The Journal of Experimental Medicine

morphic, ligands forinhibitoryKIRmolecules,areextremelypoly- attack onn target cell Building, Room259,Durham,NC 27710. Road, StevenageSG12NY,United Kingdom. 319-335-8916; E-mail:[email protected] University ofIowa,IowaCity,IA52242.Phone:319-335-8151;Fax: Address correspondencetoCharlesT.Lutz,DepartmentofPathology, Ig-like receptors(KIRs). of stimulatoryandinhibitoryreceptors,includingthekiller distinguish adaptive TandBlymphocyteimmuneresponses.To cytokines. NK NK cells killtheaberrantcellsandreleaseavarietyof rapidly totumorcells,viruses,parasites,andcertainbacteria, partoftheinnateimmune system(1).Responding important NK cellsareantigen-nonspecificlymphocytesthatan killer Ig-likereceptor. Introduction

* B.E. Mace’spresentaddressisDuke University,BryanResearch M.J. Wilson’spresentaddressisGlaxoSmithKline, GunnelsWood

Abbreviations usedinthispaper:

determining thevarietyofpeptideantigens MHC classImoleculesandpreventNKcell

aberrant fromnormalcells,NKcellsuseavariety

ormal cells(2).MHCclassImolecules,the

cytokines helporchestratesubsequent

245

InhibitoryKIRmoleculesbind

only oneortwonucleotides.Allele-specific neous expressionofmultiple Michael J. Wilson, tain clonallyrestrictedexpressionofhighlyhomologous gene expression regulation killercells, receptor natural•killerinhibitory •allelesDNAmethylation • Key words: Abstract

genes. Despiteallele-independentexpression, 2DL4 restricted NK class I. molecules, allowingnaturalkiller(NK)cellstorecognizeaberrantthathavedown-regulated Killer immunoglobulin-likereceptors(KIR)bindself–majorhistocompatibilitycomplexclassI 5 4 1 Huei-Wei Chan, and 5 Mary Carrington, Mary inhibitor, 5-aza-2 DNA Methylation Maintains Allele-speciﬁc Expression inHumanNaturalKillerCells Aza,5-aza-2 Immunology Division, ofPathology, Department University ofCambridge, CB21QP, Cambridge UnitedKingdom Basic Research Program,Basic Research SAICFrederick, NationalCancerInstitute, Frederick, MD21702 ofPathology,Department

and

Graduate Programs inImmunology andMolecularBiology, University ofIowa, Iowa City, IA52242 http://www.jem.org/cgi/doi/10.1084/jem.20021127 The JournalofExperimental Medicine

allelesandeitheroneorbothoftheclonallyrestricted

gene DNAhypomethylationinNKcellsvitroandvivo.Themethylase

KIR

cells expressvariablenumbersandcombinationsofhighlyhomologousclonally

-deoxycytidine; KIR, genes,butuniformlyexpress -deoxycytidine, induced 1 5 4 Zoya B. Kurago, John Trowsdale, 2 Maureen P. Martin, Department ofOral Pathology,Department Oral Radiology, andOral Medicine, KIR presented toCD8 presented Clonally restricted 2DL2/2DL3 bindcomplementarysetsofHLA-Callotypes. the Bw4subsetofHLA-Ballotypes.KIR2DL1and MHC classImolecules(2).Forexample,KIR3DL1binds clonally restrictedKIRmoleculesbinddistinctsubsetsof also mayenhanceNKcellsurveillancebecauseinhibitory panzees andhumans(8).The tightlypacked rodents andshowingstriking differencesbetweenchim- NK cellscontrol therapeutic purposes,itisnecessarytodeterminehow understand NKcellfunctionandregulateactivityfor specific MHCclassImolecules,oftenHLA-B(6,7).To (5). Inaddition,tumors mayselectivelydown-regulate roleforHLA-B–specificKIR3DL1/3DS1receptors potential maintaining HLA-C expression (4),consistentwitha down-regulate HLA-AandHLA-Bmoleculeswhile class Ilocioralleles.HIV-1–infectedcellsselectively cells thathaveselectivelydown-regulatedspecificMHC NK cellstobeactivatedbyvirus-infectedandtumor highly iterativewithextremely highsequencesimilarity genes.Thus,NKcellsuseDNAmethylationtomain- The •Vlm 9,Nme ,Jnay2,20 245–255 January20,2003 •Volume197,Number 2, 1, 2 4 and Charles T. Lutz KIR C.Stewart, Andrew KIR2DL4 3DL1 5 3DL1 KIR Brian E.Brian Mace, locusisrapidlyevolving,being absentfrom geneexpressioncorrelatedwithpromoter DNAhypomethylationandheteroge- allelesdifferedinthecorepromoterby KIR . WeshowthatNKclonesexpressboth KIR T cells(3).MHCclassIpolymorphism geneexpression. KIR expressionmayallowasubsetof genesandalleles. 1, 3 1 4 KIR KIR Gene

3DL1 KIR and locusis 3DL2 The Journal of Experimental Medicine kb region,the150-kb (9, 10).Indeed,exceptforthebordersandoneinternal14- clonally restricted the hypothesisthat expression inNKcellsvitroandvivo.Finally,wetest in matureNKcellsgrownvitro. Materials andMethods predicted thatclonallyrestricted tion bybothrestingandactivatedNKcells(13).Thus,itis pattern is (clones K1–K8)and3DL1 KIR 2DL4 receptorcross-linkinguniquelyactivatesIFN- phoblast cells(12).IncontrasttoinhibitoryKIRmolecules, cules, whosephysiologicalexpressionislimitedtofetaltro- 2DL4 receptorbindsHLA-GnonclassicalMHCmole- genes withadirectorthologueinchimpanzees(8).The KIR between previously described(20)andKIR 3DL1 NK-enriched donorLNKcellswere clonedbylimitingdilutionas removed byFicolldensitygradient centrifugation(Sigma-Aldrich). StemCell TechnologiesInc.).Erythrocyte-leukocyte rosetteswere leukocytes CD3,CD4,CD19,CD36,andCD66b(RosetteSep™; antibody complexesbispecificforerythrocyteglycophorinAand board. ToenrichNKcells,peripheralbloodwasincubatedwith approved bytheUniversityofIowaHumanSubjectsreview clones studiedthusfarandisoneofthefewhuman press oneorbothallelesof remains unproven. methylation Therefore, thelong-standinghypothesisthatDNA their naturallocationsinnormaldevelopmentvivo. methylation islinkedtotranscriptionalrepressionofgenesin sion (17–19).Fewdetailedstudiesshowthatpromoter a possiblecorrelationbetween additional analysis.DonorKNK cellsweresortedfor3DL1 pomethylated promotersand5 has beenquestionedandmanytissue-specificgeneshavehy- of methylationincontrollingtissue-specificgeneexpression transfected genes,andtransgenes(16–19).However,therole inactive Xchromosomeoffemalecells,imprintedgenes, methylation correlateswithpoorgenetranscriptionofthe cytosines thatarepartofthemini-palindrome,CpG.DNA Consistent withthishypothesis,mammalsmethylatemost methylation controlstissue-specificgeneexpression(14,15). regulated bydistinctmechanisms. anywhere fromonetoeight (11). NKcellclonesfromthesameindividualmayexpress both numberandidentityinaseeminglyrandompattern quence similarity, tinctive stretchofmorethan100bp.Despitehighse- An exceptiontotheseeminglyrandom Cells. In thisreport,weinvestigatewhetherNKcellclonesex- More than25yrago,twogroupsproposedthatDNA geneexpression. geneexpressiondiffersbetweenNKcellclonesin KIR Informed consentwasobtainedandhumanstudieswere KIR2DL4 controls tissue-specificgeneexpressioninvivo genesinbothcodingandnoncodingregions KIR KIR . ThisgeneisexpressedbyallNKcell KIR KIR expressionpatternsarequitestable genesareregulatedindependently. (clone K9)expressionusingmAb DX9 promotermethylationcontrols locusdoesnothaveasingledis- KIR 246 KIR regions regardlessofexpres- KIR genes.Wealsoinvestigate KIR genes.Onceestablished, Allele-specific Regulation ofNaturalKillerCellReceptor Expression methylationandgene and clones wereselectedfor

2DL4

KIR

expression

genesare

secre-

KIR

control trol fortransfectionefficiency,testplasmidwascotransfectedwith firefly luciferasereportergeneinthepGL3plasmid(22).Tocon- typed byBsaAIandSspIdigestion. and ctgtaggtccctgcaagggaaa,clonedintoplasmid.Alleleswere PCR amplifiedusinglocus-specificprimers,ttcttggtccagagggccgtt synthesized usingrandomhexamerprimers.3DL1cDNAwas treated with5-aza-2 ter, Indianapolis,IN.NK-92subclonesandYT-Indycellswere were providedbyZ.Brahmi,UniversityofIndianaMedicalCen- Inc. andsubclonedaspreviouslydescribed(21).YT-Indycells The NK-92celllinewasobtainedfromStemCellTechnologies or Z27.3.7andanalyzedinbulkclonedbylimitingdilution. cold dTTPorwith ers. Taqpolymerasewasaddedwith either amplicons werehybridizedwith selectedoligonucleotideprim- (24). Inbrief,afterbisulfitetreatment andallele-specificPCR, single nucleotideprimerextensionassayaspreviouslydescribed gtttatta. Productswereanalyzedusingthemethylation-sensitive lele-specific primers,tatgaaaaattttaatggtttattgorttatgaaaaattttaatg- taatattagaaatattat, followedbysemi-nestedamplificationwithal- locus-specific primers,cctctaaacccatatctttacctccaandggttttgttg- Alternatively, bisulfite-treateddonorKDNAwasamplifiedusing gttagtatagattttagg andcctctaaacccataactccawereusedfordonor L. ccatatctttacctcca wereusedfordonorKandNK-92.Primerstat- treatment. Primersggttttgttgtaatattagaaatattatandcctctaaac- were convertedtothymidines,consistentwithadequatebisulfite lyzed, results agree with those presented in Fig. 1. lyzed, resultsagreewiththosepresentedin aaccaagagcctgcgggac andggaagagtgatgctctaagatgg.Inallcasesana- was ggaagagtgatgctctaagatggandtheexon8–9PCRprimerswere cagctgctggttcattggat. For2DL4,thereversetranscriptionprimer and theexon7–9PCRprimersweregtatctgcagacacctgcat PCR primerswereacaaacccttcctgtctgcccandctgtgatcacgatgtccagg, verse transcriptionprimerwascagctgctggttcattggat,theexon3–4 agtggtcatcatcctcttcatc andgtgtacaagatggtatctgta.For3DL2,there- and ctgtgaccatgatcaccac,theexon7–9PCRprimerswere gatggtatctgta, theexon3PCRprimerswerettcttggtccagagggccggt above. For3DL1,thereversetranscriptionprimerwasgtgtacaa- separate polymorphicsites,usingprimersdistinctfromthoseused primers, followedbyamplificationandsequencingofoneortwo cell cloneswereretestedusinglocus-specificreversetranscription gagtgagg, respectively.WhensufficientRNAwasavailable,NK ctcga, ggtgtgaaccccgacatg,gcccctgctgaaatcagg,andacaactgatagggg- primers accttcgcttacagcccg,gggtttcctgtgacagaaacag,cgctgtggtgc- PCR amplificationofcDNAandDNAsequenceanalysis,using allele expressionwasassessedindependentlyindonorKcellsby 3DL1 and wasclonedsequenced.InallPCRclonessequenced, was normalizedtocontrolactivityaspreviouslydescribed(22). treated DNAwasPCRamplifiedusing with sodiumbisulfiteaspreviouslydescribed(23).Bisulfite- Gene segmentsendingupstreamofthetranslationstartsite using akitasdirectedbythemanufacturer(Bio-RadLaboratories). tended by0or1nucleotide.Oligonucleotides wereseparatedby sence ofotherdNTP.Underthese conditions,primerswereex- sciences) orhavebeendescribed(20). 21). mAbswereobtainedcommercially(ImmunotechorBDBio- alyzed byflowcytometryorclonedaspreviouslydescribed(20,

98.8% ofrandomcytosines(notpartCpGdinucleotides) galactosidase activitywasmeasured26–42hlaterandtest

Allele-specific Expression. Methylation Analysis. Promoter Analysis.

3DL2

galactosidase or

, and

2DL4

-deoxycytidine (Aza;Sigma-Aldrich)andan-

P-labeled dTTPandcolddCTP intheab- The 5

genes(9)wereclonedupstreamofthe

Genomic DNAwastreatedfor6–19h Renilla

RNA wasisolatedandcDNA

RACE techniquewasperformed luciferaseplasmid.Luciferaseand KIR

2DL4 3DL1 32 P-labeled dCTPand , 3DL1 -specific primers , and 3DL2 The Journal of Experimental Medicine cells. The fore, fore, mostNKcellclonesexpressedonlyoneallele.There- to monoallelic and paternalallelesareexpressed. ClonedNKcellsexhibited 3DL1 * periment (K10–K15).OneNKclone(K10)expressedthe we testedanadditionalsixclonesfromaseparatecloningex- cDNA (27 andunpublisheddata). coding sequenceisidenticaltotheprototypic3DL1 antibody bindingisunlikelybecausethedonorKcDNA tive indonorKcells.Defectivecellsurfaceexpressionor 3DL1 of the products (seebelow).Inthreebiallelicclones,expression data) andbyDNAsequenceanalysisofbulkRT-PCR nucleotide primerextensionassay(26 andunpublished allele alone.Identicalresultswereobtainedbyasingle ment (K1–K9)expressedboth Nine donorKNKcellclonesfromonecloningexperi- the two 3DL1 were 3DL1 (Fig. 1A).Incontrast,all15cloneL3cDNAplasmids and 14were3DL1 NK cloneL1cDNAplasmidsanalyzedwere3DL1 stable invitro. both from donorLshowedthreepatternsofalleleexpression: one allele,y leles, and0.0256(x designated pomethylation. Ifx x 3DL1 3DL1 cells thathypomethylateneitherallele,then0.536 3DL1 stant Imager(PackardInstrumentCo.). PAGE andincorporatedradioactivitywasquantifiedusinganIn- Fashion. 2[x 9.9% werebiallelicfor 3DL1 to estimatethepercentageofcellswith and BsaAI,whichselectivelydigest3DL1 erozygous for lated NKcellclonesfromtwoindividualswhoarehet- cloned intoplasmids.Individualplasmidsweretypedfor 3DL1 cDNAfragmentswereamplifiedbyPCRand Results 002 Because oftheskewed KIR GenesMayBeExpressedinaMonoallelicorBiallelic Calculations. alleleonly,showingthatdonorKcellsmayexpressthis 9.05y orthat90.1%of3DL1 10 sequencesfromdonorK–sorted3DL1 3DL1 alleleintheabsenceof * * 3DL1 * * alleleusagewithrestrictionendonucleases,SspI y 002, indicatingmonoallelicexpression.IndonorL 002 cDNA,respectively.3DL1 002 002 3DL1 6 cells, indicatingthatmonoallelic 3DL1 3DL1 To investigate 0.0256(x transcriptionandRNAstabilityarenotdefec- alleleonly(Fig.1A).Forexample,16ofthe allelescanbeexpressedmonoallelically inNK * 3DL1 NKAT3 alleles,the 001 andall27cloneL7cDNAplasmidswere alleleswasequal(Fig.1A),indicatingthat 3DL1 the numberofcellsthathypomethylatetwoal- The observedfractionof0.536hypomethylated geneisnotimprintedbecause bothmaternal allelesareexpressedaloneortogether. expressionafter3–5wkofgrowth invitro and y) 3DL1 * * the numberofcellsthathypomethylate 001 001, indicatingbiallelicexpression y)] Solvingthisequationindicatesthat 3DL1 the numberofcontaminating3DL1 3DL1 and hypomethylation. KIR NKB1 247 alleledistributionindonorK, alleleexpression,weiso- 3DL1 * * 3DL1 001 001 , respectively(25).KIR cells weremonoallelicand Chan etal. allele(Fig.1A).Asbe- alleleonly,andthe * 002 3DL1 allelesorthe KIR alleles,formerly NK cellswasused NK cellclones monoallelichy- expressionis * (x 001 and * * * 2y) / 001 002 002 specific geneexpressionfor typed byrestrictionfragmentlengthpolymorphism.(B)DonorKallele- are thenumberofcDNAcloneseach Shown forNKclonesfromdonorL(L1–L7)andK(K1–K15) Figure 1. 103LP ( and DNAsequencing. (K1–K9). Alleleexpressionwas determined bylocus-specificPCR AMC5 ). Eachlineshows [ ] and KIR geneexpressionismonoallelicorbiallelic.(A) NKAT4 KIR ), and alleleexpressionbyanNKcell clone 3DL1 2DL4

(

( 002

KIR-103AS

3DL1

[

allele.Plasmidswere ] and

[

001

] and

3DL2

KIR-

The Journal of Experimental Medicine count forallele-specific geneexpression,we sequenced429 To investigatewhetherpromoter polymorphismscouldac- the translationstartsites,respectively (unpublisheddata). core promoterswerewithin 262and271bpupstreamof cific. Asimilaranalysisshowed thatthe data). Thisshowsthat was notdetectableinHeLaepithelialcells(unpublished promoter/enhancer inYT-IndyNKcells(Fig.2A),but was equivalenttothatofthepositivecontrolSV-40virus translation startsite(Fig.2A). transcription startsite,starting254bpupstreamofthe the Indy NKtumorcells(Fig.2A).Theresultsindicatethat activity wasmeasured24–48haftertransfectionintoYT- inserted nexttotheluciferasereportergeneand and extendedintotheneighboring that includedtheentireregionupstreamof core promoter,wemade5 translation startsite(unpublisheddata).Tolocatethe the transcriptionstartsiteislocated37bpupstreamof nique producedastaggeredseriesofclones,suggestingthat sion withinasinglegeneticcomplex. the firstexampleofbothmonoallelicandbiallelicexpres- NK clonesexamined(Fig.1B).The KIR some. Incontrasttoresultswiththetwoclonallyrestricted will beexpressedfromthesameoroppositechromo- necessarily determinewhetheracloselylinked pression ofa leles ofthenearby tion withthe monoallelically expressedthe cant cellsurfaceexpression.NKclonesK5,K6,andK7 lelic 3DL1 mAb(unpublisheddata).Thisindicatesthatmonoal- donor LNKclonesstudiedherestainedbrightlywithanti- or biallelic(Fig.1B).ExceptforcloneK9,alldonorKand 3DL1 and3DL2RNAexpressionwaseithermonoallelic Regardless ofpossiblequantitativevariationinRNAlevels, 3DL1 RNAdespitehavinglittleornocellsurfaceprotein. available atthetimeofanalysis.Oneclone(K9)expressed RNA levelsandmonospecificanti-3DL2mAbwasnot of unselectedNKclones(11).WedidnotquantifyKIR though 3DL2isexpressedatahighlevelinonlyminority was detectedinallninedonorKclonesexamined,even primers (refertoMaterialsandMethods).KIR3DL2RNA specific cDNAprimingandtwodifferentsetsofPCR sults wereobtainedusingrandomcDNAprimingorgene- striction fragmentlengthpolymorphismassay.Similarre- completely consistentwiththeresultsofplasmidre- tively (Fig.1B).TheseresultsforclonesK1–K9were dicated monoallelicandbiallelic The presenceofoneortwosignalsatpolymorphicsitesin- cific primers,bulkRT-PCRproductsweresequenced. 3DL2 ther examineddonorK,whoisheterozygousatthe Characterization ofKIRPromoters. To investigateexpressionofother 3DL1 genes, 3DL1 , and corepromoteris217bplongrelativetothe expressionisassociatedwithbiologicallysignifi- 2DL4 KIR2DL4 AMC5 KIR loci.Afteramplificationwithgene-spe- geneononechromosomedoesnot 3DL2 allele,the expressionwasbiallelicinallnine KIR gene(Fig.1B).Therefore,ex- promoteractivityistissuespe- 248 deletions ofa2.3-kbfragment 3DL1 KIR3DL1 NKAT4 3DL1 * Allele-specific Regulation ofNaturalKillerCellReceptor Expression 2DL4 001 The 5 KIR expression,respec- KIR alleleincombina- allele,orbothal- promoteractivity 2DL4 gene.DNAwas alleles,wefur- locusprovides RACE tech- and 3DL1 KIR 3DL1 3DL2 3DL1 gene gene , denotes exon1,and The thickblacklinedenotesthecorepromoterregion,openbox AC0111501.7). The from GenBank/EMBL/DDBJunderaccessionnos. AF457597–9 and 3DL1 nucleotide polymorphismsindonorL(top)andK(bottom) independent trialsforeachconstruct.(B)Promoterand5 promoter/enhancer inanexperimentthatistypicalofthreeorfour various lengthsisshownrelativetotheactivitydirectedbySV-40 of reporterluciferaseactivitydirectedby Figure 2. methylated state. TheotherdonorKclones expressed leles andhadallfourCpGsites inapredominantlyhypo- CpG sites.ClonesK2,K3,and K4expressedboth random (control)cytosineresidue andfourrepresentative assay. Thistechniqueassayed bulkPCRpopulationsatone nor Kclones,weusedasingle nucleotideprimerextension ers and5 pressed andcontainedcompletelyhypomethylatedpromot- (Fig. 3A).InNKclonesL1andL2,bothalleleswereex- and thenonexpressedallelewasalmostuniformlymethylated the expressedallelewerealmostcompletelyhypomethylated allele wasactiveinthesecells,thepromoterand5 lelic and sequencing(29).FivedonorLNKcloneshadmonoal- the basestobedistinguishedbyPCRamplification,cloning, to uracil,butdoesnotconvert5-methylcytosine,allowing DNA Hypomethylation. cific gated whetherCpGmethylationcorrelatedwithallele-spe- the computer searchalgorithms(unpublisheddata).Because known transcriptionfactorbindingsitepredictedbytwo core promoterpolymorphicnucleotidedidnotaffectany differences innearbyregions(Fig.2B).ThesingledonorK lelic differencesinthecorepromoterand4bpinterallelic were verysimilarinbothdonors,withonly1–2bpinteral- bp surroundingthe KIR3DL1 Allele-specificGeneExpressionCorrelateswith 3DL1 3DL1 * KIR 001 and expressioninNKcells. promotercontainsaCpGisland(28),weinvesti- expression(Fig.1A).Regardlessofwhich regions. Toanalyzethemethylationstatusindo- Characterization ofthe3DL1promoter.(A)Theamount 3DL1 * 3DL1 represents thetranslationstartsite. * 002 3DL1 * alleles(thesesequencedataareavailable 002 Sodium bisulfiteconvertscytosine allelesareidenticalinthetwodonors. promoter.The 3DL1 promoterconstructsof 3DL1 regions of gene single 3DL1 alleles 3DL1 al- The Journal of Experimental Medicine patterns invitro thatmaynotreflectmethylation patterns sion InVivo. and donorKNKclones. CpG sitesinthenonexpressed correlated withallele-specific and background,promoter 5 methylated (Fig.3B).Despitedifferencesinassaymethod 3DL1 3DL1 KIR3DL1 HypomethylationCorrelates withGeneExpres- * * 001 001 allelewerelargelyhypomethylated,whereas but not Cultured cellscanadoptDNA methylation 3DL1 * 002 249 3DL1 3DL1 . CpGsitesintheexpressed region hypomethylation expressionindonorL Chan etal. * 002 allelewerehyper- 3DL1 PCR clone.OntheleftisNKcellclonenameandexpressed unmethylated CpGsites,respectively.Eachrowrepresentsaseparate * expressed both is indicatedbythedarksegmentofeachpiechart.NKclonesK2–K4 the corepromoter.Thefractionofmethylatednucleotidesateachsite region andlies5 sites (M1,M4,M6,andM8).M1isthefirstCpGsiteinamplified sites wereanalyzed:onenon-CpGcontrolcytosine(C)andfourCpG methylation-sensitive singlenucleotideprimerextensionassay.Five sents thetranscriptionstartsite. DNA sequenceanalysisofbisulfite-treatedDNA.Thearrowrepre- gene expression.(A)NKcellclonesfromdonorLwereanalyzedby Figure 3. two donors.CD56 tion inNKcellsanalyzeddirectlyexvivofromthesame sion correlatedwithpromoterand5 in vivo(30).Therefore,wetestedwhether peripheral blood NKcellshadfullyorpartially methylated bisulfite, PCRamplified,cloned, andsequenced.3DL1 spectively. DNAwasextracted, treatedwithsodium populations were99.4–98.2% and97.5–98.9%pure,re- tion ofnegativeselectionand flowcytometrysortingto 001 99.5% purity.DonorKand donor L3DL1 allele. allele(s).(B)NKcellclonesfromdonorKwereanalyzedby KIR3DL1 hypomethylationcorrelateswithallele-specific 3DL1 to thecorepromoter.M4,M6,andM8liewithin alleles.NKclonesK5–K8expressedonlythe NK cellswereenrichedbyacombina- and represent methylatedand region hypomethyla- 3DL1 and 3DL1 expres- The Journal of Experimental Medicine Hypermethylation ofsomesequencesfrom3DL1 expression inNKcellsvivo. may havecomefromacontaminating3DL1 3DL1 promoter and5 pletely hypomethylated.Thismayindicatethatcomplete 3DL1 lele-specific polymorphisms, wewereabletoidentify which isabyproductofbisulfite treatment(29).Usingal- bination eventsincreasewith fragmentedtemplateDNA, vivo ortoPCRrecombinationinvitro(31).recom- 4). Thismightbeduetoincompletegenemethylation in tion atoneendandhypomethylationtheother(Fig. 3DL1 of NKcellclones(unpublished data).Twosequencesfrom Five DNAclonesfrom3DL1 with similarfindingsforboth hypomethylated andhypermethylated heavily methylated.Of14sequencesfrom2DS4 contrast, 3DL1 3DL1 4). Thus, 7 werehypomethylatedandhypermethylated(Fig. DNA sequencesfrom2DS4 2DS4 of thetwo was expectedbecausemostNKclonesexpressedonlyone One DNAclonefromsorted3DL1 genethatencodesastimulatoryKIR.Allnine expression.Alternatively,thehypomethylatedDNA genesin28outof29sequencesanalyzed(Fig.4).In * 001/ cells weremethylatedinthe codingregionbut KIR 3DL1 * 002 recombinantDNAclones inouranalysis DNAhypomethylationcorrelateswithgene peripheral bloodNKcellscontainedboth gene hypomethylationisnotsufficientfor alleles.Similarresultswerefoundforthe 250 * 001 cells showedhypermethyla- donor FNKcellswere and Allele-specific Regulation ofNaturalKillerCellReceptor Expression * 002 3DL1 cells wascom- alleles(Fig.4). sequences, NK cells, NK cell. 2DS4 cells were evenmorestrikingfordonorF2DS4 (refer toMaterialsandMethods,Calculations).Results 53.6% ofsequencesfromdonorK3DL1 which themostsequenceinformationwasavailable, in Most PeripheralBloodNKCells. was occasionallyincomplete. Aza, aninhibitorofDNAmethylases. Usingflowcytome- hypomethylation, wetreated NK92.10tumorcellswith whether transcriptionisacause orconsequenceofDNA surface KIR gene expressionsuggeststhatthegreatmajorityofcell cells hadonlyonehypomethylated with 3DL1 promoter hypomethylationisnotsufficientforhighlevel largely hypomethylatedinthepromoter,suggestingthat of therelevantclonallyrestricted that 98.9%ofcirculating2DS4 served 50% Based onthe98.9%purityofsortedcellsandob- strong correlationbetween analyze allele-specificgeneexpressiondirectly,the hypomethylated pomethylation rateindicatesthat90.1%of3DL1 served inNKcellclones(Fig.3).A53.6% hypomethylated (Fig.4),similartothe64.3%rateob- KIR GenesHaveaMonoallelicHypomethylationPattern Methylase InhibitorInducesKIR GeneExpression. 3DL1 expression. Althoughhypomethylationcorrelated expressioninvivoandvitro,methylation 2DS4 circulating NKcellsexpress onlyoneallele quenced region.Analysisisasdescribedin Fig. 3A. The 2DS4allelesareindistinguishableinthese- position oftherest is notknown,theATGtranslationstartsiteand are indicated.Becausethetranscriptionstartsite K anddonorL3DL1 with geneexpressioninvivo.Resultsdonor Figure 4. Results withdonorF2DS4 indicated for 2DS4 hypomethylationrate,weestimate allele.Althoughwecouldnot KIR3DL1 hypomethylationcorrelates 3DL1 KIR * NK cellshadonlyone 001 hypomethylationand KIR 2DS4 and 3DL1 3DL1 and Analyzing datafor locus. exon1areindicated. and 2DS4 3DL1 NK cellswere alleleinvivo NK cellsare * 3DL1 002 NK cells. NK cells To test alleles. hy- NK The Journal of Experimental Medicine 33). Therefore,weclonedcells 72hafterdrugtreatment from areasofhighmethylation intoneighboringDNA(32, much higherAzadosesand becausemethylationspreads pected becausedemethylation isincompleteevenwith ter cessationofAzatreatment. Lossofexpressionwasex- was heterogeneous(Fig.5)and waslostfrommostcellsaf- to analyze demethylation atthe tory andstimulatory cells toexpressmultiple three mAbs.Thus,DNAmethylaseinhibitioninducesNK bound bothDX9andGL183mAbs surface protein(Fig.5B).ThemajorityofAza-treatedcells pressed bothinhibitory3DL1andstimulatory2DS4cell heterogeneous. AboutonethirdofAza-treatedcellsex- or 2DS2(detectedbyGL183mAb).KIRexpressionwas of 2DS4(detectedbyFES172mAb)and2DL2,2DL3,and/ cells wereelectronicallygatedandanalyzedforexpression press 3DL1boundbyDX9mAb(Fig.5B).The Most treatedNK92.35cells(95.9%)wereinducedtoex- three anti-KIRmAbswithnonoverlappingspecificities. treated andanalyzedbymulticolorflowcytometryusing tory andinhibitoryreceptors.NK92.35cellswereAza to expressmultipleKIRmolecules,includingbothstimula- Next, weaddressedwhethersinglecellscouldbeinduced induced onYT-Indycells(Fig.5Aandunpublisheddata). CD8 expressionwasnotinducedonNK92.10cellsbut (CD3), orBcells(CD19;Fig.5Aandunpublisheddata). molecules thataremadebyNKcellsubsets(CD16),Tcells protein expressionwasspecificbecausewedidnotdetect NK cells(unpublisheddata).InductionofcellsurfaceKIR inhibition alsoinduced induced heterogeneousKIRexpression.DNAmethylase EB6 mAbs.ThisindicatesthatDNAmethylaseinhibition GL183 mAbbutonlyaminorityboundeitherDX9or analysis, approximatelyhalfoftheAza-treatedcellsbound several otherKIRmolecules(Fig.5A).Atthetimeof 3DL1 mAbandGL183EB6mAbsthatcrossreactwith try, newKIRexpressionwasdetectedusingDX9anti- Aza inductionofKIRexpressionmayhavebeendueto KIR genedemethylation,butKIRexpression KIR KIR KIR KIR . locusoratotherloci.Wewished 251 geneexpressioninYT-Indy genes,includingbothinhibi- Chan etal. 30% boundall KIR demethylated individualKIRgenes,directlyleadingto been ruledout,theseresultssuggestthatAzatreatment gene expression.Althougheffectsonothergeneshavenot DNA methylaseinhibitionleadsto correlating withRNAexpression(Fig.6B).Therefore, the A). ThelineZ expression biasedinfavorofthe sion. LineCexpressedboth NK92.26.5 sublineswereanalyzedfor thatthe3DL1moleculewasfunctional(21).Two ing specifically inhibitedcytolysisbyNK92.26.5cells,show- growth intheabsenceofdrug.TargetcellHLA-B and and analyzedrareKIR Discussion Pax5 cally, suchas ranging immunesystemgenesarealsoexpressedmonoalleli- Recent directandindirectevidenceindicatesthatnonrear- of theotherallele,aphenomenoncalledallelicexclusion. B cellreceptororTCRalleleusuallypreventsrearrangement TCR genes.Successfulrearrangementandexpressionofone express onlyonefunctionalalleleofBcellreceptorand (35). Ithaslongbeenappreciatedthatlymphocytestypically of theimmunesystemareexpressedinamonoallelicfashion man syndromes(34).Althoughnotimprinted,severalgenes ous developmentaldefects,suchasPrader-WilliandAngel- maternal orpaternalallele.Defectsinimprintingcauseseri- genes areimprinted,withexpressionbeinglimitedtothe lelically. Asmallnumberofgrowth-controllingautosomal expressed butsomeautosomalgenesaremonoal- maintained two allelesofthe mining lymphocytedevelopmental decisions. 37, 40–45).Mostorallofthese genesareimportantindeter- The twoallelesofmostautosomalgenesarecoordinately 3DL1 IL-4 expression. transcriptionfactor,andcytokines (35–45).Individualcellsmay expresszero,one,or * 001 3DL1 Ly49 is shownonalogarithmicscale. and GL183binding(rightpanel).Fluorescenceintensity mAb binding(leftpanel)wereanalyzedforFES172 Aza-treated cells(notdepicted).CellspositiveforDX9 ing ofnonspecificmouseIgGtoeitheruntreatedor line onleftpanel), binding ofanti-KIRmAb(middlepanelanddotted Untreated NK92.35cellsshowedonlybackground (FES172), h andstainedforexpressionof3DL1(DX9),2DS4 72 2DS1(EB6). (B)NK92.35cellswereAzatreatedfor KIR2DS2 (GL183),3DL1(DX9),and2DL1 active withCD16(3G8),CD8(3B5),2DL2,2DL3, analyzed 60h peaks) orpresence(openof1 (A) Figure 5. allelewaspredominantlyhypomethylated, 3DL1 Ly49 NK92.10 cellsweregrownintheabsence(filled expressionduringseveralmonthsof and * , 002 and 2DL2,2DL3,2DS2(GL183). NKG2A NKG2A Methylase inhibitorinducesKIRexpression. clones (21).CloneNK92.26.5 allelewashypermethylatedand later byflowcytometryusingmAbre- 3DL1 which wasnearlyidenticaltobind- mouseNKcellreceptors, , IL-2 3DL1 KIR allelesbutlineZhad , and IL-1A 3DL1 demethylationand * 001 IL-4 alleleexpres- , allele(Fig.6 IL-2 genes(36, M Azaand , IFN- * 2705 , The Journal of Experimental Medicine of cellsurfaceKIR both show monoallelicexpressioninvitro.Hypomethylation of some, orbothchromosomes.Clonallyrestricted pressed fromthesamechromosome,oppositechromo- does notdeterminewhetheraseparate cells analyzeddirectlyexvivo. Baseduponthestrongcor- monoallelic hypomethylation inthemajorityofsortedNK restricted mature NKcellsoftenexpressonlyonealleleofclonally with two explain thesefindings,butwe pointoutthatourresults Z asdescribedin Fig. 3A. Fig. 1A.(B)Methylationanalysisof 2DL3 nor hadalmostcompletehypomethylation of tourlidis etal.foundthatpolyclonalNKcellsfromonedo- gene expression.Inapparentcontrasttoourwork,San- ported acorrelationbetween manuscript wasunderreview,Santourlidisetal.(47)re- identity ofexpressed cell clonesfromthesamedonorvaryinnumberand genes appeartoberegulatedindependently.IndividualNK extremely highsequencesimilarity,clonallyrestricted fraction ofNKcellsinaclonallyrestrictedfashion.Despite NK cloneswhereastheother from aberrantself.The are criticalforhumanNKcelldiscriminationofnormal 3DL1 treated NK92.26.5linesCandZ.NK92.26cellswerewith1 were grownseparatelyandanalyzedfor allele-specific expression.(A) Figure 6. M Azafor72handthenclonedbylimitingdilutionresultinginthe KIR moleculesrecognizeMHCclassIand KIR genesfromsortedKIR NK92.26.5 clone(reference 21). NK92.26.5linesCandZ alleleswasdeducedtooccurinasmallminority KIR KIR Methylase inhibitorcauses3DL1hypomethylationand genesandthatexpressionofone genesfromthreedonorsare consistentwith peripheral bloodNKcells.Whilethis KIR KIR2DL4 genes(11).Ourdatashowthat KIR3DL1 252 KIR KIR 3DL1 NK cells(47).Wecannot genesareexpressedbya geneisexpressedbyall 3DL1 alleleexpressioninAza- Allele-specific Regulation ofNaturalKillerCellReceptor Expression hypomethylationand allelesinNK92.26.5line allelesasdescribedin KIR geneisex- 3DL1 KIR KIR genes allele and KIR stricted showed unequivocalmonoallelicexpressionofclonallyre- extend thisfindingtoindividualalleles.NKcellclones tic mannerduringdevelopment. homologous genesbyindividuallymphocytesinastochas- pression reflectstheactivationoflimitedsubsetshighly Coffman andReiner(46)postulatedthatmonoallelicex- only onealleleofclonallyrestricted sion, weproposethatcirculatingNKcellstypicallyexpress mitted tospecificclonallyrestricted ture (20,48).ThisshowsthatNKcellclonesremaincom- anti-KIR mAbbindingaftershort-termorlong-termcul- ( centage ofbiallelicexpressionmayhavebeenhigher expressed ing (42,43,45).Mostinvitro–stimulatedNKcellclones relatively weaksignalingandbiallelicallyafterstrongsignal- methylation of severalmaternalalleles.Methylation ofan drome/Angelman syndrome, animprintingcentercontrols tion appearstobeakeyregulator (34).InPrader-Willisyn- quiescent neighbors. ers preventtranscribed than 2kbapart.Wespeculatethatinsulatorsorotherbarri- relation between lele-specific regulation,the its firstintron(9,10).Despitedifferencesinlocus-andal- only KIR2DL4 clonally restricted genetic complex.Itisnotsurprisingthat both monoallelicandbiallelicexpressionwithinasingle rounds ofinvitrostimulation, mature withinvitrostimulation(42–44).Aftermultiple for expressionofarecombinantmarked was stable.Incontrast,transgenicTcellsthatweresorted (this study).The pressed byallhumanNKclones(11)inabiallelicfashion terns becameclonallystable(44). and transcription machinerymustbeassembled.The T cellstimulation.Witheachroundofstimulation,the contrast, IL-2andIL-4quicklydisappearintheabsenceof ciated withthe proteins. Thus,transcriptionalmachinerymayremainasso- dent geneexpression.NKcellsconstitutivelyexpressKIR stability mightbeduetodifferencesinactivation-depen- was notstable.Thedifferencein site allele(42–44).IntheseTcellscytokinechoice and thenreculturedcouldswitchexpressiontotheoppo- We studied allele stabilityalsomaydependuponlymphocytematurity. ing supplyoftranscriptionfactors(42). leles arepostulatedtobeaccessibleandcompeteforalimit- 10 T cellsexpress NK cellssorteddirectlyexvivoretainasinglepatternof Regulation ofimprintedgenes iscomplexbutmethyla- Unlike theclonallyrestricted 33%) thanincirculatingNKcells. 6 IL-4 KIR cells. Therefore,clonallyrestricted KIR werestudiedinnaiveTcellsthatcontinuedto locusthatdoesnothaveadistinctminisatellitein 3DL1 has14kbofuniqueupstreamDNAandisthe genesafterculturefor3–5wkandgrowthto KIR KIR inamonoallelicfashionalthoughtheper- genesinmatureNKcells,whereas IL-2 KIR KIR KIR allelesthatarecontinuouslyactive.In and locusprovidesthefirstexampleof genehypomethylationandexpres- genesareregulateddifferently. KIR IL-4 2DL4 genesfromactivatingtheir genesmonoallelicallyafter IL-4 KIR and KIR KIR allelicexpressionpat- genes, KIR andcytokineallele 3DL1 genes.Ourresults KIR IL-2 KIR genesinvivo. 2DL4 andcytokine or geneslieless allelechoice 2DL4 IL-4 andthe IL-4 isex- allele IL-2 al- The Journal of Experimental Medicine and othertissues(50).Inthe ported toregulategeneexpressioninbreastepithelium study showedthatthechicken genes intheirnaturallocationsvivo.Awidelyquoted between DNAmethylationandexpressionofendogenous stable, clonallyrestrictedallele-specific showing thatNKcellsuseDNAmethylationtomaintain and alleles(thisstudy).Ourfindingsresolvethisdilemma, press distinctnumbersandcombinationsof reconcile withtheobservationthatindividualNKcellsex- tremely high pression bydemethylatingthe DNA methylasetreatmentdirectlyinduced but notothers.Instead,wefavortheinterpretationthat direct mechanismwouldhavetoinducesome ment inducedheterogeneous pression. Itshouldbepointedout,however,thatAzatreat- demethylation ofgenesthatindirectlyinfluenced pression. Induced KIR tory genes.DNAmethylaseinhibitortreatmentalsocaused multiple gene expressionarecorrelative. teins. Instead,allele-specificDNAhypomethylationand KIR region similarityarguesthatthedifferentiallyexpressed promoters arehighlysimilar(thisstudy).Closepromoter cytes (49).Recently, becomes methylatedandisnotexpressedinadulterythro- demethylated andactiveinembryonicerythrocytesbut single distinctivesequenceof ated regions,the tissue-specific geneexpression.Exceptforthreenoniter- stringent testofthehypothesisthatmethylationcontrols genes.TheKIRlocusprovidesanextremely autosomal tion controlsallele-specificexpressionofnonimprinted Igf2 CTCF proteinandallowsadistantenhancertoactonthe insulator inthe methylated insomeTcellsdespiterobustIFN- vivo (53).Incontrast,thekeypromotersitewasheavily with IFN- restriction endonucleases,hypomethylationcorrelated duction (51).Asassessedusingtwomethylation-sensitive correlated withthepresenceandabsenceofIFN- all monocyteshadamethylatedCpG,respectively,which nearly allNKcellshadanunmethylatedCpGand sion betterthanotherCpGsites(51,52).Atthissite, ylation ofthe ever, studieswith rearranginggenesare complicated by nonrearranged TCR- demethylation correlatedwith germlinetranscriptionof sary norsufficientfor Therefore, promoterhypomethylation wasneitherneces- the (54). AlthoughsomeBlymphocytesdidnotmethylate Few detailedstudiesshowaconvincingrelationship Inhibition ofDNAmethylasesup-regulatedexpression promoter.However,itwasnotclearwhethermethyla- demethylation,linkinghypomethylationandgeneex- allelesdonotbinddiscretesetsoftrans-actingpro- 53 CpGsite,theyfailedto produceIFN- KIR genes,includingbothinhibitoryandstimula- expression innaiveandmatureTcells KIR 53 CpGsitecorrelatedwithgeneexpres- H19/Igf2 sequencesimilarityhasbeendifficultto KIR 150-kb expressionmayhavebeendueto IFN- serpinb5 genes inthymocytes(55).How- locuspreventsbindingofthe 253 KIR KIR IFN- expression. IL-7–dependent genemethylationwasre- KIR 100 bp(9)and locusdoesnotcontaina geneexpression.Anyin- globin genepromoteris genesthemselves.Ex- Chan etal. promoter, hypometh- KIR geneexpression. KIR KIR genes(11) KIR KIR geneex- secretion KIR genes allele (51). pro- ex- that onceselected, dependent expressionof such as tem genesthatareexpressedinamonoallelicfashion, The samemechanismslikelyapplytootherimmunesys- mechanism thatoperatesatthelevelofindividualalleles. cytokine-activated transcriptionfactorsinduce NK cellsinvitro(59).Thissuggeststhepossibilitythat IL-15 inducedKIRexpressionbydevelopinghuman KIR the relationshipbetweenDNAmethylationpatternsand tained byDNAmethylation.Anoutstandingquestion is expression of highly homologous,mechanismsthatallowindependent gardless ofgeneexpression(58).However,5 IL-4 either mechanismmaydominateindifferentsettings.The inaccessible totrans-actingproteins(19).Itislikelythat which compactchromatinandmakeregulatoryregions methyl DNAbindinghistonedeacetylasecomplexes, acting proteins.Inaddition,DNAmethylationrecruits tion ofspecificpromotersitesblocksbindingkeytrans- expression inmouseTcellclonesvitro(52).Methyla- of promoterhypomethylationwithIFN- was reminiscentofthegeneralbutimperfectcorrelation quences showedonlypartialmethylation.Thissituation stricted demethylation. Italsoseemsprobablethatclonallyre- Because clonallyrestricted selectively down-regulatedHLAclassIlocioralleles. vated bytumorcellsandvirus-infectedthathave expression mayallowspecificNKcellsubsetstobeacti- dent expressionof class Igenesandallelesprovidesarationaleforindepen- virus-infected cellstoselectivelydown-regulateMHC MHC classImolecules.Theabilityoftumorcellsand levels ofDNAmethylation. sion. moter hypomethylationisnotsufficientforgeneexpres- exon 1andintronsites.Thissuggeststhat promoter hypomethylationbutheavymethylationofthe of Health,theMedicalResearch Council,andtheWellcome Mark Stinskifortechnicalassistance andhelpfulcomments. We thankColleenFullenkamp, PhilipLin,AndrewRusso,and the observationsthat 5 ripheral blood3DL1 tion (58).Similarly,one Th2 commitment,correlatingwithincreasingIL-4secre- the 3DL1 with NKcell that promoterand5 tion aroseinmethylatedDNA(56,57).Ourresultsshow This workwassupportedbygrants fromtheNationalInstitutes Individual KIRproteinsrecognizedistinctsetsof Most gene methylation.Afew3DL1 IL-4 promoterishypomethylatedinallCD4Tcellsre- expressionduringNKcelldevelopment.IL-2and KIR NK cellsshowedalmostcompletepromoterand Ly49 genebecomeprogressivelyhypomethylatedafter 3DL1 KIR geneexpressionmightbeinhibitedbyoverall , expressionisachievedbyastochastic KIR KIR NKG2A DNAsequencesfromperipheralblood geneexpressioninvitroandvivo. geneswouldbeexpectedtoallowin- KIR KIR NK cellsshowedalmostcomplete Ig andTCR- , and region hypomethylationcorrelate genes.Clonallyrestricted expressionpatternsaremain- 3DL1 KIR Pax5 KIR alleles.Ourresultsshow DNAsequencefrompe- genesandallelesare . 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