Peptide-specific T helper cells identified by MHC class II tetramers differentiate into several subtypes upon immunization with CAF01 adjuvanted H56 tuberculosis vaccine formulation

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Vaccine

jo u rn al h om ep a g e :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

Peptide-specific

T

helper

cells

identified

by

MHC

class

II

tetramers

differentiate

into

several

subtypes

upon

immunization

with

CAF01

adjuvanted

H56

tuberculosis

vaccine

formulation

Gennaro

Prota

_,

_Dennis

_Christensen

_,

_Peter

_Andersen

_,

_Donata

_Medaglini

_,

Annalisa

Ciabattini

a_{LaboratoriodiMicrobiologiaMolecolareeBiotecnologia(LA.M.M.B.),DipartimentodiBiotecnologieMediche,UniversitàdiSiena,Siena,Italy} b_{DepartmentofInfectiousDiseaseImmunology,StatensSerumInstitut,Copenhagen,Denmark}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20February2015 Receivedinrevisedform17July2015 Accepted9September2015 Availableonline20October2015 Keywords: CD4+_Tcellpriming MHCclassIItetramers Thelpersubsets Subcutaneousimmunization

a

b

s

t

r

a

c

t

CD4+_{T-cellprimingisanessentialstepinvaccinationduetothekeyroleofThelpercellsindriving}

botheffectorandmemoryimmuneresponses.HerewehavecharacterizedinC57BL/6micetheThelper

subtypedifferentiationamongtetramer-specificCD4+_{Tcellsprimedbysubcutaneousimmunizationwith}

thetuberculosisvaccineantigenH56plustheadjuvantCAF01.Peptide-specificpopulationidentified

bytheMHCclassIItetramersdifferentiatedintoseveralThelpersubtypesuponantigenencounter,

andthefrequencyofsubpopulationsdifferedaccordingtotheirlocalization.Th1(CXCR3+_T-bet+_),Tfh

(CXCR5+_PD-1+_Bcl-6+_)andROR␥t+_{cellswereinducedinthelymphnodesdrainingtheimmunization}

site(dLN),whileTh1cellswerethepredominantsubtypeinthespleen.Inaddition,CD4+_Tcells

co-expressingmultipleT-celllineage-specifyingtranscriptionfactorswerealsodetected.Inthelungs,most

ofthetetramer-bindingTcellswereROR␥t+_{,whileTfhandTh1cellswereabsent.Afterboosting,ahigher}

frequencyoftetramer-bindingcellsco-expressingthemarkersCD44andCD127wasdetectedcompared

toprimedcells,andcellsshowedaprevalentTh1phenotypeinbothdLNandspleens,whileTfhcellswere

significantlyreduced.Inconclusion,thesedatademonstratethatparenteralimmunizationwithH56and

CAF01elicitsadistributionofantigen-specificCD4+_{Tcellsinbothlymphoidtissuesandlungs,andgives}

risetomultipleThelpersubtypes,thatdifferdependingonlocalizationandfollowingreactivation.

©2015TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Thelpercellsplayafundamentalroleintheadaptiveimmune responseuponvaccination,sincetheyarerequiredforthe induc-tion of effector as well as memory immune responses [1–3]. CharacterizationofT-cellpriminginducedbyavaccinationstrategy iscriticalinordertodevelopoptimalprime-boostcombinations capableofelicitingthetypeofimmuneresponserequiredtofight aspecificpathogen[4].Magnitude,qualityandlocalizationof pri-maryThelper(Th)cellresponsesareoffundamentalimportance fortheevaluationofnovelvaccinecandidates.T-cellprimingcan beevaluatedasatargetforimprovingandmodulatingtheimmune responseduringvaccination[5],andasanearlypredictorofvaccine

∗ Correspondingauthorat:Università diSiena,Dipartimentodi Biotecnolo-gieMediche,LaboratoriodiMicrobiologiaMolecolareeBiotecnologia(LA.M.M.B.), PoliclinicoLeScotte,Vlottopiano1,VialeBracci,Siena53100,Italy.

Tel.:+390577233100;fax:+390577233334.

E-mailaddress:annalisa.ciabattini@unisi.it(A.Ciabattini).

immunogenicity[6,7]. CD4+ _{T-cell priminghasbeenstudied to} characterizethemechanismofactionofabroadrangeofadjuvants suchasalum[8],CpGODN[9],lipopolysaccharide[10],cholera toxin[11]oritsBsubunit(CTB)[12].

TheanalysisofThelpercellsubsetsallowsthecharacterization ofthespecializedeffectorcellselicitedbyvaccination.Upon anti-genencounter,clonallyexpandedCD4+ _{Tcells differentiateinto} variousfunctionallydefinedeffectorsubpopulations,accordingto thelocalpro-inflammatoryenvironment,thedoseandtheroute ofthevaccineused[13–15]. BesidestheTh1,Th2,Th17and T-regsubtypes,Tfollicularhelper(Tfh)cellshaveemergedinrecent yearsasanotherCD4+_{Tcellsubtypeessentialforpromotingthe} differentiationofactivatedBcellsintomemoryBcellsandlong livedplasma-cells[16].The inductionofTfhcellsis thereforea target for vaccines aimedat stimulatinga protective antibody-mediated immune response. Tfh cells express many molecules importantfortheirdifferentiationandfunction,suchasthe fol-liclehomingchemokinereceptorCXCR5,thetranscriptionalfactor B-celllymphoma-6 (Bcl6), theprogrammedcell death-1(PD-1) [17].

http://dx.doi.org/10.1016/j.vaccine.2015.09.024

0264-410X/©2015TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

6824 G.Protaetal./Vaccine33(2015)6823–6830 TheexvivostudyofprimedCD4+_{Tcellsistechnicallylimited}

duetotheverylowfrequencyofantigen-specificTcells. Antigen-specificTcellresponseshavebeendetectedbyfunctionalassays uponinvitroantigenicrestimulation.Nevertheless,since pheno-typicandfunctionalpropertiesofreactivatedcellsmaybealtered bythisrestimulationphase[18],technologiessuchastheadoptive transferofTCR-transgenicTcellsinrecipientmice[9,12,19–22,22] and,morerecently,MHCclassIItetramers[23]havebeen devel-opedandemployedtostudyT-cellpriminguponvaccination[2]. MHCclassII-peptidecomplextetramersinparticular,have pro-videdan invaluableway ofmonitoring exvivo T-cell mediated immuneresponsesandquantifyingthedevelopmentofan antigen-dependentresponseinanimalstudies[23–26].

In the present study we have identified and characterized, by using the tetramer technology, CD4+ _{T cells specific for a} peptideof the Mycobacteriumtuberculosis vaccine antigen H56 uponparenteralimmunization.H56isapromisingvaccine candi-dateconsistingof6-kDaearlysecretoryantigenictarget(ESAT-6), Ag85B,andtheproteinRv2660,thatisacomponentofvaccines currentlyin clinicaltrials (ClinicalTrials.govIdentifier numbers: NCT01967134,NCT01865487)[27].Thesolubleantigenwashere combinedwiththeadjuvantCAF01,a liposomesystemthathas beenshown toinduce combined Th1 and Th17 responses and promoterobustlong-livedmemoryimmunity[28–30];CAF01has recentlybeenalsotestedinhumanstudies[31].Themagnitudeand qualityofthepeptide-specificCD4+_{Tcellresponseelicitedby} sub-cutaneous(SC)immunizationwithH56andCAF01wasassessed byemployingMHCclassIItetramerscomplexedwithan Ag85B-derivedpeptide,intheC57BL/6mousemodel.Theexpansionof Ag85B-tetramerbindingcells,theirsubtypedifferentiationand dis-seminationtowardseffectorsiteswereanalyseduponprimingand subsequentboosting.

2. Materialsandmethods 2.1. Mice

Eight-weeksoldfemaleC57BL/6mice,purchasedfromCharles River(Lecco,Italy)weremaintainedunderspecificpathogen-free conditions at theUniversity of Siena, and treated according to nationalguidelines(DecretoLegislativo26/2014).Allanimal stud-ieswereapprovedbytheEthicsCommittee“ComitatoEticoLocale dell’AziendaOspedalieraUniversitariaSenese”andtheItalian Min-istryofHealth(number4/2011,ondate20/07/2011).

2.2. Immunizationsandsamplecollection

MicewereimmunizedbytheSCroutewiththeM.tuberculosis fusionproteinH56[27](10␮g/mouse)mixedwiththeadjuvant CAF01 [30] (250␮g dimethyldioctadecylammonium (DDA) and 50␮gtrehalosedibehenate(TDB)/mouse;StatensSerumInstitut, Denmark).SCimmunizationwasperformedatthebaseofthetail inatotalvolumeof150␮lofTRISHCl10␮M.Groupsofmicewere immunizedatday0andsacrificedonday7,orboostedatweek 4,andsacrificed7dayslater.Iliac(subiliac,medialandexternal) lymphnodes,lungsandspleenswerecollected7daysafter pri-maryorboosterimmunizationandtreatedaspreviouslydescribed [20].

2.3. Flowcytometricanalysis

Cells were incubated for 30min at 4◦C in Fc-blocking solution (complete medium plus 5␮g/ml of CD16/CD32 mAb [clone 93; eBioscience, CA, USA]) and then stained for 1h at RT with PE-conjugated I-A(b) M. tuberculosis Ag85B precur-sor 280–294 (FQDAYNAAGGHNAVF), or PE-conjugates human

classII-associatedinvariantchainpeptide(PVSKMRMARPLLMQA) tetramers(kindlyprovidedby NIHMHC TetramerCoreFacility, EmoryUniversity,Atlanta,GA)togetherwithAPC-conjugated anti-CXCR5(clone2G8,BDbiosciences,CA,USA).Surfacestainingwas performedonicewithFITC-conjugatedanti-PD-1(cloneJ43), PE-CY7-conjugatedanti-CXCR3(cloneCXCR3-73,bothfromBiolegend, CA, USA), HV500-conjugated anti-CD4 (clone RM4-5), HV450-conjugatedanti-CD44(cloneIM7,bothfromBDbiosciences)and PerCp-eFl710-conjugatedanti-CD127(cloneSB/199,eBioscience). SampleswerelabelledwithLIVE/DEADFixableNearIRDeadCell Stain Kit according to the manufacturer’sinstructions (Invitro-gen,USA).IntracellularstainingforBV605-conjugatedanti-T-bet (clone 4b10, Biolegend), PECF594-conjugated anti-Bcl-6 (clone K112-91)andPerCPCY5.5-conjugatedanti-ROR␥t(cloneQ31-378, bothfromBDbiosciences)wasperformedusingtheFoxp3staining bufferset (eBioscience)according tothe manufacturer instruc-tion.Antibodiesandtetramersweretitratedforoptimaldilution. CD44+ _tetramer+ _{cells were identified using a gating strategy} basedonlivecells,FSC×SSClymphocytecharacteristics,singlets and CD4+ _{expression; gateswere setaccordingtoFluorescence} MinusOne(FMO)controls.About106 _{cellswerestoredforeach} sampleacquiredonLSRIIflowcytometer(BDbiosciences);data analysis was performed using FlowJo software (TreeStar, OR, USA).

2.4. Cytokineassay

IFN-␥,IL-4,IL-10,IL-17A,IL-2andIL-21productionwasassessed inculturesupernatantsofrestimulatediliaclymphnodescellsby Bio-Plexcytokineimmunoassay(Bio-Rad).Lymphocyteswere cul-turedwith5␮g/mlofH56incompletemediumfor72hat37◦Cin 5%CO2.IFN-␥,IL-4,IL-10,IL-17A,IL-2cytokinesweredetectedin supernatantsusingthe5-plexassaysystem,whileIL-21was ana-lysedindividually.Cytokinedetectionwasperformedfollowingthe manufacturer’sprotocol,andanalysedbyLuminex100(Bio-Rad). Cytokineconcentrationswerecalculatedbasedonstandardcurve datausingBio-PlexManagersoftware(version4.0).

2.5. Statisticalanalysis

Two-tailedStudent’st-testwasemployedtoanalysestatistical differencesbetweenthefrequenciesofAg85B-specificCD4+_Tcells detectedinprimedandboostedgroups,andbetweenthe percent-ageofT-helpersubsetsdetectedinprimedand boostedgroups. StatisticalsignificancewasdefinedasP≤0.05.Graphpad4.0 soft-warewasusedforanalysis.

3. Results

3.1. PrimedAg85B-specificCD4+_{Tcellsdifferentiateintomultiple} Thsubtypesaccordingtotheirlocalization.

Theprimaryantigen-specificCD4+_{T-cellresponsewasstudied} afterSCimmunizationwithH56plusCAF01adjuvant inlymph nodesdraining theimmunizationsite (dLN),spleenand alsoin thelungs,thatareanessentialeffectorsite forfightingairways pathogens.CD4+_{Tcellsspecificfortheimmunodominantepitope} ofAg85B,thatispartoftheH56fusion protein,wereidentified usingAg85B280–294-complexed MHC classII tetramers.Staining specificityofAg85B280–294-complexedMHCclassIItetramerswas determined usinga controltetramer complexedwith an unre-latedantigenthatshowedalevelofstainingbelow0.02% (data notshown).Tetramer-positive(Tet+_{)Tcellsubtypeswere} charac-terized7daysafterprimingfortheexpressionofsurfacemarkers and transcription factorsindicative of Tfh,Th1, and Th17 sub-typedifferentiation.Tfhcellphenotypewasidentifiedbyassessing

Fig.1.CharacterizationofAg85B-specificCD4+_{Tcellsubpopulationsinlymphnodesdrainingtheimmunizationsite.C57BL/6micewereprimedwithH56andCAF01bythe}

SCroute,andsacrificed7dayslater.LymphocytesfromdLNwerecollected,stainedwithAg85B280–294MHCclassIItetramer,andanalysedfortheexpressionofsurfacemarkers

andintracellulartranscriptionfactorsforidentifyingdifferentsubpopulations.(A)DetectionofTet+_TcellsasCD44high_Tet-Ag85B+_{cellsgatedonliveCD4}+_lymphocytes

(a);percentageofTet+_{TcellsrespecttoCD4}+_{Tcellsisreported.GatedTet}+_{TcellswereassessedfortheexpressionofCXCR5andPD-1cells(b),anddouble-positivecells}

(indicatedasTfh,withfrequencyrespecttoTet+_{Tcells)wereanalysedforBcl-6expression(c,filledhistogram;inoveralyBcl-6expressionassessedinCD4}+_CD44−_tetramer−

cells,openhistogram).BothTfhandnon-TfhcellswereassessedfortheexpressionofCXCR3(dande,percentagesreportedarereferredtoTfhandnon-Tfhcellsrespectively), andnon-TfhCXCR3+_{cellswereanalysedfortheexpressionofT-bet(f,filledhistogram,inoverlayitsexpressionassessedinCD4}+_CD44−_tetramer−_{cells,openhistogram).}

Tet+_{TcellsweretestedforROR␥texpression(g,percentagereportedisreferredtoTet}+_{Tcells),andgatedpositivecellswereanalysedfortheexpressionofBcl-6versus}

T-bet(h,percentagesreportedineachquadrantarereferredtoROR␥t+_{cells).(B)ROR␥t(green),Bcl-6(orange),andT-bet(blue)expressionacrossCXCR5versusPD-1dot}

plotanalysis.Dotplotsrepresentasingleanimalfrom2independentexperimentswith5miceeach;about106_{cellswerestoredforeachsample.}

theexpressionofPD-1andCXCR5togetherwiththeintracellular expressionofBcl-6[33],Th1cellsweredetectedbytheexpression ofCXCR3andT-bet[34],whileTh17byROR␥t[35].Antigen-specific CD4+_{TcellswerenotdetectedusingMHCclassIItetramers} spe-cificfortheESAT6peptide(QQWNFAGIEAAASA,datanotshown), furtherconfirmingtheimmunodominanceoftheselectedAg85B epitope,asshownalsoinpreviousstudies[32].

3.2. ThelpersubpopulationsindLN

IndLNTet+_{Tcellsexpanded(0.32%oftotalCD4}+_Tcells)and differentiatedintoseveralThelpercellsubpopulations(Fig.1A). About20%ofTet+_{TcellsexpressedtheTfhsurfacemarkers} PD-1andCXCR5(Fig.1A,panelb)andthesegatedcellsexpressedat highlevelsthespecificBcl-6transcriptionfactor(Fig.1A,panelc). About40%ofgatedTfhcellsexpressedCXCR3,amarker preferen-tiallymaintainedbycellscommittedtotheTh1cellpathway[36]

(Fig.1A,paneld),buttheydidnotexpresstheTh1master regula-torfactorT-bet(datanotshown).Ithasbeenpreviouslyreported thatcirculatingCD4+_CXCR5+_{TcellscanexpressCXCR3[37],here} weshowthatalsolymphnoderesidentTfhcellscanexpressthis chemokinereceptor.Amongnon-Tfhgatedcells(Fig.1A,panelb) 60%expressedCXCR3(Fig.1A,panele),andmostofthemwere T-bet-positive(Fig.1A,panelf)indicatingthedifferentiationintoTh1 cells.CXCR3+_T-bet+_{Th1cellsthusmadeupabout27%oftheTet}+_T cells.

About 40% of Tet+ _{Tcells expressedthe Th17 master} regu-latorfactor ROR␥t(Fig.1A,panelg), andinterestingly,manyof gatedROR␥t+ _{cells co-expressedothermasterregulatorfactors,} suchas Bcl-6(22%) or T-bet (21%),while about55% were pos-itive onlyfor ROR␥t(Fig.1A, panelh). Thisanalysisshows the presenceof“transitional”phenotypesinwhichtheco-expression ofmore thanone “master”transcription factorcanbedue toa transientstatusofcellsthatwereredirectingtheirphenotypeand

6826 G.Protaetal./Vaccine33(2015)6823–6830

Fig.2. CharacterizationofAg85B-specificCD4+_{Tcellsubpopulationsinspleenandlungs.C57BL/6micewereprimedwithH56andCAF01bytheSCroute,andsacrificed}

7dayslater.LymphocytesfromSPL(A)andlungs(B)werecollected,stainedwithAg85B280–294MHCclassIItetramer,andanalysedfortheexpressionofsurfacemarkers

andintracellulartranscriptionfactorsforidentifyingdifferentsubpopulations.(A)DetectionofTet+_TcellsasCD44high_Tet-Ag85B+_{cellsgatedonliveCD4}+_{lymphocytes(a);}

percentageofTet+_{TcellsrespecttoCD4}+_{Tcellsisreported.GatedTet}+_{TcellswereassessedfortheexpressionofCXCR5andPD-1(b)anddouble-positivecells(indicatedas}

Tfh,withfrequencyrespecttoTet+_{Tcellsinbrackets)wereanalysedforBcl-6expression(c,filledhistogram;inoverlayBcl-6expressionassessedinCD4}+_CD44−_tetramer−

cells,openhistogram).Non-TfhcellswereanalysedfortheexpressionofCXCR3(paneld,percentagereportedisreferredtonon-Tfhcells),andgatedCXCR3+_cellswere

analysedfortheexpressionofT-bet(e,filledhistogram,inoverlayitsexpressionassessedinCD4+_CD44−_tetramer−_{cells,openhistogram).Tet}+_{TcellsweretestedforROR␥t}

expression(f,percentagereportedisreferredtoTet+_{Tcells),andgatedpositivecellswereanalysedfortheexpressionofBcl-6versusT-bet(g,percentagesreportedineach}

quadrantarereferredtoROR␥t+_{cells).(B)Sameanalysisdescribedin(A),performedinthelungs.Percentagesofpositivecellsarereportedineachdotplot.Dotplotsshown}

representasingleanimalfrom2independentexperimentswith5miceeach;about106_{cellswerestoredforeachsample.}

functiontowardsonespecificsubtype.CellsexpressingROR␥twere equallydistributedbetweenCXCR5andPD-1positiveornegative subpopulations,whileBcl-6andT-betshowedaclearseparation betweenthesetwo populations(Fig.1B).Thestudy ofThelper subtypesperformedwithindLNhighlightstheCD4+ _Tcell plas-ticity,andthecloserelationshipbetweendifferentThcelllineages [38,39].

3.3. Thelpersubpopulationsinspleenandlungs

Atthesametimepoint,theprofileofprimedTet+_Tcellswas analysedintospleenandlungs.ThesubpopulationsdetectedindLN wereobservedalsointhespleenbutwithdifferentfrequencies (Fig.2A).Tfhcells(PD-1+_CXCR5+_{expressingBcl-6)wereabout8%} ofTet+_{Tcells,whilethefrequencyofCXCR3}+_T-bet+_Th1cellswas around42%(Fig.2A,panelsb,d,e).About40%expressedROR␥t (Fig.2A,panelf),ofthesecells,20%co-expressedT-bet,whileabout 78%werepositiveonlyforROR␥tandonly2%co-expressedROR␥t andBcl-6(Fig.2A,panelg).

Theanalysisperformedinthelungsshowedtheabsenceofboth Tfh(CXCR5+_PD-1+_{)andTh1(CXCR5}−_PD-1−_CXCR3+_{)cells(Fig.2B,} panelsbandc).About30%ofTet+_{TcellsexpressedROR␥t,and} mostofthem(81%)didnotco-expressedothertranscriptionfactors (Fig.2B,panelsdande).ThesedatashowaprevalenceofTh1cells inthespleenandROR␥t-positivecellsinlungs.

3.4. EffectofboosterimmunizationonAg85B-specificCD4+_Tcells Theresponse ofprimedAg85B-specificCD4+ _{Tcells andthe} persistenceofThsubpopulationswerestudieduponbooster immu-nizationwith H56and CAF01 in dLN,spleen and lungs 7 days afterboosting. Asignificantincreaseinthepercentage ofTet+_T cellswasobservedinallinvestigatedorgansindicativeoftherecall response,andthefrequenciesofTet+_{Tcellsincreasedto0.56%,1%} and2.3%indLN,spleenandlungs,respectively(P<0.001,P<0.01 andP<0.05respecttoprimedgroups;Fig.3A).TheamountofTet+_T cellsafterboosterimmunizationwasabout40,000_±8,000cellsin dLN,42,000±10,000inlungs,and183,000±54,000inthespleens (meanvalue±SEMof15mice),andthefold-increaserespecttothe amountgeneratedbytheprimaryimmunizationwas7.2,4.2and 2.6inlungs,spleenanddLN,respectively(datanotshown).CD127 expression,thatisbelievedtobeindicativeofmemory,was ana-lysedinTet+_{T-cells.Afterboosterimmunizationthefrequencyof} Tet+_CD44hi_CD127+_{antigen-experiencedTcellsincreasedrespect} toprimedcells (Fig.3B).Thefrequency,aswellastheabsolute number,ofPD-1+_CXCR5+_{Tfhcellsdetecteduponpriming,} signifi-cantlydecreasedfollowingboosterimmunizationbothindLNand spleens(Fig.4A),whilethepercentageofTh1andTh17cellswere steadilymaintainedinallorgans(Fig.4A).Theanalysisoffunctional activityofTcellscollectedfromdLNcorrelatedwiththephenotype observed,asshownbytheproductionofIFN-␥andIL-17,while

Fig.3. FrequencyofAg85B-specificCD4+_{Tcellsfollowingboosterimmunization.C57BL/6micewereprimedwithH56andCAF01andboosted4weekslater.Lymphocytes}

fromdLN,SPLandlungs,collected7daysafterprimaryorboosterimmunization,werestainedwithAg85B280–294MHCclassIItetramersandsurfacemarkerantibodies.

(A)FrequenciesofTet+_{TcellsrespecttoCD4}+_{cellsfollowingprimary(gray)orboosterimmunization(black).Dataarereportedasthemean±SEMof15micefromthree}

independentexperiments.Two-tailedStudent’st-testwasusedforcomparingvalues.*P<0.05,**P<0.01,and***P<0.001.(B)CD44versusCD127expressiononCD4+

tetramer-Ag85B+_{gatedcellsfollowingprimary(left)orbooster(right)immunizationindLN(toppanels),spleen(middlepanels)andlungs(bottompanels).Percentagesof}

CD44andCD127doublepositivecellsrespecttoCD4+_{tetramer-Ag85B}+_{cellsareshown.Dotplotsrepresentasingleanimalfrom2independentexperimentswith5mice}

each;about106_{cellswerestoredforeachsample.}

thereleaseofIL-2wasindicativeoftheproliferativeresponseand activationprogramofTet+_{Tcells(Fig.4B).}

4. Discussion

Thepresentworkexplorestheantigen-specificCD4+_T prolifera-tionanddifferentiationintoThelpersuptypesfollowingparenteral immunizationwiththevaccineformulationH56andCAF01.Our datahighlightthatthepeptide-specificpopulation,identifiedby MHCclassIItetramers,produceddifferenteffectorcellpatterns upon antigen encounter, and that thefrequency of subpopula-tionsdiffered according to theirlocalization. In particular, Tfh, Th1and ROR␥t+ _{cells wereall inducedinto dLNtogether with} “transitional”subtypes thatco-expressedmultiple helperTcell lineage-specifyingtranscriptionfactors.Tfhcellswerelargely har-bouredwithinLNdrainingtheimmunizationsite,whileTh1cells werepredominantinthespleen,andROR␥t+ _{cellsin thelungs.} Therefore,ourresultsshowthatprimedThelpercellsthat recog-nizedthesamepeptide-MCHclassIIcomplex,didnotdifferentiate into a single polarized phenotype but in more subtypes, that

showedadifferentfateuponreactivation.Althoughthecytokine milieuit isknowntoplayanimportantroleindeterminingthe typesofeffectorcellsgenerated,recentevidencehasdemonstrated thatsignalsreceivedthroughtheTCRarealsoimportantforeffector celldifferentiation[26].Theinterclonalandintraclonalfunctional heterogeneityofCD4+_{Tcellsprimedbypathogensorvaccineshas} alsobeenrecentlydemonstratedbyTCRdeepsequencing[40],thus supportingtheone-cellmultiplefatesmodelofhelperTcells dif-ferentiation.TheCD4+_{T-cellresponsethatwehavetrackedusing} Ag85Bepitope-specifictetramers,canbeconsidered representa-tiveoftheresponsetowardsthewholevaccine,sinceithasbeen previouslyshowntobeanimmunodominantpeptide[32]andthe stainingwithtetramersspecificforotherepitopesincludedinH56, didnotidentifyanypopulation.

GenerationofTfhcellsisessentialfortheefficacyofvaccinesin whichantibodyresponseisstronglyrequired.Ag85B-specificTfh cellsweredetecteduponprimingwithindLN,andwithalower frequencyinthespleen.WhetherTfhcellsestablishapersistent localmemorycompartmentisstillpoorlyunderstood,asitisnot clearifTfhareterminallydifferentiatedcells orcangiveriseto

6828 G.Protaetal./Vaccine33(2015)6823–6830

B

IL-2 IFNγ IL-17 IL-4 IL-10 IL-21

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naive

primed

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Fig.4. Ag85B-specificCD4+_{Tcellsuponboosterimmunization.GroupsofC57BL/6micewereprimedwithH56andCAF01andboosted4weekslater.Lymphocytesfrom}

dLN,SPLandlungswerecollected7daysafterprimaryorboosterimmunizationandstainedwithAg85B280–294MHCclassIItetramers,surfaceandintracellularantibodies.

A.FrequenciesofTfh(CXCR5+_PD-1+_),Th1(CXCR5−_PD-1−_CXCR3+_{),andTh17(Bcl-6}−_T-bet−_ROR␥t+_{)cellsrespecttoTet}+_{TcellsindLN,SPLandlungsfollowingprimary}

(gray)orbooster(black)immunization.Barsrepresentthemeanfrequencies±SEMof10micefromtwoindependentexperiments.Frequenciesofdifferentsubpopulations werecomparedusingthetwo-tailedStudent’st-test.*P<0.05,**P<0.01,and***P<0.001.(B)IL-2,IFN␥,IL-17,IL-4,IL-10andIL-21concentrationsassessedbyBio-Plex immunoassayinculturesupernatantsoflymphocytesrestimulatedwithH56for72h,collectedfromdLNofnaive(white),primed(dotted)orboosted(gray)mice.

otherhelperTcellsubsets oncethegerminalcenter reactionis resolved[41].Ourdatashowthepresenceofantigen-experienced CD44high_CD127+_Tet+_{TcellswithindLNafterbooster} immuniza-tion,neverthelessweobserved a reduction ofCXCR5 and PD-1 double-positivecells,whileCXCR3+_{Th1cellsweresteadily} main-tained.RecentstudiesshowthatTfhcellsgiverisetomemoryT cellswithunexpectedplasticitythatallowthemtore-differentiate alsointoconventionalhelperTcells[41].Thissupportstheideathat

Tfhcellsarenotterminallydifferentiated,andmaintainaclose rela-tionshipwithotherThcelllineages.Thedevelopmentalplasticity modelofThdifferentiationisalsosupportedbythetranscription factorsanalysisthatweperformedamongTet+_Tcellsafter prim-ing.Theanalysisclearlyshowednotonlythepresenceof“defined” Thsubpopulations,suchasTfhand Th1,butalsooftransitional phenotypes,inwhichtheco-expressionofmorethanone“master” transcriptionfactorwasobserved.WedetectedCXCR5+_PD-1+_cells

thatexpressedBcl-6togetherwithROR␥t,orCXCR5−PD-1−CXCR3+ cellspositiveforbothROR␥tandT-bet.Whiletheco-expression ofROR␥tandT-bethasbeenfrequentlyreported[39],more stri-kinglyistheexpressionofROR␥twithinCXCR5+_PD-1+_Bcl-6+_cells. In general,thereis a highdegreeof uncertainty asto whether Th17cellsrepresentastable,terminallydifferentiatedThlineage. Somestudieshave highlightedthat Th17cellscanprovidehelp toBcellsandpromoteGCformationincludingisotypeswitching [42],orthatintestinalTh17cellsacquiredaTfhphenotypewithin Peyer’s patchesand inducedthedevelopmentof IgA-producing germinalcenterBcells[43].Takentogether,thesedatasuggestthat theconcomitantexpressionofROR␥tandBcl-6lineage-specifying transcriptionfactorscanbeduetoatransientstatusofcellsthat wereredirectingtheirphenotypeandfunctiontowardsonespecific subtype,inaccordancewithaplastic,morethandefined,modelof Tcelldifferentiation[39].

Following parenteral immunization, primed and boosted Ag85B-specificCD4+_{Tcellsweredetectedalsointhelungs.We} canspeculatethatmostofreactivatedcellsexitthedLNto dissem-inateintolungs,butalsothatlungtissue-residentmemorycells, generatedbyprimaryimmunization,werereactivatedinsituby boosterimmunization.Ithasbeendemonstratedthatitis tech-nicallycomplextodistinguishparenchyma-associatedfromlung bloodvasculaturecirculatingcells[44];thereforewearecurrently workingfor characterizingthe actuallocalization ofthese cells (unpublisheddata).ThephenotypicanalysisofTet+_Tcells asso-ciatedwiththelungsshowedcharacteristicsdifferentfromcells detectedinthelymphoidcompartments,asshownbytheabsence ofTfhandTh1cells.SinceCXCR3affectstraffickingofeffectorT cellstothelungsuponintranasalinfection[45,46],thelow expres-sionofCXCR3onTet+_{Tcellscouldmeanthatcellswerealready} residentinthelungs.Nevertheless,itisalsopossiblethatother chemokinescanmediateTet+_{Tcellsmigrationtothelungs,} follow-ingaparenteralimmunization.Interestingly,Ag85B-specificCD4+ TcellsdetectedinthelungsexpressedprevalentlyROR_␥tassingle mastertranscriptionfactorinlinewithpre-clinicaldataobtained withCAF01adjuvantthatobservedtheinductionofTh17cells[29]. Th17cellsplayanimportantroleinprotectiveimmunityagainst severalpathogens,andinsuchcasesitwouldbehighlyrelevant andbeneficialtoinduceTh17responses.

Inconclusion,ourresultsshowthatThelpercellsthatrecognize thesamepeptide-MCHclassIIcomplexdifferentiateintomore sub-sets,withdifferentfrequenciesaccordingtotheirlocalization.Tfh cellswereelicitedandharbouredwithinLNdrainingthe immuniza-tionsite,whileTh1werepredominantinthespleen,andROR␥t+ cellsinthelungs.Thelpercellsalsodisplayedahighdegreeof plas-ticity,asshownbyboththepresenceof“transitional”subtypesthat co-expressedmultiple“masterregulator”factors,andphenotypes ofreactivatedmemorycellsthatwerepartiallydifferentfromthose observeduponpriming.Thesedataproviderelevantknowledge aboutCD4+ _{Tcellresponseselicitedbythevaccineformulation} H56andCAF01andontheroleoftheprimingeventinaffecting thecellularresponseinthecontextofavaccinationstrategy.

Acknowledgments

TheauthorsacknowledgetheNIHTetramerCoreFacility (con-tractHHSN272201300006C)forprovisionofMHCclassIItetramers, andtheNovartisVaccineFlowCytometryFacilityforassistanceand advices.TheauthorsthankAnnaMariaCupponeandFabioFiorino fortheirexcellenttechnicalassistance.

Thisstudyhasbeencarriedoutwithfinancialsupportfromthe CommissionoftheEuropeanCommunities,Seventh Framework Programme, contract HEALTH-2011-280873 “Advanced Immu-nizationTechnologies”(ADITEC).

References

[1]SiegristCA.Vaccineimmunology.In:PlotkinAA,OrensteinWA,OffitPA, edit-ors.Vaccines.Philadelphia:ElsevierInc.;2008.p.17–36.

[2]CiabattiniA,PettiniE,MedagliniD.CD4(+)Tcellprimingasbiomarkertostudy immuneresponsetopreventivevaccines.FrontImmunol2013;4:421. [3]HusterKM,BuschV,SchiemannM,LinkemannK,KerksiekKM,WagnerH,etal.

SelectiveexpressionofIL-7receptoronmemoryTcellsidentifiesearly CD40L-dependentgenerationofdistinctCD8+memoryTcellsubsets.ProcNatlAcad SciUSA2004;101(15):5610–5.

[4]WoodlandDL.Jump-startingtheimmunesystem:prime-boostingcomesof age.TrendsImmunol2004;25(2):98–104.

[5]FiorinoF,PettiniE,PozziG,MedagliniD,CiabattiniA.Prime-booststrategiesin mucosalimmunizationaffectlocalIgAproductionandthetypeofThresponse. FrontImmunol2013;4:128.

[6]GalliG,MediniD,GorgogniE,ZeddaL,BardelliM,MalzoneC,etal. Adju-vanted H5N1 vaccine induces early CD4+ T cell response thatpredicts long-termpersistenceofprotectiveantibodylevels.ProcNatlAcadSciUSA 2009;106(10):3877–82.

[7]NayakJL,FitzgeralsTF,RichardsKA,YangH,TreanorJJ,SantAJ.CD4+T-cell expansionpredictsneutralizingantibodyresponsestomonovalent,inactivated 2009pandemicinfluenzaA(H1N1)virussubtypeH1N1vaccine.JInfectDis 2013;207(2):297–305.

[8]McKeeAS,BurchillMA,MunksMW,JinL,KapplerJW,FriedmanRS,etal. HostDNAreleasedinresponsetoaluminumadjuvantenhancesMHCclass II-mediatedantigenpresentationandprolongsCD4T-cellinteractionswith dendriticcells.ProcNatlAcadSciUSA2013;110(12):E1122–31.

[9]CiabattiniA,PettiniE,FiorinoF,ProtaG,PozziG,MedagliniD.Distributionof primedTcellsandantigen-loadedantigenpresentingcellsfollowingintranasal immunizationinmice.PLoSONE2011;6(4):e19346.

[10]McAleerJP,VellaAT.EducatingCD4Tcellswithvaccineadjuvants:lessons fromlipopolysaccharide.TrendsImmunol2010;31(11):429–35.

[11]MarksEM,HelgebyA,AnderssonJO,SchönK,LyckeNY.CD4+T-cellimmunityin thefemalegenitaltractiscriticallydependentonlocalmucosalimmunization. EurJImmunol2011;41(9):2642–53.

[12]PettiniE,CiabattiniA,PozziG,MedagliniD.AdoptivetransferoftransgenicT cellstostudymucosaladjuvants.Methods2009;49(4):340–5.

[13]Jelley-GibbsDM,StruttTM,McKinstryKK,SwainSL.Influencingthefatesof CD4Tcellsonthepathtomemory:lessonsfrominfluenza.ImmunolCellBiol 2008;86(4):343–52.

[14]LanzavecchiaA,SallustoF.Understandingthegenerationandfunctionof mem-oryTcellsubsets.CurrOpinImmunol2005;17(3):326–32.

[15]TuboNJ,JenkinsMK.TCRsignalquantityandqualityinCD4Tcell differentia-tion.TrendsImmunol2014;35(12):591–6.

[16]CrottyS.Follicular helper CD4 Tcells (TFH). AnnuRev Immunol 2011; 29:621–63.

[17]HaynesNM,AllenCD,LesleyR,AnselKM,KilleenN,CysterJG.RoleofCXCR5 andCCR7infollicularThcellpositioningandappearanceofaprogrammed celldeathgene-1highgerminalcenter-associatedsubpopulation.JImmunol 2007;179(8):5099–108.

[18]BacherP,ScheffoldA.Flow-cytometricanalysisofrareantigen-specificTcells. CytometryA2013;83(8):692–701.

[19]KearneyER,PapeKA,LohDY,JenkinsMK.Visualizationofpeptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1994;1(4):327–39.

[20]CiabattiniA,PettiniE,AndersenP,PozziG,MedagliniD.Primaryactivationof antigen-specificnaiveCD4+andCD8+Tcellsfollowingintranasalvaccination withrecombinantbacteria.InfectImmun2008;76(12):5817–25.

[21]MedagliniD,CiabattiniA,CupponeAM,CostaC,RicciS,CostalongaM,etal. InvivoactivationofnaiveCD4+Tcellsinnasalmucosa-associatedlymphoid tissuefollowingintranasalimmunizationwithrecombinantStreptococcus gor-donii.InfectImmun2006;74(5):2760–6.

[22]PettiniE,ProtaG,CiabattiniA,BoianelliA,FiorinoF,PozziG,etal.Vaginal immunizationtoelicitprimaryT-cellactivationanddissemination.PLoSONE 2013;8(12):e80545.

[23]McHeyezer-WilliamsMG,AltmanJD,DavisMM.Enumerationand charac-terizationofmemorycellsintheTHcompartment.ImmunolRev1996;150: 5–21.

[24]CrawfordF,KozonoH,WhiteJ,MarrackP,KapplerJ.Detectionof antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes.Immunity1998;8(6):675–82.

[25]PepperM,PaganAJ,IgyartoBZ,TaylorJJ,JenkinsMK.Opposingsignalsfrom theBcl6transcriptionfactorandtheinterleukin-2receptorgenerateThelper 1centralandeffectormemorycells.Immunity2011;35(4):583–95. [26]TuboNJ,PaganAJ,TaylorJJ,NelsonRW,LinehanJL,ErteltJM,etal.Singlenaive

CD4+Tcellsfromadiverserepertoireproducedifferenteffectorcelltypes duringinfection.Cell2013;153(4):785–96.

[27]AagaardC,HoangT,DietrichJ,CardonaPJ,IzzoA,DolganovG,etal.Amultistage tuberculosisvaccinethatconfersefficientprotectionbeforeandafterexposure. NatMed2011;17(2):189–94.

[28]LindenstromT,AggerEM,Korsholm KS,DarrahPA,Aagaard C,SederRA, etal.Tuberculosissubunitvaccinationprovideslong-termprotective immu-nity characterized by multifunctional CD4 memory T cells. J Immunol 2009;182(12):8047–55.

[29]Lindenstrom T, Woodworth J, Dietrich J, Aagaard C, Andersen P, Agger EM.Vaccine-inducedTh17cellsaremaintainedlong-termpostvaccination

6830 G.Protaetal./Vaccine33(2015)6823–6830 as a distinct and phenotypically stable memory subset. Infect Immun

2012;80(10):3533–44.

[30]Agger EM, RosenkrandsI, Hansen J, Brahimi K,Vandahl BS,Aagaard C, etal.Cationicliposomesformulatedwithsyntheticmycobacterial cordfac-tor(CAF01):aversatileadjuvantforvaccineswithdifferentimmunological requirements.PLoSONE2008;3(9):e3116.

[31]vanDisselJT,JoostenSA,HoffST,SoonawalaD,PrinsC,HokeyDA,etal.A novelliposomaladjuvantsystemCAF01,promoteslong-livedMycobacterium tuberculosis-specificT-cellresponsesinhuman.Vaccine2014;32:7098–107. [32]BennekovT,DietrichJ,RosenkrandsI,StryhnA,DohertyTM,AndersenP.

Alter-ationofepitoperecognitionpatterninAg85BandESAT-6hasaprofound influenceonvaccine-inducedprotectionagainstMycobacteriumtuberculosis. EurJImmunol2006;36(12):3346–55.

[33]Nurieva RI, Chung Y, Martinez GJ, Yang XO, TanakaS, Matskevitch TD, etal. Bcl6mediatesthedevelopmentof Tfollicularhelper cells.Science 2009;325(5943):1001–5.

[34]SzaboSJ,KimST,CostaGL,Zhang X,FathmanCG,GlimcherLH.Anovel transcription factor T-bet, directs Th1 lineage commitment. Cell 2000; 100(6):655–69.

[35]IvanovII,McKenzieBS,ZhouL,TadokoroCE,LepelleyA,LafailleJJ,etal.The orphannuclearreceptorRORgammatdirectsthedifferentiationprogramof proinflammatoryIL-17+Thelpercells.Cell2006;126(6):1121–33.

[36]GroomJR,LusterAD.CXCR3inTcellfunction.ExpCellRes2011;317:620–31. [37]BentebibelSE,LopezS,ObermoserG,SchmittN,MuellerC,HarrodC,etal. InductionofICOS+CXCR3+CXCR5+THcellscorrelateswithantibodyresponses toinfluenzavaccination.SciTranslMed2013;5(176):176ra32.

[38]EvansCM,JennerRG.TranscriptionfactorinterplayinThelpercell differenti-ation.BriefFunctGenomics2013;12(6):499–511.

[39]Oestreich KJ, Weinmann AS. Master regulators or lineage-specifying? Changing views on CD4+ Tcell transcription factors. Nat Rev Immunol 2012;12(11):799–804.

[40]BecattiniS,LatorreD,MeleF,FoglieriniM,DeGregorioC,CassottaA,etal.T cellimmunity.FunctionalheterogeneityofhumanmemoryCD4+Tcellclones primedbypathogensorvaccines.Science2015;347(6220):400–6.

[41]LuthjeK,KalliesA,ShimohakamadaY,BelzGT,LightA,TarlintonDM,etal.The developmentandfateoffollicularhelperTcellsdefinedbyanIL-21reporter mouse.NatImmunol2012;13(5):491–8.

[42]MitsdoerfferM,LeeY,JagerA,KimHJ,KornT,KollsJK,etal.Proinflammatory Thelpertype17cellsareeffectiveB-cellhelpers.ProcNatlAcadSciUSA 2010;107(32):14292–7.

[43]HirotaK,TurnerJE,VillaM,DuarteJH,DemengeotJ,SteinmetzOM,etal. PlasticityofTh17cellsinPeyer’spatchesisresponsiblefortheinductionof Tcell-dependentIgAresponses.NatImmunol2013;14(4):372–9.

[44]AndersonKG,SungH,SkonCN,LefrancoisL,DeisingerA,VezysV,etal.Cutting edge:intravascularstainingredefineslungCD8Tcellresponses.JImmunol 2012;189(6):2702–6.

[45]KohlmeierJE,CookenhamT,MillerSC,RobertsAD,ChristensenJP,Thomsen AR,etal.CXCR3directsantigen-specificeffectorCD4+Tcellmigrationtothe lungduringparainfluenzavirusinfection.JImmunol2009;183(7):4378–84. [46]SakaiS,KauffmanKD,SchenkelJM,McBerryCC,Mayer-BarberKD,MasopustD,

etal.Cuttingedge:controlofMycobacteriumtuberculosisinfectionbyasubset oflungparenchyma-homingCD4Tcells.JImmunol2014;192(7):2965–9.