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

项目摘要 我们克隆了TIM 3作为一种在产生IFN-γ的T细胞上差异表达的分子，并已成为一种主要的免疫调节因子。 抑制性分子是终止效应T细胞应答所必需的。Tim 3表达增加， 效应T细胞在人类慢性病毒感染和癌症中的作用，使它们功能失调。相反，在人类 在自身免疫性疾病中，效应T细胞上的Tim 3表达缺失，使它们高度亲 炎性和致病性。由于其在T细胞耗竭中的作用，Tim 3在多种临床试验中被靶向。 癌症的临床试验Tim 3也在树突状细胞（DCs）上组成性表达，然而，Tim 3的作用和功能尚不清楚。 Tim 3在DC上的作用尚不清楚，从临床试验的角度来看，理解这一点尤其重要。 正在进行的抗Tim 3抗体测试 与在T细胞中一样，Tim 3在DC中与其衔接蛋白Bat-3共表达，其中Bat-3充当分子“门”， Keeper”，其限制Tim 3信号和功能。为了理解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， 在Bat-3不存在的情况下Tim 3的信号传导使Bat-3缺陷型DC具有耐受原性，使得它们不能有效地 诱导炎性T细胞应答，并且小鼠对自身免疫的发展具有抗性。基于我们 根据初步数据，我们假设DC中未减弱的Tim 3信号传导促进致耐受性DC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性DC的发育。我们观察到 通过删除Bat-3，特别是在DC中，Tim 3的无对抗信号传导抑制了多个细胞的发展， 自身免疫性疾病，包括实验性自身免疫性脑脊髓炎（EAE）， 这个提议。使用DC中Tim 3和Bat-3的条件性“敲除”小鼠，我们建议 确定Bat-3 cKO小鼠对自身免疫的抗性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如何损失 Bat-3调节DC表型和功能。 2.确定Tim 3与Smad/TGF-β通路相互作用的分子机制， 促进致耐受性DC的产生。使用无偏倚的蛋白质组筛选来识别分子 在没有Bat-3的情况下与Tim 3尾结合，我们鉴定了Smad-2，TGF-β途径的转导子， Tim 3相互作用蛋白。这一新的观察使我们能够研究Tim 3 介导其抑制功能，特别是我们将能够确定的分子基础，蒂姆 3/Smad/TGF-β通路促进耐受原性DC的发育。使用高密度时间 Tim 3和Bat-3缺陷DC的转录分析，我们建议开发转录 Tim 3：Bat-3通路通过其介导DC中的抑制功能的网络。 拟议的研究将确定Tim 3：Bat-3途径如何使DC致耐受性，提供关键的 这些信息可以用于治疗多种人类疾病。虽然抑制Tim 3功能可以 增强慢性病毒感染和癌症的免疫反应，增强Tim 3信号可以抑制 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.