Introduction: Cytomegalovirus (CMV) is a major cause of morbidity and mortality after transplantation. Recently, we confirmed that adoptive transfer of autologous IE-1/pp65-specific T-cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV-induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. Methods & Results: In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV-specific cytokine and proliferative response compared to patients with non-recurring CMV infections. Analysis of in vitro expanded CMV-specific T cells revealed that CD25highCD39+ T cells functionally suppress CD25low effector T cells (Teff) upon epitope-specific reactivation. Additionally to this functional evidence, we could demonstrate that CD25highCD39+ T cells belong to the peripherally induced subset of Tregs (iTregs), for several reasons: (1) they are Helios negative, (2) they express a methylated foxp3 locus in contrast to natural thymus-derived Tregs (nTregs), and (3) they share the same dominant TCR-Vβ-CDR3 clones with functionally distinct CD25low Teff indicating that Teff and iTreg can differentiate from one clone with specificity to the same viral epitope. Moreover, the same clones were present in freshly isolated CD4+CD25high and CD4+CD25low T cells suggesting their in vivo generation. In contrast to nTregs, in vitro expanded CMV-specific iTregs produce IFNg upon antigen-specific re-stimulation. However, they secrete low amounts of IL-2 and are able to suppress IL-2 responses of Teff cells, like nTregs. Therefore, we were interested whether the effector and/or immunoregulatory cytokine expression of CMV-specific iTregs is merely a transient ability modulated by in vitro culturing or, in contrast, is a stably imprinted phenotype inherited from progenitor cells. As epigenetic modifications are an inheritable basis for memory of cytokine expression, we analyzed the DNA methylation status of the respective promoters of CMV-specific T cells compared to nTregs. Preliminary results suggest that culturing of CMV-specific T cells with rapamycin actively maintained methylation of the IL-2 promoter, thus blocked IL-2 production. Additionally, the epigenetic control of IL-2 seems to be distinct in iTregs vs Teff cells. Conclusion: Taken together, our findings demonstrate that peripheral iTreg generation seems to be related to frequent antigen appearance and that the suppressive impact of CMV-specific iTregs is of importance for solid organ transplanted patients suffering from CMV infections. If the methylation status of cytokine promoters is a tool to further differentiate natural from induced Tregs remains to be investigated.