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

1Department of Dermatology, University Hospital, University Duisburg-Essen, Essen, Germany2Department 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 of

immunosuppressive cells like regulatory T-cells (Tregs) and

myeloid derived suppressor cells (MDSCs) in patients with

advanced melanoma.2–5 MDSCs suppress innate and adaptive

immunity by various mechanisms.2,4,6 Recent data suggest

that elimination of MDSCs or modulation of their function

unleashes T cell anti-tumor responses.7–10 Ko et al.9 observed

that treatment of renal cell carcinoma (RCC) patients with

sunitinib, a tyrosine receptor kinase inhibitor, decreases

MDSC frequency and suppressive function. Specific

elimination of MDSCs, however, is limited by an apparently

heterogenic phenotype of these cells.6,11

In general, human MDSCs can be divided into granulo-

cytic (grMDSCs) and monocytic MDSCs (moMDSCs). This

discrimination is mainly based on morphological and pheno-

typical characteristics of grMDSCs and moMDSCs. Although

sharing the expression of common myeloid markers like

CD11b, grMDSCs are usually found to be CD151 while

moMDSCs are CD152/dim but can express CD14.2,12–16 The

phenotypical and functional differences between these two

populations are not well defined. Both subsets were reported

to use Arginase 1, reactive oxygen species (ROS) and TGF-b

to suppress innate and adaptive immunity and to promote

tumor growth by production of pro-angiogenic mole-

cules.2,4,6,17 It is widely believed that different tumors induce

distinct 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 to

suppress T-cell proliferation and Interferon-g production

while corresponding CD141HLA-DR1 cells exhibited no

suppressive properties. Although CD66b1 grMDSCs have

been described in patients with RCC,16 head and neck cancer,

lung cancer as well as urogenital cancer,18 no corresponding

MDSC subpopulation has been identified in melanoma.

Recently, Poschke et al.2 confirmed the accumulation of

CD141HLA-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: 00, 000–000 (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 and

is further complicated by the observation of Gros et al.19 who

failed to detect an elevated frequency of MDSCs in the pe-

ripheral blood of melanoma patients. This work also reported

that 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 needs

to be further clarified.

Although the mechanisms of MDSC induction by malig-

nant melanoma are still unknown, a potential influence of

effective tumor therapies on MDSC populations could be pre-

sumed. Recently, treatment of melanoma patients with

advanced disease with Vemurafenib led to impressive objec-

tive response rates. Vemurafenib is a highly specific inhibitor

of mutant B-RAFV600E, a mutation leading to constitutive

activation of the MAP Kinase pathway and found in �60%

of cutaneous melanoma.20 Despite high rates of objective

responses in patients with advanced melanoma treated with

Vemurafenib, tumors eventually become resistant. The me-

dian response duration is 6 months of therapy and long-term

responses are observed rarely.21 Vemurafenib might reduce

tumor growth not only through cell intrinsic mechanisms but

also reduce cytokine-driven induction of MDSCs in the tu-

mor microenvironment. Impairing melanoma mediated

immunosuppression would provide an ideal setting for the

successful implementation of additional immunotherapies

known to induce durable tumor regression.15,21,22 To test this

hypothesis, we explored the nature of MDSCs in patients

with melanoma and investigated the impact of Vemurafenib

on MDSCs ex vivo and in vitro.

Material and Methods

Blood samples

Peripheral blood was obtained from 25 patients with meta-

static melanoma (Stage IV, AJCC 200923), 16 patients with

Stage I–III Melanoma (AJCC) and 10 age and sex matched

healthy donors (HD). Blood was drawn before therapy in

patients with active disease (AD). All subjects with no clinical

evidence of disease (NED) were tumor free for at least 3

months at the time of phlebotomy and signed an informed

consent approved by the local ethics committee. Patients

were seen at the Department of Dermatology Essen between

December 2011 and January 2013. Clinicopathological char-

acteristics including B-RAF status in Stage IV patients are

listed in Table 1. B-RAF status in melanoma tissue was deter-

mined by Sanger sequencing.24 When no information is given

in Table 1, B-RAF status was not determined. In patients

undergoing Vemurafenib treatment, clinical course of the dis-

ease was determined every 4 weeks, computed tomography

(CT) scans of the chest and abdomen were performed every

8 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 for

MDSCs were obtained up to 2 weeks before treatment with

Vemurafenib was commenced (Visit 0, baseline). Consecutive

specimens were obtained 4–8 weeks (Visit 1) and 12–16

weeks (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 well

as anti-CD45-APC-H7 and anti-CD15-Pacific blue antibodies

from 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 obtained

from 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 from

Beckman 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 line

Ma-Mel-51 was established and maintained as described.26

Cell cultures were tested monthly for mycoplasma infection

and 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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2 Vemurafenib and MDSCs in melanoma


Generation of conditioned medium

For the generation of conditioned medium (CM), an equal

number 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 by

Roche, Grenzach-Wyhlen, Germany) or vehicle was added.

After 72 hr, supernatants were harvested and contaminating

cells were removed by centrifugation.

Surface and intracellular staining for flow cytometry

For surface staining, cells were incubated with antibodies for

30 min at 4�C. After washing, cells were fixed and permeabil-

ized using a commercially available kit (eBiocience, San

Diego, CA) according to the manufacturer’s instructions.

Without washing, cells were stained intracellularly for 30 min

at room temperature. All antibodies were pretitrated on

freshly harvested and activated PBMCs to determine optimal

working dilutions. Viability was assessed by Aqua Viability

Dye (Detected on FL-10, BD). Samples were acquired on a

Beckman 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 based

separation (MACS technology, Miltenyi, Bergisch-Gladbach,

Germany), a scheme of the experimental strategy is provided

as supporting information Figure 1. First, CD31 cells were

positively selected on LS columns after incubation with anti-

CD3 coated magnetic beads according to the manufacturer’s

instructions. Next, CD32 PBMCs were incubated with an

anti-CD66b-PE mAb for 20 min at 4�C. After washing, cells

were incubated with anti-PE coated magnetic beads according

to 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 column

afterward. From the remaining CD32CD66b2 PBMCs,

CD141 cells were negatively selected using the monocyte iso-

lation kit II (Miltenyi) according to the manufacturer’s

instructions. In a last step, CD32CD66b2CD141 cells were

separated into HLA-DR1 and HLA-DR2/low cells using

HLA-DR coupled magnetic beads (Miltenyi). The purity and

viability 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 MACS

and subsequent fluorescent activated cell sorting (FACS).

Briefly, CD141 cells were positively selected on MS columns

using anti-CD14 coated beads. CD141 cells were then stained

with anti-HLA-DR-PE and anti-CD14-PerCP5.5.

CD141HLA-DR1 and CD141HLA-DR2/low and sorted on

an ARIA I (BD) as described before.27

CFSE-based suppression assay

Suppression of proliferation of CD31 T-cells was performed

as previously described.28 Briefly, CD31 responder cells (RC)

labeled with 2mM carboxyfluorescein succinimidyl ester

(CFSE, Invitrogen, Carlsbad, CA) were cultured in complete

RPMI1640 medium in the presence of anti-CD3/anti-CD28

coated beads (Miltenyi, bead to T-cell ratio 1:2) in wells of

96-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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melanoma patients or harvested from in vitro cultures were

added to the RC at different ratios and cultures were incu-

bated in 5% CO2 in air at 37�C. Flow cytometric analyses of

CFSE 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 from

either untreated or Vemurafenib-treated Ma-Mel-51 cells on

six-well plates. PBMCs cultured in complete RPMI16406 1mM

Vemurafenib served as an additional control. PBMCs were har-

vested after 72 hr and used immediately for phenotypical and

functional characterization of moMDSCs. RC for CFSE-based

suppression assays were isolated from autologous PBMCs kept

in complete RPMI1640 until usage.

Statistical analysis

Data were analyzed by SPSS (IBM) using two-way analysis of

variance (ANOVA). p values< 0.05 were considered significant.

Results

Detection 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 patients

with melanoma.29,30 Using this panel, we were able to detect

moMDSCs (CD141HLA-DR2/low) within PBMCs of patients

with advanced melanoma. In addition, we found a population

of CD66b1Arginase11 cells in the peripheral circulation of

Stage IV melanoma patients, suggesting that these cells might

be CD66b1CD11b1CD331Arginase11CD142CD16low

grMDSCs as described by Rodriguez et al.16 in RCC (Fig. 1).

Further analysis of moMDSCs and grMDSCs found in

patients with malignant melanoma revealed a partial pheno-

typical overlap. Monocytic MDSCs were found to be CD451

Lin2HLA-DR2/lowCD141CD15dimCD66b2CD331CD11b1

Arginase12CD162/low. In contrast, granulocytic MDSCs

were CD451Lin2HLA-DR2CD142CD151CD66b1CD331

CD11b1Arginase11CD162/low (Fig. 1). Thus, moMDSCs

and grMDSCs in patients with advanced melanoma are two

distinct populations. Although moMDSCs have been

described by others, grMDSCs represent a previously

unrecognized subpopulation of human MDSCs found in

patients 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 patients

might 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 investigate

this, we performed a functional analysis of both moMDSCs and

grMDSCs. First, we developed an isolation strategy allowing the

separation of both populations from the same donor as described

in materials and methods. Importantly, our isolation approach

excludes cross-contamination of moMDSCs and grMDSCs.

When used in CFSE-based suppressor assays, both moMDSCs

and 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 and

grMDSCs (Figs. 2a and 2b). When comparing the suppressive

capacity of moMDSCs and grMDSCs, we found grMDSCs to be

more 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 PBMCs

from melanoma patients to those obtained from HD. In

Stage IV melanoma patients with AD, defined as having clin-

ical or radiological detectable metastasis, we found an

increased frequency of both moMDSCs and grMDSCs com-

pared to HD (Figs. 3a and 3b). Lower Stage (I–III) melanoma

patients with an AD at the time of blood draw did not differ

in 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 within

PBMCs of Stage I–III melanoma patients with NED at the

time of phlebotomy. This also did not differ significantly

from 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 cells6 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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Schilling et al. 5


samples from Stage IV patients with NED at the time of

blood draw. Six of these patients were rendered tumor-free

by surgery while one showed a complete response to Ipilimu-

mab. Interestingly, the frequency of grMDSCs and

moMDSCs of these patients was not increased compared to

HD 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 in

Figure 3, the frequency of moMDSCs showed a similar tend-

ency suggesting that successful treatment of melanoma

patients 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 patients

with 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 with

objective response, four patients with partial response (PR:

circle, hexagon, square and inverted triangle), one patient

with stable disease (SD: triangle) to treatment showed a

decline in moMDSC frequency (% decrease as compared to

visit 0: 10.5% to 27.5%, mean 18.4%). In one donor

(diamond) showing stable disease (SD) at Visit 1, an increase

of moMDSCs was observed (% increase as compared to Visit

0: 13.7%). In those patients still showing objective response

to 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 in

moMDSC frequency (% increase as compared to Visit 1:

8.3%). In contrast, the patient showing SD and a decrease in

moMDSC frequency at the time of Visit 1 (triangle), had PR

of 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 moMDSCs

above baseline level (19%).

Although the frequency of moMDSCs was strongly associ-

ated with the clinical course of melanoma, the behavior of

grMDSC frequency in the same patients was diverse (Fig. 4c).

In those patients who had PD at the time of Visit 2 (square

and diamond), grMDSCs showed the same course as

moMDSCs. In addition, in one of the four patients showing

PR at the time of visit 2 (triangle) a steady decline in the

grMDSC frequency was observed. However, in the other

three 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 inverted

triangle).

Vemurafenib inhibits MDSC generation in an in vitro model

of the melanoma microenvironment

To further explore the mechanisms of MDSC accumulation

in patients with advanced melanoma, we established a

model of the melanoma microenvironment using CM from

primary 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 using

CM from Ma-Mel51, flow cytometry analysis revealed the

generation of a cell population with a moMDSC phenotype

after 72 hr of culture. As shown in Figures 5a and 5b, we

found a significant induction of cells with a moMDSC phe-

notype in the presence of Ma-Mel51 CM compared to

RPMI1640 treatment. Dilution of Ma-Mel-51 CM with

RPMI1640 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 PBMCs

with a viability dye showed no difference in the frequency

of apoptotic cells from in vitro cultures in CM or

RPMI1640 (data not shown). Next, we tested moMDSCs

from 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 culture

did not (Fig. 5c). Thus, moMDSCs induced in our in vitro

model appear to be fully functional. Next, we tested the

influence of Vemurafenib on the generation of moMDSCs.

The frequency of moMDSCs generated in the presence of

CM taken from short-term Vemurafenib treated Ma-Mel-51

cells was significantly lower as compared to controls having

received CM from untreated Ma-Mel-51 (Figs. 5a and 5b).

Vemurafenib added directly to in vitro cultures containing

CM from untreated Ma-Mel51 had no impact on the gener-

ation of moMDSC. Thus, Vemurafenib inhibits the release

of 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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Discussion

In our study, we performed a detailed analysis of the nature

of MDSCs in patients with malignant melanoma and tested

the 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 multicolor

flow cytometry, we identified two distinct populations of

MDSCs in patients with advanced melanoma. Besides CD141

HLA-DR2/low moMDSCs which were previously described in

melanoma 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 the

first report examining the impact of Vemurafenib on MDSC

frequencies in vitro and in vivo.

Both monocytic and granulocytic myeloid cells analyzed

in our study can be considered MDSCs as they suppressed

autologous T-cell proliferation, although to different extents

and most likely via different mechanisms. MoMDSCs and

grMDSCs 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 events6SEM 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 cells6SEM 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 of

Class-II molecules. This means that using HLA-DR, CD11b

and CD33 for enumeration and isolation of human MDSCs

will lead to cross-contamination of these distinct subsets.14,32

To prevent this, we isolated these cells based on the markers

CD14, HLA-DR and CD66b (see Supporting Information

Fig. 1). A functional analysis comparing grMDSCs and

moMDSCs 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 a

comparative analysis of grMDSCs and moMDSCs in patients

with advanced gastrointestinal malignancies. The grMDSCs

and moMDSCs identified in our study were phenotypically

identical to those found in our study,33 despite CD15 instead

of CD66b being used for isolation. However, it can be

assumed 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 in

melanoma patients express Arginase1 on the protein level

and have significantly greater ability to suppress autologous

T-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 a

major role in regulating T-cell function in cancer patients.34

By flow cytometry, we did not detect Arginase1 protein

expression in moMDSCs. In contrast to our findings, Poschke

et al. reported an elevated expression of Arginase1 mRNA in

moMDSCs from melanoma patients as compared to healthy

donors. 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, an

Arginase inhibitor. This treatment would have affected

enzyme activity in both grMDSCs and moMDSCs. Future

studies applying isolated cell populations instead of bulk or

MDSC-depleted PBMCs will be needed to fully delineate the

mechanisms of T-cell suppression used by grMDSCs and

moMDSCs.

Although our study further clarified the nature of MDSCs

in patients with metastatic melanoma, their role in disease

progression remains complex. Accumulation of CD141HLA-

DR2/low cells in the peripheral blood of patients with

advanced melanoma was first reported by Ugurel et al.31

Later, CD141HLA-DR2/low cells were isolated by Filipazzi

et al.4 and characterized as MDSCs by showing suppressive

effects on T-cell proliferation while the corresponding

CD141HLA-DR1 population did not. Conflicting with our

study and our own data, Gros et al.19 recently reported that

both CD141HLA-DR2/low and CD141HLA-DR1 cells iso-

lated from patients with advanced melanoma have suppres-

sive properties. These authors also failed to observe an

increased frequency of CD141HLA-DR2/low in the peripheral

blood 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 blood

rather than PBMCs. As therapeutic interventions might alter

myeloid cell frequency and function,9 the discrepancies found

could also be due to the fact that Gros et al. analyzed sam-

ples from previously treated rather than therapy-na€ıve

patients with metastatic melanoma. Yet another study by

Mandruzzato 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 patients

still needs to be confirmed and the role of IL-4Ra in the dif-

ferentiation and function of murine MDSCs has recently

been questioned.13,36,37

Besides identifying an additional subpopulation of MDSCs

and comprehensively analyzing their phenotype and function,

we also evaluated the impact of Vemurafenib on MDSCs in

patients with advanced melanoma. We were able to show an

effect of Vemurafenib on MDSCs in the peripheral blood of

patient with metastatic melanoma, fitting the results reported

by Poschke et al.,2 where moMDSC frequency was associated

with disease course in Stage IV melanoma patients. In all

patients responding to Vemurafenib treatment, the moMDSC

frequency declined over time. The grMDSC frequency

changes noted in the same patients were less consistent. In

one of the three patients showing partial response to Vemur-

afenib, grMDSC frequency increased during treatment. For

all other patients, the grMDSC frequency changes corre-

sponded to the observed moMDSC frequency alterations.

Potentially the effects of Vemurafenib on the secretion of

molecules necessary for grMDSC induction or survival vary

significantly from patient to patient. As grMDSCs were previ-

ously not recognized in melanoma, the mechanisms of their

induction are still unknown. It would stand to reason that

the mechanisms and molecules responsible for grMDSC and

moMDSC induction are distinct.

To further analyze the effect of Vemurafenib on MDSCs,

we established an in vitro model of moMDSC induction. In

this model, PBMCs from HD cultured in CM from a primary

melanoma 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 cytokines

produced by human tumors, including melanoma, that are

involved in the differentiation of moMDSCs: GM-CSF, IL-

1b, IL-6, TNF-a, VEGF as well as FLT3L and TGF-b.38,39 To

what extent these or additional soluble factors are involved in

moMDSC induction in our model will have to be addressed

in future studies. Our finding that CM from Vemurafenib-

treated melanoma cells loses its ability to induce moMDSCs

strongly supports a critical role for soluble factors. RNA-in-

terference targeting B-RAFV600E has been shown to decrease

the production of VEGF, IL-6 and IL-10 by melanoma cell

lines in vitro.40 Taken together, these data support that

Vemurafenib alters the secretion profile of melanoma cells

from an immunosuppressive to a nonimmunosuppressive

composition. This fits well with the observation of Wilmott

TumorIm

munology

Schilling et al. 9


et al.21 who reported an increased tumor infiltration by

CD41 and CD81 lymphocytes in patients treated with selec-

tive B-RAF inhibitors. To further explore the impact of

Vemurafenib on myeloid cells in the melanoma microenvir-

onment, tumor samples from patients undergoing treatment

could be screened for alterations of the infiltrating myeloid

cell populations.

Both our data and work by Sumimoto et al.40 indicate

that targeting B-RAFV600E alters the cytokine secretion of

melanoma cells, thereby reducing immune evasion. Although

clinical response to Vemurafenib is observed in the majority

of 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 of

patients.18,41 Effective induction of anti-tumor immunity by

immunotherapy might be impeded by immunosuppressive

Tregs and MDSCs accumulating in the peripheral circulation

and the tumor microenvironment of melanoma patients. This

could explain the overall low response rates.4,42 Therefore,

reducing immunosuppression by MDSCs with Vemurafenib

might augment the efficacy of future immunotherapies.

In summary, in our study, a novel population of

Arginase11 grMDSCs was identified and isolated from PBMCs

of melanoma patients. They were found to be more potent sup-

pressors of T-cell proliferation than the previously described

moMDSCs. In addition, we show that Vemurafenib reduces

the generation of MDSCs both in vitro and in vivo. These find-

ings imply that the effectiveness of Vemurafenib treatment is

not only based on cell intrinsic inhibition of tumor critical on-

cogenic signaling pathways but also additionally increases anti-

tumor immunity by reducing immunosuppression. Potentially

combining established immunotherapies such as Ipilimumab

with Vemurafenib induced immunomodulation will lead to a

significant augmentation of their overall effectiveness. This

might be a promising approach to obtain long-term treatment

responses and complete remissions in high numbers of

patients.

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 by

an Interdisciplinary Grant from the University of Essen (IFORES). The

authors thank I. Moll, N. Bielefeld, B. Gutt and C. Kochs for their help.

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Vemurafenib reverses immunosuppression by myeloid derived suppressor cells

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