Epigenetic Editing of FOXP3 in Human T Cells Induces Overexpression and Is Sufficient to Create a Regulatory T Cell Phenotype in Vitro

Abstract

Defects in T cells (Tregs) have been identified in some autoimmune diseases. The development of Tregs is marked by epigenetic modifications, associated with demethylation of the FOXP3 gene. Guide RNA (gRNA) sequences targeting the human FOXP3 promoter, TSDR, and CNS1 region were designed. An epigenetic editing SUNTAG construct was used to facilitate demethylation of specific genomic regions. Constructs containing SUNTAG construct transfected by electroporation into Jurkat cells and cultured for 24 hours. FOXP3 and CTLA4 gene expression was determined by qPCR and FOXP3-TSDR and DNA methylation quantified by bisulfite pyrosequencing three days after transfection. Primary CD4+ T cells, stained with CellTrace reagent and combined with FOXP3 epigenetically-edited Jurkat cells were stimulated. Suppression of T cell division was determined by flow cytometry. All gRNAs increased FOXP3 expression. Also, epigenetic editing of FOXP3 resulted in increased expression of the Treg-related gene CTLA4. Epigenetically edited cells resulted in suppression of naïve T-cell proliferation by 20-30%. Epigenetic editing of FOXP3 using a SUNTAG construct induces DNA demethylation, overexpression, and a regulatory T cell phenotype. Our data are intriguing but need confirmation, particularly to clarify the persistence of induced DNA methylation changes and resistance to phenotype switching. If confirmed, this approach has the potential to significantly improve upon current methods of T-reg generati