Exploitation of ER Stress Induced Immune Dysfunction to Improve Immunotherapy

项目摘要 目前尚不清楚T细胞如何感知和响应实体瘤的压力，或者这种反应如何形成 CD 8肿瘤浸润淋巴细胞（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反应。PKR样内质网 蛋白激酶（PERK）是使细胞对应激反应生效的三种ER传感器之一。我们是第一个 显示肿瘤中的T细胞经历PERK导向的应激反应。使用独特的T细胞特异性PERK KO小鼠（OT-1-Lckcre-PERKf/f），我们发现PERK严重限制了T细胞控制肿瘤的能力， 增长我们现在揭示了开创性的初步数据，这些数据表明，在应对急性应激的过程中， 肿瘤微环境PERK抑制T细胞抗肿瘤代谢并限制CD 8中的蛋白质合成 TILs。此外，我们发现CD 8 TILs经历与PERK末端相关的慢性ER应激， 未折叠蛋白反应（UPR）酶ER氧化还原酶1（ERO 1a），其诱导失调的蛋白 体内平衡（蛋白质平衡）和损害肿瘤控制。该提案旨在扩大我们的初步数据， 将上述新发现发展为免疫策略，以治疗肉瘤， 对实体瘤癌症患者的影响。为了实现这里提出的目标，我们开发了一种独特的 肿瘤微环境应激试验，并寻求新的方法应用于CD 8 TIL生物学测量 在肿瘤应激下T细胞中发生的分子变化。在目标1中，我们将使用来自OT-1-Lckcre-PERKf/f的T细胞 在我们的肿瘤应激试验中，小鼠与代谢组学配对，以显示PERK是CD 8 TIL的中心调节因子， 新陈代谢.我们还将使用来自肉瘤患者的T细胞和PERK的CRISPR/Cas9体外基因编辑， 测量PERK如何影响肿瘤微环境应激下的人T细胞代谢。结果 有望重新构建和推进我们目前对CD 8 TIL代谢的理解。在目标2中，我们将使用T细胞 从OT-1-Lckcre-PERKf/f小鼠中，我们的肿瘤应激试验与核糖体测序配对，以发现 受肿瘤微环境影响的主动翻译mRNA的身份。这项工作还将测试 新的概念，即在肿瘤的压力下，替代的翻译途径是可持续的。结果 有望创造一个全新的重塑翻译范式，以改善免疫治疗。对于目标3， 创建了独特的ERO 1a-/-小鼠，以正式定义ERO 1a如何破坏CD 8 TIL中的蛋白质稳态， 体内肉瘤模型和蛋白质组学。这些目标的成功实现将彻底改变癌症领域 免疫疗法通过产生一个新的范例，细胞应激反应的主要因素， CD 8 TIL在实体瘤中的抗肿瘤功效。

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.