Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3

Regulatory T cells (Tregs), which specifically express the master transcription factor FoxP3, play a pivotal role in maintaining immunological homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases. While stimulation of naive CD4 T cells in the presence of TGF-beta and IL-2 can induce FoxP3+ Tregs in vitro (iTregs), the resulting cells are often unstable and have thus far hampered translational efforts. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalog gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCoR repressor complex as a novel, context-specific regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iTreg differentiation and function, independent of canonical Notch signaling. Repeated cytokine and T cell receptor signaling stimulation in vitro revealed that RBPJ-deficient iTregs exhibit increased stability compared to control cells through demethylation of the FOXP3 enhancer CNS2. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of histone acetylation by HDAC3. Lastly, RBPJ-ablated iTregs suppressed disease in a xenogenic model of graft versus host disease (GVHD). Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.