CRISPR screens and ILC2-targeted Boolean circuitry reveal Mef2d-potentiation of type-2 immunity [RNA-Seq]. CRISPR screens and ILC2-targeted Boolean circuitry reveal Mef2d-potentiation of type-2 immunity [RNA-Seq]

Group 2 innate lymphoid cells (ILC2s) orchestrate tissue homeostasis, allergic disorders and anti-helminth protective immunity through their production of cytokines. However, their rarity has hampered high-throughput genetic screening approaches to uncover novel ILC2 regulators in an unbiased manner. We combined expansion of progenitors from multi-reporter mouse strains with optimised ILC differentiation cultures to perform CRISPR-Cas9 screens for regulators of ILC2 development and function. Mef2d emerged as a critical regulator of GATA3 and IL-13, and was essential for type-2 immunity as demonstrated by conditional Mef2d-deficient mice, including a Boolean-ILC2-Cre (BIC) mouse strain developed to enable ILC2-specific gene deletion. Mechanistically, Mef2d bound and transcriptionally repressed the Zc3h12a locus encoding the endonuclease Regnase-1, a negative regulator of IL-33 receptor (ST2) and GATA3 expression, thereby promoting IL-33-mediated ILC2 proliferation and cytokine production. Notably, in ILC2s Mef2d also acted downstream of leukotriene C4-induced calcium-mediated signalling to translocate NFAT1 to the nucleus to promote type-2 gene transcription. These Mef2d-mediated pathways converge and feedback to potentiate type-2 immunity. Overall design: Il7rCre control or Mef2dIL7RKO mice were treated with 3 doses of IL-33 intranasally and lung ILC2s were flow purified for RNA-seq analysis.

2型固有淋巴细胞（Group 2 innate lymphoid cells, ILC2s）可通过分泌细胞因子调控组织稳态、过敏性疾病及抗蠕虫保护性免疫。然而，因其细胞丰度极低，高通量遗传筛选难以无偏地发掘新型ILC2调控因子。本研究将多报告基因小鼠品系的祖细胞扩增与优化的ILC分化培养体系相结合，针对ILC2发育与功能的调控因子开展CRISPR-Cas9筛选。结果显示，Mef2d是GATA3与IL-13表达的关键调控因子；条件性Mef2d敲除小鼠（包括本研究构建的可实现ILC2特异性基因敲除的Boolean-ILC2-Cre（BIC）小鼠品系）实验证实，Mef2d对2型免疫至关重要。机制层面，Mef2d可结合并转录抑制编码核酸内切酶Regnase-1的Zc3h12a基因座——Regnase-1是IL-33受体（ST2）与GATA3表达的负调控因子，由此促进IL-33介导的ILC2增殖与细胞因子产生。值得注意的是，在ILC2中，Mef2d还可参与白三烯C4诱导的钙介导信号通路下游过程，将NFAT1易位至细胞核以促进2型基因转录。上述Mef2d介导的通路相互汇聚并形成反馈，进而增强2型免疫。实验整体设计：分别经鼻给予Il7rCre对照小鼠与Mef2dIL7RKO小鼠3剂IL-33，随后流式纯化肺部ILC2并进行RNA测序分析。