Re-circulation of lymphocytes mediated by sphingosine-1-phosphate receptor-1 contributes to resistance against experimental infection with the protozoan parasite Trypanosoma cruzi

ContentslistsavailableatSciVerseScienceDirect

Vaccine

jo u r n al h om ep a ge : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Re-circulation

of

lymphocytes

mediated

by

sphingosine-1-phosphate

receptor-1

contributes

to

resistance

against

experimental

infection

with

the

protozoan

parasite

Trypanosoma

cruzi

Mariana

R.

Dominguez

_,

_Jonatan

_Ersching

_,

_Ramon

_Lemos

_,

_Alexandre

_V.

_Machado

_,

Oscar

Bruna-Romero

_{, Mauricio}

_M.

_Rodrigues

_,

_José

_Ronnie

_C.

_de

_Vasconcelos

a_{CentrodeTerapiaCelulareMolecular(CTCMol),UniversidadeFederaldeSãoPaulo-EscolaPaulistadeMedicina,Brazil}

b_{DepartamentodeMicrobiologia,ImunologiaeParasitologia,UniversidadeFederaldeSãoPaulo-EscolaPaulistadeMedicina,Brazil}

c_{CentrodePesquisasRenéRachou,FIOCRUZ,BeloHorizonte,MG,Brazil}

d_{DepartamentodeMicrobiologia,InstitutodeCiênciasBiológicas,UniversidadeFederaldeMinasGerais,BeloHorizonte,MG,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13December2011

Receivedinrevisedform26January2012 Accepted15February2012

Available online 28 February 2012

Keywords: CD8 Protozoan FTY720

Gene-basedvaccines

a

b

s

t

r

a

c

t

T-cellmediatedimmuneresponsesarecriticalforacquiredimmunityagainstinfectionbytheintracellular protozoanparasiteTrypanosomacruzi.Despiteitsimportance,itiscurrentlyunknownwhereprotectiveT cellsareprimedandwhethertheyneedtore-circulateinordertoexerttheiranti-parasiticeffector func-tions.Here,weshowthataftersubcutaneouschallenge,CD11c+_{-dependentspecificCD8}+_T-cellimmune responsetoimmunodominantparasiteepitopesarisesalmostsimultaneouslyinthedraininglymphnode (LN)andthespleen.However,untilday10afterinfection,weobservedaclearupregulationofactivation markersonlyonthesurfaceofCD11C+_PDCA1+_{cellspresentintheLNandnotinthespleen.Therefore,} wehypothesizedthatCD8+_{Tcellsre-circulatedrapidlyfromtheLNtothespleen.Weinvestigatedthis} phenomenonbyadministeringFTY720toT.cruzi-infectedmicetopreventegressofTcellsfromtheLNby interferingspecificallywithsignallingthroughsphingosine-1-phosphatereceptor-1.InT.cruzi-infected micereceivingFTY720,CD8T-cellimmuneresponseswerehigherinthedrainingLNandsignificantly reducedintheirspleen.Mostimportantly,FTY720increasedsusceptibilitytoinfection,asindicatedby elevatedparasitemiaandacceleratedmortality.Similarly,administrationofFTY720tomicegenetically vaccinatedwithanimmunodominantparasiteantigensignificantlyreducedtheirprotectiveimmunity, asobservedbytheparasitemiaandsurvivalofvaccinatedmice.

Weconcludedthatre-circulationoflymphocytesmediatedbysphingosine-1-phosphatereceptor-1 greatlycontributestoacquiredandvaccine-inducedprotectiveimmunityagainstexperimentalinfection withahumanprotozoanparasite.

© 2012 Elsevier Ltd.

1. Introduction

T cells are important mediators of the adaptive immune response againstinfections caused by intracellular microorgan-isms, including the digenetic intracellular protozoan parasite

Trypanosomacruzi,thecausativeagentofChagasdisease(American trypanosomiasis).Geneticdeficiencyorspecifictreatments lead-ingtothedepletionofCD4+_orCD8+_{Tcellscriticallyimpairsthe} acquiredimmunityobservedduringexperimentalmouseinfection

[1–4].Although,theanti-parasiticeffectexertedbytheTcellsis

∗Correspondingauthorsat:CTCMol,UNIFESP– EscolaPaulistadeMedicina,Rua Mirassol,207,SãoPaulo-SP,04044-010,Brazil.Tel.:+551155711095;

fax:+551155711095.

E-mailaddresses:[email protected](M.M.Rodrigues), [email protected](J.R.C.deVasconcelos).

largelymediatedbyIFN-␥,othermediatorsmayalsoparticipatein theefficienteliminationofparasitesfromthehost[1–4].

Ininbredmousestrainsorhumans,MHCclassII-restrictedCD4+ Tcells recognizemultipleantigensfromT.cruzi[5–9],whereas MHC class Ia-restricted CD8+ _{T cells are primarily specific for} immunodominantepitopesthatareexpressedbysurfaceantigens membersofalargefamilyofT.cruziproteinsnamedtrans-sialidases (TS)[1,4,9–18].Tcellsarenotonlycriticalforacquiredimmunity, buttheyarealsoimportantmediatorsofprotectiveimmunityin responsetovaccinationwithrecombinantproteins,plasmidDNA, andbacteria-andvirus-basedvaccineconstructsagainstT.cruzi [19–25].Additionally, asinthecase ofimmunityacquired dur-inginfection,IFN-␥isakeymediatorofprotectiveimmunity[25]. DespitetheimportantroleofT-cellmediatedimmuneresponses, itiscurrentlyunknownwhereprotectiveTcellsareprimedand whetherthey need tore-circulate in order to exert their anti-parasiticeffectorfunctionsduringacquiredimmuneresponses.

0264-410X/© 2012 Elsevier Ltd.

doi:10.1016/j.vaccine.2012.02.037

Open access under the Elsevier OA license.

M.R.Dominguezetal./Vaccine30 (2012) 2882–2891 2883

Withthisaim,we firstevaluatedthekineticsofCD8+ _T-cell activationintheLNand spleenfollowing asubcutaneous para-sitechallenge.Althoughthekineticsofactivationinbothlocations wereverysimilar,wedetectedthepresenceofclearlyactivated CD11C+_{PlasmacytoidDendriticCells1}+_{(PDCA-1)cellsonlyinthe} LN.CD11C+_PDCA-1+_{areknownfortheircapacitytosecretelarge} amountsoftypeIIFNuponactivation.Butmostimportantforour purposes,veryrecently,theyhavebeenimplicatedinthepriming ofCD8+_{Tcells[26].Basedonthat,wehypothesizedthatCD8}+_Tcells wereactivatedattheLNsandre-circulatedrapidlytothespleen.

To evaluate this possibility, we administered an immuno-suppressivedrug,FTY720,tointerferewithT-cell signallingvia

thesphingosine-1-phosphate receptor-1(S1P1). Thisreceptor is expressedonTcellsthatrespondtoS1P1byemigratingoutofthe thymus,LN,andbonemarrow[27–29].FollowingT-cellactivation, S1P1istransientlydownmodulated,resultinginprolonged resi-denceofTcellswithinlymphoidtissuesand improvedpriming efficacy.FTY720interfererswiththisprocess,sinceupon applica-tion,itbecomesrapidlyphosphorylatedtoFTY720-P,thusbehaving asa strongS1P1agonist.Thisresultsin sustainedinhibition of S1P1signalling,effectivelytrappingnaiveandrecentlyactivated Tcellswithinthesecondarylymphoid.AlthoughFTY720allows T-cellpriming,itefficientlyblocksmigrationofactivatedTcellsfrom theLNstotheperipheraltissuesandtherebyprecludesperipheral T-cellresponses[27–29].

Essentially,we observedthatadministration ofFTY720after challengewithT.cruziinmicethatnormallysurviveacuteinfection (C57Bl/6) or susceptible vaccinated A/Sn mice ledto a signifi-cantincrease in the susceptibilityto infection, as indicated by elevatedparasitemiaand accelerated mortality.Together,these resultscorroboratethehypothesisthatre-circulationofT lympho-cytesmediatedbyS1P1playsanimportantroleduringacquiredor vaccine-inducedprotectiveimmuneresponsestoT.cruziinfection.

2. Methods

2.1. Ethicsstatement

Thisstudywascarriedout instrictaccordancewiththe rec-ommendationsin theGuidefor theCareand UseofLaboratory AnimalsoftheBrazilianNationalCouncilof Animal Experimen-tation(http://www.cobea.org.br/).Theprotocolwasapprovedby theCommitteeontheEthicsofAnimalExperimentsofthe Institu-tionalAnimalCareandUseCommitteeattheFederalUniversityof SaoPaulo(Id#CEP0426/09).

2.2. Miceandparasites

Female8-week-oldmice(C57BL/6andA/Sn)werepurchased fromCEDEME(FederalUniversityofSãoPaulo).Transgenicmice expressingthediphtheriatoxinreceptor(DTR)undercontrolof theCD11cpromoter(CD11c-DTR)onaC57BL/6backgroundwere derivedasdescribedandweremaintainedinourcolonyas het-erozygotes[30].Blood-derivedtrypomastigotesoftheYstrainof

T.cruziwereobtainedfromA/Snmiceinfected7–8daysearlier. EachC57BL/6orA/Snmousewaschallengedsub-cutaneously(s.c.) atthebaseofthetailwithafinaldosecontaining104_–105_or150 parasites,respectively,inafinalvolumeof0.1mL.Parasite devel-opmentwasmonitoredbycountingthenumberofblood-derived trypomastigotesin5␮Loffreshbloodcollectedfromthetailvein

[10].

2.3. AdministrationofDTorFTY720

Wildtype(WT)andCD11c-DTRmiceweretreatedi.p.with2 dosesof50ng diphtheriatoxinfromCorynebacteriumdiphteriae

(DT,Sigma),48hbeforeandonthesamedayofchallenge.In addi-tion,infectedWTmiceweretreatedeveryotherday,beginningon thesamedayofinfection,withdosesof20␮gFTY720(Cayman Chemical,AnnArbor,MI)permouse(1mg/kg)inafinalvolumeof 0.2mL.Thecontrolmicewereinjectedwiththediluentonly.

2.4. Peptides

PeptideswerepurchasedfromGenscript(Piscataway,NJ).Purity wasasfollows:VNHRFTLV,97.2%andTsKb-20(ANYKFTLV),99.7%.

2.5. Recombinantplasmidsandadenoviruses

PlasmidpIgSPCl.9andthehumanreplication-defective aden-ovirustype5containingtheasp-2geneweredescribedpreviously

[22,24,25,31].Heterologousprime-boostimmunization involved primingi.m.with100␮gofplasmidDNAfollowedbyadoseofviral suspensioncontaining2×108_{plaque-formingunits(pfu)of} aden-ovirus21dayslaterinthesamelocations.Immunologicalassaysor challengeswereperformed14daysafterviralinoculation(boost).

2.6. Phenotypiccellanalysesbyflowcytometry

ThepanelofconjugatedantibodiesusedforFACSanalyseswere CD11c-FITC (clone HL3), CD19-PECy7 (clone 1D3), CD8␣-PerCP (clone53-6.7),CD86-APC(cloneGL1),CD80-APC(clone16-10A1), CD40-APC(clone3/23)allfromBD;PDCA-1-PE(cloneJF05-1C2.4.1) fromMiltenyiBiotec.Single-cellsuspensionsfromInguinallymph nodesorspleenwerestainedforsurfacemarkersonicefor20min, andthen washedtwiceinbuffercontainingPBS,0.5%BSA, and 2mMEDTAfixedin4%PBS-paraformaldehydesolutionfor10min. Atleast300,000eventswereacquiredonaBDFACSCantoIIflow cytometerandthenanalyzedwithFlowJo(TreeStar,Ashland,OR).

2.7. Sortingofcellsbyflowcytometry

PDCA-1+ _{cellswereisolatedfromLN collectedfromC57BL/6} miceinfected5daysearliers.c.with104_{T.cruziparasites.As} con-trols,weusedPDCA-1+_{cellsisolatedfromLNofnaïveC57BL/6mice} (n=15).Inguinallymphnodeswereremoved,collagenase-treated, andthesinglecellsuspensionwasstainedwiththefollowing anti-bodies:CD3PacificBlue(500A2),IAb_{FITC(25-9-17),CD11cAPCCy7} (HL3)allfromBD, and PDCA-1PE(JF05-1C2.4.1) fromMiltenyi Biotec.CD3−_IAb+_CD11c+_PDCA-1+_{cellswerethensortedinaBD} FACSAriaIIIcellsorter.

CD8+_{cellswereobtainedfromC57BL/6mice(n=2)s.c.infected} with104 _{T.cruziparasites.Spleenswereremoved15daysafter} infection.Followingredbloodcelllysis,asinglecellsuspension wasstainedwithCD8PE(53-6.7)fromBDandpositivecellswere subjectedtosortinginaBDFACSAriaIIIcellsorter.Asdetermined byFACSanalysis,thepurityoftheCD8+_was98%.

2.8. ImmunologicalT-cellassays

ExvivoELISPOT(IFN-␥)orinvivocytotoxicityassayswere per-formedexactlyasdescribedpreviously[13,25].Briefly,theinvivo

cytotoxicityassays, C57BL/6 splenocytes weredividedinto two populationsandlabeledwiththefluorogenicdye carboxyfluores-ceindiacetatesuccinimidyldiester(CFSEMolecularProbes,Eugene, Oregon,USA)atafinalconcentrationof10␮M(CFSEhigh)or1␮M (CFSElow).CFSEhigh_{cellswerepulsedfor40minat37}◦_Cwith1␮M

2884 30 (2012) 2882–2891

Fig.1. KineticsofthespecificCD8+_{T-cell-mediatedimmuneresponsesinmiceinfectedwithT.cruzi}

C57BL/6micewerechallengeds.c.with104_{blood-bornetrypomastigotesoftheYstrainofT.cruzi.Attheindicateddays,IFN-␥-producingcellswereestimatedexvivoby}

usingtheELISPOTassayinthepresenceofpeptidesVNHRFTLV(PanelA)orTsKb-20(PanelB).Theinvivocytotoxicactivitieswereestimatedagainsttargetcellscoatedwith peptidesVNHRFTLV(PanelC)orTsKb-20(D).Theresultsarepresentedasthemeanofthevaluesof4mice±SDpergroup,exceptfortheinvivocytotoxicactivityofLN cells.Inthatcase,wepooledcellsfrom4miceforanalysis.AsterisksdenotethefrequencyofSFCsinthespleenofinfectedmiceweresignificantlyhigherthantheLNcells (P<0.05).

lymphnodecellsofrecipientmicewerecollected20hafter trans-fer,fixedwith3.7%paraformaldehydeandanalyzedby FACSas describedabove.Thepercentageofspecificlysiswasdetermined usingtheformula:

1−%CFSEhigh infected/%CFSElow infected %CFSEhigh naive/%CFSElow naive ×

100%

ThesurfacemobilizationofCD107aandtheintracellular expres-sionofcytokines(IFN-␥andTNF-␣)wereevaluatedafterinvitro

cultureofsplenocytesinthepresenceorabsenceofanantigenic stimulus. Cells were washed 3 times in plain RPMI and re-suspendedincellculturemediumcontainingRPMI1640medium (pH7.4),supplementedwith10mMHepes,0.2%sodium bicarbon-ate,59mg/Lpenicillin,133mg/Lstreptomycin,and10%Hyclone fetalbovinesera(Hyclone,Logan,Utah).Theviabilityofcellswas evaluatedusing0.2%Trypan Blueexclusion dyetodiscriminate betweenliveanddeadcells.Thecellconcentrationwasadjusted to5×106_{cells/mLinacellculturemediumcontaininganti-CD28} (2␮g/mL,BD Pharmingen),brefeldinA(10␮g/mL,BD Pharmin-gen),monensin(5␮g/mL,Sigma,St.Louis,MO),andFITC-labeled anti-CD107a(Clone1D4B,2␮g/mL,BDPharmingen).Inhalfofthe cultures,VNHRFTLVpeptidewasaddedatafinalconcentrationof 10␮M.CellswerecultivatedinV-bottom96-wellplates(Corning)

inafinalvolumeof200␮Linduplicates,at37◦_Cinahumid

envi-ronmentcontaining5%CO2.Afternomorethan12hincubation, cellswereharvestedandstainedforsurfacemarkerswithPer-CP orPE-labeledanti-CD8onicefor20min.TodetectIFN-␥,or

TNF-␣byintracellularstaining(ICS),cellswerethenwashedtwicein buffercontainingPBS,0.5%BSA,and2mMEDTAandthenfixedand permeabilizedfor20minonicewith100␮LCytofix/Cytoperm(BD Pharmingen).Afterwashingtwicewith250␮Lpermwashbuffer (BD Pharmingen), the cells were stainedto detectintracellular markersusingAPCorPE-labeledanti-IFN-␥(cloneXMG1.2)and PE-labeledanti-TNF-␣(cloneMP6-XT22).Finally,cellswerewashed twice and fixed in 1% PBS-paraformaldehyde.At least 300,000 eventswereacquiredona BDFACSCantoIIflowcytometerand thenanalyzedwithFlowJo(TreeStar,Ashland,OR).

2.9. Statisticalanalysis

Valuesareexpressedasmeans±SD.Thesevalueswere com-pared using Oneway ANOVA followed by Tukey’s HSD tests (http://faculty.vassar.edu/lowry/VassarStats.html). The Logrank testwasusedtocomparemousesurvivalratesafterchallengewith

M.R.Dominguezetal./Vaccine30 (2012) 2882–2891 2885

Fig.2.CD11c+_{cellsarerequiredfortheCD8T-cellimmuneresponsesofmice}

infectedwithT.cruzi.

WTorCD11c-DTRmicewereinfecteds.c.with104_{T.cruzibloodparasites.48h}

beforeandonthedayofchallenge,WTandCD11c-DTRmiceweretreatedi.p.with 50ngofDT.Oneweeklater,IFN-␥-producingspleencellswereestimatedexvivoby ELISPOTinthepresenceofpeptidesVNHRFTLVorTsKb-20.Resultsareexpressedas means±SDof4micepergroupandarerepresentativeofexperimentsperformedat twicewithsimilarresults.Asterisksdenotethatthenumberofspotsformingcells (SFC)frominfectWTmiceweresignificantlyhigherwhencomparedtonaïveor infectedCD11c-DTRmice(P<0.01,one-wayANOVA).

3. Results

During experimentalinfectionof H-2b _{inbred mousestrains} withparasitesoftheYstrainofT.cruzi,epitopesVNHRFTLVand TsKb-20 (ANYKFTLV) are recognized by H-2Kb_{-restricted CD8}+ cytotoxicTcells.Inpreviousstudieswehavedescribedthatthe firstistheimmunodominantepitopeleadingtoahigherimmune responseandthesecondasub-dominantepitope[10,12,13].After s.c. challenge withinfective trypomastigoteforms of the para-site,detailedanalysesofthekineticsofpeptide-specificimmune responsesweredeterminedexvivobyELISPOTandinvivoby cyto-toxicityassays.Attheindicatedtimepoints,spleenorLNcellswere incubatedinvitrowithmedium(control)orpeptides(VNHRFTLV orTsKb-20).Thenumberofpeptide-specificIFN-␥secretingcells wasdeterminedbyELISPOTassay(13).Alternatively,atthe indi-catedtimepoints,targetcellswerelabeledwithCFSEandcoated withpeptidesVNHRFTLV or TsKb-20as described inSection 2. Thesecellsweretransferredtoinfectedornaïvemice.Twentyhours later,spleenorLNcellswerecollectedandtheinvivocytotoxicity estimated.

Theresultsshowedthattheeffectorpeptide-specificimmune cellsdevelopedatasimilarrateinboththedrainingLNandthe spleen(Fig.1A–D).Themaintransitionoccurredfromdays4to12 inbothorgans,forbothpeptides.

To determine the role of CD11c+ _{cells during the} expan-sion/maturationphaseoftheadaptiveimmuneresponse,weused transgenicmiceexpressingtheDTRundercontrolof theCD11c promoter.Wheninfectedmiceweresubjectedtodiphtheriatoxin (DT),thepeptide-specificimmuneresponseintheirspleen12days afterinfectionwasseverelycompromised,asmeasuredusingthe ELISPOTassay(Fig.2).TheseresultsstronglysuggestthatCD11c+ cellsareimportantforprimingofpeptide-specificcellsfollowing

T.cruziinfection.

TofurtherevaluatethesubpopulationofCD11c+_{cellsthatcould} potentiallybeinvolved inthepriming ofthepeptide-specificT

cells,westainedLNandspleencellswithantibodiestoCD11cand PDCA-1andtheactivationmarkersCD40,CD40L,CD80,andCD86 atdifferenttimesafterchallenge.AsdepictedinFig.3A,aclear upregulatedpatternofexpressionofCD40,CD80andCD86,butnot CD40L,canbeseenonthesurfaceofCD11c+_PDCA-1+_{cellsobtained} fromtheLN.Incontrast,wedetectonlytheupregulationofCD40 onCD11c+_PDCA-1+_{splenocytesatday10afterinfection(Fig.3B).}

In addition, we also stained LN and spleen cells for CD11c expressioninconjuctionwithCD8␣inadditiontotheactivation markersCD40,CD40L,andCD86atdifferenttimesafterinfection. AlimitedpatternofupregulationofexpressionofCD86canbeseen onthesurfaceofCD11c+_CD8␣+_{cellscollectedfromtheLNorspleen} ondays3–7followinginfection(Fig.4AandB).

SimilaranalyseswerealsoconductedforCD11C+_CD8a− _cells

collectedfromthespleenandLN,butwedidnotdetectan upregu-lationofexpressionoftheactivationmarkersCD40,CD40L,CD80, orCD86atanytimepointfrom3to30daysinthespleenorLN (datanotshown).

To determine whether indeed CD11c+_PDCA-1+ _{cells could} present antigen for specific CD8 lymphocytes, we purified CD11c+_PDCA-1+_{.Aftersortingthecellsfromnaïveor5-dayinfected} LNcells,weobtainedcellsthatwere95.3and83%pureas deter-minedby thePDCA-1marker (Fig. 5Aand B, respectively). For someunknownreason,duringthepurificationprocess,somecells becomenegativeforthemarkerforCD11cmarkerbutstillretained thePDCA-1marker.ThePDCA-1+ _{cellsobtainedfrommicethat} wereinfectedexpressedsignificantlyhigheramountsof MHC-II-IAb _{and CD80(Fig.5C and D,respectively).PDCA-1}+ _{cells were} used to stimulate purified CD8+ _{splenic cells obtained from T.}

cruziinfected mice.Asshown in Fig. 5E, IFN-␥ producing cells weredetectedonlywhenCD8+_{wereincubatedwithPDCA-1}+_cells obtainedfrominfectedmice.

ThefactthatCD11c+_{cellsfromthespleenexhibitalimited} acti-vationphenotypesuggestedthatperhapsmostofthespecificTcells foundinthespleenmightnotbeprimedthere.Ifthisassumptionis correct,there-circulationofTcellscouldaccountfortheCD8+_T-cell mediatedfunctionsdetectedinthisorgan.Totestwhether lym-phocytere-circulationwasresponsiblefortheimmuneresponse observedinthespleen,wetreatedinfectedmicewithFTY720.This immunosupressivedruginhibitsS1P1signalling,thusefficiently blockingre-circulationofnaïveandactivatedTcellsfromtheLNs intoperipheraltissues,therebypreventingdevelopmentperipheral T-cellresponses[27–29].

MicewereinfectedwithT.cruziparasitesandFTY720or dilu-entwereadministeredonthesamedayofchallengeandevery2 daysthereafterasdescribedinSection2.Twoweekslater,the fre-quencyofIFN-␥-producingcellswereestimatedexvivobyusing ELISPOTassayinthepresenceofpeptidesVNHRFTLVorTsKb-20. Alternatively,splenocyteswereculturedinthepresenceorabsence ofpeptidesVNHRFTLVorTsKb-20andtheexpressionofsurface CD107a,IFN-␥andTNF-␣byICS.

In infected mice,administrationof FTY720resulted in 2.52-or 3.05-fold increasesin the frequency of IFN-␥-secreting cells fromtheLNsspecificforVNHRFTLVorTsKb-20,respectively,as detectedusingtheELISPOTassay(Fig.6).Incontrast,thisincrease inthefrequencyIFN-␥-secretingpeptide-specificcellsintheLN wasaccompaniedbyasignificantdecreaseofimmuneresponses ofsplenic lymphocytes.Immune responseswereinitially deter-minedbythefrequencyofIFN-␥-producingcellsasmeasuredby theELISPOTassay(Fig.7A).ThefrequencyofIFN-␥-producingcells foundinthespleenafterFTY720administrationwasreducedby 74.55%or100%uponstimulationwithpeptidesVNHRFTLVor TsKb-20,respectively(Fig.7A).

2886 30 (2012) 2882–2891

Fig.3. ExpressionofactivationmarkersonCD11c+_andPDCA-1+_{DCsfromtheLNsorspleensfrominfectedmice}

WTC57BL/6micewereinfecteds.c.with104_{T.cruzibloodparasites.Attheindicateddaysafterinfection,LN(panelA)orsplenic(PanelB)cellswerecollectedandco-stained}

fortheexpressionofCD11c,PDCA-1,andtheindicatedmarker.

Theexpressionofthedifferentactivationmarkerswerecomparedbetweencellscollectedfrominfected(bluelines)andnaïve(redlines)mice.

peptidesinvitro.ThefrequencyofCD8+_{cellsthatwereCD107a}+_, IFN-␥+ _{or TNF-␣}+ _{wasreduced by74.61% or 84.15%after} stim-ulation withVNHRFTLV or TsKb-20, respectively (Fig. 7B). The reductionsubstantiallyaffectedallthedifferentsubpopulationsof CD8+ _{cells (Fig.7C).Theproportionsofeachpopulationdidnot} changesignificantlyinthecellscollectedfrominfectedmicethat wereadministeredornotwithFTY720(Fig.7D).

ToevaluatetheinfluenceofrestrictingT-cellre-circulationon theoutcomeofinfection,wealsomonitoredtheparasitemialevels andsurvivalofmicethatwereandwerenotsubjectedtoFTY720 overthecourseofinfection.Wefoundthatdrugexposureresulted inincreasedparasitemiaandacceleratedmortalityofinfectedmice (Fig.8AandB,respectively).Therefore,weconcludedthat lympho-cytere-circulationisindeedimportantfortheacquiredprotective immuneresponseinthismousemodelofacuteinfection.

We then sought totest thesame hypothesis by applying a distinctlydifferentapproach.Inthiscase,weusedhighly suscep-tibleA/Snmicethatweregeneticallyvaccinatedbyprimingwith

plasmidpIgSPCl.9followedbyaboosterimmunizationwith AdASP-2. We previously showed that this heterologous prime-boost regimen reproducibly conferred protective immunity against a lethalchallengewithT.cruzi[25].ImmunitywasmediatedbyCD8+ TcellsasdepletionoftheseTcellsrendersthesemicecompletely susceptibletoinfection.TheseCD8+_{Tcellsarespecificforthe} ASP-2H-Kk_{restrictedepitopesTEWETGQI,PETLGHEIorYEIVAGYI[31].} Priortochallenge,thesemiceexhibitastrongimmuneresponseto allthreeepitopes[31].

M.R.Dominguezetal./Vaccine30 (2012) 2882–2891 2887

Fig.4.ExpressionofactivationmarkersonCD11c+_andCD8-␣+_{DCsfromtheLNsorspleensofinfectedmice}

WTC57BL/6micewereinfecteds.c.with104_{T.cruzibloodparasites.Attheindicateddaysafterinfection,LN(panelA)orsplenic(PanelB)cellswerecollectedandco-stained}

fortheexpressionofCD11c,CD8-␣,andtheindicatedmarker.

Theexpressionofthedifferentactivationmarkerswerecomparedoncellscollectedfrominfected(bluelines)andnaïve(redlines)mice.

protectiveimmuneresponse,asseeninthissecondmousemodel ofacuteinfection.

4. Discussion

TheCD8+_{T-cellimmuneresponseelicitedbyT.cruziinfectionin} mostinbredmousestrainscancontrolmultiplicationofthis intra-cellularpathogenandpreclude acute-phasepathologies suchas death[1,10–17].Thetimeatwhichacquiredimmunitydevelopsis highlydependentontheparasiteload[12,32].Inourmodel,with theYstrainofT.cruzi,weobservedthattheCD8+_T-cellimmune response is only triggeredat thetime of thepeak parasitemia

[10,12].Becausethenumberofcirculatingparasitesatthistime ishigh,antigenpresentationcouldoccurinthedrainingLNorthe spleen.However,theresultsofourexperimentsthatinvolvedthe useoftheimmunosupressivedrugFTY720,incombinationwith theidentificationofactivatedCD11c+_{cells,foundmostlyintheLN,} clearlydemonstratedthattheLNsdrainingtheparasiteentrance arewherethespecificCD8+_{Tcellsareprimed.Then,theyexittheLN} andreachthespleen.Ourresultsaresimilartothoseof experimen-talvaccinationstudieswithradiation-attenuatedmalariaparasites

[33].In this case,theCD8+ _{T-cellresponseoriginatesin theLN} drainingsiteatthesiteofparasiteentranceintheskin,andthen thesecellsmigratetootherperipheralorgans.Similartoourresults, exposuretoFTY720ledtoaccumulationofspecificTcellsinthe drainingLNanda∼85%reductionofthespecificCD8+_Tcellsinthe spleen[33].Together,theseresultsprovidecompellingevidence thattheprimingofCD8+_{Tcellscantakeplaceinthelocallymphoid} tissueduringprotozoaninfection/vaccinationandthatarapid re-circulationtothespleenislikelytooccur.Asinourcase,theauthors concludethatthisrapidre-circulationduringinfectionwascritical forprotectiveimmunitymediatedbymalaria-specificCD8+_Tcells

[33].

2888 30 (2012) 2882–2891

Fig.5.PDCA-1+_{cellspurifiedfrominfectedmicepresentantigentospecificCD8}+

cells.

PDCA-1+_{cellsweresortedfromLNcellsfrommiceinfected5daysearliers.c.withT.}

cruziparasitesasdescribedinSection2.Ascontrols,weusedPDCA-1+_{cellsisolated}

fromLNofnaïvemice.

(AandB)Sortedcellsfromnaïveorinfectedmicewerestainedwithantibodiesto CD11candPDCA-1.Numbersrepresentthefrequencyofcellsineachgate. (CandD)PDCA-1+_{cellswerestainedwithantibodiestoIA}b_orCD80.

(E)PurifiedPDCA-1+_{cellsfromnaïveorinfectedmicewereusedin}

quadrupli-cateculturestostimulatespecificCD8+_{cells.Asteriskdenotesthethatnumber}

ofSFCsofCD8+_{cellsstimulatedwithPDCA-1}+_{cellspurifiedfrominfectedmice}

weresignificantlyhighertheonestimulatedwithPDCA-1+_{cellsfromnaïveanimals}

(P<0.01).

infections [34–36]. The precise reasons for these divergent responsesarenotclearbutprobablyreflectdifferencesinthe prim-ingsitesaswellas,theimmunopathologiescausedbythedifferent infectiousagents.

Inaddition totheroleof S1P1-dependentcirculationduring protective immunityacquired duringT. cruziinfection, wealso

Fig.6.AdministrationofFTY720increasesthenumberofpeptide-specificcellsin theLNofmiceinfectedwithT.cruzi.

C57BL/6micewereinfecteds.c.with105_{T.cruziblood-borneparasites.Micewere}

administeredonthesamedayofchallengeandevery2daysthereafter20␮gof FTY720ordiluenti.p.Twoweekslater,thefrequencyofIFN-␥-producingcellswere estimatedexvivobyusingELISPOTassayinthepresenceofpeptidesVNHRFTLV orTsKb-20.Theresultsarepresentedasmeans±SDof4micepergroupandare representativeofexperimentsperformedatleasttwicewithsimilarresults. Aster-isksdenotethenumberofSFCsfrominfectedmicegivenFTY720weresignificantly higherthaninfectedmicetreatedwiththediluentonly(P<0.01).

observedthatpreviouslyvaccinatedmicebecamemoresusceptible toinfectionwhensubjectedtoFTY720exposure.Forvaccination, weusedaheterologousprime-boostregimenconsistingofan ini-tialimmunizationwithplasmidDNAandaboosterimmunization withareplication-defectiverecombinanthumanadenovirustype 5(HuAd5),bothencodingtheasp-2gene.Immunityelicitedbythis vaccinationprotocolislonglivedandmediatedbyTh1CD4+_aswell asCD8+_{Tc1cells[25,31,37].}

Theheterologousprime-boostingregimenofvaccinationusing plasmidDNAandreplication-defectiverecombinantHuAd5 pro-videsprotective immunityinsomeotherimportantpre-clinical experimental modelssuch as SIV,malaria, Ebola, and Marburg viruses[38–45].Based onthesepre-clinicalexperimental mod-els,humantrialshavebeeninitiated[46–49].Ourobservationthat S1P1isimportantforprotectiveactivityofTcellsinpreviously vac-cinatedanimalsiscompletelynewandshouldbestudiedfurther intheseexperimentalmodels.

Although we measured only CD8 T-cell mediated immune responsesonly,itishighlypossiblethatthesamepatternwould happentospecificCD4+_{Tcells.ThisT-cellsub-populationisvery} important for protective immunity during to T. cruzi infection

[25]. The absence of re-circulation of both types of lympho-cytesprobablyaccountforthesub-optimalprotectiveimmunity observedafteradministrationofFTY720.Possibly,bothcells pro-mote the processes required for parasite elimination on the tissue.

ThefactthatFTY720interferewithS1P1activationmakesit theoreticallycapableofactonothercellstypesthatexpressthis receptor.However,theeffectonothercelltypesispoorlyknown atpresent.IthasbeenpreviouslydescribedthatFTY720 admin-istrationmayincreaseorreducetheactivityofregulatoryTcells

M.R.Dominguezetal./Vaccine30 (2012) 2882–2891 2889

Fig.7. AdministrationofFTY720reducesthenumberofpeptide-specificcellsinthe spleenofmiceinfectedwithT.cruzi

C57BL/6micewereinfecteds.c.with104_{T.cruziblood-borneparasites.Micewere}

administeredonthesamedayofchallengeandevery2daysthereafter20␮gof FTY720ordiluenti.p.Twoweekslater,thefrequencyofIFN-␥-producingcellswere estimatedexvivobyusingELISPOTassayinthepresenceofpeptidesVNHRFTLVor TsKb-20(PanelsAandB).

Alternatively,splenocytesfromthesemicewereculturedinthepresenceof anti-CD107aandanti-CD28,withorwithoutthepeptidesVNHRFTLVorTsKb-20.After 12h,thecellswerestainedtodetectCD8,IFN-␥,andTNF-␣(PanelsCandD).The resultsarepresentedasmeans±SDof4micepergroupandarerepresentativeof experimentsperformedatleasttwicewithsimilarresults.Asterisksdenotethatthe numberofSFCsfrominfectedmicereceivingFTY720weresignificantlylowerthan theinfectedmicetreatedwiththediluentalone(P<0.01).

AcurrentlimitationofthisexperimentalmodelforT.cruzi infec-tionisthelackofinformationonwhereCD8+ _{Tcells encounter} andeliminateparasite-infected cells;thisisanaspectthatmay be critical to fully understand immune responses. Considering

Fig.8. AdministrationofFTY720increasessusceptibilitytoT.cruziinfection C57BL/6micewereinfecteds.c.with105_{T.cruzibloodparasites.Miceweretreated}

onthesamedayofchallengeandevery2daysthereafterwith20␮gofFTY720 i.p.ordiluentonly.(A)Parasitemialevelsforeachmousegroupispresentedasthe mean±SD(n=6).Asterisksdenotethatmicefromgroupsinjectedwithdiluentonly exhibitedsignificantlylowerparasitemia(P<0.01)thananimalsreceivingFTY720. (B)Kaplan–Meiersurvivalcurvesforthemousegroupstreatedasdescribedabove (n=6).Micefromgroupsinjectedwithdiluentonlysurvivedlongerthananimals receivingFTY720(P<0.01,Logranktest).Noanimalsdiedafterthe30thdayuntil theterminationoftheexperiment.Theresultsarerepresentativeof2independent experiments.

that T. cruzi can infect many cell types and cause systemic infection, it is plausible that many tissues may serve as sites of infection and for parasite/T-cell encounters. Supporting this hypothesis,weobservedthatvaccinated animalswereresistant toT.cruzichallengebydifferentroutesofinfection(i.p.ands.c.

[25,37]).

The finding that the administration of FTY720 significantly reducesprotectiveimmunityagainstT.cruziinfectionandimpairs theprotectiveimmunityaffordedbyvaccinationmayalsohave clinicalimplicationsfortheuseofthisimmunosuppressivedrug. Certainly, its use in regions where Chagas disease is endemic shouldbedonewithcautionconsideringthepotentialincreasein susceptibilityoftreated individuals.Finally,treatmentof organ-transplantedpatientswithFTY720mayinterferewithimmunity elicitedbypreviousvaccination.

2890 30 (2012) 2882–2891

Fig.9.AdministrationofFTY720increasessusceptibilitytoT.cruziinfectionin pre-viouslyvaccinatedA/Snmice

A/Snmicewereprimedi.m.with100␮gofplasmids,pcDNA3orpIgSPCl.9.Three weekslater,thesemicewereboostedi.m.with2×108_{pfuAd␤-galorAdASP-2.}

Allmicewerechallengeds.c.with150bloodstreamtrypomastigotes.Halfofthe vaccinatedmicereceived20␮gofFTY720everyotherday,andtheotherhalf receiveddiluentonly.(A)Parasitemialevelsforeachmousegroupispresented asthemean±SD(n=5).Asterisksdenotethatmicefromgroupsimmunizedwith pIgSPCl.9/AdASP-2andwhoreceiveddiluentonlyhadsignificantlylower para-sitemiathanothermousegroups(P<0.01).(B)Kaplan–Meiersurvivalcurvesforthe mousegroupsimmunizedandchallengedasdescribedabove(n=10).Mice immu-nizedwithpIgSPCl.9/AdASP-2andreceivingdiluentonlysurvivedsignificantly longerthanthoseimmunizedwithpIgSPCl.9/AdASP-2andadministeredFTY720 (P<0.01).Theresultsshownarecombinedfrom2experiments.Noanimalsdied afterthe50thdayofchallenge.

Acknowledgments

Funding:Fundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo (2009/06820-4), The National Institute for Vaccine Technology (INCTV-CNPq),TheMillenniumInstituteforVaccineDevelopment and Technology (CNPq – 420067/2005-1) and The Millennium InstituteforGeneTherapy(Brazil).MMR,OBRandRLare recip-ientsoffellowshipsfromCNPq.MRD,JEandJRVarerecipientsof fellowshipsfromFAPESP.Conflictofinterest:Theauthorsdeclareno competinginterest.Authors’contributions:MRD,JE,RL,andJRV per-formedtheexperimentalwork;AVMandOBRprovidedessential reagents;MRD,JE,RL,MMRandJRVwereresponsiblefor concep-tionanddesign,aswellaswritingthefirstandfinalversionsofthe manuscript.Allauthorshavereadandapproveofthefinalversion ofthemanuscript.

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