Unraveling a Revealing Paradox: Why Major Histocompatibility Complex I

The Journal of Experimental Medicine

ation ofDPthymocytesintomatureCD4 trathymic peptide–MHC-IIcomplexesresultsindifferenti- MHC–peptide complexes(1).TCRengagementofin- positive TcellsbyTCRengagementofintrathymic signaled inthethymustodifferentiateintomaturesingle individual TCRsusetoengageintrathymicligands, as choice isaconsequenceofthecoreceptormoleculesthat developmental immunology.Itisthoughtthatlineage mines lineagechoiceremainsanunresolvedproblem in MHC classspecificityofsurfaceTCRcomplexesdeter- Introduction [email protected] thesda, MD20892.Phone:301-496-5461; Fax:301-496-0887;E-mail: Branch, NationalCancerInstitute, Building10,Room4B36,Be- Address correspondencetoAlfred Singer,ExperimentalImmunology Immature CD4 mature CD8 mature plexes resultsindifferentiationofDPthymocytesinto TCR engagementofintrathymicpeptide–MHC-Icom-

Abbreviations usedinthispaper:

T cells(2,3).Themechanismbywhichthe

(double positive[DP]

1709

DP,doublepositive;hCD2,human CD2.

cells duringtheirdifferentiationintoCD8 Positive SelectionofCD8 to occurattheCD4 is notnecessarilyindicativeofcommitmenttotheCD4 also demonstratesthatinitialcessationofCD8geneexpressioninMHC-I–signaledthymocytes pression, notbecauseofchangesinCD8surfaceproteinexpressionordistribution.Thisstudy e od:lineagecommitment •kineticsignalingcoreceptor reversal •positive selection Key words: lineage commitmentbutfulfillpredictionsofthekineticsignalingmodel. patibility complex(MHC)-I–signaledthymocytesparadoxicallyappearasCD4 lineage decisioninthethymusisnotknown.Animportantcluefactthatmajorhistocom- The mechanismbywhichTcellreceptorspecificitydeterminestheoutcomeofCD4/CD8 1 Singer and Alfred thymocytes appearasCD4 gene expression.Butcontrarytothisperspective,studydemonstratesthatMHC-I–signaled tain thepotentialtodifferentiateintoCD8 mocytes asCD4 the oppositecoreceptorgene.Fromthisperspective,appearanceofMHC-I–signaledthy- Abstract Remy Bosselut, “Paradoxically” Appear asCD4 Histocompatibility ComplexI–signaled Thymocytes Unraveling aRevealing Paradox: Why Major Laboratory ofImmune CellBiology and Institutes ofHealth, Bethesda, MD20892 http://www.jem.org/cgi/doi/10.1084/jem.20030170 The JournalofExperimental Medicine * ) thymocytesare T cellswhereas 8 1 lo Terry I. Guinter, 2 cells wouldbeduetoeffectsonCD8surfaceproteinexpression,not 8 (double positive)stageofdifferentiationandtoresultinsilencing 8 lo cells becauseoftransientdown-regulationCD8geneex- 2 Experimental ImmunologyExperimental Branch, NationalCancerInstitute, National gene expressionanddictatecommitmenttotheCD4 CD4 coengagementsonDPthymocytesterminateCD8 models oflineagecommitmentproposethatTCRplus eral types:instructionalandstochastic(6–10).Instructional eage choiceremainsamatterofintensecontroversy. which TCRandcoreceptorcoengagementsinfluencelin- tor molecules(4,5).However,theactualmechanismby generally requirescoengagementofsurfaceCD8corecep- whereas TCRengagementofpeptide–MHC-Icomp coengagementofsurfaceCD4coreceptormolecules requires TCR engagementofpeptide–MHC-IIcomplexesgenerally the CD8 terminate CD4geneexpression anddictatecommitmentto nals transducedbyTCRplusCD8coengagementsthat tively orquantitatively(instrengthduration)fromsig- cell lineagebytransducingsignalsthatdiffereitherqualita- randomly terminateexpression ofonecoreceptorgene, with eithercoreceptormolecule signalsDPthymocytesto eage commitmentpropose that coengagementofTCR Classical modelsoflineagecommitmentaretwogen- 2 •Vlm 9,Nme 2 ue1,20 1709–1719 June16,2003 •Volume197,Number 12, Susan O. Sharrow, T Cells T cells.Theseresultschallengeclassicalconceptsof T cells.Lineagecommitmentisgenerallythought T celllineage(11–15).Stochastic modelsoflin- 8 lo Transitional CellsDuring T celllineage,assuchthymocytesre- 2 8 lo transitional lexes T The Journal of Experimental Medicine proportion ofCD8lineagecellsinitiallyappearasCD4 23). ThisclassicalviewofCD8 CD8 expressiononCD8-committedthymocytes(9,22, signals onCD8surfaceproteinsthatresultedinreduced pothesizing thatitwasduetoadirecteffectofMHC-I onciled withclassicallineagecommitmentmodelsbyhy- naled thymocyteshadnotbeenpredicted,itcouldberec- paradoxical lossofCD8surfaceproteinsonMHC-I–sig- levels ofCD8proteinsontheircellsurface.Althoughthe express CD8coreceptorgenes,yetthesecellsdisplaylow face, and(b)CD8-committedthymocytesarethoughtto cells displayhighlevelsofCD4proteinsontheircellsur- thought tohaveterminatedCD4geneexpression,yetthese on twolevels,as:(a)CD8-committedthymocytesare tion ofintermediateCD4 ceases, intermediateCD4 TCR signalingpersists,intermediateCD4 TCR-mediated positiveselectionsignalspersistorcease. If CD4 mitment occursafterDPthymocyteshaveconvertedinto (24–26). Sokineticsignalingpostulatesthatlineagecom- T celldifferentiationinthethymus isdepictedinFig.1B. ted thymocytesasCD4 transitional cells(18–22).TheappearanceofCD8-commit- with thesepredictionsasassessmentofCD4 cells. However,experimentalobservationsarediscordant pear asCD4 therefore willloseCD8surfaceproteinexpressionandap- T celllineagehaveterminatedCD8geneexpressionand dict thatDPthymocyteshavecommittedtotheCD4 the oppositecoreceptorgene. tulated tooccurinDPthymocytesandresultsilencingof nals (16,17).Inbothmodels,lineagecommitmentispos- coreceptor moleculesresponsivetosubsequentsurvivalsig- with onlythymocytesstillexpressingmatchingTCRand CD4 CD8 DP thymocytesthathavecommittedtotheCD8 CD4 undergoing coreceptorreversal totranscriptionallybecome cause theyinitiallyterminate CD8geneexpressionbefore differentiate intoCD4 signaled thymocytesinitially appearasCD4 quently, thekineticsignalingmodelpredictsthatMHC-I– expression, referredtoas“coreceptorreversal.”Conse- cells isthesilencingofCD4andreinitiationCD8gene CD4 surfaceproteinexpressionandappearas lineage haveterminatedCD4geneexpressionandwilllose CD4 netic signalingmodel,lineagedirectionisdetermined in tential todifferentiateintoeitherCD4 CD4 CD8 geneexpressionandtranscriptionallyconvertinto mitment proposesthatsignaledDPthymocytesterminate picted inFig.1A. Determining the basisforreducedCD8surface protein As aresult,classicalmodelsoflineagecommitmentpre- In contrast,thekineticsignalingmodeloflineagecom- lo 8 8 8 8 8 T cells.Asaresult,criticalstepinthedifferentia- transitional populationsrevealedthatasignificant thymocytes. Thekineticsignaling viewofCD8 intermediate thymocytesthatstillretainthepo- intermediate thymocytes.Accordingtotheki- intermediate thymocytessimplybywhether 8 lo transitional cells.Reciprocally,signaled 8 T cells,butifTCRsignaling

lo 8

1710

transitional cellsisparadoxical

thymocytes differentiateinto

thymocytes intoCD8

T celldifferentiationisde-

Basis forReduced CD8ExpressiononMHC-I–signaled Thymocytes

or CD8

thymocytes

cells be-

T cells

T cell lo

and

8 8

naled CD4 that lowCD8surfaceproteinexpressiononMHC-I–sig- pression. Incontrast,thekineticsignalingmodelpredicts fects onCD8surfaceproteinexpression,notgeneex- challenge moreclassicalconceptsoflineagecommitment. cells. Theseresultssupportthekineticsignalingmodelbut MHC-I–signaled CD4 gene predictsthatlowCD8surfaceproteinexpressionon results inirreversiblesilencingoftheoppositecoreceptor cept thatlineagecommitmentoccursinDPthymocytesand cepts ofhowlineagecommitmentoccurs.Theclassicalcon- cell differentiationtestspredictionsmadebydifferentcon- at comparablelevels.TheCD8 Materials andMethods ment totheCD4lineage,asmanyintermediate regulation ofCD8geneexpressiondoesnotimplycommit- expression. Inaddition,thisstudydemonstratesthatdown- surface proteins,butisduetodown-regulatedCD8gene is notduetointernalizationnorslowreexpressionofCD8 fluorescence: panCD8 fluorescence: with thefollowingspecificities wereusedfordirectimmuno- and endogenousCD8 mouse linesexpressedtransgenicandendogenousCD8 erate II BD Biosciences),TCR mal facility.MHC-II 28) wereusedat8–12wkofageandmaintainedinourownani- thymocytes retaintheabilitytodifferentiateintoCD8 expression onMHC-I–signaledthymocytesduringCD8 Line 30micewerematedwithMHC-II moter activityandstrictlyparallelsCD4geneexpression(31). T cellsinLine30miceservesasafaithfulreporterofCD4pro- NY; 31).SurfaceexpressionofhCD2proteinonthymocytesand man (SkirballInstituteofBiomolecularMedicine,NewYork, (including theCD4silencerelement)wereprovidedbyD.Litt- hCD2 cDNAunderthecontrolofCD4locusregion proteins. 27), CD8 face proteinexpressiononMHC-I–signaledCD4 tempted todeterminetheactualbasisforreducedCD8sur- CD8 geneexpression.Consequently,thisstudyhasat- dogenous CD8 113.1; reference32),CD8 surface expressiononMHC-I–signaledCD4 mocytes. TheseresultsdemonstratethatreducedCD8 MHC-II MHC-II less CD8 based CD8 mating intoMHC-II transgenic CD8 transgenic of CD8 ate II added tocultureswhereindicated. (G11; Caltag).Recombinantmurine IL-7(R&DSystems)was

Line 30transgenicmiceexpressingatransgeneencoding CD8

Antibodies andReagents. Animals.

CD8

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transgenic miceconstructedwithhumanCD2(hCD2)- with transgenicCD8 Line 30mice.

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(CD8 molecules havebeendescribed(29,30).Transgenic o

transgenes encodingeitherfull-lengthCD8

Mice deficientinMHC-II(MHC-II mice. 8

mice, andwerebredwithCD8

proteinsbeingCD8

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proteins expresseddifferentallelicforms o (53-6.7; BDBiosciences),CD8

(H57-597; BDBiosciences),and hCD2

CD8

.2 (2.43;reference33),CD4(GK1.5; lo Fluorochrome-conjugated antibodies

thymocytes isduetodirectef-

, andII proteins beingCD8

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The Journal of Experimental Medicine at 6ng/mlfinalconcentration. (24, 26).Whereindicated,recombinantmurineIL-7wasadded tions wereplacedinsuspensionculturesaspreviouslydescribed mocytes. parable toCD4andCD8fluorescenceonuntreatedDPthy- expression at24h,whichwassetequalto100%andcom- CD8 fluorescenceonDPthymocytesafterfullcoreceptorre- fluorescence unitsandexpressedasapercentageofCD4 various timesafterpronasestrippingwasquantitatedintotal reexpression, CD4andCD8fluorescenceonthymocytesat transcribing (22).Forquantitativeanalysisofsurfacecoreceptor they reexpressedthecoreceptormoleculeswereactively 37 were treatedwith0.01%pronase(Calbiochem)for15minat cells. GatingonTCR Results II mocytes, weintroducedaCD8 protein expressionisreducedonMHC-I–signaledthy- existing surfaceCD4andCD8proteins(22).5 indicated, thymocyteswerepronasetreatedtoremovepre- developed attheNationalInstitutesofHealth. propidium iodidestaining.Analysiswasperformedusingsoftware son). Deadcellswereexcludedbyforwardlightscattergatingand analyzed onamulti-laserFACSVantage™SE(BectonDickin- vested, stainedwithfluorochrome-conjugatedantibodies,and press twodifferentCD8 molecules (29,30).EndogenouslyencodedCD8 encoded CD8 teins andtransgene-encodedCD8 sion ofendogenouslyencodedCD8 and down-regulatingCD8geneexpression,surfaceexpres- on DPthymocytesbytargetingCD8regulatoryelements MHC-I signalinginitiallyreducesCD8surfaceexpression two CD8proteinsareessentiallyidentical.However, if genic CD8 CD8 regulatoryelements,whereasthe endogenous CD8 CD8 However, thegenesencodingendogenousandtransgenic extracellular domainthatisresponsiblefortheirallelism. cal exceptforthesingleaminoacidchangein pressed onthecellsurfaceatsimilarlevelsandwereidenti- up-regulate TCRsurfaceexpression tobecomeTCR lated byintrathymicligands to undergopositiveselection Thymocytes. Reduced CD8SurfaceProtein Expression onMHC-I–signaled CD8 duced butsurfaceexpressionoftransgenicallyencoded CD8 surfaceproteins,bothendogenous face expressiononDPthymocytesbydirectlyaffecting (34). Thus,ifMHC-IsignalinginitiallyreducesCD8sur- utilizes heterologoushCD2promoter/enhancerelements o In VitroSuspensionCulture. To determinethemechanismbywhichCD8surface Pronase TreatmentandtheCoreceptorReexpressionAssay. Flow CytometryandElectronicCellSorting. Assessment ofCD8ProteinInternalization astheBasisfor C andthenculturedat37 mice sothatMHC-I–signaledthymocyteswouldex- .1 proteinswillnotbereduced. proteins werequitedifferentfromoneanother.The .1 proteinlevelswillbeequallyreducedasthe Immature thymocytesthathave beenstimu- .2 andtransgenicallyencodedCD8 .2 geneutilizesCD8promoterand hi cells inMHC-II 1711 molecules, i.e.,endogenously C overnightduringwhichtime Purified thymocytesubpopula- .1 transgeneintoMHC- Bosselut etal. .1 proteinswereex- .2 proteinswillbere- o mice andassessing Cells werehar- .2 andtrans- .1 transgene 10 6 /ml cells .2 pro- Where .1 hi CD4 transgenic mice:atransitionalthymocytepopulationthatis MHC-I–signaled thymocytesinbothnormalandCD8 CD8 surface expressionofendogenouslyencodedCD4and signaled DPthymocytesthatthendifferentiateintoCD8 sent inMHC-II models (A)orthekineticsignalingmodel(B).Cellsinlightgrayareab- progeny relationshipsaccordingtoeitherclassicallineagecommitment dicate coreceptorgeneexpression.Arrowsindicateproposedprecursor/ surface levelsofCD4/CD8proteinexpression.Labelsinsidethecellsin- mocytes. Labelsoutsidetheplotidentifythymocytesubpopulationsby transitional CD4 which MHC-I–signaledthymocytesdifferentiateintoCD8 Figure 1. transgenic mice,inthatendogenousCD8 pression ofendogenousCD8 T cells(20–22).Importantly,itcanbeseenthatsurfaceex- CD8 curs inCD4 the kineticsignalingmodel,TCRdisengagementofMHC-Iligandsoc- tional cellsasaresultofdiminishedorabsentCD8geneexpression.In color plotofsurfaceCD4versusCD8expressionMHC-II ferentiation ofMHC-I–signaledthymocytesisschematizedasatwo coreceptor geneexpression,andterminaldifferentiationintoCD8 coreceptor coreceptor levelsandresultsincessationofTCRsignaling,reversal initially terminateCD8geneexpressionandappearasCD4 The kineticsignalingmodelproposesthatTCR-signaledDPthymocytes of changesinCD8proteinexpression,notgeneexpression.(B) MHC-I–signaled thymocytesappearasCD4 the oppositecoreceptorgene.Asaresult,allclassicalmodelsrequirethat eage commitmentoccursinDPthymocytesandresultssilencingof signal, durationofsignal)areallbasedonthecentralparadigmthatlin- commitment models(e.g.,instruction,stochastic/selection,strengthof CD8 (Fig. 2).Insharpcontrast,surfaceexpressionoftransgenic inducing CD8 signals donotreduceCD8 identical, theseresultsindicate thatintrathymicMHC-I of transgenicCD8 molecules, MHC-Isignalsdid notaltersurfaceexpression mocytes reducedsurfaceexpressionofendogenousCD8 Thus, althoughintrathymicMHC-IsignalsinDPthy- surface CD8 sion waslowonCD8 mained highonbothCD8 endogenous andtransgenic CD8 hi CD8 molecules onCD8 molecules revealedtwomajorpopulationsof (Fig. 2).Previousstudieshavedocumentedthat Alternative schematicrepresentationsofthemechanismby 8 lo o lo thymi butpresentinnormalthymi.(A)Classicallineage transitional cellsbecauseofdiminishedsurfaceCD8 molecules onbothwild-typeandCD8 and amaturepopulationthatisCD4 protein internalization. CD8 molecules onthesamecells. Because lo lo cells aretheprogenyofMHC-I– cells andhighonCD8 surface proteinexpressionby lo transgenic thymocytesre- molecules paralleledthatof and CD8 proteins areessentially 8 lo transitional cellsbecause hi surface expres- cells (Fig.2). T cells.Dif- 8 lo hi T cells. transi- o cells thy-

The Journal of Experimental Medicine dogenous CD8 ecules onCD8 lation thatwasCD4 CD8 was onlyslightlyreducedonCD8 of eitherfull-lengthortaillessCD8 regardless ofwhetherthetransgene encoded full-lengthCD8 line) thanCD8 mocytes wascomparedforsurfaceexpressionofendogenousCD8 CD8 major populationsofMHC-I–signaledthymocytesinboth coded CD4andCD8 cells andassessingsurfaceexpressionofendogenouslyen- coreceptor expressiononinvivo–signaledcells(rightpanels).CD4versusCD8 were stainedwithapananti-CD8 expression ofendogenous,butnottransgenic,CD8 expression trathymic MHC-Isignalsmighthavereducedsurface quently, weconsideredtheunlikelypossibilitythatin- (referred toasCD8 mocytes alsoexpressasplicevariantofendogenousCD8 identical tofull-lengthendogenousCD8 In contrast,surfaceexpressionoftransgenicCD8 by thebluerectangulargateandCD8 CD8 that allsurfaceCD8 II complexes containingaCD8 mocyte populationthatwasCD4 ther thefull-lengthCD8 by fourcolorflowcytometry.ShownonwholethymuspopulationsfromeachmouseareCD4versusCD8 intrathymically signaledthymocytesup-regulatesurfaceTCRexpressionandbecome o Although transgenicCD8 CD8 hi TCR transgenic molecules(Fig.3).GatingonTCR and CD8 o mice thatlackendogenousCD8 hi thymocytes. .2 moleculeswasfoundtoparallelthatofCD8 hi (red line)TCR lo cells byselectivelyinternalizingCD8 transgenic mice:atransitionalthy- ) lackingacytosolictail(35).Conse- 8 hi molecules revealedthesametwo . Importantly,surfaceexpression complexes wouldcontainonly or taillessCD8 hi 1712 thymocytes, transgenicCD8 mAb. IncontrasttoendogenousCD8 hi chain. Weintroducedei- molecules areessentially identified bytheredrectangulargate.CD8 8 Basis forReduced CD8ExpressiononMHC-I–signaled Thymocytes lo lo transgenic molecules and amaturepopu- thymocytes relative .1 moleculesdidnotparallelthatofCD8 molecules, thy- transgene into expression so or taillessCD8 , inthatCD8 molecules wereexpressedatsimilar levelsonCD8 mol- .2 andtransgenicCD8 hi molecules thatwereexpressedatsignificantly lowerlevelsonCD8 CD8 expressed asCD8 gene intoII To assessthispossibility,weintroducedtheCD8 CD8 might havefailedtoreducesurfaceexpressionoftransgenic quently, weconsideredthatintrathymicMHC-Isignals molecules againrevealedtwomajorpopulationsofMHC- expression ofendogenouslyencodedCD4andCD8 CD8 engagements mightresultinselectiveinternalizationof modimers (30),itwasconceivablethatintrathymicMHC-I molecules withgreateraviditythandoCD8 CD8 surface CD8proteinexpressionbyselectivelyinternalizing unlikely possibilitythatintrathymicMHC-Isignalsreduce thymocytes couldexpresswouldbeCD8 cules (Fig.4).GatingonTCR composed ofendogenousand/ortransgenicCD8 to CD8 .2 expressionwaslowonCD8 molecules. expression onTCR Because surfaceCD8 staining onCD8 molecules becausetransgenicCD8 molecules wereexpressedasCD8 complexes. complexes butnotCD8 hi hi .1 moleculesontheidenticalcells.Surfaceexpressionofen- thymocytes (Fig.3).Theseresultsexcludethe o cells, wegatedonTCR , inthatCD8 CD8 hi thymocytes isshown,withCD8 o micesothattheonlyCD8complexes lo (blue line)andCD8 homodimers whereasendogenous and TCR .1 expressionwasidenticalonCD8 lo heterodimers bindtoMHC-I cells andhighonCD8 Figure 3. and transgenicCD8 the exceptionthatbothendogenous cytometric analysisasin Fig. 2,with jected tothesamefourcolorflow molecules. Thymocytesweresub- CD8 CD8 ther expressedonlyfull-length from MHC-II CD8 endogenous CD8molecules(both CD8 of full-lengthCD8 MHC-I signalsonsurfaceexpression MHC-II mocytes wereobtainedfrom hi hi lo CD8 (either withorwithoutthe Thymocytes fromMHC-II intrathymic MHC-Icomplexes. mocytes signaledinvivoby genic CD8 sion ofendogenousandtrans- Figure 2. stained forTCR and eitherCD8 CD8 CD8 thymocytes todeterminesurface cells andassessingsurface expression (leftpanels).Because and CD8 and CD8 transgenic molecules.Thy- (middle row)ortailless o (bottom row)transgenic ) expressionandanalyzed ) orCD8 mice thatexpressedonly transgene) weresurface complexes. Conse- hi E o

(red line)TCR ff CD8 hi ect o Differential expres- expression onthy- molecules were TCR heterodimers. ; toprow),or lo .2 (endogenous , CD4,CD8 o cells identified .1 (transgenic f hi intrat hi complexes mice thatei- TCR and tailless thymocytes molecules mole- trans- lo hi h 0 hi lo (blue ymic mice cells. ho- thy- and .2 , The Journal of Experimental Medicine cules wasnotreducedontheverysameCD8 CD4 then phenotypicallybecomeCD4 gular gate).CD8 CD8 modimers, surfaceexpressionofendogenousCD8 complexes onthesethymocytescouldonlybeCD8 ecules wasobviouslyreducedonCD8 (CD8 CD8 dogenously encodedCD8proteins(CD8 thymocytes displayreducedsurfaceexpressionofallen- CD8 that wasCD4 I–signaled thymocytes:atransitionalthymocytepopulation CD4 to phenotypicallyconvertintoCD4 remove bothCD4andCD8proteinsfromthecellsurface In thisproposal,TCR-signaledDPthymocytesinitially and before,changesincoreceptorgeneexpression(9,23). surface proteinexpressionthatoccurindependentlyof, mocytes undergoacomplexsetofchangesincoreceptor nation. IthasbeenproposedthatTCR-signaledDPthy- whether CD8proteinexternalizationmightbetheexpla- sion onMHC-I–signaledthymocytes,wenextconsidered tion asthebasisforreducedCD8surfaceproteinexpres- Thymocytes. Reduced CD8SurfaceProteinExpressiononMHC-I–signaled whereas surfaceexpressionoftransgenicCD8 surface thanendogenousCD8 teins face thanCD8proteins.Thus,transgenic proteins aremorerapidlyreexpressedonthecellsur- duce surfaceexpressionofCD8 the possibilitythatintrathymicMHC-Isignalsfailtore- (Fig. 4).ThedetectionofTCR ther CD8 transgene-encoded CD8 ulations areCD4versusCD8 CD4 positively selectedDPthymocytesinto transitional vert signals reduceCD8surfaceproteinexpressionandcon- cannot bethemechanismbywhichintrathymicMHC-I these resultsdemonstratethatCD8proteininternalization TCR Assessment ofCD8ProteinExternalizationastheBasisfor These resultsdemonstratethatMHC-I–signaledCD4 hi might alsobemorerapidlyreexpressedonthecell thymocytes isshownandidentifies CD8 8 8 ), butnotanytransgene-encodedCD8proteins .2 CD8 proteins buthighsurfacelevelsoftransgenicCD8 , CD8 lo lo lo .2 (endogenousCD8 cells. cells expressedlowsurfacelevelsofendogenous thymocytes inII .2 Having excludedCD8proteininternaliza- .1 surfaceexpressionwasfoundtobe similaronCD8 hi CD8 ). Becauseendogenouslyencodedand (Fig. 4).EventhoughallsurfaceCD8 .2 lo expression, CD4versusCD8 and amaturepopulationthatwas proteins areessentiallyidentical, ) orCD8 o 1713 CD8 proteins, explainingwhy o homodimers.

.1 (transgenicCD8 mice directlyexcludes 8 Bosselut etal. hi lo lo 8 .2 transitional CD4 cells becauseCD4 lo lo thymocytes that cells (mostlyCD4 .2 , CD8 lo thymocytes, .2 expression,andTCR .1 mole- .2 .2 mol- ) expressionandanalyzedbyfourcolor flowcytometry.Shownonwholethymuspop- lo , and pro- cells ho- 8 lo cells, bluerectangulargate)andCD8 .2 lo (blue line)andCD8 be theexplanationforMHC-I–signaledCD4 for differentialreexpressionratesofcoreceptorproteinsto periment wasperformed(Fig.5,AandB).Consequently, the cellsurfaceatidenticalrates,regardlessofhowthisex- pressing onlyoneortheotherCD8 and transgenicCD8 to determinereexpressionratesofendogenousCD8 CD8 ways. Weusedallele-specificanti-CD8 cellular protease,pronase.Wedidthisintwodifferent teins hadbeenstrippedawaybytreatmentwiththeextra- face proteinsonthymocytesaftersurfacecoreceptorpro- reexpression ratesoftransgenicandendogenousCD8sur- proteins (Figs.2–4).Consequently,wecomparedrelative nous andtransgenicCD8 A andB),asoriginallydescribed(23).However,endoge- teins andcouldbedetectedonthecellsurfacefirst(Fig.5, pressed onthecellsurfaceatafasterratethanCD8pro- both experimentalsituations,CD4proteinswerereex- genic CD8 mine reexpressionratesofendogenousCD8 thymocytes asCD4 coreceptor proteinexternalization. expression ofbothendogenousandtransgenicCD8 identical reexpressionrates,MHC-I–signaledCD4 teins astheirreexpressionratesareidentical.But,despite CD8 geneexpressioninpositivelyselectedDPthymocytes Lineage Commitment. down-regulation ofendogenousCD8geneexpression. the hCD2-basedtransgeneandresultinselective dogenous CD8regulatoryelementsthatarenotpresentin thymocytes mustreflecttheimpactofTCRsignalsonen- mocytes asCD4 Thus, thephenotypicappearanceofMHC-I–signaledthy- levels oftransgenicallyencodedCD8 nously encodedCD8 thymocytes onlyexhibitedlowsurfacelevelsofendoge- mocytes, CD4 transgenic CD8 Rather, thedifferenceinexpressionofendogenousand nalization norslowexternalizationofCD8surfaceproteins. We concludethattheappearanceofMHC-I–signaled Initial CessationofCD8GeneExpressionDoesNotImply expression (leftpanels).CD4versus CD8 proteins (Fig.5A),andweusedapan-CD8 .1 proteinsonDPthymocytesexpressingboth .2 8 hi lo 8 molecules onMHC-I–signaledCD4 (red line)TCR lo thymocytes wouldhavetobelowfor cells isnotduetodifferentialratesof 8 .1 proteinsonDPthymocytesex- lo proteins butexhibitedhighsurface If MHC-Isignalsdown-regulate cells isneitherduetorapidinter- .2 hi proteins werereexpressedon cells (mostlyCD4 hi MHC-II mocytes wereobtainedfrom complexes.Thy- homodimeric face expressionofCD8 mic MHC-Isignalsonsur- Figure 4. CD8 bothendogenous expressed for TCR Thymocytes weresurfacestained molecules asCD8 thymocytes (rightpanel). .2 andtransgenicCD8 protein (Fig.5B).In proteins (Figs.2–4). o

, CD4,CD8 .2 expressionongated CD8 mAbs todeter- Effect ofintrathy- cells, redrectan- .2 andtrans- o mice that complexes. 8 , andei- lo mAb pro- thy- 8 8 .2 .1 lo lo The Journal of Experimental Medicine transcribing andthecoreceptor mRNAsthattheycontain coreceptor genesthatindividual thymocytesareactively mented thatthecoreceptor reexpression assayrevealsthe 6; reference 22). Notethatwehavepreviously docu- of thecoreceptormoleculesactively beingtranscribed(Fig. teins andculturedovernight toallowsurfacereexpression move preexistingCD4andCD8surfacecoreceptorpro- in whichtheyweretreatedwithextracellularpronasetore- progeny ofCD4 consisting ofsignaledTCR thereby causingthemtobecometransitionalCD4 consisting ofsignaled(i.e.,TCR contain twodistinctsubpopulations:onesubpopulation CD4 ceptor reversalandbecometranscriptionallyCD4 MHC-I–signaled CD4 MHC-I–signaled CD4 MHC-I–signaled experimentally identifysuchsubpopulationsamong To transcriptionally CD4 before theirterminaldifferentiationintoCD8 8 lo thymocytes tothecoreceptorreexpressionassay 8 cells thathavejustundergonecore- 8 8 8 lo lo , andtheothersubpopulation thymocytes, wesubjectedsorted thymocyte populationsshould 1714 hi cells thataretheimmediate hi ) thymocytesthatare Basis forReduced CD8ExpressiononMHC-I–signaled Thymocytes T cells, 8 lo cells 8 . CD4 reexpressionwereidenticalonthymocytesfromMHC-II mum reexpressionat24h,whichwassetequalto100%.Thekineticsof tive reexpressionofCD4andCD8surfaceproteinsrelativetotheirmaxi- recognizes bothCD8 CD4 andCD8 gle cellsuspensionsat37 coreceptor proteinsbytreatmentwithextracellularpronase,culturedinsin- only transgenicCD8 proteins. (A)ThymocytesfromMHC-II Figure 5. CD8 cules wereidentical.(B)ThymocytesobtainedfromMHC-II ics ofreexpressionendogenousCD8 CD4 reexpressionoccurredmorequicklythanCD8reexpression,thekinet- sus CD8 CD8 mAbs.DisplayedarehistogramsofCD4versus CD8 suspensions at37 tracellular pronase.Thestrippedthymocyteswereculturedinsinglecell ecules werestrippedofallsurfacecoreceptorproteinsbytreatmentwithex- transgenic MHC-II nous CD8 Quantitation ofsurfacereexpressionversustimeCD4(blueline),endoge- expressing onlyendogenousCD8 gene andexpressedbothendogenousCD8 found thatCD4 (22, 24).Applyingthecoreceptorreexpressionassay,we ceptor geneexpressionstillretained thepotentialtodiffer- firms thatMHC-I–signaledTCR been signaledinvivo(Fig.6).Thus,thisexperimentcon- TCR signaled TCR pressed bothCD4andCD8(Fig.6,populationb),un- thymocytes fromMHC-II typically appearasCD4 were bothpresentamongelectronicallysortedCD4 coreceptor proteinsandtherefore wereCD4 were unsignaled(i.e.,TCR TCR subpopulations wereTCR CD4 norCD8(Fig.6).Importantly,bothoftheselatter the earlystagesofapoptosisandsoreexpressedneither c). AlsounavoidablyincludedintheCD4 reexpression assay,areCD4 Consequently, weaskedif invivoMHC-I–signaled .2, andCD8 o hi hi mice thatcontainedtheCD8 subpopulations that,asrevealed bythecoreceptor .1, reexpressionatdifferenttimesafterpronasestripping.Although CD4 .2 (redline),andtransgenicCD8 Reexpression ratesofendogenousandtransgeniccoreceptor by stainingwithanti-CD4andapananti-CD8mAbthat C forvaryingtimes,andassessedreexpressionofCD4, 8 lo lo o

.1 bystainingwithanti-CD4andallele-specificanti- DP thymocytesthatwerepresumablyin CD8 thymocytes thatreexpressed onlyCD4 .1 andCD8 .1 molecules.Thymocyteswerestrippedofsurface 8 C forvaryingtimes,andassessedreexpressionof o and CD4 mice, butaredisplayedonlyfortheformer. o 8 lo lo mice (Fig.6,populationsaand .2 isoforms.Linegraphsshowquantita- , indicatingthattheyhadnot lo 8 .2 molecules,andfromMHC-II cells actuallyconsistoftwo ) DPthymocytesthatreex- o and CD4 transgene andthereforeexpressed mice thatcarriedtheCD8 .2 andtransgenicCD8 8 hi .2 andtransgenicCD8 thymocytes thatpheno- .1 (greenline)aredisplayed. TCR hi 8 subpopulations 8 . lo .2 andCD4ver- 8 sorting gate o for core- and CD8 .1 mole- .1 mol- o trans- mice 8 lo o The Journal of Experimental Medicine CD4 pression assay(Fig.7,d),andpurifiedcellsthatwere thymocytes consistprimarilyoftwoTCR CD4 become CD4 they havetransientlyterminatedCD8geneexpressionto h). Thus,mostinvivoMHC-I–signaledCD4 expression (asrevealedbyCD8reexpression;Fig.7,fand and absenthCD2reexpression)reinitiatingCD8gene nating CD4genetranscription(asindicatedbyabsent tured overnightin37 which surfaceexpressionofthehCD2reporterproteinreflectsCD4promoteractivity(reference31).Thesortedthymocyteswere that wastranscriptionallyCD4 cell sorting(Fig.7,aandb),identifiedthesubpopulation to maintaincellviability(24,26).UnlikesortedCD4 tures withIL-7,acytokinepresentinthenormalthymus, The purifiedcellswerethenplacedintoshort-termcul- CD4 coreceptor reexpressionassayparallelsCD4andCD8mRNAexpressionasassessedbyRT-PCR(references2426). therefore consideredtohavenotbeensignaledinvivo.NotethatpreviousanalysesdocumentedCD4andCD8proteinre differentiate intoCD4 the potentialtoreversecoreceptorgeneexpressionand CD8 geneexpression.However, thisstudydocumentsthat CD8 surfaceproteins,andnot duetodown-regulationof intermediates wouldbedue to removalorredistributionof Discussion CD4 cells thatremainedDPinculture(Fig.7,gandi),most protein. Weobtainedinvivo–signaledCD4 CD4 transcriptionbysurfaceexpressionofhCD2reporter lencer elementsandthereforefaithfullyreportsinvivo hCD2 cDNAunderthecontrolofCD4enhancerandsi- ferred toasLine30;reference 31) thatiscomposed of entiate intoCD4 pearance ofMHC-I–signaled DPthymocytesasCD4 tor gene(9,10).Fromthisperspective theparadoxicalap- and resultsinpermanentsilencing oftheoppositecorecep- concept thatlineagecommitmentoccursinDPthymocytes into CD4 mocytes underwentcoreceptorreversalinvitro,converting mocytes fromsuchMHC-II used MHC-II mocytes phenotypicallyconvertintoCD4 and h). not changecoreceptorgeneexpressioninculture(Fig.7,f Most modelsoflineagecommitmentincorporatethe We concludethatintrathymicMHC-I–signaledDPthy- 8 8 8 8 subpopulation (c).SortedCD4 hCD2 cells remainlineageuncommittedastheyretain hCD2 8 cells. AminorityofCD4 o 8 mice containingareportertransgene(re- cells underwentadramaticchange,termi- by furtherelectronicsorting(Fig.7,c). C suspensioncultures,andassessedbymulticolorflowcytometryforexpressionofCD4,CD8 cells. Wealsoconcludethatmanysuch 8 cells. Toaddressthisquestion,we 8 cells. 1715 o Line 30micebyelectronic 8 8 lo hi in thecoreceptorreex- thymocytes thateitherreexpressedbothCD4andCD8(b),orneithernorCD8,wereTCR subpopulations asdeterminedbythecoreceptorreexpressionassay:aCD4 Bosselut etal. 8 hCD2 8 lo cells because cells did 8 8 lo thy- thy- 8 8 lo retain thepotentialtodifferentiateintoCD8 into CD4 basis forconversionofMHC-I–signaledDPthymocytes removal orredistributionofCD8surfaceproteinsisnotthe of MHC-I–signaledCD4 tions havebeenproposedthatcanreconciletheappearance basis forthisobservationhasbeenuncertain.Twoexplana- CD8SP Tcells(18,21,22).Nevertheless,themolecular cells, butarecellsontheirwaytodifferentiatinginto cally convertintoCD4 imply commitmenttotheCD4 Thus, initialcessationofCD8geneexpressiondoesnot MHC-I–signaled CD4 been confirmedbyothers(18,19,23,36).Importantly, dently bytwodifferentlaboratories(21,22)thathassince CD8SP Tcellswasasurprisingobservationmadeindepen- siently appearasCD4 with theeffectthatMHC-I–signaledDPthymocytestran- the cellsurfacethatarethenreexpressedatdifferentrates, to removebothCD4andCD8coreceptorproteinsfrom MHC-I–specific TCRinteractionssignalDPthymocytes second explanationismorecomplexandpositsthat thymocytes toterminateCD4geneexpression(22).The CD8coreceptor proteinsevenastheyinduceDP surface TCR interactionssignalDPthymocytestointernalize gene. ThefirstexplanationpositsthatMHC-I–specific sults inpermanentsilencingoftheoppositecoreceptor that lineagecommitmentoccursinDPthymocytesandre- CD8 in MHC-I–signaledthymocytesduringdifferentiationinto lated CD8geneexpressiontobecomeCD4 that MHC-I–signaledthymocyteshavedown-regu- tion ofCD8geneexpression.Thisstudyalsodemonstrates tions havenowbeenexcludedbythisstudy. tively expressbothcoreceptorgenes(9,23).Bothexplana- trolled byendogenousCD8 promoter/enhancerelements elements, asexpressionofendogenous CD8 coding themarecontrolledby entirelydifferentregulatory CD8 dogenous CD8 is expressedonthymocytes at similarlevelstothatofen- That manyMHC-I–signaledDPthymocytesphenotypi- This studyusedanhCD2-basedCD8 T cells. proteins areessentiallyidentical, butthegenesen- 8 lo cells, butratherthatitisduetodown-regula- .2 proteins.Endogenousand transgenic 8 lo 8 8 cells thoughtheycontinuetoac- lo lo 8 cells duringdifferentiationinto thymocytes arenotdead-end lo thymocytes withtheconcept , andTCR cells enrichedinCD4 electronically sortedtoobtain MHC-II obtainedfrom Thymocytes coreceptor reexpressionassay. sitional thymocytesbythe signaled (TCR Figure 6. contain areportertransgenein tional thymocytes.Line30mice T celllineage,asitoccurs 8 o subpopulation (a)anda Line 30micewere pronasestripped,cul- . CD4 .1 transgenethat Analysis ofMHC-I– hi expression inthe ) CD4 genes iscon- 8 8 lo transitional cells still T cells. 8 8 lo lo transi- lo tran- and The Journal of Experimental Medicine plexes, suchasthosecontainingonlytaillessCD8 ized onlyspecificsubsetsofCD8surfaceproteincom- and excludedthepossibilitythatMHC-Isignalsinternal- (37–42) whereasexpressionoftheCD8 CD4 Rather, theappearanceofMHC-I–signaled thymocytesas thymocytes convertinvivo intoCD4 events arenotthemechanism bywhichMHC-I–signaled coreceptor proteins,thisstudy demonstratesthatsuch signals stimulatetheremovaland/orreexpressionofsurface proteins (23).Consequently,regardlessofwhetherMHC-I on thecellsurfaceatafasterratethanendogenousCD8 CD8 evaluated andexcludedthepossibilitythattransgenic levels oftransgenicallyencodedCD8 enously encodedCD8 of endogenousCD8geneexpression. naled thymocytesappearedinvivoasCD4 complexes wasnotthemechanismbywhichMHC-I–sig- And todocumentthatslowexternalizationofCD8protein teins, thoseconsistingonlyofCD8 CD4 ments (29,34).ThisstudyfoundthatMHC-I–signaled trolled byheterologoushCD2promoter/enhancerele- mocytes appearedinvivoasCD4 was notthemechanismbywhichMHC-I–signaledthy- to documentthatinternalizationofCD8surfacecomplexes signals onCD8 vation thatisnotconsistentwithadirecteffectofMHC-I complexes, oronlyofCD8 8 8 proteins, likeCD4mightbereexpressed lo lo thymocytes expressedlowsurfacelevelsofendog- cells mustresultfromtransient down-regulation surface proteinexpression.Nevertheless, proteins butexpressedhighsurface 1716 homodimeric complexes. 8 Basis forReduced CD8ExpressiononMHC-I–signaled Thymocytes lo cells, weevaluated proteins, anobser- transgene iscon- 8 lo heterodimeric 8 thymocytes. lo cells, we pro- subpopulations ofTCR expression, suchCD4 study demonstratesthatdespitehavingceasedCD8gene of down-regulatedCD8geneexpression.Indeed,this also presentwithinthetransitionalCD4 the processofdifferentiatingintomatureCD8 that weretranscriptionallyCD4 population becausetheyaretheimmediateprogenyofcells CD4 mocytes phenotypicallyappearedasCD4 cordant withourconclusionthatMHC-I–signaledthy- MHC-I–signaled CD4 tor reversal intoCD4 sult, TCR kine thatispresentwithinthenormalthymus(43).Asare- of theoppositecoreceptorgene. Cessationofcoreceptor curs inDPthymocytesand results inpermanentsilencing reconciled withtheconcept thatlineagecommitmentoc- cells (25). CD4 deed down-regulatedCD8geneexpressiontobecome coreceptor geneexpression:onesubpopulationthathadin- dergo coreceptorreversaland differentiateintoCD8 imply CD4lineagecommitment,assuchcellsmayun- sion invivo–signaledthymocytesdoesnotnecessarily coreceptor reversal.Thus,cessationofCD8geneexpres- coreceptor geneexpressionpatternofCD4 Supporting thisconclusion,wefoundthatinvivo It seemstousthatthepresent resultscannotbeeasily gene expressionwasCD4 8 8 lo , andtheothersubpopulationwhosecorecep- population containedTCR hi hCD2 termine ifcoreceptorgeneexpressionwasfixed,purified fore werehCD2 mocytes thathadceasedCD8geneexpressionandthere- entiate into CD8 entiate into have ceasedCD8geneexpressionretaintheabilitytodiffer- gene expressionincultureandremainedhCD2 CD4 andCD8coreceptorgenesdidnotalter had notbeensignaledinvivoandthereforeexpressedboth enriched inCD4 Line 30mice(a)wereelectronicallysortedtoobtaincells Figure 7. (g andi). CD4, CD8 sessed bymulticolorflowcytometryforreexpressionof were thenpronasestripped,culturedovernight,andas- into hCD2 culture (c),underwentcoreceptorreversalandconverted moter activity(handi).MostCD4 the hCD2proteinthatservesasareporterofCD4pro- and g).Culturedcellswerealsoassessedforreexpressionof tured inIL-7andassessedforcoreceptorreexpression(f cells thataretranscriptionallyCD4 CD4 8 , andthehCD2reporterprotein(d).Tode- CD4 cells inthepresenceofIL-7,acyto- MHC-I–signaled CD4 8 8 8 hi cells (c)andCD4 lo cells withdifferentpatternsof cells couldundergocoreceptor CD8 8 T cells.ThymocytesfromMHC-II thymocytes consistedoftwo lo CD4 transitional thymocytes(b),which cells (fandh).Thymocytesthat 8 8 . Thatthetransitional 8 at thebeginningofIL-7 hi before undergoing thymocytes witha 8 8 8 8 lo cells (e)werecul- lo lo 8 thymocytes that transitional thy- lo cells because 8 thymocyte T cellsare 8 was con- lo CD4 and in 8 T o The Journal of Experimental Medicine diate CD4 sistent withthekineticsignalingperspectivethatinterme- coreceptor lineage.Incontrast,thepresentresultsarecon- reflect anindividualcell’s“commitment”totheopposite cessation ofcoreceptorgeneexpressionisstillconsideredto the oppositecoreceptorgene(11,14,15,44).Importantly, that lineagechoiceisrevealedbyitssubsequentsilencingof mocytes commitsthecelltoonecoreceptorlineageand that thestrengthordurationofTCRsignalinDPthy- gene expressionwithlineagecommitment,butpostulate struction modelnolongerequatecessationofcoreceptor occurred randomly.Updatedversionsoftheoriginalin- lated thattheselectionofwhichcoreceptorgenetosilence ceptor genewassilenced,whereasstochasticmodelspostu- TCR pluscoreceptorengagementsdictatedwhichcore- original instructionalmodelpostulatedthatMHC-specific to theoppositecoreceptorlineage(6,16).Forexample, gene expressionwasoriginallyequatedwithcommitment sion ofallendogenouslyencodedCD8genes(CD8 TCR signalscauseDPthymocytestodiscontinueexpres- scription. OurpresentobservationthatMHC-I–specific nals mayaffectCD8mRNAstabilityaswelltran- proteins, butitispossiblethatMHC-I–specificTCRsig- CD8 geneexpressionratherthantoeffectsonsurface signaled CD4 remains tobedetermined. gene expressionbeforeundergoinglineagecommitment TCR infactinducetransientdown-regulationofCD8 not signalstransducedbylowaffinityMHC-I–specific CD8 proteinlevelscandetectablydecline(25).Whetheror reversal andreinitiateCD8geneexpressionbeforesurface of lowligandaffinitythatthesecellsundergocoreceptor but theirTCRsignalsareofsuchshortdurationasaresult gene expressionasproposedbythekineticsignalingmodel, MHC-I–signaled cellsdotransientlydown-regulateCD8 pearing asCD4 differentiate directlyintoCD8SPTcellswithouteverap- parent affinityforintrathymicMHC-Iligandsappearto example, DPthymocytesthatexpressTCRwithlowap- through astagewheretheyappearasCD4 25). However,notallMHC-I–signaledthymocytesgo ceptor reversalanddifferentiationintoCD8SPTcells(24, MHC-I–specific signalingwhich,inturn,promotescore- thymocytes andresultsincessationofCD8-dependent sion occursduringdifferentiationofMHC-I–signaledDP signaling model,transientcessationofCD8geneexpres- in whichlineagecommitmentthenoccurs.Inthekinetic are likelyoccurring invivo–signaledDP thymocytes. coreceptor mRNAs(46,47). Wethinkthatsimilarevents transcription andmodestlydestabilizes bothCD4andCD8 thymocytes selectivelyterminates endogenousCD8gene that antibody-mediatedTCR engagementofpurifiedDP tions fromthislaboratoryhave previouslydemonstrated from thehCD2-basedtransgene.Indeed,invitroobserva- within theendogenousCD8genelocusthatareabsent dicates thatTCRsignalstargetregulatoryelementspresent CD8 This studydemonstratesthattheappearanceofMHC-I– ) withoutaffectingexpressionofCD8transgenesin- 8 8 thymocytes arelineage-uncommittedcells lo 8 thymocytes isduetodown-regulationof lo cells (36,45).Itispossiblethatsuch 1717 Bosselut etal. 8 lo cells. For and “on” andCD8geneexpression“off”(i.e.,CD4 TCR signalingwouldmaintainCD4geneexpression and CD4silencerelements)withtheresultthatpersistent lineage-specific regulatoryelements(i.e.,CD8enhancer in developingthymocytesmightblockactivationofall It isinterestingtofurtherspeculate(26)thatTCRsignals minating activationofimmatureCD8enhancerelements. basal transcriptionalstateinDPthymocytesbysimplyter- stage ofdifferentiation.Thus,TCRsignalscaninducethe tion ofimmatureDPthymocytesintomatureCD8 the CD8genelocusisknowntoshiftduringdifferentia- elements areactivated.However,enhancerusagewithin which neitherCD8enhancerelementsnorCD4silencer be CD4 pression indevelopingthymocytesmightbeconsideredto sult, the“basal”or“default”stateofcoreceptorgeneex- tivation ofaCD4silencerelement(31,48,49).Asre- CD4 geneexpressionisspecificallydown-regulatedbyac- of stage-specificCD8enhancerelements(37–42)and gene expressionisspecificallyup-regulatedbyactivation lated byfundamentallyoppositemechanisms,asCD8 of CD8andCD4coreceptorgenesappearstoberegu- tion indevelopingthymocytes,lineage-specificexpression permanent silencingoftheoppositecoreceptorgene. eage commitmentoccursinDPthymocytesandresults in kinetic signalingmodelandchallengetheconceptthatlin- References Accepted: 20April2003 Revised: 20April2003 Submitted: 31January2003 ings ofthemanuscript. We thankDrs.JonAshwellandB.J.Fowlkesfortheircriticalread- ate intoCD8 cells areabletoundergocoreceptorreversalanddifferenti- T celllineage,asmanyinvivoMHC-I–signaledCD4 gene expressiondoesnotimplycommitmenttotheCD4 deed, thisstudydemonstratesthatinitialcessationofCD8 sults fromtransientcessationofCD8geneexpression.In- ance ofMHC-I–signaledthymocytesasCD4 proposed bythekineticsignalingmodel. sulting inthymocytedifferentiationintoCD4 drive CD8geneexpressionatthemature differentiation and“mature”CD8enhancerelements gene expressionattheimmatureDPthymocytestageof cells, as“immature”CD8enhancerelementsdrive thymocytes intoCD4 of CD8geneexpressionandconversionsignaledDP thymocytes mightselectivelyresultintransientcessation lated providessomecluesastohowTCRsignalinginDP of howCD8andCD4genesaretranscriptionallyregu- transcription isstillfarfromcomplete,currentknowledge .Guidos,C.J.,I.L.Weissman,andB.Adkins.1989.Intrathy- 1. 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