Background: Epigenetic mechanisms like acetylation and methylation have been gradually appreciated to play a critical role in the generation and function of various immune cells. Previous studies have been largely focused on the induction of regulatory T cells (Tregs) by DNMT inhibitors (DNMTi, eg, 5-Aza-2'-deoxycytidine, 5-AZA), we hereby attempt to explore their influence on effector T cells (Teffs) and the underlying mechanism.

Method: The in vivo effect of DNMTi on allo-immune response was evaluated using an allogeneic mouse cardiac transplantation (BALB/C to C57BL/6 mice). T Cell proliferation, differentiation and cytokine production in the presence or absence of 5-AZA in vitro were measured by flow cytometry and ELISA. The expression of a panel of cell cycle related proteins were determined with real-time PCR and western blot. Methylation specific PCR (MSP) and Bisulfite sequencing PCR (BSP) were performed to assess the DNA methylation status in the promoter region of p15, p16, p21, p27 and Foxp3.

Results: Consecutive 5-AZA (0.25 or 0.5 mg/kg/day) administration intraperitoneally for 14 days significantly prolonged allograft survival in a dose-dependent manner (MST=45±22.6 and 92.2±10.8 days, respectively, n=5, p<0.05 vs. vehicle), associated with diminished anti-donor recall response. Further mechanistic studies revealed 5-AZA not only effectively inhibited naïve T cells proliferation, but also inhibited Th17 cell differentiation while preserving the Treg generation and function, thus deviated the immune balance towards immune regulation. Along this line, we found that 5-AZA potently inhibited T cell cycle progression as well as increased T cell apoptosis. While the protein expression of cyclins or cyclin-dependent kinases were not overtly different, several main cell cycle inhibitors (p15, p16, p21, p27) in T cells were significantly increased in the presence of 5-AZA (10uM) in vitro (p<0.05 vs. vehicle). MSP and BSP confirmed lower levels of CpG island methylation in the promoter region of p15, p16, p21 and p27, suggesting these cell cycle inhibitors are potential targets of DNMTi.Demethylation of the Foxp3 promoter was also identified.

Conclusion: Demethylation of key cell cycle inhibitors is associated with delayed T cell cycle progression, DNMTi can be a novel Treg-sparing means in the treatment of acute allograft rejection and tolerance induction.

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