Objective: The contribution of regulatory T cells (Tregs) to the pathogenesis of acute coronary syndrome (ACS) remains poorly understood. One core obstacle is the lack of Treg-specific markers. A highly conserved CpG enriched element in forkhead box P3 intron 1 (FOXP3 i 1) is unmethylated only in Tregs, and measuring the unmethylation of FOXP3 i 1 can be used to identify the role of Tregs in clinical diseases. This study investigated whether analyzing the demethylation status of FOXP3 i 1 is a more reliable means than using Treg-specific surface markers in ACS. Methods and results: We evaluated circulating Tregs percentages on different levels including cell frequencies (CD4(+)CD25(hi)FOXP3(+)Tregs and CD4(+)CD25(hi)CD45(+)naive Tregs) or FOXP3 mRNA, FOXP3 i 1 demethylation status and related cytokine secretion in 89 patients with ACS and 35 controls. FOXP3 i 1 demethylation assay showed that the amount of Tregs in ACS patients was significantly reduced than that in controls (p = 0.0005). However, flow cytometry analysis did not identify any reduction of CD4(+)CD25(hi)FOXP3(+)Tregs in ACS patients. Notably, younger patients had higher percentage of CD4(+)CD25(hi)FOXP3(+)Tregs but decreased percentage of CD4(+)CD25(hi)CD45(+)naive Tregs than either controls or older patients. Furthermore, a DNA hypomethylation agent increased the amount of CD4(+)CD25(hi)FOXP3(+)Tregs and Tregs related cytokine IL-10 and suppressed the production of pro-inflammatory cytokine interferon-g by inducing FOXP3 i 1 demethylation in vitro. Conclusions: A quantitative defect of Tregs, suggestive of decreased peripheral tolerance, could be a potential hallmark of ACS disease. Targeting FOXP3 i 1 demethylation might elevate the inhibitory activity of Tregs in ACS. (C) 2013 Elsevier Ireland Ltd. All rights reserved.