OPEN
LETTER TO THE EDITOR
Azacytidine impairs NK cell activity in AML and MDS patients undergoing MRD-based pre-emptive treatment after allogeneic stem cell transplantation
Blood Cancer Journal (2013) 3, e136; doi:http://dx.doi.org/10.1038/bcj.2013.35
Web End =10.1038/bcj.2013.35 ; published online 30 August 2013
We have recently demonstrated within a prospective trial (RELAZA1 study) that minimal residual disease (MRD)-triggered pre-emptive treatment with azacytidine (AZA) is able to prevent or at least delay hematologic relapse in patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT).1 Besides direct apoptotic effects at higher doses, AZA is considered to act as a hypomethylating agent at lower doses. Of note, hypomethylating effects not only affect cancer cells by inducing the re-expression of previously silenced tumor suppressor genes, but also inuence gene expression in bystander cells, such as T lymphocytes (T cells). In line with this hypothesis, several groups recently reported epigenetic regulation of the FOXP3 locus, the critical transcription factor, driving regulatory T-cell differentiation. AZA-induced demethylation induced upregulation of FOXP3 and expansion of T-regulatory cells (Tregs), which might also contribute to lower graft-versus-host disease rates in the setting of allogeneic HSCT.2
Besides T cells, studies investigating the impact of AZA on other immune cell populations such as dendritic cells (DCs) or natural killer (NK) cells are rather limited. As AZA is currently explored in many clinical trials in the post-allograft setting, we aimed to study its impact on NK and DCs in vitro as well as in vivo when given as pre-emptive treatment within the RELAZA1 study.1 For in vitro experiments we used AZA doses of 100 nM, 1 mM and 3 mM, based on the observation of peak plasma levels of 1 mM in humans after the application of the standard 75 mg/m2 subcutaneous AZA dose.
First we investigated the impact of AZA on DCs, which are professional antigen-presenting cells, involved in the activation of innate and adaptive antitumor responses. Functional data revealed that 6-sulfo LacNAc (slan) DCs represent a major subset of native human blood DCs, which produce various proinamma-tory cytokines and contribute to antitumor immunity by activating NK and T cells.3,4 To explore how AZA modulates the secretion of proinammatory cytokines by slanDCs in vitro, we isolated slanDCs immunomagnetically from peripheral blood of three healthy
Figure 1. Impact of AZA on immunostimulatory properties of slanDCs. (ac) Freshly isolated slanDCs from three healthy donors were cultured in the presence or absence of 100 nM, 1 mM or 3 mM
AZA. SlanDCs were maintained for 6 h and then activated by adding LPS. After additional 18 h, supernatants were collected and the concentration of TNF-a (a), IL-12 (b) and IL-6 (c) was determined by
ELISA. Columns represent meanss.e. of results obtained from three different healthy donors. For each donor the mean of triplicate determination was used. (d) Freshly isolated slanDCs were cultured for 6 h with or without 100 nM, 1 mM or 3 mM AZA, washed, and coincubated with eFluor670-stained allogeneic CD4 T cells. After 7 days, proliferation of CD4 T cells was analyzed by ow cytometry. Values represent the percentage of proliferating cells (lled) compared to non-activated cells (empty).
Citation: Blood Cancer Journal (2013) 3, e136; doi:10.1038/bcj.2013.35
& 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13 http://www.nature.com/bcj
Web End =www.nature.com/bcj
Letter to the Editor
2 donors, at high purity (490%) as described previously.3 Subsequently slanDCs were cultured with different concentrations of AZA (100 nM, 1 mM and 3 mM). After the addition of lipopolysaccharide (LPS, 1 mg/ml) for additional 18 h to stimulate cytokine secretion, supernatants were collected and
secretion of proinammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). As demonstrated in Figure 1ac, the production of tumor necrosis factor (TNF)-a, interleukin (IL)-6 and IL-12 by slanDCs was not altered by AZA. This is in accordance with data of a recent study, indicating that AZA
Figure 2. Inuence of AZA on NK cell activation. (af) Freshly isolated NK cells from up to ve healthy donors were incubated with or without 100 nM, 1 mM or 3 mM AZA and IL-2 for 5 days. (a) Supernatants were harvested and INF-g production of NK cells was determined by ELISA. The results are presented as meanss.e. of duplicate determinations. Asterisks indicate a statistically signicant difference (Pr0.05), n 3.
(b) AZA-treated and non-treated IL-2-stimulated NK cells were co-cultured with 51Cr-labeled K-562 cells at an E/T ratio of 1:1. After 4 h of incubation, chromium release was measured. The results are presented as meanss.e. of triplicate determinations. Asterisks indicate a statistically signicant difference (Pr0.05), n 3. (c) AZA-treated and non-treated IL-2-stimulated NK cells were stained using annexin V and
propidium iodide to determine apoptotic cell death by ow cytometry. The percentages of cells positive for annexin V and/or propidium iodide are indicated in the dot blots as well as the percentages of cells being negative for both agents. The results of one representative donor out of ve performed with similar results are demonstrated. (df) The expression levels of TRAIL, NKG2D and NKp46 of AZA-treated and non-treated IL-2-stimulated NK cells were determined by ow cytometry. Each dot represents the mean of mean uorescence intensity (MFI) for each surface molecule obtained from NK cells of ve healthy donors. Error bars represent the standard deviation. Asterisks indicate a statistically signicant difference (Pr0.05). (gi) NK cells were obtained from AML or MDS patients after HSCT before (d1 day 1), during (d5)
and at the end (d7) of AZA treatment cycles 1 and 2. Subsequently, expression levels of TRAIL, NKG2D and NKp46 were measured by ow cytometry. Each dot represents the mean of MFI for each surface molecule obtained from NK cells of eight patients. Error bars represent the standard deviation. Asterisks indicate a statistically signicant difference (Pr0.05).
Blood Cancer Journal & 2013 Macmillan Publishers Limited
Letter to the Editor
3
does not inuence the release of the above-mentioned proinammatory cytokines.5 But in contrast, AZA signicantly impaired the secretion of IL-27, which is known to be inversely correlated with Th17 cell activation.
In further experiments, we evaluated whether AZA alters the capacity of slanDCs to stimulate T-cell responses. For this purpose, slanDCs were again exposed to different concentrations of Aza (100 nM, 1 mM, 3 mM) and co-cultured with immunomagnetically isolated allogeneic CD4 T cells for 7 days. Proliferation was determined by 3H-thymidine incorporation. Again, the obtained results revealed no signicant effect of AZA on slan DC-induced T-cell proliferation (Figure 1d). This is in agreement with a previous nding, indicating that AZA at a concentration of 4 mM does not alter the capacity of monocyte-derived DCs to stimulate T-cell proliferation.5
Next we explored the impact of AZA on NK cells, which have an important role in the regulation of immune responses against malignant cells. To investigate the impact of AZA on the immunomodulatory ability and cytotoxic potential of NK cells, human CD56 CD3 NK cells were isolated immunomagnetically from freshly prepared peripheral blood mononuclear cells of healthy donors at a purity of 490% as assessed by ow cytometry. Isolated NK cells of each donor were then exposed in vitro to increasing concentrations of AZA (100 nM, 1 mM, 3 mM) in the presence of the activating cytokine IL-2 (300 U/ml) for 5 days. Interestingly, AZA signicantly impaired IFN-g release by IL-2-activated NK cells (Figure 2a). To explore the cytotoxic potential of NK cells we used the chromium release assay6 with the tumor cell line K562 as target cells. As depicted in Figure 2b, AZA efciently inhibited the cytotoxic activity of NK cells at a concentration of 3 mM. These results are in agreement with a previous report, indicating that AZA suppresses the cytolytic potential of a human NK cell line as well as native human NK cells.7 To get insights into the underlying mechanisms, we explored whether AZA modulates the viability of NK cells. As demonstrated in Figure 2c, AZA increased the percentage of apoptotic NK cells at a concentration of 3 mM. Further more, we investigated the impact of AZA on the expression of important NK cell receptor and cytotoxic molecules, given the fact, that some NK cell receptors are regulated by promoter methylation of the respective genes.7,8 Notably, AZA reduced the surface density of TRAIL and the activating receptors NKG2D and NKp46 on NK cells (Figure 2df). In contrast, the expression of NKp30, NKp44, perforin, granzyme-B or Fas ligand was not altered by AZA (data not shown).
To further substantiate these in vitro ndings, we investigated the effect of AZA on the expression of NK cell-activating receptors and cytotoxic molecules in vivo within the prospective RELAZA trial (NCT00422890).1 This clinical study investigated whether pre-emptive AZA treatment (75 mg/m2 for 7 days) in MDS or AML patients with MRD is a suitable tool to prevent hematologic relapse. In agreement with our in vitro results, we found a signicantly reduced expression of TRAIL, NKG2D or NKp46 on NK cells during AZA therapy in comparison to NK cells obtained before treatment (Figure 2gi) considering eight patients. Respectively, the observed reduction of NK cell-activating receptors and TRAIL during AZA treatment was seen in patients with a reduction or stable course of MRD, suggesting that the clinical effect of AZA is not mediated by NK cell activation.
In summary, despite the limited sample sizes we demonstrated that AZA does not modulate the potential of human slanDCs to produce proinammatory cytokines and promote proliferation of CD4 T cells. These results indicate that immunostimulatory properties of slanDCs, which may have an important role in tumor cell elimination are maintained by AZA. Further studies revealed
that AZA efciently inhibits IFN-g secretion and reduces cytotoxic potential of activated human NK cells. This impaired NK cell function might be explained by the potential of AZA to increase NK cell apoptosis and reduce the surface expression of the cytotoxic effector molecule TRAIL as well as the activating receptors NKG2D and NKp46 on NK cells in vitro and in vivo. Taken together these data suggest that the clinical effects of AZA in the post-transplant setting are not mediated by enhancing NK cell activity. Instead, the data indicate an inhibitory effect of AZA on NK cell function. Until the mechanisms of AZA are not fully understood, patients should be carefully selected to receive hypomethylating agents in the post-transplant setting and therapy should be given within controlled clinical trials.
CONFLICT OF INTEREST
UP has received speakers honoraria and research funding from Celgene. The remaining authors declare no conict of interest.
ACKNOWLEDGEMENTS
The technical assistance of Barbel Lbel, Karin Gnther, Ivonne Habermann and Susann Helas is greatly appreciated. This work was supported by grants from the Jose Carreras Leukemia foundation, SFB 655 of the DFG to UP and MB, and the Medical Faculty, Technical University of Dresden to RW.
C Schnefeldt1,5, K Sockel1,5, R Wehner2, S Sopper3, D Wolf4, M Wermke1, C Thiede1, U Oelschlagel1, G Ehninger1,
M Bornhaser1, U Platzbecker1 and M Schmitz2
1Medical Clinic I, University Hospital Carl Gustav Carus, Dresden, Germany;
2Institute of Immunology, Medical Faculty Carl Gustav Carus, Dresden, Germany;
3University Hospital Innsbruck, Innsbruck, Austria and
4Medical Clinic III, University Hospital Bonn, Bonn, Germany E-mail: mailto:[email protected]
Web End [email protected]
5These authors contributed equally to this work.
REFERENCES
1 Platzbecker U, Wermke M, Radke J, Oelschlaegel U, Seltmann F, Kiani A et al.
Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial. Leukemia 2012; 26: 381389.2 Goodyear OC, Dennis M, Jilani NY, Loke J, Siddique S, Ryan G et al. Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood 2012; 119: 33613369.3 Schakel K, von Kietzell M, Hansel A, Ebling A, Schulze L, Haase M et al. Human 6-sulfo LacNAc-expressing dendritic cells are principal producers of early interleukin-12 and are controlled by erythrocytes. Immunity 2006; 24: 767777.4 Schmitz M, Zhao S, Deuse Y, Schakel K, Wehner R, Wohner H et al. Tumoricidal potential of native blood dendritic cells: direct tumor cell killing and activation of NK cell-mediated cytotoxicity. J Immunol 2005; 174: 41274134.5 Frikeche J, Clavert A, Delaunay J, Brissot E, Gregoire M, Gaugler B et al. Impact of the hypomethylating agent 5-azacytidine on dendritic cells function. Exp Hematol 2011; 39: 10561063.6 Baltz KM, Krusch M, Bringmann A, Brossart P, Mayer F, Kloss M et al. Cancer immunoediting by GITR (glucocorticoid-induced TNF-related protein) ligand in humans: NK cell/tumor cell interactions. FASEB J 2007; 21: 24422454.7 Gao XN, Lin J, Wang LL, Yu L. Demethylating treatment suppresses natural killer cell cytolytic activity. Mol Immunol 2009; 46: 20642070.8 Chan HW, Kurago ZB, Stewart CA, Wilson MJ, Martin MP, Mace BE et al. DNA methylation maintains allele-specic KIR gene expression in human natural killer cells. J Exp Med 2003; 197: 245255.
This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/
Web End =http:// http://creativecommons.org/licenses/by/3.0/
Web End =creativecommons.org/licenses/by/3.0/
& 2013 Macmillan Publishers Limited Blood Cancer Journal
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright Nature Publishing Group Aug 2013