Novel mouse models for studying Tim-3 signaling

摘要 T细胞衰竭是在长期接触抗原的情况下经常观察到的一种T细胞功能状态， 例如，在一些慢性病毒感染和实体瘤中。然而，驱动和维持的信号 T细胞的衰竭仍未得到很好的了解。大量耗尽的T细胞表面蛋白标记物 现在已被鉴定，包括跨膜蛋白PD-1和TIM-3，以及其他标记。抗体 阻断PD-1抑制作用的药物现在被FDA批准用于治疗黑色素瘤和一些 肺癌，还有许多其他适应症的临床试验仍在进行中。TIM-3作为一种新技术的发展 治疗目标正在取得进展，但落后于其他目标，包括PD-1等。 尽管大量相关证据表明TIM-3的表达与T细胞功能障碍有关 在力竭过程中的激活，人们对TIM-3参与 发展和/或维持精疲力竭。这一直具有挑战性，部分原因是多个 已经确定了TIM-3的混杂配体。此外，假定的针对TIM-3的“封闭”抗体并没有 由于它们对TIM-3与其各种配体相互作用的影响而得到了彻底的表征。最后，我们有 在多种细胞类型(包括T细胞)中提供了TIM-3实际上可能增强早期信号传递的证据 通过抗原受体，提出了一些TIM-3抗体是否真的通过 TIM-3分子筛的主动交联。因此，需要新的试剂和模型系统，以允许 对TIM-3在原代T细胞中功能的更直接定义。 与PD-1或其他T细胞活性的负调控因子不同，Tim-3不包含募集T细胞活性的基序 抑制性磷酸酶。相反，我们最近的工作表明，Tim-3的表达实际上增加了信号转导 通过通常与阳性结果相关的途径，即有效的TCR/CD3介导的T细胞 激活，至少在急性情况下是这样。来自其他组织的数据表明，T细胞耗尽是由 慢性抗原刺激延长T细胞激活的效应期，以T细胞为代价 记忆。我们假设通过TIM-3胞浆尾部的信号与T细胞耗竭有关 和/或挽救与慢性T细胞激活相关的耗尽的T细胞群体中的T细胞。 这一假设将通过两个具体目标进行检验。在目标1中，并基于我们以前的 结构/功能研究，我们将产生新的小鼠模型，用于TIM-3的可诱导敲除或截断 它的细胞质尾巴。在目标2中，我们将首先确定诱导性TIM-3 KO或截断的效果， 特别是在T细胞中，对李斯特菌和LCMV的急性T细胞反应；此外，我们将确定它们的影响 在慢性LCMV-Clone 13感染中可诱导的、T细胞特异性的KO或TIM-3的截断。

Abstract T cell exhaustion is a state of T cell function often observed under conditions of chronic exposure to antigen, for example in some chronic viral infections and in solid tumors. However, the signals that drive and maintain exhaustion in T cells are still not well understood. Numerous surface protein markers of exhausted T cells have now been identified, including the transmembrane proteins PD-1 and Tim-3, among other markers. Antibodies that block the inhibitory effects of PD-1 are now licensed by the FDA for the treatment of melanoma and some lung cancers, with many more clinical trials still underway for other indications. Development of Tim-3 as a therapeutic target is progressing, but lags behind other targets, including PD-1, among others. Despite the extensive amount of correlative evidence linking Tim-3 expression to dysfunction of T cell activation during exhaustion, little is known about the mechanisms by which Tim-3 contributes to the development and/or maintenance of exhaustion. This has been challenging, in part due to the fact that multiple promiscuous ligands to Tim-3 have been identified. In addition, putative “blocking” antibodies to Tim-3 have not been thoroughly characterized for their effects on interactions of Tim-3 with its various ligands. Finally, we have provided evidence in multiple cell types (including T cells) that Tim-3 may actually enhance early signaling through antigen receptors, raising the question of whether some Tim-3 antibodies may actually function by actively crosslinking of Tim-3. There is therefore a need for new reagents and model systems that will allow for the more direct definition of Tim-3 function in primary T cells. Unlike PD-1 or other negative regulators of T cell activity, Tim-3 contains no motifs for recruitment of inhibitory phosphatases. Rather, our recent work suggests that Tim-3 expression actually increases signaling through pathways normally associated with positive outcomes, i.e. efficient TCR/CD3-mediated T cell activation, at least under acute conditions. Data from other groups indicate that T cell exhaustion results from chronic antigenic stimulation that extends the effector phase of T cell activation, at the expense of T cell memory. We hypothesize that signaling through the cytoplasmic tail of Tim-3 contributes to T cell exhaustion and/or the rescue of T cells in a population of exhausted T cells associated with chronic T cell activation. This hypothesis will be tested with two Specific Aims. In Aim 1, and based on our previous structure/function studies, we will generate novel mouse models for inducible knockout of Tim-3 or truncation of its cytoplasmic tail. In Aim 2, we will first determine the effects of inducible Tim-3 KO or truncation, specifically in T cells, on acute T cell responses to Listeria and LCMV; in addition, we will determine the effects of inducible, T cell-specific, KO or truncation of Tim-3 on chronic infection with LCMV-Clone 13.