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

项目摘要(摘要) 滤泡B辅助T细胞(TFH)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。当调节异常时，例如在系统性红斑狼疮(SLE， 狼疮)，它们推动自身反应性记忆B细胞的成熟和致病浆细胞的形成。调变 狼疮小鼠模型中T-B细胞的相互作用可以改善疾病，并承诺这种干预将 对人类狼疮有治疗作用。因此，有理由制定一个更全面的 TFH细胞分化、存活及与B细胞协同作用机制的研究 正常设置和疾病设置。在激活后，CD4T细胞表现出代谢的动态变化，以满足 它们的增殖性和效应性需求。目前对TFH细胞的新陈代谢是如何调节的了解 还不够，尽管最近的研究表明，干扰T细胞代谢编程是 在狼疮模型中有益，因为它在癌症免疫治疗中是有益的，尽管到目前为止还没有发现特定的T细胞 细胞靶点(S)。由于与其T辅助效应器对应的细胞相比，TFH细胞在独特的GC利基中工作， 我们假设这些细胞利用不同的新陈代谢程序来支持它们的功能，并有靶向 这种结节被认为是重新编程肿瘤浸润性T淋巴细胞细胞毒性的一种策略。在 首先，本项目R61期，我们将建立体内高通量sgRNA文库筛选体系， 支持用代谢组sgRNA文库转导过继转移的T细胞生成TFH细胞 这允许足够的文库覆盖率；提供了足够的敏感性，使得Cas9介导的基因 病变导致可观察到的TFH细胞表型；并允许跟踪个别病毒整合事件。至 为了实现这些目标，我们将利用一种急性阿姆斯特朗LCMV(淋巴细胞性脉络膜脑膜炎病毒) 我们用来询问TFH细胞分化的感染模型，在这个模型中，我们可以跟踪发育和 逆转录病毒shRNA基因调控过继转移的T细胞的分化。 这个模型将适用于我们的逆转录病毒sgRNA系统。我们将使用sgRNA来对抗已知的 严格调控TFH细胞发育以建立基准，以此作为评估集合筛查的基准 并确定实验装置的动态范围。我们还将生成一个新的条形码向量 跟踪单独转换的T细胞，这将提高屏幕的分辨率并提供健壮的 分析的统计力量。一旦实现了这些目标，我们将着手生成 条形码sgRNA文库并进行体内筛选。在第二个阶段，R33，我们将生成新的 使用Cas9技术验证屏幕点击与已验证目标的相关性的基因敲除动物模型 为了直接测试候选代谢基因在自身免疫性疾病中的作用，培育了易患狼疮的小鼠。

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.