Plasmacytoid Dendritic Cells Support Melanoma …...Research Article Plasmacytoid Dendritic Cells...

Research Article

Plasmacytoid Dendritic Cells Support MelanomaProgression by Promoting Th2 and Regulatory Immunitythrough OX40L and ICOSL

Caroline Aspord1,2, Marie-Therese Leccia2,3, Julie Charles2,3, and Joel Plumas1,2

AbstractEven though melanoma is considered to be one of the most immunogenic solid tumors, handling its

development remains a challenge. The basis for such escape from antitumor immune control has not yetbeen documented. Plasmacytoid dendritic cells (pDC) are emerging as crucial but still enigmatic cells incancer. In melanoma, the function of tumor-infiltrating pDCs remains poorly explored. We investigated thepathophysiologic role of pDCs in melanoma, both ex vivo from a large cohort of melanoma patients and invivo in melanoma-bearing humanized mice. pDCs were found in high proportions in cutaneous melanomaand tumor-draining lymph nodes, yet associated with poor clinical outcome. We showed that pDCsmigrating to the tumor microenvironment displayed particular features, subsequently promoting proin-flammatory Th2 and regulatory immune profiles through OX40L and ICOSL expression. Elevated frequen-cies of interleukin (IL)-5-, IL-13- and IL-10–producing T cells in patients with melanoma correlated withhigh proportions of OX40L- and ICOSL-expressing pDCs. Strikingly TARC/CCL17, MDC/CCL22, and MMP-2found in the melanoma microenvironment were associated with pDC accumulation, OX40L and ICOSLmodulation, and/or early relapse. Thus, melanoma actively exploits pDC plasticity to promote itsprogression. By identifying novel insights into the mechanism of hijacking of immunity by melanoma,our study exposes potential for new therapeutic opportunities. Cancer Immunol Res; 1(6); 402–15. �2013AACR.

IntroductionCancer cells deploy numerous strategies to escape from

the host immune system taking advantage of its plasticity. Inmelanoma, despite a high immunogenicity, an adverse evo-lution is often observed due to establishment of an immu-nosuppressive microenvironment favoring tumor progres-sion. The basis for such subversion of immunity in melano-ma has not yet been documented. However, understandingthe mechanisms underlying the lack of efficient antitumorimmunity is crucial for the development of novel therapeuticapproaches to prevent the negative outcome associated withmetastatic disease.

Dendritic cells (DC) play an essential role in initiatingand shaping antitumor immune responses (1–3). Among DCsubsets, plasmacytoid dendritic cells (pDC) are key playersin the regulation of immune responses (4, 5). Depending ontheir maturation state and on the microenvironment, pDCscan mediate immunity or tolerance. Indeed, pDCs arecritical for the development of peripheral tolerance orinduction of tolerance at mucosal sites (6). However, uponactivation, pDCs have the ability to initiate protectiveimmunity towards viruses through production of largeamounts of type I IFN and proinflammatory cytokines. Suchreactivity is based on the capacity of pDCs to sense viralnucleic acids through Toll-like receptors (TLR) 7 and 9 andtrigger downstream signaling. Therefore pDCs display ahigh functional plasticity, being able to orient immunitytowards multiple profiles depending on the surroundingsignals (5, 7).

pDCs constitute an emerging intriguing immune cell incancer. Indeed, pDCs play a pivotal role in antitumorimmunity through their ability to process and cross-presenttumor antigens to T cells (8), and subsequently induceadaptive immune responses (9). In the context of melano-ma, pDCs have been shown to prime functional immuneresponses (9–11), display direct cytotoxic activity towardstumor cells through TRAIL expression (12) or lysozymesecretion (13), or once activated, potentially achieve mela-noma tumor control through efficient priming of antitumor

Authors' Affiliations: 1Etablissement Francais du Sang Rhone-Alpes,R&D-Laboratory; 2University Joseph Fourier; INSERM, U823, Immunobiol-ogy & Immunotherapy of Cancers; 3Department of Dermatology, GrenobleUniversity Hospital, Grenoble, France

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

J. Charles and J. Plumas contributed equally to this work.

Corresponding Author: Caroline Aspord, EFS-R&D laboratory, INSERMU823, 29 avenue du Maquis Gresivaudan, 38700 LaTronche, France.Phone: 334-7642-4344; Fax: 334-7654-6918; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-13-0114-T

�2013 American Association for Cancer Research.

CancerImmunology

Research

Cancer Immunol Res; 1(6) December 2013402

on May 24, 2020. © 2013 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst September 26, 2013; DOI: 10.1158/2326-6066.CIR-13-0114-T

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responses (14–17). Infiltration of tumors by pDCs has beenfound in many types of cancers, yet associated with poorprognosis, especially in ovarian (18) and breast cancer (19).Functional alterations of pDCs have been identified (20–22)and related to the induction of immune tolerance leading totumor progression (20, 23) and pDC-dependent angiogen-esis induction (24). In melanoma, pDCs were found to berecruited to the tumor site and sentinel lymph nodes (9, 25–27). The infiltration of primary tumors by CD123þ cells hasbeen linked with a poor prognosis (28). However, themechanism by which pDCs promote the progression ofmelanoma tumors and the consequences of the specificfeatures of pDCs on subsequent adaptive immunityremained unknown.pDCs play a fundamental yet still enigmatic role in the

control of tumor development. Their plasticity endorsesthem with a powerful ability to drive effective antitumorimmunity but also with a potential to trigger tolerance andtumor progression. This challenging controversy promptedus to investigate the pathophysiologic role of pDCs in thecontext of melanoma. We delineated the role of pDCs bothex vivo from a large cohort of melanoma patients and in vivoin an innovative melanoma-bearing humanized mousemodel (29). We showed that melanoma exploits pDCs fordriving immune responses to its advantage. We identifiedfor the first time a novel pDC-triggered pathway leadingto tumor progression and early relapse in melanoma, whichoffers new opportunities to design effective antitumortherapies.

Materials and MethodsSamples from melanoma patients and healthy donorsBlood and tumor samples were obtained from 61 and 54

patients with melanoma respectively, stage I–IV. Clinical fea-tures are shown in Supplementary Tables S1 and S2. Bloodsamples were also obtained from 37 healthy donors. Peripheralblood mononuclear cells (PBMC) were purified by Ficoll-Hypaque density-gradient centrifugation (Eurobio). Tumorsamples were mechanically dilacerated and digested with 2mg/mL collagenase-D (Roche) 20 U/mL DNase (Sigma). Theresulting tumor-infiltrating cell suspensions were filtered andwashed. Small whole tumor fragments (10 mm3) were incu-bated for 24 hours in complete RPMI-1640 10% fetal calf serum(FCS) to generate tumor supernatants. All procedures wereapproved by the ethics committee of Grenoble UniversityHospital and the French Blood Agency's Institutional ReviewBoard. All participants in the study signed informed consentforms.

Melanoma cell lines and human CD34þ hematopoieticprogenitor cell isolationHumanmelanoma lines COLO829 andA375were purchased

from ATCC (LGC-Standards). Cell lines were reauthenticatedat the time of the experiments by analyzingMelA expression byflow cytometry and were regularly confirmed to be Mycoplas-ma-free by using the Mycoplasma Stain Kit (Sigma). Cultureswere performed in RPMI-1640 supplemented with 1% non-essential amino acids, 1 mmol/L sodium pyruvate (Sigma), 100

mg/mL gentamycin, and 10% FCS (Invitrogen). Human CD34þ

hematopoietic progenitor cells (HPC) were purified from cordblood using the CD34 isolation kit (Miltenyi). This procedurewas approved by the French Blood Agency's InstitutionalReview Board, and participants signed informed consentforms.

Humanized miceNOD-SCIDb2m�/� immunodeficient mice (NOD.Cg-

PrkdcSCIDb2mTm1Unc/J) were purchased from Jackson Immu-noResearch Laboratories and bred at the Plateforme de HauteTechnologie Animale (La Tronche, France). Humanized micewere constructed by intravenously xenotransplanting 1–2 �105 human CD34þ HPCs into sublethally irradiated NOD-SCIDb2m�/� mice (100–120 cGy). Four weeks later uponreconstitution of the human immune system, 10� 106 humanmelanoma tumor cells were implanted subcutaneously intothe flanks. Three days later, tumor, draining lymph nodes,control lymph nodes, spleen, and bone marrow were har-vested. Organs were eventually digested with 2 mg/mL col-lagenase-D (Roche). Resultant cell suspensions were filteredandwashed. Small whole tumor fragments (10mm3)were alsoincubated for 24 hours in complete RPMI-1640 10% FCS togenerate tumor supernatants. These studies were carried outin accordance with European Union guidelines (86/609/CEE).French National Chart guidelines and protocols were app-roved by the Ethic Committee for Animal Experimentation ofGrenoble.

Phenotypic analysisCell suspensions were suspended in PBS 2% FCS and stained

with anti-human antibodies (BD, Beckman). pDCs were iden-tified as CD45þ HLA-DRþBDCA2þ or Lin�HLA-DRþCD123þ

and myeloid dendritic cells as Lin�HLA-DRþCD11cþ. Thephenotype of pDCswas determined using anti-CD62L, -OX40L,-GITRL, ICOSL, -PDL1, -PDL2, -CD40, -CD80, -CD86 antibodiesand their isotype-matched controls. Suspensions were sub-jected to flow cytometry analysis using a FACSCalibur andCellQuest software (BD).

Response to TLR-L stimulationCell suspensions were resuspended at 1 � 106/mL in

complete RPMI-1640 10% FCS and cultured for 24 hourswith 640UHA/mL UV-formol–inactivated influenza virusstrain A/H3N2/Wisconsin/67/05 (Sanofi-Pasteur) or CpGA

ODN-2336 (10 mg/mL, Coley Pharma). The activation phe-notype of pDCs was analyzed using anti-CD40 and CD80,and -CD86 antibodies. Human soluble IFN-a, IP10, IL-6, andTNF-a production was measured in culture supernatants bya Cytometric Bead Array assay (CBA, BD). The proportion ofpDCs was evaluated in the corresponding samples by flowcytometry using anti-HLA-DR and anti-BDCA2 labeling tocalculate the relative cytokine production per pDC. The IFN-a production was also evaluated within pDCs after 6 hoursof stimulation with TLR-L in the presence of brefeldinA (1mL/mL) for the last 3 hours followed by anti-HLA-DR andanti-BDCA2 surface labelling and intracellular IFN-a stain-ing (BD).

pDCs' Hijacking by Melanoma

www.aacrjournals.org Cancer Immunol Res; 1(6) December 2013 403




E F

BA

C

HLA-DR

BDCA2

Tumor Draining LN

42.3% 43%

Melanoma-bearing Humice

HLA-DR

BDCA2

Cutaneous

tumor

Cutaneous

tumor

Lymph node

metastasis

Lymph n

ode

metasta

sis

Blood

Blood

Melanoma patients

0.43%0.53%0.89%

D

% p

DC

s w

ith

in C

D4

5+ c

ells

I+II III+IV

0.0

0.5

1.0

1.5

0.0021

Stage at sampling time

Blood

0.01

0.1

1

10

% H

LA

-DR

+ B

DC

A2

+ p

DC

s

with

in C

D4

5+ c

ells

Melanoma patients

Blood

healthy

donor

0.02 ns

0.038

Tumor DLN CLN Spleen BM

0

20

40

60

80

% H

LA

-DR

+ B

DC

A2

+ p

DC

s

with

in C

D4

5+ c

ells

0.0003< 0.0001

< 0.0001< 0.0001

< 0.0001


Median OS :

%pDC > 0.4 = 36.5 mo

%pDC < 0.4 = 96 mo

Log-rank (Mantel–Cox) test :* P = 0.0104

% pDC <0.4

%pDC >0.4

0 50 100 150 200

0

20

40

60

80

100

OS (mo)

Pe

rce

nta

ge

su

rviv

al

Figure 1. pDCs are found in high proportions in cutaneousmelanoma andDLN of tumor-bearing Humice andmelanoma patients and are associated with poorclinical outcome. A–D, cutaneous tumors, lymph nodemetastasis, and blood samples were obtained frommelanoma patients or healthy donors (HD) and theproportion of pDCs was evaluated by flow cytometry. A, representative dotplots of HLA-DRþBDCA2þ pDCs in each sample type (gated on CD45þ cells). B,percentagesof pDCs foundwithinCD45þ cells in cutaneousmelanoma (n¼ 12), lymphnodemetastasis (n¼ 28), blood (n¼ 38) of patients andbloodof healthydonors (n¼ 20). C, proportion of pDCs in blood of patients with melanoma corresponding to the stage of the disease at sampling time. D, comparative overallsurvival (OS) of patients with low (<0.4%) or high (>0.4%) tumor-infiltrating pDCs. Groups were separated using the median percentage of tumor-infiltratingpDCs: 0.4%. Comparison was done using log-rank test. E and F, humanized mice displaying a human immune system were transplanted withmelanoma cells and analyzed 3 days later for the presence of pDCs at the tumor site, in DLN, control lymph nodes (CLN), spleen, and bone marrow (BM).E, representative dotplots of HLA-DRþBDCA2þ pDCs in tumor and DLN (gated on CD45þ cells). F, percentages of pDCs found within CD45þ cells intumor, DLN, CLN, spleen, and bone marrow (n ¼ 46 Humice). P values calculated using the Mann–Whitney test.

Aspord et al.

Cancer Immunol Res; 1(6) December 2013 Cancer Immunology Research404




C

A B

D

Melanoma patients

Blood

healthy

donor

0

20

40

60

80

100

% C

D4

0+ p

DC

s w

ith

in p

DC

s

nsns0.04

0.001

CD40

0

20

40

60

80

100

% C

D8

0+ p

DC

s w

ith

in p

DC

s

0.048

nsns0.018

CD80

Melanoma patients

Blood

healthy

donor

20

0

40

60

80

100

% C

D8

6+ p

DC

s w

ith

in p

DC

s

ns

ns0.04 < 0.0001

CD86

Melanoma patients

Blood

healthy

donor

% C

D6

2L

+ p

DC

s

0.03

0.018<0.0001

0

20

40

60

80

100

Melanoma patients

C BL

Blood

healthy

donor

C BL C B

L C BL

Tumor DLN Spleen BM

0

20

40

60

80

100

% C

D6

2L

+ p

DC

s

0.0009<0.0001


CD40

Tumor DLN Spleen0

20

40

60

80

100

% C

D4

0+ p

DC

s

0.0004<0.0001

CD80

% C

D8

0+ p

DC

s

Tumor DLN Spleen0

5

10

15

20

25ns

0.0001

CD86

Tumor DLN Spleen0

20

40

60

80

100

% C

D8

6+ p

DC

s

0.00050.0002

Melanoma-bearing Humice Melanoma-bearing Humice Melanoma-bearing Humice

Figure 2. pDCs display a higher basal activation status at the tumor site compared with the DLN. The expression of CD62L (A and B) and costimulationmolecules (C andD) was analyzed on pDCs found in cutaneous tumor, lymph nodemetastasis, and blood of patients withmelanoma (A andD; n¼ 12, 28, and38, respectively), blood of healthy donors (A,D; n ¼ 20) as well as in the tumor, DLN, spleen, and bone marrow (BM) of melanoma-bearing Humice(B and C; n ¼ 16). A and B, percentages of CD62Lþ pDCs among pDCs in melanoma patients and healthy donors (A) and Humice (B). C, percentagesof pDCs expressing CD40, CD80, and CD86 among pDCs in Humice. D, percentages of expression of CD40, CD80, and CD86 on pDCs in patientswith melanoma compared with healthy donors. P values were calculated using the Mann–Whitney test.






A

B

C

−−Flu

CpGA

0

20

40

60

% I

FN

-α+ p

DC

s w

ithin

pD

Cs

*** *** ****** *** ***

0.034

0.006

0.01

0.007

Cutaneous

+ LN metastasisBlood Blood

HD

Melanoma patients

CD86CD40

−Flu

CpGA

0

20

40

60

80

100*** *** ***

*** *** ***

% C

D86

+ p

DC

s

0

1,000

2,000

3,000*** *** **

*** *** ***

MF

I C

D40

+ p

DC

s

Cutaneous


HD

Melanoma patients

Cutaneous


HD

Melanoma patients

ns ns0.0074

−FluCpGA

IP10

1

10

100

1,000

10,000IP

10 (

ng/m

L/1

05

pD

C)

*** ns *

ns ns *

< 0.0001

< 0.0001

Cutaneous


HD

Melanoma patients

IFN

-α(n

g/m

L/1

05

pD

C)

IFNα

*** ns ns* *** ***

0.001

0.0002

Cutaneous


HD

Melanoma patients

0.1

1

10

100

1,000

Figure 3. pDCs from the melanoma environment responded to TLR-L stimulation. Tumor-infiltrating cells or PBMC from patients with melanoma or healthydonors (HD) were stimulated with TLR7-L (Flu virus) or TLR9-L (CpGA). Expression of costimulatory molecules and cytokine secretion was evaluated24 hours later. A, percentages of expression (top) and mean fluorescence intensity (MFI; bottom) of CD40 and CD86 on pDCs from tumor (n ¼ 13–15)or blood (n¼ 26) of patients withmelanoma compared with healthy donors (HD; n¼ 19). B, IFN-a and IP10 weremeasured in the supernatants 24 hours later.Resultswere standardized by calculating the cytokine production per pDC in each sample (n¼ 33, 19, and 19, respectively). C, IFN-a intracellular labelingwasperformed within pDCs frommelanoma patients or healthy donors (n¼ 25, 30, and 22, respectively) P values were calculated using the two-way RMANOVAtest (straight line) or the Mann–Whitney test (dotted lines). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Aspord et al.





A

B

C

Melanoma-bearing Humice - purified pDCs + naïve CD4 T cells

IL-5

Tumor DLN Spleen BM0

200

400

600

800

IL-5

(p

g/m

L)

IL-13


300

600

900

1,200

1,500

IL-1

3 (

pg/m

L)

TNF-α


20

40

60

80

TN

F-α

(pg/m

L)

IL-10


10

100

1,000

IL-1

0 (

pg

/mL

)

IL-5

< 0.0001

LN

metastasis

patient

Blood

patient

0

200

400

600

800

1,000

IL-5

(p

g/m

L)

IL-13

0

1,000

2,000

3,000

IL-1

3 (

pg

/mL

)

IL-10

0

200

400

600

800

IL-1

0 (

pg

/mL

)

TNF-α

0

20

40

60

TN

F-α

(pg/m

L)

< 0.0001 < 0.0001 < 0.0001

Melanoma patients - purified pDCs + naïve CD4 T cells

LN

metastasis

patient

Blood

patient

LN

metastasis

patient

Blood

patient

LN

metastasis

patient

Blood

patient

Melanoma-bearing Humice – total cell suspension + naïve CD4 T cells

IL-5

Tumor DLN Spleen BM

0

100

200

300

400

500

IL-5

(p

g/m

L)

IL-13


500

1,000

1,500

IL-1

3 (

pg

/mL

)

TNF-α


50

100

150

200

TN

F-α

(p

g/m

L)

IL-2


20

40

60

IL-2

(p

g/m

L)

IFN-γ


20

40

60

80

100

IFN

-γ(p

g/m

L)

IL-10


10

20

30

40

50

IL-1

0 (

pg

/mL

)

Figure 4. pDCs from the melanoma environment drive a Th2 proinflammatory and regulatory profile. A and B, cell suspensions (A, n¼ 15–21) or purified pDCs(B, n ¼ 15–21) from tumor, DLN, spleen, and bone marrow (BM) of melanoma-bearing Humice were cocultured with allogeneic naïve CD4 T cells. Thecytokine profile was evaluated 5 days later in the supernatants. C, pDCs purified from lymph node (LN) metastasis (n¼ 17 tumor samples) and blood samples(n¼ 28) of patients with melanoma were cocultured with allogeneic naïve CD4 T cells. The cytokine profile was evaluated 5 days later in the supernatants. Pvalues were calculated using the Mann–Whitney test.






A

B

LN metastasis

patient

Blood

patient

Blood

HD

IL-5 IL-13

CD4

1.41%

0.41%

0.96%

0.46%

0.09%

CD3

0.02%

IL-10

5.85%

1.29%

0.16%

1.86% 2.07%

2.02% 1.1%

IL-5 IL-13

CD8

CD8

0.17% 0.01%

IL-10

0.15%

0.68%

11.7%

C

*

ns*

% I

L-5

+ C

D8

T c

ells

ns**

**

% I

L-1

3+ C

D8

T c

ells

ns***

***

% I

L-1

0+ C

D8

T c

ells

LN

metastasis

patient

Blood

patient

Blood

HD

0.01

0.1

1

10

0.01

0.1

1

10

0.01

0.1

1

10

100

CD8

T cells

IL-5

nsns

ns

% I

L-5

+ C

D4

T c

ells

IL-13

ns***

**

% I

L-1

3+ C

D4

T c

ells

IL-10

******

*

% I

L-1

0+ C

D4

T c

ells

0.01

0.1

1

10

0.01

0.1

1

10

0.01

0.1

1

10

CD4

T cells

LN

metastasis

patient

Blood

patient

Blood

HD

LN

metastasis

patient

Blood

patient

Blood

HD

LN

metastasis

patient

Blood

patient

Blood

HD

0

10

20

30

% F

oxP

3+

CD

25

+ C

D4

T c

ells

***

***

0.02

(Legend for Fig. 5 on following page.)

Aspord et al.





Coculture of pDCs/na€�ve CD4T cells for determination ofTh profilepDCs were purified from patients' or healthy donors' cell

suspensions using the EasySep Human pDC enrichment kit(StemCell; purity >92%) and from cell suspensions fromhumanized mice using the human BDCA4þ selection kit(Miltenyi; purity 70–98%). Human na€�ve CD4 T cells werepurified from cord blood using the Human Na€�ve Enrich-ment Kit (StemCell; purity 97–99.5%). pDCs and na€�ve CD4 Tcells were cultured in a 1:10 ratio for 5 days. T cells (1 � 106

cells/mL) were restimulated for 16 hours with 50 ng/mLphorbol 12—myristate 13—acetate (PMA) and 1 mg/mLionomycin (Sigma). In some experiments, anti-OX40L block-ing antibodies, goat IgG control antibodies (10 mg/mL; R&Dsystems), anti-ICOSL blocking antibodies, or mouse IgGcontrol antibodies (1 mg/mL; eBiosciences) were addedduring the pDC/T cocultures. Culture supernatants werecollected at day 5 and after 16 hours of restimulation. IL-5,IL-13, TNF-a, IL-10, and IFN-g cytokines were quantified bya Cytometric Bead Array assay (BD).

Intracellular cytokine staining of T cellsCell suspensions (1 � 106/mL) were stimulated with 50

ng/mL PMA and 1 mg/mL ionomycin (Sigma) for 5.5 hours inthe presence of brefeldinA (1 mL/mL) for the last 3 hours. Cellswere then surface labeled with anti-CD4, -CD8, and -CD3antibodies, and intracellular cytokine staining was performedusing anti-IL-5, IL-13, -IL-10 and IFN-g antibodies (BD).

Quantification of chemokines/Th2-prone factors intumor supernatantsCCL17/thymus and activation-regulated chemokine (TARC),

CCL22/macrophage-derived chemokine (MDC), thymic stro-mal lymphopoietin (TSLP), and matrix metalloproteinase-2(MMP-2) were quantified in tumor supernatants derived frompatients with melanoma or melanoma-bearing Humice byELISA (R&D systems).

Modulation of pDCs by rhCCL17/TARC, rhCCL22/MDC,and rhMMP-2pDCs were purified from blood of healthy donors and

cultured in the presence of IL-3 (10 ng/mL) and increasingconcentrations of rhTARC, rhMDC, or rhMMP-2. The level ofexpression ofOX40L and ICOSLonpDCswas assessed 24 hourslater using flow cytometry.

Statistical analysisThe statistical analyses were performed by Prism soft-

ware using the Mann–Whitney test, the nonparametric Utest, the Wilcoxon matched t test, the one-way ANOVA, the

two-way RM ANOVA, the Spearman correlation, and thelog-rank test.

ResultspDCs are found in high proportions in cutaneous tumorsand tumor-draining lymph nodes and are associatedwith a poor prognosis

We first evaluated the proportion of pDCs in tumors andblood samples of patients with melanoma (SupplementaryTables S1 and S2) compared with healthy donors. We foundthat pDCs infiltrated melanoma tumor tissue, with a higherproportion of pDCs in cutaneous tumors compared withlymph nodes metastasis and blood (Fig. 1A and B). Moreover,when classified according to disease stage, patients atadvanced stage III–IV melanoma displayed a lower frequencyof circulating pDCs compared with patients at early stage I–II(Fig. 1C), suggesting a preferential recruitment of pDCs to thetumor site. Strikingly, we found that tumor-infiltrating pDCswere significantly associated with a poor clinical outcome, asthe proportion of tumor-infiltrating pDCs was correlated withthe Breslow level (Supplementary Fig. S1), and most impor-tantly, a high proportion of pDCs was correlated with shortersurvival (Fig. 1D). To better investigate the crucial role of pDCsin human melanoma, we developed a melanoma-bearinghumanizedmousemodel (29) based on immunodeficientmicereconstitutedwith a human immune system following hCD34þ

HPC transplantation, and subsequently engrafted with humanmelanoma (Supplementary Fig. S2). In this model, humanpDCs were able to rapidly and massively migrate to humanmelanoma tumors and tumor-draining lymph nodes (DLN;Fig. 1E and F). In contrast, myeloid dendritic cells (mDC)represented a minor population among infiltrating cells (Sup-plementary Fig. S3A and S3B). Together, these data illuminateda preferential attraction of pDCs by melanoma and a relation-ship between the presence of pDCs at the tumor site and poorclinical outcome.

pDCs fromthedraining lymphnodesdisplay apoorbasalactivation status compared with the cutaneous tumorsite but exhibit an unaltered functionality with respect toTLR triggering

To understand how tumor-infiltrating pDCs could lead toearly relapse, we examined the migratory profile and basalactivation status of melanoma-infiltrating pDCs. Interestingly,we observed in patients with melanoma a higher proportion ofCD62L-expressing pDCs in blood compared with sites of tumormetastasis or with circulating pDCs of healthy donors (Fig. 2A),suggesting the mobilizing potential of circulating pDCs inpatients. Inmelanoma-bearingHumice, allowing amore precise

Figure5. The frequencyof IL-5-, IL-10-, and IL-13–producingCD4andCD8Tcells is elevated in patientswithmelanomacomparedwith healthy donors (HD).A, intracellular staining of IL-5, IL-10, and IL-13 within CD4 T cells (left, gated on CD4 T cells) and CD8 T cells (right, gated on CD8 T cells) infiltratingmelanoma tumors or circulating (blood) in comparison with healthy donors' PBMC, with one representative sample for each condition. B, frequencies of IL-5–, IL-13–, and IL-10–secreting CD4 T cells (top) and CD8 T cells (bottom) within total CD4 and CD8 T cells, respectively (n ¼ 34 tumor-infiltratinglymphocyte (TIL) samples, 26PBMCpatients and 16PBMChealthy donors). C, frequency of FoxP3þCD25þCD4 regulatory T cells withinCD4T cells (n¼20TIL samples, 24 PBMC patients and 16 PBMC healthy donors). P values were calculated using one-way ANOVA test (straight line) or Mann–Whitney test(dotted lines). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.






0

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Figure 6. OX40L and ICOSL expression by pDCs is respectively responsible for the Th2-biased and regulatory-prone immunity in melanoma. A,percentages of expression of OX40L, ICOSL, GITRL, PDL1, and PDL2 on pDCs from tumor, DLN, spleen, and bone marrow (BM) of melanoma-bearingHumice (n ¼ 9–17 Humice). B, percentages of expression of OX40L and ICOSL on pDCs from cutaneous tumors and lymph node (LN) metastasis orblood of melanoma patients compared with pDCs from healthy donors (HD; n ¼ 22–33 TIL, 27–49 PBMC patients, 17–20 PBMC healthy donors).(Legend continued on the following page.)


Aspord et al.




analysis of the tumor site and its associated DLN, we found ahigher proportion of CD62L-expressing pDCs at the tumor sitecompared with the DLN and spleen (Fig. 2B), suggesting amobilization of pDCs to the tumor site followed by theirmigration to the DLN. Moreover, a higher proportion of pDCsexpressing CD40, CD80, and CD86 was found at the tumor sitecompared with DLN and spleen in melanoma-bearing Humice(Fig. 2C) and in cutaneous tumors comparedwith sites of lymphnode metastasis in patients with melanoma (Fig. 2D). Together,these results clearly indicate that pDCs display a poor activationstatus when reaching DLN compared with the tumor site.We further investigated whether melanoma could affect

the ability of pDCs to respond to TLR-L stimulation, potentactivators revealing pDCs' fitness. pDCs from tumors andblood of patients with melanoma upregulated the costimu-latory molecules CD40 and CD86 in response to TLR7-L andTLR9-L stimulation in a similar manner to samples fromhealthy donors (Fig. 3A). pDCs from the tumor site appearedslightly impaired in their capacity to secrete IFN-a and IP10in response to TLR9-L stimulation compared with pDCsfrom patients' blood, but their response to TLR7-L was notaffected (Fig. 3B). In addition, when we performed intracel-lular staining of IFN-a within pDCs, the frequency of cyto-kine-producing pDCs at the tumor site was higher than thatfound in the blood of patients or of healthy donors (Fig. 3C).Therefore, whatever their activation status, pDCs from themelanoma microenvironment remain fully functional withregard to TLR triggering.

pDCs from the melanoma environment drive a Th2proinflammatory and regulatory profileNext, we determined how such tumor-induced features of

pDCs affected subsequent aspects of adaptive immunity. Inmelanoma-bearing Humice, we observed a dramatic pro-duction of IL-5, IL-13, and TNF-a in cocultures of na€�ve CD4T cells with DLN suspensions (Fig. 4A). We confirmed thatsuch a profile was directly driven by pDCs as this responsewas observed using purified pDCs (Fig. 4B). Notably, inpatients with melanoma, only pDCs isolated from tumormetastasis caused na€�ve CD4 T cells to secrete IL-5, IL-13,TNF-a, and IL-10 (Fig. 4C). Furthermore, such an immuneprofile could be seen directly in vivo as we found higherfrequencies of IL-10- and IL-13–producing CD4 and CD8 Tcells in tumor metastasis and blood of melanoma patientscompared with that of healthy donors, and a higher fre-quency of IL-5–producing CD8 T cells in blood of patientswith melanoma compared with that of healthy donors (Fig.5A and 5B). In addition, we observed a high frequency ofFoxP3þCD25hi regulatory CD4 T cells (Treg) in tumor sam-ples and an increased circulating Treg frequency in patientswith melanoma compared with healthy donors (Fig. 5C).Together, these results strongly suggest that melanoma

instructs pDCs to trigger proinflammatory and regulatoryimmune profiles.

OX40L and ICOSL expression by pDCs is responsible fortheTh2- and regulatory-prone immunity, respectively, inmelanoma

Next, we investigated the mechanism by which tumor-polarized pDCs induce such an immune profile. In melano-ma-bearing Humice, by analyzing a panel of moleculesknown to modulate immunity towards Th2 or regulatoryprofiles, we observed significantly higher proportions ofpDCs expressing OX40L and/or ICOSL at the tumor siteand DLN compared with pDCs from spleen or bone marrow(Fig. 6A). In contrast, no differential expression of GITRL,PD-L1, and PD-L2 by pDCs was observed. Notably, byassessing the relevance of these observations in patientswith melanoma, we found elevated proportions of OX40L-expressing pDCs in tumor metastasis and blood of patientswith melanoma compared with healthy donors, and higherproportions of ICOSL-expressing pDCs in tumor metastasisthan in the blood of either melanoma patients or healthydonors (Fig. 6B). Remarkably, the proportions of OX40L- andICOSL-expressing pDCs correlated with the frequencies ofIL-13–producing CD4 and CD8 T cells (Fig. 6C) and IL-10–producing CD4 T cells (Fig. 6D), respectively. To directlyinvestigate the role of OX40L and ICOSL in the pDC-inducedpolarization of immune responses, we assessed the ability oftumor-derived pDCs to drive na€�ve T cells towards thisprofile in the presence of OX40L- or ICOSL-blocking anti-bodies. Strikingly, the blocking of OX40L during pDC/Tcocultures abrogated IL-5, IL-13, and TNF-a production,whereas the blocking of ICOSL specifically inhibited IL-10secretion (Fig. 6E). Furthermore, patients with advancedmelanoma showed higher levels of OX40Lþ pDCs and Th2T cells in circulation compared with patients at an earlystage of the disease (Supplementary Fig. S4). Thus, tumor-modulated pDCs seem to drive immunity preferentiallytowards Th2 and regulatory profiles throughOX40L and ICOSLexpression.

TARC/CCL17, MDC/CCL22, and MMP-2 factors found inthe melanoma microenvironment are associated withpDC accumulation, OX40L and ICOSL upregulation, andclinical outcome

To get further insights into the mechanism of pDC mod-ulation by melanoma tumors, we examined the presence ofTh2- and regulatory-modulating factors in the tumor micro-environment. We looked for the presence of TARC, MDC,TSLP, and MMP-2 in tumor supernatants, as these factors areassociated with Th2 immunity (30, 31) and/or attraction ofTh2 (32) and Treg cells (33). In both melanoma-bearingHumice and patients with melanoma, we found the presence

(Continued.) C, correlationbetween the frequencyofOX40LþpDCsand the frequencyof IL-13–secretingCD4 (left) orCD8 (right) T cellswithinmelanomapatients'samples. D, correlation between the frequency of IL-10–secreting CD4 T cells and the frequency of ICOSLþ pDCs within melanoma patients' samples. E, pDCspurified from lymph nodemetastasis of patients with melanoma were cocultured with allogeneic naïve CD4 T cells in presence of anti-OX40L antibodies (top) oranti-ICOSL antibodies (bottom) ormatched control isotypes. Cytokine secretion was evaluated 5 days later in supernatants.P values were calculated using one-way ANOVA test (A and B), Spearman correlation (C and D), and Wilcoxon matched paired test (E). �, P < 0.05; ��, P < 0.01; ��P < 0.001.






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Figure 7. MDC/CCL22 andMMP-2 factors are found in themelanomamicroenvironment and correlate with pDC accumulation and early relapse. A and B, thefactors TARC, MDC, TSLP, andMMP-2 were quantified in tumor supernatants collected frommelanoma-bearing Humice (A, n¼ 49) and melanoma patients(B, n¼ 20). C, correlation between the frequency of tumor-infiltrating pDCs and the level of MDC (left) or MMP-2 (right) found within the same patient's tumors(n ¼ 15). D, correlation between the PFS and the level of MDC found in patients' tumors (n ¼ 10).


Aspord et al.




of TARC, MDC, and MMP-2 at the tumor site (Fig. 7A and B).Importantly, the levels of MDC and MMP-2 correlated withthe proportions of pDCs found in the corresponding samples(Fig. 7C). Furthermore, MDC, MMP-2, and TARC favored theupregulation of OX40L and ICOSL respectively on pDCs(Supplementary Fig. S5). Strikingly, we found that MDC wasinversely associated with the length of progression-free sur-vival (PFS; Fig. 7D), as a high level of MDC at the tumor sitecorrelated with early relapse. Together, our data stronglysuggest that TARC, MDC, and MMP-2 promote pDC accu-mulation and modulation at the tumor site, subsequentlytriggering immunity preferentially towards Th2 and regula-tory profiles and ultimately leading to tumor progression andearly relapse.

DiscussionpDCs are emerging as crucial but still enigmatic cells in

melanoma. Under specific conditions, pDCs are able to inducepotent tumor-specific responses, but the tumor microenviron-ment may compromise such potential and promote tumorprogression. By combining the in vivo analysis of melanoma-bearing humanizedmice and the ex vivo study of a large cohortof patients with melanoma, we identified the mechanism ofimmunity hijacking bymelanoma, bridging the observations ofthe presence of tumor-infiltrating pDCs and of Th2 andregulatory immune profiles.We showed in bothmelanoma-bearing Humice and patients

with melanoma that pDCs rapidly and massively accumulatedat the tumor site and in DLN, confirming earlier studiesperformed by immunohistochemistry analysis on tissue sec-tions (9, 25, 26). The combined analysis of pDC frequency andmigratory profile suggests a depletion of pDCs from blood andan accumulation at the cutaneous tumor site and DLN duringthe course of the disease. This is in line with the increased pDCfrequency observed in the invaded sentinel lymph node inpatients with melanoma (27) and the attraction of circulatingpDCs to the tumor site through the CCR6/CCL20 pathway (25).Moreover, infiltrating pDCs appeared to be important formelanoma development because extensive pDC infiltrationwas associated with poor clinical outcome. A negative impactof pDCs on patient outcomes has also been demonstrated inbreast and ovarian cancers (19, 21). In melanoma, our observa-tions are in accordance with a recent study describing arelation between pDC infiltration (defined as CD123þ cells)on primary melanoma sections and early relapse (28), yetwithout elucidating the cellular and molecular networksinvolved.Strikingly, we demonstrated that pDCs at the tumor site

triggered IL-5-/IL-13–secreting CD4 and CD8 Th2 cells andIL-10–secreting regulatory T cells through OX40L and ICOSLexpression, respectively. Remarkably, a proinflammatoryTh2 microenvironment has been identified by gene expres-sion profile in the sentinel lymph node (34) of patients withmetastatic melanoma and further supported by a systemicTh2-driven inflammation characterized by high plasma con-centrations of IL-4, IL-5, and IL-13 cytokines (35). Such animmune profile has been described in breast cancer but was

found to be driven by myeloid dendritic cells (29, 36). Th2cytokines may lead to melanoma tumor progression inseveral ways. Indeed, IL-13 has been involved in the sup-pression of immune surveillance and inhibition of cytotoxicT-cell functions (37). Moreover, IL-13 may also directly favormelanoma growth through signalling via IL-13Ra as mela-noma tumor cells express IL-13Ra (38). A strong associationbetween pSTAT3 expression by melanoma cells and infil-tration by pDCs has been described (28), suggesting ahelping role of pDCs in tumor progression through thetriggering of Th2 responses. IL-10þ regulatory T cells maylead to immune suppression through the suppression of Th1and cytotoxic T cells (39). Infiltrating ICOSþ regulatory Tcells have been identified in melanoma (40), and these cellsdisplay potent immunosuppressive functions in the contextof cancer (33, 41). The local interaction of ICOSþ regulatorycells with ICOSLþ pDCs may lead to their expansion withinthe tumor microenvironment.

We found TARC/CCL17 and MDC/CCL22 in the melanomamicroenvironment, chemokines that are known to be involvedin the recruitment of Th2 (31, 32, 42) and Treg to the tumor site(33, 43). Furthermore, high quantities of the proteolytic enzymeMMP-2 are also found within the melanoma microenviron-ment. Dysregulation of antitumor immunity toward an inflam-matory Th2 profile could be attributed to MMP-2 throughmyeloid dendritic cells conditioning to express OX40L (30, 44).We also demonstrated the ability of TARC, MDC, and MMP-2to upregulate the expression ofOX40L and ICOSLonpDCs. Thelevel of MDC/CCL22 at the tumor site is inversely correlatedwith PFS, reinforcing its role in the tumor microenvironment.In line with these data, the level of MDC/CCL22 reflects thedisease activity in patients with atopic dermatitis (45), aninflammatory skin disease characterized by high serum levelsof Th2-type cytokines. Therefore, melanoma developed strat-egies to simultaneously polarize pDCs to elicit Th2 andimmunosuppressive pathways, and to promote the recruit-ment of pDC-primed T cells that in turn will facilitate tumorprogression.

Even if pDCs displayed particular features within themelanoma microenvironment evidenced by a marked defectin their activation level in lymph node in contrast with thetumor site, we showed no impairment in their capacity toupregulate coactivation molecules or to produce cytokinesin response to TLR7- and TLR9-L stimulation. The intactability of melanoma-infiltrating pDCs to respond to TLRLstimulation is consistent with the potent antitumor res-ponses following imiquimod (TLR7-L) therapy in patientswith melanoma (46–48) or after CpG (TLR9-L; refs. 14, 15)treatment, both of which involved the recruitment andactivation of pDCs (16, 17, 46–48). Despite the role of pDCson tumor progression under steady state conditions, remo-bilization of pDCs using TLR agonists represents a promis-ing means to achieve melanoma tumor control by reversingthe functional hijack of pDCs.

On the basis of our previous work (25) and work fromothers (30), the present study leads to a better understand-ing of the hijacking of immunity by melanoma. pDCs expres-sing CCR6 are recruited to melanoma tumor through CCL20.






pDCs are then induced to express OX40L and ICOSL that,under MMP-2 conditioning, could drive IL-5- and IL-13–producing Th2 CD4 and CD8 T cells and IL-10–producingregulatory T cells that might migrate to the tumor sitethrough TARC/CCL17 and MDC/CCL22. This sequence leadsto tumor progression and early relapse. Melanoma activelycircumvents the immune system to facilitate its progression.By delineating the mechanisms underlying melanoma-driv-en plasticity of pDC in the melanoma microenvironmentconcomitant with their unaltered ability to respond to TLR-mediated signals, we have identified novel therapeuticoptions for patients with metastatic melanoma.

Disclosure of Potential Conflicts of InterestThe authors disclose no conflicts of interest.

Authors' ContributionsConception and design: C. Aspord, M.-T. LecciaDevelopment of methodology: C. AspordAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Aspord, J. Charles, J. Plumas

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Aspord, J. Charles, J. PlumasWriting, review, and/or revision of themanuscript: C. Aspord, M.-T. Leccia,J. Charles, J. PlumasStudy supervision: C. Aspord, M.-T. Leccia

AcknowledgmentsThe authors thank the surgeons of CHU Grenoble for their active participa-

tion in this study. The authors also thank the patients and healthy donors whoconsented to participate in this study and Stratton Beatrous for revision of themanuscript.

Grant SupportThis work was supported by the Institut National du Cancer (ACI-63-04

and canceropole 2004-05), the Etablissement Francais du Sang, the SocieteFrancaise de Dermatologie (SFD), and the company's foundation SILAB-JeanPaufique.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received August 6, 2013; revised September 12, 2013; accepted September 20,2013; published OnlineFirst September 26, 2013.

References1. Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines

against cancer. Nat Rev Immunol 2005;5:296–306.2. Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveil-

lance: immunoselection and immunosubversion. Nat Rev Immunol2006;6:715–27.

3. Finn OJ. Cancer immunology. N Engl J Med 2008;358:2704–15.4. Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in

immunity. Nat Immunol 2004;5:1219–26.5. Lande R, Gilliet M. Plasmacytoid dendritic cells: key players in the

initiation and regulation of immune responses. Ann N Y Acad Sci2010;1183:89–103.

6. Matta BM, Castellaneta A, Thomson AW. Tolerogenic plasmacytoidDC. Eur J Immunol 2010;40:2667–76.

7. Ito T, Amakawa R, Inaba M, Hori T, Ota M, Nakamura K, et al.Plasmacytoid dendritic cells regulate Th cell responses through OX40ligand and type I IFNs. J Immunol 2004;172:4253–9.

8. Villadangos JA, Young L. Antigen-presentation properties of plasma-cytoid dendritic cells. Immunity 2008;29:352–61.

9. Salio M, Cella M, Vermi W, Facchetti F, Palmowski MJ, Smith CL, et al.Plasmacytoid dendritic cells prime IFN-gamma-secreting melanoma-specificCD8 lymphocytes and are found in primarymelanoma lesions.Eur J Immunol 2003;33:1052–62.

10. Rothenfusser S, Hornung V, AyyoubM, Britsch S, Towarowski A, KrugA, et al. CpG-A and CpG-B oligonucleotides differentially enhancehuman peptide-specific primary and memory CD8þ T-cell responsesin vitro. Blood 2004;103:2162–9.

11. Liu C, Lou Y, Lizee G, Qin H, Liu S, Rabinovich B, et al. Plasmacytoiddendritic cells induceNK cell-dependent, tumor antigen-specific T cellcross-priming and tumor regression in mice. J Clin Invest 2008;118:1165–75.

12. Chaperot L, Blum A, Manches O, Lui G, Angel J, Molens JP, et al. Virusor TLR agonists induce TRAIL-mediated cytotoxic activity of plasma-cytoid dendritic cells. J Immunol 2006;176:248–55.

13. Matsui T, Connolly JE, Michnevitz M, Chaussabel D, Yu CI, Glaser C,et al. CD2 distinguishes two subsets of human plasmacytoid dendriticcells with distinct phenotype and functions. J Immunol 2009;182:6815–23.

14. Furumoto K, Soares L, Engleman EG, Merad M. Induction of potentantitumor immunity by in situ targeting of intratumoral DCs. J ClinInvest 2004;113:774–83.

15. Speiser DE, Lienard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F,et al. Rapid and strong human CD8 þT cell responses to vaccination

with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest2005;115:739–46.

16. Pashenkov M, Goess G, Wagner C, Hormann M, Jandl T, Moser A,et al. Phase II trial of a toll-like receptor 9-activating oligonucleotidein patients with metastatic melanoma. J Clin Oncol 2006;24:5716–24.

17. MolenkampBG, Sluijter BJ, van LeeuwenPA, Santegoets SJ,Meijer S,Wijnands PG, et al. Local administration of PF-3512676 CpG-B insti-gates tumor-specificCD8þ T-cell reactivity inmelanoma patients. ClinCancer Res 2008;14:4532–42.

18. Labidi-Galy SI, Treilleux I, Goddard-LeonS, Combes JD, Blay JY, Ray-Coquard I, et al. Plasmacytoid dendritic cells infiltrating ovarian cancerare associated with poor prognosis. Oncoimmunology 2012;1:380–2.

19. Treilleux I, Blay JY, Bendriss-Vermare N, Ray-Coquard I, Bachelot T,Guastalla JP, et al. Dendritic cell infiltration and prognosis of earlystage breast cancer. Clin Cancer Res 2004;10:7466–74.

20. Sisirak V, Faget J, Gobert M, Goutagny N, Vey N, Treilleux I, et al.Impaired IFN-alpha production by plasmacytoid dendritic cells favorsregulatory T-cell expansion that may contribute to breast cancerprogression. Cancer Res 2012;72:5188–97.

21. Labidi-Galy SI, Sisirak V,MeeusP, GobertM, Treilleux I, Bajard A, et al.Quantitative and functional alterations of plasmacytoid dendritic cellscontribute to immune tolerance in ovarian cancer. Cancer Res2011;71:5423–34.

22. Hartmann E, Wollenberg B, Rothenfusser S, Wagner M, Wellisch D,Mack B, et al. Identification and functional analysis of tumor-infiltratingplasmacytoid dendritic cells in head and neck cancer. Cancer Res2003;63:6478–87.

23. Wei S, Kryczek I, Zou L, Daniel B, Cheng P, Mottram P, et al. Plas-macytoid dendritic cells induce CD8 þregulatory T cells in humanovarian carcinoma. Cancer Res 2005;65:5020–6.

24. Curiel TJ, Cheng P, Mottram P, Alvarez X, Moons L, Evdemon-HoganM, et al. Dendritic cell subsets differentially regulate angiogenesis inhuman ovarian cancer. Cancer Res 2004;64:5535–8.

25. Charles J, Di Domizio J, Salameire D, Bendriss-Vermare N, Aspord C,Muhammad R, et al. Characterization of circulating dendritic cells inmelanoma: role of CCR6 in plasmacytoid dendritic cell recruitment tothe tumor. J Invest Dermatol 2010;130:1646–56.

26. Vermi W, Bonecchi R, Facchetti F, Bianchi D, Sozzani S, Festa S, et al.Recruitment of immature plasmacytoid dendritic cells (plasmacytoidmonocytes) and myeloid dendritic cells in primary cutaneous mela-nomas. J Pathol 2003;200:255–68.


Aspord et al.




27. Gerlini G, Urso C, Mariotti G, Di Gennaro P, Palli D, Brandani P, et al.Plasmacytoid dendritic cells represent a major dendritic cell subset insentinel lymph nodes of melanoma patients and accumulate in met-astatic nodes. Clin Immunol 2007;125:184–93.

28. Jensen TO, Schmidt H, Moller HJ, Donskov F, Hoyer M, SjoegrenP, et al. Intratumoral neutrophils and plasmacytoid dendriticcells indicate poor prognosis and are associated with pSTAT3expression in AJCC stage I/II melanoma. Cancer 2012;118:2476–85.

29. AspordC,Pedroza-GonzalezA,GallegosM, TindleS,BurtonEC,SuD,et al. Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4þ T cells that facilitate tumor development. J Exp Med2007;204:1037–47.

30. Godefroy E, Manches O, Dreno B, Hochman T, Rolnitzky L, LabarriereN, et al.Matrixmetalloproteinase-2 conditions humandendritic cells toprime inflammatory T(H)2 cells via an IL-12- and OX40L-dependentpathway. Cancer Cell 2011;19:333–46.

31. Soumelis V, Reche PA, Kanzler H, Yuan W, Edward G, Homey B, et al.Human epithelial cells trigger dendritic cell mediated allergic inflam-mation by producing TSLP. Nat Immunol 2002;3:673–80.

32. Hammad H, Smits HH, Ratajczak C, Nithiananthan A, Wierenga EA,Stewart GA, et al. Monocyte-derived dendritic cells exposed to Der p 1allergen enhance the recruitment of Th2 cells: major involvement of thechemokines TARC/CCL17 and MDC/CCL22. Eur Cytokine Netw2003;14:219–28.

33. Gobert M, Treilleux I, Bendriss-Vermare N, Bachelot T, Goddard-LeonS, Arfi V, et al. Regulatory T cells recruited through CCL22/CCR4 areselectively activated in lymphoid infiltrates surrounding primary breasttumors and lead to an adverse clinical outcome. Cancer Res 2009;69:2000–9.

34. Torisu-Itakura H, Lee JH, Scheri RP, Huynh Y, Ye X, Essner R, et al.Molecular characterization of inflammatory genes in sentineland nonsentinel nodes in melanoma. Clin Cancer Res 2007;13:3125–32.

35. Nevala WK, Vachon CM, Leontovich AA, Scott CG, Thompson MA,Markovic SN. Evidence of systemic Th2-driven chronic inflamma-tion in patients with metastatic melanoma. Clin Cancer Res 2009;15:1931–9.

36. Pedroza-Gonzalez A, Xu K, Wu TC, Aspord C, Tindle S, Marches F,et al. Thymic stromal lymphopoietin fosters human breast tumorgrowth by promoting type 2 inflammation. J Exp Med 2011;208:479–90.

37. Terabe M, Matsui S, Noben-Trauth N, Chen H, Watson C, DonaldsonDD, et al. NKT cell-mediated repression of tumor immunosurveillanceby IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 2000;1:515–20.

38. Badr G, Sayed D. Human melanoma cells release soluble and func-tional receptors. Egypt J Immunol 2006;13:23–31.

39. Menard C, Ghiringhelli F, Roux S, Chaput N, Mateus C, Grohmann U,et al. Ctla-4 blockade confers lymphocyte resistance to regulatoryT-cells in advanced melanoma: surrogate marker of efficacy oftremelimumab? Clin Cancer Res 2008;14:5242–9.

40. Strauss L, Bergmann C, Szczepanski MJ, Lang S, Kirkwood JM,Whiteside TL. Expression of ICOS on human melanoma-infiltratingCD4þCD25highFoxp3þ T regulatory cells: implications and impact ontumor-mediated immune suppression. J Immunol 2008;180:2967–80.

41. Faget J, Bendriss-Vermare N, Gobert M, Durand I, Olive D, Biota C,et al. ICOS-ligand expression on plasmacytoid dendritic cells supportsbreast cancer progression by promoting the accumulation of immu-nosuppressive CD4þ T cells. Cancer Res 2012;72:6130–41.

42. Liu YJ, Soumelis V, Watanabe N, Ito T, Wang YH, Malefyt RdeW, et al.TSLP: an epithelial cell cytokine that regulates T cell differentiation byconditioning dendritic cell maturation. Annu Rev Immunol 2007;25:193–219.

43. Faget J, Biota C, Bachelot T, Gobert M, Treilleux I, Goutagny N, et al.Early detection of tumor cells by innate immune cells leads to T(reg)recruitment through CCL22 production by tumor cells. Cancer Res2011;71:6143–52.

44. Godefroy E, Bhardwaj N. Dysregulation of anti-tumor immunity by thematrix metalloproteinase-2. Oncoimmunology 2012;1:109–11.

45. Hashimoto S, Nakamura K, Oyama N, Kaneko F, Tsunemi Y, Saeki H,et al. Macrophage-derived chemokine (MDC)/CCL22 produced bymonocyte derived dendritic cells reflects the disease activity inpatients with atopic dermatitis. J Dermatol Sci 2006;44:93–9.

46. DummerR,Hauschild A, Becker JC,Grob JJ, Schadendorf D, TebbsV,et al. An exploratory study of systemic administration of the toll-likereceptor-7 agonist 852A in patients with refractory metastatic mela-noma. Clin Cancer Res 2008;14:856–64.

47. Steinmann A, Funk JO, Schuler G, von den Driesch P. Topical imiqui-mod treatment of a cutaneous melanoma metastasis. J Am AcadDermatol 2000;43:555–6.

48. Palamara F, Meindl S, Holcmann M, Luhrs P, Stingl G, Sibilia M.Identification and characterization of pDC-like cells in normal mouseskin and melanomas treated with imiquimod. J Immunol 2004;173:3051–61.






2013;1:402-415. Published OnlineFirst September 26, 2013.Cancer Immunol Res Caroline Aspord, Marie-Therese Leccia, Julie Charles, et al. ICOSLPromoting Th2 and Regulatory Immunity through OX40L and Plasmacytoid Dendritic Cells Support Melanoma Progression by

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