CD84 is a regulator of the immunosuppressivemicroenvironment in Multiple Myeloma

Hadas Lewinsky, … , Steven Rosen, Idit Shachar

JCI Insight. 2021. https://doi.org/10.1172/jci.insight.141683.

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Research Hematology Oncology

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Lewinskyetal. 1

CD84isaregulatoroftheimmunosuppressive

microenvironmentinMultipleMyeloma

HadasLewinsky1*, EmineG.Gunes2,3*, KerenDavid1, LihiRadomir1,MatthiasP.Kramer1,

BiancaPellegrino1,MichalPerpinial1,JingChen8,Ting-fangHe8,AnthonyG.Mansour3,Kun-

YuTeng3, SupriyoBhattacharya9, EnricoCaserta2,3, EstelleTroadec2,3, Peter Lee8,Mingye

Feng8, JonathanKeats2,AmritaKrishnan2,3,MichaelRosenzweig2,3, JianhuaYu3,MichealA.

Caligiuri3, Yosef Cohen5, Olga Shevetz6, Shirly Becker-Herman1, Flavia Pichiorri2,3, Steven

Rosen2,3*andIditShachar1#

*Thetwofirstandlastauthorscontributedequallytothiswork

1DepartmentofImmunology,DepartmentofBiochemistry,WeizmannInstituteofScience,

Rehovot, Israel,2JudyandBernardBriskinCenter forMultipleMyelomaResearch,Cityof

Hope,Duarte,CA,USA;3DepartmentofHematologicMalignancies&TranslationalScience,

Beckman Research Institute, City of Hope, Duarte, CA, USA. 4Translational Genomics

ResearchInstitute,Phoenix,AZ,USA.5SanzMedicalCenter,LaniadoMedicalCenter,Netanya,

Israel. 6Hematology Institute, Kaplan Medical Center, Rehovot, Israel. 7Department of

ComputationalandQuantitativeMedicine,BeckmanResearchInstitute,CityofHope,Duarte,

CA, USA. 8Department of Immuno-Oncology, Beckman Research Institute, City of Hope,

Duarte, CA, USA. 9 Translational Bioinformatics, Center for Informatics, Department of

ComputationalandQuantitativeMedicine,CityofHope,Duarte,CA,USA.

#CorrespondingAuthor:IditShachar,DepartmentofImmunology,WeizmannInstituteofScience,Rehovot76100,Israel;Tel:972-8-9344257,Fax:972-8-9344141,Email:idit.shachar@weizmann.ac.il

Lewinskyetal. 2

Abstract

Multiplemyeloma(MM)ischaracterizedbyanaccumulationofmalignantplasmacells(PCs)

withinthebonemarrow(BM).TheBMmicroenvironmentsupportssurvivalofthemalignant

cells and is comprised of cellular fractions that foster myeloma development and

progression, by suppression of the immune response. Despite major progress in

understanding thebiologyandpathophysiologyofMM, thisdisease is still incurable and

requiresaggressivetreatmentwithsignificantsideeffects.CD84isaself-bindingimmuno-

receptor belonging to the signaling lymphocyte activating molecule (SLAM) family.

Previously,weshowedthatCD84bridgesbetweenchroniclymphocyticleukemiacellsand

theirmicroenvironment,andregulatesTcellfunction.Inthecurrentstudy,weinvestigated

theroleofCD84inMM.OurresultsshowthatMMcellsexpresslowlevelsofCD84.However,

these cells secrete the cytokine, macrophage migration inhibitory factor (MIF), which

induces CD84 expression on cells in their microenvironment. Its activation leads to an

elevation of expression of genes regulating differentiation to monocytic/granulocytic-

(M/G)-myeloidderivedsuppressorcells(MDSCs)andupregulationofPD-L1expressionon

MDSCs,which together suppressT cell function.DownregulationofCD84or its blocking

reduceMDSCaccumulation,resultinginelevatedTcellactivityandreducedtumorload.Our

datasuggestthatCD84mightserveasanoveltherapeutictargetinMM.

Lewinskyetal. 3

Significance

Multiple myeloma (MM) is a hematological malignancy caused by an accumulation of

malignantplasmacellsinthebonemarrow(BM).Myeloid-derivedsuppressorcells(MDSC)

havebeenshowntoplayapivotalroleinregulationofthehostimmuneresponsesinMM.

OurresultsshowthatCD84regulatesimmunesuppressionbyaffectingMDSCdifferentiation

andfunction.Thus,CD84canserveasanoveltherapeutictargetinMMbyregulatingMDSC

functionandTcellmediatedanti-tumoractivity.

Highlights

1. CD84isexpressedonmultiplemyeloma-associatedmyeloidderivedsuppressorcells

(MDSCs)andregulatestheirfunctionandTcellactivity

2. In vivo ablation or blocking of CD84 in humanpatient samples andmice reduces

MDSCnumbersandactivity,resultinginenhancedfunctionalityofTcellsandreduced

tumorload.

Lewinskyetal. 4

Introduction

Multiple myeloma (MM) is a hematological malignancy caused by an accumulation of

malignantplasmacellsinthebonemarrow(BM).TheBMmicroenvironmentisknownto

havesupportiverolesforMMsurvival,growth,andcelladhesion-mediateddrugresistance.

ThemicroenvironmentoftheBMconsistsofcellularelementssuchasosteoblasts,stromal

cells, endothelial cells, osteoclasts and immune cells, and a non-cellular compartment.

Interactions between myeloma cells and other cells that are part of the tumor

microenvironment foster myeloma development and progression by suppression of the

immune response. The interactions of MM cells with accessory cells in their

microenvironmentregulatethetransformationoftheBMmicroenvironmentintoatumor

promotingenvironmentthroughasuppressiveimmunemilieucontainingcytokines,growth

factorsandchemokines(1-3).

Although themajority of studies have focused on the BM stroma and osteoclasts inMM

pathogenesis, less attention has been given to the potential role of additional cell types

involved inmodulation of the immune response (4-6). In recent years, myeloid-derived

suppressorcells (MDSC)havebeenshowntoplayapivotal role inregulationof immune

responses in cancer. MDSCs are comprised of a heterogeneous myeloid cell population

consistingofpathologicallyactivatedmyeloidprogenitorsandimmaturemyeloidcells,with

robust immunosuppressiveactivity (7). Inhealthy individuals,MDSCsarerareorabsent,

whileanincreasednumberofMDSCshavebeenidentifiedintumors(2).

It was recently shown thatMDSCsmediate suppression of T-cell responses through the

induction of T-cell anergy and T-reg development in the MM microenvironment (8),

rendering lymphocytes unable to control tumor infiltration and progression (9). The

immunecheckpointmolecule,Programmedcelldeath1(PD-1)togetherwithitstwoligands,

programmeddeath-ligand 1 (PD-L1) and 2 (PD-L2) are crucial for themaintenance of a

malignant pro-survival microenvironment (10). In addition, the checkpoint molecules

Lymphocyte-ActivationGene3(LAG-3),CytotoxicTLymphocyteAntigen-4(CTLA-4),2B4,

Tim-3andCD160playasignificantroleinexhaustionofTcells(11).

Thesignaling lymphocyteactivationmolecule(SLAM) familyofreceptorsarecellsurface

proteinsthatmodulatetheimmuneresponse(12).CD84(SLAMF5)isamemberoftheSLAM

family,which self-associates, forminghomophilicdimers, and ismostlyexpressedby the

Lewinskyetal. 5

hematopoietic cells (13-16). Previous studies have shown that CD84 regulates a novel

survivalpathwayinChronicLymphocyticLeukemia(CLL)(17).ItbridgesbetweenCLLand

variouscellsinthemicroenvironment,mediatingtheirinteractionbothinvitroandinvivo

(18). Inaddition,cell-cell interactionsmediatedbyCD84inhumansandmiceelevatethe

expression of PD-L1 on CLL and the surrounding microenvironment, as well as the

expressionofPD-1onTcells,regulatingtheTcellresponseintheCLLmicroenvironment

(19).

Inthepresentstudy,wefocusedonunderstandingthefunctionofCD84inMMcancercells

andtheirmicroenvironment.OurresultsdemonstratethatMMcellsexpressundetectableor

low levels of CD84. The malignant cells induce expression of CD84 on cells in their

microenvironmentbysecretionofthecytokine,macrophagemigrationinhibitoryfunction

(MIF).CD84regulatesPD-L1/PD-1andexhaustionmarkerexpressiononMDSCsandTcells,

respectively,resulting in thedownregulationof the immuneresponse. Invivoblockingof

CD84leadstoreducedaccumulationofMDSCsinthetumormicroenvironment,andelevated

activityofTcells,throughthecontrolofessentialfunctionalpathwaysinMDSCsleadingto

reducedtumorload.OurresultssuggestanoveltherapeuticstrategytargetingCD84inthe

MM microenvironment as a promising approach to induce T cell mediated anti-tumor

activity.

Lewinskyetal. 6

Results

CD84isexpressedonimmunosuppressivecellsintheMMmicroenvironment

MMcellsareprotectedfromapoptosisbyimmunosuppressiveaccessorycells,whicharean

integralpartoftheirmicroenvironment(8,20).TodeterminewhetherCD84playsarolein

theMMdiseaseandtofurtherunderstandtheinteractionofthesemalignantcellswiththeir

microenvironment, we first evaluated CD84 expression on MM (gating is shown in

SupplementaryFig1A).AsshowninFig.1A,anincreaseinCD84expressionwasdetected

onMMcellscomparedtoitsexpressiononcellsfrompatientswithmonoclonalgammopathy

ofunknownsignificance(MGUS),theprecursortoMM.AnalysisoftheMMRFCoMMpassIA9

dataset(SupplementaryFig1B)revealedlowlevelsofCD84mRNAinhumanMMcelllines.

UndetectabletolowexpressionofCD84wasfoundonhumanMMcelllines(Supplementary

Fig1C-D).The5TGM1MMplasmacelllineisacommonlyusedinamousemodelforMM

which (21).AnalysisofCD84expressionon5TGM1cellsderivedfromspleenandBMof

injectedmiceshowedsignificantlyhigherexpressionlevelsofCD84onBMcells,compared

to itsexpressiononthemalignantcellsderived fromthespleen(Fig1B),suggestingthat

CD84mighthaveanimportantroleintheBMmicroenvironment.

While the elevation in the expression of CD84 on malignant cells was minimal, the

upregulation of CD84 expression on cells derived from the tumor microenvironment

compared to its expression on healthy donorswas strongly enhanced. BM stromal cells

derivedfromMMpatientsexpressedhigherlevelsofCD84comparedtocellsderivedfrom

bothpatientswithsmolderingdiseaseandhealthydonors(Fig1C).Inaddition,analysisof

BMstromacellsderivedfromthe5TGM1MMmurinemodelshowedhigherlevelsofCD84

comparedtoitsexpressiononmicenotcarryingtumors(Fig1D).Asignificantupregulation

ofCD84expressionwasdetectedonBM-(Fig1E)andPB-(Fig1F)derivedhumanmyeloid

CD14+cells(around10timeshigher)comparedtoitsexpressiononCD14-cellsandCD14+

cellsderivedfromhealthyhumandonors,suggestingthattheCD14+populationhasamore

significantroleinMMpatients.

Next,theMDSCpopulationswereanalyzed(gatingisshowninSupplementaryFig1E).An

increasedabundanceoftheM-MDSCpopulationandCD14+cellswasdetectedinPBderived

fromMMpatients(SupplementaryFig1F-H).SinceMDSCsplayanimportantroleintumor

maintenance, CD84 expression was determined on M-MDSC (CD14+, CD11B+, HLA-DR-,

Lewinskyetal. 7

CD15-)andG-MDSCs(CD15+,CD11B+,HLA-DR-,CD14-)derivedfromBMpatientsamples.

Strikingly,asignificantelevationofCD84expressionwasobservedonbothcelltypesderived

from MM patients compared to its expression on cells derived from the earlier, pre-

malignant, stage of smoldering myeloma as well as from healthy BM (Fig. 2 A-B,

SupplementaryFig.1F).TofurthervisualizeCD84expressionontheMMmicroenvironment,

weusedt-distributedstochasticneighborembedding(t-SNE),whichisamethodtovisualize

high-dimensional data by graphing similar high-dimensional points close together and

dissimilarpointawayfromeachother.AsseeninFig.2CandSupplementaryFig2,clusters

composedofM-MDSCs,G-MDSCsandsomeoftheMMcellsexpressedCD84.Thesefindings

suggestthatCD84ismainlyexpressedonMDSCsderivedfromtheBMmicroenvironmentof

MMpatients.TheelevationofitsexpressionsuggeststhatCD84mightplayaroleinthecross

talkbetweenthetumorcellsandtheirmicroenvironment.

CD84activationupregulatesPD-L1expressionintheMMmicroenvironment

CD84waspreviouslyshowntoregulateexpressionofthePD-L1immunecheckpointligand

onmalignantCLLcellsandintheirmicroenvironment(19).WethereforeanalyzedPD-L1

expressiononMMcells, and cellsderived from theirmicroenvironment.Higher levelsof

surfaceexpressionofPD-L1weredetectedonBMstromalcellsofMMpatientscomparedto

its expression on cells from healthy donors (Supplementary Fig 3A), and in MMmouse

models compared to its expression BM stromal cells derived from tumor-free mice

(SupplementaryFig3B).TodirectlydeterminewhetherCD84regulatesPD-L1expressionin

MM,bonemarrowMMandstromalcellstakenfromMMpatientswerestimulatedwithanti-

CD84–activating antibody, as previously described (18, 19). PD-L1 mRNA and surface

proteinlevelsweresignificantlyelevatedinCD84activatedBMmyelomacells(Fig.3A)and

stromalcells(Fig.3B)derivedfromthepatients.Inaddition,activationofCD84expression

onthemurine5TGM1celllineinducedPD-L1expression(Fig.3C),anditsblockingusingthe

B4 blockingmAb (Supplementary Fig 3C; (18, 19)) lowered PD-L1mRNA (Fig. 3D) and

protein (Fig. 3E) levels compared to cells treated with an isotype control. Furthermore,

reducedexpressionofCD84in5TGM1cellsusingsiCD84resultedindownregulationofPD-

L1mRNAlevelscomparedwiththecellstransfectedwithcontrolsiRNA(Fig.3F).

Lewinskyetal. 8

Next, the regulationofPD-L1expressionbyCD84wasanalyzed inCD14+cells.Elevated

levels of cell surface PD-L1 were detected on CD84 activated healthy donor-derived PB

CD14+cells(Fig.3G).

Furthermore,PD-L1mRNAlevelswereelevatedinCD84activatedM-MDSCsandG-MDSCs

fromMMpatientBMaspirates(Fig.4A-B).ToconfirmtheeffectofCD84onPD-L1,M-MDSCs

(Ly6G-,Ly6C+,CD11b+,CD11c-)-andG-MDSCs(Ly6G+,Ly6Clow,CD11b+,CD11c-)from5TGM1

injectedmicewereblockedusingtheanti-CD84antibody,B4.Followingthismanipulation,

mRNAlevels(Fig.4C-D)aswellasproteinsurfaceexpression(Fig.4E-F)ofPD-L1were

significantlyreduced.

Next,theeffectofCD84expressedonMDSCswasanalyzed.SortedG-orM-MDSCsfromMM

injectedmiceweretreatedwithB4blockingantibodyoranisotypecontrol,incubatedwith

Tcells,andco-culturedfor72hrs.AsseeninFigs.4Gand4H,blockingCD84significantly

reducedthesuppressivefunctionofMDSCs,andincreasedCD8+TcelldivisionandIFN-γ

secretion.

To identify thedownstreamevents inducedbyCD84 that regulatePD-L1expression, the

signalingcascadeinducedbyCD84inTHP1 cellswasfollowed.Tothisend,weestablished

aCD84CRISPR/Cas9systeminthemonocyticcellline,THP1,withconstitutiveknockoutof

CD84expression(SupplementaryFig.3D).LackingCD84,downregulatedPD-L1expression

on thesecells (Fig.4I). StimulatingCD84withananti-CD84activatingantibodyonCD84

expressing THP1 cells resulted in significantly elevated surface protein levels of PD-L1

comparedtocellstreatedwithIgGcontrolantibodies(SupplementaryFig3E).Inaddition,

asshowninFig.4J,levelsofpAKTandpS6whosepathwaysregulatePD-L1expression(22)

weremarkedlyincreaseduponCD84stimulation.

Next, we wished to understand whether CD84 induces an immunosuppressive

microenvironment in MM patients. PB CD14+ cells derived from healthy donors were

incubatedaloneorinthepresenceofthehumanMMcellline,MM.1SandhealthydonorT

cells,inthepresenceorabsenceofcontrolIgG2aortheB4blockingmAbs.Theexpression

ofPD-L1/PD-1andcellsurvivalwereanalyzed.TheMM.1Scelllineinducedexpressionof

PD-L1ontheCD14+cells(Fig.5A)andPD-1onTcells(Fig.5B).Thiseffectwasabrogatedin

thepresenceoftheanti-CD84blockingantibody,B4.Inaddition,blockingCD84elevatedthe

TcellmediatedkillingofMMcelllines,indicatedbyincreased7AADstainingoftheMMcell

Lewinskyetal. 9

line(MM.1SGFP+/Luc+)(Fig.5C),andincreasedchromiumreleasebyMMcellsinakilling

assay(SupplementaryFig3H).

We further analyzed the effect of CD84 blocking on primary human MM bone marrow

aspiratesfromMMpatients.BMaspiratesweretreatedinculturewitheithercontrolIgG2a

ortheB4mAbs,andthesurvivalofthecellswasanalyzed.AsseeninFig5D,blockingCD84

inducedapoptosisofthemyelomacells,leadingtoreducednumbersofthesemalignantcells.

ThistreatmentalsodownregulatedPD-L1surfaceexpressionontheMMcells(Fig.5E).In

addition, blocking CD84 reduced the numbers ofM-MDSCs (Fig. 5F) and lowered PD-L1

expressiononthesecells(Fig.5G).ThistreatmenthadalmostnoeffectonthenumberofG-

MDSCs (Fig.5H),while the levelsofPD-L1were reducedon thesecells, aswell (Fig.5I).

Furthermore,blockingCD84reducedtheexpressionoftheTcellexhaustionmarkersPD-1,

LAG-3andCTLA-4on thesurfaceofCD4andCD8Tcells, respectively (Fig5 J-K).Taken

together,theseresultsshowthatCD84regulatesimmunosuppressionandTcellactivityin

MMpatientsamples.

CD84expressioniselevatedinaMIF-dependentmannerintheMMmicroenvironment

MMcellsexpressundetectabletolowlevelsofCD84,whilecellsintheirmicroenvironment

expresshighlevelsofthisreceptorinthepresenceofthetumor.Wethereforehypothesized

thattheinductionbytheMMcellsofCD84andPD-L1expressiononthesurroundingcells

maybemediatedby secretionof cytokines.Todeterminewhethermyelomacells induce

CD84andPD-L1expressionontheirmicroenvironment,healthydonorderivedPBCD14+

cellswereincubatedaloneorinthepresenceofthreedifferenthumanMMcelllinesMM.1S,

U266andKMS11,whichdonotexpressCD84(SupplementaryFig.1C-D),andPD-L1and

CD84surfaceproteinlevelswereanalyzedafter48hrs.TheMMcelllinesinducedincreased

cellsurfacelevelsofCD84(Fig.6A)andPD-L1(Fig.6B)ontheCD14+cells.

Wehavepreviouslyshownthatthecytokine,macrophagemigrationinhibitoryfactor(MIF),

inducesCD84expression inCLLcellsbybinding to its receptorCD74(17).We therefore

sought to determinewhether the upregulated expression of CD84 on cells in the tumor

microenvironment ismodulatedbyMIF.MIFproteinexpression levels inhumanMMcell

lineswereanalyzedusingwesternblotanalysis.AsdemonstratedinFig6C,MIFwashighly

expressedinthehumanMMcelllines.

Lewinskyetal. 10

To determine whether MM cells induce CD84 and PD-L1 expression on their

microenvironmentinaMIF-dependentmanner,PBCD14+cellsfromthreedifferenthealthy

donorsortheTHP1monocyticcelllinewereincubatedaloneorinthepresenceofhuman

recombinantMIF.After48hrs,surfaceproteinlevelsofCD84andPD-L1weremeasuredon

thesecells.MIFactivationelevatedexpressionofCD84andPD-L1onthesecells(Fig6 D-E).

Next, stromatakeneither fromhealthymice(Fig6F)orMMinjectedmice(Fig6G)were

incubatedwithrecombinantmurineMIF.MIFstimulationupregulatedCD84expressionon

stromafrombothuntreatedandMM-injectedmice.

To demonstrate that MIF secreted from the malignant cells regulates CD84 and PD-L1

expressiononcellsinthetumormicroenvironment,MIFexpressionwasknockeddownin

MM.1Scells.Reductionofabout40%inMIFmRNAlevelswasobservedincellstransfected

withsiMIF(SupplementaryFig.2F-G).SupernatantcollectedfromMM.1Stransfectedwith

MIForcontrolsiRNAwasthentransferredtoisolatedCD14+PBMCsderivedfromahealthy

donor and CD84 and PD-L1 expression levels on the CD14+ cells were analyzed.

DownregulationofCD84(Fig.6H)andPD-L1(Fig.6I)cellsurfacelevelsweredetectedon

cellsincubatedwiththeconditionedmediumcontainingreducedlevelsofMIF.

TofurtherevaluatetheeffectofMIFonCD84andPD-L1expressiononCD14+cells,healthy

donor-derivedPBCD14+cellswereincubatedaloneorinthepresenceofthehumanMMcell

lineMM.1SinthepresenceorabsenceoftheMIFinhibitor,ISO-1.InhibitionofMIFsignaling

activitysignificantlyreducedcellsurfaceexpressionofCD84(Fig7A)andPD-L1(Fig7B)on

CD14+cells.Moreover,thePBMCobtainedfromhealthydonorsgainedinducedabilitytokill

MM1.SGFP+/LucandRajicellsinthepresenceofISO-1(Fig7CandSupplementaryFig3H-

I).Thus,MIFregulatesCD84andPD-L1expressionlevelsonCD14+cells.

Finally,wholeBM frommicebearingMMwere treatedwith ISO-1and thepercentageof

CD84 on G- or M-MDSCs was determined. ISO-1 treatment significantly reduced the

expressionofCD84onMDSCsderivedfrommicebearingMM(Fig7D-E).

Together,theseresultsshowthattheexpressionandfunctionofCD84areregulatedbyMIF

secretedfromthemalignantcells.

CD84regulatesfunctionalandimmunosuppressivepathwaysinhumanMMMDSCs

Lewinskyetal. 11

MDSCsplayanimportantroleinthesupportofMMcells(8).Sincethesecellshighlyexpress

CD84duringMMprogression,wenextwishedtofollowthepotentialdirectroleofCD84on

thesecells.

SortedG-MDSCsorM-MDSCsderivedfrompatientbonemarrowaspirates(Supplementary

Fig4.A-B)wereactivatedwitheitherCD84orIgGcontrolantibodies.After24hrs,RNAwas

purified,andactivationwasconfirmedbyelevationofPD-L1expression.RNAsequencing

was conducted using Mars-seq. Principal component analysis (PCA) and dendrogram

analysisshowedthatthesamplescouldbeidentifiedaseitherCD84activatedorIgGcontrol

treatmentbasedonsimilarityingeneexpressionprofiles(SupplementaryFig4C-D).CD84

activatedM-MDSCcellsdifferentiallyexpressed433genes(determinedasgeneswithap

value<0.05andabasemeanofmorethan5,afteradjustmentwiththeRpackagefdrtool).

Thesegenesweredividedintoclustersofupregulatedanddownregulatedgenes(Fig.8Aand

SupplementaryFig.4E).Eachclusterwasthenanalyzedforgeneontologypathwaysusing

EnrichR. The downregulated cluster did not contain pathways related toMDSC function

(resultsnotshown).However,intheupregulatedgenes,pathwaysrelatedtoMDSCfunction

wereidentified(Fig.8B,SupplementaryFig4F),includinggenesrelatedtoROSproduction,

Ragereceptorbinding,andabnormalTcellselection.

A more detailed analysis revealed genes including S100A9, CYBA (p22phox) and CCL5,

related to the above pathways (Fig 8C). It was previously shown that in the absence of

S100A9,theMDSCpopulationisreduced,resultinginreducedtumorsizeintumorbearing

mice, while its overexpression leads to an accumulation of MDSCs and impaired DC

differentiation(23).CYBA(P22phox)isasubunitoftheNADPHoxidase(NOX2),andNOX2

is the main source of ROS, used by MDSCs to suppress T cells (24). Further, CCL5 has

previously been shown to play an important role in tumor progression, by inhibiting

infiltrationofCD8+TcellsandincreasingthemigrationofMDSCsintothetumorarea(25,

26).WenextvalidatedtheregulationofexpressionofthesegenesbyCD84inCD84activated

sortedhumanM-MDSCsfromMMbonemarrowaspirates.S100A9mRNA(Supplementary

Fig.4G)andCYBAmRNA(SupplementaryFig4H)wereupregulatedinCD84activatedcells.

BlockingCD84onwholeculturesofprimarypatientMMbonemarrow,reducedS100A9(Fig.

8D) and CYBA (Fig. 8E) in M-MDSC cells. Moreover, blocking CD84 by the B4 mAb

significantlydecreasedthemRNAandproteinexpressionofS100A9(Fig.8F)andCYBA(Fig.

Lewinskyetal. 12

8G)inmouseM-MDSCsfrom5TGM1injectedmice.Together,thissuggeststhatCD84affects

pathwaysregulatingthefunctionalityofM-MDSCs.

Activation of G-MDSC cells resulted in fewer differentially expressed genes. Principal

componentanalysis (PCA)anddendrogramanalysis showed that the sequencedsamples

couldbe separated into eitherCD84activatedor IgG control treatment groupsbasedon

similarityingeneexpressionprofiles(SupplementaryFig4I-J).Thesegenesweredivided

intoupregulatedanddownregulatedclusters,andanalyzed(Fig9AandSupplementaryFig

4K).Whilethedownregulatedclusterswerenotassociatedwithnoteworthypathways(not

shown),theupregulatedclusteredgeneswererelatedtotheHIF1αnetworkandabnormal

neutrophilphysiology(Fig9BandSupplementaryFig4L).Wethenfollowedseveralspecific

upregulated genes, CMTM6,HIF1α (included due to upregulated pathway gene ontology

terms, although p value=0.18), EP300, and BCL2A1 (Fig 9C). CMTM6 is a known PD-L1

regulatoronmanycancercelltypes(27). ThetranscriptionfactorHIF1α,togetherwithits

co-activator EP300, regulatesmany genes involved in the hypoxia response. Previously,

HIF1αhasbeenshowntohavearolebothindifferentiationandPD-L1expressiononMDSCs

(28).OverexpressingBCL2A1specificallyonG-MDSCswasshowntoincreasethesurvivalof

G-MDSCsandtheirsuppressivecapabilities(29).

TheexpressionofthesegenesfollowingCD84activationwasvalidatedbyRT-qPCR.CMTM6

(SupplementaryFig.4M),BCL2A1(SupplementaryFig.4N)andHIF1α(SupplementaryFig.

4O) mRNA levels were upregulated in sorted G-MDSCs from human MM patients.

Furthermore,blockingCD84usingtheB4antibodyonwholeculturesofprimarypatientMM

bonemarrow,alsoreducedtheexpressionofCMTM6(Fig.9D),BCL2A1(Fig.9E)andHIF1α

(Fig. 9F).Moreover, blocking CD84 by B4mAb, reduced RNA and protein expression of

CMTM-6(Fig.9G)andBCL2A1(Fig.9H)inmouseG-MDSCs.Inconclusion,CD84regulates

pathways that are important for both MDSC development and suppressive function. In

addition,CD84servesdifferent,yetimportant,rolesinthetwoMDSCsubsets.

TheinvivoroleofCD84inMM

TodeterminetheinvivoroleofCD84inMM,5TGM1murineMMcellsandMM.1Shuman

myeloma cells were I.V. injected into syngeneic immune-competent KaLwRij mice

(SupplementaryFig.5A,Fig.10A)orintoimmuno-deficientNSGmice(SupplementaryFig.

Lewinskyetal. 13

5B),respectively, leadingtoasignificantincreaseinMDSCnumbersinthePB(Fig.10Aand

SupplementaryFig.5C),toincreasedCD84expression(Fig10BandSupplementaryFig5D)

onMDSC cell surface and to increased PD-1 cell surface expression onBMCD8+T cells

(SupplementaryFig5E).

TodirectlyanalyzetheroleofCD84expressedonthemicroenvironmentinvivo,chimeric

miceweregenerated.CD84-/-orWTmicewerelethallyirradiatedandinjectedwithKaLwRij

BMcells.After2months,5TGM1cellswereinjectedintothesemice,andafteranadditional

3weeks, themicewere sacrificed, and their BM, blood and spleenswere harvested and

examined(SupplementaryFig5F).DeficiencyofCD84onstromacellsresultedinareduction

in tumor load in the bonemarrow (Fig. 10C) and spleen (Fig. 10D), resulting in smaller

spleensize(SupplementaryFig5G)aswellasreducedIgG2bintheblood(Fig.10E).PD-L1

expressionontheMMcellswasdownregulatedinbothcompartments(Fig.10F).Analysisof

theMDSCpopulationsrevealedthatmicedeficientinCD84ontheirstromacellsshoweda

significantreductionintheabundanceofM-MDSCs(Fig.10G),whichexpressedlowerlevels

ofPD-L1(Fig.10H).

Furthermore,lowerlevelsofG-MDSCswereobservedintheBM(Fig.11A).PD-L1cellsurface

expression on these cells was downregulated, as well (Fig. 11B). These results further

emphasizetheroleofCD84asaregulatorofMDSCnumberandfunction.AnalysisofTcells

derived from the chimeric mice lacking CD84 on stroma revealed reduced levels of

exhaustionmarkers, among themPD-1, aswell as an increase in IL-2, IFNγ,GRZMBand

LAMP-1intheBM(Fig.11C),andspleen(Fig.11D-E).Thus,CD84expressedonstromacells

in the tumor microenvironment regulates immunosuppression through MDSCs and

functionalityofTcells,whichtogetherresultinthesupportofMMcellviability.

We furtheranalyzed theroleofCD84 inMMinCD84-/-mice thatwerebackcrossedwith

C57BL/KaLwrij mice for 6-7 generations. 5TGM1 cells were I.V. injected to the CD84-/-

/KaLwrij or C57BL/KaLwrij mice and mice survival was followed. CD84 deficiency

significantly enhanced the survival of the injected mice (Fig 12A). Furthermore, mice

sacrificedondays25-28daysshowedasignificantlyreducedtumorloadintheBM(Fig.12B)

and spleen (Supplementary Fig. 6 A-B), and their spleen size was smaller (Fig. 12C).

Furthermore, PD-L1 cell surface levels were reduced on MM cells in the spleen

(SupplementaryFig.6C).AnalysisofMDSCsinthesebackcrossedmicerevealedareduced

Lewinskyetal. 14

number of M-MDSCs in the BM (Fig. 12D), spleen (Supplementary Fig. 6D) and blood

(SupplementaryFig.6E).DeficiencyofCD84resulted in lower levelsofPD-L1(Fig.12E),

S100A9(Fig.12F)andCYBA(Fig.12G)comparedtotheirexpressiononWTBMM-MDSCs.

TheexpressionofPD-L1wasalsoreducedonPBM-MDSCslackingCD84(Supplementary

Fig.6F).

CD84-deficientG-MDSCswerereducedinthespleenandblood(SupplementaryFig.6G-H),

whilenochangewasfoundintheBM(Fig13A).TheCD84-deficientBMG-MDSCsexpressed

significantlyreducedlevelsofPD-L1,BCL2A1andCMTM6comparedtoWTG-MDSCs(Fig

13B-D),andbloodG-MDSCsexpressedlowerlevelsofPD-L1onthesecells(Supplementary

Fig 6I). Thus, deficiency of CD84 on MDSCs reduces the number of MDSCs and their

immunosuppressivefunctionsinvivo.

Next,theeffectofCD84onBMTcellswasstudied.BMCD84-/-Tcellsexpressedreduced

levels of the exhaustion markers PD-1, LAG-3, CTLA-1, 2B4 and KLRG1 (Fig. 13E,

SupplementaryFig.6J-L,andrepresentativehistogramsinSupplementaryFig6M),whileno

changewasobservedintheexpressionofthesemarkersonsplenicTcells(Supplementary

Fig6N).Inaddition,theCD84-deficientsplenic(Fig13F)andBM(Fig13G)Tcellselevated

the expression of cytotoxic factors. Thus, CD84 deficiency in the environment of mice

injectedwithMMresultsinasignificantreductioninMDSCimmunosuppression,resulting

inasignificantincreaseinTcellactivity,leadingtoasignificantreductionintumorload.

Therapeuticinterferenceusingtheanti-CD84blockingantibody

TodeterminewhetherCD84canserveasapotentialtargetfortreatmentofMM,itsactivity

wasblocked invivoby injectionwith theB4,anti-CD84blockingmAb.5TGM1cellswere

injectedintotwogroupsofsyngeneicKaLwRijmice,and2weekslaterthemicewereinjected

witheitherIgG2acontrolmAborB4anti-CD84(SupplementaryFig,7A).2daysafterthelast

of the five injections, mice were sacrificed, and tumor load, MDSCs number and PD-L1

expression, as well as T cell functionality were examined. B4 mAb treatment led to a

significantdecreaseinmalignantcellnumbersintheBM(Fig.14A),inspleensizeandcell

number(Fig.14BandSupplementaryFig7B-C),and in levelsof IgG2b intheblood(Fig.

14C), leadingtoincreasedsurvivalofthemice(Fig.14D).Furthermore,whiletheisotype

Lewinskyetal. 15

controlmAbinjectedmicewereallparalyzedbyday31,40%ofmAbB4injectedmicewere

stillabletowalk(Fig.14E).

Next,theMDSCpopulationswereanalyzed.BlockingCD84reducedtheaccumulationofM-

MDSCsinthebonemarrow(Fig.14F)andledtodecreasedexpressionofPD-L1onthese

cellsinBM(Fig.14G),blood(SupplementaryFig.7D)andspleen(SupplementaryFig.7E-F).

NodifferenceinthenumberofG-MDSCcellswasdetectedintheBM(SupplementaryFig.

7G); however, PD-L1 expression levels on these BM, spleen and blood-derived cellswas

reduced(Fig14HandSupplementaryFig7H-J).

Next,asignificantelevationintheexpressionofTcellexhaustionmarkersonBM(Fig.14I)

andspleen(Fig.14J,withrepresentativehistogramsshowninSupplementaryFig.7K)and

splenicCD8productionofthecytokinesIL-2andIFNγ,andofthemarkersofcytotoxicity

GRZMBandLAMP-1(Fig.14K)wasobservedinB4mAbtreatedmice.Noeffectwasobserved

onthenumberofTregs(SupplementaryFig.7L)orPD-1expression(SupplementaryFig.

7M)onTregsintheBM,showingthatCD8+cellsaretheprimaryaffectedTcellpopulation.

Finally,thedosedependenteffectoftheB4mAbtreatmentwasanalyzed.AsseeninFig15A,

thepercentageofmyelomacellsinthebonemarrowdecreasedby50percentinmicetreated

with30µgB4/mouse,andbycloseto75percentinmicetreatedwith200µgB4/mouse,

comparedtomicetreatedwith200µg/mouseIgG2acontrol,withasimilartrendfoundin

thespleen(SupplementaryFig.8A-B).Inaddition,whilethelowestdoseofB4mAbdidnot

affectPD-L1expressiononthe5TGM1cellsthemselves(datanotshown),thehigherdose

had an effect in the BM (Fig. 15B) and spleen (Supplementary Fig. 8C). Similarly, the

abundanceofBMM-MDSCs (Fig.15C), splenicM-MDSCs (SupplementaryFig.8D),BMG-

MDSCs(Fig.15D),PD-L1expressiononM-MDSCs(SupplementaryFig.8E),splenicG-MDSCs

(SupplementaryFig.8F)andPD-L1expressiononG-MDSCs(SupplementaryFig.7G)was

alsoaffectedinadosedependentmanner,thoughbonemarrowG-MDSCsonlydecreased

relativetocontrolatthehigherdoseof200µgB4/mouse.Moreover,toshowtheeffectof

CD84blockingonMDSC function,Arginase-1and iNOSexpression levelswereexamined.

MDSCderivedArginase-1and iNOSareknownTcell suppressionpathways thatoperate

throughmetabolismofL-Arginine(30,31).Thehigherdoseof200µgB4/mousereduced

Arginase-1expression inG-andM-MDSCs(Fig.15E-Fwithrepresentativehistograms in

Lewinskyetal. 16

SupplementaryFig.8H-I)andiNOSinM-MDSCs(Fig.15Gwithrepresentativehistogramsin

Supplementary Fig. 8J). In addition, T cells were analyzed under these conditions. All

exhaustionmarkersdecreasedinresponsetotreatment,inadose-dependentmanner(Fig.

15H).

Taken together, blockingCD84, canbeused to reduce the expansionofMDSCs and thus

decreasetheimmunosuppressivemicroenvironment,resultinginincreasedTcellactivation,

whichinturnleadstoadecreaseintumorload.

Lewinskyetal. 17

Discussion

Multiplemyelomaisconsideredanincurabledisease,despitetheimprovedunderstanding

ofitsunderlyingdiseasebiologyandthesuggestionofpotentialtherapies.Theroleofthe

microenvironmentandimmunefunctionhavebeenextensivelystudiedinthisdisease.The

main dysregulated subsets within the MMmicroenvironment are Tregs, Th17, DCs and

MDSCs (32). T cells display an exhausted phenotype in this malignancy, showing

downregulatedexpressionofactivatingreceptorsandupregulatedexpressionofexhaustion

markers,suchasPD-1(33,34).Moreover,uponstimulation,TcellsfromMM-bearingmice

expressreducedlevelsofLAMP-1,aproteinmarkingdegranulationofcytotoxicvesicles,and

secretesignificantlylessIFNγthantheircounterpartsfromhealthymice,furtherindicating

theirreducedanti-tumorcapabilities(35).

The cell surface receptor CD84 mediates the interaction of CLL cells with their

microenvironment (18), and regulates PD-L1 expression on CLL cells and their

microenvironment,resultingintheregulationofTcellsintheBM(19).Thecurrentstudy

focusedontheexpressionandfunctionofCD84onMMcellsandtheMMmicroenvironment.

Weshowthatthecytokinemacrophagemigrationinhibitoryfactor(MIF)released

fromtheMMcellsupregulatesCD84expressiononcellsintheirmicroenvironment,

andthatPD-L1expression is inducedonthesecells. Inaddition,Tcellsderived fromthe

tumormicroenvironmentexpresselevatedlevelsofPD-1,whichinhibitstheirfunctionality

and controls their immunosuppressive behavior. Our results demonstrate that blocking

CD84reducesPD-L1expressiononcellsintheMMmicroenvironment,andPD-1expression,

inadditiontootherTcellexhaustionmarkerssuchas2B4,CTLA-4,KLRG-1andLAG-3,onT

cells,resultinginelevatedTcell immunityleadingtoasignificantdecreaseintumorload

bothinaninvivomousemodelandinpatient-derivedsamples.

BothM-andG-MDSCsaccumulateinMMpatientsandaffecttumorprogression(36).MDSCs

are classified as a heterogeneous collection of immunosuppressive cells derived from

myeloid progenitors. These cells are mostly composed of granulocytic- and monocytic

MDSCs, and are an important component in many different types of tumor

microenvironments.TheysupportthetumorbysuppressingtheTcell-antitumorresponse,

mediated by reactive oxygen species, arginase andnitric acid synthase (37). In addition,

thesecellsexpresshighlevelsofPD-L1inthetumortissue(37,38).

Lewinskyetal. 18

Our results showan important regulatory role forCD84 in the functionofMDSCs. CD84

controlstheexpressionofS100A9inthesecells.S100A8andS100A9havebeenshownto

regulatesthedifferentiationofMDSCsattheexpenseofmaturecells,suchasdendriticcells

andmacrophages. In addition, S100A8 and S100A9 also act as chemoattractants for the

MDSCs(39).Furthermore,CD84upregulatestheexpressionofCYBAandPD-L1onM-MDSCs

in the environment of themalignant cells. The NADPH oxidase complex is composed of

p40phox, p47phox, p67phox, gp91phox and CYBA (p22phox), which generate reactive oxygen

species (ROS) (40).Theexpressionof thesesubunits isupregulated inM-MDSCs isolated

from non-small cell lung cancer patients controlling the immune suppression of T cells

mediatedbyROS(41).Inaddition,theelevateddifferentiationofMDSCsinducedbyS100A9

hasbeenattributedtoROSandNADPHoxidase,therebylinkingCYBAandS100A9(42),as

essentialpathwaysforM-MDSCfunction.

In G-MDSCs, CD84 induces the expression HIF1a and CMTM6, BCL2A1 and PD-L1. The

transcriptionfactorHIF-1ahasbeenimplicatedasaregulatorofbothdifferentiationand

PD-L1expressioninthesecellsbydirectlybindinghypoxiaresponseelementsinthePD-L1

promotorinMDSCs(28,43).StabilizingHIF1αexpressiondecreasesbothproliferationand

IFNg secretion of T cells, and upregulates known suppressive pathways in MDSCs (43).

BCL2A1, a member of the anti-apoptotic BCL2 family, has a pro-survival function. Its

overexpressioninG-MDSCssignificantlyincreasesthesuppressionofTcellproliferationand

IFNgsecretionresultinginelevatedtumorload(29).

OurstudyshowsthatMMcellsinduceexpressionofCD84oncellsintheirmicroenvironment

bysecretionofthecytokineMIF.BlockingorknockingoutCD84inmiceleadstoareduction

in the levels of bothM- andG-MDSCs, aswell as reduction in their immunosuppressive

pathways, leadingtoaninducedanti-tumorresponsebyTcellsandareductionintumor

load. In addition, treating mice or human MM patient samples with anti-CD84 blocking

antibodyreducestheamountofMDSCsandtheirimmunosuppression,leadingtoanincrease

intheanti-tumorresponseofTcells,resultinginreductionintheamountoftumorcellsina

dosedependentmanner,andincreasedmousesurvival.

Together,theseresultssuggestthatCD84playsakeyroleinregulatingthefunctionofMDSCs

duringMMprogression,and itsblockadecanreducethenumberof tumorcellsandtheir

Lewinskyetal. 19

immunosuppressiveproperties,evenaftertheMMtumorhasalreadybeenestablished;this

rendersCD84anattractivetargettotreatimmunosuppression,leadingtoanincreaseinthe

Tcellmediatedanti-tumorresponseinMM.

Finally,treatmentofMMinjectedmicewithbothanti-PD1andanti-PD-L1hasbeenshown

to completely eliminate myeloma cells in MM mice. Initial studies of single-agent PD-1

blockadedidnotresult inaclinicalbenefit.ResultsofthephaseI/IIandIIItrialsofPD-1

blockadewithubiquitinationandproteasomaldegradationofIkarosfamilyproteinsI(MiD

) anddexamethasone inMMhad identified apatientpopulationwithpotential toderive

clinicalbenefitfromthistreatmentapproach.However,clinicaltrialsincludinganti-PD-1in

combination with other treatments have been paused or halted due to serious adverse

eventscausedbythetreatment(44).

Further,micelackingPD-1orPD-L1spontaneouslydevelopautoimmunediseases(45,46),

whilemice lackingCD84havebeendescribedasgrossly indistinguishable fromWTmice

(47). Our results show that CD84 can regulate PD-1, PD-L1 and additional exhaustion

markers.WethereforebelievethatblockingCD84mayreducetheimmunosuppressionthat

accompaniesMMwithfeweradverseevents.

Lewinskyetal. 20

MaterialsandMethods

Mice

C57BL/KaLwRijHsd(Harlan)CD84deficient(CD84-/-)(47)micewereusedat6-8weeksof

age.Themicewereageandsexmatchedineachexperiment.

Cells

The5TGM1mouse cell linewasakindgift fromProf.Oyajobi at theUniversityofTexas

HealthScienceCenteratSanAntonio,Texas.Thecellswereculturedin10%RPMIwith10%

FCS.

MMprimary cells-A list ofpatient characteristics isprovided inSupplementaryTable1.

HealthyBMsamplesaredefinedassampleswithoutanyevidenceofabnormality,aschecked

BMsmearandbiopsy.

HumanMMcell lines(MM.1S,NCI-H929,KMS-11,RPMI-8226,MM.1RandU266),andthe

THP1 monocytic cell line were purchased from ATCC; L363 cells were purchased from

GermanCollectionofMicroorganismsandCellCultures.HumanMMcelllinesandTHP1were

culturedin10%RPMI-1640,100IU/mlpenicillinand100µg/mlstreptomycin.

PrimarymousestromalcellswerecollectedfromthefemurandtibiabyflushingouttheBM,

andseededon24wellplatesonFCS.After1hr,10%FCSRPMImediumwasaddedandthe

cellsweremaintainedbywashingofnonadherentcellsandweeklychangingofthe10%FCS

RPMImedium.

RNAisolationandqRT-PCR

Total cellularRNA frompatients ormouseMDSCswaspurifiedusing thedirect-zolRNA

purification kit (Zymogen). cDNA was prepared using the qScript cDNA Synthesis Kit

(Quantabio).Quantitativerealtime-PCRwasperformedusingSYBRgreen(Roche).Primers

arelistedinSupplementaryTable2.

Total cellularRNA fromtheTHP1cell linewasextractedbyTRIZOLreagent followedby

preparation of cDNA. Quantitative real time-PCR (qRT-PCR) was performed using the

TaqMan method according to the manufacturer’s instructions. The TaqMan probes for

mRNA quantification were purchased from Applied Biosystems, and all reactions were

Lewinskyetal. 21

performed in triplicate. OAZ1 was used as endogenous control to normalize CD84

expression.

Stainingforflowcytometry

CellswerestainedusingspecificantibodiesaslistedinSupplementaryTable3.Intracellular

stainingofTcells,wasperformedaspreviouslydescribed(19).Forintracellularstainingof

MDSCs, cells were stained for surface markers and then fixed with either the

Fixation/Permeabilization Solution Kit (BD Bioscience) for cytoplasmic proteins, or

Fixation/PermeabilizationConcentrateandDiluent(Thermofisher)fornuclearproteins.

Westernblot

Protein lysis,separationandtransferwereperformedaspreviouslydescribed(19).Blots

wereprobedusingantibodiesagainstbeta-Actin(SantaCruz)asahousekeepinggene,anti-

CD84(SantaCruz),anti-phosphoAKT,anti-phosphoS6(Cellsignaling),andanti-PD-L1.The

bandswerethendetectedusingECLWesternBlottingSubstrate.

CD84stimulationofhumancells

CD84stimulationwasperformedaspreviouslydescribed(17-19).

CD84blockingbytheB4mAboncells

CD84blockingwasperformedaspreviouslydescribed(17-19).

MIFstimulationonhumancells

MIF stimulation was performed using CD14+ cells isolated from primary samples of

peripheralbloodwithCD14microbeadsaccordingtothemanufacturer'sinstructions.1×106

CD14+ purified cells were incubated in RPMI medium containing 200 ng/ml of human

recombinantMIFat37°Cfor18–24hrs.

Mousebonemarrowstromal cellswere first starvedwith0.1%RPMImedium for3hrs,

incubatedwith100ng/ml ofmouse recombinantMIF at 37°C for 48hrs, and thereafter

stainedforflowcytometry.

Lewinskyetal. 22

MIFblockingbytheISO-1MIFinhibitoryonhumancells

ForMIFblocking,1×106isolatedCD14+cellswereincubatedfor48hrsin1ml1%FCSRPMI

mediumcontaining100μMof theMIFantagonist, ISO-1(Sigma- Aldrich, catalognumber:

478336-92-4).

Co-cultureofCD14+cellswithhumanMMcelllines.

HumanperipheralbloodCD14+cellswereisolatedusingCD14microbeads(catalognumber:

130-050-201,Miltenyl)accordingto themanufacturer's instructions. IsolatedCD14+ cells

wereco-culturedwithhumanmyelomacelllinesfor48hrs.Followingculture,CD14+cells,

stainedwithantibodies,wereanalyzedusingflowcytometry.

RNAsequencing

Sorted G-MDSCs orM-MDSCswere treatedwith activating CD84 antibody or control, as

describedabove,andRNAwasthenpurified.AbulkadaptationoftheMARS-Seqprotocol

(48)wasutilizedtogeneratetheRNA-SeqlibrariesforexpressionprofilingofbothG-MDSCs

andM-MDSCs.

CD84activatedgenesinG-MDSCwereclassifiedasdifferentiallyexpressedbasedonacut-

offofp-value<0.05beforecorrectionswithabasemean>=5,andalog2foldchangeof0.58/-

0.58(1.5or-1.5foldofIgGvalues).CD84activatedM-MDSCgenesthatwereclassifiedas

differentiallyexpressedbythecut-offofp-value<0.05afterp-valuecorrections,withabase

mean>=5andalog2foldchangeof0.58/-0.58(1.5or-1.5foldofIgGvalues).

AnalysisandvisualizationofRNA-seq

HeatmapandvolcanoplotswerepreparedusingtheparametersstatedaboveusingRstudio

version3.6.2andthelibrariesdendextend,pheatmapandggplot2.Geneontologyanalysis

wasconductedwithRstudioversion3.6.2usingthedatabaseEnrichR.

Principal component analysis anddendrogramanalysis of sampleswas performedusing

Spyder version3.3.6Python3.7with the librariesnumpy,matplotlib.pyplot, sklearn and

scipy.

Chimericmice

Lewinskyetal. 23

6-8weekoldWTorCD84−/−micewere lethally irradiatedwith1050Rad.Thenextday,

1x106C57BL/KaLwRijmousebonemarrowcellswereinjectedintothetailveinofeachof

themice.After60days,1x1065TGM1murineMMcellswereinjectedintothetailveinofthe

WTandCD84−/−mice,and3weekslater,miceweresacrificedandtheirBM,spleenandPB

wereisolated.

InvivoCD84inhibition

C57BL/KaLwRijmicewereinjectedtothetailveinwith1x1065TGM1murineMMcells.After

14days,themicewereseparatedintotwoorthreegroupsandadministeredeitherB4mAb

ortheIgG2aisotypecontrol(Biolegend)atdosesbetween30μgand200μgeveryotherday

intothetailvein,foratotalof5injections,or11injectionsforlongtermstudies.

VirusandcellularproductionofCRISPRlibrary

TheCRISPRmanipulationswereperformedusingamethodsimilarto(49).Thefollowing

sgRNA sequences were used: Control: GAACGUAGAAAUUCCCAUUU; CD84:

GAGUCAGAAUUGUUGCUGAC.

Chromium-51ReleaseAssay

MM.1S and Raji cells were labeled withChromium 51 (Cr-51) by incubating 1×106cells

in100µCi ofCr-51 for1.5hours at 37°C. The labeled cells werewashed 3 times and re-

suspendedin complete RPMI medium, and plated in 96-well round bottom plates in

triplicates.Theneffectorcellswereaddedat40∶1,20∶1and10∶1effector-to-targetratios

withB4andISO-1orIgG2aandDMSOcontrolsandincubatedat37°Cfor16hours.Cr-51

releasewasdetectedaspreviouslydescribed(50).

Suppressionassay

MDSCsuppressionassaywasperformedaspreviouslydescribed(51).

Statistics

AnalysisofdatawasperformedusingGraphPadPrism(Version8.0,GraphPadSoftware,Inc.,

LaJolla,CA,USA).Formostexperiments,themean ± standarderrorisprovided,andatleast

Lewinskyetal. 24

three experimentswereperformed. Formouse experiments, due todifferences in tumor

load,resultswerenormalized ineachexperiment to theaverageof thecontrolmice,and

resultsarethereforeshownasfoldchange;thisdidnotaffectthepercentageofimmunecells,

suchasMDSCs,ineachmouse,whichareshownasactualpercentages.

Totestforsignificance,eitherone-ortwo-sidedStudent’st-testwasuseddependingonthe

experiment,unlessstatedotherwise.Resultswereconsideredsignificantatapvalueof≤

0.05.

Studyapproval

AllanimalprocedureswereapprovedbytheAnimalResearchCommitteeattheWeizmann

InstituteofScience.

Primarypatient-derivedMMorhealthysamplescellswereprovidedincompliancewiththe

institutionalreviewboardsoftheparticipatinghospitals.

Acknowledgments

TheauthorswishtothankmembersoftheShacharlabforfruitfuldiscussionandTaliShalit

and the staff of the Nancy and Stephen Grand Israel National Center for Personalized

Medicine.WealsowishtothankDomenicoViolaforhishelpinthestudy.Thisresearchwas

supported by the Israel Cancer Research Foundation (ICRF) Judy and Bernie Briskin

Foundation,andYeda.I.S.istheincumbentoftheDr.MortonandAnnKleimanProfessorial

Chair.H.L.wassupportedbyKungligavetenskapsakademin’sfoundationGretaochRagnar

Müllersresestipendiefond.

Theauthorsdeclarenocompetinginterests

Authorcontributions

H.L.-designedresearch,performedexperiments,analyzeddataandwrotethepaper.

E.G.G.-designedresearch,performedexperiments,analyzeddataandwrotethepaper.

K.D.-designedresearch,performedexperiments,andanalyzeddata.

L.R.-designedresearch,performedexperiments,andanalyzeddata.

Lewinskyetal. 25

M.P.K.-designedresearchandanalyzeddata.

M.P.-performedexperiments.

B.P.-Performedexperiments.

J.C-designedresearch,andanalyzeddata.

T.-F.H.-designedresearchandanalyzeddata.

A.G.M.-designedresearchandanalyzeddata.

K.-Y.T.-designedresearchandanalyzeddata.

S.B.-designedresearchandanalyzeddata.

E.C.-performedexperimentsandanalyzeddata.

E.T.-performedexperiments

P.L.-designedresearchandanalyzeddata.

M.F.-designedresearchandanalyzeddata.

J.K.-Providedessentialreagent.

A.K.-Providedessentialreagent.

M.R.-Providedessentialreagent.

J.Y.-Providedessentialreagent.

M.A.C.-Providedessentialreagent.

O.S.-Providedessentialreagent.

Y.C.-Providedessentialreagent.

S.B-H.-designedresearchandanalyzeddata.

F.P.-designedresearchandanalyzeddata.

S.R.-designedresearch,analyzeddataandwrotethepaper.

I.S.-designedresearch,analyzeddataandwrotethepaper.

Lewinskyetal. 26

References

1. ManierS,SaccoA,LeleuX,GhobrialIM,andRoccaroAM.Bonemarrowmicroenvironmentin

multiplemyelomaprogression.JBiomedBiotechnol.2012;2012:157496.

2. Gorgun GT, Whitehill G, Anderson JL, Hideshima T, Maguire C, Laubach J, et al. Tumor-

promoting immune-suppressive myeloid-derived suppressor cells in the multiple myeloma

microenvironmentinhumans.Blood.2013;121(15):2975-87.

3. Stewart TJ, and Abrams SI. How tumours escape mass destruction. Oncogene.

2008;27(45):5894-903.

4. MeadsMB,HazlehurstLA,andDaltonWS.Thebonemarrowmicroenvironmentasatumor

sanctuaryandcontributortodrugresistance.ClinicalCancerResearch.2008;14(9):2519-26.

5. Yaccoby S. Osteoblastogenesis and tumor growth in myeloma. Leuk Lymphoma.

2010;51(2):213-20.

6. ReaganMR,andGhobrialIM.Multiplemyelomamesenchymalstemcells:characterization,

origin,andtumor-promotingeffects.ClinCancerRes.2012;18(2):342-9.

7. GabrilovichDI,andNagarajS.Myeloid-derivedsuppressorcellsasregulatorsoftheimmune

system.NatRevImmunol.2009;9(3):162-74.

8. MalekE,deLimaM,LetterioJJ,KimBG,FinkeJH,DriscollJJ,etal.Myeloid-derivedsuppressor

cells:Thegreenlightformyelomaimmuneescape.BloodRev.2016;30(5):341-8.

9. McKinneyEF,andSmithKG.Tcellexhaustionandimmune-mediateddisease-thepotential

fortherapeuticexhaustion.CurrOpinImmunol.2016;43:74-80.

10. GreavesP,andGribbenJG.TheroleofB7familymoleculesinhematologicmalignancy.Blood.

2013;121(5):734-44.

11. PaukenKE,andWherryEJ.SnapShot:TCellExhaustion.Cell.2015;163(4):1038-e1.

12. CalpeS,WangNH,RomeroX,BergerSB,LanyiA,EngelP, et al.TheSLAMandSAPgene

families control innate and adaptive immune responses. Advances in Immunology, Vol 97.

2008;97:177-250.

Lewinskyetal. 27

13. SintesJ,RomeroX,deSalortJ,TerhorstC,andEngelP.MouseCD84isapan-leukocytecell-

surfacemolecule thatmodulates LPS-induced cytokine secretion bymacrophages. J Leukoc Biol.

2010;88:687-97.

14. MartinM,RomeroX,delaFuenteMA,TovarV,ZapaterN,EspluguesE,etal.CD84functions

asahomophilicadhesionmoleculeandenhancesIFN-gammasecretion:AdhesionismediatedbyIg-

likedomain1.JournalofImmunology.2001;167(7):3668-76.

15. Romero X, Zapater N, Calvo M, Kalko SG, de la Fuente MA, Tovar V, et al. CD229 (Ly9)

lymphocyte cell surface receptor interacts homophilically through its N-terminal domain and

relocalizestotheimmunologicalsynapse.JournalofImmunology.2005;174(11):7033-42.

16. YanQR,MalashkevichVN,FedorovA,FedorovE,CaoE,Lary JW,etal. StructureofCD84

providesinsightintoSLAMfamilyfunction.ProceedingsoftheNationalAcademyofSciencesofthe

UnitedStatesofAmerica.2007;104(25):10583-8.

17. Binsky-EhrenreichI,MaromA,SobottaMC,ShvidelL,BerrebiA,Hazan-HalevyI,etal.CD84

isasurvivalreceptorforCLLcells.Oncogene.2014;33:1006-16.

18. Marom A, Barak AF, Kramer MP, Lewinsky H, Binsky-Ehrenreich I, Cohen S, et al. CD84

mediatesCLL-microenvironmentinteractions.Oncogene.2017;36(5):628-38.

19. LewinskyH,BarakAF,HuberV,KramerMP,RadomirL,SeverL,etal.CD84regulatesPD-

1/PD-L1expressionandfunctioninchroniclymphocyticleukemia.JClinInvest.2018;128(12):5465-

78.

20. Herlihy SE, Lin C, and Nefedova Y. Bonemarrowmyeloid cells in regulation of multiple

myelomaprogression.CancerImmunolImmunother.2017;66(8):1007-14.

21. LwinST,EdwardsCM,andSilbermannR.Preclinicalanimalmodelsofmultiplemyeloma.

BonekeyRep.2016;5:772.

22. Lastwika KJ,WilsonW, 3rd, Li QK, Norris J, Xu H, Ghazarian SR, et al. Control of PD-L1

Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer.

CancerRes.2016;76(2):227-38.

23. ChengP,NefedovaY,MieleL,OsborneBA,andGabrilovichD.Notchsignalingisnecessarybut

notsufficientfordifferentiationofdendriticcells.Blood.2003;102(12):3980-8.

Lewinskyetal. 28

24. Corzo CA, Cotter MJ, Cheng P, Cheng F, Kusmartsev S, Sotomayor E, et al. Mechanism

regulatingreactiveoxygenspeciesintumor-inducedmyeloid-derivedsuppressorcells. JImmunol.

2009;182(9):5693-701.

25. FlemingV,HuX,WeberR,NagibinV,GrothC,AltevogtP,etal.TargetingMyeloid-Derived

SuppressorCellstoBypassTumor-InducedImmunosuppression.FrontImmunol.2018;9:398.

26. Zhang S, Zhong M, Wang C, Xu Y, Gao WQ, and Zhang Y. CCL5-deficiency enhances

intratumoralinfiltrationofCD8(+)Tcellsincolorectalcancer.CellDeathDis.2018;9(7):766.

27. BurrML,SparbierCE,ChanYC,WilliamsonJC,WoodsK,BeavisPA,etal.CMTM6maintains

theexpressionofPD-L1andregulatesanti-tumourimmunity.Nature.2017;549(7670):101-5.

28. NomanMZ,DesantisG,JanjiB,HasmimM,KarrayS,DessenP,etal.PD-L1isanoveldirect

targetofHIF-1alpha,anditsblockadeunderhypoxiaenhancedMDSC-mediatedTcellactivation.J

ExpMed.2014;211(5):781-90.

29. Medina-EcheverzJ,ArandaF,andBerraondoP.Myeloid-derivedcellsarekeytargetsoftumor

immunotherapy.Oncoimmunology.2014;3:e28398.

30. RodriguezPC,andOchoaAC.Arginineregulationbymyeloidderivedsuppressorcellsand

toleranceincancer:mechanismsandtherapeuticperspectives.ImmunolRev.2008;222:180-91.

31. BronteV,andZanovelloP.RegulationofimmuneresponsesbyL-argininemetabolism.Nat

RevImmunol.2005;5(8):641-54.

32. RomanoA,ConticelloC,CavalliM,VetroC,LaFauciA,ParrinelloNL,etal. Immunological

dysregulationinmultiplemyelomamicroenvironment.BiomedResInt.2014;2014:198539.

33. PittariG,VagoL,FestucciaM,BoniniC,MudawiD,GiacconeL,etal.RestoringNaturalKiller

CellImmunityagainstMultipleMyelomaintheEraofNewDrugs.FrontImmunol.2017;8:1444.

34. Gorgun G, Samur MK, Cowens KB, Paula S, Bianchi G, Anderson JE, et al. Lenalidomide

Enhances Immune Checkpoint Blockade-Induced Immune Response in Multiple Myeloma. Clin

CancerRes.2015;21(20):4607-18.

35. Zelle-RieserC,ThangavadivelS,BiedermannR,BrunnerA,StoitznerP,WillenbacherE,etal.

Tcellsinmultiplemyelomadisplayfeaturesofexhaustionandsenescenceatthetumorsite.JHematol

Oncol.2016;9(1):116.

Lewinskyetal. 29

36. Favaloro J, Liyadipitiya T, Brown R, Yang S, Suen H,Woodland N, et al. Myeloid derived

suppressorcellsarenumerically,functionallyandphenotypicallydifferentinpatientswithmultiple

myeloma.LeukLymphoma.2014;55(12):2893-900.

37. Consonni FM, Porta C, Marino A, Pandolfo C, Mola S, Bleve A, et al. Myeloid-Derived

SuppressorCells:DuctileTargetsinDisease.FrontImmunol.2019;10:949.

38. LuC,ReddPS,LeeJR,SavageN,andLiuK.TheexpressionprofilesandregulationofPD-L1in

tumor-inducedmyeloid-derivedsuppressorcells.Oncoimmunology.2016;5(12):e1247135.

39. Ostrand-Rosenberg S, and Fenselau C. Myeloid-Derived Suppressor Cells: Immune-

Suppressive Cells That Impair Antitumor Immunity and Are Sculpted by Their Environment. J

Immunol.2018;200(2):422-31.

40. BabiorBM.NADPHoxidase:anupdate.Blood.1999;93(5):1464-76.

41. Singel KL, and Segal BH. NOX2-dependent regulation of inflammation. Clin Sci (Lond).

2016;130(7):479-90.

42. ChengP,CorzoCA,LuettekeN,YuB,Nagaraj S,BuiMM,et al. Inhibitionofdendritic cell

differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by

S100A9protein.JExpMed.2008;205(10):2235-49.

43. CorzoCA,CondamineT,LuL,CotterMJ,YounJI,ChengP,etal.HIF-1alpharegulatesfunction

anddifferentiationofmyeloid-derivedsuppressorcellsinthetumormicroenvironment.JExpMed.

2010;207(11):2439-53.

44. LesokhinAM,BalS,andBadrosAZ.LessonsLearnedfromCheckpointBlockadeTargeting

PD-1inMultipleMyeloma.CancerImmunolRes.2019;7(8):1224-9.

45. NishimuraH,NoseM,HiaiH,MinatoN,andHonjoT.Developmentoflupus-likeautoimmune

diseasesbydisruptionofthePD-1geneencodinganITIMmotif-carryingimmunoreceptor.Immunity.

1999;11(2):141-51.

46. KeirME,LiangSC,GuleriaI,LatchmanYE,QipoA,AlbackerLA,etal.TissueexpressionofPD-

L1mediatesperipheralTcelltolerance.JExpMed.2006;203(4):883-95.

47. CannonsJL,QiH,LuKT,DuttaM,Gomez-RodriguezJ,ChengJ,etal.OptimalGerminalCenter

ResponsesRequireaMultistageTCell:BCellAdhesionProcessInvolvingIntegrins,SLAM-Associated

Protein,andCD84.Immunity.2010;32(2):253-65.

Lewinskyetal. 30

48. Keren-Shaul H, Kenigsberg E, Jaitin DA, David E, Paul F, Tanay A, et al.MARS-seq2.0: an

experimentalandanalyticalpipelineforindexedsortingcombinedwithsingle-cellRNAsequencing.

NatProtoc.2019;14(6):1841-62.

49. SanjanaNE,ShalemO,andZhangF.Improvedvectorsandgenome-widelibrariesforCRISPR

screening.NatMethods.2014;11(8):783-4.

50. KarimiMA,LeeE,BachmannMH,SalicioniAM,BehrensEM,KambayashiT,etal.Measuring

cytotoxicitybybioluminescenceimagingoutperformsthestandardchromium-51releaseassay.PLoS

One.2014;9(2):e89357.

51. SolitoS,PintonL,DeSanctisF,UgelS,BronteV,MandruzzatoS,etal.MethodstoMeasure

MDSCImmuneSuppressiveActivityInVitroandInVivo.CurrProtocImmunol.2019;124(1):e61.

A B

E

Figure 1

C D

10.6%

0-104 104

CD84

0

20

40

60

80

100

IsoAnti-CD84

20.5%

0 1040

20

40

60

80

100

IsoAnti-CD84

MMMGUS

F

CD14-

HD-PB CD14 + HD-PB

CD84

80.7%

HealthyCD14-

HealthyCD14+

MMCD14-

MMCD14+

0

20

40

60

80

100 *******

3.95%

CD14 -MM-PB CD14 + MM-PB

94.3%17.4%

23.5%

0 103

CD84

0

20

40

60

80

100

WT

5TGM1 inj

Stroma

Iso

HealthyCD14-

HealthyCD14+

MMCD14-

MMCD14+

0

10

20

40

60

80

100 ********

CD14- MM-BM

c3.95% 80.7% 7.7% 88.88%

CD14 -

HD-BMCD14 +

HD-BMCD14 -

MM-BM

CD14 +

MM-BM

CD84

CD84

Spleen

BM5TGM1

19.8%

102 103 104 1050

20

40

60

80

100

Healthy MM

CD84

%12.8% 26.0%

Healthypatient

Smolderingpatient

MMpatient

0

10

20

30*

***ns

Spleen BM0

5

10

15

20

25 *

WT 5TGM1 inj0

5

10

15

20

25 ***

HealthyCD138+CD38+

MGUSCD138+CD38+

SmolderingCD138+CD38+

MMCD138+CD38+

05

10152025303540455055 *

0.1449

Lewinskyetal. 31

Figure 1- CD84 is expressed on immunosuppressive cells in the MM

microenvironment

(A-D)CD84expressionwasanalyzedbyFACSon(A)MGUS,smolderingdisease,andpatient-

derivedCD138+CD38+MMcellsfromtheBM.Representativehistograms,withpercentages

ofthepositivelystainedcells,areshownontheright(n=3-10,p=0.44,one-wayANOVA).(B)

CD138+5TGM1cellstakenfromspleenandBMof5TGM1injectedC57BL/KaLwRijWTmice.

ArepresentativehistogramdemonstratingthepercentofCD84+BMandspleen5TGM1cells,

is shown on the right (n=7-8). (C) MM BM stroma cells from MM grown in culture.

Representativehistograms,showingthepercentofCD84positivecells,areshownonthe

right(n=3-6).(D)StromaderivedfromBMaspiratesof5TGM1injectedC57BL/KaLwRijWT

or non-injectedmice grown in culture. A representative histogram, with percentages of

CD84+stromacellsfrominjectedmiceisshownontheright(n=3-7).(E-F)CD14+cellsfrom

MM or healthy bone marrow aspirates (E), or MM or healthy peripheral blood (F).

Representativehistograms,indicatingpercentagesofpositivelystainedcells,areshownon

theright(n=7-16).

A

C

B

Healthy Smoldering MM0

20

40

60

80

100 *********

Healthy Smoldering MM0

10

20

30

40

50*

ns

**

99.3%

0 103 104

CD84

0

20

40

60

80

100 M-MDSC

HealthySmoldering

Iso

Myeloma

28.1%

0 103

CD84

0

20

40

60

80

100

HealthySmoldering

Iso

Myeloma

G-MDSC

tSNE 1

CD138+/CD38+

CD84+

CD14+/CD15-

M-MDSCs

CD15+/CD14-

G-MDSCs

Figure 2

Lewinskyetal. 32

Figure2-CD84isexpressedonMDSCsintheMMmicroenvironment

(A)HumanBM-derived(CD14+,CD11B+,CD15-,HLA-DR-)M-MDSCs.Arepresentativeplot,

showingpercentagesCD84postivieMMderivedM-MDSCs,isshownontheright(n=3-8).

(B) Human BM derived (CD15+, CD11B+, CD14-, HLA-DR-) G-MDSCs. A representative

histogram,andplotshowingpercentG-MDSCsexpressingCD84,isshownontheright(n=3-

8).(C)t-distributedstochasticneighborembedding(tSNE)plotsidentifyingCD14+cells,M-

MDSC,CD15+ cells,G-MDSCs,MMcells andCD84expression.Representative t-SNEplots

basedonCD14+,CD15+,M-MDSC,G-MDSC,MMandCD84+cellsfromtheMMpatientBM

samples(t-SNEwasrunwithperplexity=30,1000iterations,and800000livecellswere

randomlyselected).ThecellsarecoloredaccordingtotheexpressionlevelofCD14+/CD15-

for CD14; CD14+/CD15-/CD11b+/HLA-DR low/- for M-MDSC; CD15+/CD14- for CD15;

CD15+/CD14-/CD11b+/HLA-DRlow/-forG-MDSC;CD138+/CD38+forMMcells,andCD84

markers.

*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, with either unpaired T-test for pairwise

comparisonsorone-wayANOVAwithHolm-Sidakmultiplecorrectionstestforthreegroups.

A

E

Figure 3

IgG CD840.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 *

IgG CD840.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 *

IgG CD840.0

0.5

1.0

1.5

2.0 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

gG2a B40

10

20

30

40

50

60

70 *

IgG CD840.00.51.01.52.02.53.03.54.04.5 *

IgG CD840.0

0.5

1.0

1.5

2.0

2.5 *

siCTRL siCD840.00

0.25

0.50

0.75

1.00

1.25 *

siCTRL siCD840.00

0.25

0.50

0.75

1.00

1.25 *

B

C D F

47.3%

0 1040

20

40

60

80

100

PD-L1

MM

IgG

CD84

Iso

14.2%

0-103 103 1040

20

40

60

80

100

PD-L1

BM Stroma

IgG

CD84

Iso

29.0%

0-104 104 1050

20

40

60

80

100

PD-L1

5TGM1

IgG

CD84

Iso

IgG CD840.0

0.5

1.0

1.5

2.0

2.5 ***

G

IgG CD840

500

1000

1500

2000

2500 *

Lewinskyetal. 33

Figure3-CD84upregulatesPD-L1expressioninMMcellsandtheirmicroenvironment

(A)SortedprimaryMMcells(formRNAanalysis)ortotalprimaryBMstainedtoidentifyMM

cells (forproteinanalysis) frompatientswere stimulated with anti-CD84activating or

controlantibodies.PD-L1mRNA(leftgraph)orprotein(rightgraph)weredeterminedby

qRT-PCRorFACSanalysisrespectively.Arepresentativehistogram,withpercentofCD84

positivecells,isshown.(B)BonemarrowaspiratesfromprimaryMMpatientswereseeded

andgrownuntilaconfluentadherentlayerwasformed.Thereafter,CD84wasactivatedwith

anti-CD84orcontrolantibodies,andRNA(leftgraph)andprotein(rightgraph)expression

was analyzed using qPCR and flow cytometry, respectively. A representative histogram,

demonstratingthepercentofCD84positivecells,isshownontheright(n=3-5).(C)5TGM1

cellswereactivatedwithanti-CD84orcontrolantibodies.PD-L1messagewasanalyzedby

qRT-PCR (n=3). (D-E) 5TGM1 cells were incubated with antagonistic anti-CD84 B4 or

control antibodies. PD-L1mRNA (D) or protein (E)were analyzed by qRT-PCR or flow

cytometry,respectively.Representativehistogramsareshownontheright(n=3).

(F)5TGM1cellsweretreatedwitheithersiCTRLorsiCD84;RNAwaspurified,andanalyzed

byqPCRforCD84(leftgraph)andPD-L1(rightgraph)mRNAlevels(n=3).(G)PrimaryPB

CD14+cellsfromhealthydonorsweretreatedwithanti-CD84activatingantibodyorcontrol

antibodies.PD-L1mRNA(leftgraph)orprotein(rightgraph)levelswereanalyzedbyqRT-

PCRandflowcytometry,respectively(n=3-5).

24.6%

0-104 1040

20

40

60

80

100

PD-L1

M-MDSC

IgG

CD84

Iso

29.3%

0-104 1040

20

40

60

80

100

PD-L1

G-MDSC

IgG

CD84

Iso

IgG2a B40.0

0.5

1.0

1.5

2.0 *

IgG2a B4

0.0

0.5

1.0

1.5

2.0

2.5 *

IgG2a B40.00.51.01.52.02.53.03.54.04.55.05.56.06.5 *

IgG2a B40

1

2

3

4

5 *

+CD3+M - MDSCs+IgG

+CD 3 +M MDSCs+B4

+CD3 +G - MDSCs+IgG

+CD3 +G- MDSCs+B 4

Dividing18.4%

102 103 104 1050

20

40

60

80 IgG2a

B4

Unactivated

Dividing47.9%

102 103 104 1050

20

40

60

80

100

IgG2a

B4

Unactivated

IFNγ10.7%

IFNγ4.20%

CD84

p-mTOR

PD-L1

p-S6

p-AKT

β-Actin

THP1

CRISPRCRISPRNon-targetNon-target

CRISPRCD84

0

10

20

30 *

CRISPRNon-target

CRISPRCD84

0

10

20

30

40 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

D

E

C

F

G H

I J

Figure 4

100

IFNγ21.2%

IFNγ13.2%

IgG CD840

1

2

3

4

5

6

7*

A

IgG CD840.00.51.01.52.02.53.03.54.04.5

*B

Lewinskyetal. 34

Figure4-CD84upregulatesPD-L1expressioninMDSCs

(A-B)SortedprimaryM-MDSCs(CD14+,CD15-,CD11b+,HLA-DR-)(A),andG-MDSCs(CD15+,

CD14-,CD11b+,HLA-DR–)(B)frombonemarrowaspiratesofMMsampleswereanalyzedfor

PD-L1messagebyqRT-PCR.(C-F)Sorted(mRNA)orwholebonemarrow(protein)primary

M-MDSCs (LY6C+, LY6G-, CD11b+, CD11C-) (C, E), and G-MDSCs (LY6G+, LY6Clow, CD11b+,

CD11C-)(D,F),fromBMaspiratestakenfrom5TGM1injectedmicewereincubatedinthe

presenceofanti-CD84(B4)blockingorisotypecontrolantibodiesandanalyzedforPD-L1

mRNA (C-D) and protein (E-F) by qRT-PCR or FACS analysis, respectively (n=4).

RepresentativehistogramsareshowninLandM.(G-H)SortedM-(G)andG-MDSCs(H)were

treatedwithanti-CD84B4 inhibitoryor controlantibody, co-cultured for72hrswithWT

wholespleensataratioof1:4,andthepercentageofdividingCD8+TcellsandIFNγwas

analyzed thereafter (n=4-7, *p<0.05). (I) LentiCRISPR-v2 CRISPR/Cas9 system was

establishedintheTHP1celllinewithconstitutiveknockoutofCD84expression,andprotein

levelsofCD84andPD-L1weredeterminedbyflowcytometry(n=3).(J)THP1cellswere

activatedwithanti-CD84orcontrolantibodies,andCD84,p-mTOR,pAKT,pS6,andPD-L1

expressionlevelsweredeterminedbywesternblottinganalysis.

*p<0.05,**p<0.01,***p<0.001,witheitheroneortwotailedunpairedorpairedT-testfor

pairwisecomparisons.

A

D

B

C

J

E F G

H

I

K

Figure 5

19.6%

0-104 104

105

0

20

40

60

80

100

PD-L1

MM

IgG2a

B4

Iso

9.33%

0 104

PD-L1

0

20

40

60

80

100 M-MDSC

IgG2a

B4

0

1

2

3 * *Isotype Ctrl

CD14+/MM.1S/B4

CD14+/MM.1S

CD14+

PD-L1

CD3+

CD3+/CD14+/MM.1S

CD3+ /CD14+ /MM.1S/B4

PD-1

32.47% 52.68% 37.90%

0

20

40

60 * *

0

20

40

60

80

100 *

7AAD

Max Killing Min Killing

97.1% 8.7% 27.3% 36.6%

IgG B4

29.2%

0 103 104

PDL1

0

20

40

60

80

100G-MDSC

IgG2a

B4

4.17%

0 104

CTLA-4

0

20

40

60

80

100

14.9%

0-103 103

PD-1

0

20

40

60

80

100 CD8

IgG2a

B4

CD8

IgG2a

B4

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2 **

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 **

0.00

0.25

0.50

0.75

1.00

1.25

CTLA4 LAG3 PD-1

***

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2 ***

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

0.00

0.25

0.50

0.75

1.00

1.25

CTLA4 PD-1

** ****

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2

1.21%

0 104 105

LAG-3

0

20

40

60

80

100

8.85%

0-103 103

PD-1

0

20

40

60

80

100

2.41%

0 104

CTLA-4

0

20

40

60

80

100CD4

IgG2a

B4

CD4

IgG2a

B4

CD4

IgG2a

B4

Annexin-V46.5%

live52.1%

0-103 103 104 1050

20

40

60

80

100

Annexin-V37.8%

live61.2%

0-103 103 104 1050

20

40

60

80

100MM MM

IgG B4

I

Lewinskyetal. 35

Figure5-BlockingCD84inducesTcell-mediatedkillingofhumanMMcells

(A-B)PBCD14+(A)orT(B)cellsfromahealthydonorwereculturedaloneorwithMM.1S

MMcelllinecellsataratioof1:2,inthepresenceorabsenceoftheanti-CD84-inhibitory(B4)

orcontrolantibodies.After48hrs,PD-L1(A),orPD-1(B)cellsurfacelevelsweredetermined

byflowcytometry(n=3).Representativeplotsareshownontheright.(C)PurifiedPBMCs

fromhealthydonorsweretreatedwithIgGorB4antibodies.After24hrs,thecellswereco-

culturedwithGFP+MM.1Sataratioof6:1for16hrs.Thepercentageof7AADstainingon

GFP+MM.1Scellswasdeterminedbyflowcytometry.Representativehistogramsareshown

ontheright(n=3).(D-I)PrimaryMMbonemarrowaspiratesweretreatedwithB4orIgG2a

antibodies.After48hrs,thepercentageofMMcells(Dleftgraph)andAnnexin-Vstaining(D

right graph) as well as PD-L1 expression (E) were determined by flow cytometry.

Representativehistograms,showingthepercentofofAnnexin-VpositivecellsorB4treated

cells,areshownontheright(n=4-6).(F-I)M-(F-G),andG-(H-I)MDSCswereincubatedwith

controlorB4antibodies.Percentageofcells(F,H),andPD-L1expression(G, I) following

treatmentweredeterminedbyFACS.Representativeplotsorhistogramsareshownonthe

right(n=4-5).(J-K)ThepercentageofCTLA-4,LAG-3andPD-1onCD4+(H)andCD8+(I)T

cells fromwholeMMbonemarrowaspiratestreatedwithB4orIgG2acontrolantibodies

wasanalyzedusingflowcytometry.Representativehistogramsareshownontheright(n=5-

7). *p<0.05, **p<0.01, ****p<0.0001, with unpaired or paired T-test for pairwise

comparisons.

Lewinskyetal. 36

Figure 6- CD84 expression is regulated in a MIF-dependent manner in the MM

microenvironment

(A-B) PB CD14+ cells derived from healthy volunteers were cultured alone or with the

humanmyelomacelllines(MM.1S,U266,KMS11)ataratioof1:2.After48hrs,theCD14+

cells were analyzed by flow cytometry for CD84 (A) and PD-L1 (B) expression (n=3).

Representativehistogramsareshownontheright.(C)MIFproteinexpressionwasanalyzed

inthehumanMMcelllinesNCI-H929,MM.1S,U266,RPMI-8226,L363,MM.1RandRajicell

line(positivecontrol)bywesternblotanalysis.(D-E)PBCD14+cellsfromhealthydonors

(D)orTHP1cells(E)wereculturedwithorwithout200ng/mlhrMIf.After48hrs,thecells

wereanalyzedforCD84andPD-L1expressionbyflowcytometry(n=3).(F-G)Bonemarrow

fromC57BL/KaLwRijWTmice(F)or5TGM1injectedC57BL/KaLwRijmice(G)wasgrown

untilanalmostconfluentadherentcell layerwasformed.Thereafter,100ng/mlMIFwas

added. After 48hrs, the cellswere harvested and analyzed by flow cytometry (excluding

CD45+andCD138+cells)forCD84expression.(n=3-5).(H-I)MM.1Scellsweretransfected

usingMIForcontrolsiRNA(10µM/mL).After48hrs,themediumwasreplacedwithfresh

culturemedium, and cells grown an additional 72hrs for collectionof supernatant.After

72hrs,thesupernatantsofthecellsweretransferredtoPBCD14+cellsderivedfromhealthy

donors.After48hrs, theCD14+cellswereharvestedandanalyzedby flowcytometry for

CD84(H),andPD-L1(I)expression(n=5).

Lewinskyetal. 37

Figure7-BlockingMIFreducesCD84expressionintheMMmicroenvironment

(A-B)PBCD14+cellsfromhealthydonorswereco-culturedwithMM.1Sataratioof1:2in

presenceorabsenceofMIFinhibitor(ISO-1).After48hrs,CD14+cellswereanalyzedfor

CD84 (A) and PD-L1 (B) expression by flow cytometry (n=3). (C) Purified PBMCs from

healthydonorsweretreatedwithorwithoutISO-1.After24h,thecellswereco-cultured

withGFP+MM.1Sataratioof6:1,andthepercentof7AADstainingonGFP+MM.1Scellswas

determined16hrs laterby flowcytometry.Representativehistogramsareshownon the

right (n=3).(D-E)WholeBMcultures frommicebearingMMwere treatedwith ISO-1or

control;CD84expressionwasdetermined48hrslateronM-MDSCs(D)orG-MDSCs(E)by

flowcytometry.Representativehistograms,withpercentagesdisplayingtheISO-1treated

cells,areshownontheright(n=3-4,*<0.05).*p<0.05,**p<0.01,****p<0.0001,withunpaired

orpairedT-testforpairwisecomparisons.

B

E

Figure 8

AA C D

ADAM8

ELF2

CSF3R

CARD19

CCL5

CCNDBP1

ID2 JUNB

CCDC15

PRDM1

CLLU1

SCARB2

CXCL16

AZI2

TCF4

NPC2

CYBA

S100A9

0

3

6

9

12

−2.5 0.0 2.5Foldchange (log2)

GenesDownnsUp

−log10(pvalue)

rRNA bindingzinc ion binding

transition metal ion bindingmRNA 5'−UTR binding

mRNA bindingubiquitin protein ligase binding

ubiquitin−like protein ligase bindingcadherin binding

actin bindingprotein kinase binding

alpha−mannosidase activitymannosidase activity

metal ion bindingN−acyltransferase activity

RAGE receptor bindingkinase binding

immunoglobulin bindingNADH dehydrogenase activity

0.0 0.5 1.0 1.5

CCDC15PRDM1

TCF4

AZI2

ELF2

CLLU1

CCNDBP1

CCL5

CSF3R

CARD19

SCARB2

CYBA

ADAM8

S100A9

JUNB

CXCL16

ID2

NPC2

−3

−2

−1

0

1

2

3

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 ***

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

F G

27.4%

0-104 104 105

S100A9

0

20

40

60

80

100M-MDSC

IgG2a

B4

47.3%

102 103 104 1050

20

40

60

80

100

CYBA

M-MDSC

IgG2a

B4

40.7%

0-104 104 105

S100A9

0

20

40

60

80

100M-MDSC

IgG2a

B4

52.5%

0 103 104

CYBA

0

20

40

60

80

100 M-MDSC

IgG2a

B4

Lewinskyetal. 38

Figure8-CD84regulatesfunctionalandimmunosuppressivepathwaysinhumanMM

M-MDSCs

(A-C)SortedBMM-MDSCs(CD14+,CD15-,HLA-DR-,CD11B+)wereactivatedwithanti-CD84

or IgG1k antibodies. After 24 hrs, RNAwas purified and sequenced usingMars-seq. (A)

VolcanoplotforM-MDSCgeneswithgreyindicatingnonDEgenes,andDEexpressedgenes

showneitherinblue,fordownregulated,orred,forupregulated,genes(DEgeneshadap

valueof<0.05afteradjustment,andabasemeanofmorethan5afteradjustmentwiththeR

package,fdrtool,n=4).(B)HighlightedgeneontologytermsfromtheupregulatedDEgenes

ofM-MDSCs(MolecularFunction,EnricheR).(C)Heatmapdepictinghighlightedgenesfrom

theDEgenes,witheachcolumnrepresentingthelog2CD84activatedtoIgGcontrolratio

valueofasinglepatient(n=4).(D-E)PurifiedprimaryMMBMsampleswereincubatedwith

B4orIgG2aantibodies.After48hrs,M-MDSCswereanalyzedforS100A9(D)(n=6)orCYBA

(E) protein expression (n=6). (F-G) Sorted primary M-MDSCs (message) or whole BM

cultures (protein) frommurine5TGM1 injectedmicewere incubatedwithB4 or control

antibodies.After24(mRNA,left)or48hrs(protein,right)cellswereanalyzedforS100A9

(F)andCYBA(G)expression.Representativehistogramsareshownontheright(n=3-4).

HIPK1

DOCK11

EMB

MT2A

CARHSP1

SASH1

HBB

ELANE

CST7

BDP1

ID2

HIF1A

BCL2A1

CMTM6

EP300

IFNGR1

LYST

0

1

2

3

4

−5.0 −2.5 0.0 2.5Foldchange (log2)

GenesDownnsUp

−log10(pvalue)

decreased metastatic potential

decreased macrophage cell number

altered tumor susceptibility

increased susceptibility to bacterial infection

increased susceptibility to parasitic infection

abnormal epicardium morphology

increased susceptibility to fungal infection

impaired macrophage phagocytosis

abnormal T−helper 1 physiology

0 1 2 3 4 5 6

abnormal neutrophil physiology CST7

HBB

BDP1

ELANE

CARHSP1

SASH1

HIF1A

BCL2A1

ID2

EP300

IFNGR1

HIPK1

DOCK11

MT2A

CMTM6

EMB

LYST

−2

−1

0

1

2

3

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

27.4%

0 102 103 104

BCL2A1

0

20

40

60

80

100G-MDSC

IgG2a

B4

Iso

BA C D

E

F G

H

IgG2a B4

0.00

0.25

0.50

0.75

1.00

1.25 *

37.1%

0-103 103 104 1050

50K

100K

150K

200K

250K

45.3%

0-103 103 104 1050

50K

100K

150K

200K

250K

0.84%

0-103 103 104 1050

50K

100K

150K

200K

250K

HIF1α

G-MDSCB4

G-MDSCIgG

G-MDSCIso

Figure 9

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

4.85%

0 104 105

CMTM6

0

20

40

60

80

100G-MDSC

IgG2a

B4

Iso

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 *

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

14.5%

0 104

BCL2A1

0

20

40

60

80

100G-MDSC

IgG2a

B4

Iso

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25 **

49.6 %

0 102

103

104

0

-103

103

104

105

39.0 %

0 102

103

104

0

-103

103

104

105

0 %

0 102 103 104

0

-103

103

104

105

CMTM-6

G-MDSCB4

G-MDSCIgG2a

G-MDSCIso

Lewinskyetal. 39

Figure9-CD84regulatesfunctionalandimmunosuppressivepathwaysinhumanMM

G-MDSCs

(A-C)SortedBMG-MDSCs(CD15+,CD14-,HLA-DR-,CD11B+)wereactivatedwithanti-CD84

or IgG1k antibodies. After 24 hrs RNAwas purified and sequenced using Mars-seq. (A)

VolcanoplotforG-MDSCgeneswithgreyindicatingnon-DEgenes,andDEexpressedgenes

showneitherinblue,fordownregulated,orinred,forupregulated,genes.(B)Highlighted

gene ontology terms from the upregulated DE genes of G-MDSCs (MGI Mammalian

Phenotype2017,EnricheR).(C)HeatmapdepictinghighlightedgenesfromtheDEgenes,

withlog2CD84activatedtoIgGcontrolratiovaluesshownforeachpatient(n=3).

(D-F)PrimaryMMbonemarrowaspiratesweretreatedwithB4orIgG2aantibodies.After

48hrs,G-MDSCswereanalyzed forCMTM6 (n=4) (D),BCL2A1 (n=5) (E), andHIF1α (F)

proteinlevels.Representativeplotsareshownonintheright.(G-H)Primarymurinesorted

G-MDSCs5TGM1injectedmicewereblockedwithB4orIgG2aantibodies.RNAwaspurified

after24hrs,andcellswereanalyzed forCMTM6(G)andBCL2A1(H)message(left)and

protein(right)expression.Representativehistogramsareshownontheright(n=3-4).

*p<0.05,**p<0.01,***p<0.001,withpairedT-testforpairwisecomparisons.

A

Figure 10

B

C D E F

HG

WT CD84 -/-0.00

0.25

0.50

0.75

1.00

1.25

1.50 *

WT CD84 -/-0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75 *

WTCD84 -/-0

4

8

12

16 ****

WT CD84 -/-0.00

0.25

0.50

0.75

1.00

1.25

1.50 *

WT CD84 -/-0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75 **

WTCD84 -/-0.00

0.25

0.50

0.75

1.00

1.25

1.50 *

WT CD84 -/-0.0

0.4

0.8

1.2

1.6 **

WTCD84 -/-0.00

0.25

0.50

0.75

1.00

1.25 *

4.27%

0 104 105

0

-103

103

104

10514.1%

0 104 105

CD19

0

-103

103

104

105

CD13

8

WT chimera CD84 -/- chimera

G-MDSC

M-MDSC

0-103 103 104 105

0

-103

103

104

105

G-MDSC

M-MDSC

0-103 103 104 105

Ly6G

0

-103

103

104

105

Ly6C

12.3 %

9.38 % 4.29 %

4.29 %

WT chimera CD84 -/- chimera

41.9%

0 104 1050

20

40

60

80

100

PD-L1

MM

WTCD84 -/-

Iso

27.0%

0 104 1050

20

40

60

80

100

PD-L1

M-MDSC

WT CD84 -/-

Iso

0

20

40

60

80

100

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

0 7 14 210

20

40

60

80

100%

M-M

DSC

in P

B

*** ns **

****

0

20

40

60

80

100

** ns *****

0

20

40

60

80

100

** **** *****

G-M

DSC

in P

B%

% C

D84

on

G-M

DSC

in P

B

% C

D84

on

M-M

DSC

in P

B

Days0 7 14 21

Days0 7 14 21

Days0 7 14 21

Days

Lewinskyetal. 40

Figure10-M-MDSCexpansionandsuppressioninvivoisdependentonCD84

(A-B) 5TGM1 cellswere I.V. injected into syngeneic immune-competent C57BL/KaLwRij

mice.PBsampleswerecollectedandanalyzedafter1week,2weeks,and3weeksbyweekly

submandibularbleedingaftertheinjection.ThepercentagesofM-MDSCs,G-MDSCs(A)and

CD84 expression onM-MDSCs andG-MDSCs (B)were analyzed by flow cytometry (n=5,

**p<0.01,***p<0.001,****p<0.0001).(C-H)WTorCD84-/-micewereirradiatedwith1050

Radandinjectedwith1x106C57BL/KaLwRijBMcellsafter1day.After60days,themice

wereinjectedwith1x1065TGM1cells.Afteranadditional21days,themiceweresacrificed,

andtheirbonemarrow,bloodandspleensanalyzed.(C-D)PercentofMMcellsinthebone

marrow(C),andspleen(D).(E)IgG2bantibodiesintheblood.(F)PD-L1proteinlevelson

thesurfaceofMMfrombonemarrow(Leftgraph)andspleen(Rightgraph).Representative

histogramoftheBMisshown(n=9-14,*p<0.05,**p<0.01).(G)PercentofM-MDSCsinthe

bonemarrow.Representativedotplotsshownontheright(n=14,****p<0.0001).(H)PD-L1

on the surface of M-MDSCs from bone marrow (left graph) and spleen (right graph).

Representativehistogram,ofBMisshowninthemiddle(n=6-10,*p<0.05).

WT CD84-/-0

4

8

12

16

20 ***

0.00.40.81.21.62.02.42.83.23.6

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

* * * * *

WT CD84-/-0.00

0.25

0.50

0.75

1.00

1.25

1.50 *

0.0

0.5

1.0

1.5

2.0

2.5 ***

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

****** ** **

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

IL- 2 IFNγ GRZMB LAMP- 1

* ** *** **

29.4%

0 104 1050

20

40

60

80

100

PD-L1

G-MDSCWT CD84-/-

Iso

13.3%

0 103 1040

20

40

60

80

100

4.56%

0 103 1050

20

40

60

80

100

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

CD8+

WT CD84-/-

Iso Iso CD8+

WT CD84-/-

4.05%

0 103 1040

20

40

60

80

100

3.76%

0-103 103 104 1050

20

40

60

80

100

9.43%

3 3 4 50

20

40

60

80

100CD8+

WT CD84-/-

CD8+

WT CD84-/-

CD8+

WTCD84-/-

Iso Iso Iso Iso

13.4%

0 104

IFNγ

0

50K

100K

150K

200K

250K

8.35%

0 104

IFNγ

0

50K

100K

150K

200K

250K

18.4%

0 104

GRZMB

0

50K

100K

150K

200K

250K

31.3%

0 104

GRZMB

0

50K

100K

150K

200K

250K

WT chimera CD84-/- chimera WT chimera CD84-/- chimeraCD8+ CD8+ CD8+ CD8+

1.46%

0-103 103 104 1050

50K

100K

150K

200K

250K

GRZMB

Unactivated

1.20%

0-103 103 104 1050

50K

100K

150K

200K

250K

IFNγ

Unactivated

Figure 11

A B C

D

E

Lewinskyetal. 41

Figure11-G-MDSCexpansionandsuppressioninvivoisdependentonCD84

(A-E) WT or CD84-/- mice were irradiated with 1050 Rad and injected with 1x106

C57BL/KaLwRij BM cells after 1 day. After 60 days, themice were injected with 1x106

5TGM1cells.Afteranadditional21days,themiceweresacrificed,andtheirbonemarrow,

bloodandspleensanalyzed.(A)PercentofBMG-MDSCs(n=12-13,***p<0.001).(B)PD-L1

expressiononthesurfaceofBMG-MDSCs.Representativehistogram,isshownontheright

(n=7-10,*p<0.05).(C-E)Bonemarrow(C)andspleen(D)-derivedTcellswereanalyzedfor

PD-1, LAG-3, CTLA-4, 2B4 and KLRG-1. Representative histograms, with percentages

displaying the CD84-/- cells, are shown on the right (M; n=13-17, *p<0.05) (N; n=11-15,

**p<0.01,***p<0.001).(E)SplenicTcellswereactivatedfor24hrsusinganti-CD3antibody.

Brefeldin-Awasaddedtothecultureforthelast2hrs.Representativedotplotsareshown

ontheright(n=13-17,*p<0.05,**p<0.01,***p<0.001).

A

0 80

20

40

60

80

100

28 30 32 34 36 38

*

Days

WT

CD84 -/-

WT CD84-/-0.00

0.25

0.50

0.75

1.00

1.25

1.50 ****

BMM

8.69%

0-103

103

104

0

-103

103

104

105MM

23.2%

0-103

103

104

CD19

0

-103

103

104

105

WT CD84-/-

C

CD84-/-WTWT CD84-/-0.0

2.5

5.0

7.5

10.0

12.5

15.0 **D

G-MDSC

M-MDSC

0 104 105

Ly6G

0

-103

103

104

105

G-MDSC

M-MDSC

0 104 105

0

-103

103

104

105

26.4 %

12.6 %

23.2 %

5.28 %

WT CD84-/-

WT CD84-/-0

10

20

30

40

50

60

70

80 *E

54.2%

102 103 104 1050

20

40

60

80

100

PD-L1

M-MDSC

WTCD84-/-

Iso

WT CD84-/-0102030405060708090

100 *F

27.8%

0 104 1050

20

40

60

80

100

S100A9

M-MDSC

WTCD84-/-

FMO

WT CD84-/-0

10

20

30

40

50

60

70 ***

G

19.7%

0 104 105

CYBA

0

20

40

60

80

100 M-MDSCWTCD84-/-

Figure 12

Lewinskyetal. 42

Figure12-LossofCD84reducesM-MDSCexpansionandMMtumorload.

CD84-/-micebackcrossedtoC57BL/KaLwRijfor6-7generationsandC57BL/KaLwRijmice

wereinjectedwith5TGM1cells.After28daysforallexperimentsexceptthesurvivalstudy,

themicewere sacrificed and their bonemarrow, blood and spleenswere analyzed. (A)

Survival curve (n=8-9, *p<0.05, Log-rank test). (B) Percentage of MM cells in the bone

marrow(n=9-10,****p<0.0001).Representativedotplotsareshown.(C)Spleensderived

fromtheWTandCD84-/-mice.(D)PercentageofBMM-MDSCs.Representativedotplotsare

shownontheright,(n=5-6,**p<0.01).(E-G)PD-L1(E),S100A9(F),andCYBA(G)protein

levelsweredeterminedinbonemarrowM-MDSCsbyFACS.Representativehistograms,are

shownontheright(n=5-11,*p<0.05,**p<0.01,***p<0.001).

Figure 13

WT CD84-/-0

10

20

30

40 ns

A

WT CD84-/-0102030405060708090 ****

B

41.8%

102 103 104 1050

20

40

60

80

100

PD-L1

G-MDSC

WTCD84-/-

Iso

WT CD84-/-0102030405060708090 *

C

22.2%

103 104 105

BCL2A1

0

20

40

60

80

100G-MDSC

WTCD84-/-

Iso

WT CD84-/-0

10

20

30

40

50

60

70 *D

19.8%

0 103 104 105

CMTM6

0

20

40

60

80

100G-MDSC

WTCD84-/-

Iso

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

* * ***E

0

1

2

3

4

5

6

7

GRZMB IFNγ Lamp-1 IL-2

** * * *

FWTCD84-/- 26.6%

0 102 103 104

50K

100K

150K

25.1%

0 102 103

50K

100K

150K

9.29%

0 102 103

50K

100K

150K

9.73%

0 102 103 104

50K

100K

150KWTCD8+

GRZMB

CD84-/-

CD8+

GRZMB

WTCD8+

IFNγ

CD84-/-

CD8+

IFNγ

CD8+

IFNγ 3.72%

0 102 103

50K

100K

150KUnactivated

0%

2 3 4

50K

100K

150KUnactivatedCD8+

GRZMB

G

9.81%

0-103 103 104 1050

50K

100K

150K

200K

250K

20.6%

0-103 103 104 1050

50K

100K

150K

200K

250K

0%

0-103 103 104 1050

50K

100K

150K

200K

250KWTCD8+

LAMP-1

CD84-/-

CD8+

LAMP-1

UnactivatedCD8+

LAMP-1

0

1

2

3

4

GRZMB IFNγ Lamp-1 IL-2

* * * *

WTCD84-/-

Lewinskyetal. 43

Figure13-LossofCD84reducesG-MDSCexpansionandTcellsuppression.

CD84-/-micebackcrossedtoC57BL/KaLwRijfor6-7generationsandC57BL/KaLwRijmice

were injectedwith 5TGM1 cells. After 28 days, themicewere sacrificed and their bone

marrow,bloodandspleenswereanalyzed.(A)PercentageofBMG-MDSCs.(B-D)PD-L1(B),

BCL2A1(C),CMTM6(D)proteinlevelsweredeterminedonbonemarrowG-MDSCsbyFACS.

Representative histograms, are shown on the right (n=4-11, *p<0.05, ****p<0.0001). (E)

Bonemarrow-derivedTcellsanalyzedforPD-1,LAG-3,CTLA-4,2B4andKLRG-1cellsurface

levelsbyFACS(n=6-9,*p<0.05).(F-G)Splenic(M)orBM(N)Tcellswereactivatedfor24

hrswithanti-CD3inthepresenceoftheBrefeldin-Aduringthelast2hours.GRZMB,IFNγ,

LAMP-1andIL-2proteinlevelswereanalyzedbyFACS.RepresentativedotplotsofGRZMB

andIFNγareshown(n=4-11,*p<0.05,**p<0.01).

IgG2a B40.000.250.500.751.001.251.501.752.002.25 **

IgG2a B40.00

0.25

0.50

0.75

1.00

1.25

1.50 **

0 5 26 28 30 32 34 360

102030405060708090

100

Days

B4IgG2a*

0 5 26 28 30 32 340

102030405060708090

100

Days

B4IgG2a**

IgG2a B40.0

2.5

5.0

7.5

10.0 **

IgG2a B405

10152025303540455055

%P

D-L

1on

M-M

DS

Cin

BM

**

24.2%

0 103 104 1050

20

40

60

80

100 IgG2aB4

PDL-1B4

0

10

20

30

40

50

60

70 **

%P

D-L

1on

G-M

DS

Cin

BM

47.5%

0 103 104 1050

20

40

60

80

100 IgG2aB4

PDL-1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

GRZMB IFNg

**** *** *

Lamp-1 IL-2

*

IFNg20.3%

0 103 1040

50K

100K

150K

200K

250K

GRZMB12.0%

0-103 1030

50K

100K

150K

200K

250K

IFNg9.17%

0 103 1040

50K

100K

150K

200K

250K

30μg IgG2a

GRZMB28.7%

0-103 1030

50K

100K

150K

200K

250K30μg B4

Granzyme B

INFγ

SS

C

CD8+ CD8+

CD8+ CD8+

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

***** ***

0.00

0.25

0.50

0.75

1.00

1.25

1.50

PD-1 LAG-3 CTLA-4 2B4 KLRG-1

* **** * *

%B

MC

D3+

+ T

cel

ls

(X-f

old

of Ig

G2a

)

A

Figure 14

D E F

G H

I J K

B C

IgG2aB4

IgG2aB4

GRZMB0.42%

0-103 1030

50K

100K

150K

200K

250K CD8

IFNg0.62%

0 103 1040

50K

100K

150K

200K

250K CD8

G-MDSC

M-MDSC

0 104 105

0-10

310

3

104

105

G-MDSC

M-MDSC

0 104 105

Ly6G

0-10

310

3

104

105

9.86%

28.6%

6.22%

27.1%

30 μg B430 μg IgG

MM22.0%

0-103 103 104

CD19

0

103

104

105 MM

11.0%

0-103 103 104

0

103

104

10530 μg B430 μg IgG

IgG2a B40.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 *

IgG2aB4

+

+

Lewinskyetal. 44

Figure14-B4hasatherapeuticeffectonMMinjectedmice.

(A-K)C57BL/KaLwRijmicewereinjectedwith1x1065TGM1cells.After2weeks,themice

weredividedintotwogroupsandtreatedwith30µgB4anti-CD84blockingantibody,or

IgG2acontrol.(A-C)Percentof5TGM1cellsinthebonemarrow(A),andspleen(B)andfold

ofchanceinserumIgG2b(C),followingfivetreatmentswithB4.Representativedotplotsare

shownontheright (n=9-12). (D-E)Survivalcurve(D)andpercentofparalyzedmice(E)

(n=10-11). (F-G) Percent of BMM-MDSCs (F) and PD-L1 expressed on their surface (G)

followingfiveinjections.Representativedotplotisshownontheright(n=12;9).(H)Percent

of PD-L1 on G-MDSCs in the bone marrow following five injections. Representative

histogram,areshownintheright(n=7).(I-J)Bonemarrow(I),andspleen(J)-derivedTcells

wereanalyzedforPD-1,LAG-3,CTLA-4,2B4andKLRG-1byFACS(n=9-13).(K)SplenicT

cellswereactivatedfor24hrswithanti-CD3togetherwithBrefeldin-Aduringthelast2hrs.

GRZMB,IFNγ,LAMP-1andIL-2proteinlevelswereanalyzedbyFACSwithrepresentative

dotplots(n=7-12).

Lewinskyetal. 45

Figure15-B4reducestumorload,MDSCsandTcellsuppressioninadosedependent

manner.

(A-H)C57BL/KaLwRijmicewereinjectedwith1x1065TGM1cells.After2weeks,themice

weredividedintothreegroupsandtreatedwithfiveinjectionsof90or200μgofB4,or200

μg IgG2a. (A) Percentage of 5TGM1 cells in the bonemarrow following five treatments.

Representativedotplotshownontheright(n=4-7).(B)PercentofPD-L1on5TGM1cellsin

thebonemarrowfollowingfivetreatments,(n=4-6).(C)PercentofM-MDSCs inthebone

marrowfollowingfiveinjections.Representativedotplotsareshown(n=4-6).(D)Percent

ofG-MDSCsinthebonemarrowfollowingfiveinjections.Representativedotplotshownin

(C)(n=4-6).(E-F)Arginase-1stainingonBMM-MDSCs(E),andG-MDSCs(F)followingfive

injectionsof200μgB4or200μgIgG2a(n=5-7,**p<0.01).(G)iNOSstainingonBMM-MDSCs

followingfiveinjectionsof200μgB4or200μgIgG2a(n=5-7,**p<0.01).(H)Bonemarrow-

derived T cells analyzed for PD-1, LAG-3, CTLA-4, 2B4 and KLRG-1. Representative

histogramsareshownontheright (n=4-8). *p<0.05,**p<0.01,***p<0.001,****p<0.0001,

withunpairedT-testforpairwisecomparisons,one-wayANOVAwithHolm-Sidakmultiple

correctionstestforthreegroups.