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Adoptive transfer of regulatory T cells (T regs ) is therapeutic in type 1 diabetes (T1D) mouse models. T regs that are specific for pancreatic islets are more potent than polyclonal T regs in preventing disease. However, the frequency of antigen-specific natural T regs is extremely low, and ex vivo expansion may destabilize T regs , leading to an effector phenotype. Here, we generated durable, antigen-specific engineered T regs (EngT regs ) from primary human CD4 + T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4 + T cells isolated from patients with T1D, we generated islet-specific EngT regs that suppressed effector T cell (T eff ) proliferation and cytokine production. EngT regs suppressed T effs recognizing the same islet antigen in addition to bystander T effs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngT regs homed to the pancreas and blocked diabetes triggered by islet-specific T effs or diabetogenic polyclonal T effs in recipient mice. These data demonstrate the potential of antigen-specific EngT regs as a targeted therapy for preventing T1D.

Thymic regulatory T cells (tT regs ) are potent inhibitors of autoreactive immune responses, and loss of tT reg function results in fatal autoimmune disease. Defects in tT reg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of T reg as cell therapies to establish immune tolerance. Because tT regs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous T reg -like cells from bulk CD4 + T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tT reg Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edT regs , manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tT reg Both human and murine edT regs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edT reg with robust in vitro and in vivo functional activity. Last, edT reg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edT reg production may permit broad future clinical application., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)