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RESEARCH ARTICLE RESEARCH ARTICLE
Immune Cell–Poor Melanomas Benefi t from PD-1 Blockade after Targeted Type I IFN Activation Tobias Bald 1 , Jennifer Landsberg 1 , Dorys Lopez-Ramos 1 , Marcel Renn 1 , Nicole Glodde 1 , Philipp Jansen 1 , Evelyn Gaffal 1 , Julia Steitz 4 , Rene Tolba 4 , Ulrich Kalinke 5 , Andreas Limmer 2 , Göran Jönsson 6 , Michael Hölzel 3 , and Thomas Tüting 1
on March 1, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 3, 2014; DOI: 10.1158/2159-8290.CD-13-0458
JUNE 2014�CANCER DISCOVERY | 675
ABSTRACT Infi ltration of human melanomas with cytotoxic immune cells correlates with spon-
taneous type I IFN activation and a favorable prognosis. Therapeutic blockade of
immune-inhibitory receptors in patients with preexisting lymphocytic infi ltrates prolongs survival, but
new complementary strategies are needed to activate cellular antitumor immunity in immune cell–poor
melanomas. Here, we show that primary melanomas in Hgf-Cdk4 R24C mice, which imitate human immune
cell–poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peri-
tumoral injections of immunostimulatory RNA initiated a cytotoxic infl ammatory response in the tumor
microenvironment and signifi cantly impaired tumor growth. This critically required the coordinated
induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-
mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 recep-
tors further prolonged the survival. These results highlight important interconnections between type I
IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combina-
tion immunotherapies.
SIGNIFICANCE: Using a genetically engineered mouse melanoma model, we demonstrate that tar-
geted activation of the type I IFN system with immunostimulatory RNA in combination with blockade
of immune-inhibitory receptors is a rational strategy to expose immune cell–poor tumors to cellular
immune surveillance. Cancer Discov; 4(6); 674–87. ©2014 AACR.
Authors’ Affi liations: 1 Laboratory of Experimental Dermatology, Depart-ment of Dermatology and Allergy, 2 Department of Orthopaedics and Trauma Surgery, 3 Unit for RNA Biology, Department of Clinical Chemis-try and Clinical Pharmacology, University of Bonn, Bonn; 4 Institute for Laboratory Animal Science, University Hospital, RWTH Aachen Univer-sity, Aachen; 5 Twincore, Centre for Experimental and Clinical Infection Research, Hannover, Germany; and 6 Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden
Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Author: Thomas Tüting, Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University of Bonn, Sigmund-Freud-Straße 25, 53105 Bonn, Germany. Phone: 49-228-287-19257; Fax: 49-228-287-19393; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-13-0458
©2014 American Association for Cancer Research.
INTRODUCTION Primary and metastatic human melanomas show consider-
able variability in the composition, density, and distribution
of tumor-infi ltrating immune cells in different patients. In
agreement with the tumor immune-surveillance theory, several
studies found a correlation among the presence of T cells in
primary melanomas, the expression of MHC class I molecules
on tumor cells, and a favorable prognosis ( 1, 2 ). Conversely, the
absence of tumor-infi ltrating T cells in primary melanomas
was associated with an increased risk for metastatic spread into
the sentinel lymph nodes and decreased survival ( 3, 4 ). The
underlying mechanisms that recruit immune cells and regu-
late their function in the tumor microenvironment are poorly
understood. Previously, we described an association between
the presence of granzyme B–expressing T lymphocytes and a
locally activated type I IFN system, indicated by expression of
the antiviral protein MxA, in primary melanomas of patients
showing signs of spontaneous regression ( 5 ). The rationale
for our analyses was derived from our observations in experi-
mental mouse models with transplantable tumors, including
the B16 melanoma, where local transgenic expression of IFNαcould augment CTL responses in the tumor microenviron-
ment ( 6–8 ). More recently, it was shown that activation of the
host type I IFN system is indeed a critical requirement for the
innate immune recognition of transplanted B16 melanomas
through signaling on CD8α + dendritic cells (DC), which then
initiate adaptive cellular immunity ( 9, 10 ).
However, melanomas also frequently progress despite T-cell
infi ltration. This was originally thought to be due to the Dar-
winian selection of tumor-cell variants that escape immune
destruction ( 11 ). As an alternative explanation, it was found
that T cells lose their effector functions in the immunosup-
pressive microenvironment of tumors in which regulatory
immune cell subpopulations accumulate ( 12 ). More recently,
dynamic adaptive changes of both tumor and immune cells
caused by infl ammatory cytokines have been described that
contribute to the immune escape of melanoma ( 13–15 ).
Prominent among these is the upregulation of the immune-
inhibitory receptor PD-L1 on melanoma cells in response to
T cell–derived IFNγ, which in turn engages PD-1 on T cells
and attenuates their effector functions ( 16, 17 ). Blockade of
the interaction between PD-L1 and PD-1 can reactivate effec-
tor functions of melanoma-specifi c T cells both in mouse and
man, demonstrating the critical importance of this immuno-
regulatory mechanism in tumor tissue ( 18–21 ).
Given the success of new immunotherapies that abrogate
the immune-inhibitory PD-L1–PD-1 interactions in patients
with melanoma with preexisting antitumor immunity ( 16 ,
20 ), the treatment of patients with melanomas lacking T-cell
infi ltrates (“immune cell–poor melanomas”) has emerged as
a major clinical challenge. We experimentally addressed this
issue in the genetically engineered Hgf-Cdk4 R24C mouse model
on March 1, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 3, 2014; DOI: 10.1158/2159-8290.CD-13-0458
676 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Bald et al.RESEARCH ARTICLE
in which primary melanomas histomorphologically imitate
human pigmented melanomas with little immune cell infi l-
tration and metastasize early in lymph nodes and lungs ( 22 ).
In our work, we investigated three principal hypotheses: (i)
immune cell–poor primary melanomas evade innate type I
IFN–dependent immune surveillance and thereby avoid the
induction of antitumoral CTL immunity; (ii) targeted activa-
tion of type I IFNs can establish cellular immune surveillance;
(iii) type I IFNs simultaneously activate immune-inhibitory
PD-L1–PD-1 receptor interactions, and therapeutic blockade
of this pathway further augments tumor immune surveillance.
RESULTS Immune Cell–Poor Melanomas Evade Type I IFN–Dependent Immune Surveillance and Editing
On the basis of our immunohistochemical observation
that regressive primary melanomas with extensive T-cell infi l-
tration stain positive for markers of an activated type I IFN
system ( 5 ), we expected that immune cell–poor melanomas
lacking T cells would show only low expression levels of type
I IFN–regulated genes. A bioinformatic analysis of genome-
wide transcriptomic data for 223 primary melanomas ( 23 )
indeed showed that the expression of CD3D and other T-cell
transcripts directly correlated with the expression of a set of
genes that are regulated by type I IFNs (Pearson correlation
coeffi cient r = 0.6) in melanoma cells ( Fig. 1A and B and Sup-
plementary Table S1). The type I IFN response signature was
generated from a publicly available dataset of IFNα-treated
human melanoma cells ( 24 ). Furthermore, high expression
of type I IFN–responsive genes or CD3D was associated with
an increased relapse-free survival, consistent with the idea
that preexisting antitumoral immune responses determine
a favorable prognosis in patients with melanoma ( Fig. 1C ).
In turn, the absence of IFN-regulated genes in immune
cell–poor melanomas suggested that these tumors escape
Figure 1. Immune cell–poor pigmented primary human melanomas show low expression of type I IFN–regulated genes and are morphologically imi-tated in the Hgf-Cdk4 R24C mouse melanoma model. A, representative histomorphology of immune cell–poor (left) and immune cell–rich (right) pigmented primary cutaneous human melanomas in H&E-stained sections. Red arrows, immune cell infi ltrates. B, heatmap showing expression levels for type I IFN–regulated and T cell–related genes (CD3) in a clinically annotated cohort of 223 human primary melanomas. Samples are ordered by increasing CD3 transcript levels. C, corresponding progression-free survival in the indicated patient subgroups. Unbiased median expression value cutoffs were used for patient subgroup classifi cation. P values were determined by a log-rank test. D, histomorphology of primary cutaneous melanomas in Hgf-Cdk4 R24C mice showing the typical immune cell–poor pigmented phenotype in H&E-stained sections (left), and schematic diagram depicting the genetic alterations in Hgf-Cdk4 R24C mice (right).
Immune cell–poor pigmented
primary human melanoma
Immune cell–rich pigmented
primary human melanoma
50 μm 50 μm
A
Immune cell–poor pigmented
primary mouse melanoma
D
Growth factor
signaling
Cell-cycle
control
Cdk4R24C
G1 S
Cyclin D1
tkAErk
Ras Raf
p16/Ink4a
MetHgf
PI3K
C High, n = 105
Low, n = 104
CD3D
Time (y)
0
20
40
60
80
100
0
20
40
60
80
100
High, n = 104
P = 3.15 × 10–8
The Hgf-Cdk4R24C mouse model
Low, n = 103
Re
lap
se
-fre
e s
urv
iva
l (%
)
P = 0.0375
IFN response signature
CD
3
Type I IF
N r
esponse s
ignatu
re
Primary melanomas, n = 223ACACBBIRC3BST2CXCL1CXCL2DDX60DHX58GBP1HERC5IFI27IFI44IFI44LIFI6IFIH1IFIT3IFITM1IRF7ISG15ISG20LGALS9MX1OAS1OAS2PARP12RASGRP3SAMD9SERPING1SLC15A3SP110STAT1XAF1CD3DCD3ELCKCD247
20 4 6 8 10Time (y)
20 4 6 8 10
Log2 ratio
–3 3 0
B
on March 1, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 3, 2014; DOI: 10.1158/2159-8290.CD-13-0458
JUNE 2014�CANCER DISCOVERY | 677
Combination Immunotherapy for Immune Cell–Poor Melanomas RESEARCH ARTICLE
the immune-surveillance function of the type I IFN system
and thereby avoid the recruitment of immune cells and the
subsequent induction of protective tumor-specifi c cytotoxic
immune responses. Primary cutaneous melanomas in Hgf-
Cdk4 R24C mice morphologically imitate immune cell–poor
pigmented primary human melanomas ( Fig. 1D ) and show
very low expression of type I IFN–regulated genes. This exper-
imental system therefore allowed us to investigate whether
malignant transformation indeed takes place without the
induction of type I IFN–dependent activation of cellular
antitumor immunity.
We therefore crossed Hgf-Cdk4 R24C into the Ifnar1 −/−
background to obtain melanoma-prone mice that lack a
functional type I IFN system. Cohorts of 8-week-old Ifnar1 -
competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice then
received a single epicutaneous application of the carcinogen
7,12-dimethylbenz(a)anthracene (DMBA) on the back skin
( Fig. 2A ) to accelerate and synchronize the development of pri-
mary melanomas ( 13 ). We found that DMBA-initiated primary
melanomas appeared with the same growth kinetics and mul-
tiplicity in the skin of Ifnar1 -defi cient and Ifnar1 -competent
Hgf-Cdk4 R24C mice ( Fig. 2B ). Accordingly, DMBA-exposed
Ifnar1 -defi cient and Ifnar1 -competent Hgf-Cdk4 R24C mice
showed largely identical survival curves ( Fig. 2C ). These results
indicated that the development of primary melanomas in this
experimental system was not affected by type I IFN–dependent
immune surveillance.
In subsequent experiments, we injected cohorts of Ifnar1 -
competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice subcuta-
neously with the carcinogen methylcholanthrene (MCA) to
induce primary fi brosarcomas ( Fig. 2D ). In line with previ-
ously reported experimental work in this well-established
model for IFN-dependent cancer immune surveillance ( 25 ),
we found that fi brosarcomas developed with decreased
latency and increased penetrance in Ifnar1 -defi cient com-
pared with Ifnar1 -competent Hgf-Cdk4 R24C mice ( Fig. 2E ),
resulting in shorter survival ( Fig. 2F ). This ruled out the pos-
sibility that the anti-infl ammatory properties of hepatocyte
growth factor ( HGF ), which can promote a tolerogenic DC
phenotype and the expansion of regulatory T cells ( 26, 27 ),
precluded immune surveillance by the type I IFN system in
Hgf-Cdk4 R24C mice.
Figure 2. Primary melanomas in Hgf-Cdk4 R24C mice escape type I IFN–mediated immune surveillance. A and D, experimental protocol for the induction of primary cutaneous melanomas (A) or sarcomas (D) in cohorts of Ifnar1 -competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice with a single epicutaneous applica-tion of DMBA or a single subcutaneous injection of MCA, respectively. B and E, tumor growth kinetics of the largest DMBA-induced melanoma (B) and of MCA-induced sarcomas (E) in representative cohorts of 5 individual Ifnar1 -competent (top) and Ifnar1 -defi cient (bottom) mice over time. C and F, correspond-ing Kaplan–Meier survival curves of melanoma-bearing (C) or sarcoma-bearing (F) Ifnar1 -competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice. G, representative CD45-stained sections of a primary DMBA-induced melanoma (left) and a primary MCA-induced sarcoma (right) in Ifnar1 -competent Hgf-Cdk4 R24C mice. H, fl ow cytometric quantifi cation of tumor-infi ltrating immune cells in primary Hgf-Cdk4 R24C melanomas and sarcomas (left and middle; mean ± SEM; n = 12) and cor-responding real-time PCR analysis of IFN-induced genes (right; mean ± SEM; n = 12; *, P < 0.05).
4
6
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0 70 140 2
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Tum
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Days after DMBA treatment Days after MCA injection
A Monitor
tumor development
DMBA
epicutaneously
140 56 Age (d)
Ifnar1-deficient Hgf-Cdk4R24C
Ifnar1-competent Hgf-Cdk4R24C
CD45 100 μm CD45 100 μm
0
50
100
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% S
urv
ival
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Ifnar1-deficient Hgf-Cdk4R24C
Ifnar1-competent Hgf-Cdk4R24C
Monitor
tumor development
MCA
subcutaneously
140 56 Age (d)
0
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100
0 70 140
% S
urv
ival
25
75
Rel. e
xpre
ssio
n
10–4
10–3
10–2
10–1
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Irf7
Cxcl1
0
Ccl5
Melanoma Sarcoma
* *
*
B
C
G
H
Days after DMBA treatment Days after MCA injection
D
E
F
% c
ells
in the tum
or
0
5
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15
20
CD
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+
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ells
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*
* *
on March 1, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 3, 2014; DOI: 10.1158/2159-8290.CD-13-0458
678 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Bald et al.RESEARCH ARTICLE
To understand the divergent role of the type I IFN system
in the pathogenesis of primary melanomas and fi brosarco-
mas, we performed further morphologic, cellular, and molecu-
lar investigations. Immunohistopathologic analyses revealed
increased numbers of tumor-infi ltrating CD45 + immune cells
in primary MCA-induced fi brosarcomas when compared with
primary DMBA-induced melanomas in Hgf-Cdk4 R24C mice ( Fig.
2G ). This difference was further quantifi ed and confi rmed to
be signifi cant by fl ow cytometric analyses of tumor-cell suspen-
sions ( Fig. 2H , left). Fibrosarcomas showed increased numbers
of tumor-infi ltrating CD8 + T cells ( Fig. 2H , middle) and sig-
nifi cantly higher expression levels of type I IFN–regulated genes
( Fig. 2H , right). Taken together, these results demonstrated that
melanomas and fi brosarcomas interact with the host immune
system in fundamentally different ways, although they were
both induced by chemically closely related potent carcinogens.
It was previously described that MCA-induced fi bro-
sarcomas derived from Ifnar1 -defi cient mice show a highly
immunogenic, “unedited” phenotype and therefore do not
grow progressively when transplanted onto Ifnar1 -competent
mice ( 25 ). Because primary Hgf-Cdk4 R24C melanomas did not
activate type I IFNs, we hypothesized that they would also
escape the type I IFN–dependent immune-editing process.
To test this hypothesis, we transplanted fi ve different Ifnar1 -
competent and fi ve different Ifnar1 -defi cient DMBA-induced
primary Hgf-Cdk4 R24C melanomas onto groups of three Ifnar1 -
competent, Ifnar1 -defi cient, and Rag2 -defi cient syngeneic
C57BL/6 mice ( Fig. 3A ). Each transplanted melanoma grew
progressively in all three strains of mice ( Fig. 3B and C ), indi-
cating the poor immunogenicity of both Ifnar1 -competent
and Ifnar1 -defi cient melanomas. Interestingly, a subset of
Ifnar1 -competent and Ifnar1 -defi cient melanoma cells grew
more rapidly in Ifnar1 -defi cient mice when compared with
Ifnar1 -competent or Rag2 -defi cient mice, pointing toward the
previously described role for the host type I IFN system in
transplanted melanoma ( 9 ).
Figure 3. Both Ifnar1 -competent (CT) and Ifnar1 -defi cient (IFT) Hgf-Cdk4 R24C melanomas grow pro-gressively when transplanted in Ifnar1 -competent and Ifnar1 -defi cient hosts. A, experimental proto-col for transplantation of primary DMBA-induced melanomas from 5 individual Ifnar1 -competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice onto groups of 3 syngeneic Ifnar1 -competent, Ifnar1 -defi cient, and Rag2 -defi cient C57BL/6 mice. Tumor develop-ment was monitored over time. B, time to tumor onset of Ifnar1 -competent Hgf-Cdk4 R24C melanomas in the indicated genotypes grouped according to similar (left) or faster (right) growth in Ifnar1 -defi cient recipient mice. C, corresponding data for Ifnar1 -defi cient Hgf-Cdk4 R24C melanomas. WT, wild-type.
3 Ifnar1-competent
3 Ifnar1-deficient
WT
Ifnar
1–/–
Rag
2–/–
30
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60
90
120 IFT-2
3 Rag2-deficient
A
B
Tum
or
onset (d
) Tum
or
onset (d
)
WT
Ifnar
1–/–
Rag
2–/–
CT-1
30
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WT
Ifnar
1–/–
Rag
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Ifnar
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Ifnar
1–/–
Rag
2–/–
WT
Ifnar
1–/–
Rag
2–/–
IFT-4 IFT-5
Similar growth in Ifnar1-deficient recipients
5 Ifnar1-competent (CT)
Hgf-Cdk4R24C melanomas
Ifnar1-deficient Hgf-Cdk4R24C melanomas
Ifnar1-competent Hgf-Cdk4R24C melanomas
Similar growth in Ifnar1-deficient recipients
WT
Ifnar
1–/–
Rag
2–/–
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Ifnar
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2–/–
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Faster growth in Ifnar1-deficient recipients
WT
Ifnar
1–/–
Rag
2–/–
WT
Ifnar
1–/–
R
ag2–
/–
IFT-1 IFT-3
Faster growth in Ifnar1-deficient recipients
Tx Monitor
tumor development
5 Ifnar1-deficient (IFT)
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Combination Immunotherapy for Immune Cell–Poor Melanomas RESEARCH ARTICLE
Targeted Type I IFN Activation Establishes Immune Surveillance and Impairs Melanoma Growth
We hypothesized that therapeutic activation of type I IFNs
in the microenvironment of primary Hgf-Cdk4 R24C melanomas
could alert innate immune surveillance and thereby delay
tumor growth. As an experimental strategy, we used the proto-
typic immunostimulatory RNA polyinosinic:polycytidylic
acid [poly(I:C)], which triggers the innate viral recognition
receptors TLR3 and MDA5 and effi ciently stimulates the
type I IFN system ( 28, 29 ). Peritumoral injections of estab-
lished DMBA-induced primary Hgf-Cdk4 R24C melanomas with
poly(I:C) for 2 weeks strongly induced the expression of type
I IFN–regulated genes and promoted the recruitment of
immune cells into the tumor microenvironment ( Fig. 4A and
B ). Prolonged treatment with poly(I:C) considerably delayed
the growth of primary melanomas and increased survival
( Fig. 4C and D ). This effect was completely abrogated in
Ifnar1 -defi cient Hgf-Cdk4 R24C mice ( Fig. 4E ). Thus, targeted
type I IFN activation with poly(I:C) exposed immune cell–
poor Hgf-Cdk4 R24C melanomas to the surveillance functions
of cellular immunity.
To investigate the role of the type I IFN system in tumor
versus host cells, we used the slowly growing transplantable
Hgf-Cdk4 R24C melanoma cell line HCmel3, which morpho-
logically imitates primary Hgf-Cdk4 R24C melanomas ( 13 ) and
shows similar low expression levels of type I IFN–regulated
genes. Short-term treatment of established HCmel3 melano-
mas with poly(I:C) for 2 weeks ( Fig. 4F ) also stimulated type
I IFN–regulated genes and increased the number of immune
cells in the tumor microenvironment to a similar extent
when compared with primary melanomas ( Fig. 4B and G ).
Figure 4. Peritumoral injections of poly(I:C) induce type I IFN–dependent cytotoxic immunity and delay the growth of primary and transplanted Hgf-Cdk4 R24C melanomas. Primary DMBA-induced (A–F) and intracutaneously (i.c.) transplanted (F–J) Hgf-Cdk4 R24C melanoma model. A and F, experimental protocols for short-term treatment of melanomas with poly(I:C). B and G, fold increase of mRNA expression levels for the indicated genes (left) and of the percentage of tumor-infi ltrating immune cells (right) in primary and transplanted Hgf-Cdk4 R24C melanomas compared with controls (mean ± SEM; n = 6). C and H, experimental protocols for long-term treatment of melanomas with poly(I:C). D and I, tumor growth kinetics of melanomas in individual mice treated as indicated (left, middle), and mean survival in each cohort (right; mean ± SEM; n = 6; **, P < 0.01). Similar results were obtained in two inde-pendent treatment cohorts. E and J, corresponding tumor growth kinetics and mean survival in Ifnar1 -defi cient cohorts of mice treated as indicated ( n = 6). Similar results were obtained in two independent treatment cohorts.
0
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Ifnar1-deficient Hgf-Cdk4R24C mice
Ifnar1-competent Hgf-CdkR24C mice
Tu
mo
r d
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ete
r (m
m)
% cells % cells
Ifnar1-deficient C57BL/6 mice
Ifnar1-competent C57BL/6 mice
ctrl poly (I:C)
0
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)
Grz
b
Per
f
Cd3
Klrb
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240
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)
ctrl 0
60
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240
Me
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m)
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poly(I:C)
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poly(I:C)
poly (I:C)
poly (I:C)
D I
Days after DMBA treatment Days after i.c. injection of HCmel3
Hgf-CdkR24C
mice C57BL/6
mice
Ifnar1-competent
Ifnar1-deficient
Hgf-Cdk R24C
mice
C57BL/6
mice
**
**
4
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10
70 140 210 2
poly(I:C)
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Bald et al.RESEARCH ARTICLE
Prolonged poly(I:C) treatment delayed the growth of trans-
planted HCmel3 melanomas and signifi cantly increased sur-
vival ( Fig. 4H and I ). Transplanted HCmel3 cells grow with
similar kinetics in Ifnar1 -competent and Ifnar1 -defi cient mice
( Fig. 4I and J , left), demonstrating that this melanoma cell line
does not spontaneously engage the host type I IFN system in
the transplantation setting. Importantly, the therapeutic effi -
cacy of poly(I:C) was completely abrogated in Ifnar1 -defi cient
C57BL/6 mice ( Fig. 4J ), demonstrating the critical require-
ment for a functional type I IFN system in host cells.
In primary and transplanted MCA-induced sarcomas,
Ifnar1 signaling in host hematopoietic cells was shown to be
critical for the induction of antitumor immune responses
( 25 ). The activation of natural killer (NK) cell responses with
poly(I:C) requires signaling through Ifnar1 not only in NK
cells but also in other immune cell types, most importantly
in DCs ( 30 ). To experimentally dissect the contribution
of Ifnar1 signaling in various immune cell subsets to the
observed antitumor effects, we treated HCmel3 cells with
poly(I:C) in mice with cell type–specifi c conditional deletion
of the Ifnar1 gene ( 31–33 ). We found that the therapeutic
activity of poly(I:C) was largely abrogated in mice lacking
the Ifnar1 gene specifi cally in CD11c + , LysM + , CD4 + , or Nc1r +
cells, which are primarily expressed in DCs, macrophages/
neutrophils, T cells, and NK cells, respectively ( Fig. 5A ).
These results demonstrate a requirement for the coordinated
activation of type I IFN responses in all of these different
immune cell subsets to obtain the full antitumor effi cacy of
poly(I:C).
Next, we explored the antitumor effector mechanisms
induced by targeted activation of the type I IFN system in
our experimental model. Antibody-mediated depletion of
NK cells largely abrogated and depletion of CD8 + T cells
severely compromised the effi cacy of poly(I:C) treatment
( Fig. 5B ). This result is consistent with the notion that
NK-cell cytotoxicity is required early to keep melanomas in
Figure 5. The therapeutic effi cacy of poly(I:C) requires a functional type I IFN system in DCs, mac-rophages/neutrophils, NK cells, and T cells. A, experi-mental protocol for treatment of HCmel3 melanomas in Ifnar1 fl /fl , Ifnar1 ΔLysM , Ifnar1 ΔCD11c , Ifnar1 ΔCD4 , and Ifnar1 ΔNc1r mice (top) and Kaplan–Meier survival curves in groups of 5 mice with the indicated geno-types (middle, bottom). Similar results were obtained in two independent experiments. B, experimental protocol (top) and Kaplan–Meier survival curves in groups of 5 mice treated as indicated (bottom). Similar results were obtained in two independent experiments. i.c., intracutaneous.
Ifnar1ΔLysM
0
25
50
75
100
0 35 70 105 140
Ifnar1ΔCD11c
0
25
50
75
100
0 35 70 105 140
Ifnar1ΔNc1r
0
25
50
75
100
0 35 70 105 140
Control
poly(I:C) + Ctrl IgG
poly(I:C) + anti-CD8
poly(I:C) + anti-NK1.1
poly(I:C) + anti-IFNγ
Days after i.c. injection of HCmel3
HCmel3 Control or poly(I:C)
0 70 67 28 24 Days
% S
urv
ival
Control or poly(I:C)
0 70 67 28 24 Days
Days after i.c. injection of HCmel3
% S
urv
ival
A
B
0
25
50
75
100
0 35 70 105 140
0
25
50
75
100
0 35 70 105 140
Ifnar1fl/fl
% S
urv
ival
Ctrl IgG, anti-CD8, anti-NK1.1 or anti-IFNγ mAb
0
25
50
75
100
0 35 70 105 140
Ifnar1ΔCD4
% S
urv
ival
HCmel3
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Combination Immunotherapy for Immune Cell–Poor Melanomas RESEARCH ARTICLE
check and promote the subsequent development of CD8 +
T cell–mediated immunity. Both NK cells and CD8 + T
cells exert their antitumor activity at least in part through
the secretion of IFNγ ( 34, 35 ). Antibody-mediated block-
ade of IFNγ completely abolished the antitumor immune
responses induced by poly(I:C) ( Fig. 5B ). Taken together,
our fi ndings indicate that targeted activation of the type I
IFN system in immune cell–poor Hgf-Cdk4 R24C melanomas
induces effective immune cell surveillance through activa-
tion of NK and CD8 + T cells and subsequent production
of IFNγ.
Combination Immunotherapy with Poly(I:C) and a Blocking Anti–PD-1 mAb Prolongs Survival
Treatment of primary and transplanted Hgf-Cdk4 R24C
melanomas with poly(I:C) restrains their growth for sev-
eral weeks but eventually fails ( Fig. 4D and I ). Both type I
and II IFNs upregulated the expression of PD-L1 on the
Hgf-Cdk4 R24C melanoma cell line HCmel3 in vitro ( Fig. 6A ).
We also observed a signifi cant upregulation of Pdl1
mRNA expression levels in poly(I:C)–treated compared with
untreated Hgf-Cdk4 R24C mouse melanomas that correlated
with the upregulation of type I IFN–regulated genes such as
Irf7 ( Fig. 6B ). Melanoma-bearing mice treated with poly(I:C)
also showed increased numbers of PD-1–expressing CD8 + T
cells in the peripheral blood ( Fig. 6C ), indicating an activa-
tion of the immune-inhibitory PD-L1–PD-1 signaling axis as
a counter-regulatory mechanism to attenuate effector func-
tions of both T and NK cells ( 17, 18 , 36 ).
We therefore reasoned that therapeutic blockade of PD-1
signaling would further augment and sustain the antitu-
mor activity of targeted type I IFN activation with poly(I:C).
Indeed , injections of a PD-1–blocking monoclonal antibody
(mAb) together with poly(I:C) were able to cause partial
regression in established HCmel3 mouse melanomas and
signifi cantly prolonged the survival compared with poly(I:C)
treatment alone (126 ± 16 vs. 97 ± 13 days; Fig. 6D–F ). Injec-
tions of anti–PD-1 mAb alone did not show any treatment
effect, consistent with our fi nding that Hgf-Cdk4 R24C mouse
melanomas escape cellular immune surveillance. Thus, tar-
geted activation of type I IFNs in combination with blockade
of the IFN-induced immune-inhibitory PD-L1–PD-1 signal-
ing pathway represents a rational strategy to expose immune
cell–poor tumors to prolong immune surveillance. Finally,
a detailed toxicologic study revealed that intracutaneous
injections of poly(I:C) alone or in combination with intraperi-
toneal injections of PD-1–blocking mAbs only caused local
skin infl ammatory responses, without substantial treatment-
related acute toxic side effects affecting vital organ structure
and function (Supplementary Figs. S1 and S2).
Our experimental fi ndings suggested that the expression of
PD-L1 in melanoma tissues correlates with the expression of
CD3 and type I IFN–responsive genes. A poor-quality probe
precluded the analysis of PD-L1 expression in the primary
Figure 6. Antibody-mediated blockade of the immune-inhibitory PD-1–PD-L1 signaling pathway prolongs survival in poly(I:C)-treated mice. A, upregula-tion of MHC class I (MHCI) and PD-L1 on the surface of HCmel3 melanoma cells following exposure to IFNα or IFNγ in vitro . B, correlation of relative mRNA expression levels for PD-L1 and IRF7 in untreated and poly(I:C)-treated HCmel3 melanoma samples determined by quantitative reverse transcriptase PCR (qRT-PCR ). C, representative histograms showing the percentage of PD-1–expressing CD8 + T cells (left) and cumulative data for a correlation of PD-1 expression on CD8 + T cells in the blood of HCmel3-bearing mice treated as indicated (right). D, experimental protocol for combination therapy for trans-planted HCmel3 melanomas with poly(I:C) and anti–PD-1 antibody. E, representative tumor growth kinetics of HCmel3 melanomas in individual mice treated as indicated ( n = 5). Similar results were obtained in at least two independent treatment cohorts. F, corresponding Kaplan–Meier survival curves (left) and mean survival of mice treated as indicated (right); mean ± SEM; n = 10; *, P < 0.05; n.s., nonsignifi cant; IgG, immunoglobulin G; i.c., intracutaneous.
Control IFNα IFNγP
D-L
1
MHCI
A
91% 2%
15
15
10
0
5 0
% PD-1+ cells in CD8+ T cells
% C
D8
+ T
cells
in P
BM
C
10
5
PD-1 in CD45+CD8+ gate
Control poly(I:C)
1.5% 10.4%
HCmel3
0 105 102 28 24 Days
0
25
50
75
100
0 70 140 210
% S
urv
ival
Days after i.c. injection of HCmel3
Ctrl IgG
poly(I:C) +Ctrl IgG
poly(I:C) +aPD-1
D
Days after i.c. injection of HCmel3
0
5
10
15
20
0 70 140 210
0
5
10
15
20
0 70 140 210
Ctrl
0
5
10
15
20
0 70 140 210
aPD-1
0
5
10
15
20
0 70 140 210
Tum
or
dia
mete
r (m
m)
poly(I:C) + aPD-1
poly(I:C) + Ctrl IgG E
C
F
B
aPD-1
Mean s
urv
ival (d
)
poly(I:C) +Ctrl IgG
aPD1 mAb
poly(I:C) +aPD-1
Ctrl IgG
0
50
100
150
200 *
n.s.
95%
10–2
10–3
10–4
10–1 10–010–2
Control poly(I:C)
IRF7 expression
PD
-L1
expre
ssio
n
Control poly(I:C)
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Bald et al.RESEARCH ARTICLE
melanoma dataset shown in Fig. 1B . Therefore, we interro-
gated two additional publicly available datasets of either pri-
mary melanomas (GSE15605) or melanoma metastasis [The
Cancer Genome Atlas (TCGA) skin cutaneous melanoma]
that were generated using a different microarray platform
( 37 ) or RNA sequencing (RNA-seq ), respectively. Indeed, this
analysis confi rmed our hypothesis, as we found a strong
correlation among CD3D , IFN-responsive genes, and PD-L1
expression in both datasets irrespective of the genomics plat-
form ( Fig. 7A and B ). Clinical follow-up data were available
for the TCGA melanoma metastasis cohort, and we classi-
fi ed samples by unbiased median expression value cutoffs as
described for the primary melanoma cohort shown in Fig. 1B .
Consistently, high expression levels of CD3D , IFN-responsive
genes, and, importantly, also PD-L1 were associated with a
favorable disease course ( Fig. 7C ).
DISCUSSION In our work, we experimentally investigated the role of type
I IFN–dependent tumor immune surveillance in a geneti-
cally engineered mouse model of melanoma. By crossing
the Hgf-Cdk4 R24C mouse strain onto the Ifnar1 -defi cient back-
ground, we show that immune cell–poor primary melanomas
do not spontaneously activate the immune-surveillance and
immune-editing functions of the endogenous type I IFN sys-
tem. Because Hgf-Cdk4 R24C mouse melanomas imitate immune
cell–poor human primary melanomas with a bad prognosis,
our results suggest that this subset of tumors also evades type
I IFN–dependent immune surveillance.
In contrast to DMBA-induced melanomas, we found that
primary MCA-induced sarcomas spontaneously activated the
host type I IFN system in Hgf-Cdk4 R24C mice, confi rming a
previously published report ( 25 ). Because both tumor types
were induced by chemically related and highly potent car-
cinogens, one would expect a similar spectrum of tumor
antigens due to genetic mutations. The divergent interaction
of primary melanomas and sarcomas with the innate immune
system might therefore refl ect the different immunologic
properties of the cells of origin, for example, melanocytes
versus fi broblasts. It is tempting to speculate that malig-
nant fi brosarcoma cells dictate an immunologically much
more active microenvironment as they express higher levels
of MHC class I molecules and secrete increased amounts of
Figure 7. PD-L1 expression correlates with T-cell markers and an IFN response signature in human melanomas. A, bottom, heatmap of primary melanoma samples ( n = 46; GSE15605) ordered by increasing T-cell marker gene levels (CD3) and visualization of corresponding IFN response signature gene expression. The color code represents log 2 -transformed and mean-centered expression values generated with the Affymetrix Hgu133plus microar-ray platform (bottom). Corresponding barplot of PD-L1 levels and trend line of the IFN response signature matched to the samples shown in the heatmap below. Pearson correlation coeffi cient ( r ) is indicated (top). B, PD-L1 , IFN response signature, and CD3D expression in human melanoma metastasis ( n = 248) from the TCGA melanoma dataset (skin cutaneous melanoma; SKCM). Pearson correlation coeffi cients ( r ) are indicated. Expression values represent log 2 -transformed normalized RNA-seq reads generated with the Illumina platform. C, Kaplan–Meier analysis of overall survival (calculated as years to death or years to last follow-up) using the TCGA cohort (melanoma metastasis; n = 248) and median expression value cutoffs for CD3D , the IFN response signature, and PD-L1. P values were determined by a log-rank test.
B
PD
-L1,
expre
ssio
n
(RN
A-s
eq log
2)
r = 0.73
C
CD3D
IFN response signature
PD-L1
High, n = 124
Low, n = 124
High, n = 124
Low, n = 124
High, n = 124
Low, n = 124
Overa
ll surv
ival (%
) O
vera
ll surv
ival (%
)
P = 0.00533
P = 0.00387
P = 0.00509
TCGA melanoma metastasis
Overa
ll surv
ival (%
)
PD
-L1,
expre
ssio
n
(RN
A-s
eq log
2)
IFN signature expression
(RNA-seq log2)
0 2 4 6 8 10
2
0
4
6
8
10
2
0
4
6
8
10
6 7 8 9 10
r = 0.71
r = 0.72
0 2 4 6 8 10
IFN
sig
natu
re e
xpre
ssio
n
(RN
A-s
eq
log
2)
6
7
8
9
10
CD3D expression
(RNA-seq log2)
CD3D expression
(RNA-seq log2)
5 4 3 2 1 0
Years to death or last follow-up
5 4 3 2 1 0
5 4 3 2 1 0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
PD-L1
r = 0.75
IFN response signature
CD3 low CD3high
CD
3
Typ
e I IF
N r
esp
on
se
sig
na
ture
0 4–4
Log2 ratio
Primary melanomas, n = 46 (GSE15605)
A
n = 23 n = 23
PD
-L1
exp
ressio
n (
log
2)
–3
–2
–1
0
1
2
3
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Combination Immunotherapy for Immune Cell–Poor Melanomas RESEARCH ARTICLE
proinfl ammatory chemokines and cytokines when compared
with malignant melanoma cells. Our observations are in line
with recent reports in genetically engineered mouse models in
which malignant transformation in lung epithelial cells ( 38 )
or muscle cells ( 39 ) was driven by the same genetic events (e.g.,
introduction of oncogenic Kras and simultaneous Trp53 dele-
tion), but primary lung carcinomas and muscle fi brosarcomas
interacted with the immune system in fundamentally differ-
ent ways (e.g., tolerance induction vs. immune surveillance
and editing). Together, these experimental fi ndings emphasize
that the spontaneous immune response to cancer is highly
diverse and depends on contextual elements, including the cell
of origin, the nature of the local immune system, and the type
of genetic changes that drive malignant transformation ( 40 ).
Poly(I:C) is a prototypic immunostimulatory RNA that
potently stimulates innate pattern recognition receptors for viral
RNA in macrophages and DCs, leading to the induction of type I
IFNs and the activation of innate and adaptive cellular immune
responses ( 29 , 41 , 42 ). Our experimental results in a genetically
engineered mouse model show that poly(I:C) can alert the cel-
lular immune system to nascent primary cutaneous melano-
mas that evade IFN-dependent immune surveillance. Using the
Ifnar1 -competent transplantable Hgf-Cdk4 R24C melanoma cell
line HCmel3 that does not spontaneously activate type I IFNs
and mice with conditional deletion of the Ifnar1 gene in differ-
ent immune cell subsets, including macrophages/neutrophils,
DCs, NK, and T cells, we demonstrate that poly(I:C) induces the
coordinate type I IFN–dependent activation of all these cell types
to promote effective tumor immunity. In the effector phase, this
depends on the presence of both NK cells and CD8 + T cells and
the production of IFNγ, in line with a large body of experimen-
tal evidence in different tumor models ( 34, 35 , 43, 44 ). These
fi ndings underscore the critical importance of a functional type
I IFN system in cells of the host immune system that was also
found in experimental models in which tumor cells spontane-
ously activated type I IFN responses after transplantation ( 25 ).
Here, type I IFN–dependent activation of DC subsets that are
specialized for antigen cross-presentation was required for effec-
tive induction of antitumor immunity ( 9 , 32 ).
Targeted activation of the type I IFN system in the microen-
vironment of immune cell–poor Hgf-Cdk4 R24C mouse melano-
mas with poly(I:C) was associated with cytotoxic immune cell
recruitment, subsequent upregulation of PD-L1 expression in
tumor tissue, and an increased expression of PD-1 on periph-
eral blood CD8 + T cells. Because type I and type II IFNs upregu-
late PD-L1 expression on melanoma cells in vitro , and because
poly(I:C) induces cellular antitumor immunity that critically
depends on type I and type II IFNs in vivo , we hypothesized
that the interaction between melanoma and T cells through
PD-L1 and PD-1 receptors represents an adaptive resistance
program to IFN-driven cytotoxic immunity that attenuates
effector functions of T and NK cells. Our observation that anti-
body-mediated PD-1 blockade prolonged the survival of mice
only in combination with poly(I:C) but not given as a mono-
therapy demonstrates that activation of the type I IFN system
leads to subsequent functional activation of the PD-L1–PD-1
immune-inhibitory signaling axis in immune cell–poor Hgf-
Cdk4 R24C melanomas. Because PD-L1 is expressed not only on
melanoma cells but also on accessory cells in the tumor stroma
(such as fi broblasts and DCs), the relative contribution of these
cell types for PD-1–mediated interaction with T cells will have
to be experimentally resolved in future work. This would have
to include studies in other experimental systems to confi rm
the generality of our fi ndings beyond the Hgf-Cdk4 R24C mouse
melanoma model used in our work.
Recent clinical trials demonstrated that blockade of the
immune-inhibitory PD-L1–PD-1 pathway can achieve high
response rates in some patients with advanced metastatic
melanoma and other types of cancers ( 19–21 ). PD-L1–PD-1
blockade seemed to be particularly effective in patients with
melanoma with an ongoing cellular immune response ( 20 ).
Our experimental results confi rm the notion that upregula-
tion of the PD-1–PD-L1 signaling axis in tumor tissue, as
a consequence of type I IFN activation and invasion by NK
and T cells, predicts therapeutic benefi t from therapeutic
PD-L1–PD-1 blockade alone. We therefore propose that the
expression of PD-L1 and type I IFN–responsive genes in
tumor tissues could serve as a sensitive biomarker for patient
stratifi cation in clinical trials investigating PD-1–PD-L1 anti-
body-containing regimens. RNA-seq data from the TCGA
melanoma project indicate a comparatively low abundance
of PD-L1 mRNA (lower third) relative to all other detected
reference transcripts (data not shown). Taking our survival
analysis into account, it is therefore conceivable that PD-L1
levels below the current detection threshold of immunohis-
tochemistry are functionally and clinically relevant and may
explain discrepancies addressing the prognostic and predic-
tive value of PD-L1 expression.
Approximately one third of all metastatic melanomas are
only poorly infi ltrated with immune cells ( 45 ). Consistent
with recently published work, our bioinformatic analysis
revealed that patients with these immune cell–poor melano-
mas, in which type I IFN–regulated genes, T cell–related
genes, and PD-L1 are expressed at low levels, had a com-
paratively poor prognosis ( 23 , 46–48 ). Hence, there is an
obvious need for new therapeutic strategies in this patient
subgroup. Our observation that treatment with a combina-
tion of poly(I:C) and a blocking anti–PD-1 mAb prolonged
the survival of mice with immune cell–poor melanoma not
only highlights the critical importance of immune-inhibitory
PD-L1–PD-1 interactions in vivo but also provides a preclini-
cal proof-of-concept that targeted type I IFN activation is a
rational strategy to increase the therapeutic benefi t of PD-1–
PD-L1 blockade for patients with immune cell–poor melano-
mas as well. These insights underscore the clinical relevance
of our work and provide a rationale for further experimental
investigations to develop similar combination treatment pro-
tocols. These may further augment cytotoxic immunity by
additionally targeting other IFN-driven counter-regulatory
mechanisms that attenuate NK- and T-cell effector functions
in the tumor microenvironment ( 49 ).
METHODS Mice
Wild-type, Ifnar1 −/− and Rag2 −/− C57BL/6 mice were purchased
from The Jackson Laboratory. Ifnar1 −/− C57BL/6 mice were crossed
with melanoma-prone Hgf-Cdk4 R24C mice to obtain Ifnar1 −/− × HGF-
CDK4 R24C mice. Ifnar1 fl /fl , LysM-Cre × Ifnar1 fl /fl ( Ifnar1 ΔLysM ) CD11c-
Cre × Ifnar1 fl /fl ( Ifnar1 ΔCD11c ), CD4-Cre × Ifnar1 fl /fl ( Ifnar1 ΔCD4 ), and
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Bald et al.RESEARCH ARTICLE
Nc1r-Cre × Ifnar1 fl /fl ( Ifnar1 ΔNc1r ) mice on the C57BL/6 background
were bred as described previously ( 31–33 ). All animal experiments
were approved by the local government authorities (LANUV, NRW,
Germany) and performed according to the institutional and national
guidelines for the care and use of laboratory animals.
Induction and Analysis of Primary Melanomas and Primary Sarcomas
The development of primary melanomas on the shaved back skins
of 8-week-old Ifnar1 -competent and Ifnar1 -defi cient Hgf-Cdk4 R24C mice
was accelerated and synchronized by a single epicutaneous applica-
tion of 100 nmol DMBA as described previously ( 13 ). Alternatively,
mice received a single subcutaneous injection of 100 μg MCA into the
fl ank to induce fi brosarcomas. Tumor development was monitored
by inspection, palpation, and digital photography. Tumor sizes were
measured weekly using a vernier calliper and recorded as mean diam-
eter. Mice were sacrifi ced when progressively growing melanomas or
sarcomas exceeded 10 mm or when signs of illness were observed.
Serial Tumor Transplantation Primary DMBA-induced melanomas from Ifnar1 -competent (CT)
and Ifnar1 -defi cient (IFT) Hgf-Cdk4 R24C mice were serially trans-
planted onto Ifnar1 -competent, Ifnar1 -defi cient, or Rag2 -defi cient
syngeneic C57BL/6 mice. For this, tumors were excised, dissociated
mechanically, fi ltered through 70-μm cell strainers (BD Biosciences),
and washed in PBS. A total of 2 × 10 5 cells were injected intracuta-
neously into the fl ank, and tumor development was monitored by
inspection and palpation. Tumor onset was defi ned as the day when a
tumor reached 2 mm in diameter and grew progressively. Tumor sizes
were measured weekly and recorded as mean diameter.
HCmel3 Tumor Transplantation The HCmel3 melanoma cell line was generated from a primary
Hgf-Cdk4 R24C melanoma as described previously ( 13 ). Groups of syn-
geneic C57BL/6 mice were injected intracutaneously with 4 × 10 5
HCmel3 melanoma cells into the fl ank, and tumor size was meas-
ured weekly and recorded as mean diameter in millimeters. Mice
with tumors exceeding 20 mm were sacrifi ced. Experiments were
performed in groups of 5 or more mice and repeated at least twice.
Tumor Treatment When primary or transplanted melanomas became palpable, twice
weekly peritumoral injections with 50 μg poly(I:C) (Invivogen) were
performed. Therapeutic blockade of PD-1 was performed by twice
weekly intraperitoneal injections of 250 μg rat anti-mouse PD-1
IgG2a (clone RMP1-14; BioXcell) or control-rat IgG2a mAb (clone
2A3; BioXcell). Antibody-mediated depletion of CD8 + T cells or
NK cells and neutralization of IFNγ was performed by twice-weekly
intraperitoneal injections of 200 μg rat anti-mouse CD8 IgG2a (clone
2.43; BioXcell), 200 μg rat anti-mouse Nk1.1 IgG2a (clone PK136;
BioXcell), or 100 μg rat anti-mouse IFNγ IgG1 (clone XMG1.2; BioX-
cell), respectively. Control groups again received 200 μg/mouse of the
irrelevant rat IgG2a mAb (clone 2A3; BioXcell).
Repeated-Dose Acute Toxicity Study To evaluate the potential toxicity of the combination treatment
with 50 μg poly(I:C) intracutaneously and 250 μg anti–PD-1 anti-
bodies intraperitoneally, groups of C57BL/6 mice were treated twice
weekly for 4 weeks. A control group of mice was injected with identi-
cal volumes of PBS intracutaneously and intraperitoneally. The body
weight and general health were observed and documented for 28
days. At necropsy 4 days after the last therapeutic dose, blood was
collected for clinical chemistry and hematologic analyses by retro-
orbital puncture. Various biochemical parameters were measured
in the sera with the Vitros 250 (Ortho-Clinical Diagnostics) clinical
chemistry automated system. Counts of white blood cells, red blood
cells, and platelets as well as hemoglobin levels were determined in
EDTA-blood using the Celltac α (Nihon Khoden Europe) instru-
ment. In addition, vital internal organs were isolated, weighed, fi xed
in formalin, and embedded in paraffi n for subsequent histopatho-
logic analyses. Relative organ weights were calculated as a percentage
of total body weight. Hematoxylin and eosin (H&E)–stained sections
of several organs were scored for pathologic alterations, including
degenerative changes and immune cell infi ltration.
Histology and Immunohistology Mouse tumors were immersed in a zinc-based fi xative (BD Pharmin-
gen) and human melanoma samples in buffered paraformaldehyde
(DAKO). Informed consent to use melanoma biopsy material for sci-
entifi c purposes was obtained from all patients. Tissues were embed-
ded in paraffi n and sections were stained with H&E according to the
standard protocols. Immunohistochemistry was performed with rat
anti-mouse CD45 mAb (BD Biosciences), followed by enzyme-conju-
gated secondary antibodies and the LSAB-2 color development system
(DAKO). Heavily pigmented mouse melanomas were bleached before
staining (20 minutes at 37°C in 30% H 2 O 2 and 0.5% KOH, 20 seconds
in 1% acetic acid and 5 minutes in TRIS buffer). Stained sections were
examined with a Leica DMLB microscope. Images were acquired with
a JVC digital camera KY-75FU and processed with Adobe Photoshop.
Flow Cytometry Melanoma-infi ltrating immune cells were isolated and stained
with fl uorochrome-conjugated mAbs specifi c for mouse CD45,
CD11b, CD8, Nk1.1, and MHC I (all from BD Pharmingen) accord-
ing to the standard procedures. Surface expression of PD-L1 and
MHC I on HCmel3 melanoma cells was analyzed with fl uorochrome-
conjugated mAbs specifi c for PD-L1 and MHC I (both BD Pharmin-
gen) according to the standard procedures. Data were acquired with
a FACSCanto Flow Cytometer (BD Biosciences) and analyzed with
FlowJo software (TreeStar, V7.6.5 for Windows).
Real-Time Reverse Transcriptase PCR Tumor samples were harvested and immediately snap-frozen in
liquid nitrogen. Total RNA was isolated using TRI Reagent (Sigma-
Aldrich) and purifi ed using RNeasy columns (Qiagen). Reverse
transcription was performed with the SuperScript II system and
oligo-dT18 primers (Invitrogen). Real-time PCR analysis was per-
formed with diluted cDNA and Fast SYBR Green Master Mix
(Applied Biosystems) using a 7500 Real-time PCR system (Applied
Biosystems). Sequences of primers:
• Irf7 (F: CCAGTTGATCCGCATAAGGT; R:AGCATTGCTGAGGCT
CACTT);
• Cxcl10 (F: GCCGTCATTTTCTGCCTCAT; R:GCTTCCCTATGGCC
CTCATT);
• Ccl5 (F: TGCCTCACCATATGGCTCG; R: GCACTTGCTGCTGGT
GTAGA);
• Cd3 (F: GAACCAGTGTAGAGTTGACGTG R: CCAGGTGCTTAT
CATGCTTCTG);
• Klrb (F: TTGTTCAGTTAATTTAGAGTGCCC; AGCAAAGTGGC
TCCTTTTCTAC);
• Grzb (F: CTCCAATGACATCATGCTGC; R:TGGCTTCACATTGA
CATTGC);
• Perf (F: TGAGAAGACCTATCAGGACC; R:AAGTCAAGGTGGAG
TGGAGG);
• Pdl1 (F: AGTATGGCAGCAACGTCACG; R:TCCTTTTCCCAGTA
CACCACTA); and
• Ubc (F: AGGCAAGACCATCACCTTGGACG; R:CCATCACACCCA
AGAACAAGCACA).
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JUNE 2014�CANCER DISCOVERY | 685
Combination Immunotherapy for Immune Cell–Poor Melanomas RESEARCH ARTICLE
Relative expression to the reference gene Ubc was calculated with
the Δ C t method using the following equations: ΔC t (sample) = C t
(target) − C t (reference); relative quantity = 2 −ΔC1 .
Cell Culture and Treatment of HCmel3 Melanoma Cells HCmel3 melanoma cells were generated from primary Hgf-Cdk4 R24C
mouse melanomas in our laboratory and cultured in complete RPMI-
1640 medium containing 10% fetal calf serum (FCS ; Biochrome), 2
mmol/L L -glutamine (Gibco), 10 mmol/L nonessential amino acids
(Gibco), 1 mmol/L HEPES (Gibco), 20 μmol/L 2-mercaptoethanol,
100 IU/mL penicillin, and 100 mg/mL streptomycin (Invitrogen).
HCmel3 cells were authenticated by genomic PCR for the Hgf trans-
gene and the Cdk4 R24C knockin alleles. Melanoma cells were seeded in
6-well plates and treated with 1,000 U/mL recombinant mouse IFNα
(PBL) or IFNγ (Peprotech). After 24 hours of stimulation, surface
expression of MHC I and PD-L1 was analyzed.
Statistical Analyses Statistical analyses of experimental results were evaluated with the
GraphPad Prism 4 software. Two-tailed Student t test analyses were
performed as indicated. Results were considered statistically signifi -
cant when *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Bioinformatic Analyses of Gene Expression Array Data for Human Melanomas
We used the R programming environment and the Bioconductor
platform for our bioinformatic analysis. The GSE19428 dataset was
used to identify a core signature of type I IFNα-induced genes across
fi ve human melanoma cell lines (referred to as type I IFN response
signature). The gene expression data (GSE19428_series_matrix.txt)
were downloaded as normalized data using global scaling with
a trimmed mean target intensity of each array set to 100 ( 24 ).
Expression data were log 2 -transformed, and the top 50 differentially
expressed genes were identifi ed by comparing mean expression values
of IFNα-treated cells versus untreated control cells. This type I IFN–
responsive gene set from melanoma cell lines was used in the further
analyses of the human melanoma tissue samples.
The gene expression dataset (Illumina WG-DASL array platform)
of 223 primary melanomas was previously described ( 23 ). A median
expression cutoff value for CD3D expression as a T-cell marker was
used to analyze relapse-free survival of the CD3D high versus the CD3D low
subgroup. The mean expression of the IFN-induced gene set was used
to defi ne IFN signature high and IFN signature low subgroups using an
unbiased median expression cutoff value. Relapse-free survival was
determined by the Kaplan–Meier analysis and signifi cance was assessed
by a log-rank test. A gene probe for PD-L1 ( CD274 ) on the Illumina
WG-DASL array platform failed our quality control and was consid-
ered as not reliable. Correlations among PD-L1 , IFN signature, and
CD3D expression were determined using two independent melanoma
datasets and genomics platforms: (i) primary melanomas, GSE15605,
Hgu133plus2 Affymetrix microarray platform ( 37 ); (ii) TCGA skin
cutaneous melanoma (SKCM), melanoma metastasis, Ilumina RNA-
seq platform (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp).
Primary melanoma samples ( n = 46) were selected from the GSE15605
dataset, and raw CEL fi les were normalized by Robust Multi-Array Aver-
age (RMA). Gene expression values were log 2 -transformed and mean
centered for heatmap visualization. The gene probe for PD-L1 ( CD274 )
was 227458_at. Expression values of the type I IFN response signature
genes were averaged (mean) and scaled for the barplot representation.
CD3 high and CD3l ow were defi ned by the median cutoff. RNA-seq–based
gene expression data of the TCGA melanoma samples (SKCM) for
CD3D, PD-L1 ( CD274 ), and the type I IFN response signature were
retrieved through the CGDS server of the cBioportal hosted by the
Memorial Sloan-Kettering Cancer Center (New York, NY) using the
R-package cgdsr ( 50 ). The TCGA clinical annotation data fi le was
downloaded (January 2014) from the TCGA Data Portal (https://
tcga-data.nci.nih.gov/tcga/) using the Data Matrix download option.
We used the data columns “vital status,” “days to death,” “days to last
follow-up,” and “tumor tissue site” to select samples from melanoma
metastasis and to analyze survival in cohorts stratifi ed by median gene
expression level cutoffs. Primary melanomas were excluded because of
low case numbers and short prospective clinical follow-up. We included
samples only from regional lymph node metastasis, regional cuta-
neous or subcutaneous metastasis (including satellite and in-transit
metastasis), and distant metastasis at various anatomic sites such as
trunk, extremities, and head/neck region. We obtained a total of 248
samples with clinical annotation and RNA-seq gene expression data.
For convenience, the survival data provided as “days to death” and
“days to last follow-up” were transformed to “years to death” and “years
to last follow-up.” Within this selected TGCA metastasis cohort, the
clinical follow-up of many cases started well before the sample collec-
tion and molecular characterization of the respective metastatic lesion
that occurred later in the course of the disease. As exemplifi cation, the
cohort contains many samples from metastatic lesions of patients that
were initially diagnosed with a nonmetastatic, for example, stage I or II,
melanoma several years ago, but have developed a melanoma metastasis
later in the course of their disease. Hence, survival (“days to death or
last follow-up”) refl ects a combination of retrospective and prospec-
tive survival data as initiation of the clinical follow-up and serves as an
assessment of the overall disease course.
RNA-seq read counts were log 2 normalized, and unbiased median
gene expression value cutoffs were applied for the analysis of high/
low gene expression subgroups and their potential associations with
overall disease outcome (“days to death or last follow-up”). Expres-
sion values of the type I IFN response signature genes were averaged
(mean) before calculation of the median expression cutoff value.
Overall survival was calculated by the Kaplan–Meier method and
signifi cance was determined by the log-rank test.
Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.
Authors’ Contributions Conception and design: T. Bald, M. Renn, M. Hölzel, T. Tüting
Development of methodology: T. Bald, J. Landsberg, P. Jansen,
T. Tüting
Acquisition of data (provided animals, acquired and man-
aged patients, provided facilities, etc.): J. Landsberg, M. Renn,
N. Glodde, P. Jansen, E. Gaffal, J. Steitz, R. Tolba, A. Limmer, T. Tüting
Analysis and interpretation of data (e.g., statistical analy-
sis, biostatistics, computational analysis): T. Bald, N. Glodde,
P. Jansen, E. Gaffal, J. Steitz, R. Tolba, A. Limmer, G. Jönsson,
M. Hölzel, T. Tüting
Writing, review, and/or revision of the manuscript: T. Bald,
J. Landsberg, P. Jansen, J. Steitz, R. Tolba, A. Limmer, M. Hölzel, T. Tüting
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): N. Glodde, E. Gaffal,
U. Kalinke, T. Tüting
Study supervision: T. Tüting
Performance of experimental work and participation in discus-
sions: D. Lopez-Ramos
Design, performance, and analysis of animal study for toxicity
evaluation: J. Steitz
Performance of the repeated dose toxicity studies: R. Tolba
Acknowledgments The authors thank Glenn Merlino and Mariano Barbacid for
providing genetically engineered mice, and Sandra Bald, Alexander
Sporleder, Pia Aymanns, Tanja Arzt, and Corina Lemke for managing
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686 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Bald et al.RESEARCH ARTICLE
the mouse colony and performing histopathology, immunohisto-
pathology, and quantitative PCR.
Grant Support This research was supported in part by grants from the DFG (A12
in the SFB832 and A22 in the SFB704 to T. Tüting), DFG SFB832
core support and DFG HO 4281/2-1 (to M. Hölzelm), and by
BONFOR (to J. Landsberg). Both M. Hölzelm and T. Tüting are
members of the DFG excellence cluster Immunosensation.
Received August 1, 2013; revised February 26, 2014; accepted
February 26, 2014; published OnlineFirst March 3, 2014.
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2014;4:674-687. Published OnlineFirst March 3, 2014.Cancer Discovery Tobias Bald, Jennifer Landsberg, Dorys Lopez-Ramos, et al. Targeted Type I IFN Activation
Poor Melanomas Benefit from PD-1 Blockade after−Immune Cell
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