Tumor Immunoediting by NKp46

Moran Elboim, Roi Gazit, Chamutal Gur, Hormas Ghadially,

This information is current as
of April 18, 2011
Gili Betser-Cohen and Ofer Mandelboim
J Immunol 2010;184;5637-5644; Prepublished online 19 April
2010;
doi:10.4049/jimmunol.0901644
http://www.jimmunol.org/content/184/10/5637

References This article cites 37 articles, 13 of which can be accessed free at:
http://www.jimmunol.org/content/184/10/5637.full.html#ref-list-1

Downloaded from www.jimmunol.org on April 18, 2011

Article cited in:
http://www.jimmunol.org/content/184/10/5637.full.html#related-urls

Subscriptions Information about subscribing to The Journal of Immunology is online at

http://www.jimmunol.org/subscriptions
Permissions Submit copyright permission requests at
http://www.aai.org/ji/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at
http://www.jimmunol.org/etoc/subscriptions.shtml/

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
9650 Rockville Pike, Bethesda, MD 20814-3994.
Copyright ©2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
 The Journal of Immunology

Tumor Immunoediting by NKp46

Moran Elboim,* Roi Gazit,† Chamutal Gur,* Hormas Ghadially,* Gili Betser-Cohen,* and
Ofer Mandelboim*
NK cells interact with a wide variety of hazardous cells including pathogen-infected and tumor cells. NKp46 is a specific NK killer
receptor that recognizes various influenza hemagglutinins and unknown tumor ligands. It was recently shown that NKp46 plays
a significant role in the in vivo eradication of tumor cells; however, the role played by NKp46 in vivo with regard to tumor de-
velopment is still unclear. In this study, we used the 3-methylcholanthrene (MCA)-induced fibrosarcoma model in NKp46-deficient
mice to test the NKp46 recognition of carcinogen-induced tumors. We show that although the rate of MCA-induced tumor formation
was similar in the presence and in the absence of NKp46, the expression of its unknown ligands was NKp46 dependent. The unknown
NKp46 ligands were nearly absent in tumors that originated in wild-type mice, whereas they were detected in tumors that originated
in the NKp46-deficient mice. We demonstrate that the interactions between NKp46 and its MCA tumor-derived ligands lead to the
secretion of IFN-g but not to the elimination of the MCA-derived tumor cells. In addition, we show that the in vivo growth of
MCA-derived tumor cells expressing high levels of the NKp46 ligands is NKp46 and IFN-g dependent. Thus, we present in this
study a novel NKp46-mediated mechanism of tumor editing. The Journal of Immunology, 2010, 184: 5637–5644.

Downloaded from www.jimmunol.org on April 18, 2011

N
atural killer cells are innate lymphocytes able to quickly
respond to a wide range of transformed and pathogen-
infected cells (1–3). The activity of NK cells is controlled
by a balance of signals delivered by inhibitory and activating re-
ceptors (1, 4). The main activating receptors include NKp46,
NKp44, and NKp30 (collectively known as the natural cytotoxicity
receptors [NCRs]), NKp80, 2B4, and NKG2D (5, 6). In mice,
NKG2D and 2B4 are present; however, the only functional member
of the NCRs is the mouse ortholog of the human NKp46, named
NCR1 (5, 7). Only a few ligands were identified for the activating
NK receptors. These ligands are either tumor derived, such as B7-
H6, an NKp30 ligand, stress induced, such as the ligands for
NKG2D, or self-ligands, such as AICL for NKp80 and BAT3 for
NKp30 (6, 8–11). With regard to the NCRs, we have demonstrated
that the viral hemagglutinin (HA) proteins of various influenza
strains interact with the human NKp44 and NKp46 and with the
mouse NCR1 and that these interactions lead to increased killing of
influenza-infected cells (7, 12, 13). In addition, we showed that
NKp30 interacts with the HCMV’s pp65 protein, leading to the
inhibition of killing by NK cells (14).
Our group knocked out the Ncr1 gene and inserted the GFP re-
porter gene instead (7). Almost all NK cells in the knockout (KO)
and heterozygous mice are labeled green, and the function of the
*Lautenberg Center for General and Tumor Immunology, Institute for Medical
Research Israel Canada, Hebrew University-Hadassah Medical School, Jerusalem,
Israel; and †Immune Disease Institute, Harvard Stem Cell Institute, Harvard Medical
School, Boston, MA 02115
Received for publication May 27, 2009. Accepted for publication March 14, 2010.
This work was supported by grants from the Israeli Science Foundation, the Israeli
Science Foundation (Morasha), a Croatia Israel research grant, a MOST-DKFZ re-
search grant, the European Consortium (Grant MRTN-CT-2005), the Rosetrees Trust,
the Israel Cancer Association (Grant 20100003), and the Association for International
Cancer Research (all to O.M). O.M is a Crown Professor of Molecular Immunology.
Address correspondence and reprint requests to Prof. Ofer Mandelboim, Lautenberg
Center for General and Tumor Immunology, Institute for Medical Research Israel
Canada, Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israel.
E-mail address: oferm@ekmd.huji.ac.il
Abbreviations used in this paper: HA, hemagglutinin; NCR, natural cytotoxicity re-
ceptor; poly(I):poly(C), polyinosinic:polycytidylic acid.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
NCR1 receptor in the KO mice is impaired (7). We have demonstrated
that the NCR1 KO mice are sensitive to influenza infection, thus
confirming in vivo the importance of the NKp46/NCR1-HA inter-
actions. Although the function of NK cells against viral infections was
studied in detail (1, 15, 16), only limited number of studies in-
vestigated the in vivo function of NKp46 against tumors, mainly by
using tumor cell lines (17, 18). Thus, whether NKp46 is also important
for the growth of carcinogen-induced tumors and what would be the
effect of the NKp46 on tumor immunoediting was not investigated.
The immune response against developing tumors was studied in
detail using the MCA model (19, 20). Smyth and colleagues (20)
define three stages of tumor immunoediting: elimination, equilib-
rium, and escape. They demonstrated that the major immune pop-
ulations involved in tumor immunoediting are T cells. The in-
volvement of NK cells in immunoediting has so far been studied
only in relation to NKG2D and DNAM1 (19, 21, 22). It was shown
that the MCA-induced tumors were more frequently observed
in mice treated with blocking anti-NKG2D Abs (22). In contrast,
another report failed to observe an increased incidence of MCA-
induced sarcomas in NKG2D-deficient mice (19). Moreover, be-
cause NKG2D is also expressed by several T cell subsets (10),
the effect of a specific NK cell receptor such as NKp46 on the de-
velopment of carcinogen-induced tumors and on tumor immuno-
editing remains obscure. By using the MCA-induced carcinogenesis
model in Ncr1-deficient mice, we demonstrate in this study a tumor
immunoediting mechanism aimed at reducing the NCR1-dependent
IFN-g secretion during tumor development.
Materials and Methods
Mice
All experiments were performed using 8- to 12-wk-old C57BL/6 mice. The
generation of the Ncr1gfp/gfp mice was described previously (7). IFN-g–
deficient mice (B6.129S7-Ifngtm1Ts/J) were obtained from The Jackson
Laboratory (Bar Harbor, ME). All experiments were performed in the
specific pathogen-free unit of the Hadassah Medical School (Ein Kerem,
Jerusalem), according to the guidelines of the ethical committee.
Carcinogenesis
Mice were injected i.m. with 50, 25, or 5 mg MCA (Sigma-Aldrich,
St. Louis, MO) emulsified in olive oil by heating in boiling water. Tumor
growth was examined weekly for a period of at least 200 d.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901644
5638
Generation of MCA-induced cell lines
Two weeks after tumor appearance, the tumors were removed, and part of
the tumor was taken for digestion by trypsin for 5 min. Following this
trypsin treatment, tumors were mashed in a culture dish, and RPMI 1640
medium was added. After an overnight incubation in 37˚C and 5% CO2,
tumor clumps were removed, and the remaining cells were used as tumor-
derived cell lines that were grown in RPMI 1640 medium supplemented
with 10% FCS and 1% sodium pyruvate, glutamine, nonessential amino
acids, and penicillin-streptomycin solution.
Fusion proteins and flow cytometry
The NCR1-Ig, NKp46-Ig, and NKG2D-Ig fusion proteins were generated in
COS-7 cells and purified by affinity chromatography using a protein G
column, as described previously (13). The staining of cell lines was vi-
sualized using a secondary PE-conjugated goat anti-human Abs (Jackson
ImmunoResearch Laboratories, West Grove, PA).
BW reporter assay
A total of 50,000 BW or BW-NKp46-z cells were coincubated at 1:1 ratio
with 50,000 irradiated (3000 rad) specified cells of various cell lines for
48 h at 37˚C and 5% CO2. Supernatants were collected and the levels of
IL-2 were quantified by using commercially available anti-mouse IL-2
mAbs (BD Pharmingen, San Diego, CA) and standard ELISA.2332
Growth of MCA tumor cell lines
All experiments were performed by using 8- to 12-wk-old C57BL/6 mice.
Groups of 12 Ncr1+/+, Ncr1gfp/gfp, or IFN-g–deficient mice were injected
s.c. with 5 3 105 of the various MCA-induced tumor cells, and tumor size
was measured every 2–3 d after injection using a micrometer.
NK cell depletion
Mice were injected i.v with 200 mg anti-NK1.1 Ab (PK136) in 200 ml sterile
PBS. Ab was injected into the tail vein every 7 d during the experiment.
Sterile PBS was injected as a control. Depletion was verified every 3 d after
injection.
In vitro cytotoxicity assays
For the in vitro assay, 200 mg polyinosinic:polycytidylic acid [poly(I):poly
(C)] (Sigma-Aldrich) were injected i.p., and splenocytes were removed
18 h later. Target cells were labeled with [35S]methionine and incubated
for 5 h at 37˚C with effector splenocytes at various E:T ratios. Specific
killing was calculated as described previously (23).
In vivo lung clearance assay
For the in vivo clearance assay, we used a published fluorescence labeling
method with a modification to avoid possible influence of intrinsic GFP
present in the Ncr1gfp/gfp mice. Target cells were labeled with Vybrant Dil
cell labeling solution (Molecular Probes, Carlsbad, CA), and control HeLa
FIGURE 1. The absence of NCR1 has
no effect on MCA tumor growth. Mice
were injected with 50 mg MCA (Ncr1+/+,
n = 11; Ncr1gfp/gfp, n = 12) (A), with 25 mg
MCA (Ncr1+/+, n = 12; Ncr1gfp/gfp, n = 12)
(B), or with 5 mg MCA (Ncr1+/+, n = 16;
Ncr1gfp/gfp, n = 22) (C). Figure shows per-
centage of tumor-positive mice following
injection. One representative experiment is
shown out of three performed.
TUMOR IMMUNOEDITING BY NKp46
cells were labeled with Vybrant DiD. A total of 2 3 106 cells of each of the
cell type were mixed in 400 ml PBS and injected into the tail vein of C57BL/6
Ncr1+/+ or Ncr1gfp/gfp mice. Lungs were explanted 3 h later. Single-cell
suspensions were obtained by using a cell strainer, and fluorescence was
analyzed by flow cytometry. The ratio of the tested MCA-induced cell lines
target cells to the internal control HeLa cells was calculated.
CD107a assays
Mice were injected i.p with 200 mg poly(I):poly(C), and spleens were
removed 18 h later. NK cells were isolated from splenocytes using the
mouse NK isolation kit (Miltenyi Biotec, Auburn, CA) and an autoMACS
instrument according to the manufacturer’s instruction. NK cells (5 3 105)
were coincubated with the indicated target cells in a ratio of 1:1 in the
presence of 0.1 mg of an allophycocyanin-conjugated CD107a Ab (1D4B;
Southern Biotechnology Associates, Birmingham, AL) for 2 h. Afterward,
cells were washed and analyzed by flow cytometry. To analyze the in-
volvement of NKG2D, saturating amounts of blocking Abs to NKG2D
(C7; a gift from W. Yokoyama, Department of Medicine, Washington
University School of Medicine, St. Louis, MO) were added to the ex-
periments before the start of the coculture.
Cytokine secretion assay
Mice were injected i.p. with 200 mg poly(I):poly(C), and spleens were
removed 18 h later, splenocytes were extracted, and NK cells were isolated
as described above. NK cells (50,000) were coincubated with the indicated
Downloaded from www.jimmunol.org on April 18, 2011
target cells at a ratio of 1:1 for 48 h at 37˚C and 5% CO2. Supernatants
were collected and IFN-g levels were determined using a commercially
available ELISA assay (BD Pharmingen).
Results
NCR1 plays no significant role in the development of
MCA-induced fibrosarcomas
It was previously demonstrated that NCR1 is involved in the in vivo
killing of some tumor cell lines (7, 17, 18). To address the role played
by NCR1 in carcinogen-induced tumors, we used the MCA-induced
fibrocarcinoma model. NCR1 KO-Ncr1gfp/gfp and wild-type (WT)-
Ncr1+/+ littermates were injected with MCA, and fibrosarcomas
arose at the site of injection after a long latency period. Fig. 1
demonstrates the timeline of tumor progression in KO and WT mice.
In MCA-injected mice (50 mg/mouse), tumors began to appear in the
Ncr1gfp/gfp mice 78 d postinjection, whereas in the Ncr1+/+ mice
tumors appeared ∼20 d later (Fig. 1A). When lower doses of
MCA were injected (25 mg/mouse), tumors started to appear in the
Ncr1gfp/gfp mice at ∼88 d postinjection, whereas in the Ncr1+/+ mice,
tumors were observed ∼10 d later (Fig. 1B). Finally, early initial
tumor growth was also observed in the Ncr1gfp/gfp injected with
The Journal of Immunology
5 mg/mouse MCA (Fig. 1C). The differences observed between
the Ncr1gfp/gfp and the Ncr1+/+ were noted only in one to two mice,
and in most cases, there were no significant differences in the
tumor growth rates between the Ncr1gfp/gfp and Ncr1+/+ mice (Fig.
1). Thus, we concluded that NCR1 plays no significant role in
preventing the development of the MCA-induced fibrosarcomas.
NKp46-mediated immunoediting
To further investigate the role played by NCR1 in MCA-induced
tumor cell recognition, we generated cell lines from the primary
tumors of both Ncr1gfp/gfp and Ncr1+/+ genotypes. Cell lines with
a WT or KO suffix were originated from Ncr1+/+ or Ncr1gfp/gfp
mice, respectively. Because the NCR1 ligands are unknown, we
studied whether NCR1 recognizes ligands on the MCA-induced
cell lines by using NCR1-Ig and NKp46-Ig fusion proteins. For
this purpose, the extracellular portion of each receptor was fused
to the Fc of human IgG1, as described previously (13). As can be
seen in Fig. 2A, all cell lines were recognized by NCR1-Ig fusion
protein. Remarkably, we noticed that all cell lines that originated
in the WT mice expressed low levels of NCR1 ligands, whereas
cell lines that originated in NCR1-deficient mice expressed, in
general, higher levels of NCR1 ligands (Fig. 2A). Several KO cell
lines such as 19KO, 70KO, 79KO, and 99KO expressed lower
levels of NCR1 ligands compared with the other KO cell lines.
However, on average, the levels of the NCR1 ligands were higher
in the Ncr1gfp/gfp mice as compared with their WT littermates (Fig.
2A). Furthermore, all cell lines expressed low levels of NKG2D
ligands, irrespective of whether they were generated in the WT or
in the Ncr1gfp/gfp mice (Fig. 2B and data not shown).
To further investigate whether indeed in the presence of NCR1 the
emerging tumors express less NCR1 ligands, we used an additional
assay using reporter cell lines. BW cells are mouse thymoma tumor
cells that had been transfected with constructs encoding a chimeric
FIGURE 2. Expression of NCR1 and NKG2D ligands on the MCA-induced tumors. A, Binding of NCR1-Ig to the MCA-induced cell lines and average
of all cell lines originated in Ncr1+/+ versus all cell lines originated in Ncr1gfp/gfp. pp , 0.005; t test; the differences are statistically significant. B, Binding
of NKG2D-Ig to the MCA-induced cell lines. Gray shading, Background staining with secondary Ab only; dark lines, specific staining with the indicated
fusion protein. The various tumor cell lines are indicated. Data from one of four independent experiments are shown.
5639
protein in which the transmembrane and intracellular tail of the mouse
CD3z-chain is fused to the extracellular portion of NKp46 protein.
In this system, triggering the chimeric protein by its ligand will lead
to the secretion of IL-2 (thus, the unknown NCR1 ligands, if present,
should induce IL-2 production). Because we observed that the hu-
man NKp46-Ig recognizes the MCA tumor ligands similarly as
NCR1-Ig (examples are shown in Fig. 3A and data not shown), we
used the BW cells expressing the human NKp46 protein fused to
z-chain to address the functional relevance of the NCR1 ligands.
Importantly, and in agreement with the NCR1-Ig binding results
(Fig. 2), only the tumors that originated in the NCR1-deficient mice
were able to stimulate IL-2 production and secretion to a level
similar to that observed after cross-linking the NKp46-z chimeric
receptor by a mAb (Fig. 3B). In contrast, all tumors that were iso-
lated from WT mice failed to induce IL-2 secretion (Fig. 3B). These
observations imply on the existence of NCR1-mediaed tumor im-
munoediting process. As expected, because of tumor heterogeneity,
not all tumors that originated in the Ncr1gfp/gfp mice induced NKp46-
mediated IL-2 secretion. Importantly, a direct correlation was ob-
served between the expression of the NCR1 ligands detected by the
NCR1-Ig fusion protein (Fig. 2A) and the stimulation of the IL-2
Downloaded from www.jimmunol.org on April 18, 2011
production. The KO cell lines, which demonstrated low levels of
NCR1 ligands expression (19KO, 70KO, 79KO, and 99KO; Fig.
2A), did not stimulate IL-2 secretion whereas others, which express
high levels of the NCR1 ligands, did (Fig. 3B). Thus, in the presence
of NCR1, the developing tumors undergo an immunoediting process
aiming at reducing NKp46 ligands expression.
Increased immunogenicity of the Ncr1gfp/gfp-derived cell lines
Becausethe NCR1ligands are downregulatedwhentumorsgrowinthe
WT mice facing NCR1 pressure, we assumed that these ligands might
influence tumor growth. We therefore injected MCA-induced tumor
cell lines that express either high or low levels of the NCR1 ligands into
5640 TUMOR IMMUNOEDITING BY NKp46

Downloaded from www.jimmunol.org on April 18, 2011

FIGURE 3. NKp46-mediated immunoediting. A, Binding of NKp46-Ig and NCR1-Ig to the MCA-induced cell lines. Gray shading, background staining
with secondary Ab only; dark lines, specific staining with the indicated fusion protein. The various tumor cell lines are indicated. Data from one of three
independent experiments are shown. B, IL-2 secretion from BW-NKp46 cell lines incubated with the various targets indicated in the x-axis. Anti-NKp46
mAb (461.G1 clone) was used as positive control. pp , 0.05; t test; the differences are statistically significant.

Ncr1gfp/gfp and Ncr1+/+ mice. Tumor sizes were measured for a period
of 3 wk until the tumors had grown too big and the mice had to be
sacrificed because of ethical reasons.
In accordance with our prediction, the tumors that originated
in the absence of NCR1 (such as 60KO, 20KO, and 82KO) and
expressed the putative cellular NCR1 ligands, grew faster and
reached a bigger mass in Ncr1gfp/gfp mice as compared with the
Ncr1+/+ mice (Fig. 4A). In contrast, tumors that originated in the
Ncr1+/+ mice facing the NCR1-dependent immunoediting pro-
cess, such as cell lines 2WT, 3WT, and 40WT, demonstrated
similar growth kinetics in WT and in the NCR1-deficient mice
(Fig. 4A). Importantly, a direct correlation was observed between
the NCR1-Ig staining (Fig. 2), the BW reporter assays (Fig. 3) and
the in vivo growth of the different tumors. No differences in tumor
growth rates were observed when low levels of NCR1 ligands
were present (even with the 19KO, which expresses low levels of
NCR1 ligands), whereas differences were noticed when tumors
expressed high levels of the unknown ligands (Fig. 4A). The
growth rate differences were NCR1 dependent and NK cell de-
pendent as when NK cells were depleted by injection of anti-
NK1.1 Abs, the tumor growth rates of the WT and Ncr1gfp/gfp mice
were similar (Fig. 4B).
The killing of all MCA-induced tumors is NKG2D but not NCR1
dependent
Next we sought to determine the mechanism responsible for the
differences in the tumor growth. We first performed in vitro killing
assays using splenocytes isolated from either Ncr1gfp/gfp or Ncr1+/+
mice and incubated them with the MCA-induced tumor cell lines.
Ncr1+/+ and Ncr1gfp/gfp mice were preactivated by poly(I):poly(C)
before performing the experiments, because in the absence of such
stimulation, the basal killing of target cells by NK cells was
minimal (data not shown). As can be seen in Fig. 5A, all of the
MCA-induced tumor cells were killed by activated NK cells, but
no differences were observed in the percentages of specific killing
among the various NK cells derived either from Ncr1gfp/gfp, from
Ncr1+/+, or from the Ncr1gfp/+ mice (data not shown).
We next assumed that it might be that the poly(I):poly(C) acti-
vation of NK cells, which results in increased killing, abrogate the
differences that exists between the Ncr1gfp/gfp and the Ncr1+/+ mice.
We therefore used the in vivo lung clearance assay in which NK
cells are not manipulated. Fluorescence-labeled tumors cells were
injected into the tail vein of the various mice. Because the injection
of cells might vary between different mice and as we aimed to
compare between the ability of the Ncr1gfp/gfp and Ncr1+/+ mice to
kill tumor cells in vivo, each injection was controlled by reference
cells such as HeLa cells. As can be seen in Fig. 5B, no differences
were observed between the Ncr1gfp/gfp and Ncr1+/+ mice, even in
the in vivo lung clearance killing assays. Moreover, the cell line
2WT, which expresses low levels of the NCR1 ligands, was killed
more efficiently in vivo, as compared with line 82KO (Fig. 5B),
further indicating that NCR1 plays no significant role in the killing
of the fibrosarcoma tumors. The PD1.6 tumor cell line, which is
killed in vitro and in vivo by NK cells in an NCR1-dependent
manner (17), was used as a positive control and, indeed, it exhibited
increased killing by the Ncr1+/+ NK cells. Thus, we concluded that
NCR1 plays no role in the MCA tumor killing.
It was previously shown that NKG2D is involved in the killing of
MCA-induced tumors (22). To test the role of NKG2D in the
presence and in the absence of NCR1, we stained the NK cells from
Ncr1gfp/gfp and heterozygous-Ncr1gfp/+ mice for CD107a (LAMP-1,
a marker for cytoplasmic granules release), after incubating the NK
cells with the various MCA-induced tumors cell lines. We used the
Ncr1gfp/gfp and the Ncr1+/gfp mice, because in these mice, NK cells
can be visualized specifically and accurately, as a GFP reporter gene
was introduced into the Ncr1 locus (7). The Ncr1+/gfp mice are
The Journal of Immunology 5641

FIGURE 4. Increased growth of the

MCA tumors originated in the NCR1-
deficient mice. The indicated MCA-
induced tumor cell lines were injected
s.c. into Ncr1gfp/gfp and Ncr1+/+ mice
(500,000 cells/mouse). Tumor size was
measured during a 3-wk period. A,
Tumor growth in mice injected with
cell lines originated in Ncr1gfp/gfp mice,
and tumor growth in mice injected
with cell lines originated in Ncr1+/+
mice. B, NK cells were depleted 2 d
before the injection of tumor cell lines.
Upper panel, Tumor growth in mice
injected with the 60KO line. Lower
panel, Tumor growth in mice injected
with line 2WT. Shown is one represen-
tative experiment of three performed.

Downloaded from www.jimmunol.org on April 18, 2011

normal and behave similarly to the Ncr1+/+ mice (7, 24, 25). As
above, no significant difference in the surface expression of
CD107a was observed between the NK cells derived either from the
Ncr1+/gfp or from the Ncr1gfp/gfp mice (Fig. 5C). Importantly, the
killing of all MCA-induced cell lines, irrespective of whether they
were obtained from the Ncr1gfp/gfp or Ncr1+/+ mice, was NKG2D
dependent as it was blocked by an anti-NKG2D mAb (Fig. 5C).
Thus, NKG2D and not NCR1 is involved in the killing of the MCA-
induced tumor by NK cells.
NCR1 ligands trigger IFN-g secretion
Activation of NK cells could result in direct cytotoxicity and/or in
cytokine secretion, mainly IFN-g. Because we observed that
NCR1 plays no significant role in the direct killing of the MCA-

FIGURE 5. The killing of all MCA-induced tumor lines is NKG2D dependent. A, Target cells were radioactively labeled and then incubated with NK
cells from Ncr1+/+ and Ncr1gfp/gfp mice for 5 h at an E:T. ratio of 100:1. Data from one of three independent experiments are shown. Values are mean 6 SD
for triplicate samples. B, In vivo cytotoxicity assay. Vybrant Dil-labeled MCA-induced tumor cell lines (2WT mice and 82KO) or the PD1.6 cell line was
injected together with a control Vybrant DiD-labeled HeLa cells into the tail vein of Ncr1+/+ and Ncr1gfp/gfp mice. Cells in the lungs were quantified 3 h
after injection by using the flow cytometry. Data from one of three independent experiments are shown. Values are mean 6 SD for triplicate samples. C,
Ncr1gfp/gfp and Ncr1+/gfp mice were incubated for 2 h with MCA-induced tumor cell lines and with YAC-1 or HeLa cells, in the presence or absence of
a blocking Ab for NKG2D. The CD107a levels on NK cells were quantified using the flow cytometry. Data from one of three independent experiments are
shown. pp , 0.05; t test; the differences are statistically significant.
5642
FIGURE 6. IFN-g secretion is in-
duced by MCA tumor cell lines origi-
nated in the absence of NCR1. IFN-g
secretion of poly(I):poly(C)-activated
NK cells from mice of different geno-
types in response to a 48-h incubation
with different target cells. Values are
mean 6 SD for duplicate samples. Data
from one of three independent experi-
ments are shown. pp , 0.05; t test; the
differences between the Ncr1gfp/gfp and
Ncr1+/gfp mice, which are statistically
significant.
induced tumors (Fig. 5), our next goal was to determine whether
the NCR1 ligands are involved in IFN-g secretion by NK cells.
NK cells were extracted from poly(I):poly(C)-activated spleno-
cytes of both Ncr1+/gfp and Ncr1gfp/gfp mice and were incubated
with MCA-induced tumor cell lines or control tumor cells lines.
Supernatants were collected 48 h later and tested for the presence
of IFN-g by ELISA. Significantly higher levels of IFN-g were
observed when NK cells derived from the Ncr1+/gfp mice were
incubated with target cells that originated in the Ncr1gfp/gfp mice
(Fig. 6). IFN-g secretion was not enhanced above the background
level when NK cells were incubated with tumor cell lines that
originated in the Ncr1+/+ mice or with cell lines that originated in
the Ncr1gfp/gfp mice but express low levels of the NCR1 ligands
(Fig. 6). When HeLa cells, which do not express the murine
NKp46 ligands, were incubated with the NK cells no IFN-g se-
cretion was observed.
The Ncr1gfp/gfp NK cells were not defective in their IFN-g pro-
duction, because equal amounts of IFN-g were detected when YAC-
1 cells were incubated with NK cells derived from both genotypes.
Because IFN-g secretion was not detected when NCR1 ligands
were absent (Fig. 6), we concluded that the MCA-induced tumors
are killed mainly in an NKG2D-dependent manner (Fig. 5),
whereas the production of cytokines is mainly NCR1 dependent.
To demonstrate in vivo that the unknown NCR1 ligands are in-
volved in the secretion of IFN-g by NK cells, we injected the various
MCA-induced tumor cell lines into IFN-g–deficient mice
(B6.129S7-Ifngtm1Ts/J). As predicted, in the IFN-g–deficient mice,
TUMOR IMMUNOEDITING BY NKp46
the tumor sizes were similar between the Ncr1gfp/gfp and the IFN-g–
deficient mice in all MCA-induced cell lines (Fig. 7). As above,
reduced tumor growth rates were observed in the WT mice when
MCA-induced tumors expressing high levels of NCR1 ligands were
Downloaded from www.jimmunol.org on April 18, 2011
used (Fig. 7).
Taken together, our results demonstrate a novel NCR1-de-
pendent tumor immunoediting process of MCA-induced tumors,
which probably evolved to diminished IFN-g secretion.
Discussion
The MCA-induced fibrosarcoma model has been used to establish
principles of tumor immunosurveillance and immunoediting (19, 26,
27). It was previously reported that MCA- induced tumors were
more frequent in mice treated for a long period of time with blocking
NKG2D Abs (22). In contrast, another report failed to observe an
increased incidence of MCA-induced sarcomas in NKG2D-deficient
mice (19). The reasons for these discrepancies are not completely
understood; however, because NKG2D is not expressed exclusively
by NK cells, it is still unclear whether the absence of an NK specific
killer receptor will affect MCA-induced tumor growth.
By using the MCA model in the Ncr1gfp/gfp gene-targeted mice,
we were able to address two important issues. The first is whether
NCR1 is crucial for immune surveillance of MCA-induced fi-
brosarcomas in vivo and the second is whether there is a selective
pressure that will favor the downregulation of the unknown NCR1
ligands (i.e., immunoediting). In this paper, we report that al-
though NCR1 does not play a significant role in preventing the
FIGURE 7. Tumor growth in the
IFN-g–deficient mice. The indicated
MCA-induced tumor cell lines were
injected s.c. into Ncr1gfp/gfp, Ncr1+/+,
and IFN-g–deficient mice (500,000
cells/mouse). Tumor size was mea-
sured during a 3-wk period. Shown is
one representative experiment of
three performed.
The Journal of Immunology
formation of MCA-induced fibrosarcomas, the developing tumors,
facing the NCR1 pressure, downregulate its unknown ligands.
Many different functions are assigned to NK cells (28), and it
was even demonstrated that NK cells possess some adoptive im-
munity features (29–31). In addition to their cytolytic ability, a well-
established function of NK cells is cytokine secretion (32). IFN-g is
a prototypic NK cell cytokine that was shown to be essential for the
rejection of transplanted tumor cells and for the decrease in MCA-
induced tumor growth (33). Importantly, we show that in the MCA-
model, IFN-g secretion by NK cells is NCR1 dependent, because
cell lines that originated in Ncr1+/+ mice and express low levels of
the NCR1 ligands could not trigger the IFN-g secretion by NK cells.
As expected, because of tumor heterogeneity, not all cell lines that
originated in the Ncr1gfp/gfp expressed high levels of the NCR1 li-
gands. In general, however, we observed higher levels of NCR1 li-
gands in tumors developed in vivo in the absence of NCR1, and in all
cases, a direct correlation was observed between the expression of
the NCR1 ligands (as detected by NCR1-Ig staining or BW reporter
assays), IFN-g secretion, and the in vivo growth of tumors. Thus, in
the presence of NCR1, developing tumor undergo an immunoediting
process to reduce the NCR1 ligands expression, probably to affect
IFN-g secreted by NK cells.
The unknown NCR1 ligands seem to have a dual role, either
inducing cytokine secretion (as shown in this paper for the MCA-
induced tumors) or promoting cytolytic activity by NK cells (as
shown for some lymphomas [17]). Several reasons could account for
this observation. It is possible that various ligands exist for NCR1,
some of which activate cytokine secretion, some are involved in NK
cytotoxicity, and others might promote both activities. Alternatively,
it is possible that the same ligands are involved in NK cytotoxicity
and cytokine production, and the outcome of the NK cell response
and the magnitude of the effect are determined by additional factors,
which might be cell specific. It has indeed been demonstrated that
the activation of NKp46 could not induce cytotoxicity of freshly
isolated NK cells but rather requires the ligation of additional re-
ceptors to exert the NK activity. However, in activated NK cells, the
engagement of NKp46 alone triggers cytotoxicity (34). The only
ligands identified so far for NKp46/NCR1 are various viral he-
magglutinins (HAs) (7, 13). It was shown that NK cells kill virus-
infected melanoma cells such as 1106mel through the interactions
between the NKp46 receptor and the viral HA (13). In contrast, when
dendritic cells where infected with the influenza virus, the in-
teraction between NKp46 and HA led to IFN-g secretion and not to
cytotoxicity (35). Finally, we recently demonstrated that the inter-
actions between NKp46 and the HA protein H1 could directly lead
to cytotoxicity, whereas when NKp46 interacts with the H5 HA,
additional receptors were needed to enhance the killing (36). Thus,
even when NKp46 is triggered by the same HA ligand, the functional
outcome of such activation might be different.
We show in this paper that in the MCA-induced model, the
NKp46 ligands are involved in IFN-g secretion. We and other
showed that NKp46 is important for melanoma and lymphoma
eradication (17, 18), and recently, we demonstrated that the
NKp46 ligands are also involved in type 1 diabetes development
(37). Clearly, the identification of the unknown NKp46 ligand is of
special importance. We think that the MCA-induced tumor cell
lines generated in this study, which express high and low levels of
the unknown NKp46 ligands, are not only a good example of
tumor immunoediting but also that these cell lines might also be
a valuable tool for the NKp46 ligand identification.
Disclosures
The authors have no financial conflicts of interest.
5643
References
1. Arnon, T. I., G. Markel, and O. Mandelboim. 2006. Tumor and viral recognition
by natural killer cells receptors. Semin. Cancer Biol. 16: 348–358.
2. Lodoen, M. B., and L. L. Lanier. 2006. Natural killer cells as an initial defense
against pathogens. Curr. Opin. Immunol. 18: 391–398.
3. Newman, K. C., and E. M. Riley. 2007. Whatever turns you on: accessory-cell–
dependent activation of NK cells by pathogens. Nat. Rev. Immunol. 7: 279–291.
4. Achdout, H., I. Manaster, and O. Mandelboim. 2008. Influenza virus infection
augments NK cell inhibition through reorganization of major histocompatibility
complex class I proteins. J. Virol. 82: 8030–8037.
5. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari,
R. Biassoni, and L. Moretta. 2001. Activating receptors and coreceptors involved
in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19: 197–223.
6. Welte, S., S. Kuttruff, I. Waldhauer, and A. Steinle. 2006. Mutual activation of
natural killer cells and monocytes mediated by NKp80-AICL interaction. Nat.
Immunol. 7: 1334–1342.
7. Gazit, R., R. Gruda, M. Elboim, T. I. Arnon, G. Katz, H. Achdout, J. Hanna,
U. Qimron, G. Landau, E. Greenbaum, et al. 2006. Lethal influenza infection in the
absence of the natural killer cell receptor gene Ncr1. Nat. Immunol. 7: 517–523.
8. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and T. Spies.
1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-
inducible MICA. Science 285: 727–729.
9. Brandt, C. S., M. Baratin, E. C. Yi, J. Kennedy, Z. Gao, B. Fox, B. Haldeman,
C. D. Ostrander, T. Kaifu, C. Chabannon, et al. 2009. The B7 family member
B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30
in humans. J. Exp. Med. 206: 1495–1503.
10. Ljunggren, H. G. 2008. Cancer immunosurveillance: NKG2D breaks cover.
Immunity 28: 492–494.
Downloaded from www.jimmunol.org on April 18, 2011
11. Pogge von Strandmann, E., V. R. Simhadri, B. von Tresckow, S. Sasse,
K. S. Reiners, H. P. Hansen, A. Rothe, B. Böll, V. L. Simhadri, P. Borchmann,
et al. 2007. Human leukocyte antigen-B-associated transcript 3 is released from
tumor cells and engages the NKp30 receptor on natural killer cells. Immunity 27:
965–974.
12. Arnon, T. I., M. Lev, G. Katz, Y. Chernobrov, A. Porgador, and O. Mandelboim.
2001. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur. J.
Immunol. 31: 2680–2689.
13. Mandelboim, O., N. Lieberman, M. Lev, L. Paul, T. I. Arnon, Y. Bushkin,
D. M. Davis, J. L. Strominger, J. W. Yewdell, and A. Porgador. 2001. Recog-
nition of haemagglutinins on virus-infected cells by NKp46 activates lysis by
human NK cells. Nature 409: 1055–1060.
14. Arnon, T. I., H. Achdout, O. Levi, G. Markel, N. Saleh, G. Katz, R. Gazit,
T. Gonen-Gross, J. Hanna, E. Nahari, et al. 2005. Inhibition of the NKp30 ac-
tivating receptor by pp65 of human cytomegalovirus. Nat. Immunol. 6: 515–523.
15. Jonjić, S., M. Babić, B. Polić, and A. Krmpotić. 2008. Immune evasion of natural
killer cells by viruses. Curr. Opin. Immunol. 20: 30–38.
16. Lanier, L. L. 2008. Evolutionary struggles between NK cells and viruses. Nat.
Rev. Immunol. 8: 259–268.
17. Halfteck, G. G., M. Elboim, C. Gur, H. Achdout, H. Ghadially, and O. Mandelboim.
2009. Enhanced in vivo growth of lymphoma tumors in the absence of the NK-
activating receptor NKp46/NCR1. J. Immunol. 182: 2221–2230.
18. Lakshmikanth, T., S. Burke, T. H. Ali, S. Kimpfler, F. Ursini, L. Ruggeri,
M. Capanni, V. Umansky, A. Paschen, A. Sucker, et al. 2009. NCRs and DNAM-1
mediate NK cell recognition and lysis of human and mouse melanoma cell lines
in vitro and in vivo. J. Clin. Invest. 119: 1251–1263.
19. Guerra, N., Y. X. Tan, N. T. Joncker, A. Choy, F. Gallardo, N. Xiong,
S. Knoblaugh, D. Cado, N. M. Greenberg, N. R. Greenberg, and D. H. Raulet.
2008. NKG2D-deficient mice are defective in tumor surveillance in models of
spontaneous malignancy. [Published erratum appears in 2008 Immunity 28: 723.]
Immunity 28: 571–580.
20. Koebel, C. M., W. Vermi, J. B. Swann, N. Zerafa, S. J. Rodig, L. J. Old,
M. J. Smyth, and R. D. Schreiber. 2007. Adaptive immunity maintains occult
cancer in an equilibrium state. Nature 450: 903–907.
21. Iguchi-Manaka, A., H. Kai, Y. Yamashita, K. Shibata, S. Tahara-Hanaoka,
S. Honda, T. Yasui, H. Kikutani, K. Shibuya, and A. Shibuya. 2008. Accelerated
tumor growth in mice deficient in DNAM-1 receptor. J. Exp. Med. 205: 2959–2964.
22. Smyth, M. J., J. Swann, E. Cretney, N. Zerafa, W. M. Yokoyama, and
Y. Hayakawa. 2005. NKG2D function protects the host from tumor initiation. J.
Exp. Med. 202: 583–588.
23. Mandelboim, O., H. T. Reyburn, M. Valés-Gómez, L. Pazmany, M. Colonna,
G. Borsellino, and J. L. Strominger. 1996. Protection from lysis by natural killer
cells of group 1 and 2 specificity is mediated by residue 80 in human histo-
compatibility leukocyte antigen C alleles and also occurs with empty major
histocompatibility complex molecules. J. Exp. Med. 184: 913–922.
24. Beuneu, H., J. Deguine, B. Breart, O. Mandelboim, J. P. Di Santo, and P. Bousso.
2009. Dynamic behavior of NK cells during activation in lymph nodes. Blood
114: 3227–3234.
25. Satoh-Takayama, N., C. A. Vosshenrich, S. Lesjean-Pottier, S. Sawa, M. Lochner,
F. Rattis, J. J. Mention, K. Thiam, N. Cerf-Bensussan, O. Mandelboim, et al. 2008.
Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that
provide innate mucosal immune defense. Immunity 29: 958–970.
26. Sozmen, M., R. Tunca, and S. Dag Erginsoy. 2008. Cyclin A expression is as-
sociated with apoptosis and mitosis in murine 3-methylcholanthrene-induced
fibrosarcomas. Exp. Toxicol. Pathol. 61: 41–49.
27. Swann, J. B., M. D. Vesely, A. Silva, J. Sharkey, S. Akira, R. D. Schreiber, and
M. J. Smyth. 2008. Demonstration of inflammation-induced cancer and cancer
immunoediting during primary tumorigenesis. Proc. Natl. Acad. Sci. USA 105:
652–656.
5644
28. Di Santo, J. P. 2008. Natural killer cells: diversity in search of a niche. Nat.
Immunol. 9: 473–475.
29. Cooper, M. A., J. M. Elliott, P. A. Keyel, L. Yang, J. A. Carrero, and
W. M. Yokoyama. 2009. Cytokine-induced memory-like natural killer cells.
Proc. Natl. Acad. Sci. USA 106: 1915–1919.
30. O’Leary, J. G., M. Goodarzi, D. L. Drayton, and U. H. von Andrian. 2006.
T cell- and B cell-independent adaptive immunity mediated by natural killer
cells. Nat. Immunol. 7: 507–516.
31. Sun, J. C., J. N. Beilke, and L. L. Lanier. 2009. Adaptive immune features of
natural killer cells. Nature 457: 557–561.
32. Bryceson, Y. T., and E. O. Long. 2008. Line of attack: NK cell specificity and
integration of signals. Curr. Opin. Immunol. 20: 344–352.
33. Blankenstein, T., and Z. Qin. 2003. The role of IFN-g in tumor transplantation
immunity and inhibition of chemical carcinogenesis. Curr. Opin. Immunol. 15:
148–154.
TUMOR IMMUNOEDITING BY NKp46
34. Bryceson, Y. T., M. E. March, H. G. Ljunggren, and E. O. Long. 2006. Synergy
among receptors on resting NK cells for the activation of natural cytotoxicity and
cytokine secretion. Blood 107: 159–166.
35. Draghi, M., A. Pashine, B. Sanjanwala, K. Gendzekhadze, C. Cantoni,
D. Cosman, A. Moretta, N. M. Valiante, and P. Parham. 2007. NKp46 and
NKG2D recognition of infected dendritic cells is necessary for NK cell activa-
tion in the human response to influenza infection. J. Immunol. 178: 2688–2698.
36. Achdout, H., T. Meningher, S. Hirsh, A. Glasner, Y. Bar-On, C. Gur,
A. Porgador, M. Mendelson, M. Mandelboim, and O. Mandelboim. 2010.
Killing of avian and swine influenza by natural killer cells. J. Virol. 84: 3993–
4001.
37. Gur, C., A. Porgador, M. Elboim, R. Gazit, S. Mizrahi, N. Stern-Ginossar,
H. Achdout, H. Ghadially, Y. Dor, T. Nir, et al. 2010. The activating receptor NKp46
is essential for the development of type 1 diabetes. Nat. Immunol. 11: 121–128.
Downloaded from www.jimmunol.org on April 18, 2011