Increasing the cytotoxicity of T cells in response to PD-1 blockade

项目摘要： 细胞毒性T淋巴细胞（CTL）能够杀死全身的肿瘤细胞，并且可以被刺激。 通过一种称为PD-1检查点阻断的免疫疗法来实现这一点。由于未知的原因，大多数 给予抗PD-1治疗的患者对治疗无反应。因此，长期目标是确定 与PD-1阻断失败相关的因素，以便制定可以显著改善 答复率。这个特定应用程序的总体目标是确定潜在的机制 即使在抗PD-1治疗后，也能阻止CTL杀死它们的肿瘤细胞靶。NKG 7是一个专业的 由活化的T细胞表达的与细胞毒性颗粒膜相关的蛋白质。降低 在对PD-1阻断无应答的患者的CD 8 + T细胞中观察到NKG 7水平。这导致 核心假设是缺乏细胞毒性关键介质的适当表达的CD 8 + T细胞， 例如NKG 7，不能对PD-1阻断疗法作出反应。中心假设将通过追求来检验- 有两个具体目标：1）确定CD 8 + T细胞中NKG 7表达水平的降低如何导致治疗失败。 抗PD-1治疗的效果; 2）确定影响存在于PD-1中的功能性NKG 7的量的机制。 CD 8 + T细胞。在第一个目标下，NKG 7将在两种细胞的CD 8 + T细胞内被敲低或过表达。 人和小鼠来源。然后使用体外和体内细胞毒性测定来攻击这些T细胞， 确定NKG 7水平的调节如何影响CTL的颗粒释放和肿瘤细胞杀伤。在证券交易委员会- 第二个目的是探索选择性剪接作为一种控制细胞内功能性NKG 7水平的机制。 CD 8 + T细胞。RT-PCR将用于确认异常NKG 7 mRNA剪接瓦里体的相对丰度， 基线时患者CD 8 + T细胞中的蚂蚁与抗PD-1治疗的最终失败相关。在体外作为- Says也将开始剖析上游信号在这些剪接变化中可能发挥的作用。亲- 提出的研究是创新的，因为它侧重于确定细胞毒活性所必需的关键因素， CTL，以及该过程中的缺陷如何导致PD-1阻断失败。预计捐款将 因为它将有助于提高对PD-1阻断的应答率，这是一种治疗方法， 具有巨大的潜力，可以诱导针对多种癌症类型的有效免疫攻击， 每年都能提高成千上万病人的存活率。

Project Summary: Cytotoxic T lymphocytes (CTL’s) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. For unknown reasons, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, the long-term goal is to identify key factors associated with failure of PD-1 blockade in order to develop strategies that can significantly improve response rates. The overall objective for this specific application is to determine the underlying mechanisms that prevent CTL’s from killing their tumor cell targets, even after anti-PD-1 therapy is on board. NKG7 is a pro- tein expressed by activated T cells which is associated with the membrane of cytotoxic granules. Decreased levels of NKG7 were noted in the CD8+ T cells of patients who did not respond to PD-1 blockade. This leads to the central hypothesis that CD8+ T cells that lack appropriate expression of crucial mediators of cytotoxicity, such as NKG7, are unable to respond to PD-1 blocking therapy. The central hypothesis will be tested by pursu- ing two specific aims: 1) Determine how decreased NKG7 expression levels in CD8+ T cells could lead to fail- ure of anti-PD-1 therapy; and 2) Identify mechanisms that influence the amount of functional NKG7 present in CD8+ T cells. Under the first aim, NKG7 will be knocked-down or over-expressed within CD8+ T cells of both human and mouse origin. These T cells will then be challenged using in vitro and in vivo cytotoxicity assays to determine how modulation of NKG7 levels affects granule release and tumor cell killing by CTL’s. In the sec- ond aim, alternative splicing will be explored as a mechanism that controls the levels of functional NKG7 within CD8+ T cells. RT-PCR will be use to confirm that the relative abundance of aberrant NKG7 mRNA splice vari- ants in the CD8+ T cells of patients at baseline correlates with eventual failure of anti-PD-1 therapy. In vitro as- says will also begin to dissect what upstream signaling may play a role in these splicing changes. The pro- posed research is innovative because it focuses on identifying key factors necessary for the cytotoxic activity of CTL’s, and how defects in this process could lead to failure of PD-1 blockade. The contribution is expected to be significant because it will contribute to the goal of increasing response rates to PD-1 blockade, a therapy with tremendous potential to induce an effective immune attack against a wide range of cancer types and in- crease the survival rate of thousands of patients every year.