Article

Activation of p38 Mitogen-Activated Protein Kinase Drives Dendritic Cells to Become Tolerogenic in Ret Transgenic Mice Spontaneously Developing Melanoma

Skin Cancer Unit, German Cancer Research Center and University Hospital Mannheim, Heidelberg, Germany.
Clinical Cancer Research (Impact Factor: 8.72). 07/2009; 15(13):4382-90. DOI: 10.1158/1078-0432.CCR-09-0399
Source: PubMed

ABSTRACT

The purpose of the study was to investigate signaling molecules involved in the acquisition of tolerogenic properties by dendritic cells (DC) in ret transgenic mice with spontaneous melanoma progression and to target these molecules to overcome the barrier for effective melanoma immunotherapy.
DC functions and expression patterns of p38 mitogen-activated protein kinase (MAPK) in DCs were evaluated in a ret transgenic murine cutaneous melanoma model, which shows high similarity to human cutaneous melanoma with respect to clinical development. In contrast to transplantation melanoma models (like B16), this model allows the study of melanoma progression under conditions of natural interactions between tumor and host cells over time.
We showed a strong tumor infiltration with immature DCs and a reduction in the number of mature DCs in lymphoid organs during melanoma progression. DCs from melanoma-bearing mice secreted significantly more interleukin 10 and less interleukin 12p70, and showed a decreased capacity to activate T cells compared with DCs from tumor-free animals. Observed DC dysfunction was linked to considerable activation of p38 MAPK. Inhibition of its activity in spleen DCs from tumor-bearing mice led to normalization of their cytokine secretion pattern and T-cell stimulation capacity.
Our data show a critical role of constitutively activated p38 MAPK in the acquirement of tolerogenic pattern by DCs during melanoma progression that contributes to the suppression of antitumor T-cell immune responses. We suggest that new strategies of melanoma immunotherapy can include inhibitors of p38 MAPK activity in DCs.

Full-text

Available from: Dirk Schadendorf
Activation of p38 Mitogen-Activated Protein Kinase Drives
Dendritic Cells to Become Tolerogenic in Ret Tr a n s g e n i c
Mice Spontaneously Developing Melanoma
Fang Zhao,
1
Christine Falk,
2
Wolfram Osen,
1
Masashi Kato,
3
Dirk Schadendorf,
1
andViktor Umansky
1
Abstract
Purpose:The purpose of the study was to investigate signaling molecules involved in the acqui-
sition of tolerogenic properties by dendritic cells (DC) in ret transgenic mice with spontaneous
melanoma progression and to target these molecules to overcome the barrier for effective mela-
noma immunotherapy.
Experimental Design: DC functions and expression patterns of p38 mitogen-activated protein
kinase (MAPK) in DCs were evaluated in a ret transgenic murine cutaneous melanoma model,
which shows high similarity to human cutaneous melanoma with respect to clinical development.
In contrast to transplantation melanoma models (like B16), this model allows the study of mela-
noma progression under conditions of natural interactions between tumor and host cells over
time.
Results:We showed a strong tumor infiltration with immature DCs and a reduction in the number
of mature DCs in lymphoid organs during melanoma progression. DCs from melanoma-bearing
mice secreted significantly more interleukin 10 and less interleukin 12p70, and showed a de-
creased capacity to activateT cells compared with DCs from tumor-free animals. Observed DC
dysfunction was linked to considerable activation of p38 MAPK. Inhibition of its activity in spleen
DCs from tumor-bearing mice led to normalization of their cytokine secretion pattern and T-cell
stimulation capacity.
Conclusions: Our data show a critical role of constitutively activated p38 MAPK in the acquire-
ment of tolerogenic pattern by DCs during melanoma progression that contributes to the suppres-
sion of antitumor T-cell immune responses. We suggest that new strategies of melanoma
immunotherapy can include inhibitors of p38 MAPK activity in DCs.
Dendritic cells (DC) are viewed as important regulators of
effective adaptive immune responses against various tumors,
including melanoma (1, 2). Considering high intrinsic mela-
noma immunogenicity (3, 4), DCs were loaded with defined
melanoma antigens and applied for antitumor immunother-
apy. Thus, such DCs were able to reject B16 melanoma and to
stimulate antitumor T-cell reactions in mice (5, 6). Vaccination
of melanoma patients could also result in the induction of
cytotoxic tumor-specific CD8 T cells and in positive clinical
effects in some patients (2, 7, 8). Despite the initial promising
data, the overall results of clinical studies are not satisfactory
(9). Insufficient antitumor reactivity could be due to the
different mechanisms dealing with structural and functional
changes both in tumor and stroma cells. Tumor-derived factors,
like transforming growth factor (TGF)-h, interleukin (IL)-6, IL-
10, and vascular endothelial growth factor (VEGF) have been
described to induce tolerogenic DCs (10, 11), which stimulated
the expansion of T
H
2 cells or regulatory T cells. In particular, IL-
10 accumulated in metastatic lymph nodes from melanoma
patients blocked DC maturation, which resulted in the
induction of anergic T cells and/or regulatory T cells (12). In
addition, some human tumors stimulated the expression of
inhibitory molecule B7-H4 on the DC surface, contributing
thereby to the generation of an immunosuppressive tumor
microenvironment (13). Furthermore, B16 mouse melanoma
cells could recruit DCs to draining lymph nodes and stimulate
them to produce IL-10 and TGF-h, which led to the regulatory
T-cell expansion (14).
There is thus an urgent need to develop innovative
immunotherapeutic strategies that avoid tolerogenic effects
mediated by DCs in the tumor-bearing host. In contrast to
transplantation models (e.g., B16), in which tumor-host
interactions are not comparable with the clinical situation, a
Cancer T herap y: Preclinical
Authors’ Affiliations:
1
Skin Cancer Unit, German Cancer Research Center and
University Hospital Mannheim;
2
Immunomonitoring Unit, National Center forTumor
Diseases and Institute for Immunology, Heidelberg, Germany ; and
3
Unit of
Environmental Health Sciences, Department of Biomedical Sciences, College of
Life and Health Sciences, Chubu University, Aichi, Japan
Received2/16/09;revised3/25/09; accepted 3/26/09;published OnlineFirst6/23/09.
Grant support: Dr. Mildred Scheel Foundation for Cancer Research grant106096,
the Initiative andNetworking Fundof the Helmholtz Associationwithinthe Helmholtz
Alliance on Immunotherapy of Cancer (V. Umansky and D. Schadendorf) and the
Framework Program of the EUgrant 518234 (D. Schadendorf andW. Osen).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section1734 solely to indicate this fact.
Note: Current address for D. Schadendorf: Department of Dermatology, University
Hospital Essen, Essen 45122, Germany.
Requests for reprints:Viktor Umansky, Skin Cancer Unit (G300), German Cancer
Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany. Phone:
49-621-3833773; Fax: 49-621-3832163; E-mail: v.umansky
@
dkfz.de.
F 2009 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-09-0399
www.aacrjournals.orgClin Cancer Res 2009;15(13) July 1, 2009 4382
Page 1
recently described MT/ret transgenic mouse model closely
resembles human melanoma with respect to tumor genetics,
histopathology, and clinical development (15, 16). Mice
expressing the human ret transgene in melanocytes develop
spontaneously malignant cutaneous melanoma metastasizing
to the lymph nodes, lungs, brain, kidney, and spleen (16). This
metastatic profile is similar to that observed in melanoma
patients (17).
In the present study, we investigated alterations in DC
functions during tumor progression with the use of a ret
transgenic mouse model. We found that the DC population in
tumor-bearing mice was characterized by decreased amounts of
mature cells, higher IL-10 and lower IL-12 production, and by a
diminished capacity to stimulate T cells compared with DCs
from mice without macroscopic tumors or nontransgenic
littermates. DCs from melanoma mice showed an increased
expression of phosphorylated forms of p38 MAPK, signal
transducers and activators of transcription 3 (STAT3), and small
mothers against decapentaplegic homologue 3 (Smad3).
Inhibition of p38 MAPK activity in DCs resulted in the
reconstitution of their cytokine secretion profile and T-cell
stimulation capacity. Therefore, constitutive activation of p38
MAPK by tumor-derived factors could induce a DC cytokine
profile, which led to hampered antitumor T-cell responses and
melanoma progression. Our study suggests that p38 MAPK
inhibition in DCs emerges as a promising ingredient of new
strategies for melanoma immunotherapy.
Materials and Methods
Mice. Animals (C57BL/6 background) expressing human ret proto-
oncogene in melanocytes under the control of mouse metallothionein I
promoter/enhancer (15) were kindly provided by Dr. Izuma Naka-
shima (Department of Biomedical Sciences, Chubu University, Aichi,
Japan). OT-I mice, which express a transgenic T-cell receptor (Va2/Vh5)
specific for the ovalbumin-derived peptide SIINFEKL, were kindly
provided by Dr. Bernd Arnold (German Cancer Research Center,
Heidelberg, Germany). All mice were crossed and kept under specific
pathogen-free conditions in the animal facility of the German Cancer
Research Center. Experiments were done in accordance with govern-
ment and institutional guidelines and regulations. The survival and
general performance of mice were monitored daily. Spontaneous tumor
development was assessed macroscopically.
Antibodies and reagents. The media used were Ficoll (PAA) as well
as RPMI 1640 (PAA) supplemented with 10% FCS (PAN Biotech) and
50 Amol/L h-mercaptoethanol (Sigma). The rat anti-mouse directly
conjugated monoclonal antibodies (mAb; I-A
d
/I-E
d
-FITC, CD40-PE,
CD80-PE, CD86-PE, CD11c-biotin, CD45.2-PerCP-Cy5.5, and isotype-
matched control mAbs), streptavidin-allophycocyanin, and the mixture
of mAbs against mouse Fc-receptors (Fc-block) used for the fluorescence-
activated cell sorting staining were purchased from BD Biosciences.
Purified rabbit antibodies against murine phosphorylated p38 MAPK,
STAT3, Smad3, and extracellular signal-regulated kinase (Erk1/2) MAPK
(Cell Signaling Technology), and Alexa Fluor 488 conjugated secondary
mAbs against rabbit IgG (Molecular Probes) were also used for flow
cytometry. Neutralizing rat anti-mouse mAbs for IL-6 and TGF-h1, as
well as purified goat antibodies against mouse VEGF were from R&D
Systems. SB203580, a specific inhibitor of p38 MAPK, and lipopolysac-
charide were purchased from Sigma. CpG oligodeoxynucleotide 1668
was purchased from MWG. The ovalbumin-derived peptide SIINFEKL
was synthesized in the core facility of the German Cancer Research
Center. Ovalbumin-specific CD8
+
T-cell line was established upon
peptide immunization of C57BL/6 mice by SIINFEKL together with T-
helper peptide (aa 128-140) derived from hepatitis B virus core antigen.
Preparation of single cell suspensions. Fresh bone marrow, spleen,
lymph node, and tumor samples were immediately transferred into
PBS, cut into small pieces, and filtered through a cell strainer. Bone
marrow and spleen samples were depleted of erythrocytes by
ammonium chloride lysis.
DC isolation ex vivo. Single spleen cell suspension was digested
in PBS supplemented with 2% FCS, 5 mg/mL DNase I (Sigma), and
10 mg/mL collagenase IV (Life Technologies) for 30 min at room
temperature. Digested cells were then centrifuged at 400 g for 30 min in
Ficoll followed by positive selection of DCs by CD11c MicroBeads
isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol.
To test phosphorylation levels of transcription factors (p38 MAPK,
STAT3, Smad3, and Erk1/2 MAPK) in DCs, ex vivo isolated splenocytes
were directly fixed in 2% paraformaldehyde for 10 min at 37jC and
permeabilized in 100% methanol for 30 min on ice followed by
extensive washing with PBS. In some experiments, splenocytes were
incubated for 15 min at 37jC in the RPMI 1640 medium supplemented
with 10% FCS and 50 Amol/L h-mercaptoethanol, and containing
supernatants from cultured Ret melanoma cells (Ret conditioned
medium; 50%, volume for volume) followed by fixation and
permeabilization as described above.
DC generation from the bone marrow. DCs were generated as
previously described (18) with some modifications. Briefly, 2
10
6
bone marrow cells from ret transgenic mice or nontransgenic
littermates were incubated for 8 d in RPMI 1640 medium supple-
mented with 50 Amol/L h-mercaptoethanol (Merck), 10 ng/mL
recombinant mouse granulocyte macrophage colony-stimulating factor
(eBioscience), and 10 ng/mL recombinant mouse IL-4 (R&D Systems).
In some experiments, Ret conditioned medium was added to the
culture medium (50%, volume for volume) used for the generation of
DCs from nontransgenic littermates. In addition, some culture samples
were supplemented with neutralizing antibodies for IL-6, VEGF, or TGF-
h1 (1.0, 0.4, and 1.0 Ag/mL, respectively).
Flow cytometry. Single cell suspensions were treated with Fc-block
and mAbs for 20 min at 4jC. To measure the expression of
phosphorylated transcription factors, cells were incubated at room
temperature with respective primary mAbs for 1 h and with secondary
mAbs for 30 min. Acquisition was done by four-color flow cytometry
Translational Relevance
Despite the well-known melanoma immunogenicity, the
results of immunotherapy trials are not satisfactory. Insuf-
ficient antitumor reactivity could be due to tolerogenic
dendritic cells (DC), which induce the anergy of tumor-
specific T cells and expansion of immunosuppressive regu-
latoryT cells. However, these data were obtained with the
use of transplantation tumor models, in which the tumor
development and tumor-host interactions are not compa-
rable with the clinical situation. Moreover, the role of p38
mitogen-activated protein kinase (MAPK) was contro-
versially discussed. In this study, we used ret transgenic
mice spontaneously developing melanoma, which re-
sembles human melanoma in histopathology and clinical
development. Mechanistic investigations revealed that
tumor-derived factors induced in tumor-bearing mice
the p38 MAPK activation linked to DC dysfunctions. Inhi-
bition of p38 MAPK activity in these DCs resulted in
the normalization of their T-cell stimulation capacity.
We suggest that novel melanoma immunotherapies can
include p38 MAPK inhibitors to neutralize the immuno-
suppressive tumor microenvironment.
Tolerogenic Dendritic Cells i nTransgenic Mice with Melanoma
www.aacrjournals.org Clin Cancer Res 2009;15(13) July 1, 20094383
Page 2
with the use of FACSCalibur with CellQuest software or FACSCanto II
with FACSDiva software (both BD Biosciences) with dead cell exclusion
based on scatter profile or propidium iodide inclusion. FlowJo software
(Tree Star) was used to analyze at least 100,000 events. Data were
expressed as dot plots or histograms.
Reverse transcription-PCR. Total RNA was extracted from primary
tumors, Ret melanoma cells, and B16F10 melanoma cells with the use
of a TRIzol reagent (Invitrogen) according to the manufacturer’s
instruction. cDNA was synthesized with the use of Moloney murine
leukemia virus reverse transcriptase (Invitrogen) and random hexamers
(Amersham Biosciences) for 2 h at 42jC. Reverse transcription products
were amplified by PCR.
Detection of cytokines and growth factors. DCs generated from the
bone marrow or ex vivo isolated DCs (5
10
5
cells/mL) were
stimulated with 1 Ag/mL lipopolysaccharide or 3 Ag/mL CpG1668 for
24 h. Concentrations of IL-12p70 and IL-10 in supernatants were
determined with the use of respective ELISA kits (both BD
Biosciences) according to the manufacturer’s protocols. In some
experiments, ex vivo isolated spleen DCs were first incubated with the
p38 MAPK inhibitor SB203580 (10 Amol/L) at 37jC for 1 h, and
then stimulated with CpG1668 for 24 h followed by measurement of
IL-12p70 and IL-10 via ELISA. In other experiments, isolated spleen
DCs were incubated with or without SB203580 (10 Amol/L) during
the loading with the ovalbumin-derived peptide SIINFEKL for 1 h.
After extensive washing, DCs were cocultured with CD8
+
T cells
isolated from OT-I mice for 3 d (T cell/DC ratio, 5:1), followed by
measurement of IFN-g levels in supernatants with the use of an
ELISA kit (BD Biosciences). To measure tumor-derived factors,
primary tumors and Ret tumor cells were treated by lysis solution
(Bio-Rad) followed by centrifugation at 4,500 g for 6 min at 4jC.
Protein concentration was determined through Bradford assay and
adjusted to 500 Ag/mL with the use of serum diluent (both Bio-Rad).
Amounts of IL-6 and VEGF in tumor lysates and murine serum were
measured with the use of multiplex technology (Bio-Rad), and levels
of TGF-h1 in these samples were tested by a single plex kit
(Millipore) according to the manufacturer’s protocols.
IFN-g enzyme-linked immunosorbent spot assay. The assay was done
as previously described (19) with modifications. Briefly, ex vivo isolated
spleen DCs were loaded with SIINFEKL (100 ng/mL) for 1 h at 37jC,
washed, and cocultured with the ovalbumin-specific CD8
+
T-cell line
for 40 h (T cell/DC ratio, 10:1). In another set of experiments, CD8
+
T cells were isolated from spleens of OT-I mice with the use of a CD8
+
T-cell isolation kit (Miltenyi Biotec) and coincubated with SIINFEKL-
pulsed DCs for 72 h with the use of the same T cell/DC ratio. The
number of IFN-g producing cells was detected with Bioreader 3000
(Biosys).
Data analysis. Statistical analyses were done with the use of
parametric (Student’s ttest) and nonparametric (Mann-Whitney Utest)
tests. A value of P < 0.05 was considered statistically significant.
Results
Decrease in DC numbers in lymphoid organs of tumor-bearing
mice and accumulation of immature DCs in advanced tumors. In
this study, we used transgenic mice overexpressing the human
oncogene ret (15) backcrossed at least six times with C57BL/6
wild-type mice. After a short latency (20-70 days of age),
around 25% of all transgenic mice develop skin tumors on the
face (nose, ears, eyes, and neck), back, or on the tail. Tumor-
bearing mice developed metastases in the lymph nodes, lungs,
liver, and brain.
We investigated CD11c
+
MHCII
+
DCs in the spleen, bone
marrow, and lymph nodes of ret transgenic mice with the use
of flow cytometry (Fig. 1A to D). Compared with non-
transgenic littermates and/or ret transgenic mice without
visible tumors (control groups), transgenic animals with
Fig. 1. Analysis of DCs in the spleen,
bone marrow, and lymph nodes of
ret transgenic mice. Cells from mice with
macroscopic tumors (ret tu), without visible
tumors (ret), and from nontransgenic
littermates (wild type) were stained with
mAbs for CD11c, MHC class II, and the
leukocyte marker CD45.2 followed by flow
cytometry. A, accumulative data for DCs
in the spleen and bone marrow are
expressed as the percentage within
leukocytes. B, accumulative data for DCs in
lymph nodes with macroscopic metastases
(ret tu met) and without visible metastatic
lesions (ret tu non-met) expressed as the
percentage within leukocytes. C, amounts
of mature CD86
+
DCs in the spleen and
bone marrow expressed as the percentage
among leukocytes. D, numbers of CD86
+
DCs in lymph nodes with macroscopic
metastases and without metastases
expressed as the percentage within
leukocytes. Data, means F SE from 5 to
20 mice. *, P
< 0.05; lines, significant
differences between groups.
Cancer Therapy: Preclinical
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Page 3
macroscopic tumors displayed in the spleen and bone marrow
a significant decrease in total DC numbers within CD45.2
+
leukocytes (P < 0.05; Fig. 1A). Interestingly, in the bone
marrow, this decrease was observed already in tumor-free
transgenic mice compared with wild-type littermates. More-
over, DC amounts in metastatic lymph nodes were signifi-
cantly lower than those in nonmetastatic lymph nodes from
the same tumor-bearing mice or in lymph nodes from control
groups (P < 0.05; Fig. 1B). We found also a profound
diminution in numbers of CD86
+
DCs in all investigated
lymphoid organs compared with control groups (P < 0.05;
Fig. 1C and D). Furthermore, the level of CD86 expression on
DCs was also reduced (data not shown). Similar changes were
revealed in the distribution and expression of CD40 and
CD80 costimulatory molecules on these DCs (data not
shown). Thus, lymphoid organs from tumor-bearing mice
show a systemic decrease in DC numbers and expression of
costimulatory molecules. No statistical correlation between
observed alterations and mouse age, tumor weight, or the
dynamics of tumor growth was found (data not shown).
Next we studied DCs infiltrating primary skin tumors as a
subset of tumor-infiltrating CD45.2
+
leukocytes (Fig. 2A to C).
Increasing proportions of tumor-infiltrating DCs among tumor-
infiltrating leukocytes were found to correlate with the
increasing weight of primary melanomas (r
2
= 0.18; P < 0.05;
Fig. 2B). Importantly, in larger tumors, significantly higher
amounts of tumor-infiltrating DCs displayed immature phe-
notype according to the CD40 expression profile compared
with DCs infiltrating smaller tumors (r
2
= 0.31; P < 0.05;
Fig. 2C). These findings suggest that, in the process of tumor
progression, the suppressive tumor microenvironment can
block DC development at the immature stage.
Impaired DC functions in ret transgenic tumor-bearing
mice. We investigated the secretion of IL-12p70 and IL-10 by
ex vivo isolated spleen DCs after their stimulation with CpG
1668 with the use of an ELISA assay. DCs from ret transgenic
mice with macroscopical tumors secreted significantly less IL-
12p70 than DCs from mice of both control groups (P < 0.05;
Fig. 3A). Moreover, the amount of IL-10 produced by tumor
DCs was significantly increased compared with DCs from
nontransgenic littermates (P < 0.05; Fig. 3B). This indicates an
immunosuppressive profile of cytokines produced by DCs from
tumor-bearing animals.
To address the question of whether the T-cell stimulating
capacity of DCs from mice with macroscopic tumors was also
impaired, ex vivo isolated spleen DCs were pulsed with the
ovalbumin peptide SIINFEKL and cocultured with CD8
+
T
lymphocytes isolated from OT-I mice followed by the
detection of IFN-g producing cells via enzyme-linked im-
munosorbent spot assay. As shown in Fig. 3C, significantly
reduced numbers of T cells were able to secrete IFN-g after
stimulation with DCs from mice with macroscopic tumors
compared with DCs from nontransgenic littermates (P <
0.05). In another set of experiments, SIINFEKL-pulsed spleen
DCs were coincubated with ovalbumin-specific CD8
+
T cells
generated from ovalbumin-immunized mice. We detected
substantially lower amounts of IFN-g producing T cells in
the presence of DCs from mice with macroscopic tumors
than in samples with DCs from control mice (P < 0.05;
Fig. 3D). Taken together, the data on cytokine secretion and
T-cell activation capacity suggest that DCs from transgenic
tumor-bearing mice display a tolerogenic pattern.
Tumor-derived factors promote generation of DCs with a
tolerogenic profile. To investigate which tumor-derived fac-
tors could be involved in the development of tolerogenic
DCs during melanoma progression, we first examined the
expression of some cytokines and growth factors, like IL-6,
IL-10, VEGF, and TGF-h1, by reverse transcription-PCR at the
Fig. 2. Tumor infiltration with DCs is
dependent on tumor progression. Single cell
suspensions prepared from mouse
tumors were stained with mAbs for CD11c,
MHC class II, CD45.2, and CD40. A,
representative dot plots are shown. B and C,
the weight of each tumor sample (n =37)
was plotted against the percentage of TIDCs
within CD45.2
+
TILs (B) or against the
percentage of CD40
+
mature DCs within
TIDCs (C). The correlation between the two
variables was calculated through a linear
regression analysis.TIDC, tumor-infiltrating
DC; TIL, tumor-infiltrating leukocytes.
Tolerogenic Dendritic Cells i nTransgenic Mice with Melanoma
www.aacrjournals.org Clin Cancer Res 2009;15(13) July 1, 20094385
Page 4
mRNA level in the cell line, which was established from
primary skin melanomas isolated from ret transgenic mice
(Ret melanoma cells). Whereas IL-6, VEGF, and TGF-h1
mRNAs were found in this cell line, the IL-10 mRNA
expression was not detected (data not shown). Considerable
amounts of VEGF and TGF-h1 proteins were shown in
supernatants from cultured Ret melanoma cells by ELISA
(data not shown). Next we analyzed the expression of all
four above-mentioned factors in primary tumors removed
from transgenic mice both at the mRNA and protein levels.
Primary melanomas also expressed IL-6, VEGF, and TGF-h1
mRNAs, whereas IL-10 mRNA was not detectable (data not
shown). At the protein level, we showed IL-6, VEGF, and
TGF-h1 production in primary tumors with the use of bio-
plex assay. Notably, the amount of VEGF displayed a
significant positive correlation with the tumor weight (r
2
=
0.56; P < 0.05; Fig. 4A). Moreover, concentrations of IL-6
and VEGF were significantly elevated in the serum from
transgenic tumor-bearing mice compared with wild-type
littermates (P < 0.05; Fig. 4B).
To investigate a direct effect of these tumor-derived factors
on DCs, supernatants from cultured Ret melanoma cell (Ret
conditioned medium) were mixed with the culture medium
(50%, volume for volume) used for the DC generation from
normal bone marrow precursors. DCs generated under these
conditions secreted significantly lower amounts of IL-12p70
upon lipopolysaccharide stimulation than DCs generated in
the normal DC medium (P < 0.05; Fig. 4C). Furthermore,
after adding neutralizing antibodies for IL-6, VEGF, or TGF-
h1 to the DC medium supplemented with Ret conditioned
medium, we found a significant increase in IL-12p70
production compared with DCs generated without these
antibodies (P < 0.05; Fig. 4C).
p38 MAPK plays a key role in the development of tolerogenic
DCs in tumor-bearing mice. Having shown a critical impor-
tance of IL-6, VEGF, and TGF-h1 for the acquirement of
tolerogenic properties by DCs from tumor-bearing mice, we
then addressed the question of which transcription factors
regulating the functions of these substances are responsible
for observed DC impairments. Phosphorylation levels of p38
MAPK, STAT3, Smad3, and Erk1/2 MAPK were examined in
freshly isolated spleen DCs by flow cytometry (Fig. 5A). To
prevent possible artificial effects induced by the DC isolation,
spleen cells were immediately fixed in paraformaldehyde and
permeabilized in ice-cold methanol followed by stainings
with mAbs for CD11c and phosphorylated transcription
factors. We found that DCs from transgenic mice with
macroscopic tumors displayed a significant up-regulation in
the expression of phosphorylated p38 MAPK, STAT3, and
Smad3 compared with those in DCs from wild-type
littermates (P < 0.05), whereas the level of phosphorylated
Erk1/2 MAPK remained unchanged (Fig. 5A and B). To test if
tumor-derived factors can directly modulate the p38 MAPK
expression, normal spleen DCs were incubated in the RPMI
1640 medium supplemented with Ret conditioned medium
(50%, volume for volume). The expression of p38 MAPK was
significantly increased compared with DCs cultured in the
normal RPMI 1640 medium (P < 0.05; Fig. 5C).
We next assessed whether the inhibition of p38 MAPK
activity could reverse the altered pattern of cytokine produc-
tion shown by DCs from transgenic tumor-bearing mice.
Freshly isolated spleen DCs were incubated in the presence of
SB203580, a p38 MAPK specific inhibitor, followed by the
CpG stimulation. The suppression of p38 MAPK activity led
to the profound decrease in IL-10 production compared with
untreated cells (P < 0.01; Fig. 6A). To examine if SB203580
could restore the impaired capacity of DCs from transgenic
melanoma-bearing mice to stimulate T cells, freshly isolated
spleen DCs were loaded with SIINFEKL in the presence of
SB203580. After washing out the inhibitor, tumor DCs were
cocultured for 3 days with CD8
+
T cells isolated from spleens
of OT-I mice followed by the measurement of IFN-g in the
supernatant via ELISA. DC pretreatment with SB203580
significantly stimulated IFN-g production by T cells (P <
0.05; Fig. 6B). Thus, inhibition of the p38 MAPK activity in
DCs from tumor-bearing mice led to the decrease in IL-10
production and to the restoration of the DC ability to
stimulate CD8
+
T cells.
Taken together, our data suggest a crucial role of p38 MAPK
activity in the generation of DCs with a tolerogenic pattern in
ret transgenic tumor-bearing mice.
Fig. 3. Cytokine secretion and T-cell stimulation by DCs from tumor-bearing mice.
A and B, spleen DCs simultaneously isolated ex vivo from tumor-bearing (ret tu),
tumor-free (ret), and wild-type mice (WT) were stimulated with CpG1668 for 24 h,
followed by detection of IL-12p70 (A)andIL-10(B) in supernatants with the use
of ELISA. Data, mean F SE from 10 mice per experimental group. *, P
< 0.05;
differences between indicated groups. C and D, CD11c
+
DCs were isolated through
positive selection, loaded with the ovalbumin peptide SIINFEKL at 37jCfor1h,
and cocultured for 3 d with CD8
+
Tcells isolated from spleens of OT-I mice (C),
or cocultured with an ovalbumin-specificT-cell line for 40 h (D). T-cell activation
was evaluated by spot numbers in the IFN-g enzyme-linked immunosorbent spot
assay. Means F SE from four animals per experimental group are shown. *, P
< 0.05;
differences between indicated groups.
Cancer Therapy: Preclinical
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Page 5
Discussion
In the present study, we focused on the investigation of the
molecular mechanisms of acquisition of tolerogenic properties
by DCs in ret transgenic mice with spontaneous skin melanoma
with high similarity to human melanoma (15, 16). In contrast
to transplantation mouse melanoma models (e.g., B16), this
transgenic model provides an opportunity to study mecha-
nisms of melanoma-induced immunosuppression and thera-
peutic strategies for its neutralization under conditions relevant
to the clinical situation.
First, we examined total MHCII
+
CD11c
+
DC amounts in the
spleen and bone marrow, and found a remarkable reduction of
these cells in ret transgenic tumor-bearing mice. Numerous
clinical studies reported a dramatic decrease in DC numbers in
the peripheral blood of patients with squamous cell carcinoma
of the head and neck, lung cancer, myeloma, invasive breast
cancer, hepatocellular carcinoma, and leukemia (20 25).
Moreover, the presence of metastases resulted in a more
profound decrease in the numbers of circulating peripheral
blood DCs in cancer patients (25, 26). In agreement with these
reports, we showed a significant decrease in DC amounts in
metastatic lymph nodes from tumor-bearing mice compared
with those in nonmetastatic lymph nodes from the same mice
or animals of both control groups.
An observed systemic reduction in DC numbers could be due
to the induction of apoptosis in DCs and/or their precursors by
tumor cells or soluble tumor-derived factors (2729). How-
ever, we were not able to observe DC apoptosis upon
generation from normal bone marrow precursors in the
presence of melanoma-derived conditioned medium or after
incubation of normal spleen DCs in this medium.
4
Another
mechanism of the decreased DC frequency in tumor-bearing
mice may be attributed to the inhibition of their maturation
at a certain stage. It is known that mature DCs are characterized
by high levels of costimulatory molecules, like CD80, CD86,
and CD40. Investigating these markers on DCs from tumor-
bearing transgenic mice, we found a significant decrease in
the numbers of mature DCs expressing CD40, CD80, and CD86
in all studied lymphoid organs. Moreover, the growth of pri-
mary melanomas correlated with the accumulation of tumor-
infiltrating DCs with the immature phenotype. These findings
are in agreement with observations made in cancer patients
(30 33). Blocking of normal DC differentiation and matura-
tion can result also in the accumulation of cells with the
properties of myeloid-derived suppressor cells (32). This
heterogeneous population of myeloid cells has been recently
found to induce a dramatic suppression of T-cell functions
in mouse tumor models and in cancer patients (34, 35). We
observed an accumulation of CD11b
+
Gr-1
+
myeloid-derived
suppressor cells in primary tumors and lymphoid organs
from transgenic mice
4
that may be responsible for the loss of
mature DCs.
Investigating the functional properties of DCs ex vivo isolated
from spleens of transgenic tumor-bearing mice, we found less
IL-12p70 and more IL-10 production upon appropriate
stimulation than DCs from nontransgenic littermates. IL-12
has been described to be a critical cytokine for T-cell
stimulation and for DC maturation and survival (36, 37). In
contrast, IL-10 is known to stimulate regulatoryT-cell differ-
entiation and induce T-cell anergy, and is characteristic for
tolerogenic antigen-presenting cells (32, 38). Indeed, we
showed here that coincubation of ovalbumin-specific CD8
+
T
cells with SIINFEKL-loaded DCs isolated from ret transgenic
mice with macroscopic tumors led to the drastic reduction in
IFN-g producing cells in an enzyme-linked immunosorbent
spot assay compared with T lymphocytes activated by normal
DCs pulsed with the peptide.
Fig. 4. Tumor-derived cytokines and growth factors induce down-regulation of IL-12 production by DCs. A, VEGF concentrations was measured in tumor lysates with the use
of bio-plex technology, plotted against the weight of tumors (n = 15), and expressed as picograms per milligram protein. The correlation between the two variables was
calculated through a linear regression analysis. B, amounts ofVEGF and IL-6 were detected in serum of tumor-bearing and healthy mice. Data (means F SE) from 6 to
12 mice per group are expressed as picograms per milliliter. *, P
< 0.05; differences between indicated groups. C, DCs were generated from bone marrow precursors of
nontransgenic mice in the presence of supernatants from cultured Ret melanoma cells (Ret conditioned medium; 50%, volume for volume) and/or neutralizing antibodies
for IL-6 (a-IL-6),VEGF (a-VEGF), or TGF-h1(a-TGF-h1) followed by lipopolysaccharide stimulation for 24 h. IL-12p70 levels were tested in supernatants via ELISA. Results
(means F SE) of four independent experiments are expressed as pg/10
6
cells. *, P < 0.05; differences between indicated groups.
4
Unpublished data.
Tolerogenic Dendritic Cells i nTransgenic Mice with Melanoma
www.aacrjournals.org Clin Cancer Res 2009;15(13) July 1, 20094387
Page 6
Numerous growth factors and cytokines, like VEGF, TGF-h,
IL-6, and IL-10, produced by human and mouse tumor cells
have been reported to hamper DC maturation in vitro (11,
3941). Tumor-derived VEGF can affect the early stage of DC
maturation in the bone marrow and recruit immature DC
from the bone marrow to the tumor microenvironment (32,
39, 41). Moreover, IL-6 knockout in ret transgenic mice
resulted in the decrease of melanoma incidence and tumor
size, indicating an importance of IL-6 for tumor progression
in this melanoma model (42). In our studies, VEGF and IL-6
were found to be produced both in primary tumors and in
Ret melanoma cells. We observed also significantly up-
regulated serum levels of IL-6 and VEGF in transgenic
tumor-bearing mice. Furthermore, DCs from these animals
displayed an up-regulated surface expression of the IL-6
receptor a.
4
Finally, neutralizing antibodies for IL-6 or VEGF
were shown to reduce the inhibitory effect of Ret conditioned
medium on IL-12 production by stimulated DCs.
Although the list of tumor-derived factors involved in the
impairment of DC functions is getting longer, they may
utilize similar transcription factors and protein kinases, in
particular STAT3 or p38 MAPK. Constitutive STAT3 activa-
tion in many human and mouse tumors was reported to
inhibit DC maturation via STAT3 up-regulation that led to
the impairment of antitumor T-cell responses (32, 43). In
accordance with these data, we found a significant elevation
of STAT3 expression in DCs isolated from ret transgenic
tumor-bearing mice. In this melanoma model, we detected
even a more considerable increase of the phosphorylated
p38 MAPK expression. Furthermore, DC treatment with a
specific p38 MAPK inhibitor SB203580 resulted in the
decrease in IL-10 production and in the capability to
stimulate ovalbumin-specific T cells. A role of p38 MAPK
in DC tolerogenic functions during tumor progression has
been controversially discussed in recent publications. Thus,
Escors et al. (44) showed that constitutive activation of p38
MAPK in mouse DCs resulted in their maturation and
stimulation of antitumor T-cell responses. On the other
hand, in agreement with our findings reported here on the
clinically relevant melanoma model, Wang et al. (45) found
that the phenotype and T-cell stimulatory capacity of
Fig. 5. Tumor-derived factors induce up-regulation of the expression of phosphorylated p38 MAPK in DCs. Spleen cells from tumor-bearing, tumor-free, or wild-type mice
were fixed in paraformaldehyde and permeabilized in ice-cold methanol. A, expression of phosphorylated p38 MAPK (p-p38), STAT3 (p-STAT3), Smad3 (p-Smad3), and
Erk1/2 MAPK (p-Erk1/2) was evaluated in CD11c
+
DCs after staining with respective primary and secondary mAbs with the use of flow cytometry. As a negative control
(con), we used CD11c
+
DCs stained only with secondary mAbs. Results from one representative experiment of three are shown. B, accumulative data for transcription factors
in spleen DCs (mean F SE; 4-6 mice per group) are expressed as MFI ratio (MFI of experimental samples/MFI of respective negative controls). *, P
< 0.05; differences
between indicated groups. C, splenocytes from normal mice were first incubated for15 min at 37jC in the medium supplemented with Ret conditioned medium (50%, volume
for volume), then fixed and permeabilized as described above. Expression of p-p38 MAPK was evaluated in CD11c
+
DCs with the use of flow cytometry. Results (mean F
SE; 4-6 mice per group) are expressed as MFI ratio. *, P
< 0.05; differences between indicated groups. MFI, mean fluorescence intensity.
Fig. 6. Inhibition of p38 MAPK in DCs from tumor-bearing mice induces
down-regulation of IL-10 production and increasesT-cell stimulation. A, spleen
CD11c
+
DCs were isolated from tumor-bearing mice with the use of MicroBeads
isolation kit, incubated with or without p38 MAPK inhibitor SB203850 (10 Amol/L)
for1h,andstimulatedwithCpG1668foranother24h.IL-10production
was measured in supernatants by ELISA. Data, mean F SE from five independent
experiments. *, P
< 0.05; differences between indicated groups. D, freshly isolated
spleen DCs were incubated with or without SB203580 during the loading with the
ovalbumin peptide SIINFEKL for 1h. After washing, DCs were cocultured with
CD8
+
Tcells from OT-I mice for 3 d, followed by measurement of IFN-g in
supernatants by ELISA. Means F SE from three independent experiments are
depicted. *, P
< 0.05; differences between indicated groups.
Cancer Therapy: Preclinical
www.aacrjournals.orgClin Cancer Res 2009;15(13) July 1, 2009 4388
Page 7
monocyte-derived DCs in patients with multiple myeloma
were considerably impaired and that they could be restored
by inhibiting p38 MAPK activity in progenitor cells. In
addition, suppression of p38 MAPK signaling in murine DCs
was reported to enhance their ability for IL-12 production,
to attenuate regulatory T-cell induction, and to stimulate
the antitumor therapeutic efficacy of DCs pulsed with tumor
antigens (46). Application of p38 MAPK inhibitors for
melanoma immunotherapy in vivo will require a thorough
examination of their effects on host T-cell antitumor
reactions because the role of p38 MAPK in these cells is
not completely clear. Whereas p38 activation was shown to
be important for T-cell development and effector functions
(47), other publications showed that p38 activity was a
prerequisite for the regulatory T-cell stimulation (48) or for
apoptotic CD8
+
T-cell death (49). The antitumor effects of
the p38 MAPK inhibitor in ret transgenic melanoma model
in vivo are currently under investigation.
In conclusion, our findings provide evidence that consti-
tutive activation of p38 MAPK is responsible for turning of
DCs to display a tolerogenic profile during melanoma
progression. We showed that suppression of p38 MAPK
activity in DCs from ret tumor-bearing mice could recon-
stitute their impaired cytokine secretion and ability to
stimulate T cells, suggesting thereby that such normalization
of signaling pathways in DCs can represent an effective
immunotherapeutic strategy in melanoma patients.
Disclosure of Potential Conflicts of Interests
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Izumi Nakashima for initially providing ret transgenic mice, Bernd
Arnold for providing OT-I mice, Axel Benner for help with the statistical analysis,
and Kathrin Frank for excellent technical assistance.
Tolerogenic Dendritic Cells i nTransgenic Mice with Melanoma
www.aacrjournals.org Clin Cancer Res 2009;15(13) July 1, 20094389
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  • Source
    • "The transfectants E. G7 (EL4 cells expressing chicken ovalbumin) [43], 2F11 (RMA cells expressing the HPV16-derived oncoprotein E7) [44] and RMA/TRP-2 (expressing human tyrosinase related protein 2) (kindly provided by A. Paschen), were grown in the same medium containing 0.8 μg/ml G418 (Gibco). The following antigen-specific CTL lines were used: OVA-specific CTL line [45] recognizing the H2K b -restricted OVA-derived epitope aa257-264 (SIINFEKL) [46]; E7-specific CTL line [47], reacting against the E7-derived D b -restricted epitope RAHYNI VTF (amino acids, aa, 49–57 [48], and a TRP-2-specific CTL line obtained upon TRP-2-specfic DNA immunization of C57BL/6 mice (Osen, unpublished), recognizing the epitope SVYDFFVWL (aa 180–188) [49] . All CTL lines were expanded in vitro upon periodical restimulation with the transfectant clones expressing the respective target antigen in CTL medium, according to the protocol described in [50]. "
    [Show abstract] [Hide abstract] ABSTRACT: The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.
    Full-text · Article · Sep 2015 · PLoS ONE
  • Source
    • "Notably, Jackson and colleagues demonstrated that blockade of MEK 1/2 and ERK MAPK signaling restores tumor-mediated inhibition of DC function and promotes IL-12 production and Th1 T cell responses, whereas inhibition of p38 MAPK increases signal transduction through ERK 1/2 and blocks IL-12 production (17). In similar vein, p38 MAPK signaling in DC up-regulates IL-10 expression and induces tolerance in a mouse model of melanoma, resulting in suppression of anti-tumor T cell response, whereas inhibition of p38 signaling restored the ability of DC to stimulate T cell responses (18). The observation that p38 inhibition or MEK/ERK activation restores DC function in myeloma patients provides further evidence that p38 blockade may be of therapeutic benefit (19). "
    [Show abstract] [Hide abstract] ABSTRACT: Clinical optimism for dendritic cell vaccination against ovarian cancer has been tempered by the knowledge that tumors avail themselves of multiple mechanisms of immune evasion, thus blunting the efficacy of therapeutic vaccination. Mechanisms of immune suppression include infiltration by regulatory T cells (Treg) and myeloid suppressor cell populations, expression of co-inhibitory receptors, and expression of indoleamine 2,3-dioxygenase (IDO). Expression of both B7-H1 and IDO are associated with differentiation and recruitment of Treg, and clinical studies have shown that each of these mechanisms correlates independently with increased morbidity and mortality in ovarian cancer patients. In sharp contrast, recent studies have indicated that Th17 cell infiltration in ovarian cancer correlates with improved patient outcomes and prolonged overall survival. Given that IDO plays a pivotal role in the balance between Treg and Th17 immunity, elucidation of the mechanisms that regulate IDO activity and immune suppression may lead to novel adjuvants to boost the clinical efficacy of dendritic cell vaccination against ovarian cancer and other malignancies.
    Full-text · Article · Nov 2013 · Frontiers in Immunology
  • Source
    • "In contrast, no difference was detected in the function of TAM between the cSHP-2 KO and WT mice (supplementary Fig. S5). It has been well documented that various cytokines, chemokines, and growth factors including IL-6, IL-10, TGF-β are needed to drive MDSC migration into tumor lesions and to keep their suppressive phenotype in tumor-bearing host303132. An accumulation of functionally active MDSC in melanoma lesions and lymphoid organs was reported to be strongly associated with T-cell anergy33. "
    [Show abstract] [Hide abstract] ABSTRACT: The Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) has been reported to have both tumor-promoting and tumor-suppressing roles in tumorigenesis. However, the role of SHP-2 in tumor immunity remains unclear. Here we observed progressively lower levels of phosphorylated SHP-2 in tumor-associated CD4(+) T cells during melanoma development in a murine model. Similarly, the levels of phosphorylated SHP-2 in the CD4(+) T cells of human melanoma specimens revealed a decrease paralleling cancer development. The CD4(+) T cell-specific deletion of SHP-2 promoted melanoma metastasis in mice. Furthermore, SHP-2 deficiency in CD4(+) T cells resulted in the increased release of inflammatory cytokines, especially IL-6, and the enhanced accumulation of tumor-promoting myeloid-derived suppressor cells (MDSCs) in tumor-bearing mice. An IL-6-neutralizing antibody reduced MDSC accumulation and inhibited tumor growth in CD4(+) T-cell-specific SHP-2-knockout mice. Our results suggest that SHP-2 in CD4(+) T cells plays an important role in preventing melanoma progression and metastasis.
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