Vemurafenib reverses immunosuppression by myeloid derived suppressor cells

Vemurafenib reverses immunosuppression by myeloid derivedsuppressor cells

Bastian Schilling1, Antje Sucker1, Klaus Griewank1, Fang Zhao1, Benjamin Weide2, Andr�e G€orgens3, Bernd Giebel3,

Dirk Schadendorf1 and Annette Paschen1

1 Department of Dermatology, University Hospital, University Duisburg-Essen, Essen, Germany2 Department of Dermatology, University Medical Center, University of T€ubingen, T€ubingen, Germany3 Institute of Transfusion Medicine, University Hospital, University Duisburg-Essen, Essen, Germany

Myeloid derived suppressor cells (MDSCs) suppress innate and adaptive immunity, thereby limiting anti-tumor immune

responses in cancer patients. In patients with advanced melanoma, the phenotype and function of MDSCs remains controver-

sial. In our study, we further explored two distinct subpopulations of MDSCs and investigated the impact of Vemurafenib on

these cells. Flow cytometry analysis revealed that in comparison to healthy donors and patients with localized disease, PBMCs

from patients with metastatic melanoma showed an increased frequency of CD141HLA-DR2/low monocytic MDSCs (moMDSCs)

and of a previously unrecognized population of CD142CD66b1Arginase11 granulocytic MDSCs (grMDSCs). In vitro, both popu-

lations suppressed autologous T-cell proliferation, which was tested in CFSE-based proliferation assays. Vemurafenib treat-

ment of melanoma patients reduced the frequency of both moMDSCs and grMDSCs. According to our in vivo finding,

conditioned medium (CM) from Vemurafenib treated melanoma cells was less active in inducing moMDSCs in vitro than CM

from untreated melanoma cells. In conclusion, patients with advanced melanoma show increased levels of moMDSCs, and of

a population of CD142CD66b1Arginase11 grMDSCs. Both MDSCs are distinct populations capable of suppressing autologous

T-cell responses independently of each other. In vitro as well as in vivo, Vemurafenib inhibits the generation of human

moMDSCs. Thus, Vemurafenib decreases immunosuppression in patients with advanced melanoma, indicating its potential as

part of future immunotherapies.

Despite the development of specific anti-tumor immunity,metastatic melanoma remains a disease with poor prognosis.1

This might be, at least in part, due to an accumulation ofimmunosuppressive cells like regulatory T-cells (Tregs) andmyeloid derived suppressor cells (MDSCs) in patients withadvanced melanoma.2–5 MDSCs suppress innate and adaptiveimmunity by various mechanisms.2,4,6 Recent data suggestthat elimination of MDSCs or modulation of their functionunleashes T cell anti-tumor responses.7–10 Ko et al.9 observedthat treatment of renal cell carcinoma (RCC) patients withsunitinib, a tyrosine receptor kinase inhibitor, decreasesMDSC frequency and suppressive function. Specific

elimination of MDSCs, however, is limited by an apparentlyheterogenic phenotype of these cells.6,11

In general, human MDSCs can be divided into granulo-cytic (grMDSCs) and monocytic MDSCs (moMDSCs). Thisdiscrimination is mainly based on morphological and pheno-typical characteristics of grMDSCs and moMDSCs. Althoughsharing the expression of common myeloid markers likeCD11b, grMDSCs are usually found to be CD151 whilemoMDSCs are CD152/dim but can express CD14.2,12–16 Thephenotypical and functional differences between these twopopulations are not well defined. Both subsets were reportedto use Arginase 1, reactive oxygen species (ROS) and TGF-bto suppress innate and adaptive immunity and to promotetumor growth by production of pro-angiogenic mole-cules.2,4,6,17 It is widely believed that different tumors inducedistinct MDSC subpopulations.6

In the peripheral blood of patients with metastatic mela-noma, Filipazzi et al.4 were the first to report an accumula-tion of CD141HLA-DR2/low moMDSCs using TGF-b tosuppress T-cell proliferation and Interferon-g productionwhile corresponding CD141HLA-DR1 cells exhibited nosuppressive properties. Although CD66b1 grMDSCs havebeen described in patients with RCC,16 head and neck cancer,lung cancer as well as urogenital cancer,18 no correspondingMDSC subpopulation has been identified in melanoma.Recently, Poschke et al.2 confirmed the accumulation ofCD141HLA-DR2/low moMDSCs in patients with advanced

Key words: melanoma, vemurafenib, MDSC

Additional Supporting Information may be found in the online

version of this article.

Grant sponsor: Helmholtz-Gemeinschaft Deutscher

Forschungszentren (HGF) “Alliance on Immunotherapy of Cancer”

and Interdisciplinary Grant from the University of Essen (IFORES)

DOI: 10.1002/ijc.28168

History: Received 22 Nov 2012; Accepted 8 Mar 2013; Online 23

Mar 2013

Correspondence to: Bastian Schilling, Department of Dermatology,

University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany,

Tel.: 149-201-72383590; E-mail: [email protected]

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Int. J. Cancer: 133, 1653–1664 (2013) VC 2013 UICC

International Journal of Cancer

IJC

melanoma and further characterized these cells as Stat3high,CD801, CD831 and DC-sign1. In contrast to Filipazzi et al.,this publication described Arginase 1 and ROS as mecha-nisms of suppression of CD141HLA-DR2/low moMDSCs.2

The reason for these discordant findings remains unclear andis further complicated by the observation of Gros et al.19 whofailed to detect an elevated frequency of MDSCs in the pe-ripheral blood of melanoma patients. This work also reportedthat both CD141HLA-DR2/low as well as CD141HLA-DR1

MDSCs suppress autologous T-cell proliferation.19 Consider-ing this conflicting data, it became apparent, that the pheno-type and function of MDSCs in malignant melanoma needsto be further clarified.

Although the mechanisms of MDSC induction by malig-nant melanoma are still unknown, a potential influence ofeffective tumor therapies on MDSC populations could be pre-sumed. Recently, treatment of melanoma patients withadvanced disease with Vemurafenib led to impressive objec-tive response rates. Vemurafenib is a highly specific inhibitorof mutant B-RAFV600E, a mutation leading to constitutiveactivation of the MAP Kinase pathway and found in �60%of cutaneous melanoma.20 Despite high rates of objectiveresponses in patients with advanced melanoma treated withVemurafenib, tumors eventually become resistant. The me-dian response duration is 6 months of therapy and long-termresponses are observed rarely.21 Vemurafenib might reducetumor growth not only through cell intrinsic mechanisms butalso reduce cytokine-driven induction of MDSCs in the tu-mor microenvironment. Impairing melanoma mediatedimmunosuppression would provide an ideal setting for thesuccessful implementation of additional immunotherapiesknown to induce durable tumor regression.15,21,22 To test thishypothesis, we explored the nature of MDSCs in patientswith melanoma and investigated the impact of Vemurafenibon MDSCs ex vivo and in vitro.

Material and MethodsBlood samples

Peripheral blood was obtained from 25 patients with meta-static melanoma (Stage IV, AJCC 200923), 16 patients withStage I–III Melanoma (AJCC) and 10 age and sex matchedhealthy donors (HD). Blood was drawn before therapy inpatients with active disease (AD). All subjects with no clinicalevidence of disease (NED) were tumor free for at least 3

months at the time of phlebotomy and signed an informedconsent approved by the local ethics committee. Patientswere seen at the Department of Dermatology Essen betweenDecember 2011 and January 2013. Clinicopathological char-acteristics including B-RAF status in Stage IV patients arelisted in Table 1. B-RAF status in melanoma tissue was deter-mined by Sanger sequencing.24 When no information is givenin Table 1, B-RAF status was not determined. In patientsundergoing Vemurafenib treatment, clinical course of the dis-ease was determined every 4 weeks, computed tomography(CT) scans of the chest and abdomen were performed every8 weeks or if progress of melanoma was suspected clinically.CT scans were analyzed according to RECIST criteria.25 Pe-ripheral blood mononuclear cells (PBMCs) analyzed forMDSCs were obtained up to 2 weeks before treatment withVemurafenib was commenced (Visit 0, baseline). Consecutivespecimens were obtained 4–8 weeks (Visit 1) and 12–16weeks (Visit 2) after initialization of treatment.

Antibodies

The following fluorochrome-labeled monoclonal antibodies(mAbs) purchased from Beckman Coulter (Krefeld, Germany)were used: anti-CD3-PE, anti-CD4-PE-Cy7, anti-CD8-APC,anti-CD19-PE, anti-HLA-DR-ECD and -PE. Anti-CD11b-Alexa Flour 700, anti-CD66b-PE and Alexa Flour 647, as wellas anti-CD45-APC-H7 and anti-CD15-Pacific blue antibodiesfrom BD Pharmigen (Heidelberg, Germany). Anti-Arginase-1-FITC polyclonal Ab was from R&D Systems (Minneapolis,MN). Anti-CD33-PECy7 and anti-CD56-PE were obtainedfrom eBioscience while anti-CD14-PerCP5.5 and anti-CD16-Pacific Blue were purchased from Biolegend (San Diego, CA).CD3, CD19 and CD56 were used as a lymphocytic linage cock-tail (Lin). Appropriate isotype controls were purchased fromBeckman Coulter and BD.

Isolation of PBMCs

PBMCs were isolated by centrifugation on Lympoprep(Alexis Shield) from heparinized venous blood.

Cell lines

The BRAFV600E mutant primary human melanoma cell lineMa-Mel-51 was established and maintained as described.26

Cell cultures were tested monthly for mycoplasma infectionand always found to be negative.

What’s new?

Censoring of the immune system, leading to its inability to mount an effective attack against tumor cells, is suspected to con-

tribute to the advance of melanoma. The restrained response may be the result of two distinct populations of myeloid derived

suppressor cells (MDSCs), as reported here. Monocytic MDSCs and a population of previously unrecognized Arginase11 granu-

locytic MDSCs were detected at elevated frequencies in patients with metastatic melanoma. Frequencies of both subtypes

declined in patients with clinical response to the enzyme inhibitor vemurafenib, which was further found to block in vitro gen-

eration of monocytic MDSCs.

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1654 Vemurafenib and MDSCs in melanoma


Generation of conditioned medium

For the generation of conditioned medium (CM), an equalnumber of Ma-Mel-51 cells were seeded in culture flasks. Af-ter 24 hr, medium was removed completely and fresh me-dium containing 1mM Vemurafenib (kindly provided byRoche, Grenzach-Wyhlen, Germany) or vehicle was added.After 72 hr, supernatants were harvested and contaminatingcells were removed by centrifugation.

Surface and intracellular staining for flow cytometry

For surface staining, cells were incubated with antibodies for30 min at 4�C. After washing, cells were fixed and permeabil-ized using a commercially available kit (eBiocience, SanDiego, CA) according to the manufacturer’s instructions.Without washing, cells were stained intracellularly for 30 minat room temperature. All antibodies were pretitrated onfreshly harvested and activated PBMCs to determine optimalworking dilutions. Viability was assessed by Aqua ViabilityDye (Detected on FL-10, BD). Samples were acquired on aBeckman Coulter Gallios flow cytometer, and data were ana-lyzed using the KaluzaVR software (Beckman Coulter).

Cell sorting

MDSCs were isolated from PBMCs by magnetic bead basedseparation (MACS technology, Miltenyi, Bergisch-Gladbach,Germany), a scheme of the experimental strategy is providedas supporting information Figure 1. First, CD31 cells werepositively selected on LS columns after incubation with anti-CD3 coated magnetic beads according to the manufacturer’sinstructions. Next, CD32 PBMCs were incubated with an

anti-CD66b-PE mAb for 20 min at 4�C. After washing, cellswere incubated with anti-PE coated magnetic beads accordingto the manufacturer’s instructions. CD66b1 cells were posi-tively selected using MS columns. To improve purity, posi-tively selected cells were run on a second MS columnafterward. From the remaining CD32CD66b2 PBMCs,CD141 cells were negatively selected using the monocyte iso-lation kit II (Miltenyi) according to the manufacturer’sinstructions. In a last step, CD32CD66b2CD141 cells wereseparated into HLA-DR1 and HLA-DR2/low cells usingHLA-DR coupled magnetic beads (Miltenyi). The purity andviability of all MACS preparations routinely exceeded 90%.No cross-contamination of the moMDSC and grMDSC frac-tion was detectable by flow cytometry.

From in vitro cultures, moMDSCs were isolated by MACSand subsequent fluorescent activated cell sorting (FACS).Briefly, CD141 cells were positively selected on MS columnsusing anti-CD14 coated beads. CD141 cells were then stainedwith anti-HLA-DR-PE and anti-CD14-PerCP5.5.CD141HLA-DR1 and CD141HLA-DR2/low and sorted onan ARIA I (BD) as described before.27

CFSE-based suppression assay

Suppression of proliferation of CD31 T-cells was performedas previously described.28 Briefly, CD31 responder cells (RC)labeled with 2mM carboxyfluorescein succinimidyl ester(CFSE, Invitrogen, Carlsbad, CA) were cultured in completeRPMI1640 medium in the presence of anti-CD3/anti-CD28coated beads (Miltenyi, bead to T-cell ratio 1:2) in wells of96-well plates (105/well). MDSCs freshly isolated from

Table 1. Clinicopathological characteristics of patients enrolled in our study

Patients Subgroup Age (range in years) Sex (male:female) Number of patients

AD Stage I–III 30–79 4:3 7

Stage I 1:0 1

Stage II 1:1 2

Stage III 2:2 4

NED Stage I–III 25–71 5:4 9

Stage I 2:2 4

Stage II 1:1 2

Stage III 2:1 3

B-RAF Status

V600E1 wt

AD Stage IV 32–82 13:8 21 13 8

M1a 1:0 1 1 0

M1b 0

M1c 12:8 20 12 8

NED Stage IV 42–77 1:6 7

M1a 0:1 1

M1b 0:1 1

M1c 1:4 5 3

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Schilling et al. 1655


melanoma patients or harvested from in vitro cultures wereadded to the RC at different ratios and cultures were incu-bated in 5% CO2 in air at 37�C. Flow cytometric analyses ofCFSE dilution was performed on Day 5 of culture.

In vitro generation of MDSCs

A total of 43 106 PBMCs from HD were cultured in CM fromeither untreated or Vemurafenib-treated Ma-Mel-51 cells onsix-well plates. PBMCs cultured in complete RPMI16406 1mMVemurafenib served as an additional control. PBMCs were har-vested after 72 hr and used immediately for phenotypical andfunctional characterization of moMDSCs. RC for CFSE-basedsuppression assays were isolated from autologous PBMCs keptin complete RPMI1640 until usage.

Statistical analysis

Data were analyzed by SPSS (IBM) using two-way analysis ofvariance (ANOVA). p values< 0.05 were considered significant.

ResultsDetection of moMDSCs and grMDSCs in patients with

advanced melanoma

A nine-color staining panel was designed to identify previ-ously described populations of human MDSCs in patientswith melanoma.29,30 Using this panel, we were able to detect

moMDSCs (CD141HLA-DR2/low) within PBMCs of patientswith advanced melanoma. In addition, we found a populationof CD66b1Arginase11 cells in the peripheral circulation ofStage IV melanoma patients, suggesting that these cells mightbe CD66b1CD11b1CD331Arginase11CD142CD16low

grMDSCs as described by Rodriguez et al.16 in RCC (Fig. 1).Further analysis of moMDSCs and grMDSCs found inpatients with malignant melanoma revealed a partial pheno-typical overlap. Monocytic MDSCs were found to be CD451

Lin2HLA-DR2/lowCD141CD15dimCD66b2CD331CD11b1

Arginase12CD162/low. In contrast, granulocytic MDSCswere CD451Lin2HLA-DR2CD142CD151CD66b1CD331

CD11b1Arginase11CD162/low (Fig. 1). Thus, moMDSCsand grMDSCs in patients with advanced melanoma are twodistinct populations. Although moMDSCs have beendescribed by others, grMDSCs represent a previouslyunrecognized subpopulation of human MDSCs found inpatients with melanoma.

Granulocytic and monocytic MDSCs from melanoma

patients suppress autologous T-cell proliferation

independently

As the phenotype of human MDSCs might be malignancy-de-pendent, CD66b1Arginase11 cells found in melanoma patientsmight not have the same function as CD66b1 grMDSCs

Figure 1. Granulocytic and monocytic MDSCs are phenotypical distinct populations. Density plots and histograms showing the gating strat-

egy and phenotype of grMDSCs and moMDSCs. After initial gating on CD451Lin2HLA-DR2/low leukocytes, grMDSCs were found to be

CD66b1CD142Arginase11CD11b1CD331 while moMDSCs showed expression of CD14, CD11b and CD33 but not CD66b and Arginase1.

CD15 is expressed weakly on moMDSCs while grMDSCs are CD151. Both grMDSCs and moMDSCs are mostly CD16 negative. Representative

data from one AD Stage IV melanoma patient is shown. Mean fluorescence intensity (MFI), side scatter (SS). [Color figure can be viewed in

the online issue, which is available at wileyonlinelibrary.com.]

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described in patients with other types of cancer. To investigatethis, we performed a functional analysis of both moMDSCs andgrMDSCs. First, we developed an isolation strategy allowing theseparation of both populations from the same donor as describedin materials and methods. Importantly, our isolation approachexcludes cross-contamination of moMDSCs and grMDSCs.When used in CFSE-based suppressor assays, both moMDSCsand grMDSCs suppressed proliferation of autologous CD31 T-cells. As shown in Figure 2, we found a dose-dependent suppres-sion of both CD41 and CD81 T-cells by moMDSCs andgrMDSCs (Figs. 2a and 2b). When comparing the suppressivecapacity of moMDSCs and grMDSCs, we found grMDSCs to bemore potent inhibitors of autologous T-cell proliferation (Fig.2c). Bulk CD66b2 as well as CD141HLA-DR1 cells used as con-trols did not suppress autologous T-cell proliferation.

Increased frequency of granulocytic and monocytic MDSCs

in patients with advanced melanoma

Next, we compared the frequency of MDSCs within PBMCsfrom melanoma patients to those obtained from HD. InStage IV melanoma patients with AD, defined as having clin-ical or radiological detectable metastasis, we found anincreased frequency of both moMDSCs and grMDSCs com-pared to HD (Figs. 3a and 3b). Lower Stage (I–III) melanomapatients with an AD at the time of blood draw did not differin the detectable frequency of both grMDSCs and moMDSCs,as compared to HD. As an additional control, we also deter-mined the frequency of grMDSCs and moMDSCs withinPBMCs of Stage I–III melanoma patients with NED at thetime of phlebotomy. This also did not differ significantlyfrom HD. Furthermore, we could analyze a small number of

Figure 2. Granulocytic and monocytic MDSCs suppress autologous T-cell proliferation. In autologous CFSE-based proliferation assays,

grMDSCs and moMDSCs inhibit T-cell proliferation. On Day 5 of co-culture, T-cell proliferation was measured by CFSE dilution. (a) Represen-

tative density plots showing suppression of autologous CD41 and CD81 cells by moMDSCs in a dose-dependent manner. (b) Representative

density plots showing suppression of autologous CD41 and CD81 cells by grMDSCs. (c) Summarized data from three independent CFSE

assays using moMDSCs and grMDSCs from three different Stage IV melanoma patients. Mean frequency of CD31CFSEdim cells 6 standard

error of the mean (SEM) shown.*p<0.05, **p<0.01, ***p<0.001. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

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samples from Stage IV patients with NED at the time ofblood draw. Six of these patients were rendered tumor-freeby surgery while one showed a complete response to Ipilimu-mab. Interestingly, the frequency of grMDSCs andmoMDSCs of these patients was not increased compared toHD and Stage I–III melanoma patients. In addition, the fre-quency of grMDSCs in Stage IV NED patients was signifi-cantly lower than in Stage IV AD patients. As shown inFigure 3, the frequency of moMDSCs showed a similar tend-ency suggesting that successful treatment of melanomapatients decreases MDSC frequencies, linking both popula-tions directly to the presence of advanced melanoma.

Vemurafenib alters the frequency of granulocytic and

monocytic MDSCs in patients with advanced melanoma

To investigate the in vivo effects of Vemurafenib on MDSCs,we analyzed serial blood samples obtained from six patientswith advanced melanoma harboring a B-RAFV600E mutation.All patients were therapy-na€ıve and received 960 mg Vemur-afenib daily. As shown in Figure 4b, five of six patients withobjective response, four patients with partial response (PR:circle, hexagon, square and inverted triangle), one patientwith stable disease (SD: triangle) to treatment showed adecline in moMDSC frequency (% decrease as compared tovisit 0: 10.5% to 27.5%, mean 18.4%). In one donor

(diamond) showing stable disease (SD) at Visit 1, an increaseof moMDSCs was observed (% increase as compared to Visit0: 13.7%). In those patients still showing objective responseto treatment at the time of Visit 2, moMDSC frequency fur-ther declined or stabilized as compared to Visit 1. However,the patient showing SD and an increase in moMDSC fre-quency at the time of Visit 1 (diamond), had progressive dis-ease (PD) at Visit 2 and showed a further increase inmoMDSC frequency (% increase as compared to Visit 1:8.3%). In contrast, the patient showing SD and a decrease inmoMDSC frequency at the time of Visit 1 (triangle), had PRof melanoma at the time of Visit 2. Finally, one patient(square), who had a decline in moMDSC frequency (215%)and PR at Visit 1, showed PD and an increase of moMDSCsabove baseline level (19%).

Although the frequency of moMDSCs was strongly associ-ated with the clinical course of melanoma, the behavior ofgrMDSC frequency in the same patients was diverse (Fig. 4c).In those patients who had PD at the time of Visit 2 (squareand diamond), grMDSCs showed the same course asmoMDSCs. In addition, in one of the four patients showingPR at the time of visit 2 (triangle) a steady decline in thegrMDSC frequency was observed. However, in the otherthree patients with PR at Visit 2, the course of grMDSC fre-quency showed a different tendency than that observed for

Figure 3. Elevated frequencies of granulocytic and monocytic MDSCs in patients with advanced melanoma. Freshly obtained PBMCs of HD

and patients with melanoma were stained and analyzed by flow cytometry. Frequency of grMDSCs is expressed as percentage of

CD66b1Arginase11CD162/low within the CD451 gate. Frequency of moMDSCs is expressed as percentage of CD141HLA-DR2/low of all

CD141 events. PBMCs of ten HD, seven Stage I–III melanoma patients with an AD, nine Stage I–III melanoma patients with NED at the time

of blood draw, 21 Stage IV melanoma patients with AD and seven Stage IV melanoma patients with NED were analyzed by flow cytometry.

Patients with metastatic melanoma show a significantly increased frequency of both moMDSCs and grMDSCs. In Stage IV patients with

NED, MDSC levels are similar to those found in HD. Bars indicate means, whiskers show 95% confidence interval. *p<0.05, ***p<0.001.

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moMDSCs in the same donors (circle, hexagon and invertedtriangle).

Vemurafenib inhibits MDSC generation in an in vitro model

of the melanoma microenvironment

To further explore the mechanisms of MDSC accumulationin patients with advanced melanoma, we established amodel of the melanoma microenvironment using CM fromprimary human melanoma cell Ma-Mel-51. In this model,PBMCs from HD were incubated with CM from Ma-Mel-51 cells treated with or without Vemurafenib. When usingCM from Ma-Mel51, flow cytometry analysis revealed thegeneration of a cell population with a moMDSC phenotypeafter 72 hr of culture. As shown in Figures 5a and 5b, wefound a significant induction of cells with a moMDSC phe-notype in the presence of Ma-Mel51 CM compared toRPMI1640 treatment. Dilution of Ma-Mel-51 CM withRPMI1640 reduced the generation of moMDSCs in vitro(Data not shown). Changes in the phenotype of CD141

cells were not caused by cell death as staining of PBMCswith a viability dye showed no difference in the frequencyof apoptotic cells from in vitro cultures in CM orRPMI1640 (data not shown). Next, we tested moMDSCsfrom in vitro cultures for their functionality. In CFSE pro-liferation assays, FACS-separated moMDSCs from Ma-Mel-51 CM in vitro cultures suppressed autologous T-cell prolif-eration while CD141HLA-DR1 cells from the same culturedid not (Fig. 5c). Thus, moMDSCs induced in our in vitromodel appear to be fully functional. Next, we tested theinfluence of Vemurafenib on the generation of moMDSCs.The frequency of moMDSCs generated in the presence ofCM taken from short-term Vemurafenib treated Ma-Mel-51cells was significantly lower as compared to controls havingreceived CM from untreated Ma-Mel-51 (Figs. 5a and 5b).Vemurafenib added directly to in vitro cultures containingCM from untreated Ma-Mel51 had no impact on the gener-ation of moMDSC. Thus, Vemurafenib inhibits the releaseof soluble factors from melanoma cells involved in the gen-eration of moMDSC in vitro.

Figure 4. MDSC accumulation is reversed by Vemurafenib in vivo. Serial analysis of PBMCs from patients with advanced melanoma harbor-

ing a B-RAFV600E mutation undergoing treatment with Vemurafenib. (a) Representative density plots showing the frequency of moMDSCs

and grMDSCs in one patient before treatment as well as 8 and 16 weeks after starting treatment with 960 mg Vemurafenib daily. (b and c)

Line graphs showing the time course of moMDSC and grMDSC frequencies in six patients with advanced melanoma. Each symbol repre-

sents one individual donor. (b) Clinical course of the disease is indicated by PD (progressive disease), stable disease (SD) and partial

response (PR) according to RECIST criteria. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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DiscussionIn our study, we performed a detailed analysis of the natureof MDSCs in patients with malignant melanoma and testedthe impact of Vemurafenib on MDSCs in vitro and in vivo.Using an elaborated panel of known markers for the detec-tion and characterization of human MDSCs in multicolorflow cytometry, we identified two distinct populations ofMDSCs in patients with advanced melanoma. Besides CD141

HLA-DR2/low moMDSCs which were previously described inmelanoma patients by other groups,2,4,31 we identified an

additional Arginase1-expressing, CD451Lin2HLA-DR2

CD142CD151CD66b1CD331CD11b1CD162/low granulo-cytic MDSC population. To our knowledge, we also offer thefirst report examining the impact of Vemurafenib on MDSCfrequencies in vitro and in vivo.

Both monocytic and granulocytic myeloid cells analyzedin our study can be considered MDSCs as they suppressedautologous T-cell proliferation, although to different extentsand most likely via different mechanisms. MoMDSCs andgrMDSCs both lack the expression of lymphocytic markers

Figure 5. MDSC generation is inhibited by Vemurafenib in vitro. (a) Representative density plots showing moMDSC induction in vitro. CM

from Ma-Mel-51 induces cells with the phenotype of moMDSCs while RPMI1640 (6 Vemurafenib 1 mM) does not. When CM from Vemurafe-

nib-treated Ma-Mel-51 cells is used, the induction of moMDSCs is decreased significantly. Adding Vemurafenib (1mM) into cultures contain-

ing CM from untreated Ma-Mel51 melanoma cells has no effect on the phenotype of CD141 cells. (b) Summarized data from four

independent experiments showing the effect of Vemurafenib on moMDSC generation in vitro. Mean frequency of CD141HLA-DR2/low of all

CD141 events 6 SEM is shown. (c) moMDSCs generated by CM from Ma-Mel-51 are fully functional. CFSE-based proliferation assays were

performed with FACS separated moMDSCs and CD141HLA-DR1 cells from in vitro culture. Although moMDSCs from in vitro cultures sup-

press autologous T-cell proliferation, CD141HLA-DR1 cells from the same culture do not. PBMCs were harvested after 72 hr of culture and

used for phenotypical and functional studies. Mean frequency of CD31CFSEdim cells 6 SEM from three independent experiments shown.

*p<0.05, ***p<0.001. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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while expressing comparable levels of CD11b and CD33.Also, both populations express no or only low levels ofClass-II molecules. This means that using HLA-DR, CD11band CD33 for enumeration and isolation of human MDSCswill lead to cross-contamination of these distinct subsets.14,32

To prevent this, we isolated these cells based on the markersCD14, HLA-DR and CD66b (see Supporting InformationFig. 1). A functional analysis comparing grMDSCs andmoMDSCs showed grMDSCs having a greater ability to sup-press autologous T-cell proliferation. Recently, similar obser-vations were made by Duffy et al. who performed acomparative analysis of grMDSCs and moMDSCs in patientswith advanced gastrointestinal malignancies. The grMDSCsand moMDSCs identified in our study were phenotypicallyidentical to those found in our study,33 despite CD15 insteadof CD66b being used for isolation. However, it can beassumed that the MDSC subpopulations analyzed were simi-lar, as CD66b and CD15 are co-expressed by grMDSCs.16

Our findings noted that CD66b1CD162/low grMDSCs inmelanoma patients express Arginase1 on the protein leveland have significantly greater ability to suppress autologousT-cell proliferation than moMDSCs. These findings are simi-lar to those described by Rodriguez et al.16 in RCC. L-Argi-nine metabolism controlled by myeloid cells seems to play amajor role in regulating T-cell function in cancer patients.34

By flow cytometry, we did not detect Arginase1 proteinexpression in moMDSCs. In contrast to our findings, Poschkeet al. reported an elevated expression of Arginase1 mRNA inmoMDSCs from melanoma patients as compared to healthydonors. An analysis at the protein level was not performed,which might explain the discrepancy in our observation.2

The authors demonstrated the functional relevance of Argi-nase1 activity by treating whole PBMCs with nor-NOHA, anArginase inhibitor. This treatment would have affectedenzyme activity in both grMDSCs and moMDSCs. Futurestudies applying isolated cell populations instead of bulk orMDSC-depleted PBMCs will be needed to fully delineate themechanisms of T-cell suppression used by grMDSCs andmoMDSCs.

Although our study further clarified the nature of MDSCsin patients with metastatic melanoma, their role in diseaseprogression remains complex. Accumulation of CD141HLA-DR2/low cells in the peripheral blood of patients withadvanced melanoma was first reported by Ugurel et al.31

Later, CD141HLA-DR2/low cells were isolated by Filipazziet al.4 and characterized as MDSCs by showing suppressiveeffects on T-cell proliferation while the correspondingCD141HLA-DR1 population did not. Conflicting with ourstudy and our own data, Gros et al.19 recently reported thatboth CD141HLA-DR2/low and CD141HLA-DR1 cells iso-lated from patients with advanced melanoma have suppres-sive properties. These authors also failed to observe anincreased frequency of CD141HLA-DR2/low in the peripheralblood of patients with advanced melanoma. This controver-sial finding might be explained by the fact that the frequency

of CD141HLA-DR2/low cells was determined in whole bloodrather than PBMCs. As therapeutic interventions might altermyeloid cell frequency and function,9 the discrepancies foundcould also be due to the fact that Gros et al. analyzed sam-ples from previously treated rather than therapy-na€ıvepatients with metastatic melanoma. Yet another study byMandruzzato et al.35 reports an accumulation of IL-4 recep-tor alpha chain (IL-4Ra) expressing CD141 and CD151

MDSCs in patients with advanced melanoma. However, IL-4Ra expression on human MDSCs found in cancer patientsstill needs to be confirmed and the role of IL-4Ra in the dif-ferentiation and function of murine MDSCs has recentlybeen questioned.13,36,37

Besides identifying an additional subpopulation of MDSCsand comprehensively analyzing their phenotype and function,we also evaluated the impact of Vemurafenib on MDSCs inpatients with advanced melanoma. We were able to show aneffect of Vemurafenib on MDSCs in the peripheral blood ofpatient with metastatic melanoma, fitting the results reportedby Poschke et al.,2 where moMDSC frequency was associatedwith disease course in Stage IV melanoma patients. In allpatients responding to Vemurafenib treatment, the moMDSCfrequency declined over time. The grMDSC frequencychanges noted in the same patients were less consistent. Inone of the three patients showing partial response to Vemur-afenib, grMDSC frequency increased during treatment. Forall other patients, the grMDSC frequency changes corre-sponded to the observed moMDSC frequency alterations.Potentially the effects of Vemurafenib on the secretion ofmolecules necessary for grMDSC induction or survival varysignificantly from patient to patient. As grMDSCs were previ-ously not recognized in melanoma, the mechanisms of theirinduction are still unknown. It would stand to reason thatthe mechanisms and molecules responsible for grMDSC andmoMDSC induction are distinct.

To further analyze the effect of Vemurafenib on MDSCs,we established an in vitro model of moMDSC induction. Inthis model, PBMCs from HD cultured in CM from a primarymelanoma cell line gave rise to fully functional CD141HLA-DR2/low moMDSCs which suppress autologous T-cell prolif-eration. Recent data by the Epstein group identified cytokinesproduced by human tumors, including melanoma, that areinvolved in the differentiation of moMDSCs: GM-CSF, IL-1b, IL-6, TNF-a, VEGF as well as FLT3L and TGF-b.38,39 Towhat extent these or additional soluble factors are involved inmoMDSC induction in our model will have to be addressedin future studies. Our finding that CM from Vemurafenib-treated melanoma cells loses its ability to induce moMDSCsstrongly supports a critical role for soluble factors. RNA-in-terference targeting B-RAFV600E has been shown to decreasethe production of VEGF, IL-6 and IL-10 by melanoma celllines in vitro.40 Taken together, these data support thatVemurafenib alters the secretion profile of melanoma cellsfrom an immunosuppressive to a nonimmunosuppressivecomposition. This fits well with the observation of Wilmott

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et al.21 who reported an increased tumor infiltration byCD41 and CD81 lymphocytes in patients treated with selec-tive B-RAF inhibitors. To further explore the impact ofVemurafenib on myeloid cells in the melanoma microenvir-onment, tumor samples from patients undergoing treatmentcould be screened for alterations of the infiltrating myeloidcell populations.

Both our data and work by Sumimoto et al.40 indicatethat targeting B-RAFV600E alters the cytokine secretion ofmelanoma cells, thereby reducing immune evasion. Althoughclinical response to Vemurafenib is observed in the majorityof treated patients, long-lasting regression of metastatic mela-noma is rarely seen. In contrast, immunotherapy of mela-noma using IL-2 or Ipilimumab, an anti-CTLA4 antibody,causes long-lasting regression in a small number ofpatients.18,41 Effective induction of anti-tumor immunity byimmunotherapy might be impeded by immunosuppressiveTregs and MDSCs accumulating in the peripheral circulationand the tumor microenvironment of melanoma patients. Thiscould explain the overall low response rates.4,42 Therefore,reducing immunosuppression by MDSCs with Vemurafenibmight augment the efficacy of future immunotherapies.

In summary, in our study, a novel population ofArginase11 grMDSCs was identified and isolated from PBMCsof melanoma patients. They were found to be more potent sup-pressors of T-cell proliferation than the previously describedmoMDSCs. In addition, we show that Vemurafenib reducesthe generation of MDSCs both in vitro and in vivo. These find-ings imply that the effectiveness of Vemurafenib treatment isnot only based on cell intrinsic inhibition of tumor critical on-cogenic signaling pathways but also additionally increases anti-tumor immunity by reducing immunosuppression. Potentiallycombining established immunotherapies such as Ipilimumabwith Vemurafenib induced immunomodulation will lead to asignificant augmentation of their overall effectiveness. Thismight be a promising approach to obtain long-term treatmentresponses and complete remissions in high numbers ofpatients.

AcknowledgementsResearch described in this article was supported in part by Helmholtz-Gemeinschaft Deutscher Forschungszentren (HGF) “Alliance on Immuno-therapy of Cancer” to Annette Paschen; Bastian Schilling was supported byan Interdisciplinary Grant from the University of Essen (IFORES). Theauthors thank I. Moll, N. Bielefeld, B. Gutt and C. Kochs for their help.

References

1. Boon T, Coulie PG, Van den Eynde BJ, et al.Human T cell responses against melanoma. AnnuRev Immunol 2006;24:175–208.

2. Poschke I, Mougiakakos D, Hansson J, et al.Immature immunosuppressive CD141HLA-DR-/low cells in melanoma patients are Stat3hi andoverexpress CD80, CD83, and DC-sign. CancerRes 2010;70:4335–45.

3. Nuber N, Curioni-Fontecedro A, Matter C, et al.Fine analysis of spontaneous MAGE-C1/CT7-specific immunity in melanoma patients. ProcNatl Acad Sci USA 2010;107:15187–92.

4. Filipazzi P, Valenti R, Huber V, et al.Identification of a new subset of myeloidsuppressor cells in peripheral blood of melanomapatients with modulation by a granulocyte-macrophage colony-stimulation factor-basedantitumor vaccine. J Clin Oncol 2007;25:2546–53.

5. Umansky V, Sevko A. Overcomingimmunosuppression in the melanomamicroenvironment induced by chronicinflammation. Cancer Immunol Immunother2012;61:275–82.

6. Greten TF, Manns MP, Korangy F. Myeloidderived suppressor cells in human diseases. IntImmunopharmacol 2011;11:802–6.

7. Vincent J, Mignot G, Chalmin F, et al. 5-Fluorouracil selectively kills tumor-associatedmyeloid-derived suppressor cells resulting inenhanced T cell-dependent antitumor immunity.Cancer Res 2010;70:3052–61.

8. Kao J, Ko EC, Eisenstein S, et al. Targetingimmune suppressing myeloid-derived suppressorcells in oncology. Crit Rev Oncol Hematol 2011;77:12–9.

9. Ko JS, Zea AH, Rini BI, et al. Sunitinib mediatesreversal of myeloid-derived suppressor cellaccumulation in renal cell carcinoma patients.Clin Cancer Res 2009;15:2148–57.

10. Umansky V, Sevko A. Melanoma-inducedimmunosuppression and its neutralization. SeminCancer Biol 2012;22:319–26.

11. Ostrand-Rosenberg S, Sinha P. Myeloid-derivedsuppressor cells: linking inflammation andcancer. J Immunol 2009;182:4499–506.

12. Eruslanov E, Neuberger M, Daurkin I, et al.Circulating and tumor-infiltrating myeloid cellsubsets in patients with bladder cancer. Int JCancer 2012;130:1109–19.

13. Gallina G, Dolcetti L, Serafini P, et al. Tumorsinduce a subset of inflammatory monocytes withimmunosuppressive activity on CD81 T cells.J Clin Invest 2006;116:2777–90.

14. Diaz-Montero CM, Salem ML, Nishimura MI,et al. Increased circulating myeloid-derivedsuppressor cells correlate with clinical cancerstage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy. CancerImmunol Immunother 2009;58:49–59.

15. Brandau S, Trellakis S, Bruderek K, et al.Myeloid-derived suppressor cells in the peripheralblood of cancer patients contain a subset ofimmature neutrophils with impaired migratoryproperties. J Leuk Biol 2011;89:311–7.

16. Rodriguez PC, Ernstoff MS, Hernandez C, et al.Arginase I-producing myeloid-derived suppressorcells in renal cell carcinoma are a subpopulationof activated granulocytes. Cancer Res 2009;69:1553–60.

17. Tartour E, Pere H, Maillere B, et al. Angiogenesisand immunity: a bidirectional link potentiallyrelevant for the monitoring of antiangiogenictherapy and the development of novel therapeuticcombination with immunotherapy. CancerMetastasis Rev 2011;30:83–95.

18. Smith FO, Downey SG, Klapper JA, et al.Treatment of metastatic melanoma usinginterleukin-2 alone or in conjunction withvaccines. Clin Cancer Res 2008;14:5610–8.

19. Gros A, Turcotte S, Wunderlich JR, et al.Myeloid cells obtained from the blood but notfrom the tumor can suppress T-cell proliferationin patients with melanoma. Clin Cancer Res 2012;18:5212–23.

20. Curtin JA, Fridlyand J, Kageshita T, et al.Distinct sets of genetic alterations in melanoma.N Engl J Med 2005;353:2135–47.

21. Wilmott JS, Long GV, Howle JR, et al. SelectiveBRAF inhibitors induce marked T-cell infiltrationinto human metastatic melanoma. Clin CancerRes 2012;18:1386–94.

22. Blank CU, Hooijkaas AI, Haanen JB, et al.Combination of targeted therapy andimmunotherapy in melanoma. Cancer ImmunolImmunother 2011;60:1359–71.

23. Balch CM, Gershenwald JE, Soong SJ, et al. Finalversion of 2009 AJCC melanoma staging andclassification. J Clin Oncol 2009;27:6199–206.

24. Zimmer L, Hillen U, Livingstone E, et al.Atypical melanocytic proliferations and newprimary melanomas in patients with advancedmelanoma undergoing selective BRAF inhibition.J Clin Oncol 2012;30:2375–83.

25. Therasse P, Arbuck SG, Eisenhauer EA, et al.New guidelines to evaluate the response totreatment in solid tumors. EuropeanOrganization for Research and Treatment ofCancer, National Cancer Institute of the UnitedStates, National Cancer Institute of Canada.J Natl Cancer Inst 2000;92:205–16.

26. Ugurel S, Thirumaran RK, Bloethner S, et al. B-RAF and N-RAS mutations are preserved duringshort time in vitro propagation and differentiallyimpact prognosis. PLoS One 2007;2:e236.

27. Hoechst B, Ormandy LA, Ballmaier M, et al. Anew population of myeloid-derived suppressorcells in hepatocellular carcinoma patients inducesCD4(1)CD25(1)Foxp3(1) T cells.Gastroenterology 2008;135:234–43.

Tum

orIm

mun

olog

y



28. Schilling B, Harasymczuk M, Schuler P, et al.IRX-2, a novel biologic, favors the expansion of Teffector over T regulatory cells in a human tumormicroenvironment model. J Mol Med (Berl) 2012;90:139–47.

29. Dumitru CA, Moses K, Trellakis S, et al.Neutrophils and granulocytic myeloid-derivedsuppressor cells: immunophenotyping, cellbiology and clinical relevance in humanoncology. Cancer Immunol Immunother 2012;61:1155–67.

30. Tang S, Lotze MT. The power of negativethinking: which cells limit tumor immunity? ClinCancer Res 2012;18:5157–9.

31. Ugurel S, Uhlig D, Pfohler C, et al. Down-regulation of HLA class II and costimulatoryCD86/B7-2 on circulating monocytes frommelanoma patients. Cancer Immunol Immunother2004;53:551–9.

32. Nagaraj S, Youn JI, Weber H, et al. Anti-inflammatory triterpenoid blocks immunesuppressive function of MDSCs and improvesimmune response in cancer. Clin Cancer Res2010;16:1812–23.

33. Duffy A, Zhao F, Haile L, et al. Comparativeanalysis of monocytic and granulocytic myeloid-derived suppressor cell subsets in patients withgastrointestinal malignancies. Cancer ImmunolImmunother 2013;62:299–307.

34. Raber P, Ochoa AC, Rodriguez PC. Metabolismof l-arginine by myeloid-derived suppressor cellsin cancer: mechanisms of T cell suppression andtherapeutic perspectives. Immunol Invest 2012;41:614–34.

35. Mandruzzato S, Solito S, Falisi E, et al.IL4Ralpha1 myeloid-derived suppressor cellexpansion in cancer patients. J Immunol 2009;182:6562–8.

36. Roth F, De La Fuente AC, Vella JL, et al.Aptamer-mediated blockade of IL4Ralphatriggers apoptosis of MDSCs and limitstumor progression. Cancer Res 2012;72:1373–83.

37. Sinha P, Parker KH, Horn L, et al. Tumor-induced myeloid-derived suppressor cellfunction is independent of IFN-gammaand IL-4Ralpha. Eur J Immunol 2012;42:2052–9.

38. Lechner MG, Megiel C, Russell SM, et al.Functional characterization of human Cd331

and Cd11b1 myeloid-derived suppressor cellsubsets induced from peripheral bloodmononuclear cells co-cultured with a diverse setof human tumor cell lines. J Transl Med 2011;9:90.

39. Lechner MG, Liebertz DJ, Epstein AL.Characterization of cytokine-induced myeloid-derived suppressor cells from normal humanperipheral blood mononuclear cells. J Immunol2010;185:2273–84.

40. Sumimoto H, Imabayashi F, Iwata T, et al. TheBRAF-MAPK signaling pathway is essential forcancer-immune evasion in human melanomacells. J Exp Med 2006;203:1651–6.

41. Hodi FS, O’Day SJ, McDermott DF, et al.Improved survival with ipilimumab in patientswith metastatic melanoma. N Engl J Med 2010;363:711–23.

42. Ahmadzadeh M, Rosenberg SA. IL-2administration increases CD41 CD25(hi)Foxp31 regulatory T cells in cancer patients.Blood 2006;107:2409–14.

Tum

orIm

mun

olog

y



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Vemurafenib reverses immunosuppression by myeloid derived suppressor cells