CRISPR screen in regulatory T cells reveals modulators of Foxp3

Regulatory T (T_reg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity¹. Conversely, T_reg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties², 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.