Expoitation of ER Stress Induced Immune Dysfunction to Improve Immunotherapy

项目总结 目前尚不清楚T细胞如何感知和响应实体瘤的压力，或者这种反应是如何形成的 CD8肿瘤浸润性淋巴细胞(TIL)功能障碍。内质网(ER)是一种细胞器 以感知环境压力，并通过启动下游信号级联来做出反应。类PKR内质网 激酶(PERK)是细胞对应激做出反应的三种内质网感受器之一。我们是第一个 表明肿瘤中的T细胞经历了PERK导向的应激反应。使用独特的T细胞特有的福利 KO小鼠(OT-1-Lockcre-PERKf/f)我们发现PERK严重限制T细胞控制肿瘤的能力 成长。我们现在公布突破性的初步数据，这些数据表明，在应对 肿瘤微环境PERK抑制T细胞抗肿瘤代谢及限制CD8蛋白质合成 TILS。此外，我们发现CD8TIL经历了与PERK终末相关的慢性内质网应激 未折叠蛋白反应(UPR)酶ER氧化还原酶1(ERO1a)诱导蛋白失调 动态平衡(蛋白质平衡)和损害肿瘤控制。这项建议旨在扩大我们的初步数据和 将上述新发现发展为治疗肉瘤的免疫治疗策略 对实体肿瘤癌症患者的影响。为了实现这里提出的目标，我们开发了一种独特的 肿瘤微环境应激检测及应用于CD8TIL生物学检测的新途径 T细胞在肿瘤应激状态下发生的分子变化。在目标1中，我们将使用OT-1-Lockcre-PERKf/f中的T细胞 在我们的肿瘤应激试验中，小鼠与代谢组学相结合，表明PERK是CD8 TIL的中心调节因子 新陈代谢。我们还将使用肉瘤患者的T细胞和CRISPR/Cas9对PERK进行体外基因编辑 测量在肿瘤微环境压力下，PERK如何影响人类T细胞的代谢。结果是 期望重塑和推进我们目前对CD8 TIL代谢的理解。在目标2中，我们将使用T细胞 在我们的肿瘤应激试验中结合核糖体测序从OT-1-Lockcre-PERKf/f小鼠中发现 主动翻译受肿瘤微环境影响的mRNAs的身份。这项工作也将检验 新的概念认为，在肿瘤的压力下，替代的翻译途径是可持续的。结果是 期望创造一种激进的重塑翻译的新范式，以改进免疫治疗。对于目标3，我们 已经创造了独特的ERO1a-/-小鼠，以正式定义ERO1a如何破坏CD8 TIL中的蛋白平衡 体内肉瘤模型和蛋白质组学。AIMS的成功完成将给癌症领域带来革命性的变化 通过产生一种新的细胞应激反应范式作为主要因素的免疫治疗 CD8TIL在实体瘤中的抗肿瘤作用

PROJECT SUMMARY It is not known how T cells sense and respond to the stress of solid tumors, or how such responses shape dysfunction in CD8 tumor infiltrating lymphocytes (TILs). The endoplasmic reticulum (ER) is an organelle primed to sense environmental stress and to respond through initiation of downstream signal cascades. PKR-like ER kinase (PERK) is one of three ER sensors that enacts the cell response to stress. We were the first group to show that T cells in tumors experience the PERK-directed stress response. Using unique T cell-specific PERK KO mice (OT-1-Lckcre-PERKf/f) we discovered that PERK severely restricts the ability of T cells to control tumor growth. We now reveal groundbreaking preliminary data that illustrate that in response to the acute stress of the tumor microenvironment PERK prohibits T cell anti-tumor metabolism and restricts protein synthesis in CD8 TILs. Moreover, we show that CD8 TILs experience chronic ER stress associated with the PERK terminal unfolded protein response (UPR) enzyme ER oxidoreductase 1 (ERO1a) that induces dysregulated protein homeostasis (proteostasis) and impairs tumor control. This proposal aims to expand our preliminary data and develop the abovementioned novel discoveries into immunotherapeutic strategies to treat sarcomas with global implications for solid tumor cancer patients. To accomplish the aims proposed here we have developed a unique tumor microenvironment stress assay and sought out new approaches to apply to CD8 TIL biology to measure molecular changes that occur in T cells under tumor stress. In Aim 1 we will use T cells from OT-1-Lckcre-PERKf/f mice in our tumor stress assay paired with metabolomics to show that PERK is the central regulator of CD8 TIL metabolism. We will also use T cells from sarcoma patients and CRISPR/Cas9 in vitro gene editing of PERK to measure how PERK affects metabolism of human T cells under tumor microenvironment stress. The results are expected to reframe and advance our current understanding of CD8 TIL metabolism. In Aim 2 we will use T cells from OT-1-Lckcre-PERKf/f mice in our tumor stress assay paired with ribosome sequencing to discover the identity of actively translating mRNAs that are impacted by the tumor microenvironment. This work will also test the new concept that alternative routes of translation are sustainable in the stress of tumors. The results are expected to create a radical new paradigm of remodeling translation to improve immunotherapy. For Aim 3 we have created unique ERO1a-/- mice to formally define how ERO1a undermines proteostasis in CD8 TILs using an in vivo sarcoma model and proteomics. Successful completion of the aims will revolutionize the field of cancer immunotherapy by generating a new paradigm for the cell stress response as the primary factor that dismantles CD8 TIL anti-tumor efficacy in solid tumors.