The instability of the regulatory T (Treg) cell lineage precludes their therapeutic potential in transplantation and autoimmune diseases. Herein, we invented a novel approach to stabilize the Foxp3 expression and suppressor function of TGF-β/IL-2 induced Treg (iTreg) cells using 3 compounds (3C), including a pan HDAC inhibitor NaB, a Histone methyltransferase G9a inhibitor UC46, and vitamin C. We found that during iTreg cell polarization NaB and UC46 synergistically enhanced the chromatin accessibility and the recruitment of DNA demethylase TETs at the Foxp3 CNS2 locus, while vitamin C enhanced TET enzyme activity to enforce active DNA demethylation. The 3C treatment further prevented the subsequent re-methylation at the Foxp3 CNS2 locus by inhibiting the IRF4 and p-STAT6 mediated DNMT recruitment. Hence, 3C conditioned iTreg (3C-iTreg) cells persistently expressed high levels of Foxp3 and exerted potent suppressive function, associated with the complete demethylation of the Foxp3 CNS2 locus. Upon in vivo adoptive transfer, virtually all 3C-iTreg cells maintained high Foxp3 expression within the 6-month study period. Next, we determined the therapeutic function of 3C-iTreg cells using three distinct models. In an experimental autoimmune encephalomyelitis (EAE) model, iTreg and 3C-iTreg cells were derived from the MOG-specific 2D2 TCR transgenic CD4⁺ T cells. Adoptive transfer of 2.5 million 3C-iTreg but not iTreg cells almost completely prevented the EAE development in mice immunized with the MOG_35-55 peptide. In a colitis model, iTreg and 3C-iTreg cells were derived from polyclonal CD4⁺ T cells. Adoptive transfer of 0.5 million 3C-iTreg but not iTreg cells completely abrogated 1 million transferred CD4⁺CD45RB^high cell-mediated weight loss and colon inflammation in Rag1^-/- mice. In a Balb/c-to-Rag1^-/-.B6 skin transplantation model, polyclonal alloreactive iTreg and 3C-iTreg cells were derived from B6 CD4⁺ T cells upon stimulation with Balb/c splenocytes. Adoptive transfer of 0.1 million 3C-iTreg but not iTreg cells completely prevented 0.2 million transferred CD4⁺Foxp^– B6 T cell-mediated skin graft rejection. Taken together, 3C treatment stabilizes iTreg cell lineage through inducing an epigenetically fixed state for Foxp3 expression. 3C-iTreg cells have a great therapeutic potential in transplantation and autoimmune diseases.

CITATION INFORMATION: Xiao X., Zhang X., Li J., Chen S., Lan P., Dou Y., Chen W., Li X. A 3C Approach Drives Epigenetic Fixation of Foxp3 Expression and Stabilizes Treg Cell Lineage and Therapeutic Function Am J Transplant. 2017;17 (suppl 3).

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