Generating a novel conditional knockout mouse for a super-enhancer that controls cytokine responsiveness

项目摘要 这个建议集中在一个新的超级增强子上，我们最初发现它可以协调一个非原型的T 细胞应答，其中效应T细胞被触发以产生关键的1型细胞因子干扰素γ（IFN-γ）。 g）独立于T细胞受体，通过用已知的精确细胞因子组合刺激， 激活某些先天免疫细胞特别地，效应T细胞响应于STAT-1而合成IFN-g。 活化细胞因子（IL-2或IL-12）加IL-1家族成员（IL-33、IL-36或IL-18）。虽然这样的“天生” T细胞应答尚未被广泛研究，它们已被证明有助于宿主防御 此外，它还可能促进癌症免疫治疗。在分析分子机制时， 为了支持先天性T细胞反应性，我们在小鼠1号染色体上发现了一个新的超级增强子 促进STAT激活的精氨酸介导的编码IL-33受体的基因转录 IL-18。重要的是，人类GWAS研究确定了该区域（人类2号染色体上）的SNP， IL-33调节的病理学哮喘和变态反应以及肺癌。此外，我们还创作了一部小说 缺乏这种超级增强子的CRISPR-Cas9基因组编辑小鼠，其常规的CD 8和CD 4 T细胞 先天能力受损。此外，缺乏超级增强子的Foxp 3 + T调节细胞表现出 降低IL-33受体的表达，这是已知的赋予抑制功能。这些全球超级- 增强子敲除小鼠在没有免疫治疗的情况下也表现出较慢的肿瘤生长速率，以及 增加对内毒素休克的抵抗力，这表明超级增强子也可以调节 其他免疫细胞类型。该项目将产生一种新的条件性超级增强子敲除小鼠 该菌株将有助于研究精确定义超级增强子在不同免疫细胞类型中的作用 以及受IL-33或IL-18影响的生理和治疗环境。

Project Summary This proposal centers on a novel super-enhancer that we initially discovered to coordinate a non-prototypical T cell response in which effector T cells are triggered to produce the key type 1 cytokine interferon gamma (IFN- g), independently of the T cell receptor, via stimulation with precise cytokine combinations that are known to activate certain innate immune cells. In particular, effector T cells synthesize IFN-g in response to a STAT- activating cytokine (IL-2 or IL-12) plus an IL-1 family member (IL-33, IL-36 or IL-18). Although such "innate-like" T cell responses have not been extensively studied, they have been shown to facilitate host defense against various infections, and may also boost cancer immunotherapy. In analyzing the molecular mechanisms underpinning innate-like T cell responsiveness, we identified a novel super-enhancer on mouse chromosome 1 that facilitates STAT-activating cytokine-mediated transcription of the genes encoding the receptors for IL-33 and IL-18. Importantly, human GWAS studies identified SNPs in this region (on human chromosome 2) linked to the IL-33-regulated pathologies asthma and allergy as well as lung cancer. Further, we generated a novel CRISPR-Cas9 genome-edited mouse lacking this super-enhancer, whose conventional CD8 and CD4 T cells have impaired innate-like capacity. Additionally, Foxp3+ T regulatory cells lacking the super-enhancer exhibit reduced expression of the IL-33 receptor, that is known to confer suppressor function. These global super- enhancer knockout mice also exhibit slower rates of tumor growth in the absence of immunotherapy, as well as increased resistance to endotoxin shock, suggesting that the super-enhancer may also regulate the function of additional immune cell types. This project will generate a novel conditional super-enhancer knockout mouse strain that will facilitate studies to precisely define the role of the super-enhancer in different immune cell types and physiologic and therapeutic settings impacted by IL-33 or IL-18.