MIFIsNecessaryforLate-StageMelanomaPatient MDSC Immune … · Research Article...

Research Article

MIF Is Necessary for Late-StageMelanomaPatientMDSC Immune Suppression and DifferentiationKavitha Yaddanapudi1,2,3, Beatriz E. Rendon1, Gwyneth Lamont1, Eun Jung Kim1,Numan Al Rayyan1, Jamaal Richie1, Sabrin Albeituni2, Sabine Waigel1, Ashley Wise2, andRobert A. Mitchell1,2,3

Abstract

Highly aggressive cancers "entrain" innate and adaptiveimmune cells to suppress antitumor lymphocyte responses.Circulating myeloid-derived suppressor cells (MDSC) consti-tute the bulk of monocytic immunosuppressive activity in late-stage melanoma patients. Previous studies revealed thatmonocyte-derived macrophage migration inhibitory factor(MIF) is necessary for the immunosuppressive function oftumor-associated macrophages and MDSCs in mouse modelsof melanoma. In the current study, we sought to determinewhether MIF contributes to human melanoma MDSC induc-tion and T-cell immunosuppression using melanoma patient-derived MDSCs and an ex vivo coculture model of humanmelanoma-induced MDSC. We now report that circulating

MDSCs isolated from late-stage melanoma patients are reliantupon MIF for suppression of antigen-independent T-cellactivation and that MIF is necessary for maximal reactiveoxygen species generation in these cells. Moreover, inhibitionof MIF results in a functional reversion from immunosuppres-sive MDSC to an immunostimulatory dendritic cell (DC)–likephenotype that is at least partly due to reductions in MDSCprostaglandin E2 (PGE2). These findings indicate that mono-cyte-derived MIF is centrally involved in human monocyticMDSC induction/immunosuppressive function and that ther-apeutic targeting of MIF may provide a novel means of induc-ing antitumor DC responses in late-stage melanoma patients.Cancer Immunol Res; 4(2); 101–12. �2015 AACR.

IntroductionStage IV melanoma is a highly aggressive and resistance-prone

malignancy that carries a 5-year survival rate of approximately15%. Melanoma cells are unusually immunogenic and, conse-quently, are adept at inducing host innate and adaptiveimmunosuppressivemechanisms that, collectively, serve to atten-uate antitumor lymphocyte responses (1). Adaptive cell types andeffectors involved in melanoma-associated immune suppressioninclude regulatory T lymphocytes (Treg), cytotoxic T lymphocyteantigen-4 (CTLA-4), and programmed cell death 1 (PD-1)—ofwhich the latter two are currently being evaluated as therapeutictargets in late-stage melanoma patients (2).

It is becoming increasingly evident that tumor-entrainedinnate immune effector cells—e.g., tumor-associated macro-phages (TAM), tumor-associated neutrophils (TAN), tolerogenicdendritic cells (DC), andMDSCs—also provide highly significant

degrees of immune escape to aggressive malignancies (3–7). Inpatients with advancedmelanoma, circulatingmonocyticMDSCsprovide the bulk of monocyte-associated immune suppression(8), negatively affect patient survival, and inversely correlate withthe presence of functional antigen-specific T cells (9).

Previous studies from our laboratory established a novel func-tional role for monocyte-derived macrophage migration inhibi-tory factor (MIF) in dictating alternative activation phenotypes inmouse melanoma TAMs; loss or inhibition of MIF reduces mel-anoma TAM- andMDSC-mediated immune suppression (10). Ina related study using the 4T1 mouse model of breast cancer,Simpson and colleagues showed that tumor-derived MIF pro-motes MDSC accumulation and immunosuppressive activity(11). Reconstitution of wild-type MIF cDNA into 4T1MIF shRNAknockdown cells, but not an enzymatically inactive MIF mutant(proline-2 to serine-2, P2S) cDNA, was capable of reconstitutingtumor-derived, MIF-dependent MDSC induction. This result wasin line with our finding that small-molecule inhibitors of MIF'senzymatic activity fully phenocopy MIF deficiency in their abilityto dictate the immunosuppressive activities ofmonocytes/macro-phages in tumor-bearing hosts (10).

Studies by theDranoff laboratory have identifiedMIF as a targetof naturally developing auto-antibodies in late-stage melanomapatients who had successfully responded to a trial immunother-apy consisting of autologous GM-CSF secreting tumor cell vac-cines followed by CTLA-4 blockade (ipilimumab; ref. 12). MIFauto-antibodies disrupted MIF-dependent effects on humanmonocytes/macrophages, suggesting that the beneficial effects ofthese MIF-targeting auto-antibodies in advanced melanomapatients are due to inhibition of MIF-dependent innate immunestromal cell phenotypes. Although this finding suggests a

1Molecular Targets Program, JG Brown Cancer Center, University ofLouisville, Louisville, Kentucky. 2Department of Microbiology andImmunology, University of Louisville, Louisville, Kentucky. 3Depart-ment of Medicine, University of Louisville, Louisville, Kentucky.

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

Corresponding Authors: Robert A. Mitchell, University of Louisville, Clinical andTranslational ResearchBuilding, Suite 404, 505SouthHancock Street, Louisville,KY 40202. Phone: 502-852-7698; Fax: 502-852-3661; E-mail:[email protected]; and Kavitha Yaddanapudi,[email protected]

doi: 10.1158/2326-6066.CIR-15-0070-T

�2015 American Association for Cancer Research.

CancerImmunologyResearch

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clinically relevant role for MIF in human melanoma diseaseprogression/survival, no studies have directly investigated thefunctional and/or mechanistic contributions of MIF to innateimmune cell–mediated immune suppression in melanomapatients.

Using our well-characterized, small-molecule MIF enzymaticantagonist (4-iodo-6-phenylpyrimidine, 4-IPP; refs. 10, 13–15),we investigated MIF contributions to human melanoma MDSCinduction, phenotype, differentiation status, and mechanisticeffectors. We show that human MDSCs derived from late-stagemelanoma patients and those induced in vitro by tumor cells relyon MIF to suppress T-cell activation. MIF reliance correspondswith reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2)/PGE2 production elicited by MDSCs. Unexpectedly, whenMDSC-derived MIF is inhibited during short-term ex vivo cultureof MDSCs, their differentiation is redirected toward a more DC-like phenotype. These MIF-inhibited monocytic MDSCs induceantigen-specific T-cell stimulatory function in these cells.

Together, our results support a crucial protumorigenic con-tribution by MIF to the immune suppression and differentia-tion of circulating melanoma MDSCs and provide justificationfor therapeutic targeting of MIF in patients with advancedmelanoma.

Materials and MethodsPatient samples and cell lines

Peripheral bloodwas collected from27patientswithmetastaticmelanoma stage III to IV, and from12 healthy donors. Melanomapatients included in this studywere not undergoing therapywhentheir samples were collected, and they all had progressive disease.Patient samples were collected after informed consent wasobtained by staff of the JG Brown Cancer Center Biorepositoryand covered under University of Louisville IRB protocol number08.0388. The melanoma cell line A375 (ATCC CRL-1619) waspurchased fromATCC andmaintained inDMEM containing 10%(v/v) FBS.We do not culture this cell line longer than 6 to 8weeks,and all of our stocks come from thawed vials that were frozen atpassage 2 after they were received from ATCC. The A375 cell linewas authenticated by the ATCC cell bank using short tandemrepeat profiling.

MiceWild-type male C57BL/6 mice (MIFþ/þ) were obtained from

Harlan Laboratories. OT-I and OT-II transgenic mice wereobtained from The Jackson Laboratory. All mice were handledwith the approval of the Institutional Animal Care and UseCommittee at the University of Louisville (Louisville, KY).

MDSC isolation and 4-IPP treatmentMonocytic CD14þ MDSCs from melanoma patients were

isolated from peripheral blood mononuclear cells (PBMC) usinganti-CD14 magnetic microbeads and the autoMACS Pro Separa-tor (Miltenyi Biotec), per manufacturer's instructions. Onemillion MDSCs were plated in complete Iscove's modified Dul-becco's medium (IMDM; supplemented with 10% human ABserum; Sigma-Aldrich; 2 mmol/L L-glutamine, and penicillin/streptomycin) per well in a 6-well plate (BD Falcon) and treatedwith 4-IPP (50 mmol/L) or DMSO (vehicle control) for 24 hours.For functional experiments, autologous T cells were isolated fromPBMCs using the Pan T-cell Isolation Kit (Miltenyi Biotec).

In vitro generation of human MDSCsCD14þ cells (1 � 106) isolated from PBMCs obtained from

healthy donors were cocultured with 5� 105 A375 tumor cells incomplete IMDMper well in a 6-well plate (16). Tumor/monocytecocultures were treated twice with 4-IPP (100 mmol/L on day 0and 50 mmol/L on day 2) or 0.1% DMSO (vehicle control). A375cocultured monocytes (both untreated and 4-IPP treated) andcontrol monocytes cultured without tumor cells were harvestedby gently scraping after 64 to68hours of culture andCD11bþ cellswere purified. Details for cell isolation techniques used areprovided in the Supplemental Methods section.

Mouse bone marrow–derived MDSCsTibias and femurs fromMIFþ/þ andMIF�/�C57BL/6micewere

removed using sterile techniques, and bone marrow was flushed.To obtain bonemarrow–derivedMDSCs, bonemarrow cells werecultured for 4 days with GM-CSF (40 ng/mL), and IL6 (40 ng/mL)cytokines, as previously described (17).MIFþ/þ andMIF�/� bonemarrow cultures were treated with 0.1% DMSO (vehicle control)or with 4-IPP (50 mmol/L) during the last 48 hours of the cultureperiod. For functional assays, CD11bþGR1þ bone marrowMDSCs were isolated from bone marrow cultures using CD11bandGR1microbeads followed bymagnetic separation (Miltenyi).

Antibodies and flow cytometryUntreated or 4-IPP–treated melanoma patient MDSCs and

tumor cell line–inducedMDSCs (A375-MDSC)were stainedwithanti-human antibodies according to the manufacturer's recom-mendations. Details on flow cytometry staining and antibodypanels are provided in the Supplemental Methods section and inSupplementary Table S1.

Functional studiesTo evaluate the suppressive functions of melanoma patient–

derived MDSCs and A375-MDSCs, autologous T cells werelabeled with 5 mmol/L carboxyfluorescein succinimidyl ester(CFSE; Invitrogen) and seeded at 100,000 cells per well in a96-well U-bottom plate. For patient samples, freshly purifiedCD14þ cells or CD14þ cells that were pretreated with or without4-IPP for 24 hours were added to T cells at ratios of 2:1, 1:1, or 1:2.T cells were activated by the addition of anti-CD3/CD28 mAb-coated beads (Invitrogen) per well for 4 days. T-cell activationwasmeasured by flow cytometry, and IFNg concentrations in thesupernatants were determined by ELISA. Controls included non-activated T cells or T cells activated with beads alone. For A375-MDSCs, CD11bþ monocytes, or CD11bþHLA-DR� cells purifiedfrom tumor cocultures with or without 4-IPP treatment for 64hours were added to T cells at ratios of 1:2 or 1:4, and T-cellactivation was measured as above.

DC phenotype and functionMelanoma patient MDSCs and A375-MDSCs were either

untreated or treated with 4-IPP (50 mmol/L), PGE2 (10 mmol/L),or with 4-IPP plus PGE2 for 72 hours and were analyzed for theexpression of human DC markers by flow cytometry. For mouseDC phenotype studies, bone marrow MDSCs from MIFþ/þ andMIF�/� mice were cultured for 48 hours and analyzed for mouseDC marker expression by flow cytometry. DC function in A375-MDSCs was analyzed using Tetanus Toxoid (TT) antigen presen-tation assays. A375-MDSCs were either untreated or treated with

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4-IPP (50 mmol/L) or 4-IPP (50 mmol/L) plus PGE2 (10 mmol/L)for 72 hours. MDSCs were added to autologous, CFSE-labeledT cells at ratios of 1:5 (20,000 MDSCs) or 1:10 (10,000 MDSCs).T cells were activated with 1.0 mg/mL of TT per well for 5 days.T-cell proliferation wasmeasured by flow cytometry. DC functionin mouse bone marrow MDSCs was determined using the OT-IITCR transgenic mice and ovalbumin (OVA) antigen presenta-tion. MIFþ/þ and MIF�/� bone marrow MDSCs harvested after48-hour culture period were added to CFSE-labeled CD4þ T cellspurified from OT-II splenocytes at 1:5 and 1:10 ratio in thepresence of 200 mg/mL of OVA (Sigma-Aldrich) per well for5 days. CD4þ T-cell activation was estimated by flow cytometry.

Quantitative PCR analysisTotal RNA and real-time analysis was performed as previously

described (10). Taqman probes (Applied Biosystems) for genes18S (Hs99999901.s1; VIC),MIF (Hs00236988_g1; FAM),COX-2(Hs00153133_m1; FAM), and NOX4 (Hs00418356_m1; FAM)were used according to the manufacturer's instructions.

Microarray analysisTotal RNA from cultured monocytes, vehicle-treated, and 4-

IPP–treated A375-MDSCs were isolated, and microarray analysiswas performed according to the manufacturer's instructions.Details of the instrumental set-up and analysis are described inthe Supplemental Methods section. The microarray datasets dis-cussed in the current study have been deposited in NCBI's GeneExpression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo) and areaccessible through GEO Series accession number GSE73333.

ROS detectionThe oxidation-sensitive dye DCF-DA was used to measure ROS

production in untreated or 4-IPP–treated melanoma patientMDSCs or A375-MDSCs. Details of ROS estimation by flowcytometry are provided in the Supplemental Methods section.

Western blottingLysates of cultured monocytes and A375-MDSCs were probed

with antibodies that recognize human MIF and human GAPDH(Santa Cruz Biotechnology, Inc.).

ELISAsCytokines weremeasured by ELISA in supernatants from T-cell:

MDSC cocultures and from MDSC cultures. ELISA kits used werethe human IFNg and PGE2 kits obtained from R&D Systems.

Statistical analysisGraphPad Prism 5.0 software (GraphPad Prism Software, Inc.)

was used for all statistical analyses. Two-group comparisonsbetween control and test samples (groups compared are indicatedin the respective figures) were done by two-tailed Student t tests.Multiple data comparisons were derived by one-way ANOVAfollowed by the Tukey post hoc test. For all tests, statistical signif-icance was assumed where P < 0.05.

ResultsCirculating CD14þHLADR�/low MDSCs with potent immuno-

regulatory activities have been identified in the peripheral bloodof patients with ovarian cancer (18), hepatocellular cancer (19),and late-stage melanoma (20–22). In an effort to extend our

previous findings that monocytic cell–derived MIF providesfunctional contributions to MDSC immunosuppressive activity(10), we first analyzed the frequency and phenotype of circu-lating monocytic MDSCs in stage III/IV metastatic melanomapatients using multicolor FACS analysis. Representative dotplots for one of the melanoma patients and one of the normaldonors are included in the study to illustrate the gating strategyused (Fig. 1A). The percentage of circulating lineage�(Lin�)CD14þCD11bþCD33þHLADR�/low monocytic MDSCs is signif-icantly elevated inmelanoma patients' freshly isolated peripheralblood compared with that in normal donors (Fig. 1A and B;refs. 21, 22).

Consistent with prior studies (21, 22), in our study purifiedCD14þ melanoma monocytes exhibited potent inhibitory activ-ity against autologous T-cell activation (Fig. 2A and B) and IFNgproduction (Fig. 2C) induced by anti-CD3/anti-CD28, comparedwith cultured CD14þ monocytes from normal donors (Fig. 2D–

F). This finding is also consistent with prior studies (8) demon-strating that both HLADRþ and HLADR�/lo CD14þ circulatingmyeloid cell populations represent highly immunsuppressiveMDSCs. We next determined whether inhibition of melanomaMDSC MIF with our small-molecule MIF enzymatic antagonist,4-IPP (10, 13–15), affected MDSC immunosuppressive activity.Treatment of melanoma patient–derived CD14þ monocyticMDSCs with 4-IPP for 24 hours significantly reduced their T-cellinhibitory activity (Fig. 2G and H). No toxicity or loss of viabilitywas observed in MDSCs treated with either 4-IPP or vehicle(Supplementary Fig. S1A), although there was a slight decreasein MDSC suppressive activity compared with freshly isolatedMDSCs (compare Fig. 2A with Fig. 2G). This loss of T-cell sup-pressive activity in short-term cytokine-free cultures of MDSCs islikely a result of ex vivo culture in the absence of tumor-derivedMDSC polarizing factors.

Because we were interested in pursuing validation and mech-anism-based studies—both of which necessitate greater num-bers of cells than would be practical using patient-derivedperipheral blood samples—we established an in vitro model ofmelanoma cell line–induced MDSCs that faithfully recapitu-lates patient-derived CD14þHLADR�/low monocytic MDSCphenotype and function (16). This model utilizes a coculturesystem consisting of A375 human melanoma cells and normaldonor CD14þ monocytes cocultured for approximately 68hours (16). We characterized the phenotype of the monocyticMDSC-like cells induced during the A375-monocyte coculturewith multicolor flow cytometry. The percentage of CD14þ

CD11bþCD33þHLADR�/low cells was substantially increasedin A375-monocyte cocultures in comparison with that in mono-cytes cultured in the absence of melanoma cells (SupplementaryFig. S2). Furthermore, CD11bþ cells purified from the A375-monocyte cocultures exhibited a significant reduction in HLA-DR and increases in CD14, CD33, PD-L1, and DC-SIGN mar-kers (Supplementary Fig. S3A and S3B)—an expression signa-ture that closely corresponds to monocytic MDSCs isolatedfrom late-stage melanoma patients (16, 21, 23). Although werefer to the CD11bþ cells isolated from the cocultures of A375cells and monocyte as "A375-MDSCs," these cells represent aheterogeneous population of cells, similar to the MDSCs iso-lated from melanoma patients (8). MIF mRNA and proteinexpression was increased in A375-MDSCs compared withmonocytes cultured without tumor cells for the same periodof time (Supplementary Fig. S3C and S3D).

Monocytic MDSC Induction by MIF

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To determine whether MIF inhibition during MDSC inductioninfluences the acquisition of MDSC phenotype/function, 4-IPPwas added at the beginning of the A375:monocyte coculture.Changes in cell-surface marker expression of A375-MDSCs andrelative T-cell suppressive activity were assessed 68 hours later.MIF inhibition during the MDSC induction phase by melanomacells resulted in reductions of CD14, CD33, and PD-L1 and anincrease in DC-SIGN expression, whereas HLA-DR or CD11cexpression was not significantly altered (Supplementary Figs.S3A and S3B).

With respect to functional immunosuppressive activities,A375-MDSCs were potent suppressors of autologous T-cell acti-vation and IFNg production compared with fresh monocytes andtumor cell–free cultured monocytes, whereas A375-MDSCs from4-IPP–treated cocultures (A375-MDSCs þ 4-IPP) possessed littleto no suppressive activity on T-cell proliferation/IFNg production(Fig. 3). The induction of suppressive function in monocytes

relied upon direct cell contact with the tumor cells: Monocytescultured in the presence of A375 tumor cell–conditioned mediadid not suppress autologous T-cell activation (SupplementaryFig. S4A). This finding is consistent with previous observationsusing the same A375-monocyte coculture model system toinduce MDSCs (16). To evaluate whether the diminished sup-pressive activity observed with MDSCs from 4-IPP–treated A375-monocyte cocultures was simply due to a reduced number ofMDSCspresent in the 4-IPP–treated cultures, we isolatedCD11bþ

HLA-DR� MDSCs from both untreated and 4-IPP–treated A375-monocyte cocultures and compared their respective immuno-suppressive functions. CD11bþHLA-DR� MDSCs from 4-IPP–treated cocultures were significantly less suppressive thanCD11bþHLA-DR� MDSCs from untreated cocultures (Supple-mentary Fig. S4B). Neither melanoma nor monocyte cell viabilitywas significantly affected by the presence of 4-IPP during cocul-ture (Supplementary Fig. S1B), but the possibility that 4-IPP may

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Figure 1.CD14þHLA-DR�/low MDSCs are increased in the peripheral blood of patients with advanced melanoma. A, flow cytometry evaluation of expression ofLineage (Lin; CD3/CD19), CD11b, CD14, HLA-DR, and CD33 in PBMCs obtained from normal donors (ND) and melanoma patients (MEL). An exampleof representative dot plots after excluding aggregates and dead cells is shown (top). Numbers indicate percentages from the populations gated.Names above FACS plots indicate the population gated that was analyzed. Markers analyzed are indicated in the axis of each FACS plot. Thegating strategy used to analyze the samples is illustrated. Gates were set based on isotype controls. B, bar graph showing the percentage of CD14þ

CD11bþHLA-DR�/lo MDSCs in late-stage melanoma patients (MEL; n ¼ 5) versus healthy donors' (ND; n ¼ 5) PBMCs. Data, average � SEM of fiveindependent experiments. � , P� 0.05.

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be influencing the expression/secretion of tumor-derived MDSC-polarizing factors, including active, tumor cell–derived MIF, wasnot ruled out (11).

To determine whether MIF was necessary for the suppressivefunction of established A375-MDSCs, isolated A375-MDSCsfrom A375:monocyte cocultures were treated with 4-IPP for 24hours. This treatment partially attenuated established A375-MDSC inhibition of T-cell proliferation (Supplementary Fig.S5A and S5B), suggesting that MDSC-derived MIF was necessaryfor maximal MDSC immunosuppressive functions. The effects of4-IPP recapitulated those previously observed in establishedmurine MDSCs (10).

To validate these observations, we turned to a murine in vitromodel of bone marrow–derived MDSC induction using GM-CSF and IL6 (17). Similar to 4-IPP treatment during the induc-tion of human MDSCs by a melanoma cell line (Fig. 3), murinebone marrow MDSCs fromMIF-deficient mice were significant-ly less immunosuppressive than their wild-type counterparts(Supplementary Fig. S6C). MIF-deficient bone marrow MDSCsexpressed less of the prototypical murine MDSC marker GR1and more CD11c when compared with bone marrow MDSCsderived from MIF wild-type mice—a finding suggestive of abroader defect in MDSC induction associated with loss of MIF

(Supplementary Fig. S6A and S6B). To rule out the possibilitythat the lower suppressive activity observed with MIF-deficientbone marrow MDSCs was not simply due to fewer MDSCspresent in the differentiated MIF-deficient bone marrowMDSCs, we isolated CD11bþGR1þ MDSCs from both MIFwild-type and MIF-deficient bone marrow cultures and com-pared their immunosuppressive functions. CD11bþGR1þ

MDSCs from wild-type bone marrow MDSCs exhibited potentinhibitory activity on antigen-specific T-cell proliferation, com-pared with MIF-deficient CD11bþGR1þ MDSCs (Supplemen-tary Fig. S7A). When 4-IPP was added during differentiation ofwild-type CD11bþGR1þ bone marrow MDSCs, they were sig-nificantly less immunosuppressive compared than vehicle-trea-ted CD11bþGR1þ bone marrow MDSCs (Supplementary Fig.S7B)—effectively phenocopying 4-IPP–treated human MDSCsand MIF-deficient bone marrow MDSCs (Supplementary Fig.S7A). To confirm that 4-IPP treatment had no off-target effects,MIF-deficient bone marrow MDSCs were treated with 4-IPP orvehicle control. No difference in immunosuppressive activitywas observed between vehicle control and 4-IPP–treated MIF-deficient bone marrow MDSCs (Supplementary Fig. 7A), con-firming the lack of any residual in vitro 4-IPP activity in theabsence of its target, MIF.

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Figure 2.Melanoma MDSCs suppress autologous T-cell activation in an MIF-dependent manner. A–C, melanoma patient-derived CD14þ MDSCs were culturedwith CFSE-labeled autologous T cells and anti-CD3/anti-CD28 beads for 4 days and T-cell activationwas determined. Representative histograms (A) and bar graphsshowing the percentage of proliferated T cells (B) and IFNg production (C). D–F, healthy donor CD14þ monocytes were cultured with CFSE-labeled autologousT cells and anti-CD3/anti-CD28 beads for 4 days and T-cell activation was determined. Representative histograms (D) and bar graphs showing the percentage ofproliferated T cells (E) and IFNg production (F). G and H, melanoma MDSCs were pretreated with or without 50 mmol/L 4-IPP for 24 hours and then addedto CFSE-labeled autologous T cells and anti-CD3/anti-CD28 beads for 4 days. Representative histograms (G) and bar graphs (H) showing the percentage ofproliferated T cells. Data, average � SEM of three independent experiments. �� , P � 0.005; �� , P � 0.0005.






To identify potential mechanistic effectors and/or pathwaysassociated with melanoma monocytic MDSCs, we performedmRNA microarray analyses on cultured monocytes and onA375-MDSCs obtained from either untreated or 4-IPP–treatedA375:monocyte cocultures. Expression profiles from A375-MDSCs were markedly different from those for cultured mono-cytes (Supplementary Fig. S8A).

WhenMIFwas inhibited during A375-MDSC induction, a largesubset of gene products reverted back to levels observed inmonocytes cultured in the absence of tumor cells (SupplementaryFig. S8B). Inflammatory cytokines, chemokines/chemokinereceptors,matrixmetalloproteases, angiogenic growth factors andarachidonic acid/prostaglandin-generating enzymes were all dif-ferentially expressed in A375-MDSCs and restored to "normal"expression by MIF inhibition (Supplementary Fig. S8C).

One gene product of particular interest is that of the NADPHoxidase 4 enzyme (NOX4; Supplementary Fig. 8C). BecauseNADPH oxidases are centrally involved in mediating MDSCimmunosuppressive activities (21, 24), we next validated byqPCR that NOX4 is induced in A375-MDSCs, but not in A375-MDSCs from4-IPP–treated cocultures (Fig. 4A).NADPHoxidases

convert molecular oxygen into superoxide anion upon activationby protein kinase 3 (25), so we next evaluated the relative abilityof phorbol myristic acid (PMA) to induce dichlorofluorescein(DCF)-detectable ROS in A375-MDSCs. DCF fluorescence wasmore strongly induced by PMA in A375-MDSCs than in A375-MDSCs obtained from 4-IPP–treated cocultures (Figs. 4B and C).In accordance with published results (21), CD14þ monocytesfrom freshly isolated PBMC from melanoma patients had moreROS than healthy donor CD14þ monocytes (Supplementary Fig.S1C), and treatment of isolatedmelanomaMDSCs with 4-IPP for24 hours significantly reduced DCF-detectable ROS in these cells(Fig. 4D and E).

Murine MDSCs, when cultured in the presence of appropriategrowth factors, can differentiate into DCs (26, 27). Humanmelanoma MDSCs are characterized by higher levels of the DCmarkers, CD80, CD83, and DC-SIGN compared with normalhuman monocytes (21). Short-term, cytokine-free culture ofhuman melanoma MDSCs moderately increases the expressionof DC markers—including and especially HLA-DR—but withoutloss of CD14 expression. The retention of CD14 expression onthese cells is indicative of a lack of lineage-specific differentiation

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Figure 3.MIF inhibition during melanoma cell line–induced MDSC reduces MDSC suppressive activity. A–C, autologous CFSE-labeled T cells were cultured in the presence offresh healthy donor monocytes (fresh mono), with monocytes cultured for 64 hours in the absence of melanoma cells (cultured mono), or with monocytescocultured with A375 cells in the absence (A375-MDSC), or presence of 4-IPP (A375-MDSC þ 4-IPP; 100 mmol/L, day 0 and 50 mmol/L, day 2). T cells wereactivated with anti-CD3/anti-CD28 beads in the absence or presence of the indicated monocytes/MDSCs for 4 days. Representative histograms (A) and bargraphs showing the percentage of proliferated CFSE-labeled T cells (B) and IFNg production (C). Data, average � SEM of three independent experiments.�� , P � 0.005; �� , P � 0.0005.

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(21). In an effort to determine whether MIF inhibition influencesMDSC ! DC differentiation phenotypes, purified MDSCs frommelanoma patients were cultured in the presence and absenceof 4-IPP for 72 hours (Fig. 5). Treatment with 4-IPP resulted insignificant increases in the percentage of cells (Fig. 5B, top) andrelative expression (MFI; Fig. 5B, bottom) of DC markers CD80,CD83, CD86, CD40, and perhaps more importantly, significantreductions in CD14 and PD-L1 on HLA-DRþ MDSCs.

We next sought to recapitulate these findings using MDSCsderived from the A375:monocyte coculture model. We tested twoindependent models (please see the diagram, Supplementary Fig.S9) for different timing of MIF inhibition by 4-IPP: (i) culturingA375 with monocyte MDSCs in the presence of 4-IPP during theinduction phase, followed by culturing purified A375-MDSCs foran additional 72 hours with no other treatments (4-IPP duringMDSC induction ¼ A375-MDSC þ 4-IPP; Supplementary Fig.S9B), and (ii) addition of 4-IPP after MDSC induction during the72-hour differentiation phase (4-IPP after MDSC induction ¼A375-MDSCs treated with 4-IPP; Supplementary Fig. S9C).Changes in DC marker upregulation were similar, whether 4-IPPwas added during MDSC induction (A375-MDSC þ 4-IPP) orafter MDSC induction (A375-MDSCs treated with 4-IPP) com-paredwith control, untreated A375-MDSCs (Fig. 6A). Specifically,markers associated with DCs—CD80, CD83, CD40, CD1A,CD86, and CD11c—showed a trend toward increased expressionin both 4-IPP treatment conditions of HLA-DRþ A375-MDSCs,although not all conditions resulted in statistically significantincreases (Fig. 6A).

To determine whether these phenotypic marker changes cor-respond to an increase in antigen-specific T-cell functionalresponses, established A375-MDSCs were cultured with or with-out 4-IPP for 72 hours (per Supplementary Fig. S9C) followed byassessment of tetanus toxoid-induced T-cell activation. Neithernormal donor-cultured monocytes (Mono:T) nor untreatedA375-MDSCs could induce tetanus toxoid–specific T-cell activa-tion to any appreciable extent (Fig. 6B and C). In contrast, 4-IPP–treatedA375-MDSCs induced an approximately 4-fold increase intetanus toxoid–specific T-cell proliferation, indicating that MIFinhibition promotes established MDSC differentiation thatresults in antigen-specific T-cell responses.

We next validated these findings in the murine MIF-deficientmodel, using bone marrow MDSCs. Like human melanomaMDSCs treated with 4-IPP, murine MIF-deficient bone marrowMDSCs cultured for 48 hours in cytokine-free media expresselevated CD80, CD83, CD86, and MHC II-IA-IE compared withMIF wild-type bone marrow MDSCs (Supplementary Fig. S10Aand S10B). These MIF-deficient bone marrow MDSCs couldinduce ovalbumin-specific CD4þ T-cell proliferation to a signif-icantly greater extent than bone marrow MDSCs from MIFwild-type mice (Supplementary Fig. S10C and S10D). Together,these findings indicate that loss or inhibition of MIF promotesMDSC differentiation toward a more DC-like cell phenotype thatresults in noticeably improved T cell–mediated antigen-specificimmune responses.

Prostaglandin E2 (PGE2) is a critical determinant of MDSCimmunosuppressive activity and, perhaps more importantly,

Figure 4.MIF maintains NADPH oxidase 4 (NOX4) expression and ROS levels in melanoma MDSCs. A, quantitative PCR analysis of NOX4 mRNA in healthy donor monocytes(n ¼ 3) cultured for 64 hours in the absence (cultured monocytes) or presence of A375 cells. A375:monocyte cocultures were either untreated (A375-MDSCs)or treated with 4-IPP (A375-MDSCsþ4-IPP; 100 mmol/L, day 0 and 50 mmol/L, day 2). Data, average � SEM of triplicate samples. B and C, representativehistogram (B) and bar graph (C) of mean fluorescent intensities (MFI) of DCF-detectable ROS in untreated and PMA-treated A375-MDSCs and A375-MDSCþ 4-IPP.D and E, DCF-detectable ROS levels in CD14þ late-stage melanoma patient-derived MDSCs (n ¼ 3) that were pretreated with either DMSO or 4-IPP(50 mmol/L) for 24 hours. Representative histogram (D) and bar graph (E) showing expression of DCF-detectable ROS in melanoma MDSCs and4-IPP–treated MDSCs. Data, average � SEM of three independent experiments. �� , P � 0.005; �� , P � 0.0005.






can redirect the differentiation of human DC toward function-ally stable MDSCs (16, 28, 29). PGE2 is generated from aprostaglandin synthase 2 (PTGS2, aka COX-2)–dependentconversion of arachidonic acid (released as a product of phos-pholipase A2 catalysis) to PGH2, which is then converted toPGE2 by prostandin E synthase (PTGES). Because the expres-sion of cytosolic PLA2 (PLA2G4), COX-2 (PTGS2), and PTGESare all increased in A375-MDSCs in an MIF-dependent man-ner (Supplementary Fig. S8C), we next sought to determinewhether reductions in PGE2 in 4-IPP–treated melanomaMDSCs were mechanistically linked to MDSC differentiationtoward DC-like cells.

As COX-2 is generally considered to be the rate-limitingstep associated with PGE2 production and release, we firstdetermined whether COX-2 expression is elevated in MDSCsfrom patients with late-stage melanoma. The average mRNAexpression of COX-2 in peripheral blood CD14þ cells isolatedfrom advanced melanoma patients (n ¼ 5) was approximately10-fold greater than that of CD14þ cells isolated from normaldonors (n ¼ 5; Fig. 7A). Inhibition of MIF with 4-IPP in bothpatient-derived MDSCs (Fig. 7B) and A375-MDSCs (Fig. 7C)significantly reduced COX-2 mRNA expression, consistent with

several studies that demonstrated a central regulatory rolefor MIF in dictating COX-2 expression (30–32). The reducedCOX-2 expression in melanoma patient–derived MDSCs trea-ted with 4-IPP established that both A375-MDSCs and A375:monocyte cocultures correlated with significant reductions inPGE2 concentrations (Fig. 7D–F).

Next, we asked whether reconstituting PGE2 to 4-IPP–treatedMDSC cultures was sufficient to reverse the effects of MIF inhi-bition onMDSC!DC-like differentiation (Fig. 7G andH). PGE2added to 4-IPP–treatedmelanomaMDSCs efficiently reduced the4-IPP–mediated increases in both the proportion of HLADRþ

MDSCs (% positive cells; Fig. 7G) and the expression (MFI; Fig.7H) of CD80, CD83, and CD40 markers. It also increased the 4-IPP–dependent reductions in percentages and expression ofCD14 on HLADRþ MDSCs (Fig. 7G and H). Consistent with theobserved reversion in immunophenotype, PGE2 reconstitutionof 4-IPP–treated MDSCs effectively inhibited their ability toinduce tetanus toxoid–specific T-cell proliferation (Supplemen-tary Fig. S11). Taken together, these data suggest that MIF is animportant and previously unrecognized determinant of humanmelanomamonocytic MDSC induction and immunosuppressivefunction. Perhaps more importantly, inhibition of MIF in

Figure 5.MIF inhibition induces a DC phenotype in melanoma patient–derived MDSCs. A and B, CD14þ melanoma patient–derived MDSCs (n ¼ 3) were cultured ex vivofor 72 hours in the absence or presence of 4-IPP (50 mmol/L) and analyzed for the expression of DC markers by flow cytometry. Representative dot plots (A)and bar graphs showing percentages and mean fluorescent intensities (MFI; B) of DC marker expression on HLADRþ MDSC and 4-IPP–treated MDSCs.Data, average � SEM of three independent experiments. � , P � 0.05; �� , P � 0.005.

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established melanoma MDSCs induces the differentiation ofimmunosuppressive MDSCs into cells with DC-like phenotypeand function. These findings provide compelling justification andrationale for therapeutic targeting of MIF in immunosuppressivehuman malignancies.

DiscussionOur data describe the important functional contributionmade

by MIF to humanmonocytic MDSCs. We show that MIF is neces-sary for CD14þHLADRlow MDSC induction, immunosuppres-sion, and in vitro differentiation. Using CD14þ MDSCs derivedfrom advanced-stage melanoma patients, we show that thesmall-molecule MIF antagonist 4-IPP strongly reduced MDSC-mediated suppression of T-cell activation and IFNg production.MIF inhibition in short-term, cytokine-free MDSC cultures led tothe reduction of MDSC-associated cell-surface markers and theinduction of DC markers. MIF inhibitor–treated MDSCs in vitroacquire antigen-specific T-cell stimulatory potential, suggesting afunctionally immunosuppressive MDSC! immunostimulatory,DC-like differentiation by MIF antagonism. MIF-deficient mouseMDSCs phenocopy this DC differentiation and acquisition of DCantigen-presentation functionality. It will be of interest to deter-

mine if thematuration status and immunostimulatory capacity ofthese DC-like cells can be further influenced by culturing themwith DC maturation–inducing cytokine cocktails such as TNFa/IL1b/IL6 (33, 34).

In cancer patients, defective DCs have been implicated inpromoting tumor growth and adversely affecting antitumor effi-cacy of vaccines. Inhibition of VEGF signaling with VEGF-Traptreatment improves DC differentiation/maturation in cancerpatients; these effects, however, are insufficient to improve anti-gen-specific immune responses (35). This lack of immuneresponse is linked to the increased presence of MDSCs in theperipheral blood of the treated patients. Our findings that a safe,bioavailable, and highly efficacious in vivo pharmacologic MIFinhibitor is sufficient to induce MDSC differentiation into func-tionally immunostimulatory DCs suggest that a multifacetedapproach combining anti-MIF therapeutics with established DCmaturation strategies could be highly effective in the treatment oflate-stage cancer patients (35).

Although several studies describe important functional con-tributions by MIF to murine innate immune tumor stromal cellphenotypes, none of these previous studies—including our own(10)—have identified mechanistic effectors and signaling path-ways of MIF-dependent functions (11, 36). In an attempt to

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Figure 6.MIF inhibition inmelanoma cell line–educatedMDSCs induces an immunostimulatory DC phenotype and function. CD11bþMDSCswere isolated from untreated or 4-IPP–treated A375:monocyte cocultures (A375-MDSC or A375-MDSCþ4-IPP). Isolated cells were cultured ex vivo for an additional 72 hours. Alternatively,4-IPP (50 mmol/L) was added to established A375-MDSCs for a 72-hour culture period (A375-MDSC treated with 4-IPP). Cells were then analyzed for theexpression of DC markers by flow cytometry. A, bar graphs showing the percentages of DC marker expressing HLADRþ cells in indicated cells after 72-hourculture period. B and C, control monocytes and established A375-MDSCs were cultured ex vivo for 72 hours in the absence or presence of 4-IPP (50 mmol/L;as shown in Supplementary Fig. 9C). Indicated cells were added to autologous CFSE-labeled T cells in the presence of Tetanus Toxoid (TT; 1.0 mg/mL)for 5 days. Representative histograms (B) and bar graphs (C) showing the percentage of proliferated T cells. Data from B, average � SEM of two independentexperiments. � , P � 0.05; �� , P � 0.005.






identify downstream MIF effectors that could be responsible forMIF-dependent phenotypic and/or functional contributions tohuman MDSCs, we did a microarray analysis of normal mono-cytes, A375-MDSCs, and A375-MDSCs þ 4-IPP cells. Althoughseveral candidate effector mRNAs were identified that are poten-tially regulated by MIF, we initially chose to focus on ROS- andPGE2-regulatory gene products for the following reasons: (i) ROSare a necessary component of MDSC-dependent immune sup-pression (21, 24, 26); (ii) COX-2 inhibitors attenuate humanmonocyticMDSC immunosuppression (16); (iii) COX-2–depen-dent PGE2 generation maintains MDSC phenotype and functionwhile inhibiting DC development (28, 29); and (iv) MIF is welldocumented to regulate ROS-regulatory and PGE2-regulatorymechanisms in a variety of cell types (31, 37–39). Our currentfindings indicate that MIF is an important determinant of severalPGE2-regulatory enzymes' expression—most notably, COX-2.MIF inhibitor treatment reduces MDSC PGE2 levels and exoge-

nously reconstituted PGE2 maintains MDSC marker expressionwhile reducing DC-like phenotype in these cells. These findingssuggest thatMIFmaintainsMDSC suppressive phenotype, at leastin part, via PGE2 production.

It is less clear how MIF mechanistically dictates MDSC immu-nosuppressive activity. Our results clearly indicate an importantfunctional role for MIF in maintaining NOX-4 expression andDCF-detectable ROS inMDSCs. At the same time,MIF is centrallyimportant to COX-2 expression andmaintaining PGE2 levels thatare necessary for MDSC immunosuppressive function (16). It islikely that both of these effector mechanisms (ROS and PGE2maintenance)—and potentially others—are involved in MIF-dependent MDSC suppression of T-cell activation, but what isless clear is how MIF regulates such a broad array of immuno-suppressive and differentiation-regulating gene products.Although we are currently evaluating the signaling requirementsfor the MIF receptor CD74 (39), in MIF-dependent MDSC

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Figure 7.MIF maintains melanoma MDSC suppressive phenotype through COX-2/PGE2 production. A–C, COX-2 mRNA expression was analyzed using qPCR.Bar graphs showing the relative mRNA expression of COX-2 in freshly isolated CD14þ cells from melanoma patients (MEL; n ¼ 7) vs. healthy donors (ND;n ¼ 5; A), in melanoma patient–derived MDSCs pretreated for 24 hours with DMSO or 4-IPP (50 mmol/L) (B), and in A375-MDSCs obtained fromuntreated or 4-IPP–treated A375:monocyte cocultures (C). D–F, PGE2 levels in supernatants from melanoma patient–derived MDSCs treated for 24 hourswith DMSO or 4-IPP (D), from A375-MDSCs treated for 24 hours with DMSO or 4-IPP (E), and from A375-MDSCs obtained from untreated or 4-IPP–treatedA375:monocyte cocultures (F). G and H, melanoma patient-derived CD14þ MDSCs (MEL; n ¼ 2) were either untreated or treated with 4-IPP (50 mmol/L),PGE2 (10 mmol/L) or with 4-IPP þ PGE2 for 72 hours and analyzed for DC marker expression. Percentages (G) and MFI (H) of DC marker-expressingHLADRþ cells in MDSC, MDSC þ 4-IPP, MDSC þ PGE2, and MDSC þ 4-IPP þ PGE2 cells. Data, average � SEM of two independent experiments.� , P � 0.05; �� , P � 0.005; �� , P � 0.0005.

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phenotypes, we cannot rule out the possibility that MIF's influ-ences on these cellsmay be receptor independent. This is based onthe fact that enzymatically inactive, CD74-binding competent,N-terminal proline MIF mutants are entirely unable to reconsti-tute MIF-dependent MDSC induction/function (11, 40). This,coupled with the fact that the MIF enzymatic inhibitor 4-IPPreportedly has little to no MIF:CD74 antagonist activity (41) butvery effectively phenocopies MIF deficiency inmonocytes/macro-phages (current study and ref. 10), is highly suggestive of a CD74-independent signaling function. If this is, in fact, the case, alter-native mechanisms for MIF-dependent modulation of MDSCfunctionality include alternative outside-in signaling via non-cognate MIF receptors (42, 43) or an intracellular mechanism ofaction via known (44), or presently unknown, pathways. It will beimportant to identify the precise mechanism of action goingforward as it will not only provide important information regard-ing a seemingly central node of control for MDSC immunoreg-ulatory functions in both mice and humans, but also because itcould point to a previously unknown function for the highlydruggable enzymatic active site of MIF.

AlthoughMIFhasbeen shown to regulateMDSC induction andsuppressive activity in murine models of cancer (10, 11), theseresults illuminate the functional contribution by MIF to humanMDSCs. Our findings introduce a role for MIF in regulatinghuman melanoma MDSC differentiation. Our data demonstrat-ing that 4-IPP effectively reduces in vitro MDSC immunosup-pression while increasing DC-like antigen-specific T-cellresponses suggest that in vivo MIF therapeutic targeting maysimultaneously reduce cancer-induced MDSC-mediated T-cellinactivation while enhancing antitumor antigen-specific T-cellresponses in metastatic melanoma patients.

It is not yet known whether current therapies that targetadaptive immune tumor suppressive checkpoints, such as anti–CTLA-4 (ipilimumab) and anti–PD-1 (pembrolizumab or nivo-lumab), could act in synergy with 4-IPP targeting of MIF. Someevidence supports the former possibility: A trial immunotherapyconsisting of ipilimumab (in combination with irradiated, GM-CSF-expressing, autologous tumor cells) provokes a humoralresponse resulting in the elicitation of clinically relevant anti-MIFautoantibodies (12). Given that the targeting of CTLA-4 with

ipilimumab is efficacious in patients with metastatic melanoma(45, 46), it is possible that combinatorial targeting of adaptiveimmunosuppressive mechanisms (anti–CTLA-4) and innateimmunosuppressive mechanisms (anti-MIF) may providesynergistic clinical responses in patients with advanced-stagemelanoma.

Disclosure of Potential Conflicts of InterestR.A. Mitchell is an inventor on patents pertaining to 4-IPP as an anticancer

therapeutic agent targeting MIF.

Authors' ContributionsConception and design: K. Yaddanapudi, R.A. MitchellDevelopment of methodology: K. Yaddanapudi, G. Lamont, R.A. MitchellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):G. Lamont, E.J. Kim, N. Al Rayyan, J. Richie, S. Waigel,A. WiseAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Yaddanapudi, G. Lamont, S. Waigel, R.A. MitchellWriting, review, and/or revision of the manuscript: K. Yaddanapudi, R.A.MitchellAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.E. Rendon, G. Lamont, N. Al Rayyan, J. Richie,S. AlbeituniStudy supervision: K, Yaddanapudi, R.A. Mitchell

AcknowledgmentsThe authors thankMelissa B. Hall for collecting and providing themwith de-

identified melanoma patient peripheral blood samples. The authors also thankDrs. Jun Yan and Jill Suttles for helpful discussions and insightful comments onthe manuscript.

Grant SupportThis work was supported in part by NIH CA186661 to R.A. Mitchell and K.

Yaddanapudi and NIH CA102285 to R.A. Mitchell.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 12, 2015; revised September 22, 2015; accepted October 16,2015; published OnlineFirst November 24, 2015.

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Yaddanapudi et al.




2016;4:101-112. Published OnlineFirst November 24, 2015.Cancer Immunol Res Kavitha Yaddanapudi, Beatriz E. Rendon, Gwyneth Lamont, et al. Suppression and DifferentiationMIF Is Necessary for Late-Stage Melanoma Patient MDSC Immune

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