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Function, and Modulation of NK Cell
Acute Myeloid Leukemia: Expression,
B Ligand in
Receptor Activator for NF-
Grosse-Hovest and Helmut Rainer Salih
Wende, Miyuki Azuma, Pascal Schneider, Ludger
Steinbacher, Alexandra Malinovska, Constantin Maximilian
Benjamin Joachim Schmiedel, Tina Nuebling, Julia
2013; 190:821-831; Prepublished online 14
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The Journal of Immunology
Receptor Activator for NF-kB Ligand in Acute Myeloid
Leukemia: Expression, Function, and Modulation of NK Cell
Benjamin Joachim Schmiedel,* Tina Nuebling,* Julia Steinbacher,* Alexandra Malinovska,*
Constantin Maximilian Wende,* Miyuki Azuma,†Pascal Schneider,‡
Ludger Grosse-Hovest,xand Helmut Rainer Salih*
The TNF family member receptor activator for NF-kB ligand (RANKL) and its receptors RANK and osteoprotegerin are key
regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied
the involvement of RANK–RANKL interaction in NK cell–mediated immunosurveillance of acute myeloid leukemia (AML).
Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling
via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML
pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-
leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated
RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab
resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by
AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity
with a more pronounced effect on IFN-g production compared with cytotoxicity. Together, our data unravel a previously unknown
function of the RANK–RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutral-
ization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.
nology, 2013, 190: 821–831.
lignancies and in particular acute myeloid leukemia (AML) is
highlighted by studies on haploidentical stem cell transplantation
\(SCT), where the recipient’s leukemia cells fail to inhibit donor
NK cells via killer Ig-like receptors (KIRs), and KIR disparity is
associated with powerful graft versus leukemia effects and better
clinical outcome (2–4). The observation that leukemia cells may
downregulate HLA class I molecules (5, 6), presumably to escape
adaptive immunity, suggests that NK cells are also involved in
The Journal of Immu-
atural killer cells are cytotoxic lymphocytes that play
an important role in anti-tumor immunity (1). Their in-
volvement in immunosurveillance of hematopoietic ma-
controlling leukemia in an autologous setting. This is also sup-
ported by the observation that NK cell counts and activity are
reduced in patients with leukemia and that activity levels of au-
tologous NK cells are associated with survival of leukemia patients
(7–9). Because NK cell reactivity is governed by a balance of mul-
tiple inhibitory and activating receptors, interaction of NK cells
and leukemia cells is dependent on various immunoregulatory mol-
ecules far beyond HLA class I–specific inhibitory KIR receptors
(10, 11). Among others, many members of the TNF/TNFR family
influence NK cell activation, and several TNF/TNFR family
members have been found to be expressed on AML cells and in-
fluence anti-leukemia reactivity of NK cells that express their
respective counterpart (12–14).
The TNFR family member receptor activator for NF-kB (RANK;
TNFRSF11A) and its ligand (receptor activator for NF-kB ligand;
RANKL) are mainly known for their key role in regulating bone
metabolism (15, 16) but were also found to influence the inter-
action of dendritic cells (DCs) and T cells as well as the path-
ophysiology of hematopoietic malignancies and metastasis of solid
tumors (17–25). Notably, available data indicate that RANK is
also expressed on NK cells (26), but to date nothing is known
regarding the functional relevance of RANK–RANKL interaction
for NK cell reactivity. We report in this study that AML cells
express RANKL in a high proportion of cases and that NK cells
of AML patients display upregulated expression of its counterpart
RANK. To account for the fact that several TNF/TNFR family
members may mediate different effects in mice and men (14, 27,
28), we set out to study the role of RANKL in AML pathophys-
iology by using primary malignant cells of leukemia patients and
examined the functional relevance of RANK–RANKL interaction
for NK cell immunosurveillance by using PBMCs of healthy
*Department of Hematology and Oncology, Eberhard Karls University, 72076 Tue-
bingen, Germany;†Department of Molecular Immunology, Tokyo Medical and Den-
tal University, Tokyo 133-8510, Japan;‡Department of Biochemistry, University of
Lausanne, Epalinges, CH-1066 Switzerland; andxDepartment of Immunology, Eber-
hard Karls University, 72076 Tuebingen, Germany
Received for publication June 28, 2012. Accepted for publication November 9, 2012.
This work was supported by grants from Deutsche Forschungsgemeinschaft
(SA1360/7-1, SFB685 TPA7), Wilhelm Sander-Stiftung (2007.115.3), and Deutsche
Krebshilfe (109620). P.S. is supported by grants from the Swiss National Science
Address correspondence and reprint requests to Prof. Helmut Rainer Salih, Depart-
ment of Hematology and Oncology, Eberhard Karls University, Otfried-Mueller
Strasse 10, 72076 Tuebingen, Germany. E-mail address: Helmut.Salih@med.uni-
The online version of this article contains supplemental material.
Abbreviations used in this article: AML, acute myeloid leukemia; CLL, chronic
lymphoid leukemia; DC, dendritic cell; FAB, French–American–British classifica-
tion; KIR, killer Ig-like receptor; mRANKL, membrane-bound RANKL; RANK,
receptor activator for NF-kB; RANKL, receptor activator for NF-kB ligand; SCT,
stem cell transplantation; SFI, specific fluorescence index; sRANKL, soluble
by guest on December 30, 2015
multiple inhibitory and activating receptors that mediate their
effects by at least partially different molecular mechanisms (10,
46). Induction of NK cell cytotoxicity and cytokine release are
distinct events and selectively controlled (47). In addition, the
assays used for analysis of cytotoxicity and cytokine production
differed with regard to the time NK cells were cultured with their
leukemia targets. The molecular mechanisms by which TNFR
family members in general and RANK in particular influence NK
reactivity are yet largely undefined, and it is thus possible that
RANK activation may in fact mediate more pronounced effects on
NK cell cytokine production compared with cytotoxicity, which
requires further elucidation. Notably, beyond affecting NK cells,
AML-expressed RANKL could also influence other components
of immunity, as RANK protein was detected, among others, on
the surface of DCs and T cells. With regard to its immune-modify-
ing properties in the interaction of the latter cell types, RANK–
RANKL interaction has been shown to enhance survival and the
immunostimulatory capacity of DCs and to modulate effector
T cell activation. In contrast, the RANK–RANKL system has been
shown to control numbers and function of regulatory T cells
(reviewed in Ref. 48). Further work is required to elucidate
whether and how RANKL expression, besides modulating NK cell
anti-leukemia reactivity, directly or via altered DC function, also
affects T cell immunity in AML.
Overall, we provide the first evidence, to our knowledge, for
the nvolvement of RANKL in AML pathophysiology. Our data
indicate that RANKL also subverts NK cell immunosurveillance,
which can be prevented by the clinically available RANKL
Ab Denosumab, at least in RANKL-positive AML cases, which
accounted for ∼two-thirds of the patients in our analyses. Notably,
species-specific functional differences, which were observed by
us and others with regard to the function of other TNFR family
members (13, 14, 27, 28, 49), were excluded in our study by using
human NK cells and primary leukemia cells of AML patients. Our
data suggest that modulation of RANK–RANKL interaction may
serve to prevent NK cell suppression thereby holding promise to
optimize the reactivity of autologous or allogeneic NK cells in
The authors have no financial conflicts of interest.
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