Elucidating inflammation-induced cell fate decisions in primary human Tregs

Abstract Adoptive regulatory T-cell (Treg) therapy has remarkable efficacy in promoting immune tolerance in preclinical models of transplantation and autoimmune disease. However, lineage-tracing studies have revealed that Tregs can undergo epigenetic reprogramming in chronically inflamed tissue environments, resulting in the acquisition of proinflammatory functions and the capacity to exacerbate autoimmunity. Despite intense interests in developing Treg therapy for humans, inflammation-induced human Treg lineage-decommitment remains poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven human Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)-2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. Single-cell transcriptomic and chromatin accessibility analyses of human Tregs undergoing destabilization revealed a gradual closing of a Treg-specific element (A) and a reciprocal opening of a competing element (B) at the IRF4 locus. CRISPR-mediated excision of the B element (IRF4ΔB) preserved Treg identity and enhanced in vitro suppressive activity following prolonged inflammatory exposure. Ongoing experiments aim to evaluate the in vivo function and stability of IRF4ΔB Tregs in a xenogeneic graft-versus-host model. These findings may facilitate the design of Treg therapeutics with greater potency and improved safety.