immune cell–poor melanomas beneﬁ t from pd-1 blockade ...primary human melanoma immune...

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

http://cancerdiscovery.aacrjournals.org/

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




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

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primary mouse melanoma

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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).

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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.

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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.

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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


% 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






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


Ctrl IgG

poly(I:C) +Ctrl IgG

poly(I:C) +aPD-1

D


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)






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


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


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






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






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).






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






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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