CD200 Receptor Restriction of Myeloid Cell Responses Antagonizes Antiviral Immunity and

20 Pages·2017·0.95 MB·English

RESEARCHARTICLE
CD200 Receptor Restriction of Myeloid Cell
Responses Antagonizes Antiviral Immunity
and Facilitates Cytomegalovirus Persistence
within Mucosal Tissue
GabrielleStack1,EmmaJones1,MorganMarsden1,MariaA.Stacey1,RobertJ.Snelgrove2,
PaulLacaze3,LauraC.Jacques1¤,SimoneM.Cuff1,RichardJ.Stanton1,Awen
M.Gallimore1,TracyHussell4,GavinW.G.Wilkinson1,PeterGhazal3,PhilipR.Taylor1,
IanR.Humphreys1,5*
a11111
1InstituteofInfectionandImmunity,SchoolofMedicine,CardiffUniversity,HeathPark,Cardiff,United
Kingdom,2LeukocyteBiologySection,NationalHeartandLungInstitute,ImperialCollegeLondon,London,
UnitedKingdom,3DivisionofPathwayMedicineandEdinburghInfectiousDiseases,Universityof
Edinburgh,Edinburgh,UnitedKingdom,4ManchesterCollaborativeCentreforInflammationResearch
(MCCIR),UniversityofManchester,Manchester,UnitedKingdom,5WellcomeTrustSangerInstitute,
Hinxton,Cambridgeshire,UnitedKingdom
¤Currentaddress:InstituteofInfectionandGlobalHealth,UniversityofLiverpool,Liverpool,UnitedKingdom
OPENACCESS
* [email protected]
Citation:StackG,JonesE,MarsdenM,StaceyMA,
SnelgroveRJ,LacazeP,etal.(2015)CD200
ReceptorRestrictionofMyeloidCellResponses Abstract
AntagonizesAntiviralImmunityandFacilitates
CytomegalovirusPersistencewithinMucosalTissue.
CD200receptor(CD200R)negativelyregulatesperipheralandmucosalinnateimmune
PLoSPathog11(2):e1004641.doi:10.1371/journal.
ppat.1004641 responses.Viruses,includingherpesviruses,haveacquiredfunctionalCD200orthologs,
implyingthatviralexploitationofthispathwayisevolutionaryadvantageous.However,the
Editor:AnnB.Hill,OregonHealthSciences
University,UNITEDSTATES rolethatCD200Rsignalingplaysduringherpesvirusinfectioninvivorequiresclarification.
Utilizingthemurinecytomegalovirus(MCMV)model,wedemonstratethatCD200Rfacili-
Received:June19,2014
tatesviruspersistencewithinmucosaltissue.Specifically,MCMVinfectionofCD200R-
Accepted:December22,2014
deficientmice(CD200R-/-)elicitedheightenedmucosalvirus-specificCD4Tcellresponses
Published:February5,2015
thatrestrictedviruspersistenceinthesalivaryglands.CD200Rdidnotdirectlyinhibitlympho-
Copyright:©2015Stacketal.Thisisanopen cyteeffectorfunction.Instead,CD200R-/-miceexhibitedenhancedAPCaccumulationthatin
accessarticledistributedunderthetermsofthe
themucosawasaconsequenceofelevatedcellularproliferation.AlthoughMCMVdoesnot
CreativeCommonsAttributionLicense,whichpermits
encodeanobviousCD200homolog,productivereplicationinmacrophagesinducedexpres-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare sionofcellularCD200.CD200fromhematopoieticandnon-hematopoieticcellscontributed
credited. independentlytosuppressionofantiviralcontrolinvivo.TheseresultshighlighttheCD200-
DataAvailabilityStatement:Allrelevantdataare CD200Rpathwayasanimportantregulatorofantiviralimmunityduringcytomegalovirusin-
withinthepaperanditsSupportingInformationfiles. fectionthatisexploitedbyMCMVtoestablishchronicitywithinmucosaltissue.
Funding:ThisworkwassupportedbyaWellcome
TrustSeniorResearchFellowshipinBasic
BiomedicalScience(WT098026MA,toIRH),anMRC
SeniorFellowship(G0601617/1toPRT)anda
AuthorSummary
President’sScholarship(toGS).PGwassupported
bytheWellcomeTrust(WT066784)andbythe Immuneinhibitoryreceptors,includingCD200receptor(CD200R),canlimitimmunere-
BBSRC/EPSRC(BB/D019621/1).PLwassupported sponsesinthemucosatorestrictreactivitytotheplethoraofharmlessantigensthat
byaBBSRCstudentship.Thefundershadnorolein
PLOSPathogens|DOI:10.1371/journal.ppat.1004641 February5,2015 1/20
CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
studydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. mucosalsurfacesarecontinuallyexposedto.However,virusesmayexploitthesesuppres-
sivemechanismstoenabletheirpersistenceandspread.Manyviruses,includingherpesvi-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. ruses,haveacquiredfunctionalhomologsofCD200,theligandofCD200R,implyingthat
viralexploitationofthispathwayisevolutionaryadvantageous.Wenowshowthattheβ-
herpesvirusmurinecytomegalovirus(MCMV)takesadvantageoftheCD200Rinhibitory
pathwaytopersistwithinamucosalsiteofMCMVpersistence,thesalivaryglands.Mice
deficientinCD200Rmountedelevatedantiviralimmuneresponsesthatweredrivenby
theincreaseddivisionandaccumulationofmyeloidcellsthatfunctiontoorchestratethe
generationofantiviraleffectorimmuneresponses.Interestingly,MCMVinfectionofmye-
loidcellsup-regulatedCD200expression.Thus,MCMVexploitstheCD200pathwayto
persistwithinmucosaltissue.
Introduction
CD200RisanImmunoglobulinsuperfamilyfamilymemberthatisexpressedbyhematopoietic
cells,withnotablyhighexpressiononmyeloidcells[1].TheligandofCD200R,CD200(OX2),
isbroadlyexpressedbycellsofhematopoieticandnon-hematopoieticorigins[2].Theprimary
functionoftheCD200Rpathwayistolimitimmunereactivity.CD200-CD200Rinteractions
induceaunidirectionalinhibitorysignalwithinCD200R-bearingcellsthatismediatedbytyro-
sinemotifsinthecytoplasmicdomainofCD200RthatrecruitDOK2andRasGAP,resultingin
inhibitionoftheERKpathway[3–6].
TheCD200Rpathwaynegativelyregulatesmyeloidcellhomeostasisintheperiphery[7],
andinthepulmonary[8]and,toalesserextent,theintestinal[9]mucosa.CD200Rsignaling
limitstherapidonsetofexperimentalautoimmuneencephalomyelitis[3,7]andrestrains
bacterial-inducedinflammation[10].Importantly,CD200Ralsorestrictsviral-inducedinflam-
mationduringrespiratoryinfluenzainfection[9,11]andherpessimplexvirus(HSV)infection
ofthecornea[11].However,CD200RalsorestrictsIFN-dependentcontrolofcoronavirus
infectionviaregulationofTLR7[12]andcontrolofintracranialHSVinfection[13],demon-
stratingthatthisinhibitoryreceptorcanimpingeonprotectiveantiviralimmunity.
Duringevolution,numerousherpesviruseshaveacquiredproteinswiththepotentialtoin-
duceimmuneinhibitoryreceptorsignaling[14].Forexample,humancytomegalovirus
(HCMV)encodesafunctionalhomologoftheinhibitorycytokineinterleukin-10(IL-10)[15].
RhesusCMVlackingitsIL-10homologinducesincreasedvirus-specificimmuneresponses
[16],andIL-10Rsignalingduringmurinecytomegalovirus(MCMV)infectionantagonizesan-
tiviralimmunityandfacilitatesviruspersistence[17–19].Thus,thesestudiesprovideinvivo
experimentalevidencesupportingarationaleforCMVexploitationofhostimmune
regulatorypathways.
IntriguinglyHCMVUL119–121proteinsdisplayhomologytohumanCD200[20],al-
thoughitiscurrentlyunknownwhethertheyinduceinhibitorysignalingthroughCD200R.
However,numerousherpesvirusesareknowntoencodefunctionalCD200orthologs
(vCD200s)implyingthatexploitationofthisinhibitorypathwayispotentiallyadvantageous
forherpesviruses.Themostwell-characterizedvCD200istheKaposi’ssarcoma-associatedher-
pesvirus(KSHV)proteinK14,whichsuppressestheactivationofneutrophils[21],basophils
andNKcells[22],Tcells[23]andmacrophages[24]invitro.Furthermore,theEnglishisolate
ofratcytomegalovirus(RCMV-E)encodesaCD200homolog(e127)capableofbinding
CD200R[25,26].
PLOSPathogens|DOI:10.1371/journal.ppat.1004641 February5,2015 2/20
CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
DespitethepossibleimportanceoftheCD200-CD200Rpathwayinmodulatinganti-CMV
immunity,howitinfluencesantiviralimmuneresponsesandvirusreplicationduringinfection
invivorequiresclarification.Toinvestigatethis,westudiedMCMVinfectioninwildtypemice
andmicelackingCD200R.ExperimentsrevealedapivotalroleforCD200Rregulationofmye-
loidcellresponsesinlimitingantiviralCD4Tcellresponses.WeprovideevidencethatMCMV
exploitstheCD200-CD200Rpathwaytofacilitatepersistentinfectionwithinmucosaltissue.
Results
CD200RpromotesMCMVpersistenceinthesalivaryglands
MCMVreplicatesinnumerousorgans,includingthespleen,liverandlungs,duringacutein-
fection,priortodisseminationtothesalivaryglands(SGs),inwhichMCMVreplicatesfor1–2
months[27,28].WehypothesizedthatCD200RsignalingmayfacilitateMCMVreplicationin
vivo.Totestthis,wildtypeC57BL/6(wt)andCD200R-/-micewereinfectedwithMCMVand
virusloadmeasured.PeakacuteMCMVreplicationatday4post-infection(pi)inthespleen
(Fig.1A)andliver(Fig.1B)wasunalteredbyCD200Rdeficiency.However,CD200R-/-mice
exhibitedareducedburdenofreplicatingvirus(Fig.1A)inthespleen7dayspi.
WenextinvestigatedwhetherCD200RpromotedMCMVpersistence.Inourmodel,repli-
catingvirusisfirstdetectableintheSGsatday7pi.VirusloadinwtandCD200R-/-miceday
7piwascomparable(Fig.1C),suggestingthatimprovedantiviralcontrolinspleensof
CD200R-/-mice(Fig.1A)didnotinfluencedisseminationtotheSGsandassociatedbrownfat
inwhichMCMVreplicatesatthistime-point[29].Crucially,however,CD200R-/-micere-
strictedpersistentMCMVreplicationintheSGs14dayspi,andmoreCD200R-/-micecleared
MCMVbyday33piascomparedtowtcontrols(Fig.1C).Thus,intactCD200Rduringchronic
infectionpromotedviruspersistencewithinthismucosalorgan.
CD200andCD200RareexpressedduringMCMVinfection
ConsistentwithbiologicalimpactofCD200RwithintheSGs,weobservedsignificantCD200R
expressionbyCD11c+MHCII+salivarygland(SG)APCs(referredtohereafterasSG-APCs,
Fig.1D&E),whicharephenotypicallyindicativeoftissue-residentmacrophages[30],andNK
cells(Fig.1D)butnotCD4andCD8Tcells(Fig.1D).CD200RexpressionbySGmyeloidcells
wasnotablyhigherthanspleniccounterparts(Fig.1E),demonstratingenhancedexpressionof
CD200Rinmucosalversusnon-mucosalsitesofMCMVinfection.CD200Rexpressionbymy-
eloidcellsinbothcompartmentswasrelativelystableduringinfection,withaslightreduction
intheintensityofCD200Rexpression4dayspipriortorecoverytosteady-statelevelsby
14days(Fig.1E).Interleukin-10(IL-10)isexpressedintheSGsinresponsetoMCMVinfec-
tionandpromotesviruspersistence[18,31].AlthoughIL-10inducesCD200Rexpressionby
macrophagesinvitro[8],MCMV-infectedIL-10-/-miceexhibitednoalterationsinCD200R
expressionbymyeloidcellsduringinfection(Fig.1F).Thus,CD200Rwasexpressedduring
infectionbutwasnotsignificantlyupregulatedinresponsetoMCMV,byeitheranIL-10-
dependentorindependentmechanism.
Unlikecertainherpesviruses[14,24],MCMVdoesnotencodeanobviousvCD200[32].
WithininfectedSGs,CD200+cellswerepredominantlylargeCD31+cells(Fig.2A,isotypecon-
trols:S1AFig.)thatwereEpCAM-(Fig.2B),suggestiveofendothelialcellorigin,andnot
EpCAM+acinarepithelialcellsinwhichMCMVreplicatesduringthepersistentphaseofinfec-
tion[33].CD200+cellsdidnotexpressalphasmoothmuscleactin(S1BFig.),alsodemonstrat-
ingthesecellswerenotmyoepithelialcells.Interestingly,CD200+cellswereoftenobservedin
ring-likestructuresaroundacinarepithelialcells(Fig.2B),indicativeofcapillarynetworksthat
surroundacini[34].CD200+CD31+cellsweredetectableinnaïveSGs(S1CFig.),andwe
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
Figure1.CD200RpromotesMCMVpersistenceinthemucosa.(A-C)MCMVtitresinthespleen(A),liver
(B)andSGs(C)weremeasured4and7(A&B)or7,14and33(C)dayspi.Individualmiceandmedianare
shown.Horizontaldashedlinesdepictthelowerlimitofdetection.(D)HistogramoverlaysofCD200R
expressionbySGleukocytesatday7pi.Blueline=wt,shaded=CD200R-/-.(E&F)Intensityofsurface
CD200RexpressionbyAPCs(E)isolatedfromtheSGsandspleensofwtmiceisrepresentedasmedian
fluorescentintensity(MFI).Resultsrepresentthemean+/-SEMof3mice.(F)WtandIL-10-/-micewere
infectedwithMCMVandon7or14dayspiCD200RexpressionbySG-APCswasanalyzedbyflow
cytometry.FluorescenceMinusOne(FMO)control=SG-APCsfrominfectedwtmice7(left)or14(right)
dayspi.Plotsarerepresentativeof3–4mice.Alldataisrepresentativeof2–3separateexperiments.
doi:10.1371/journal.ppat.1004641.g001
observednonotableincreaseintheintensityofCD200expressionbyCD31+cellswithinin-
fectedtissue.InadditiontoabundantCD200+CD31+cells,amorescarcepopulationof
CD200+CD45+cellswasalsodetectablewithintheSGsindicatingthepresenceofCD200ona
hematopoieticcelltype(s)(Fig.2C).AnalysisbyflowcytometryrevealedsignificantCD200ex-
pressionbyAPCsandTcellswithintheSGsandspleen(Fig.2D&E)duringMCMVinfection.
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
Figure2.CD200isexpressedduringinvivoMCMVinfection.(A-B)WtmicewereinfectedwithMCMV
andSGsharvested7dayspi.(A)CD31(green)andCD200(red)co-localizationinSGs.(B)DistinctCD200
(red)expressionfromEpCAM+(green)acinarepithelialcells(top).IsotypecontrolsforCD200(RatIgG2a-
Biotin)andEpCAM(RabbitIgG),andthesecondaryantibodiesStreptavidin555andAlexaFluor488anti-
rabbitIgG(Bottom).(C)Hematopoieticcell(green[CD45])expressionofCD200(red).(A-C)Magnification=
63x,whitescalebars=20μm.(D)RepresentativehistogramoverlaysofCD200expressionbySG-APCs,
CD4andCD8TcellsandNKcellsintheSGsatday7pi.FMOcontrolsareshownfromcellsisolatedatday7
pi.(E)CD200expressionbySGandsplenicmyeloidcellpopulationswasassessedovertime.SplenicDCs:
CD11c+MHCII+;splenicmacs:F4/80+CD11b+.Dataisrepresentedasmean±SEMof3mice/group
representing4experiments.
doi:10.1371/journal.ppat.1004641.g002
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
MCMVinfectionofmacrophagesup-regulatesCD200
Interestingly,wenotedthatCD200expressionbyAPCpopulationsintheSGsandspleenwas
inducedabovebaselineuponinfection(Fig.2D&E).WehypothesizedthatMCMVinfectionof
myeloidcellsmaydirectlyinfluenceCD200expression.Weinfectedmyeloidcellpopulations
invitrousingamultiplicityofinfectionof1thatleadstoaninfectionefficiencyoflessthan
60%(seeFig.3Aforexample),enablingustocomparesurfaceCD200proteinlevelsonunin-
fectedandinfectedcellsfromthesamewellofatissuecultureplate,asidentifiedbyflowcyto-
metricdetectionoftheintracellularMCMVm06protein.Infectionofbonemarrow-derived
macrophages(BM-DM,Fig.3A–C)andsplenicmacrophages(Fig.3B)up-regulatedCD200
(Fig.3A–C).Importantly,weobservedamarkedincreaseinCD200expressionbyinfected
(m06+)ascomparedwithuninfected(m06-)macrophagesderivedfromthesamewells
(Fig.3A–C),suggestingthatCD200ispreferentiallyup-regulatedbymacrophagesinwhich
MCMVisactivelyreplicating.
InfectionofBM-DMwithinfluenzadidnottriggerCD200expression(Fig.3D),demon-
stratingthatCD200up-regulationisnotagenericmacrophageresponsetoviruses.However,
CD200expressionisinducedbyligationofTLRs,includingTLR3andTLR9[10];bothof
whicharetriggeredbyMCMV[35,36].Inaccordance,TLR3ligationbyPolyI:Cinducedsub-
stantialCD200mRNAexpressionbymacrophagesinanIFNβ-dependentmanner(Fig.3E).
ToinvestigatewhetherMCMVinductionofCd200transcriptionrequiredproductivereplica-
tion,wecomparedexpressionfollowingmacrophageinfectionwithIE3knockoutreplication-
deficientMCMV(ΔIE3)[37]andreplication-sufficientwtMCMV(pSM3fr).Macrophageex-
posuretoΔIE3MCMVinducedasmall,transientinductionofCD200mRNAinanIFNβ-
dependentmanner(Fig.3F),consistentwithTLR-mediatedinductionofCD200triggereddur-
ingincompleteMCMVreplication,andthemoderateCD200proteinexpressionbyuninfected
(m06-)macrophagesderivedfrominfectedcellcultures(Fig.3C).Incontrast,replicating
MCMVinducedsubstantialandprolongedCD200mRNAexpressionindependentlyofIFNβ
(Fig.3G).Furthermore,inhibitionofviralDNApolymerasewithphosphonoaceticacid(PAA)
antagonizedMCMV-inducedCD200expressioninBM-DMs(Fig.3H),againdemonstrating
therequirementforproductivevirusreplicationinthisprocess.Importantly,weobservedthat
SG-APCsdidnotsupportMCMVreplicationinvitroinaccordancewiththeabsenceof
detectableinfectioninvivo[38],andMCMVinfectionofsplenicDCsdidnotfurtherinduce
CD200expression(Fig.3B).Thus,thesedatasuggestedthatmyeloidcellsup-regulatedCD200
duringMCMVinfectionand,inthecaseofmacrophagesinsecondarylymphoidtissues,
MCMVinducesCD200expressionindependentlyofTLRstimulationduringproductive
replication.
CD200derivedfromhematopoieticandnon-hematopoieticcells
promotesMCMVpersistence
GiventhatCD200expressionbybothhematopoieticandnon-hematopoieticcellswasob-
servedinMCMV-infectedmice,wesoughttounderstandwhichcellularcompartmentwasre-
sponsibleforinhibitingantiviralimmunity.Wemadebonemarrowchimerasderivedfromwt
miceormicedeficientofCD200,generatingmicelackingCD200withinthehematopoietic
and/orradiation-resistant(non-hematopoietic)compartment.Wethenstudiedvirusloadin
SGs14dayspost-MCMVinfection.Interestingly,deletingCD200fromeithercompartmentre-
ducedvirusloadascomparedtowt>wtmice(Fig.4),demonstratingthatCD200expressedby
hematopoieticandnon-hematopoieticcellsbothdeliveredimmunesuppressivesignalsthat
promotedMCMVpersistence.
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
Figure3.MCMVinducesCD200expressionbymacrophages.(A-C)Bonemarrow-derivedmacrophages
(BM-DMs)wereinfectedwithpSM3frMCMVandCD200expressionwasassessed.(A)Representative
bivariantflowcytometryplotsofMCMVinfection(m06+)versusCD200(top)andCD80(bottom)24hrspi.
(B)RepresentativehistogramsofCD200expressionbyinfected(m06+,blue)anduninfected/virusexposed
cellsfromthesamewell(m06-,red).BM-DMs(top),splenicF4/80+macrophages(middle)andsplenic
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
CD11chiMHCIIhiDCs(bottom).FMOsarecellsfrominfectedcellcultures(containinginfectedand
uninfectedcells).(C)CD200expressionbyMCMV-infected(m06+),MCMV-exposed(m06-)andΔIE3
MCMV-infectedBM-DMswascomparedtoexpressionbymock-infectedcells.Dataareexpressedas
medianfluorescenceintensity(MFI)andareexpressedasmean+SEMof2replicates.Resultsrepresent
2–5experiments.(D)BM-DMswereinfectedornotwithinfluenzastrainPR8(MOI:1),andCD200expression
wasassessed24hrslaterbyflowcytometry.FMOsarefromuninfectedmacrophages.Datafrom1of2
experimentsisshown.(E-G)BM-DMsweretreatedwith(E)PolyI:Corinfectedwith(F)replicationdeficient
ΔIE3MCMVor(G)wtMCMV(pSM3fr)andcd200expressionassessedbymicroarray.(H)BM-DMswere
infectedwithwtMCMV+/-PAA,andCD200expressionwasassessed24hrslater.Dataisshownasa
representativehistogram(of2separateexperiments)depictingCD200expressionbyuninfected/virus
exposedcells(m06-,red),infectedcells(m06+,blue)andPAA-treatedm06+cells(orange).Grey=m06+
untreatedcellsstainedwithisotypecontrol(ratIgG2a-PE).
doi:10.1371/journal.ppat.1004641.g003
CD200Rrestrictsmyeloidcellaccumulationandproliferation
WeassessedtheimpactofCD200Rdeficiencyonvirus-inducedmyeloidcellresponses.Re-
ducedMCMVpersistenceinCD200R-/-micewasaccompaniedbyaccumulationofsplenic
DCs14dayspi(Fig.5A).TheinabilityofSG-APCstocross-presentantigentoCD8Tcells(in
combinationwithMCMVdown-regulationofMHCclassI)hasbeenshowntoberesponsible
forthelackofCD8Tcell-mediatedcontrolofMCMVreplicationintheSGs,demonstrating
thatlocalantigen-presentingfunctionofSG-APCsisacriticaldeterminantofprotectiveTcell
immunityduringMCMVpersistence[38].Interestingly,SG-APCaccumulationininfected
CD200R-/-micewassubstantiallyincreased14dayspi(Fig.5B&C).However,SG-APCnum-
berswerecomparableinwtandCD200R-/-micefollowingresolutionofMCMVinfectionin
Figure4.CD200expressedbyhematopoieticandnon-hematopoieticcellsfacilitateMCMV
persistence.Mixedwt/CD200-/-bonemarrowchimerasweregeneratedandinfectedwithMCMV.14days
later,replicatingvirusloadintheSGswasassessedbyplaqueassay.Individualmiceandmedianareshown.
Horizontaldashedlinedepictsthelowerlimitofdetection.Datafrom3mergedexperimentsisshown.Similar
resultswereobservedinafourthexperimentsbutdatawasomittedduetointer-experimentalvariation—
medianvaluesforallgroupswere~1loglowerthanmediansofdatadepictedintheFigure.
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
Figure5.CD200RrestrictsmyeloidcellaccumulationandSG-APCproliferation.(A)Time-courseofaccumulationofsplenicCD11c+MHCII+cellsinwt
andCD200R-/-mice.Mean+SEMof4–11mice/groupisshown.(B)RepresentativeflowcytometryplotsofCD11c+MHCII+SG-APCsinwt(left)and
CD200R-/-(right)atday7pi.(C)NumbersofSG-APCsovertime.Mean+SEMof6mice/groupisshown.(D)Proliferating(EDU+)SG-APCswereassessed
byflowcytometry.Mean+SEMof3–6mice/groupisshown.(E)RepresentativebivariantflowcytometryplotsofEDUincorporationbySG-APCsinwt(left)
orCD200R-/-(right)miceatday7pi.DataisgatedonliveCD11c+MHCII+cells.Allresultsrepresentatleast2independentexperiments.(F&G)Wtmice
wereinfectedwithMCMVandSGsharvested7dayspi.(F)MHCII(green)expressingcellsadjacenttolargeCD200+(red)endothelialcells.(G)MHCII
(green)expressingcellsadjacenttoCD31+(red)endothelialcells.SectionswerecounterstainedwithTOTO-3(blue)todetectDNA.Magnification=63x,
whitescalebars=20μm.
doi:10.1371/journal.ppat.1004641.g005
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CD200RRestrictsMyeloidCellOrchestrationofAnti-CMVImmunity
ourmodel(48dayspi,S2AFig.).Thus,CD200Rrestrictedmucosalmyeloidcellaccumulation
duringearlytime-pointsofSGinfectionratherthaninfluencingmyeloidcellturnoverduring
theresolutionphaseofinfection.
Tissueresidentmacrophagesproliferateinresponsetoinflammatorystimuli[39,40].Thus,
wemeasuredSG-APCproliferationbeforeandafterMCMVinfection7dayspi.Lowlevelsof
SG-APChomeostaticproliferationweremeasuredinnaïvewtandCD200R-/-mice(Fig.5D).
However,infection-inducedSG-APCproliferationwasfurtherelevatedinCD200R-/-miceas
comparedtowtmiceday7pi(Fig.5D&E),atimeatwhichCD200Rwasexpressedbythese
cellsinwtmice(Fig.1D&E).ThissuggestedthatincreasedSG-APCaccumulationin
CD200R-/-micewasaconsequenceofheightenedproliferation.Importantly,visualizationof
MHCII+cellswithintheSGsrevealedthatMHCII+cellswereconsistentlylocatedadjacentto
largeCD200+cells7days(Fig.5F)and14days(S2BFig.)pi.InaccordancewithCD200ex-
pressionbyCD31+cells(Fig.2A),MHCII+cellswerealsoobservedsurroundingCD31+ves-
sels(Fig.5G),suggestingthattissue-residentMHCII+SG-APCinteractionswithCD200-
bearingendothelialcellsrestrictsinfection-inducedcellularproliferation.Insupportofthis
conclusion,chimericmicelackingCD200onlyinnon-hematopoieticcellsexhibitedincreased
SG-APCaccumulation(S2CFig.).Furthermore,improvedcontrolofMCMVinthesemice
(Fig.4)inadditiontotheabsenceofanimpactofnon-hematopoieticcell-derivedCD200on
splenicDCresponses(S2DFig.)pointstowardsaroleforlocalSG-APCexpansionindeter-
miningcontrolofMCMVreplicationinthemucosa.
CD4TcellslimitviruspersistenceinCD200R-/-mice
CD4TcellsarecriticaleffectorcellsinthecontrolofMCMVpersistencethataffordprotection
viaexpressionofIFNγ[38,41].DespitetheabsenceofmeasurableTcellexpressionof
CD200R(Fig.1D),SG-infiltratingCD4TcellsinCD200R-/-miceexhibitedincreasedactiva-
tion,indicatedbyCD69andCD25up-regulation10dayspi(Fig.6A&B).Enrichmentof
CD25hiCD4TcellswerenotobservedineitherwtorCD200R-/-mice(Fig.6A),consistent
withtheabsenceofregulatoryTcellinfiltrationintotheSGsinresponsetoMCMV[31].Im-
portantly,IFNγ+virus-specificCD4TcellnumberswereelevatedintheSGsofCD200R-/-
miceby14dayspi(Fig.6C).InadditiontoactivatedTcells,CD4+tissue-residentmemoryT
cellsalsoexpressCD69[42].Interestingly,whereasCD69expressionbySGCD4Tcellswasel-
evatedinCD200R-/-mice14(Fig.6D)and30(S3AFig.)dayspi,elevatedprolongedexpression
ofCD25byCD200R-/-SGCD4Tcellswasnotobserved(S3BFig.),implyingthatCD200R
mayalsorestricttheaccumulationand/orretentionofCD4Tcellswithatissue-residentmem-
ory-likephenotype.Furthermore,MHCII+SG-APCsthatproliferateandaccumulatetohigher
numbersinCD200R-/-mice(Fig.5B–E)co-localizedwithCD4Tcells(Fig.6E),suggesting
thatelevatedmyeloidcellresponseswithintheSGsofCD200R-/-miceenhancedmucosalCD4
Tcellresponses.Inaddition,elevatedsplenicDCnumbers14dayspiinCD200R-/-mice
(Fig.5A)wereaccompaniedbyanincreaseinvirus-specificCD4Tcellsinthisorganatthis
time(Fig.6F).ThesedatathereforesuggestedthatCD200Rrestrictedperipheralandmucosal
CD4Tcellresponsivenessduringviruspersistencethroughlocalizedregulationoftissue
APCaccumulation.
WenextinvestigatedwhetherelevatedCD4TcellresponsesrestrictedMCMVpersistence
inCD200R-/-mice.DepletionofCD4TcellsabrogatedtheimprovedcontrolofMCMVin
CD200R-/-mice(Fig.6G),whichisconsistentbothwiththeestablishedroleforCD4Tcellsin
limitingMCMVpersistenceintheSGs[38,41],andtheconclusionthatMCMVexploits
CD200Rtofacilitatepersistencepredominantlybyantagonizingproliferationandaccumula-
tionofMHCclassII-bearingmyeloidcells.
PLOSPathogens|DOI:10.1371/journal.ppat.1004641 February5,2015 10/20