Differential effects of immunosuppressive drugs on DNA methylation in T cells

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

Chair(s): Drs. K.C.A. Geneugelijk & prof. dr. C. van Kooten

Thursday 9 march 2017

15:42 - 15:54h at Hendrik Marsmanzaal

Categories: Parallel - Basaal

Parallel session: Parallelsessie XV - Basaal - Moleculaire analyses en het adaptieve immuunsysteem in orgaantransplantatie

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DNA methylation controls cell functions by regulating gene expression. Changes in DNA methylation of immune-related genes can influence the immune response after transplantation. Here, we investigated the effect of tacrolimus and mycophenolic acid (MPA), two commonly prescribed immunosuppressive drugs, on changes in promoter DNA methylation of the pro-inflammatory cytokine interferon-gamma (IFN-γ) during immune activation in T cells.

Pure total T cells, naive (CCR7⁺CD45RO^-) T cells and memory (CD45RO⁺ and CCR7^-CD45RO^-) T cells were stimulated separately for 3 days with α-CD3/CD28, and in combination with tacrolimus (10 ng/mL) or MPA (0.2 µg/mL). DNA methylation was quantified on two CpG sites (CpG-54 and CpG-186) in the IFN-γpromoter region using pyrosequencing analysis. Flow cytometry was used to analyze T cell differentiation and IFN-γ protein production.

DNA methylation of IFN-γin total T cells significantly increased after stimulation from 46.6% to 54.1% at day 3 (p=0.001). Addition of tacrolimus or MPA did not affect this increase in methylation. To determine whether this observation is the result of T cell differentiation, we studied IFN-γDNA methylation in isolated naive and memory T cells. After activation, naive T cells differentiated towards a memory-like phenotype (CD45RO+) and in parallel a decrease in IFN-γDNA methylation from 79.3% to 69.8% (p=0.002) was found. Immunosuppressive drugs significantly inhibited the differentiation of naive T cells towards CD45RO+ cells (p=0.02) and differentially affected the DNA methylation changes. MPA neutralized the effect of stimulation (80.7% at day 0 to 78.2% at day 3) whilst tacrolimus showed a similar decrease after stimulation. DNA methylation and differentiation of the isolated memory population were unaffected by immunosuppressive drugs. IFN-γ protein production by these cells was significantly blocked by tacrolimus but not by MPA.

DNA methylation of IFN-γ was influenced by MPA but not by tacrolimus while the differentiation of T cells was inhibited by both immunosuppressive drugs. Changes in DNA methylation as a result of immunosuppressive medication can occur independently of changes in cell phenotype and these do not necessarily follow the same dynamics after immune activation.