小柯机器人

美国西北大学Deyu Fang、加州大学旧金山分校Alexander Marson等研究人员合作，利用调节性T细胞（Treg）的CRISPR筛选鉴定出Foxp3的调节因子。2020年4月29日，《自然》在线发表了这一成果。

研究人员开发了一个基于CRISPR的汇集筛选平台，可用于筛选原代小鼠Treg细胞中的表型，并应用该技术对约500个核因子进行了功能丧失的靶向筛选，从而鉴定了促进或破坏Foxp3表达的基因调控程序。

研究人员确定了Foxp3表达的几种调节因子，包括泛素特异性肽酶22（Usp22）和Ring指蛋白20（Rnf20）。Usp22是SAGA染色质修饰复合物的去泛素化模块的成员，被发现是稳定Foxp3表达的阳性调节因子。而筛选显示Rnf20（一种E3泛素连接酶）可以充当Foxp3的负调节因子。

小鼠中Usp22的Treg特异性敲除降低了Foxp3蛋白水平，并导致其抑制功能缺陷，从而导致自发性自身免疫，但在多种癌症模型中均能防止肿瘤生长。可以通过敲除Rnf20来挽救Usp22缺陷型Treg细胞中的Foxp3不稳定，从而揭示Treg细胞中的泛素蛋白开关。

这些结果揭示了以前未知的Foxp3调节因子，并证明了一种筛选方法，其可以广泛应用于发现Treg免疫疗法用于治疗癌症和自身免疫性疾病的新靶标。

据了解，Treg是控制免疫应答和维持体内平衡所必需的，但它是抗肿瘤免疫的主要障碍。相反，以正常转录因子Foxp3的丧失和促炎为特征的Treg不稳定性可以促进自身免疫和/或促进更有效的肿瘤免疫。对调节Foxp3途径的全面理解可能会产生靶向自身免疫性疾病和癌症的有效Treg疗法。新的功能遗传工具可能用于系统性剖析调节Foxp3表达的基因调控程序。

附：英文原文

Title: CRISPR screen in regulatory T cells reveals modulators of Foxp3

Author: Jessica T. Cortez, Elena Montauti, Eric Shifrut, Jovylyn Gatchalian, Yusi Zhang, Oren Shaked, Yuanming Xu, Theodore L. Roth, Dimitre R. Simeonov, Yana Zhang, Siqi Chen, Zhongmei Li, Jonathan M. Woo, Josephine Ho, Ian A. Vogel, Grace Y. Prator, Bin Zhang, Youjin Lee, Zhaolin Sun, Igal Ifergan, Frdric Van Gool, Diana C. Hargreaves, Jeffrey A. Bluestone, Alexander Marson, Deyu Fang

Issue&Volume: 2020-04-29

Abstract: Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg 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 Treg 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. Treg-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 Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

DOI: 10.1038/s41586-020-2246-4

Source: https://www.nature.com/articles/s41586-020-2246-4

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html