Multi-phenotype CRISPR-Cas9 Screen Identifies p38 Kinase as a Target for Adoptive Immunotherapies. Cancer cell T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions. 10.1016/j.ccell.2020.05.004

CRISPR screen in regulatory T cells reveals modulators of Foxp3. Cortez Jessica T,Montauti Elena,Shifrut Eric,Gatchalian Jovylyn,Zhang Yusi,Shaked Oren,Xu Yuanming,Roth Theodore L,Simeonov Dimitre R,Zhang Yana,Chen Siqi,Li Zhongmei,Woo Jonathan M,Ho Josephine,Vogel Ian A,Prator Grace Y,Zhang Bin,Lee Youjin,Sun Zhaolin,Ifergan Igal,Van Gool Frédéric,Hargreaves Diana C,Bluestone Jeffrey A,Marson Alexander,Fang Deyu Nature Regulatory T (T) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity. Conversely, T instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties, can promote autoimmunity and/or facilitate more effective tumour immunity. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective T 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 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-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 cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in T 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 immunotherapies for cancer and autoimmune disease. 10.1038/s41586-020-2246-4

A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function. Immunity Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function. 10.1016/j.immuni.2020.06.011