Generation of islet antigen-specific engineered Treg for use in T1D therapy via homology-directed gene editing of conventional CD4+ T cells

Abstract

Abstract Adoptive transfer of regulatory T cells (Treg) is therapeutic in T1D mouse models. Notably, Treg specific for pancreatic islets are more potent than polyclonal Treg in blocking disease. However, the frequency of antigen-specific Treg is extremely low and ex vivo expansion has the potential to destabilize Treg leading to an effector phenotype. Here, we developed methods to generate durable, antigen-specific engineered (ed)Treg from primary human CD4+ T cells using a combination of lentiviral TCR transduction and FOXP3 homology-directed repair (HDR)-editing. Using TCRs derived from clonally expanded CD4+ T cells in T1D, we generated islet-specific edTreg that exhibit a Treg-like phenotype. Islet-specific edTreg effectively suppress proliferation and cytokine production by islet-specific effector T cells (Teff). Notably, edTreg suppress Teff recognizing the identical peptide as well as bystander Teff recognizing alternative islet antigens. Consistent with this, islet-specific edTreg suppress polyclonal islet-specific T cells derived from PBMC. Further, edTreg expressing TCR with high avidity have superior suppressive capacity to those expressing TCR with low avidity. To directly assess the capacity of edTreg to modulate T1D in vivo, we established an identical HDR-editing strategy in islet-specific murine cells. Adoptively transferred islet-specific mu-edTreg homed to the pancreas and blocked diabetes triggered by islet-specific Teff in recipient mice. Collectively, our approach enables the production of edTreg specific to pancreatic islets with the capacity to efficiently suppress islet specific responses. This approach has the capacity to deliver targeted islet specific therapy to treat or prevent T1D.