Research Article 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity Thomas Stübig, 1 Anita Badbaran, 1 Tim Luetkens, 2 York Hildebrandt, 1 Djordje Atanackovic, 2 Thomas M. C. Binder, 3 Boris Fehse, 1 and Nicolaus Kröger 1 1 Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany 2 Department of Oncology/Hematology, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany 3 Department for Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany Correspondence should be addressed to Nicolaus Kr¨ oger; nkroeger@uke.de Received 6 December 2013; Accepted 28 January 2014; Published 13 March 2014 Academic Editor: Beatrice Gaugler Copyright © 2014 homas St¨ ubig et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity efects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and low cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza ater alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-+ T-helper 1 cells (h1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data conirm the increase of Treg compartment, while CD8+ T-efector cell numbers were reduced. 5-Aza treatment results in a shit from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinlammatory h1-cells, indicating a strong inhibition of tumor- speciic T-cell immunity by 5-Aza. 1. Introduction Methylation plays a central role in the epigenetic regulation of gene expression [1]. Cancer cells in particular use hyper- methylation to switch of a vast number of genes, responsible for growth inhibition, diferentiation, and apoptosis [2]. Treatment induced diferentiation in myeloid malignancies was reported to exhibit substantial clinical beneit and, accordingly, demethylating drugs like 5-Azacytidine (5-Aza) have been introduced into the therapy of myelodysplastic syndrome (MDS) [3] and acute myeloid leukemia (AML) [4]. Ater cellular uptake, 5-Aza is phosphorylated to 5-aza- 2  -deoxycytidine-5  -triphosphate and subsequently is incor- porated into the DNA, to inhibit the methylating enzyme DNA methyltransferase [5]. Supplementary to its efects on genes responsible for cell growth and diferentiation, 5-Aza was found to upregulate tumor-associated antigens, such as cancer-testis antigens (CTA), potentially augmenting immune recognition of malignancies [6–8]. Several small studies have recently introduced simultaneous application of 5-Aza combined with donor lymphocyte infusions in AML patients [9–12]. However, due to its broad mechanism of action, 5-Aza may have an impact on the quality of antitumor immunity in various ways, as reported by a recent study describing its immunosuppressive properties in mice [13]. Like most eukaryotic cells, CD4+ T-cells use epigenetic mechanisms to regulate lineage commitment [14]. Particu- larly transcription factor FoxP3, as a master regulator of reg- ulatory T-cells [15], has been described to be strongly reg- ulated by methylation [16, 17]. Even though our knowledge on epigenetic regulation in CD8+ T-cells is still limited, memory function and Interferon gamma (IFN-) production Hindawi Publishing Corporation Mediators of Inflammation Volume 2014, Article ID 418292, 12 pages http://dx.doi.org/10.1155/2014/418292