Endosomal trafficking of open Major Histocompatibility Class I conformers - Implications for presentation of endocytosed antigens

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Endosomal trafﬁcking of open Major Histocompatibility Class I

conformers—Implications for presentation of endocytosed antigens ^夽

Hana Mahmutefendi ´c

^a

, Gordana Blagojevi ´c Zagorac

^a

, Maja Ili ´c Tomaˇs

^a

, Marcus Groettrup

^b

, Frank Momburg

^c

, Pero Luˇcin

^a,∗

aUniversityofRijeka,FacultyofMedicine,DepartmentofPhysiologyandImmunology,B.Branchetta20,51000Rijeka,Croatia

bUniversityofKonstanz,DepartmentofBiology/Immunology,PostboxM661,Universitaetsstr.10,D-78457Konstanz,Germany

cDivisionofTranslationalImmunology,GermanCancerResearchCenterandNCT,ImNeuenheimerFeld280,69120Heidelberg,Germany

a r t i c l e i n f o

Keywords:

Endocytosis

MajorHistocompatibilityClassIproteins OpenMHC-Iconformers

Antigenpresentation Cross-presentation Lateendosomes

a b s t r a c t

MajorHistocompatibilityClassI(MHC-I)moleculesarepresentatthecellsurfaceeitherasfullycon- formedtrimolecularcomplexescomposedofheavychain,beta-2-microglobulin(␤2m)andantigenic peptideorasvariousopenforms,devoidofthepeptideand/or␤2m.Whiletheroleoffullyconformed MHC-Iiswellstudied,thephysiologicalroleofopenconformersisneglected.Wehaveshownthatfully conformedMHC-IandopenMHC-IconformerssegregateatthePMandduringendosomaltrafﬁcking resultingintheexclusionofopenMHC-Ifromtheearlyendosomal/juxtanuclearrecyclingroute.Asa result,openMHC-Iconformersareinternalizedwithahigherratethanfullyconformedcounterparts.

AlthoughthemajorityofinternalizedopenMHC-Iisdirectedintotheacidiclateendosomal(LE)compartments,onlyafractionofthemisdegraded.Namely,asigniﬁcantfractionofopenMHC-Iispresent inasubsetofLEswiththecapacityofrecyclingand/orexocytosis.Therefore,itshouldbeexamined whetherexogenouspeptideloadingmayoccurduringtravelingofMHC-IproteinsthroughLEcompart- ments,especiallyinasubsetoflessacidicLEsthatdetachfromthecoreofperinuclearacidicLEsand migratetowardthecellperiphery.GiventhattheacidicLEenvironmentisnotfavorableforpeptide loading,anendosomalcompartmentwiththerecyclingcapacityandlessacidicenvironmentthatallows stabilizationofnewlyformedtrimolecularcomplexesispropersiteforexogenouspeptideloading.We proposethataLEcompartmentwhichcollectandretainopenMHC-Iconformersshouldbetakeninto considerationasasiteofexogenouspeptideloading.

1. Introduction

MajorHistocompatibilityClassI(MHC-I)moleculesarepresent atthecellsurfaceandintheintracellularmembraneouscompart- mentsas fully conformedtrimolecular complexes composed of heavychain,peptideand ␤-2microglobulin (fullMHC-I)andas openMHC-Iconformers(heavychainwithoutorwithveryweakly bound␤-2microglobulin).AmajorityofopenMHC-Iconformers localizeinsidethecellinmembraneouscompartmentthatbelong totheendosomalsystem.AftermorethantwodecadesofMHC-I

Abbreviations: EE,earlyendosome;eL^d,emptyL^d;fL^d,fullL^d;IR,internalizationrate;MHC-I,MajorHistocompatibilityClassI;PM,plasmamembrane;LE,late endosome;TEE,tubularearlyendosomes;TfR,transferrinreceptor;VEE,vacuolar earlyendosomes.

夽ThisarticlebelongstoSpecialIssueonAntigenProcessingandPresentation.

∗Correspondingauthorat:DepartmentofPhysiologyandImmunology,Univer- sityofRijeka,FacultyofMedicine,Bra ´ceBranchetta20,51000Rijeka,Croatia.

Tel.:+38551406500;fax:+38551675699.

E-mailaddresses:pero.lucin@uniri.hr,pero@uniri.hr(P.Luˇcin).

research,physiologicalimplicationsofopenMHC-Iconformersare stillpoorlyunderstoodandtheirexistenceatthecellsurfaceand intheendosomalsystemisnotproperlyvalidated.Still,itisnot knownhowandwherecellsurfaceandendosomalopenMHC-I conformers are generated,especially in thelight of wellestab- lishedendoplasmicreticulum(ER)qualitycontrolmachinerythat recognizeemptyMHC-Iasunfoldedandeitherretrievesthemin theERuntil theybecomepeptideloadedorredirect theminto thecytosolforproteasomaldegradation(reviewedbyDonaldson andWilliams,2009).Itis,thus,anopenquestionwhethersimilar qualitycontrolmachineryoperatesatthecellsurfaceandinthe endosomalsystem.Furthermore,itisnotclearhowtheybehave intheendosomalsystem, whataresortingprinciplesthatdrive theirendosomal trafﬁcking and what are implications for pro- cessofalternativeantigenpresentation.Therefore,physiological importanceofopenMHC-Iconformersshouldbeclariﬁednotonly for peptideloadingin theendosomal systemand regulationof theimmuneresponsebutalsoforunderstandingofproteinsor- tingintheendosomalhemisphere ofintracellularmembranous organelles.Here, wewilldescribeourcurrentunderstandingof

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https://dx.doi.org/10.1016/j.molimm.2012.10.008

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endosomaltrafﬁckingofopenMHC-Iconformersanddiscusstheir potentialroleinpresentationofendocytosedantigen.

2. Currentstatus

OpenMHC-IconformersofmurineandhumanclassicalMHC-I allelesarepresentatthecellsurfaceofvariouscelllines(Zagorac etal.,2012).Theirexpressionis2–5timeslowerthanexpressionof fullyconformedMHC-Icounterpartsandincreaseswiththeover- allincreaseofMHC-Iexpression,i.e.afterIFN-gammatreatmentor afteraberrantexpressionbytransfection.Insomecells,theamount ofopenMHC-I conformersatthecell surfacemayoverridethe amountoffullyconformedMHC-IafterIFN-gammatreatment.

Surface MHC-I proteins are internalized by the constitutive endocyticuptakeoftheplasmamembrane(PM)(Donaldsonand Williams,2009)whichisvariableindifferentcells,rangingfrom 50% to 180% membrane equivalent every hour (Huotari and Helenius,2011).Internalizationrate(IR)ofmembrane proteins, i.e.theirlossfromthecellsurface,isresultantoftheirendocytic rate(rateoftranslocationofaPMproteinintothecellinteriorby endocyticuptake)andtherecyclingrate(rateofreturnofendocy- tosedPMproteinfromcellinteriorbacktothePM).Thus,proteins that donot recyclewillhave thesame endocyticand internalizationrate.FullyconformedMHC-Iproteinsinternalizewiththe rate0.002–0.004min⁻¹(0.2–0.4%lossofinitiallysurfaceexpressed moleculesperminute,i.e.12–24%perhour)whichis5–8-foldlower thanIRoftheiropenforms(0.011–0.022min⁻¹),resultingintheir 5–8timeslongerhalf-lifeatthecellsurface(Zagoracetal.,2012).

ThisdifferenceintheIRssuggestssegregationwithinmembranes, distinctendocyticrouteanddistinctrecyclingefﬁciencyofMHC-I conformers.

FullyconformedMHC-IproteinsandopenMHC-Iconformers segregatealreadyatthePMbypartitioningintodistinctmembrane environment(Fig.1A):fullyconformedMHC-Imoleculeslocalizein thelipid-disorderedmembranecompositionwhereasopenMHC-I conformerspartitionintothelipid-organizedmembraneenviron- ment(Mahmutefendi ´cetal.,2011;Zagoracetal.,2012).Thus,itcan beexpectedthattheirconstitutiveendocyticuptakeengagesdiffer- entendocyticmachinery,althoughbothfullyconformedMHC-Iand openMHC-Iareconstitutivelyendocytosedbyamechanismthatdo notrequireclathrinanddynamin(DonaldsonandWilliams,2009).

Thisissupportedbytheobservationthattheyarenotcapturedinto thesameendocyticinvaginationsandprimaryendocyticcarriers (Mahmutefendi ´c etal.,2011).Uponendocytosis,primaryendo- cyticcarriersloadedwithfullyconformedMHC-IandopenMHC-I conformersconvergeintosameearlyendosomesbutmaintainthe preferencefordistinctmembranecompositionandpartitioninto distinctmembranedomains(Mahmutefendi ´cetal.,2011;Zagorac etal.,2012).

Inearlyendosomes(EEs),complexorganelleswithtubularand vacuolardomains(HuotariandHelenius,2011),MHC-Iconformers continue tosegregate.Approximately one-thirdof endocytosed fullyconformedMHC-IproteinssegregateintotubularEEs(TEEs), accessingearly endosomalrecycling routetogetherwith transferrinreceptor(TfR),whereasallopenMHC-Iconformersremain invacuolardomainofEEs(VEEs)togetherwiththerestoffully conformedMHC-I(Fig.1A).WithprogressionofVEEstowardthe cellcenter,roughlyahalfoffullyconformedMHC-Iproteinsthat remainedinVEEscontinuetosegregateintotubularextensions, which localize in the juxtanuclear area and collect almost all endocytosed TfR but not openMHC-I conformers. These endo- somesare knownasjuxtanuclear recyclingcompartment (JRC).

TherestoffullyconformedMHC-Iproteinsremainstogetherwith openMHC-IconformersinEEA1-positiveVEEs(Fig.1A),localize intheperinucleararea,growinsizewithtimeand matureinto lateendosomes(Mahmutefendi ´cetal.,2011).Thus,asubstantial

fractionof endocytosedfullyconformedMHC-I proteinsrecycle viaTEEsandtubularJRC,whereasnoopenMHC-Iconformerswere detectedtorecyclevia this route(Mahmutefendi ´c etal., 2011;

Zagorac et al., 2012). Although fully conformed MHC-I recycle withthesimilarrateasTfR,this ratecannotexplain theirIRin relationwiththemorerapid constitutivemembrane uptake. In addition, constitutive membrane uptake is also higher then IR of openMHC-I conformers. Thus, rapid recyclingroute of both fully conformed MHC-I and open MHC-I should be considered and examined. Both fully conformed MHC-I and open MHC-I internalizewithmuchhigherratewhenrecyclingisinhibitedby aluminumﬂuoride(Zagoracetal.,2012),whichinhibitsrecycling intheentireendosomalsystem,includingrapidrecycling.

Aspreviously explained, endocytosed openMHC-I conformers are sorted in the perinuclearregion intovacuolar EEs that enlarge, mature into acidic LEs (Fig. 1A) and acquire major LE markersLamp1 andLamp2.Although it isbelieved that MHC-I proteinswhichreachLEcompartmentsarepredestinedfordegra- dation,ourrecent studies infibroblastic cell linesshowed that endocytosedopenMHC-Iconformersaresegregatedandretained in LE compartment outside the bulk of acidic perinuclear LEs (Fig.1A).Giventhattheseendosomesmaintaintheirmultivesicular and vacuolarnature,we propose thatthis compartment repre- sentsaLEsubsetwithrecyclingcapacity(HuotariandHelenius, 2011).Asaresult,theinternalLEsvesicleswillbeshedoutinto the extracellular environment as exosomes, while the limiting membraneofLEwillbecomeintegralpartofthePMwhichwill resultsinrecyclingofassociatedcargoproteins(Fig.1A).Therefore, thedestinyofmoleculesthat havereachedLEscouldbemulti- ple:retention,degradation,exocytosis,orrecycling.Inlinewith this,weobservedthat acidicandLamp1^high perinuclearLEsare accessedbyinternalizedEGF-EGFR,cargo destinedfor degradation,andthat50–60%ofinternalizedopenL^disdegraded.Therest ofinternalizedopenL^disredirectedintolessacidicandLamp1^low vacuolarendosomesoutsidetheperinuclearareaatthecellperiph- ery andvery oftenin extensionsof fibroblasts(Mahmutefendi ć etal.,manuscriptinpreparation).These,peripheralLamp1^lowLEs are accessed by fluid phase cargo (dextran), internalized open MHC-Iconformers,andarefreeofdegradationcargo(EGF-EGFR).

Accordingly, we have found that 10–15% of internalized open L^disrecycledviaLEroute(Mahmutefendi ´cetal.,manuscriptin preparation).

Acidic endosomal compartments play an important role in alternative antigenpresentation. It hasbeenshown that endosomal acidiﬁcation is necessaryfor processing of longantigens (membraneassociatedornot)asasourceforalternativeantigen presentation.Thisendosomalprocessingpathwayfunctionsnot onlyinTAP-deﬁcientbutalsoinwildtypenonprofessionalAPCs (Tiwarietal.,2007).

Forunderstandingofmechanismsleadingtocross-presentation andalternativepresentationpathwaysinvolving“recycling”endo- somesitisessentialtofullyunderstandtheendocytictrafﬁckingof MHC-Iproteins,theirbehaviorintheacidicenvironmentofendo- somesandconsidertheroleofopenMHC-Iconformers.Inorder totestforsensitivityofMHC-Iproteinsinendosomalenvironment weexaminedtheratiooffullandopenMHC-Imolecules(murine H2-L^dallele)atthecellsurfaceexposedtovariousacidiccondi- tionsfor30min(Fig.1BandC).AsshowninFig.1B,verysmall amountoffL^disconvertedintoeL^dwhenthepHismaintained above6.0(pH ofEEsandrecyclingendosomes),whereasreduc- tionofpHbelow6.0(whichoccursinLEs)facilitatedconversionof fL^dintoeL^d.ProlongedexposuretopH6.0alsoconvertedfL^dinto eL^d(Fig.1C).SimilarwasobservedalsoforhumanMHC-Ialleles (datanotshown).Therefore,iffullyconformedMHC-Iproteinsare exposedtopH6.0for30min,verylittleofthemwilllosethepeptide andbecameempty,readytoacquirefreepeptide.Fullyconformed

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Fig.1.EndocyticroutesandsegregationofMHC-Iproteinsintheendosomalsystem.(A)SchematicrepresentationofendosomaltraffickingoffullyconformedMHC-I proteins(loadedpeptideisrepresentedbybluecircles)andopenMHC-Iconformers(withoutpeptide).(B)CellsurfaceexpressionoffullyconformedH2-L^d(fL^d)andpeptide- emptyH2-L^dconformers(eL^d)after30minexposureofP815cellstovariouspHenvironments.Datarepresentmeanfluorescenceintensity(determinedbyflowcytometry) afterstainingwithprimaryantibodies(30-5-7and64-3-7,respectively)andFITC-conjugatedsecondaryantibodysubtractedforfluorescenceintensityofcellsexposedto secondaryreagentonly(MFI).(C)CellsurfaceexpressionoffL^dandeL^dafter2hexposureofP815celltovariouspHenvironments.EE,earlyendosomes;TEE,tubular earlyendosomes;VEE,vacuolarearlyendosomes;RE,recyclingendosomes;JRC,juxtanuclearrecyclingcompartment;LE,lateendosomes;LY,lysosomes;DRM,detergent resistancemembranes.

MHC-Iproteins,remaininEEsfor15–20minand,therefore,one cannotexpecttheirconversionintoemptyformandloadingwith afreepeptide.Similarly,itisunlikelythatconversionandpeptide loadingcanoccurinrecyclingendosomeswherepHisevenhigher.

Thus,thepreferredoptionswouldbepeptideexchange,eitherwith peptidethatisofahigherafﬁnityandendocytosedinahighcon- centrationintoEEorsimultaneoustrafﬁckingofpeptideandMHC-I intoLEfromwhereMHC-Imoleculeswithexchangedpeptidewill recycletothecellsurface.

3. Futureperspectives

UnderstandingofendosomalsortingoffullyconformedMHC-I proteinsandopenMHC-Iconformershasseveralimportantimpli- cationsinunderstandingproteinsortingintheendosomalsystem, organization of the quality control in the endosomal system, function and organization of late endosomal compartments,

biogenesis and physiology of MHC-I proteins and, conse- quently, the understanding of peptide loading in the vacuolar pathway.

Properunderstandingofthepeptideloadingintheendosomal systemwillrequireidentiﬁcationoftheendosomalcompartment wherepeptideloadingandexchangecanoccur.Suchacompart- ment(i)shouldbeabletoretainMHC-Iproteins,especiallyopen MHC-Iconformers,(ii)shouldbeabletoacquirepeptideseither endocytosedbyﬂuidphaseuptakeorbyproteolyticdegradationin acidicendosomes,(iii)shouldbeabletoexchangecargowithacidic LEsandprotectpeptidesfromextensivedegradation,(iv)should beabletoreduceacidityinordertoallowproperfoldingofloaded MHC-Iproteins,and(v)shouldbeabletoreturnloadedMHC-Ipro- teinsbacktothecellsurface.Thus,itispossiblethatalternative peptideloadingtakesplaceduringtravelingofMHC-Imolecules throughclassicalacidLEsandasubsequentsortingintosubsetof lessacidicLEsthataretargetedtothePM.Theacidicenvironmentof

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LEswouldpartiallydisrupttheconformationofMHC-Imolecules, especiallyalongpeptidebindinggrove,therebyallowingthenew peptidetobebound.Finally,thelessacidicpHinthe“recycling”

LEswouldallowstabilizationofthosenewlyformedtrimolecular MHC-Icomplexes.

Amajorunderstandingofcross-presentationcamefromstudies ondendriticcellsandmacrophageswhicharekeyplayersintheini- tiatingoftheimmuneresponseinthelymphnodes.However,very littleisknownabouttheroleofopenMHC-Iconformersinthese cells.Thusstudiesperformedinfibroblasts,mastocytomacellsand carcinomacelllinesregarding endosomalMHC-I traffickingand sorting(Mahmutefendi ćetal.,2011;Zagoracetal.,2012)should be confirmed on professional antigen presenting cells isolated exvivo.

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