CRISPR screen in regulatory T cells reveals modulators of Foxp3
CRISPR screen in regulatory T cells reveals modulators of Foxp3
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DOI：
10.1038/s41586-020-2246-4
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发表时间：
2020-04-29
期刊：
影响因子：
64.8
通讯作者：
Fang, Deyu
中科院分区：
文献类型：
--
作者：
Cortez, Jessica T.;Montauti, Elena;Fang, Deyu
调节性T（T-reg）细胞是控制免疫应答和维持体内平衡所必需的，但却是抗肿瘤免疫的重要屏障（1）。相反，以主转录因子Foxp 3的缺失和促炎特性的获得为特征的T-reg不稳定性（2）可以促进自身免疫和/或促进更有效的肿瘤免疫（3，4）。全面了解调节Foxp 3的途径可能会导致更有效的自身免疫性疾病和癌症的T-reg疗法。新的功能性遗传工具的可用性，使系统解剖的基因调控程序，调节Foxp 3表达的可能性。在这里，我们开发了一种基于CRISPR的合并筛选平台，用于原代小鼠T-reg细胞中的表型，并应用该技术对约500种核因子进行靶向功能丧失筛选，以确定促进或破坏Foxp 3表达的基因调控程序。我们鉴定了几种Foxp 3表达的调节剂，包括泛素特异性肽酶22（Usp 22）和环指蛋白20（Rnf 20）。Usp 22是佐贺染色质修饰复合物的去泛素化模块的成员，被揭示为稳定Foxp 3表达的正调节剂;而筛选表明Rnf 20，E3泛素连接酶，可以作为Foxp 3的负调节剂。T-reg特异性消融小鼠中的Usp 22降低了Foxp 3蛋白水平，并导致其抑制功能缺陷，导致自发性自身免疫，但在多种癌症模型中保护了肿瘤生长。在Usp 22缺陷的T-reg细胞中，Foxp 3的不稳定可以通过Rnf 20的消融来挽救，揭示了T-reg细胞中的相互泛素开关。这些结果揭示了以前未知的Foxp 3调节剂，并证明了一种筛选方法，可以广泛应用于发现癌症和自身免疫性疾病的T-reg免疫疗法的新靶点。基于CRISPR的筛选平台用于鉴定以前未表征的调节调节T细胞特异性主转录因子Foxp 3的基因，表明该筛选方法可广泛应用于发现参与自身免疫和对癌症的免疫应答的其他基因。
Regulatory T (T-reg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity(1). Conversely, T-reg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties(2), can promote autoimmunity and/or facilitate more effective tumour immunity(3,4). A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective T-reg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse T-reg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. T-reg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient T-reg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in T-reg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for T-reg immunotherapies for cancer and autoimmune disease.A CRISPR-based screening platform was used to identify previously uncharacterized genes that regulate the regulatory T cell-specific master transcription factor Foxp3, indicating that this screening method may be broadly applicable for the discovery of other genes involved in autoimmunity and immune responses to cancer.