🎗Functional Investigation of Induced Tregs Generated via Locus-Specific Epigenome Reprogramming of Effector T Cells

Introduction: The long-term success of kidney transplantation remains hindered by chronic rejection outcomes and adverse effects of systemic immunosuppression. Regulatory T cells (Tregs), as key mediators of immune tolerance, are shown to promote allograft tolerance and prolong graft survival in preclinical models. However, clinical translation is constrained by the scarcity of endogenous Tregs and their susceptibility to functional instability, whereby prolonged exposure to inflammatory cues can erode suppressive capacity and promote conversion towards an effector-like T cell phenotype. 
T cell fate and therapeutic performance are intrinsically governed by the epigenetic landscape. In Tregs, the Treg-specific demethylation region (TSDR) within the FOXP3 locus is a key determinant of lineage stability, acting as an epigenetic ‘lock-in’ that drives Treg genetic programmes and sustains constitutive suppressive function. Harnessing this key feature of Treg identity, we aim to recapitulate this TSDR-specific pattern in effector populations to produce therapeutically robust Tregs that resist phenotypic plasticity. 
Methods: Here, we employed a transient mRNA-based TET1-dCas9 epigenetic editing platform to achieve targeted demethylation of the FOXP3-TSDR in primary human T cells, generating epigenetically engineered Tregs (EpiTregs) (Fig. 1).  Editing efficacy and phenotypic reprogramming were first evaluated in vitro. Two complementary in vivo approaches were subsequently utilised to evaluate the functional capability and stability at different timescales: a short-term proliferation assay (peritoneal lavage) and humanised skin transplantation model. Harvested skin grafts were brought through spatial transcriptomics profiling to characterise the molecular landscape of skin rejection upon adoptive transfer of EpiTregs. 

Results: Targeted TSDR demethylation in EpiTregs resulted in robust and sustained induced FOXP3 expression and acquisition of an anti-inflammatory phenotype that persisted beyond 28 days (Fig. 2a). EpiTregs demonstrated potent suppressive activity against leukocytes in vitro and in vivo. In the humanized skin transplantation models, EpiTregs maintained suppressive capacity up to 20 days post-adoptive transfer and significantly attenuated localised inflammatory responses (Fig. 2b). 

Discussion: This TET1-dCas9 epigenetic editing approach demonstrates that transient, locus-specific epigenetic modulation can induce durable phenotypic reprogramming. The ‘hit-and-run’ strategy avoids risks associated with viral integration including prolonged off-target effects and insertional mutagenesis, while enabling the scalable generation of stable and functionally robust therapeutic Treg products from readily accessible starting populations. These findings support the potential of targeted epigenome editing as a clinically translatable platform for next-generation cellular therapies in transplantation.