Abstract
The “master transcription factor” FOXP3 regulates the differentiation, homeostasis, and suppressor function of CD4+ regulatory T (Treg) cells, which are critical in maintaining immune tolerance. Epigenetic regulation of FOXP3 expression has been demonstrated to be important to Treg cell development, but the induction of human Treg cells through epigenetic modification has not been clearly described. We report that the combination of the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and suboptimal T cell receptor (TCR) stimulation promoted CD4+CD25hFOXP3+ T cell induction from human CD4+CD25− T cells. 5-Aza treatment enhanced the expression of Treg cell signature genes, such as CD25, FOXP3, CTLA-4, and GITR, in CD4+CD25h cells. Moreover, 5-Aza-treated CD4+CD25h T cells showed potent suppressive activity in a cell contact-dependent manner and reduced methylation in the Treg-specific demethylated region (TSDR) in the FOXP3 gene. The analysis of cytokine production revealed that CD4+CD25− T cells with 5-Aza treatment produced comparable levels of interferon (IFN)-γ and transforming growth factor (TGF)-β, but less IL-10 and more IL-2, when compared to cells without 5-Aza treatment. The increased IL-2 was indispensible to the enhanced FOXP3 expression in 5-Aza-treated CD4+CD25h cells. Finally, 5-Aza-treated CD4+CD25h T cells could be expanded with IL-2 supplementation alone and maintained FOXP3 expression and suppressor function through the expansion. Our findings demonstrate that DNA demethylation can enhance the induction of human Treg cells and promise to solve one of the challenges with using Treg cells in therapeutic approaches.
Highlights
The subset of CD4+ T lymphocytes called regulatory T (Treg) cells have been demonstrated to prevent autoimmune disease and transplant rejection in human and experimental animal models [1, 2]
Suboptimal T cell receptor (TCR) Stimulation Combined with 5-Aza Treatment Induced More Human CD4+CD25hFOXP3+ T Cells from CD4+CD25− T Cells
To confirm that CD25 expression correlated to Treg cells, we measured the levels of the Treg master transcription factor FOXP3
Summary
The subset of CD4+ T lymphocytes called regulatory T (Treg) cells have been demonstrated to prevent autoimmune disease and transplant rejection in human and experimental animal models [1, 2]. Natural Treg (nTreg) cells are CD4+ T cells with strong T cell receptor (TCR) signaling that survive negative selection, develop, and mature. 5-Azacytidine Promotes Human Treg Induction in the thymus. Adaptive Treg cells are induced in the periphery following specific tolerogenic stimulation [3]. The forkhead winged-helix transcription factor FOXP3 has been shown to be critical for Treg cell differentiation and function. Ectopic expression of FOXP3 confers suppressive function and activates Treg cell signature genes, such as Il2ra (CD25), Ctla (CTLA-4), and Tnfrsf (GITR), in peripheral CD4+CD25− T cells [4, 5]. Activation of human CD4+CD25− T cells through TCR stimulation results in transient, low level expression of FOXP3 without conferring suppressive activity [6, 7], indicating that FOXP3 must be constitutively expressed to maintain Treg cell function
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