An in vivo CRISPR-Cas9 genetic screen in murine primary T cells to discover metabolic regulators of follicular B helper T (Tfh) cell differentiation

项目概要（摘要） 滤泡B辅助性T（Tfh）细胞是正常免疫反应所必需的，促进记忆的发展 B细胞和长寿浆细胞。当异常调节时，例如在系统性红斑狼疮（SLE）中， 狼疮），它们驱动自身反应性记忆B细胞成熟和致病性浆细胞形成。调制 在狼疮鼠模型中，T-B细胞相互作用可以改善疾病，这种干预有望 对人类狼疮有治疗作用因此，有理由制定一个更全面的 了解Tfh细胞分化、存活和与B细胞协作的机制， 正常和疾病设置。活化后，CD 4 T细胞表现出代谢的动态变化，以满足 他们的增殖和效应需求。目前对Tfh细胞中代谢如何调节的理解是 尽管最近的研究表明，干扰T细胞代谢编程是不够的， 在狼疮模型中有益，因为它在癌症免疫治疗中是有益的，尽管迄今为止还没有鉴定出特异性T细胞。 细胞靶点。由于Tfh细胞与其T辅助效应物对应物相比在独特的GC小生境中运作， 我们假设这些细胞利用不同的代谢程序来为其功能提供燃料， 这些节点被提出作为在肿瘤浸润性T淋巴细胞中重编程细胞毒性的策略。在 首先，本项目的R61阶段，我们将建立体内、高通量的sgRNA文库筛选系统， 支持从过继转移的T细胞产生Tfh细胞，用代谢组sgRNA文库转导 其允许足够的文库覆盖;提供足够的灵敏度，使得Cas9介导的遗传修饰能够被用于基因工程。 损伤导致可观察到的Tfh细胞表型;并允许追踪单个病毒整合事件。到 实现这些目标，我们将利用急性阿姆斯特朗LCMV（淋巴细胞性脉络丛脑膜炎病毒） 我们已经使用感染模型来询问Tfh细胞分化，其中我们可以跟踪发育， 用表达shRNA的逆转录病毒遗传操作的过继转移的T细胞的分化。 该模型将适用于我们的逆转录病毒sgRNA系统。我们将使用sgRNA对抗已知的基因， 严格调节Tfh细胞发育，以建立评估合并筛选的基准 并定义实验装置的动态范围。我们还将生成一个新的条形码载体， 跟踪单个转导的T细胞，这将提高屏幕的分辨率，并提供强大的 分析的统计功效。一旦这些目标实现，我们将着手制定 条形码化的sgRNA文库并进行体内筛选。在第二阶段，R33阶段，我们将产生新的 使用Cas9技术的敲除动物模型，以验证筛选命中与经验证的靶标的相关性 为了直接测试候选代谢基因在自身免疫性疾病中的作用，

PROJECT SUMMARY (ABSTRACT) Follicular B helper T (Tfh) cells are required for normal immune responses, promoting development of memory B cells and long-lived plasma cells. When aberrantly regulated, such as in systemic lupus erythematosus (SLE, lupus), they drive maturation of autoreactive memory B cell and pathogenic plasma cell formation. Modulation of T-B cell interactions in murine models of lupus ameliorates disease, with promise that such intervention will be therapeutically beneficial in human lupus. Thus, it is reasonable to develop a more comprehensive understanding of the mechanisms that govern Tfh cell differentiation, survival, and collaboration with B cells in normal and disease settings. Upon activation, CD4 T cells exhibit dynamic changes in metabolism to meet their proliferative and effector needs. The understanding of how metabolism is regulated in Tfh cells is currently insufficient, although recent work has shown that interference with T cell metabolic programming is as beneficial in lupus models as it is in cancer immunotherapy, albeit without identification to-date of the specific T cell target(s). As Tfh cells operate in the unique GC niche in comparison to their T helper effector counterparts, we hypothesize that these cells utilize different programs of metabolism to fuel their function, with targeting such nodes proposed as a strategy for reprogramming cytotoxicity in tumor-infiltrating T lymphocytes. In the first, R61 phase of this project, we will establish in vivo, high-throughput sgRNA library screening system that supports Tfh cell generation from adoptively transferred T cells, transduced with a metabolome sgRNA library which allows for sufficient library coverage; provides enough sensitivity such that Cas9-mediated genetic lesions lead to observable Tfh cell phenotypes; and permits tracking of individual viral integration events. To achieve these goals, we will take advantage of an acute Armstrong LCMV (lymphocytic choriomeningitis virus) infection model we have used to interrogate Tfh cell differentiation, in which we can track development and differentiation of adoptively transferred T cells genetically manipulated with shRNA expressing retroviruses. This model will be adapted to our retroviral sgRNA system. We will use sgRNA against genes known to critically regulate Tfh cell development to establish benchmarks against which to evaluate the pooled screen and to define the dynamic range of the experimental setup. We also will generate a new barcoded vector to track individually transduced T cells, which will both enhance resolution of the screen and provide robust statistical power for the analysis. Once these goals have been achieved, we will proceed to generating the barcoded sgRNA library and conducting the in vivo screen. In the second, R33 phase, we will generate novel knockout animal models using using Cas9 technology to validate relevance of screen hits, with verified targets bred to lupus-prone mice in order to directly test the role of candidate metabolic genes in autoimmune disease.