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Differential Effects of Immunosuppressive Drugs on DNA Methylation in T Cells

Peters, Fleur1; Peeters, Annemiek MA1; Hofland, Leo J.2; Betjes, Michiel GH1; Baan, Carla C.1; Boer, Karin1

doi: 10.1097/

1Nephrology and Transplantation, Erasmus MC, University Medical Center, Rotterdam, Netherlands; 2Endocrinology, Erasmus MC, University Medical Center, Rotterdam, Netherlands.

Introduction: The immune response after transplantation can be affected by DNA methylation – an epigenetic modification that regulates gene expression, including that of immune-related genes. Since the methylation of gene promoters generally acts to repress gene transcription, we wanted to know whether immunosuppressive drugs might control T cell function in this manner. In activated T cells, we therefore determined the effects of tacrolimus and mycophenolic acid (MPA) on DNA methylation of the gene promoter region of interferon-gamma (IFN-γ) – a pro-inflammatory cytokine gene.

Methods: Naive T cells (CCR7+CD45RO-) and memory T cells (CD45RO+ and CCR7-CD45RO-) isolated from healthy donors were stimulated for 3 days with α-CD3/CD28 in the presence or absence of tacrolimus (10 ng/mL) or MPA (0.2 μg/mL). DNA methylation was quantified by pyrosequencing at two CpG sites (CpG-54 and CpG-186) in the IFN-γ promoter region. T cell differentiation and IFN-γ protein production were analyzed by flow cytometry.

Results: After stimulation, naive T cells differentiated into a memory-like phenotype (CD45RO+) and this differentiation was inhibited by both immunosuppressive drugs (p=0.02). In terms of IFN γ DNA methylation, the average percentage at the two sites decreased in naive T cells from 79.3% to 69.8% (p=0.002) after stimulation. The two immunosuppressive drugs had different effects on this reduction in DNA methylation. While tacrolimus had no effect, MPA neutralized the effect of stimulation (80.7% before, and, 78.2% after stimulation). In memory T cells, DNA methylation and differentiation were unaffected by the immunosuppressive drugs. IFN-γ protein production by the memory cells on day 1 was significantly blocked by tacrolimus but not by MPA.

Conclusions: In stimulated T cells, IFN-γ DNA methylation was affected by MPA but not by tacrolimus, while T cell differentiation was inhibited by both immunosuppressive drugs. These results suggest that immunosuppressive drugs induce changes in DNA methylation independently of changes in cell phenotype. Therefore, we conclude that differentiation and DNA methylation do not necessarily follow the same dynamics after immune activation of T cells.

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