Interferon-gamma DNA methylation is affected by mycophenolic acid but not by tacrolimus after T-cell activation

Immunosuppressive drug therapy is required to treat patients with autoimmune disease and patients who have undergone organ transplantation. The main targets of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA; the active metabolite of mycophenolate mofetil) are T cells. It is currently unknown whether these immunosuppressive drugs have an effect on DNA methylation-an epigenetic regulator of cellular function. Here, we determined the effect of tacrolimus and MPA on DNA methylation of the gene promoter region of interferon gamma (IFNγ), a pro-inflammatory cytokine. Total T cells, naive T cells (CCR7+CD45RO-), and memory T cells (CD45RO+ and CCR7-CD45RO-) were isolated from CMV seropositive healthy controls and stimulated with α-CD3/CD28 in the presence or absence of tacrolimus or MPA. DNA methylation of the IFNγ promoter region was quantified by pyrosequencing at 4 h, days 1, 3, and 4 after stimulation. In parallel, T-cell differentiation, and IFNγ protein production were analyzed by flow cytometry at days 1 and 3 after stimulation. Our results show that MPA induced changes in IFNγ DNA methylation of naive T cells; MPA counteracted the decrease in methylation after stimulation. Tacrolimus did not affect IFNγ DNA methylation of naive T cells. In the memory T cells, both immunosuppressive drugs did not affect IFNγ DNA methylation. Differentiation of naive T cells into a central-memory-like phenotype (CD45RO+) was inhibited by both immunosuppressive drugs, while differentiation of memory T cells remained unaffected by both MPA and tacrolimus. IFNγ protein production was suppressed by tacrolimus. Our results demonstrate that MPA influenced IFNγ DNA methylation of naive T cells after stimulation of T cells, while tacrolimus had no effect. Both tacrolimus and MPA did not affect IFNγ DNA methylation of memory T cells

Variations in DNA methylation of interferon gamma and programmed death 1 in allograft rejection after kidney transplantation

Background: The role of DNA methylation in the regulation of the anti-donor-directed immune response after organ transplantation is unknown. Here, we studied the methylation of two mediators of the immune response: the pro-inflammatory cytokine interferon γ (IFNγ) and the inhibitory receptor programmed death 1 (PD1) in naïve and memory CD8+ T cell subsets in kidney transplant recipients receiving immunosuppressive medication. Both recipients experiencing an episode of acute allograft rejection (rejectors) as well as recipients without rejection (non-rejectors) were included. Results: CpGs in the promoter regions of both IFNγ and PD1 were significantly (p < 0.001) higher methylated in the naïve CD8+ T cells compared to the memory T cell subsets. The methylation status of both IFNγ and PD1 inversely c

Disrupted regulation of serpinB9 in circulating T cells is associated with an increased risk for post-transplant skin cancer

Cutaneous squamous cell carcinoma (cSCC) is a serious complication after organ transplantation and patients benefit from an early risk assessment. We hypothesized that functional differences in circulating T cells may represent risk factors for post-transplant cSCC development. Here, we analysed genome-wide DNA methylation of circulating T cells of kidney transplant recipients before the clinical onset of cSCC, to identify differences associated with post-transplant cSCC development. This analysis identified higher DNA methylation of SERPINB9, which is an intracellular inhibitor of granzyme B, a protein that induces apoptosis in target cells. High DNA methylation of SERPINB9 in circulating T cells was confirmed in a second patient cohort during recurrent cSCC, indicating that high SERPINB9 methylation represents a persistent risk

Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer

Abstract Background Cutaneous squamous cell carcinoma (cSCC) occurs 65–200 times more in immunosuppressed organ transplant patients than in the general population. T cells, which are targeted by the given immunosuppressive drugs, are involved in anti-tumor immune surveillance and are functionally regulated by DNA methylation. Prior to kidney transplantation, we aim to discover differentially methylated regions (DMRs) in T cells involved in de novo post-transplant cSCC development. Methods We matched 27 kidney transplant patients with a future de novo cSCC after transplantation to 27 kidney transplant patients without cSCC and studied genome-wide DNA methylation of T cells prior to transplantation. From 11 out of the 27 cSCC patients, the DNA methylation of T cells after transplantation was also examined to assess stability of the observed differences in DNA methylation. Raw methylation values obtained with the 450k array were confirmed with pyrosequencing. Results We found 16 DMRs between patients with a future cSCC and those who do not develop this complication after transplantation. The majority of the DMRs were located in regulatory genomic regions such as flanking bivalent transcription start sites and bivalent enhancer regions, and most of the DMRs contained CpG islands. Examples of genes annotated to the DMRs are ZNF577, coding for a zinc-finger protein, and FLOT1, coding for a protein involved in T cell migration. The longitudinal analysis revealed that DNA methylation of 9 DMRs changed significantly after transplantation. DNA methylation of 5 out of 16 DMRs was relatively stable, with a variation in beta-value lower than 0.05 for at least 50% of the CpG sites within that region. Conclusions This is the first study demonstrating that DNA methylation of T cells from patients with a future de novo post-transplant cSCC is different from patients without cSCC. These results were obtained before transplantation, a clinically relevant time point for cSCC risk assessment. Several DNA methylation profiles remained relatively stable after transplantation, concluding that these are minimally affected by the transplantation and possibly have a lasting effect on post-transplant cSCC development