調節性T細胞CRISPR篩選揭示Foxp3調節因子
作者：
小柯機器人發布時間：2020/4/30 13:11:12
美國西北大學Deyu Fang、加州大學舊金山分校Alexander Marson等研究人員合作，利用調節性T細胞（Treg）的CRISPR篩選鑑定出Foxp3的調節因子。2020年4月29日，《自然》在線發表了這一成果。
研究人員開發了一個基於CRISPR的匯集篩選平臺，可用於篩選原代小鼠Treg細胞中的表型，並應用該技術對約500個核因子進行了功能喪失的靶向篩選，從而鑑定了促進或破壞Foxp3表達的基因調控程序。
研究人員確定了Foxp3表達的幾種調節因子，包括泛素特異性肽酶22（Usp22）和Ring指蛋白20（Rnf20）。Usp22是SAGA染色質修飾複合物的去泛素化模塊的成員，被發現是穩定Foxp3表達的陽性調節因子。而篩選顯示Rnf20（一種E3泛素連接酶）可以充當Foxp3的負調節因子。
小鼠中Usp22的Treg特異性敲除降低了Foxp3蛋白水平，並導致其抑制功能缺陷，從而導致自發性自身免疫，但在多種癌症模型中均能防止腫瘤生長。可以通過敲除Rnf20來挽救Usp22缺陷型Treg細胞中的Foxp3不穩定，從而揭示Treg細胞中的泛素蛋白開關。
這些結果揭示了以前未知的Foxp3調節因子，並證明了一種篩選方法，其可以廣泛應用於發現Treg免疫療法用於治療癌症和自身免疫性疾病的新靶標。
據了解，Treg是控制免疫應答和維持體內平衡所必需的，但它是抗腫瘤免疫的主要障礙。相反，以正常轉錄因子Foxp3的喪失和促炎為特徵的Treg不穩定性可以促進自身免疫和/或促進更有效的腫瘤免疫。對調節Foxp3途徑的全面理解可能會產生靶向自身免疫性疾病和癌症的有效Treg療法。新的功能遺傳工具可能用於系統性剖析調節Foxp3表達的基因調控程序。
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