Regulatory T (Treg) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases1–3. Although stimulation of naive CD4+ T cells in the presence of TGFβ and IL-2 can induce FOXP3+ Treg cells in vitro (iTreg cells), the resulting cells are often unstable and have thus far hampered translational efforts4–6. 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 catalogue 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 negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iTreg differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iTreg cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iTreg cells more effectively suppressed xenogeneic graft-versus-host disease than control iTreg cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.
