule differentially expressed on IFN-g producing T cells and has emerged as a major inhibitory molecule necessary for the termination of effector T cell responses. Tim 3 expression is increased on effector T cells in human chronic viral infections and cancers, rendering them dysfunctional. In contrast, in human autoimmune diseases, there is loss of Tim 3 expression on effector T cells, rendering them highly pro- inflammatory and pathogenic. Because of its role in T cell exhaustion, Tim 3 is being targeted in multiple clinical trials for cancer. Tim 3 is also expressed constitutively on dendritic cells (DCs), however, the role and function of Tim 3 on DCs is not well understood and this is especially important to understand, in the view of clinical trials that are underway with anti-Tim 3 antibodies. As in T cells, Tim 3 is co-expressed in DCs with its adapter protein Bat-3, where Bat-3 acts as a molecular “gate- keeper”, that restricts Tim 3 signaling and function. To understand the function of Tim 3 in DCs, we have generated conditional “knock-out” mice of both Tim 3 and Bat-3 in DCs. Initial studies indicate that unrestricted signaling of Tim 3 in the absence of Bat-3, makes Bat-3-deficient DCs tolerogenic such that they do not effectively induce inflammatory T cell responses and the mice are resistant to development of autoimmunity. Based on our preliminary data, we hypothesize that unabated Tim 3 signaling in DCs promotes generation of tolerogenic DCs. To address this hypothesis, we propose two specific aims: 1. Determine how the Tim 3/Bat-3 interaction regulates development of tolerogenic DCs. We have observed that unopposed signaling of Tim 3, by deleting Bat-3, specifically in DCs inhibits development of multiple autoimmune diseases including Experimental Autoimmune Encephalomyelitis (EAE) which is the focus of this proposal. Using conditional “knock-out” mice for both Tim 3 and Bat-3 in DCs, we propose to determine whether resistance to autoimmunity in Bat-3 cKO mice is partly or completely restored by deletion of Tim 3 from the same set of DCs. Furthermore, this will also allow us to determine how loss of Bat-3 regulates DC phenotype and function. 2. Determine the molecular mechanism by which interaction of Tim 3 and the Smad/TGF-b pathway promotes the generation of tolerogenic DCs. Using an unbiased proteomic screen to identify molecules that bind to the Tim 3 tail in the absence of Bat-3, we identified Smad-2, a transducer of TGF-b pathway, as a Tim 3 interacting protein. This novel observation allows us to study the mechanism by which Tim 3 mediates its inhibitory function, specifically we will be able to determine the molecular basis by which Tim 3/Smad/TGF-b pathway promotes the development of tolerogenic DCs. Using high density temporal transcriptional analysis of the Tim 3 and Bat-3 deficient DCs, we propose to develop transcriptional networks by which the Tim 3:Bat-3 pathway mediates its inhibitory function in DCs. The proposed studies will identify how the Tim 3:Bat-3 pathway makes DCs tolerogenic, providing critical information that could be exploited to benefit multiple human diseases. While repressing Tim 3 function could augment immune responses in chronic viral infections and cancer, boosting Tim 3 signals could dampen autoimmune diseases and promote antigen specific tolerance.