Tumor Biology and Immunology

Melanoma Extracellular Vesicles GenerateImmunosuppressive Myeloid Cells byUpregulating PD-L1 via TLR4 SignalingViktor Fleming1,2,3, Xiaoying Hu1,2, C�eline Weller1,2, Rebekka Weber1,2,3,Christopher Groth1,2, Zeno Riester1,2, Laura H€user1,2, Qian Sun1,2, Vasyl Nagibin1,2,Carsten Kirschning4, Vincenzo Bronte5, Jochen Utikal1,2, Peter Altevogt1,2, andViktor Umansky1,2

Abstract

Tumor cell–derived extracellular vesicles (EV) convert nor-mal myeloid cells into myeloid-derived suppressor cells(MDSC), inhibiting antitumor immune responses. Here, weshow that EV from Ret mouse melanoma cells upregulate theexpression of programmed cell death ligand 1 (PD-L1) onmouse immature myeloid cells (IMC), leading to suppressionof T-cell activation. PD-L1 expression and the immunosup-pressive potential of EV-generated MDSC were dependent onthe expression of Toll-like receptors (TLR). IMC from Tlr4�/�

mice failed to increase T-cell PD-L1 expression and immuno-suppression with Ret-EV treatment, and this effect was depen-dent on heat-shock protein 86 (HSP86) as HSP86-deficientRet cells couldnot stimulatePD-L1expressiononnormal IMC;

IMC from Tlr2�/� and Tlr7�/� mice demonstrated similarresults, although to a lesser extent. HSP86-deficient Ret cellsslowed tumor progression in vivo associated with decreasedfrequency of tumor-infiltrating PD-L1þCD11bþGr1þ MDSC.EV from human melanoma cells upregulated PD-L1 andimmunosuppression of normal monocytes dependent onHSP86. These findings highlight a novel EV-mediated mech-anism of MDSC generation from normal myeloid cells, sug-gesting the importance of EV targeting for tumor therapy.

Significance: These findings validate the importance ofTLR4 signaling in reprogramming normal myeloid cells intofunctional myeloid-derived suppressor cells.

IntroductionMalignant melanoma is characterized by a rapid progression,

metastasis to distant organs, and poor survival of patients (1).Despite the therapeutic success achieved by the immune check-point inhibitors such as antibodies targeting cytotoxic T-lym-phocyte-associated protein 4 (CTLA-4) and programmed celldeath protein 1 (PD-1), most patients fail to respond totreatment (2). One of the major reasons for this poor responserate is the development of immunosuppressive tumor micro-

environment (TME), in which myeloid-derived suppressorcells (MDSC) play a crucial role (3–6). They accumulate inpreclinical melanoma mouse models and melanoma patientsand strongly inhibit antitumor functions of T and naturalkiller (NK) cells, promoting tumor progression (4–8). MDSCrepresent a heterogeneous population of monocytic and poly-morphonuclear cells that are derived from immature myeloidcells (IMC) and activated by soluble inflammatory factorsconstantly produced by tumor and host cells (3–8). One ofthe major mechanisms of MDSC-mediated immunosuppres-sion is linked to the upregulation of programmed cell deathligand 1 (PD-L1) interacting with its receptor PD-1 expressedon tumor-infiltrating T cells (3–5, 9).

It has been recently demonstrated that MDSC could be alsoderived from IMC or differentiated myeloid cells by the exposureto extracellular vesicles (EV) secreted by tumor cells (10–14). Theterm EV is currently applied to define all kinds of vesicles releasedby various cell types including erythrocytes, platelets, leukocytes,and cancer cells (15). The process of EV secretion is particularlyactive in proliferating cells such as cancer cells (16). Small vesicles(50–150 nm) are released from the cell surface (microvesicles) orfrom the endosomal system (exosomes). By the fusion of lateendosome with the plasma membrane, exosomes are secretedinto the extracellular space (17, 18). In contrast, apoptotic vesicles(bodies) are larger (1,000–5,000nm)and canbe separatedby sizeand density from smaller vesicles (17, 18). Depending on the cellorigin, EV contain different biologically active molecules such asproteins, mRNA, miRNA, and lipids and are considered as med-iators of intercellular communication (14, 19, 20).

1Clinical Cooperation Unit Dermato-Oncology, German Cancer Research Center(DKFZ), Heidelberg, Germany. 2Department of Dermatology, Venereology andAllergology, University Medical Center Mannheim, Ruprecht-Karl University ofHeidelberg, Mannheim, Germany. 3Faculty of Biosciences, Ruprecht-Karl Uni-versity of Heidelberg, Heidelberg, Germany. 4Institute for Medical Microbiology,University Hospital Essen, Essen, Germany. 5Department of Medicine, VeronaUniversity Hospital, Verona, Italy.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

V. Fleming and X. Hu equally contributed to this article.

Corresponding Author: Viktor Umansky, German Cancer Research Center(DKFZ) and University Hospital Mannheim, Im Neuenheimer Feld 280, 69120Heidelberg, Germany. Phone: 49-621-383-3773; Fax: 49-621-383-2163; E-mail:v.umansky@dkfz.de

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�2019 American Association for Cancer Research.

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Although the conversion of IMC into immunosuppressiveMDSC by tumor-derived EV was already described, themolecularmechanism underlying MDSC induction and EV involvement inthe generation of the TME is poorly understood. We suggestedpreviously that both the EV-mediated signaling via myeloid cellreceptors and the delivery of biologically active molecules by EV(cargo function) might be important in this process (8, 20). Here,we investigated the molecular mechanisms of interactionbetween normal myeloid cells (IMC and monocytes) and mela-noma-derived EV in mouse and human setting, leading to thegeneration of immunosuppressive MDSC. Murine EV were iso-lated from the Ret melanoma cell line (Ret-EV) that was estab-lished from skin melanomas isolated from RET transgenicmice (21). This model closely resembles human melanoma interms of clinical development and tumor–stroma interac-tions (22). We found that Ret-EV induced the upregulationof PD-L1 expression on bone marrow (BM)–derived murineIMC and immortalized myeloid suppressor cell line MSC-2 (23). EV-treated IMC strongly suppressed the activation ofCD8þ T cells, which was restored by antibodies against PD-L1blocking the PD-1/PD-L1 axis. Importantly, the observed effectswere mediated by Toll-like receptor (TLR) signaling (because Ret-EV failed to induce immunosuppressive capacity of IMC fromTlr4�/�, Tlr2�/�, and Tlr7�/� mice) as well as by the heat-shockprotein 86 (HSP86) expressed on Ret-EV. Moreover, normalhuman CD14þ monocytes exposed to EV produced by humanmelanoma cells also displayed elevated PD-L1 expression in aHSP86/TLR4-dependent manner and acquired a strong immu-nosuppressive capacity, blocking T-cell activation. Our resultssuggest that EV-induced PD-L1 expression mediates the conver-sion of normal myeloid cells into immunosuppressive MDSC intumor-bearing hosts via TLR signaling, indicating a promisingtarget for cancer immunotherapy.

Materials and MethodsMice

Healthy C57BL/6 mice (6–8 weeks) were purchasedfrom Charles River. Tlr4�/�, Myd88�/�, and Myd88�/�/Trif�/�

C57BL/6 mice were delivered from University of Essen(Germany). Tlr2�/� and Tlr7�/� C57BL/6 mice were kindlyprovided by Beatrix Schumak (University of Bonn, Germany)and Stefan Bauer (University of Marburg, Germany). Micewere kept under pathogen-free conditions in the animal facilityof the University Medical Center (Mannheim, Germany).Animal studies have been conducted in accordance with anInstitutional Animal Care and Use Committee.

Cell cultureMurine Ret melanoma cell line was established from skin

melanomas isolated from RET transgenic mice (22) and culturedin RPMI-1640 (Gibco) supplemented with 10% heat-inactivatedFBS (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich).Human melanoma cell lines HT-144 and SK-MEL-28 obtainedfrom the ATCC were cultured in DMEM supplemented with0.1 mmol/L b-mercaptoethanol (both from Gibco), 10% heat-inactivated FBS, and 1% penicillin/streptomycin. Immortalizedmyeloid suppressor cell lines MSC-1 and -2 (23) were kindlyprovided by Stefano Ugel (University of Verona, Italy) and grownin RPMI-1640 supplemented with 10 mmol/L sodium pyruvate(Gibco), 10% heat-inactivated FBS, and 1% penicillin/strepto-

mycin. All cell lines were maintained under 5% CO2 at 37�C androutinely tested for Mycoplasma contamination using the Myco-plasma Detection Kit for Conventional PCR (Minerva Biolabs).Cells were at passages not greater than sevenbetween thawing anduse in the described experiments.

Reagents and antibodiesLentiviral shRNA plasmid DNA for stable knockdown of

HSP86 and Mission TRC pLKO.5-puro nonmammalianshRNA control plasmid, as well as KNK-437 (an inhibitor ofinducible HSP), actinomycin D, Bay11-7082 (an inhibitor ofNF-kB), and 5-(N,N-Dimethyl) amiloride hydrochloride(DMA) were purchased from Sigma-Aldrich. FcR BlockingReagent was provided by BD Biosciences. Dynabeads mouseand human T-Activator CD3/CD28 were from Thermo FisherScientific. Anti-human CD14 MicroBeads and CD8þ T cellIsolation Kit were purchased from Miltenyi Biotec. Anti-mouse mAbs CD9, PD-L1, and isotype control mAbs (IgG2a)were obtained from Thermo Fisher Scientific; CD81, p65,phospho-p65 (S536), STAT3, p-STAT3 (Y705), and calreticulinwere provided by Cell Signaling Technology; Histon H3and GAPDH were from Biolegend; ALIX were from SantaCruz Biotechnology. Fluorochrome-conjugated anti-mousemAbs CD11b-APC-Cy7, Gr1-PE-Cy7, and PD-L1-BV421 werepurchased by BD Biosciences. Anti-human mAbs PD-L1(405.9A11; Cell Signaling Technology) and ALIX were provid-ed by Cell Signaling Technology; GAPDH and HSP90 wereobtained from Biolegend; CD9 were from BD Biosciences;CD81 from Invitrogen. Anti-human TLR2- and TLR4-neutral-izing mAbs and TLR agonists (LPS, PAM3CSK4, and R848)were obtained from InvivoGen. Fluorochrome-conjugated anti-human mAbs CD14-FITC and PD-L1-PE-Cy7 were from BDBiosciences.

Isolation of murine cardiac fibroblastsFibroblasts were obtained as previously described (24).

Briefly, mouse hearts depleted from blood and mechanicallydisrupted in cold Hank's Balanced Salt Solution containing 100U/mL collagenase and 0.1% trypsin were added followed by aconstant shaking at 37�C for 10 minutes. After centrifugation at300 g, 4�C for 5 minutes, cells were resuspended in DMEM/F12medium (Gibco), containing 10% FBS and 1% penicillin/streptomycin, and plated into cell culture dishes. Two hourslater, only fibroblasts were adhered, and the medium waschanged.

Isolation of EV secreted by murine and humanmelanoma cellsas well as mouse fibroblasts

EV secreted by these cell types were isolated as describedbefore (25, 26). Briefly, serum-free conditioned mediumfrom cultured tumor cells or fibroblasts was sterile filteredthrough 0.22 mmol/L Steritops (Merck Millipore) followed bysize exclusion filtration through Amicon-Ultra-15, 100 kDa(Merck Millipore) at 3,500 g for 30 minutes. EV were pelletedfrom supernatants by ultracentrifugation at 100,000 g for 90minutes (Sorvall Discovery) and resuspended in sterile PBS. Thenumber/size distribution of particle was analyzed byNTA (Nano-sight). For this, EV samples were prepared at the concentration 5ng/mL, and particles weremeasured 5 times for 1minute. Each EVpreparation was tested for endotoxins by limulus amebocytelysate assay (Thermo Fisher Scientific).

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Isolation of EV from melanoma patientsEV were isolated from patients' plasma after informed

written consent (ethics committee approval 2010-318M-MA) asdescribed (27). Briefly, heparinized blood sampleswere subjectedto the density gradient centrifugation using Biocoll (Biochrom).After removal of peripheral blood mononuclear cells (PBMC),plasma was collected, aliquoted, and stored at �80�C. For EVisolation, plasma was thawed, diluted with cold PBS, and filteredthrough a 0.22 mmSyringe Filter (Merck). The filtered plasmawascentrifuged at 10,000 g for 30minutes to remove debris and thenpelleted by ultracentrifugation at 100,000 g for 70 minutes. Thepellet was resuspended in sterile-filtered PBS and frozen at�20�Cuntil use. In some experiments, plasma was depleted from EV byultracentrifugation at 100,000 g.

Cell proliferation assayRet melanoma cells with stably knocked down HSP86

(shHSP86) or treated with scrambled sequence shRNA construct(shSCR) were seeded in 96-well plates (Gibco) at a density of2,500 cells/well. Alamar blue (10% of the culture medium vol-ume) was added after cell attachment for 4 hours followed bythe measurement of fluorescence at an emission wavelength of535 nmol/L and an excitation wavelength of 590 nmol/L using aSpectraMax M5 microplate reader (Tecan Infinite F200 PRO).Cells were incubated further for 24, 48, and 72hours; Alamar bluewas added for 4 hours before the end of each time point, and thefluorescence was measured.

Electron microscopyThe analysis was performed as described before (27). Briefly,

Ret-EV were stained with anti-CD81 mAbs (dilution 1:50) fol-lowed by the treatment with Protein A-Gold (10 nm) kindlyprovided by George Posthuma (University Medical CenterUtrecht, Utrecht, the Netherlands). Images were taken with theelectron microscope EM910 (Carl Zeiss) at 80 kV and wereregistered with a CCD-Camera (TRS-system, Tr€ondle) using themanufacturer's software ImageSP.

Isolation and culture of CD11bþGr1þ IMC in vitroCD11bþGr1þ cells were isolated from the BM of healthy 6- to

8-week-old C57BL/6 mice using MDSC Isolation kit (MiltenyiBiotec) with the purity above 90%. Note that 106 cells werecultured in RPMI-1640 supplemented with 10% FBS depletedfrom EV, 10 mmol/L sodium pyruvate (Gibco), 1% penicillin/streptomycin, and 0.05 mmol/L b-mercaptoethanol with or with-out EV (50 mg/mL) in a total volume of 0.1 mL for 16 hoursfollowed by various analyses.

Isolation and culture of human CD14þ monocytesBuffy coats isolated from healthy donors were purchased from

theGermanRedCross Blood Service BadenW€urttemberg-Hessen.PBMC were isolated by density gradient centrifugation usingBiocoll (Biochrom). Then, CD14þ monocytes were sorted byanti-human CD14 MicroBeads according to the manufacturer'sprotocol. Monocytes (2 � 105) were cultured in RPMI-1640supplemented with 10% FBS depleted from EV, 10 mmol/Lsodium pyruvate (Gibco), 1% penicillin/streptomycin, and0.05 mmol/L b-mercaptoethanol with or without EV (15 mg/mL)in a total volume of 0.1 mL for 16 hours followed by FACSanalysis.

Coculture experimentsEffects of EV derived from Ret melanoma cells or cardiac

fibroblasts were evaluated using a transwell system. Note that2� 105 cells were incubated for 24 hours at 37�Cwith or without15 mmol/L DMA. After 24 hours, IMC were added to the upperchamber of a polycarbonate Transwell culture insert (Costar)witha pore size of 0.4 mm (Sarstedt) and cocultured for another24 hours. Then, IMC were analyzed by flow cytometry.

Flow cytometryCells were treated with FcR Blocking Reagent and stained with

mAbs for 30 minutes at 4�C. Acquisition was performed bymulticolor flow cytometry using FACSCanto II with FACSDiva6.0 software (BD Biosciences). Dead cell exclusion was based onthe scatter profile. The compensation was performed with BDCompBeads set (BD Biosciences) using the manufacturer'sinstructions. FlowJo software (Tree Star) was used to analyze atleast 100,000 events.

ImmunoblottingMurine IMC or immortalized myeloid suppressor cells

MSC-1 and -2 were incubated with Ret-EV (50 mg/mL) for16 hours in a total volume of 0.1 mL. In some experiments,MSC-2 cells were treated with Ret-EV in the presence of 1 mg/mLactinomycin D or preincubated for 3 hours with the NF-kBinhibitor Bay (5 or 10 mmol/L). Then, cell pellets were solu-bilized for 30 minutes on ice in lysis buffer (250 mmol/LNaCl, 50 mmol/L HEPES, 0.5% NP-40, 10% glycerol, 2 mmol/LEDTA, 10 mmol/L NaF, 1 mmol/L Na-orthovanadate, 1 mmol/LPMSF, and 10 mg/mL of each leupeptin and aprotinin) orradioimmunoprecipitation assay buffer (Merck) containing 1%protease inhibitor (Calbiochem). Lysates were cleared by centri-fugation and boiled with reducing or nonreducing SDSloading buffer. Protein concentrations were determined usingPierce BCA Protein Assay Kit (Thermo Fisher Scientific). Sampleswere separated on SDS-PAGE gels and transferred to polyvinyli-dene fluoridemembranes using iBlot2 (Thermo Fisher Scientific).After blocking with 3% BSA in Tween-20/TBS, membraneswere treated with primary antibodies followed by horseradishperoxidase–conjugated secondary antibody and ECL detection(Thermo Fisher Scientific).

In vitro CD8þ T-cell proliferation and IFNg secretion assayNote that 105 CD11bþGr1þ IMC from the BM of wild-type or

TLR-deficient mice were treated with 50 mg/mL EV in a totalvolume of 0.1 mL for 16 hours at 37�C. Then cells were washedtwice at 300 g for 5 minutes to remove the rest of the EV andtreated with PD-L1–neutralizing or isotype control mAbs for 15minutes followed by centrifugation at 300 g for 5 minutes. CD8þ

T cells were isolated from spleens of healthy C57BL/6 mice usingthe na€�ve CD8þ T cell Isolation Kit (Miltenyi Biotec) according tothe manufacturer's instructions and labeled with 1 mmol/L car-boxyfluorescein succinimidyl ester (CFSE). After washing CFSEout by the centrifugation at 300 g for 5 minutes, cells werestimulated with Dynabeads mouse T-Activator CD3/CD28(Thermo Fisher Scientific) and incubated with CD11bþGr1þ

cells at indicated ratios for 72 hours. T-cell proliferation wasevaluated by CFSE dilution by flow cytometry. In addition, IFNgsecretion was evaluated in the coculture supernatants using theMouse IFN-g ELISA MAX Standard kit (Biolegend) according tothe manufacturer's instructions.

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In vitro proliferation assay of human T cellsNote that 2� 105CD14þmonocytes fromhealthy donorswere

treated with 0.5 to 50 mg/mL EV in a total volume of 0.2 mLfor 16 hours at 37�C. Then cells were washed twice at 300 g for5 minutes to remove the rest of EV. CD8þ T cells were obtainedfrom healthy donors using the CD8þ T cell Isolation Kit, culturedin X-VIVO 20 serum-free medium (Biozym) for 16 hours at 37�Cand labeled with 1 mmol/L CFSE. After washing CFSE out by thecentrifugation at 300 g for 5 minutes, CD8þ T cells were stimu-lated with Dynabeads human T-Activator CD3/CD28 (ThermoFisher Scientific) and cocultured with CD14þ monocytes at indi-cated ratios for 72 hours. T-cell proliferation was evaluated byflow cytometry.

Transduction with lentiviral particlesHEK293T cells were used for lentiviral particle production.

For transfection, plasmid containing respective shRNA (11 mg)was incubated with the packaging plasmids VSV-G (5.5 mg)and pCMV-dR 8.91 (8.25 mg) in DMEM and X-treme GENE(Roche) solution for 30 minutes and added to HEK293Tproducer cells. After incubation for 12 hours, the supernatantwas discarded. Upon further culture for 12, 24, and 36 hours,the supernatant was collected and virus particles were concen-trated by ultracentrifugation. Then mouse Ret or human SK-MEL-28 melanoma cells were incubated with concentratedvirus for 24 hours. Upon the first infection, Ret or SK-MEL-28 cells were reinfected with the same virus in fresh medium,and, after 48 hours of transduction, the cells were washed twicewith PBS and cultured. To select transduced cells, 2 mg/mLpuromycin was added for 3 days. The following plasmidswere used: plasmid shRNA HSP86: KD 1: TRCN0000321086;KD 2: TRCN0000321084; KD 3: TRCN0000321007; KD 4:TRCN0000321085; KD 5: TRCN0000321083 and control plas-mid: Mission TRC pLKO.5-puro nonmammalian shRNA con-trol plasmid.

Tumor growth in vivoC57BL/6 mice were injected s.c. with 5 � 105 Ret cells trans-

duced with shRNA HSP86 (shHSP86 Ret) or with scrambledcontrol (shSCR Ret). Tumor development was monitored dailyby measuring tumor diameter.

Quantitative real-time PCRThe method was performed as described before (26). Briefly,

total RNA was isolated by Trizol and transcribed with SensiFastcDNA Synthesis Kit (Bioline) followed by the quantification by aNanoDrop Spectrophotometer (ND-2000; Thermo Fisher Scien-tific). Primers were designed using Primer3 web tool and wereproduced by Metabion. S18RNA was applied as an internalstandard. The sequences of primers used are shown in Supple-mentary Table S1.

Statistical analysisStatistical analyses were performed using GraphPad Prism

(GraphPad Software) on at least three independent experimentsif not indicated differently. Data were analyzed with a one-wayANOVA test for multiple groups or an unpaired two-tailed Stu-dent t test for two groups. A value of P < 0.05 was consideredstatistically significant.

ResultsEV from murine Ret melanoma cells are taken up by IMC

We isolated EV from the supernatant of murine Ret melanomacells (Ret-EV) using ultrafiltration followed by ultracentrifuga-tion (25, 26). Nanoparticle tracking analysis (NTA) showed thatthe size of Ret-EV was 99.1 nm� 7.2 nm (Fig. 1A). Moreover, EVexpressed typical markers such as CD9, CD81, and ALIX but werenegative for calreticulin, a marker of endoplasmic reticulum(ER; Fig. 1B). Interestingly, the immunosuppressive moleculePD-L1 was found in Ret cell lysate. However, it was only weaklydetectable in Ret-EV (Fig. 1B). In addition, isolated EV werevisualized by electron microscopy using immunogold stainingfor CD81 as an EV marker (Fig. 1C). To investigate the uptake bymyeloid cells, EV were labeled with CFSE and incubated withimmortalized myeloid suppressor cell line MSC-1 (23). Fluores-cent microscopy analysis revealed that labeled Ret-EV were inter-nalized and localized inside the cells (Fig. 1D). Furthermore,using flow cytometry, we observed the uptake of CSFE-labeledRet-EV by CD11bþGr1þ IMC isolated from the BM of C57BL/6mice (Fig. 1E).

EV from murine Ret melanoma cells induce PD-L1 expressionon murine IMC

To test the effects of Ret-EV onmyeloid cells in vitro, we isolatedmurine BM-derived CD11bþGr1þ IMC and exposed them to Ret-EV. In accordance with previous reports (10, 12, 26), we observeda significant upregulation of the expression of inflammatory andimmunosuppressivemediators such as IL1b, IL6, IL10, TNFa, andcyclooxygenase-2 (COX-2) in IMCmeasured byRT-PCR (Fig. 2A).Importantly, we detected also a strong upregulation of PD-L1both at the mRNA and protein levels (Fig. 2A and B). To excludepossible contamination by endotoxins, we applied the limulusamebocyte lysate assay and found Ret-EV samples free ofendotoxin.

Using flow cytometry, we confirmed Ret-EV–mediated ele-vation of the frequency of PD-L1 expressing IMC and the levelof its expression measured by the median fluorescence intensity(MFI; Fig. 2C and D). In parallel, CD11bþGr1þ IMC weretreated with EV isolated from normal cardiac fibroblasts(Fibro-EV). No significant increase in PD-L1 expression wasobserved, suggesting that this PD-L1 modulation was due toRet-EV (Fig. 2C and D).

To exclude possible changes in EV samples due to the isolationprocedure, we established a coculture system. Retmelanoma cellsor fibroblasts were seeded into 24-well plates followed by treat-ment with DMA, an Hþ proton-pump inhibitor known to blockEV secretion (12). After 24 hours of incubation, IMCwere put intothe upper compartment of transwell chambers to exclude anydirect cell–cell contact. The PD-L1 expression on IMC was mea-sured after 48 hours. As shown in Fig. 2E and F, DMA-treated Retcells induced significantly lower elevation of PD-L1 expressionthannontreated Ret cells. As expected, the incubationwith cardiacfibroblasts did not change the frequency of PD-L1–expressingIMC.

Next, we investigated if tumor-derived EV could also stimulatePD-L1 expression on myeloid cells in vivo. To this end, wetransduced Ret cells with the vector-expressing EV marker CD81linked to GFP and injected them into syngeneic C57BL/6 mice tofollow the EV production and uptake in vivo. Fourteen days later,tumor single-cell suspensions were prepared and flow cytometry

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analysis revealed the presence of CD11bþGr1þMDSC expressingGFP, suggesting that CD11bþGr1þ cells acquired CD81-GFP viathe uptake of Ret-EV (Fig. 2G). Importantly, GFPþCD11bþGr1þ

MDSC showed a tendency for higher intensity of PD-L1 expres-sion as compared with their GFP� counterparts (Fig. 2H).

EV-treated IMC acquired immunosuppressive capacity viaPD-L1 upregulation

Next, we studied the possibility that EV-educatedCD11bþGr1þ

IMC not only increased PD-L1 expression but also affected theactivation of T lymphocytes. Upon incubation with Ret-EV for16 hours, IMC were added to purified autologous mouse spleenCD8þ T cells that were labeled with CFSE and activated with anti-CD3/CD28 antibodies. We observed a dose-dependent inhibi-tion of T-cell proliferation in the presence of EV-educated IMCmeasured by flow cytometry (Fig. 3A). Importantly, blockingantibodies against PD-L1 could significantly prevent such sup-pression of T-cell proliferation (Fig. 3B). In addition, the produc-tion of IFNg by stimulated T cells was found to be restored in thepresence of PD-L1–blocking antibodies (Fig. 3C). These resultssuggest a pivotal role of PD-L1 expression induced on EV-treatedmyeloid cells in their inhibition of T lymphocyte functions.Because the immunosuppressive ability represents a key featureof MDSC, in which PD-L1 expression plays an importantrole (3–5, 9), such myeloid cells could be considered to beconverted to MDSC (3, 4) under the influence of melanoma-derived EV.

PD-L1 upregulation is induced by NF-kB signaling inMSC-2 cells

We observed that Ret-EV increased in vitro the expression ofPD-L1 on the murine MSC-2 but not MSC-1 cells (Supplemen-tary Fig. S1A). Studying the mechanism of this upregulation, wefound that such PD-L1 elevation on immortalized myeloidsuppressor cell line MSC-2 was completely abrogated in thepresence of actinomycin D, an inhibitor of mRNA synthesis(Supplementary Fig. S1B). This observation suggests that Ret-EV rather trigger the synthesis of PD-L1 de novo than deliver thisprotein to myeloid cells. Deciphering Ret-EV–induced signal-ing, we demonstrated a strong activation of transcription factorNF-kB as detected by phosphorylation of the p65 subunit inMSC-2 cells shown via Western blot (Supplementary Fig. S1C).Next, MSC-2 cells were incubated with the NF-kB inhibitorBay11-7082. We noticed a dose-dependent suppression of theinduction of PD-L1 expression, suggesting a critical role of NF-kB signaling in this induction mediated by Ret-EV (Supple-mentary Fig. S1D).

TLR signaling is involved in Ret-EV–mediated PD-L1upregulation

Next, we isolated IMC from C57BL/6 mice deficient for theexpression of adapter protein myeloid differentiation primaryresponse 88 (MyD88) involved in the TLR/NF-kB signaling. Inaddition, IMCwere obtained frommice deficient for bothMyD88and TIR-domain-containing adapter-inducing interferon-b

Figure 1.

Characterization of EV derived from Ret murine melanoma cells. EV were isolated by filtration and ultracentrifugation. A, NTA of Ret-EV. B, Representativesample showing the expression of EVmarkers (CD9, CD81, and ALIX) as well as PD-L1 detected byWestern blot analysis. ERmarker calreticulin was used as anegative control. C, EVmarker CD81 detected by immunogold labeling and electron microscopy.D and E, Ret-EV were labeled with CFSE and incubated withmurine MSC-1 cells or BM-derived IMC for 16 hours. The internalization of CFSE-EV by MSC-1 cells was measured by confocal microscopy (D) and by IMC via flowcytometry (E).

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(TRIF), another adapter protein that participates in TLR/NF-kBsignaling. As expected, the treatmentwithRet-EV failed to increasethe frequency of PD-L1þCD11bþGr1þ IMC from both types ofknockout mice (Supplementary Fig. S1E) as well as the PD-L1expression on these cells expressed as MFI (SupplementaryFig. S1F).

To identify which TLR could be involved in PD-L1 upregula-tion, CD11bþGr1þ IMC were treated with various TLR ligandssuch as Pam3/CSK4 (TLR2), LPS (TLR4), and R848 (TLR7/8).We found that all three ligands were able to induce PD-L1expression on these cells (Fig. 4A). Moreover, IMC from

Tlr2�/�, Tlr4�/�, or Tlr7�/� mice displayed significantly lowerupregulation of PD-L1 expression upon the incubation withRet-EV as compared with their counterparts from wild-typemice (Fig. 4B). However, the strongest reduction after thetreatment was observed in IMC from Tlr4�/� mice, showingalmost no elevation of the frequency of PD-L1þ cells and theintensity of PD-L1 expression (Fig. 4B and C). In agreementwith the protein expression data, the induction of PD-L1 mRNAby Ret-EV was also completely abolished in IMCs from Tlr4�/�

mice (Fig. 4D) and to a less extent in cells from Tlr2�/� andTlr7�/� mice (Supplementary Fig. S2A). Importantly, IMC from

Figure 2.

EV-dependent PD-L1 upregulation onmurine IMC. IMC isolated from the BM of wild-type C57BL/6 mice were treated with Ret-EV for 3 hours (RT-PCRexperiments) or 16 hours (flow cytometry experiments). PBS-treated cells were used as a control. A, Expression of different cytokines and PD-L1 in IMCmeasuredby RT-PCR is presented as normalized to 18S RNA. B, RepresentativeWestern blot analysis showing PD-L1 expression in IMC upon Ret-EV treatment. C andD,PD-L1 expression on IMC treated for 16 hours with Ret-EV or EV isolated from cardiac fibroblasts (Fibro-EV) was evaluated by flow cytometry. Data are presentedas the percentage of PD-L1þ IMC within total IMC (C) or the level of PD-L1 expressionmeasured as MFI (D; mean� SEM; n¼ 4). E and F, Ret melanoma cells orcardiac fibroblasts were coincubated with IMC for 24 hours in the presence or absence of the inhibitor of EV secretion, DMA (15 mmol/L), using a transwell system.The analysis of PD-L1 expression on IMCwas performed by flow cytometry. Data are shown as the percentage of PD-L1þ IMC among total IMC (E) or the level ofPD-L1 expression as MFI (F; mean� SEM; n¼ 4). Ret cells were transduced with vector-expressing CD81 linked to GFP or control vector. Transduced cells wereinjected into C57BL/6mice. Upon 2 weeks, tumors were isolated and MDSC were evaluated by flow cytometry. G, Representative dot plots for GFP expression intumor-infiltrating CD11bþGr1þ are shown. H, Expression of PD-L1 on GFPþ and GFP� tumor MDSC is presented as MFI (mean� SEM; n¼ 3). � , P < 0.05;�� , P < 0.01; ��� , P < 0.001.

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Tlr4�/� mice (in contrast to IMC from wild-type mice) wereunable to inhibit T-cell proliferation upon the treatment withRet-EV (Fig. 4E). Similar results were obtained using IMC fromTlr2�/� or Tlr7�/� mice (Supplementary Fig. S2B), suggesting acritical role of TLR for the induction of immunosuppressiveactivity of myeloid cells by tumor-derived EV.

PD-L1 induction ismediated by theHSP86 expressed onRet-EVSome HSP on EV were previously described as potent stimu-

lators of TLR signaling (12, 28–30). We examined various pre-parations of Ret-EV for the expression of different TLR ligandssuch as HSP86, HSP72, HSP60, and HMGB1 by Western blotanalysis. Only HSP86 was consistently observed in all Ret-EVsamples (Fig. 5A). To determine ifHSP86was expressed on the EVsurface, we adsorbed them on latex beads and stained for HSP86followed by flow cytometry. HSP86was found to be expressed onthe surface of Ret-EV (Fig. 5B). Next, we treated Ret melanomacells with KNK-437, a potent inhibitor of the synthesis of induc-ible HSP, and proved a dose-dependent reduction in HSP86expression in these cells (Fig. 5C). EV isolated from KNK-437–treated (KNK-EV) or control (DMSO-treated) Ret cells were incu-bated with CD11bþGr1þ IMC derived from wild-type mice.

Interestingly, KNK-EV induced significantly lower upregulationof the frequency of PD-L1þ cells (Fig. 5D) and the intensity of PD-L1 expression (Fig. 5E) than EV isolated from control Ret cells.Importantly, the NTA revealed no differences between KNK-EVand control Ret-EV regarding EV concentration and size (Fig. 5F).

In another set of experiments, theHSP86 expression in Ret cellswas stably knocked down using lentivirus-mediated shRNAdeliv-ery (shHSP86Ret). Then these cellswere cultured in24-well platesfollowed by the addition of BM-derived CD11bþGr1þ IMC asdescribed above (see Fig. 2E). Coculture with shHSP86 cellsmediated only very slight upregulation of PD-L1 expression onIMC that was significantly lower than that induced by Ret cellstreated with scrambled sequence shRNA construct (shSCRRet; Fig. 5G and H). Furthermore, wild-type IMC were treatedwith EV isolated from shHSP86 (shHSP86 Ret-EV) or shSCR(shSCR Ret-EV) Ret cells followed by IMC incubation with acti-vated autologous CD8þ T cells. As shown in Fig. 5I, IMC treatedwith shHSP86 Ret-EV (in contrast to IMC-incubated shSCR Ret-EV)werenot able to inhibit T-cell proliferation. Collectively, thesefindings indicate that HSP86 represents an essential componentfor TLR-mediated signaling leading to the upregulation of PD-L1expression on IMC treated with Ret-EV.

Figure 3.

EV-treated IMC show immunosuppressive capacity mediated by PD-L1. IMC isolated from the BM of C57BL/6 mice were incubated with Ret-EV for 16 hours. Afterwashing out the rest of EV, cells were treated with PD-L1–neutralizing or isotype control mAbs (Iso) for 15 minutes, followed by the coincubation with normalspleen CD8þ T cells labeled with CFSE and stimulated with anti-CD3/CD28 Dynabeads for 72 hours. T-cell proliferation was evaluated by flow cytometry.A, Inhibition of CD8þ T-cell proliferation by EV-treated IMC at indicated IMC:T-cell ratio. Data are presented as the percentage of divided T cells (mean� SEM;n¼ 6). B and C, Proliferation (B) and IFNg secretion (C) of stimulated CD8þ T cells upon blocking PD-L1 on IMC (IMC:T-cell ratio¼ 1:1; mean� SEM; n¼ 3).� , P < 0.05; �� , P < 0.01.

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Depletion of HSP86 in Ret melanoma cells impairs tumorgrowth and reduces PD-L1 expression on MDSC

To study the impact ofHSP86 expression in tumor cells on theirgrowth in vivo, we injected shHSP86-Ret cells into C57BL/6 mice.Tumor growth was found to be delayed as compared with miceinoculated with control shSCR-Ret cells (Fig. 6A). We tested alsoshHSP86-Ret and shSCR-Ret cells for their proliferation capacityin vitro and found no difference between the two cell types(Fig. 6B), indicating that the growth inhibition of shHSP86-Retcells in vivowas not due to the impaired cell division.However, weobserved a decrease in the frequency of CD11bþGr1þ MDSCinfiltrating HSP86-deficient tumors (Fig. 6C). In addition, thefrequency of PD-L1þ MDSC within the total MDSC populationwas significantly reduced as compared with MDSC-infiltratingcontrol Ret tumors (Fig. 6D). A similar strong reduction in thefrequency of PD-L1þ MDSC was also observed among totalMDSC in the BM (Fig. 6E).

These data suggest that HSP86 from Ret melanoma cells couldbe expressed in Ret-EV and play an important role in the gener-ation of immunosuppressive MDSC in tumor-bearing mice.

EV derived from human melanoma upregulate PD-L1expression and induce immunosuppressive properties innormal human CD14þ monocytes

Next, we addressed the question of whether also humanmelanoma–derived EV could induce PD-L1 expression on mye-loid cells. Therefore, we isolated EV from the human melanoma

cell line HT-144 (HT-144-EV) using the protocol described aboveformouse Retmelanoma cells. HT144-EVwere characterized by asize of approximately 120 nmmeasured by NTA (SupplementaryFig. S3A), the expression of typical EVmarkers, and the absence ofthe ER marker calreticulin (Supplementary Fig. S3B). Similarly tomouse melanoma–derived EV, human HT-144-EV also inducedan upregulation of several inflammatory and immunosuppres-sive factors as well as PD-L1 expression in CD14þ monocytesisolated from healthy donors measured by RT-PCR (Fig. 7A).Importantly, a strong increase in the frequency of PD-L1þ cellsand in the intensity of its expression was also confirmed at theprotein level (Fig. 7B; Supplementary Fig. S4A). Moreover, wefound that EV isolated from another human melanoma cell lineSK-MEL-28 (SK-MEL-28-EV) induced a similar upregulation ofPD-L1 expression on healthymonocytes (Fig. 7B; SupplementaryFig. S4A).

Next, we investigated if EV-educated CD14þ monocytes couldacquire an immunosuppressive potential. To this end, similar toourmouse experiments, normal humanmonocytes were exposedto HT-144-EV followed by coculture with activated human CD8þ

T cells labeled with CFSE. These experiments confirmed thatCD14þ monocytes educated by melanoma-derived EV couldstrongly inhibit CD8þ T-cell proliferation, which was dependenton the EV concentration and the ratio between EV-educatedmonocytes and T cells (Fig. 7C).

In the murine system, TLR4 and (to a lesser extent) TLR2 werefound to be the main triggers of EV-dependent PD-L1

Figure 4.

Ret-EV stimulate PD-L1 upregulation via TLR signaling in myeloid cells. IMC were isolated from the BM of wild-type and TLR-deficient C57BL/6 mice and treatedwith Ret-EV (50 mg/mL) or with 10 ng/mL Pam3/CSK4 (TLR2 agonist) or 50 ng/mL LPS (TLR4 agonist) or 10 ng/mL R848 (TLR7/8 agonist) for 16 hours,followed by flow cytometry. A–C, PD-L1 expression is presented as the percentage of PD-L1þ IMC within total IMC (A and B) or as the level of PD-1 expression onIMCmeasured as MFI (C; mean� SEM; n¼ 4). D, Expression of PD-L1 in IMC fromwild-type and Tlr4�/�mice treated with Ret-EV was measured by RT-PCR andnormalized to 18S RNA expression. E, Proliferation of CD8þ T cells incubated with IMC that were isolated fromwild-type or Tlr4�/�mice and treated with Ret-EV(IMC:T-cell ratio¼ 1:1). Data are presented as the percentage of divided T cells (mean� SEM; n¼ 6). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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upregulationonmyeloid cells. To testwhether these TLRwere alsoinvolved in the EV-dependent upregulation of PD-L1 on humanCD14þ monocytes, we incubated them with blocking anti-TLR4and anti-TLR2 antibodies prior to the HT-144-EV treatment. Likein our mouse experiments, inhibiting TLR4 signaling resulted inthe strongest abrogation of EV-mediated stimulation of the PD-L1expression on monocytes (Fig. 7D). Interestingly, both lysates ofHT-144 melanoma cells and EV isolated from these cells dis-played very weak PD-L1 expression (Fig. 7E), suggesting that theobserved PD-L1 induction was rather a result of new proteinsynthesis mediated by TLR signaling.

Next, we studied whether HSP could play a role in the EV-induced PD-L1 upregulation in human monocytes. HT-144and SK-MEL-28 melanoma cells as well as HT-144-EV and SK-MEL-28-EV were characterized by the expression of HSP86(Fig. 7E). Similar to mouse experiments, the HSP86 expressionin HT-144 cells was stably knocked down using lentivirus-mediated shRNA delivery followed by the isolation of EV fromthese cells (shHSP86HT-144 EV). Importantly, the cell lysate and

shHSP86HT-144 EV showed a drastic reduction in the expressionof HSP86 (Supplementary Fig. S5A and S5B). The coculture ofCD14þ monocytes with these EV led to a significantly lowerelevation of the frequency (Fig. 7F) and intensity (SupplementaryFig. S4B) of PD-L1 expression as compared with monocytesincubated with EV isolated fromHT-144 cells treated with scram-bled sequence shRNA construct (shSCR HT-144 EV). Further-more, monocytes incubated with shHSP86 HT-144 EV weresignificantly less efficient in the inhibition of T-cell proliferationthan monocytes treated with shSCR HT-144 EV (Fig. 7G).

To address the question ofwhether EV circulating inmelanomapatients could induce the same effect on CD14þmonocytes fromhealthy donors, EV were purified from the plasma of stage IVmelanoma patients followed by their coculture with monocytes.We found a strong increase in the frequency of PD-L1þ cells ascompared with these values in nontreated monocytes (Fig. 7H).The level of PD-L1 expression under these conditions was alsosignificantly elevated (Supplementary Fig. S4C). Importantly,monocytes incubated with plasma devoid of EV (containing only

Figure 5.

Hsp86 is critical for EV-mediated PD-L1 upregulation on IMC.A, The expression of HSP86, HSP72, HSP60, and HMGB1 in Ret cell lysate and different preparationsof Ret-EV was measured byWestern blot. B, Ret-EV were coupled to latex beads and analyzed by flow cytometry. Results present the staining with anti-HSP86antibodies (gray) and with isotype control antibodies (black). C, Ret cells were treated with the indicated concentrations of KNK-437. The expression of HSP86 incell lysates was analyzed byWestern blot. D and E, IMC were treated with EV isolated from KNK437-treated (KNK-EV) or -untreated Ret cells (Ret-EV). PD-L1expression was determined by flow cytometry and is shown as the percentage of PD-L1þ IMC among total IMC (D) or as the level of PD-1 expression as MFI (E;mean� SEM; n¼ 3). F, NTA of KNK-EV and Ret-EV showing the size and concentration of EV. G andH, Ret cells were transduced with shHSP86 (shHSP86 Ret)or respective scrambled control (shSCR86 Ret). IMC were coincubated with both cell types using a transwell system. PD-L1 expression was evaluated by flowcytometry and is presented as the percentage of PD-L1þ IMC within total IMC (G) and as the level of PD-1 expression as MFI (H; mean� SEM; n¼ 4). I,Wild-typeIMCwere treated with EV isolated from shHSP86 (shHSP86 Ret-EV) and shSCR (shSCR Ret-EV) Ret cells. Then treated IMC were added to activated CD8þ T cells(IMC:T-cell ratio¼ 1:1). Data are presented as the percentage of divided T cells (mean� SEM; n¼ 6). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

Induction of Immunosuppression by Extracellular Vesicles

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soluble factors) significantly lower upregulation of PD-L1 expres-sion (Fig. 7H; Supplementary Fig. S4C).

Taken together, our results suggest that the interaction ofmelanoma-derived EV with myeloid cells in both human andmurine systems leads to the upregulation of PD-L1 expression onthese cells and to the acquisition of their capacity to inhibit T-cellactivity.

DiscussionAlthough initial reports demonstrated that EV released

by tumor cells could be a unique source of tumor-associatedantigens and stimulate T-cell–mediated antitumor immuneresponses (28, 29), numerous studies over the last decade havedemonstrated that tumor-derived EV play a significant role in

tumor progression (16, 17, 30, 31). EV have been reported to beimportant for preparing metastatic niches and for the develop-ment of therapy resistance (13, 31, 32). Furthermore, the poten-tial of tumor-derived EV to dampen immune functions of the hosthas been brought into focus. In particular, tumor EV have beenfound to impair the function of T cells and NK cells (19, 32, 33),induce regulatory T cells (34), and convert normal myeloid cellsinto potent immunosuppressive MDSC (10, 12–14, 35).

However, the molecular mechanism of such EV-mediatedMDSC generation from normal myeloid cells is still unclear.Here, we found that normal murine IMC strongly upregulatedPD-L1 and acquire the capacity to inhibit T-cell functions uponthe treatment with EV derived from Ret melanoma cells, suggest-ing that these EV-educated cells could be defined as MDSC (3).Furthermore, blocking PD-L1 led to a substantial attenuation of

Figure 6.

Reduced growth of Ret cellsdeficient in HSP86 in vivo. shHSP86Ret cells or shSCR Ret cells(scramble control) were injecteds.c. into C57BL/6mice. A, Tumorgrowth is presented as tumordiameter. B, shHSP86 Ret cells andshSCR Ret cells were incubatedwith Alamar blue, and the metabolicactivity was measured viaspectrometry. C–E, At day 14, tumorand BM cell single-cell suspensionswere analyzed by flow cytometry.C,Data on tumor-infiltrating MDSCare shown as the percentage ofthese cells among total leukocytes.PD-L1 expression is presented asthe percentage of PD-L1þMDSC intumors (D) and in the BM (E; mean� SEM; n¼ 4). � , P < 0.05.

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their immunosuppressive potential, indicating an importance ofPD-L1 induction in the conversion of IMC into MDSC.

PD-L1 expression was demonstrated to be involved in MDSC-mediated T-cell inhibition through the binding to PD-1 expressedon effector T cells (3–5, 9). Several recent publications reportedthat tumor-derived EV could express PD-L1 at the surface anddirectly signal to T cells, thereby inhibiting their functions andmediating immune evasion (33, 34, 36–38). Moreover, PD-L1expressiononEV fromheadandneck cancer patientswas reported

to correlate positivelywithdisease progression (32, 39).However,we found a weak expression of PD-L1 in EV isolated frommouseand human melanoma cells. Moreover, Ret-EV could induce astrong PD-L1 upregulation at the protein andmRNA levels in IMCand MSC-2 but not in MSC-1 cells. In addition, blocking mRNAsynthesis by actinomycin D in MSC-2 cells led to a completeabrogation of PD-L1 expression. All these data indicate that, inour system, PD-L1 was rather induced de novo via myeloid cellreceptors but not transferred by EV to these cells.

Figure 7.

Humanmelanoma EV induce immunosuppressive activity of normal monocytes via PD-L1 upregulation. EV were isolated from human HT-144 melanoma cells(HT-144-EV) by filtration and ultracentrifugation, followed by incubation with CD14þmonocytes from healthy donors for 16 hours. Untreated cells were used as acontrol. A, Expression of cytokines and PD-L1 in monocytes was measured via RT-PCR and is expressed as normalized to b-actin. B and C, PD-L1 expression onCD14þmonocytes treated with HT-144-EV and EV from SK-MEL-28 melanoma cells (SK-MEL-28-EV) was detected by flow cytometry. Results are presented asthe percentage of PD-L1þCD14þmonocytes within total CD14þmonocytes (B; mean� SEM; n¼ 6). C, CD14þmonocytes were cocultured with HT-144-EV for16 hours, followed by incubation with T cells labeled with CFSE and stimulated with anti-CD3/CD28 Dynabeads for 72 hours. Inhibition of T-cell proliferation wasevaluated by flow cytometry at indicated IMC:T-cell ratio. Data are shown as the percentage of divided T cells (mean� SEM; n¼ 6). D,Monocytes were treatedwith HT144-EV for 16 hours alone or in the presence of the indicated TLR-blocking mAbs or anti-human IgG (Iso). PD-L1 expression was measured via flowcytometry and is expressed as the percentage of PD-L1þmonocytes within total monocytes (mean� SEM; n¼ 6). E, RepresentativeWestern blot analysisshowing the expression of HSP86 and PD-L1 in lysates from HT-144 and SK-MEL-28 cells as well as HT-144-EV and SK-MEL-28-EV. F, SK-MEL-28 melanoma cellswere transduced with shHSP86 or respective scrambled control (shSCR86), followed by EV isolation (shHSP86-EV and shSCR86-EV, respectively). Monocyteswere incubated with both EV types for 16 hours, followed by the evaluation of PD-L1 expression by flow cytometry. Data are shown as the percentage of PD-L1þ

cells within total monocytes (mean� SEM; n¼ 6). G, EV-treated CD14þmonocytes were incubated with activated T cells for 72 hours. Results are presentedas the percentage of proliferated T cells (mean� SEM; n¼ 6). H, Normal CD14þmonocytes were incubated with EV from plasma of untreated stage IVmelanoma patients or with plasma of these patients without EV. Data are presented as the percentage of PD-L1þCD14þmonocytes within total monocytes(mean� SEM; n¼ 6). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

Induction of Immunosuppression by Extracellular Vesicles

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HSP are known as chaperons for controlling the correct foldingof newly synthesized proteins. However, under certain conditionssuch as stress or cancer, they can be released and act as damage-associated molecular patterns, activating the immune system viabinding to TLR (40). HSP have been previously described to beexpressed onEV (15, 16). Thus, EV-mediatedHSP72was found tostimulate MDSC functions through STAT3 activation and cyto-kine secretionmediatedbyTLR2 signaling (12). Several otherHSPsuch as HSP70, HSP90, or HSP105 were also reported to beassociated with EV and to trigger TLR2 or TLR4 (41–43). It wasdemonstrated that TLR2 could be activated via exosomal prosta-glandin E2 (11). In addition, EV-induced stimulation of PD-L1expression on monocytes could be mediated by TLR7 signal-ing (35). Here, we identified the HSP86/TLR4 axis as a majordriver of PD-L1 upregulation, although the contribution ofTLR2 or TLR7 could not be excluded. In agreement with our data,Cheng and colleagues (44) reported that EV derived from hepa-tocellular carcinoma cells induced PD-L1 expression in humanmonocytic cell line THP-1 and mouse macrophage cell lineRAW264.7; however, the authors did not address the mechanismfor this induction. Because they observed a reduction of EV-mediated PD-L1 stimulation by treating tumor cells with mela-tonin, whichwas previously reported to decrease the induction ofseveral HSP after oxidative stress (45), we believe that HSP86might stimulate PD-L1 expression also in their system. Further-more, it has been recently demonstrated that EV isolated fromglioblastoma stem cells induced PD-L1 on normal humanmono-cytes via STAT3 activation (46).

We have recently reported that a well-defined set of miRNAexpressed inmelanoma-derived EV could convert normal humanmonocytes into MDSC (47). Because certain miRNAs were pre-viously demonstrated to activate endosomal TLR such as TLR7and TLR8 (48), it is conceivable that the miRNA described byHuber and colleagues (47)might contribute toMDSC conversionby signaling via TLRs. Thus, it seems that differentmolecules (e.g.,lipids, RNA, proteins) could stimulate diverse TLR, leading to theinduction of PD-L1 expression.

Investigating PD-L1 upregulation on myeloid cells by mel-anoma-derived EV in vivo, we injected Ret cells transduced withthe CD81-GFP vector into wild-type mice. We demonstratedthe presence of CD11bþGr1þ MDSC expressing GFP in theTME, suggesting that MDSC obtained CD81-GFP most likelyvia the uptake of Ret-EV. The ex vivo isolated, GFPþ MDSCshowed a tendency for higher intensity of PD-L1 expression ascompared with their GFP� counterparts. Although we cannotexclude that MDSC acquire GFP signal via phagocytosis, theseresults are in line with our previous publication, using a Cre-Lox system to track the uptake of EV secreted by Lewis lungcarcinoma cells (14), and suggest that the induction of PD-L1on MDSC could also happen in vivo.

HSP in cancer have emerged as novel therapeutic targets. Ithas been recently demonstrated that HSP90 inhibitionenhanced cancer immunotherapy by upregulating IFNresponse genes (49). Furthermore, inhibiting HSP90 in mela-noma cells prevented the induction of functional MDSC, dis-playing an impaired capacity to inhibit T-cell proliferationin vitro (50). In the mouse MCA205 sarcoma model, theapplication of HSP90 inhibitor 17-DMAG in vivo resulted inslower tumor development and reduced levels of MDSC in theTME (51). We reported here that HSP86 inhibition or knock-

down in Ret cells or human melanoma cell lines abolished theability of released EV to induce PD-L1 upregulation. Althoughthis observation is most likely due to the depletion of HSP86from the EV surface, we cannot rule out the possibilitythat the HSP machinery is involved in the sorting of variouscompounds into EV. Future experiments are needed to clarifythis point.

Taken together, our data highlight the molecular mechanismof the conversion of normal mouse and human myeloid cellsinto MDSC by melanoma-derived EV. It involves the triggeringof mainly TLR4 on myeloid cells by inducible HSP86 in EV,leading to NF-kB activation and upregulation of PD-L1 expres-sion. Blocking TLR4 signaling or directly PD-L1 expressionresulted in the abrogation of the immunosuppressive capacityof EV-educated myeloid cells, suggesting a critical role for thisPD-L1–inducing pathway in the acquisition of immunosup-pressive properties. Based on characteristics of these EV such assize, and expression of an endocytic marker ALIX, it is probablethat exosomes rather than microvesicles are mainly responsiblefor the described effects. Our data suggest the possibility ofMDSC generation in cancer not only by inhibiting IMC differ-entiation via inflammatory mediators (3–8) but also by thereprogramming of normal myeloid cells through tumor-derived EV that could be supported by the high plasticity ofthese cells in tumor-bearing hosts (52).

Disclosure of Potential Conflicts of InterestJ. Utikal reports receiving honoraria from the speakers' bureau of, and is a

consultant/advisory board member for, Melanoma. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: V. Fleming, X. Hu, P. Altevogt, V. UmanskyDevelopment of methodology: V. Fleming, X. Hu, R. Weber, C. Groth,V. Nagibin, P. Altevogt, V. UmanskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Fleming, X. Hu, C. Weller, R. Weber, C. Groth,Z. Riester, L. H€user, V. Nagibin, C. Kirschning, V. UmanskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Fleming, X. Hu, C. Weller, R. Weber, C. Groth,L. H€user, V. Bronte, J. Utikal, P. Altevogt, V. UmanskyWriting, review, and/or revision of the manuscript: V. Fleming, X. Hu,R. Weber, C. Groth, Z. Riester, J. Utikal, P. Altevogt, V. UmanskyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Sun, J. Utikal, V. UmanskyStudy supervision: P. Altevogt, V. UmanskyOther (punctual text editing and TLR-specific inputting): C. Kirschning

AcknowledgmentsThe authors thank S. Uhlig for help with cell sorting and E. Cordell

for help with article preparation. This work was supported, in part, bygrants from the German Research Council (RTG2099 to J. Utikal andV. Umansky), the DKFZ-MOST Cooperation in Cancer Research (CA181 toV. Umansky), and the Italian Association for Cancer Research (IG grant18603 and Special Program Molecular Clinical Oncology 5 per mille, 12182to V. Bronte).

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 indicatethis fact.

Received January 5, 2019; revised June 12, 2019; accepted July 19, 2019;published first July 23, 2019.

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Cancer Res; 79(18) September 15, 2019 Cancer Research4728

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2019;79:4715-4728. Published OnlineFirst July 23, 2019.Cancer Res   Viktor Fleming, Xiaoying Hu, Céline Weller, et al.   Myeloid Cells by Upregulating PD-L1 via TLR4 SignalingMelanoma Extracellular Vesicles Generate Immunosuppressive

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