Basaal I - P39 - Immunosuppressive medication and DNA methylation of the Inter­feron-gamma promoter in T cells

F.S. Peters, A.M.A. Peeters, L.J. Hofland, M.G.H. Betjes, K. Boer, C.C. Baan

Chair(s): dr. Michiel Betjes, Erasmus MC, Rotterdam

Thursday 10 march 2016

13:00 - 13:30h at Foyer

Categories: Postersessie

Parallel session: Postersessies XI - Opgesplitst in 3 tijdblokken en 3 categoriëen (klinisch, basaal, donatie)

Introduction:
DNA methylation is responsible for the control of cell function by regulation of gene expression, high methylation in the promoter region is associated with gene silencing. Methylation changes in immune-related genes can influence the immune response after transplantation. In this study, we first investigated the changes in DNA methylation of the pro-inflammatory cytokine interferon-gamma (IFN-γ) during immune activation and second analyzed the effect of the commonly used immunosuppressant tacrolimus.

Patients, materials and methods:
Pure CD3+ T cells were isolated from total PBMCs by MACS negative selection. These T cells were stimulated for 7 days with anti-CD3/CD28, in combination with 10-6 M 5’-aza-2’-deoxycytidine, a demethylating agent, or with 10 ng/mL tacrolimus. Cells were harvested at day 0, 1, 3, 4, 7 and after bisulfite conversion and subsequent PCR, DNA methylation was quantified on two CpG sites (CpG-54 and CpG-186) in the IFN-γ promoter region using pyrosequencing analysis. Flow cytometry was used to analyze IFN-γ protein production.

Results:
Stimulation significantly increased the percentage DNA methylation of the IFN-γ promoter from day 0 to day 4 (CpG-54; median: 52% range: (39%-66%) to 64% (54%-75%) with p=0.003, CpG-186: 42% (31%-52%) to 51% (35%-56%) with p=0.04). Addition of tacrolimus did not significantly change the DNA methylation. 5’-Aza-2’-deoxycytidine, which served as a positive control, lead to a significant decrease in DNA methylation from day 0 to day 4 (CpG-54: 52% (41%-66%) to 31% (23%-36%) with p=0.002, CpG-186: 46% (31%-52%) to 22% (18%-32%) with p=0.0006). As expected IFN-γ protein production was completely blocked in the presence of tacrolimus.

Conclusions:
Based on these data we conclude that following immune activation the DNA methylation of IFN-γ significantly increased and that this change occurs within 4 days after stimulation. Suppression of IFN-γ protein production in the presence of tacrolimus is not due to a change in DNA methylation after immune activation. Therapeutic concentrations of the immunosuppressant tacrolimus do not alter the methylation status of IFN-γ during immune activation.