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

项目概要 我们将 TIM 3 克隆为在产生 IFN-g 的 T 细胞上差异表达的分子，并已成为主要的 终止效应 T 细胞反应所必需的抑制分子。 Tim 3 表达增加 人类慢性病毒感染和癌症中的效应 T 细胞，使其功能失调。相比之下，在人类 自身免疫性疾病中，效应 T 细胞上 Tim 3 表达缺失，使其高度亲 炎症性和致病性。由于其在 T 细胞耗竭中的作用，Tim 3 正在成为多个临床研究的目标 癌症试验。 Tim 3 也在树突状细胞 (DC) 上组成型表达，然而，其作用和功能 Tim 3 对 DC 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 抗 Tim 3 抗体正在进行中。 与 T 细胞一样，Tim 3 在 DC 中与其接头蛋白 Bat-3 共表达，其中 Bat-3 充当分子“门 keeper”，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 中产生了 Tim 3 和 Bat-3 的条件“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 的信号传导使 Bat-3 缺陷的 DC 产生耐受性，使得它们不能有效地发挥作用。 诱导炎症 T 细胞反应，并且小鼠能够抵抗自身免疫的发展。基于我们的 根据初步数据，我们假设 DC 中未减弱的 Tim 3 信号传导促进了耐受性 DC 的产生。 为了解决这个假设，我们提出两个具体目标： 1. 确定 Tim 3/Bat-3 相互作用如何调节耐受性 DC 的发育。我们观察到 通过删除 Bat-3，Tim 3 的无阻碍信号传导（特别是在 DC 中）抑制了多种细胞的发育 自身免疫性疾病，包括重点关注的实验性自身免疫性脑脊髓炎（EAE） 的这项建议。在 DC 中使用 Tim 3 和 Bat-3 的条件“敲除”小鼠，我们建议 确定 Bat-3 cKO 小鼠对自身免疫的抵抗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-b通路相互作用的分子机制 促进耐受性 DC 的产生。使用无偏见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，我们鉴定出 Smad-2，TGF-b 途径的转导子， 作为 Tim 3 相互作用蛋白。这一新颖的观察使我们能够研究 Tim 3 的机制 介导其抑制功能，具体来说，我们将能够确定蒂姆的分子基础 3/Smad/TGF-b 途径促进耐受性 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 通路通过该网络介导其在 DC 中的抑制功能。 拟议的研究将确定 Tim 3:Bat-3 通路如何使 DC 产生耐受性，从而提供关键的 可以利用这些信息来有益于多种人类疾病。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 信号可能抑制 自身免疫性疾病并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.