WisconsinRegionalPrimateResearchCenterandDepartmentofObstetricsandGynecologyUniversityofWisconsinMadisonWI53715andFredHutchinsonCancerResearchCenterSeattleWA98104ReceivedforpublicationOctober26 ID: 847139
Download Pdf The PPT/PDF document "ASolubleIsoformoftheRhesusMonkeyNonclass..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
1 ASolubleIsoformoftheRhesusMonkeyNonclass
ASolubleIsoformoftheRhesusMonkeyNonclassicalMHCClassIMoleculeMamu-AGIsExpressedinthePlacentaandtheTestisAndyF.Ryan,*RichardL.Grendell,*DanielE.Geraghty,andThaddeusG.GolosThenonclassicalMHCclassIlocusisexpressedprimarilyintheplacenta,althoughothersitesofexpressionhavebeennotedinnormalandpathologicalsituations.Inaddition,solubleHLA-Gisoformshavebeendetectedintheserumofpregnantandnonpregnantwomenaswellasmen.TherhesusmonkeyplacentaexpressesanovelnonclassicalMHCclassImoleculeMamu-AG, *WisconsinRegionalPrimateResearchCenterandDepartmentofObstetricsandGynecology,UniversityofWisconsin,Madison,WI53715;andFredHutchinsonCancerResearchCenter,Seattle,WA98104ReceivedforpublicationOctober26,2001.AcceptedforpublicationMay10,2002.Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpagecharges.Thisarticlemustthereforebeherebymarkedinaccordancewith18U.S.C.Section1734solelytoindicatethisfact. peptideofHLA-G.Expressioninthetestiswasstrikinglycellspe-c,suggestingthattherhesusmonkeywillbeanexcellentmodeltostudythephysiologyofasolublenonclasicalMHCclassIMaterialsandMethodsAnimalsandtissuesAdultfemalerhesusmonkeys(Macacamulatta)usedfortimedmatingswerefromthecolonymaintainedatWisconsinRegionalPrimateResearchCenter.Rhesusmonkeyplacentaltissueswereobtainedbycesareansectionaswehavepreviouslydescribed(26).Othertissueswereobtainedfromhealthyanimalseuthanizedinotherstudies.AllsurgicalprocedureswereperformedinaccordancewiththeNationalInstitutesofHealthGuidefortheCareandUseofLaboratoryAnimalsandunderapprovaloftheUni-versityofWisconsinGraduateSchoolanimalcareandusecommittee.RT-PCR,cloning,andsequencingInRT-PCRexperiments,1gtotalRNAwasreversetranscribedtocDNAusinganoligo(dT)primerandMoloneymurineleukemiavirusreversetranscriptase(PerkinElmer,FosterCity,CA).ThecDNAwasampliedfor34cycles,witheachcycleconsistingof94Cfor30s,55Cfor30s,andCat1min,inareactionmixturecontaining10mMTris-HCl,1.5mM,50mMKCl,0.25mMofeachdNTP,and1.25UAmpliTaqDNApolymerase(PerkinElmer).Primersusedfortheamplicationandsequenc-ingofrhesusMHCclassIcDNAsaredepictedinTableI.AmplireactionsusingastemplateRTreactionstowhichnoRNAornoreversetranscriptasehadbeenaddedservedasnegativecontrols.ThePCRprod-uctswereseparatedon3%agarosegels,isolatedwiththeGeneCleanIIkit(BIO101,Vista,CA),andsubclonedusingaTAcloningkit(Invitrogen,Carlsbad,CA).SubclonedfragmentsweresequencedusinganABI377automatedsequencingmachineandtheBigDyeTerminatormix(PEAp-pliedBiosystems,FosterCity,CA).Tissuescollectedatsurgeryornecropsywereimmediatelypreparedforfrozensections.Afterxingin2%paraformaldehydefor4h,thetissueswerewashedwithPBStwicefor20mineachtime.Theywerethende-hydratedin9%sucrosefor4hand20%sucroseovernight.ThetissueswerethenembeddedinOCTmountingmedium(SakuraFinetekU.S.A.,Torrance,CA)andfrozeninisopentanecooledwithdryiceandethanol.ThemousemAbs25D3,16G1,andgoatanti--microglobulin(DAKO,Carpinteria,CA)wereusedataconcentrationof5g/ml.W6/32wasusedat1g/ml.AmouseIgG1Ab(Sigma-Aldrich,St.Louis,MO)orgoatpolyclonalIgGantiserum(SantaCruzBiotechnology,SantaCruz,CA)wereusedasnegativecontrolsatthesameconcentrationsastheprimaryAbs.Immunostainingwasperformedaspreviouslyreported(23,24).Pep-tideblockingexperimentswereperformedonplacentalandtesticularsec-tionswith16G1byincubatingtheAbwithitsimmunizingpeptideoracpeptide,GnRH,asacontrol.TheAbwasmixedwitheitherpeptideatmolarconcentrationsof4/1and10/1(peptide/Ab)andincubatedfor30minat0C.Duringthistime,thesectionswereblockedwithanimalserum.AfterAbneutralization,themixwasaddedtothesectionsfor1h.16G1aloneandmouseIgGwereusedasadditionalcontrols.Theremain-deroftheprocedurewasperformedasdescribedabove.Peroxidaseandalkalinephosphatasekitswereusedalongwithdiamin
2 obenzidene,VectorNovaRed,VectorRed,andVe
obenzidene,VectorNovaRed,VectorRed,andVectorBluesubstratekits(VectorLaboratories,Burlingame,CA).Someslideswerecounterstainedwithhematoxylin.Insituhybridization(ISH)ForISH,placentalandtesticulartissueswerepreparedforparafnsections.Tissueswerexedin2%paraformaldehydefor4handthenembeddedinn.Ten-micrometersectionswerecutandprocessedaspreviouslydescribed(23).Adigoxigenin-labeled141-bpRNAprobederivedfromexon2of,asdescribedpreviously(23),wasgeneratedforISH(digoxigeninlabelingkit,Roche,Indianapolis,IN).TranscriptionofsenseandantisenseRNAwasconrmedbydotblotandgelelectrophoresis.Hybridizationbuffercontaining400g/mlprobewasappliedtosections,whichwerethencoveredwithaglasscoversliptopreventdrying,andincubatedat55Cforupto40h.Slideswerewashedextensivelytoensurechybridization(23).Theslideswerewashedin2SSCwith0.1%SDS(0.3Msodiumchlorideand0.03Msodiumcitrate)for30min,placedin2SSCat60Cfor1h,0.2SSCatroomtemperaturefor15min,andSSCat60Cfor1h.Sectionswereblockedwith25%goatseruminPBSfor30minandthenincubatedwithalkalinephosphataseconjugatedanti-digoxigenin(1/2000;Roche)for2h.SectionswerewashedinTBSfor2,10,and10min.Slideswereincubatedindetectionbufferfor5minandthencoveredwiththesubstrateBM-purple(Roche)overnight.RecombinantMamu-AG5expressionandWesternblottingTheRNAforMamu-AG5wasampliedusingRT-PCRandprimersinexons2and6containingthestartcodonandthestopcodon,respectively(TableI,primers6and7).Thesequenceforanenterokinasecleavagesite(GACGATGACGATAAG)andhemagglutinin(HA)proteintag(TACCCATACGATGTTCCGGATTACGCTAGCCTC)werethenaddedtotheregionofthetranscriptbyasecondroundofPCR.TheresultingcDNAwasdirectlysequencedandsubclonedintopGEMvector(Promega,Mad-ison,WI)forsubsequenttransfectioninto293cells.293cellsweretran-sientlytransfectedaccordingtoastandardcalciumphosphatetransfectionprocedure,andcellswereharvestedafter48hofculture,lysed,andpre-paredforWesternblotting.Proteinsampleswerepreparedfromculturedtransfected293cellsbylysisin25mMTris-phosphate(pH7.8),2mMDTT,2mM1,2-diaminocyclohexane--tetraaceticacid,10%glycerol,and1%TritonX-100,(E1531;Promega).Sampleswerequan-titatedusingtheBradfordassay.DiscontinuousSDS-PAGEwasperformedusinga10%acrylamideresolvinggelanda3.9%stackinggel.Fiftyto100gcellextractwasfractionatedalongwithRainbowmarkers(RPN800;Amersham,ArlingtonHeights,IL)toestimatem.w.Afterelectrophoresis,proteinswereelectroblottedontoapolyvinylidenediuoridemembrane(Bio-Rad,Hercules,CA).ThemembranewaswashedtwicewithTBSandblockedwitha0.2%nonfatdrymilk/TBSsolutionovernightat4C.Fol-lowingblocking,themembranewaswashedthreetimeswitha0.1% Abbreviationsusedinthispaper:ISH,insituhybridization;HA,hemagglutinin.TableI.PrimersusedforsolubleMamu-AGRT-PCR NucleotideNo.-AGAACATGAAGACCGCGACACAGACCTA-3-GACCCCCCCAAGACAAAT-3-CTCACCTTGAGATGGGGTAAAG-3-TGGGAAAAGAGGGGAAGGTGAGGGGTCC-3-CAGCCTGAGAGTAGCTCCCGCC-3-ATGGCGGTCATGGCGCCCCGAACC-3-AAGGTCTCCAGAGAGGCTCCG-3PrimerslocationisschematicallyillustratedinFig.1Allprimersarelistedinthe5to3NucleotidenumbersarewithrespecttothesequenceofMamu-AG*0302(Ref.22,GenBankaccessionno.U84879).Thesixnucleotidesatthe3terminus(GTAAAG)arederivedfromintron4ofMamu-AG(GenBankaccessionno.Thenucleotidenumbersrefertotheintron4sequence(GenBankaccessionno.AY059404).Forprimer7,thesequenceshownreectstheintron4sequencesusedtoamplifyaMamu-AG5cDNA,numberedaccordingtointron4sequence(GenBankaccessionno.AY059404).Theprimeralsocontainedthissequenceatits5end:CGTATGGTACTTATCGTCATCGTC,whichencodedpartoftheHAepitopetag(nt110)andtheenterokinaserecognitionsite(nt674SOLUBLEMHCINRHESUSPLACENTAANDTESTIS Tween20/TBSsolution,thenincubatedwiththeanti-HAAb(0.5Roche)or16G1(5g/ml)for2hatroomtemperature.Themembraneswerewashedthreetimesfor10mineachtimeandthe
3 nincubatedwithalkalinephosphatase-conjug
nincubatedwithalkalinephosphatase-conjugatedgoatanti-mousesecondaryAb(1/3000;Bio-Rad)for1.5h.ThemembranewaswashedthreetimesandincubatedwithImmun-Starchemiluminescentsubstratesolution(Bio-Rad)for5min,andimmunoreactivebandswerevisualizedbyautoradiography.RT-PCRrevealsexpressionofatranscriptencodingasolubleWeinvestigatedwhethertheplacentaexpressesatranscriptforretainingintron4,aswiththesolublescriptspreviouslyreportedinthehumanplacentaandothertissues.First,weampliedreversedtranscribedcDNAwithprimers2and5locatedinthefourthandsixthexons,respectively,reasoningthatsincethemRNAwasonlydetectablebyRT-PCRinhumanpla-centa,wemightndthesamesituationwiththerhesusplacenta.Fig.1demonstratesthatseveralampliconsdependentonreversetranscriptionwereobtainedfromrhesusplacentalRNA.WhereasthemajorbandseeninFig.1primarilycontainedtheexpectedsequencesofthefourth,fth,andsixthexons,cloningandse-quencingofthebandindicatedbyanarrowrevealedthepresenceofanintronhighlyhomologoustothehumanintron(seebelow).TheintronsequencefromthismRNAwasconrmedtorepresentintron4initsentiretybysequencingPCRproductam-edfromrhesusgenomicDNA(notshown).Mostsigniastopcodonwasnotedatthesamelocationasthatforsoluble(Fig.1).Sincetheintron4-containingPCRampliconmightbederivedfromasplicingintermediate,wesoughttocon-rmthesplicingatexon-intronjunctions5fromthefourthintronregioninputativesolubletranscripts.Forthisexperi-mentwedesigneddownstreamprimer4withinthefourthintron,andupstreamprimer1withinthesecondexon(1domain)todeterminewhethertranscriptscontainingthefourthintronalsocontainedotherupstreamintrons.WeclonedandsequencedsevenplacentalcDNAsampliedbytheseprimersfromthreedifferentplacentas,andnonewasfoundtocontainintron2or3(notshown).WeconcludedthatthesolublecDNAsrepresentmaturemRNAsretainingonlythefourthintron.Inaddition,theseclonesallcontainedexons2,3,and4,demonstratingthatinourhandsonlyamRNAhomologoustoisdetectedintheplacenta.ThepredictedaminoacidsequencesforthefourthintronofMamu-AGandthefthintroncarboxylterminusweredetermined.AlignmentofMamu-AGintron4peptidewiththatofHLA-Grevealedthatthepredictedaminoacidsequenceswere86%iden-ticalbetweensolubleMamuAGandsolubleHLA-Gintron4-de-rivedcarboxyltermini(Fig.1).Theaminoacidsubstitutionsbe-tweenrhesusandhumanintron4peptidesweremostlyconservative.Sequencingofplacenta-derivedampliconsalsore-sultedintheidenticationofanadditionalnovelcDNA,whichretained18nucleotidesderivedfromthe3endofthefthintron(Fig.1,Mamu-AGv5).AlloftheseclonesderivedfromplacentalmRNAlackedintron4.Theentireintron5wasalsoclonedandsequencedbyPCRfromrhesusgenomicDNA,andwas442bpinlength(notshown).Theinclusionoftheintron5fragmentresultedinaninsertionofanovelsix-aminoacidcarboxyl-terminalexten-sionN-terminaltothetwoaminoacidsencodedbyexon6(Fig.).WearenotawareofanyreportsofasimilarsplicevariantinhumanMHCclassImRNAs.SolubleMamu-AGmRNAinrhesusnonplacentaltissuesOurpreviousstudiesoftheexpressionofMamu-AGdemonstratedthatthemRNAisexpressedinavarietyoftissues,withthehighestnonplacentallevelofmRNAinthetestes(25).Thosestudieswereconductedwitharibonucleaseprotectionassayse-lectiveforthe1domain(exon2).Were-evaluatedanumberofrhesusnonplacentaltissuesbyRT-PCRwithprimers3and5thatwouldselectivelyamplifymRNAscontainingtheexon4/intron4junction.Fig.1presentsrepresentativeresultsfromseveraltissues.Asexpected,placentaltissueshadabundantintronmRNA,whileinmosttissuesthisisoformwasundetectable(eye,adrenal,lymphnodes,skeletalmuscle,skin,heart,lung,liver,andpancreas;notshown)orbarelydetect-able(ovary,Fig.1;kidneyandintestine,notshown).Severaltissueshadamoderate,butconsistent,expressionofintron4-con-tainingmRNA,includingamnioticmembranes,testes,spleen(Fig.)andthyroid(datanot
4 shown).Althoughtoourknowledgetheretentio
shown).Althoughtoourknowledgetheretentionofintron4isuniqueinhumanstoHLA-Gandisnotknowninclassicalloci,thequestionaroseofwhetherthePCRfragmentsampliedrepresentedrhesusclassicalloci.SequencingofmultiplePCR-generatedclonesfrombothtesticularandplacentalRNAindicatedthatalltranscriptsfromthesetissuescontainedanentireintron4,andallcontainedthediagnosticTAAstopcodoninexon6.Fourofthesixtesticularandoneofthreespleen(butnoneof19placental)clonesse-quencedthatretainedintron4alsocontained18ntofthe3endofintron5discussedabove.Sincethestopcodonwaslocatedwithinintron4,theintron5sequencesdonotcontributetothecodingsequenceforthesenonplacentaltranscripts.Severalampliconsfromthyroidandspleenalsowerefoundtocontainthestopcodondiagnosticfor(Fig.2)(22,27,28).Surprisingly,onetranscriptfromspleenandtwofromthyroidwereapparentlyde-rivedfromclassicalMHCclassImolecule(s),sincetheydidnotcontainthediagnosticstopcodon(Fig.2).Furtherworkwillberequiredtounderstandthesignicanceofthisobservation.WesternblotsconÞrmthatAb16G1recognizesMamu-AG5WewishedtoexploretheexpressionofsolubleMamu-AGiso-formsinrhesusmonkeytissueswiththeAb16G1raisedagainstthecarboxyl-terminalregionofsolubleHLA-Gencodedbyintron4sequences.ToevaluaterecognitionofMamu-AG5by16G1,aMamu-AG5cDNAwasclonedintoanexpressionvectorcontain-ingsequencesencodinganin-frameHAtagatthe3endoftheMamu-AG5cDNA.293cellsweretransientlytransfectedwiththeconstruct,andcellextractsoftransfectedandnaivecellswereanalyzedbyWesternblot.Parallelanalysiswasperformedwith16G1andanAbagainsttheHAepitope(Fig.3).Fig.3stratesthatextractsoftransfected,butnotnave,cellsexpressedthreeimmunoreactivebandsof39kDaasidentiedbytheanti-HAAb.Fig.3demonstratesthatanidenticaltrioofbandswasidentiedbyAb16G1intransfected,butnotnave,cells.ImmunocytochemicallocalizationofplacentalandtesticularWenextconductedimmunocytochemicalanalysisforMamu-AGinthesetissues,evaluatingthreeplacentasandtestesandepi-didymifromvedifferentanimals.WeusedanmAb(25D3)againstMamu-AGwehavepreviouslydescribed(24),aswellasanmAbagainsttheintron4peptideofHLA-G(16G1)(12).Con-sistentwithpreviousreports,25D3identiedMamu-AGonthevilloussyncytialapicalmembranes(Fig.4,),intheprox-imalcolumns(Fig.4),andinthecytotrophoblasticshelldelin-eatingthematernal-fetalinterface(Fig.4).Mesenchymalcellsinthecoreofthevilli(Fig.4)aswellasthematernaldeciduaanduteruswereconsistentlynegative.TheJournalofImmunology 676SOLUBLEMHCINRHESUSPLACENTAANDTESTIS Bycontrast,immunostainingwiththe16G1Abwaslocalizedtothesyncytia,villouscytotrophoblasts,andsomevillousmesenchy-malcellsofday36placentas(Fig.4,,and).Stainingwasoftenpronouncedonthesyncytialbordertowardtheintervillousspaceandthecytotrophoblasticedgefacingthecoreofthevilli(Fig.4),suggestingcytoplasmiclocalization.Diffusestainingwasalsoobservedthroughoutbothvilloussyncytialandcytotropho-blasticcelllayers(Fig.4,,and).Largecellswithabundantcytoplasmwithinthevilli,presumablyHofbauercells,frequentlydemonstratedpositivestainingwith16G1(Fig.4).Bloodvesselswithinthevillousstromawerealsopositivelystained(Fig.4Extravillouscytotrophoblasts,notablyinthecytotrophoblasticshell,werenotstainedby16G1,unliketheresultswith25D3(compareserialsectionsFig.4,;alsoFig.4).Thespeciofimmunostainingwasdemonstratedbytheabsenceofimmuno-stainingwhenthe16G1primaryAbwaspreincubatedwiththeimmunizingpeptide(Fig.4),butnotwithanunrelatedpeptide(Fig.4).Anonspecicprimaryantiserumlikewisegavenopos-itivestaining(Fig.4Wenextconductedsimilarimmunostainingexperimentswithrhesustestes.25D3staininginthetestiswasminimalandwaslocalizedexclusivelytooccasionalcellsintheinterstitiumsur-roundingtheseminiferoustubules(Fig.5).GermcellsandSer-tolicellswithinthetubulewereconsistentlyneg
5 ative.Althoughonlyoccasionalinterstitial
ative.Althoughonlyoccasionalinterstitialcellswere25D3positive,thesecellswerefoundtobeconsistentlypositiveonserialsections,occasion-allyappearinginsmallaggregates.Othersporadiccellsweretyp-icallywithinperitubularregions,andimmunostainingofserialsec-tionssuggestedthatatleastsomeofthesecellswerepositiveforCD68,indicatingtheirlikelyidentityasmacrophages(Fig.5Serialsections(Fig.5,)demonstrateinterstitialmacrophagesthatappeartocoexpressMamu-AG,solubleMamu-AG,andCD68.EpidydimalstructuresandmaturespermwerenotpositiveforMamu-AGwith25D3(notshown). FIGURE2.Alignmentofintron4,exon5,intron5,andexon6nucleotidesequencesclonedbyRT-PCRfromrhesusplacenta,testis,spleen,andthyroid.Onerepresen-tativeplacentalsequenceisincluded,andonlynucleo-tidesthatdifferfromaconsensussequenceareshown. FIGURE1.Cloningandsequenceanalysisofsoluble,Schematicdiagramoftheexonorganizationof,indicatingthe3domains,transmembranedomain(Tm),stopcodon(TAA),andPCRprimerdesignationsandapproximatelocations.,RT-PCRamplicationofampliconsfromrhesusplacental(Pl)RNA.PrimersinthefourthandfthexonswereusedwithplacentalRNA.,AmplicationswherenoRNAwasaddedtotheRTreaction.Thearrowindicatesthebandfoundtocontaintheampliconharboringintron4.,Nucleotidesequenceofsplicevariantretainingintron4,alignedwiththeintron4mRNAsequence.Portionsofexons4and5andtheputativestopcodonsinintron4are,Predictedaminoacidsequenceoftheintron4-derivedcarboxylterminalofsolubleMamu-AGalignedwithsolubleHLA-G.,Alignmentofthenucleotidesequenceofretainingaportionofintron5(Mamu-AGv5).,Alignmentofthecarboxylterminusofmembrane-boundMamu-AG()andMamu-AGv5().G,RT-PCRofsolubleisoformmRNAfromrhesusnonplacentaltissues.Primersattheexon4/intron4boundaryandjust3oftheTAAstopcodonwereused.Asterisksindicateamplicons.mk,Sizemarkers;Amn,amnioticmembrane;Ov,ovary;Spl,spleen;Pl,placenta;neg,noRNAinRTreaction.ThethreereactionswereconductedwithRTreactionslackingRT,demonstratingthathighm.w.bandsarosefromnonspecicPCRand/orgenomicDNA.lowerpanel,RT-PCRofsamplescorrespondingto,ampliedforG3PDHtodemonstraterelativeRNAloading.TheJournalofImmunology Analysisofrhesustesteswith16G1revealedprominentimmu-nostainingintheseminiferoustubules,withonlyoccasionalpos-itivecellsintheinterstitium(Fig.5,,and).Stainingwasnotablyfoundinthemuralaspectofthetubularcompartment,throughtheentirecross-sectionoftheseminiferoustubules,sug-gestingstainingofSertolicells(e.g.,Fig.5;seealsoFig.6).IndividualSertolicellswereidentiedbytheirpositionpassingbetweengermcellsandtheirpointed,irregularnuclei(Figs.56),withcytoplasmicprocessesextendingtowardthelumenandsurroundingthenucleiofadvancedgermcells.Stainingwasnotnotedinprimaryspermatocytesinearlymetaphaseduringmeiosis,edbytheirdistinctspeckledchromatinstructure(S1,Fig.6).However,16G1immunostainingwasabundantinlatestageprimaryandsecondaryspermatocytesandspermatids,withdis-tinctlycondensednuclei(Sd,Fig.6).Variablestainingwasob-servedinspermatozoaandresidualbodies.Spermatozoaundergo-ingspermiogenesiswerepositivelystained,whilethosethatappearedtohavecompletedtheprocessandwerefreeinthelumenweregenerallynotstained(Fig.5F).Residualbodiesfreeinthelumenweresometimesstainedby16G1.Stainingwasnotob-servedintheepidydimiorinmaturesperm.Asummaryofim-munostainingresultsispresentedinTableII.Thediscrepancybetween25D3and16G1immunostaininginthetestiswasbothintriguingandpuzzling.Inparticular,itissur-prisingthat25D3didnotdetectMamu-AGwithintheseminifer-oustubules.Sincethe25D3Abonlyrecognizessurface-boundMamu-AGcomplexedwith-microglobulin,itseemedpossiblethat16G1immunostainingisdetectingsolubleMamu-AGHchainthatisnotcomplexedwith-microglobulin.Immunostainingrmedthishypothesis(Fig.5,),sincewhileabundant-microglobulinwas
6 identiedwithintheinterstitialtissue,thes
identiedwithintheinterstitialtissue,theseminiferoustubulesweredevoidofimmunostaining.Interest-ingly,SertolicellsdonotexpressWealsoevaluatedseveraltissuespreviouslyshowntoexpresslowlevelsofMamu-AGmRNA(25).Adrenal,spleen,ovary,kidney,heart,andpituitarysamplesdidnotrevealanyMamu-AGexpressionbyimmunostainingwith25D3or16G1(datanotshown).Mamu-AGISHImmunocytochemicalanalysesindicatedthatthescriptswedetectedbyRT-PCRwereexpressedintheseminiferoustubules.ToconrmthatthetubulescontainmRNA,weconductedISHwithaprobeforthe1domain,withwhichwehavepreviouslydenedconditionsforlocus-specicISH(23).Aswehavepreviouslyreported,thevilloussyncytiotrophoblastsandtrophoblasticshellconsistentlyexpressedmRNA(Fig.).Inaddition,villouscytotrophoblastsandoccasionalcellswithinthecoreofthevilliwerepositive(Fig.7).Maternalde-ciduaanduterinetissuewerealwaysnegative,andnohybridiza-tionwasobservedwiththesenseprobe(Fig.7ISHwithrhesustesteslocalizedmRNAmainlyintheseminiferoustubules(Fig.7).Mostcellsintheprocessofsper-matogenesishybridizedwiththeantisenseprobe.Cellsoccupyingthemuralcompartmentoftheseminiferoustubule,primarilysper-matogoniaandSertolicells,hadrelativelylowerhybridizationsig-nalcomparedwithspermatocytes(Fig.7).Highermagni(Fig.7)revealedthatspermatogoniacontainedlessmRNAthanthegermcellsclosertothelumen,i.e.,primarysper-matocytesandspermatids.SertolicellsweredifculttoidentifywithoutcounterstainingduringISH.Hybridizationwithspermato-zoawasuncommon,butwasoccasionallynoted.Ingeneral,alltheseminiferoustubulesinthetestissectionsdemonstratedsomede-greeofmRNAexpression.Afewofthesurroundinginterstitialcellsofthetestis,possiblymacrophagesorLeydigcells,werepositiveformRNA(Fig.7).However,theirstainingintensitywasgenerallyobservedtobelowerthanthatingermcells.Sectionsincubatedwiththesenseproberevealednostainingintestes(Fig.7Finally,weconductedRT-PCRformRNAexons6withsamplesofejaculatedrhesussperm.Cloningandse-quencingofampliedcDNAsrevealedthatbothwholesemenandfractionspermcontainedmRNA;howevernotranscriptscontainingintron4weredetected.WehavedemonstratedtheexpressionofamRNAencodingasol-ubleformofMamu-AG,anonclassicalMHCclassImoleculeexpressedathighlevelsintherhesusmonkeyplacenta.ThemRNAisexpressedinthetestesandseveralothernonplacentaltissuesatmoderatelevels.ImmunostainingwithanAbrecognizingthesolubleHLA-Gcarboxyl-terminalpeptideshowedthattheex-pressionofsolubleMamu-AGintheplacentaoverlappedbutwasnotidenticalwiththatofcellsurfaceMamu-AG.WhilewefoundonlyoccasionalcellsexpressingcellsurfaceMamu-AGinthetes-tes,wealsodemonstratedwidespread,althoughapparentlycell-selective,expressionofsolubleMamu-AGintheseminiferoustu-bulesofthetestes.OnlyafewothertissueshaddetectablesolublemRNA,andnoimmunostainingwasnotedoutsidetheplacentaandtestesinthetissuesevaluated. FIGURE3.WesternblotandimmunocytochemicalanalysisofMamu-AG5expressedinheterologouscells.,LysatesofMamu-AG5-trans-fected293cells(lane1)ornaivecells(lane2)analyzedbyWesternblotwithanAbagainsttheHAtag.,CelllysatesfromMamu-AG5-trans-fectedcells(lane3,50lane4,200g)ornaivecells(lane5)analyzedwithAb16G1againstsolubleHLA-G. FIGURE4.ImmunohistochemistryofMamu-AGintherhesusplacenta.,and,mAb25D3immunostainingformembrane-boundMamu-AG(brown),counterstainedwithhematoxylin(blue).Notethestrongsyncytialstainingandthelackoflocalizationinthecoreofvilli(,and,mAb16G1immunostainingforintron4peptide(brown)counterstainedwithhematoxylin(blue).Notethediffusestainingpatterninthecytotrophobcomparedwith25D3,includingoccasionalcellsinthecoreofthevilli()andavillousstromalvesselaswell(,16G1preincubatedwithitsimmunizingpeptide(4/1).Lackofstainingdemonstratesthespecicityof16G1againstitsimmunizingpeptide.,16G1incubatedwitha
7 nonspecipeptide,GnRH(4/1).Positiveredsta
nonspecipeptide,GnRH(4/1).Positiveredstainingonthissectionagainrevealsthat16G1issMamu-AGspecicandnotinhibitedbyanunrelatedpeptide.SectionincubatedwithnonspecicmouseIgG.Thelackofstainingiscomparabletothatin,Serialsectionscomparingthestainingpatternsof25D3and16G1.Notethat25D3clearlystainstheextravillouscytotrophoblasts(EVCT),whereas16G1doesnot.,Higherpowerviewofthespecicmembranestainingof25D3onEVCT.Scalebar:,60,and,100m;E,200,20678SOLUBLEMHCINRHESUSPLACENTAANDTESTIS TheJournalofImmunology SolubleMHCclassImoleculescanbegeneratedbyseveralmechanisms.Moleculescanbeshedfromthecellsurface,orsur-face-boundmoleculesmaybeliberatedbyextracellularproteases(29).Inaddition,alternativesplicingcangenerateMHCclassIisoformsthatarereleasedfromthecell.ClassicalMHCtranscriptslackingexon5,whichencodesthetransmembranedomainandthereforewillbereleasedfromexpressingcells,havebeeniden-edinthehumanandthemouse(3032).Althoughtherole(s)ofsolubleMHCisnotcompletelydened,itmaybeinvolvedinimmunoregulationandtoleranceinthesettingoforgantransplan-tation(33).AnovelmechanismforgeneratingasolubleMHCclassImol-eculewasrevealedinstudiesofHLA-G,anonclassicalmoleculeexpressedathighlevelsinthehumanplacenta.Exon5encodesthemembrane-spanningdomain,andexons68encodetheshortcy-toplasmicdomain,whichcontainsastopcodon18aaupstreamoftheconventionalMHCclassImoleculestopcodon.Antranscriptthatincludedthefourthintronwasrstidentiedinpla-centalRNA(12,13).Translationoftheintronresultsinanin-framestopcodonthatterminatestheproteinupstreamofthetrans-membranedomain.Theresultingproteinhasanovelcarboxylterminusandisabletocomplexwith-microglobulin,butwould FIGURE5.Immunocytochemicallocal-izationofMamu-AGintherhesustes-,mAb16G1.,16G1in-cubatedwithitsimmunizingpeptide,16G1incubatedwithGnRH,Doublelabelingof16G1(paleredtubulesinthissection,compare)and-microglobulin(blue).,mAbW6/32,stainingallclassIMHCmoleculesassociatedwithmicroglobulinisexclusivelylocalizedtotheinterstitialcells.,25D3immu-nostainingisrestrictedtoafewinter-stitialcellsofthetestis.,mAbagainstCD68localizesmacrophages,whicharerelativelyabundantinthein-terstitiuminthetestis.,MouseIgG,showingalackofnonspecicstaining.,mAbanti-vimentin,whichstainsbroblasts,endothelialcells,andothercellsofmesenchymalorigin,localizesinterstitialcellsandSertolicells.Serialsectionsshowingthesamere-gionincubatedwith16G1,25D3,CD68,andmouseIgG,respectively.Scalebar:,100,40,200,20680SOLUBLEMHCINRHESUSPLACENTAANDTESTIS bereleasedfromthecellduetothelackofatransmembranedo-main(12,13).Inadditiontotheplacenta(14,34),solubleHLA-GhasalsobeenreportedinCD4Tcells(20),activatedmonocytes(35),thymicepithelium(36),andlungtumorcells(37).Itisintriguingthatlike,thegenealsoisalterna-tivelysplicedtogiverisetoasolublemolecule.Theretentionofthisnovelsplicingpatternisanadditionalfacetofthemolecularbiologyofthelocusthatsuggestsaclosefunctionalhomologywith.Thesplicepatternsofarecom-plex,withvariouscombinationsofexonsidentiedinhumannor-malandtransformedcells(12,13,3840).However,themecha-nismsthatcontrolsplicingpatternsindifferentcelltypesarenotunderstood.Whetherthemechanismsthatdirecttheretentionofintron4inMamu-AGarerelatedtothosecontrollingHLA-Ggeneexpressionremainstobedetermined.AdifferencenotedbetweenthesolubleHLA-GandsolubleMamu-AGisoformsisthatwhereasthepredominantsolubleHLA-GisoformmaybeHLA-G6(18),wedidnotdetectmRNAintherhesusplacenta,althoughtherhesusclearlyexpressesmembrane-boundmRNA(22,25).Alternatively,perhapstheplacentaisnottheprimarysourceofcirculatingHLA-G.AnmAb(16G1)directedatthesolubleHLA-Gcarboxyl-ter-minustail(12)hasbeenusedtostudytheexpressionofsolubleHLA-Gisoformsinthehumanplacenta.UsingthisAb,solubleHLA-Ghasbeenlocalizedwithinthe
8 extravilloustrophoblastsandhasbeenreport
extravilloustrophoblastsandhasbeenreportedinassociationwithhumanvillouscytotropho-blastsaswellasinsomecaseswiththesyncytiotrophoblasts(5,14)andvillousendothelialcells(34).Finally,somemacrophageswithintheplacentalvilliwerealsoseentoexpresssolubleHLA-G(14).Itisintriguingthatourcurrentresultsgenerallyparalleltheseobservationsinthehumanplacenta.SolubleMamu-AGisalsonotednotonlyinsyncytiotrophoblasts,butwithincellsofthevil-lousstroma,apparentlyincludingHofbauercells,andvillouscy-totrophoblastsoftherhesusplacenta.Ourpreviousstudieshaveshownthat-microglobulinisreadilydetectedwithinrhesussyncytiotrophoblastsandinvillousstromalcells,butverylowstainingisfoundwithinvillouscytotrophoblasts(23).Theimmunostainingpatternobtainedwith16G1wouldsuggestthatvillouscytotropho-blasts,butnotsyncytiotrophoblastsorHofbauercells,arelikelytoproducefreeHchainMamu-AG.Recently,severalgroupshavesoughttoestablishwhethersolubleHLA-Gispresentintheserumofpregnantwomen(1518).ELISA-basedassaysusingvariousAbcaptureanddetectionapproacheshaveindicatedthepresenceofsolubleHLA-Gnotonlyintheserumofpregnantwomen,butalsoinnonpregnantwomenandinmenaswell.WhethersolubleMamu-AGisdetectableintheserumoftherhesusmonkeyre-mainstobeinvestigated.ThepatternofexpressionofsolubleMamu-AGexpressionintheplacentaoverlapsbutisnotidenticalwiththatofthemem-brane-boundmolecule.Thismayreecttheputativetargetsfor FIGURE6.Highmagnicationofaseminiferoustu-bulestainedwith16G1(brown)andcounterstainedwithhematoxylin(blue).AfewoftheSertolicells(St),sper-matogonia(Sg),primaryspermatocytes(S),spermatids(Sd),anddevelopingspermatozoa(Sz)aswellasthebasementmembrane(BM)areindicated.Scalebar,20TableII.SummaryofMamu-AGexpressioninrhesusplacentaandtestis Mamu-AGMamu-AG5TestisPlacentaTestisPlacentaIHCSertolicellsVillousCTBVillousCTBPrimaryspermatocytesExtravillousCTBPrimaryspermatocytesExtravillousCTBCTBcolumnsCTBcolumnsVillousstromaVillousstromaNDND,nodetectableexpression;,scarceexpression;,detectableexpression;,moderateexpression;,highexpression;STB,syncytiotrophoblasts;CTB,cytotrophoblast;ND,experimentnotdone;IHC,immunohistochemistry;ISH,insituhybridization.RT-PCRresultsforMamu-AGarefromRef.25.TheJournalofImmunology thesetwoisoforms.SolubleMamu-AGimmunoactivitywasrela-tivelylowinrhesusextravillouscytotrophoblasts,whereasmem-brane-boundMamu-AGwasreadilydetected.Onemightspeculatethatmembrane-boundHLA-GandMamu-AGonextravilloustro-phoblastswillligatereceptorsonadjacentdecidualleukocytestoregulatetheiractivity,whereasasolublecirculatingmoleculemaymodulatematernalimmunecellactivitybothlocally,withinthedecidua,aswellassystemically.Inthissituationthesyncytiotro-phoblastsareamoreeffectivewaytoreleaseasolublemoleculedirectlyintothematernalcirculation.TheexpressionofsolubleHLA-Gbythesyncytiotrophoblastsremainssomewhatcontrover-sial(5,14,41),anddetectionmaybedependentontheconditionsusedforxation,embeddingandprocessingaswellastheAbsused.Indeed,conictingresultsonplacentalandextraplacentallocalizationofHLA-Gsupportbothfurtherstudyaswellasthedevelopmentofappropriateanimalmodels.ThepatternofsolubleMHCclassIexpressionintherhesustestiswasunexpected.HLA-Gproteinwasnotdetectedinhumantestesinpreviousstudies(5,41),althoughmRNAexpressionhasbeenreportedinsome,butnotall,studies(42,43).Inourstudieswiththerhesus,whereastherewasonlyveryrarelydetectablemembrane-boundMamu-AGwithintheseminiferoustubules(asdeterminedby25D3staining),itwasclearthatabundantsolubleproteinwaspresentinselectedgermcellpopulations,colocalizedwithMamu-AGmRNA.Thelackof-microglobulinwithintherhesusseminiferoustubulessufcientlyexplainsthelackofcellsurfaceprotein,anobservationmadepreviouslywiththehumantestisandthepan-MHCclas
9 sIAbW6/32(43).FreeclassIHchainwasdetecte
sIAbW6/32(43).FreeclassIHchainwasdetectedinsomespermatocytepopulationsinhumans(43).However,itseemsunlikelythattesticularsolubleHLA-GmaybethesourceofcirculatingserumHLA-Ginmen,sincethepatencyoftheblood-testisbarrierwouldprecludethisrouteofckingofthemolecule.Inaddition,theassaysusedtodetectsolubleHLA-GtypicallydetecttheclassI--microglobulincomplex(1518),andimmunostainingofhumantesteswith16G1didnotdetectsolubleHLA-Gprotein(41).Thus,anothersiteofex-pressionofhumansolubleHLA-Gseemslikely.ItisintriguingtospeculateonafunctionforseminalsolubleMamu-AGinthefemalereproductivetract,perhapssuppressinganinnateimmuneresponsewithinthevaginaortheuterustomaleleukocyteswithintheejaculate.AroleincontrollingTcellex-pansion,blockingCTLactivity,andinducingapoptosisinallo-reactiveTcellshasbeensuggestedforsolubleHLA-G(19,20,44).Alternatively,solubleMamu-AGmightalsohelppreventanti-spermimmuneresponsesfollowingminorbreaksintheblood-testisbarrieroftheseminiferousbasementmembrane.Inonetu-bulewenotedhighexpressionofmembrane-boundMamu-AGonacellularaggregate(notshown)thatalsostainedpositivelyforthemacrophagemarkerCD68.TheexpressionofHLA-Ghasbeennotedinmacrophagesinvadingpsoriaticskinlesions(45)andinactivatedmacrophagesanddendriticcellsinlungtumors(34,46).Althoughtheexpressionofsolubleormembrane-boundHLA-Ginplacentalmacrophagesisnotfullyresolved(5,14,41),therhesusmonkeymaybeanexperimentalmodelinwhichtoexplorethefunctionalsignicanceofsolublenonclassicalMHCclassImol-eculesinvivo.WethankStephenG.EiseleandtheReproductiveServicesUnitfortimedmatingsandrhesussemensamples,theveterinarystaffoftheWisconsin FIGURE7.ISHfor,and,Placenta()andtestis()sectionshybridizedwiththeantisenseprobethatdetects1domainofthe,Expressionisnotedinthecytotrophoblasticshell(cs)andvillous,HighermagnitionofplacentalISH.Thearrowheadpointstoapositivemesenchymalcellofthevillousstroma.mRNAispresentinmanycellsoftheseminiferoustubule.,Highermagni-cationoftestisISH.Spermatogonia(Sg)arelesspositivelystainedthanthegermcellsclosertothelumen,e.g.,pri-maryspermatocytes(S)andspermatids(Sd).,Correspondingsec-tionshybridizedwiththesenseproberevealingalackofnonspecichybrid-ization.Scalebar:,100,20,40,600682SOLUBLEMHCINRHESUSPLACENTAANDTESTIS RegionalPrimateResearchCenterforsurgicalassistance,membersoftheGoloslaboratoryforcriticalreadingofthemanuscript,CarlosQuian-Su-arez(GeorgetownUniversity,Washington,D.C.)foradviceonhistologicalinterpretation,andStevenBuschforeditorialassistance.WeacknowledgetheassistanceofGeorgeFlentkeandtheMicroanatomyCoreoftheUni-versityofWisconsinDevelopmentalToxicologyCenterforhelpfultrain-ingandadviceforriboprobepreparation.1.Kovats,S.,E.K.Main,C.Librach,M.Stubblebine,S.J.Fisher,andR.DeMars.1990.AclassIantigen,HLA-G,expressedinhumantrophoblasts.Science248:2.Loke,Y.W.,andA.King.2000.Decidualnatural-killer-cellinteractionwithtrophoblast:cytolysisorcytokineproduction?Biochem.Soc.28:196.3.Hunt,J.S.,M.G.Petroff,P.Morales,P.Sedlmayr,D.E.Geraghty,andC.Ober.2000.HLA-Ginreproduction:studiesonthematernal-fetalinterface.Hum.Im-munol.61:1113.4.LeBouteiller,P.2000.HLA-Ginthehumanplacenta:expressionandpotentialBiochem.Soc.Trans.28:208.5.Paul,P.,N.Rouas-Freiss,P.Moreau,F.A.Cabestre,C.Menier,I.Khalil-Daher,C.Pangault,M.Onno,R.Fauchet,J.Martinez-Laso,etal.2000.HLA-G,-E,-Fpreworkshop:toolsandprotocolsforanalysisofnon-classicalclassIgenestran-scriptionandproteinexpression.Hum.Immunol.61:1177.6.Carosella,E.D.,P.Moreau,S.Aractingi,andN.Rouas-Freiss.2001.HLA-G:ashieldagainstinammatoryaggression.TrendsImmunol.22:553.7.Ponte,M.,C.Cantoni,R.Biassoni,A.Tradori-Cappai,G.Bentivoglio,C.Vitale,S.Bertone,A.Moretta,andM.C.Mingari.1999.InhibitoryreceptorssensingHLA-G1molecules
10 inpregnancy:decidua-associatednaturalkil
inpregnancy:decidua-associatednaturalkillercellsexpressLIR-1andCD94/NKG2Aandacquirep49,anHLA-G1-specicreceptor.Natl.Acad.Sci.USA96:5343.8.Cantoni,C.,S.Verdiani,M.Falco,A.Pessino,R.Cilli,R.Conte,D.Pende,M.Ponte,M.S.Mikaelsson,L.Moretta,etal.1998.p49,aputativeHLAclasscinhibitoryreceptorbelongingtotheimmunoglobulinsuperfamily.J.Immunol.28:1980.9.Colonna,M.,J.Samaridis,M.Cella,L.Angman,R.L.Allen,C.A.OR.Dunbar,G.S.Ogg,V.Cerundolo,andA.Relink.1998.Humanmyelomono-cyticcellsexpressaninhibitoryreceptorforclassicalandnonclassicalMHCclassImolecules.J.Immunol.160:3096.10.Navarro,F.,M.Llano,T.Bellon,M.Colonna,D.E.Geraghty,andM.Lopez-Botet.1999.TheILT2(LIR1)andCD94/NKG2ANKcellreceptorsrespectivelyrecognizeHLA-G1andHLA-Emoleculesco-expressedontargetEur.J.Immunol.29:277.11.Rajagopalan,S.,andE.O.Long.1999.Ahumanhistocompatibilityleukocyteantigen(HLA)-G-specicreceptorexpressedonallnaturalkillercells.J.Exp.Med.189:1093.12.Fujii,T.,A.Ishitani,andD.E.Geraghty.1994.AsolubleformoftheHLA-Gantigenisencodedbyamessengerribonucleicacidcontainingintron4.J.Im-munol.153:5516.13.Moreau,P.,E.Carosella,M.Teyssier,S.Prost,E.Gluckman,J.Dausset,andM.Kirszenbaum.1995.SolubleHLA-Gmolecule:analternativelysplicedHLA-GmRNAformcandidatetoencodeitinperipheralbloodmononuclearcellsandhumantrophoblasts.Hum.Immunol.43:231.14.Chu,W.,M.E.Fant,D.E.Geraghty,andJ.S.Hunt.1998.SolubleHLA-Ginhumanplacentas:synthesisintrophoblastsandinterferon--activatedmacro-phagesbutnotplacentalHum.Immunol.59:435.15.Hamai,Y.,T.Fujii,A.Miki,D.E.Geraghty,I.Harada,Y.Takai,S.Kozuma,O.Tsutsumi,andY.Taketani.1999.Quantitativeassessmentofhumanleukocyteantigen-Gproteininamnioticuidbyadouble-determinantenzyme-linkedim-munosorbentassayusinganti-humanleukocyteantigen-G-specicantibodyAm.J.Reprod.Immunol.41:293.16.Fournel,S.,M.Aguerre-Girr,A.Campan,L.Salauze,A.Berrebi,Y.-C.Lone,F.Lenfant,andP.LeBouteiller.1999.SolubleHLA-G:puricationfromeu-karyotictransfectedcellsanddetectionbyaspecicELISA.Am.J.Reprod.Immunol.42:22.17.Rebmann,V.,K.Pfeiffer,M.Passler,S.Ferrone,S.Maier,E.Weiss,andH.Grosse-Wilde.1999.DetectionofsolubleHLA-GmoleculesinplasmaandTissueAntigens53:14.18.Hunt,J.S.,L.Jadhav,W.Chu,D.E.Geraghty,andC.Ober.2000.SolubleHLA-Gcirculatesinmaternalbloodduringpregnancy.Am.J.Obstet.Gynecol.19.Fournel,S.,M.Aguerre-Girre,X.Huc,F.Lenfant,A.Alam,A.Toubert,B.A.,andP.LeBouteiller.2000.Cuttingedge:solubleHLA-G1triggersCD95/CD95ligand-mediatedapoptosisinactivatedCD8cellsbyinteractingwithCD8.J.Immunol.164:6100.20.Lila,N.,N.Rouas-Freiss,J.Dausset,A.Carpentier,andE.D.Carosella.2001.SolubleHLA-Gproteinsecretedbyallo-specicCD4Tcellssuppressestheallo-proliferativeresponse:aCD4Tcellregulatorymechanism.Proc.Natl.Acad.Sci.USA98:12150.21.Marchal-Bras-Goncalves,R.,N.Rouas-Freiss,F.Connan,J.Choppin,J.Dausset,E.D.Carosella,M.Kirszenbaum,andJ.Guillet.2001.AsolubleHLA-Gproteinthatinhibitsnaturalkillercell-mediatedcytotoxicity.Transplant.Proc.33:2355.22.Boyson,J.E.,K.K.Iwanaga,T.G.Golos,andD.I.Watkins.1997.IdentiofanovelMHCclassIgene,intheplacentaofprimatewithanJ.Immunol.159:3311.23.Slukvin,I.I.,J.E.Boyson,D.I.Watkins,andT.G.Golos.1998.Therhesusmonkeyanalogueofhumanlymphocyteantigen-Gisexpressedprimarilyinthevilloussyncytiotrophoblast.Biol.Reprod.58:728.24.Slukvin,I.I.,D.P.Lunn,D.I.Watkins,andT.G.Golos.2000.Placentalex-pressionofthenonclassicalMHCclassImoleculeMamu-AGatimplantationintherhesusmonkey.Proc.Natl.Acad.Sci.USA97:9104.25.Slukvin,I.I.,D.I.Watkins,andT.G.Golos.1999.TissuedistributionofthemRNAforarhesusmonkeyclassIbmoleculeMamu-AG.TissueAntigens53:26.Golos,T.G.,R.R.Handrow,M.Durning,J.M.Fisher,andJ.K.Rilling.1992.Regulationofchorionicgonadotropin-andchorionicsomatomammotropinmes-sengerribon
11 ucleicacidexpressionby8-bromo-adenosine3
ucleicacidexpressionby8-bromo-adenosine3anddexamethasoneinculturedrhesusmonkeysyncytiotrophoblasts.ogy131:89.27.Boyson,J.E.,K.K.Iwanaga,J.A.Urvater,A.L.Hughes,T.G.Golos,andD.I.Watkins.1999.EvolutionofanewnonclassicalMHCclassIlocusintwoOldWorldprimatespecies.Immunogenetics49:86.28.Urvater,J.A.,N.Otting,J.H.Loehrke,R.Rudersdorf,I.I.Slukvin,M.S.Piekarczyk,T.G.Golos,A.L.Hughes,R.E.Bontrop,andD.I.Watkins.2000.Mamu-I:anovelprimateMHCclassIB-relatedlocuswithunusuallylowJ.Immunol.164:1386.29.Demaria,S.,L.D.DeVito-Haynes,R.D.Salter,W.J.Burlingham,andY.Bushkin.1999.Peptide-conformedm-freeclassIheavychainsareintermediatesingenerationofsolubleHLAbythemembrane-boundmetalloprotein-Hum.Immunol.60:1216.30.Krangel,M.S.1985.UnusualRNAsplicinggeneratesasecretedformofHLA-A2inamutagenizedBlymphoblastoidcellline.EMBOJ.4:1205.31.Krangel,M.S.1986.SecretionofHLA-Aand-BantigensviaanalternativeRNAsplicingpathway.J.Exp.Med.163:1173.32.Ulker,N.,K.D.Lewis,L.E.Hood,andI.Stroynowski.1990.ActivatedTcellstranscribeanalternativelysplicedmRNAencodingasolubleformofQa-2an-EMBOJ.9:3839.33.Burlingham,W.J.2000.SolubleMHC,immunoregulation,andtolerance:aprogressreport.Hum.Immunol.61:1316.34.Blaschitz,A.,F.Lenfant,V.Mallet,M.Hartmann,A.Bensussan,D.E.Geraghty,P.LeBouteiller,andG.Dohr.1997.EndothelialcellsinchorionicfetalvesselsrsttrimesterplacentaexpressHLA-G.Eur.J.Immunol.27:3380.35.Onno,M.,G.LeFriec,C.Pangault,L.Amiot,V.Guilloux,B.Drenou,S.Caulet-Maugendre,P.Andre,andR.Fauchet.2000.ModulationofHLA-Gantigensexpressioninmyelomonocyticcells.Hum.Immunol.61:1086.36.Mallet,V.,A.Blaschitz,L.Crisa,C.Schmitt,S.Fournel,A.King,Y.W.Loke,G.Dohr,andP.LeBouteiller.1999.HLA-Ginthehumanthymus:asubpopu-lationofmedullaryepithelialbutnotCD83dendriticcellsexpressesHLA-Gasamembrane-boundandsolubleprotein.Intl.Immunol.11:889.37.Urosevic,M.,M.O.Kurrer,J.Kamarashev,B.Mueller,W.Weder,G.Burg,R.A.Stahel,R.Dummer,andA.Trojan.2001.HumanleukocyteantigenGup-regulationinlungcancerassociateswithhigh-gradehistology,humanleuko-cyteantigenclassIlossandinterleukin-10production.Am.J.Pathol.159:817.38.Ishitani,A.,andD.E.Geraghty.1992.AlternativesplicingofHLA-GtranscriptsyieldsproteinswithprimarystructuresresemblingbothclassIandclassIIan-Proc.Natl.Acad.Sci.USA89:2669.39.Riteau,B.,N.Rouas-Freiss,C.Menier,P.Paul,J.Dausset,andE.D.Carosella.2001.HLA-G2,-G3,and-G4isoformsexpressedasnonmaturecellsurfacegly-coproteinsinhibitNKandantigen-specicCTLcytolysis.J.Immunol.166:5018.40.Kirszenbaum,M.,P.Moreau,E.Gluckman,J.Dausset,andE.Carosella.1994.AnalternativelysplicedformofHLA-GmRNAinhumantrophoblastsandev-idenceforthepresenceofHLA-Gtranscriptinadultlymphocytes.Proc.Natl.Acad.Sci.USA91:4209.41.Blaschitz,A.,H.Hutter,V.Leitner,S.Pilz,R.Wintersteiger,G.Dohr,andP.Sedlmayr.2000.ReactionpatternsofmonoclonalantibodiestoHLA-Ginhumantissuesandoncelllines:acomparativestudy.Hum.Immunol.61:1074.42.Fiszer,D.,M.Ulbrecht,N.Fernandez,J.P.Johnson,E.H.Weiss,andM.Kurpisz.1997.AnalysisofHLAclassIbgeneexpressioninmalegameto-geniccells.Eur.J.Immunol.27:1691.43.Hutter,H.,andG.Dohr.1998.HLAexpressiononimmatureandmaturehumangermcells.J.Reprod.Immunol38:101.44.Kanai,T.,T.Fujii,S.Kozuma,T.Yamashita,A.Miki,A.Kikuchi,andY.Taketani.2001.SolubleHLA-Ginuencesthereleaseofcytokinesfromal-logeneicperipheralbloodmononuclearcellsinculture.Mol.Hum.Reprod.45.Aractingi,S.,N.Briand,C.LeDanff,M.Viguier,H.Bachelez,L.Michel,L.Dubertret,andE.D.Carosella.2001.HLA-GandNKreceptorareexpressedinpsoriaticskin.Am.J.Pathol.159:71.46.Pangault,C.,G.LeFriec,S.Caulet-Maugendre,H.Lena,L.Amiot,V.Guilloux,M.Onno,andR.Fauchet.2002.LungmacrophagesanddendriticcellsexpressHLA-Gmoleculesinpulmonarydiseases.Hum.Immunol.63:83TheJournalofImmunolog