Increased phenotypic and functional stability of human allospecific induced Tregs is associated with Vitamin C-mediated FOXP3 TSDR demethylation

Introduction: -induced Tregs (iTregs) for managing inflammatory diseases and promoting tolerance, their translation to clinical use is limited by concerns over phenotypic and functional instability, largely due to an epigenetic profile distinct from thymic Tregs. Methods: To address this, we generated and expanded iTregs in the presence of Vitamin C, known to activate TET enzymes that demethylate critical gene regions such as FOXP3. Antigen-specific Tregs were derived from allogeneic co-cultures of monocyte-derived dendritic cells and naïve T cells. After 7 days, allo-Tregs were isolated by FACS and further expanded for 3 weeks with IL-2, TGF-β, rapamycin, with or without Vitamin C supplementation. Results: T cell proliferation, even in the presence of pro-inflammatory cytokines. Notably, this proinflammatory milieu did not trigger the production of intracellular cytokines IL-17 and IFN-γ by allo-iTregs, confirming their sustained phenotypic and functional stability. Advanced analysis demonstrated that allo-iTregs possess a unique profile, setting them apart from conventional T cells. Pyrosequencing of the FOXP3 TSDR revealed reduced CpG methylation in Vitamin C-treated iTregs (60.9% at day 21 and 43.5% at day 28) compared to untreated allo-iTregs (85.9% at day 21 and 80.5% at day 28) and naïve T cells (92.8%). In addition, transcriptomic analysis demonstrated that the core Treg transcriptional signature remained largely intact with Vitamin C treatment, irrespective of cytokine exposure, whereas the major transcriptional changes were associated with activation, proliferation, and cell cycle regulation. Discussion: In summary, Vitamin C enhances both the phenotypic and functional stability of allospecific iTregs, even under proinflammatory conditions, correlating with increased TSDR demethylation, while preserving the transcription of key Treg genes. These results suggest that Vitamin C-treated allospecific iTregs are superior candidates for immunotherapy strategies to promote long-term tolerance in transplant recipients.