DTIC ADA534331: In Vivo Humoral Immune Responses to Isolated Pneumococcal Polysaccharides Are Dependent on the Presence of Associated TLR Ligands

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TheJournalofImmunology
In Vivo Humoral Immune Responses to Isolated Pneumococcal
Polysaccharides Are Dependent on the Presence of Associated
TLR Ligands1
GoutamSen,2 AbdulQ.Khan,2 QuanyiChen, and CliffordM.Snapper3
WedeterminedwhetherTcell-independentIgisotyperesponsestoisolatedpneumococcalpolysaccharides(PPS)requiredTLR
signalinginvivo.IgGanti-PPSresponsestoPPS3,PPS14,andC-polysaccharide(C-PS)werevirtuallyundetectableinTLR2(cid:1)/(cid:1)
mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations
inducedIL-6andTNF-(cid:2)fromwild-type,butnotTLR2(cid:1)/(cid:1),macrophages.TLR2activitywasdistinctfromthatofPPS,inthat
itwasphenolextractable.Immunizationofwild-typemicewithphenol-extractedPPS14alsoresultedinamarkedreductioninthe
IgG,althoughnottheIgM-anti-PPS14,responsecomparedwithuntreatedPPS14.Thecommercial23-valentPPSvaccine,Pneu-
movax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the
vaccineinmice.Finally,thecommercialpneumococcalconjugatevaccine,Prevnar,containedaTLR2ligand(s)thatsubstantially
enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly
suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral
immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappre-
ciatedcopurifiedand/orcontaminatingTLRligandsinPPSvaccinepreparations. TheJournalofImmunology,2005,175:3084–
3091.
T
he mechanism by which isolated polysaccharide (PS;4 T rapidlyinducedinvivoinresponsetoTLRligands.Collectively,
cell-independent type 2 (TI-2)) Ags induce Ig responses these in vitro data suggested that in vivo Ig responses to PS Ags
invivoispoorlyunderstood.Previousstudiesusinganin required at least two distinct signals, but the nature of the differ-
vitropolyclonalmodelformultivalentmembraneIg(mIg)cross- entiativesignal(signal2)invivoinresponsetoisolatedPSAgsor
linking in response to PS Ags (i.e., multiple anti-IgD Abs conju- PSAgsexpressedbyintactbacteriahasremainedobscure.
gated to dextran ((cid:1)(cid:2)-dex)) (1) indicated that multivalent mIg Severalstudieshavedemonstratedtheabilityofisolatedbacte-
cross-linking alone or in concert with CD40-mediated activation rial capsular PS to induce cytokine release in vitro (3–6). How-
induced vigorous proliferation, but no Ig secretion or isotype ever,thenatureofthisstimulatoryactivityanditscognatereceptor
switching by highly purified B cells (2). However, addition of aswellasitspotentialroleinmodulatinganti-PSresponsesinvivo
various TLR ligands, such as bacterial lipoprotein (TLR2), Neis- have not been elucidated. The recent discovery of TLRs and the
serial porins (TLR2), unmethylated CpG-containing oligode- creation of mice genetically deficient in either specific TLR or
oxynucleotides(TLR9),orLPS(TLR4)to(cid:1)(cid:2)-dex-activatedBcell TLRadaptorproteins(7,8)hasprovidedauniqueopportunityto
culturesinducedsubstantialIgsecretionandisotypeswitching(2). test the hypothesis that TLR signaling is a critical costimulus for
TherequirementforTLRligandscouldbereplacedbyadditionof anti-PS Ig responses in vivo. Distinct TLR have been described,
variouscytokines,suchasIL-5orIL-2incombinationwithIL-3, each with a characteristic specificity for one or more microbial
GM-CSF, or IFN-(cid:3). Of interest, some of these cytokines can be ligands. Ligand engagement of TLR on the surface or within the
cytoplasm of immune cells typically results in cytokine secretion
and/orcellularmaturation,therebypromotinginnateimmunity,as
Department of Pathology, Uniformed Services University of the Health Sciences,
Bethesda,MD20814 wellasshapingtheadaptiveimmuneresponse.Innateimmunityto
intactStreptococcuspneumoniae(Pn)hasrecentlybeenshownto
ReceivedforpublicationApril28,2005.AcceptedforpublicationJune24,2005.
dependonTLRsignaling.Inparticular,aroleforTLR2hasbeen
Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage
charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordance demonstrated, probably due to the expression by Pn of various
with18U.S.C.Section1734solelytoindicatethisfact. TLR2ligands,suchaslipoteichoicacid(LTA),peptidoglycan,and
1ThisstudywassupportedbyNationalInstitutesofHealthGrant1R01AI49192and lipoprotein (9–11). However, additional TLR have been impli-
theUniformedServicesUniversityoftheHealthSciencesDean’sResearchandEd-
cated in Pn-mediated immunity, including TLR4 in response to
ucationEndowmentFund.Theopinionsandassertionscontainedhereinarethepri-
vateonesoftheauthorsandarenottobeconstruedasofficialorreflectingtheviews pneumolysin (12). A potential role for pneumococcal DNA
oftheDepartmentofDefenseortheUniformedServicesUniversityoftheHealth (TLR9)orRNA(TLR7/8)inPn-inducedimmunityhasnot,asyet,
Sciences.
beendemonstrated.Recently,wereportedthatTLR2playsacrit-
2G.S.andA.Q.K.contributedequallytothiswork.
icalroleinpromotinganinvivotype1humoralimmuneresponse
3AddresscorrespondenceandreprintrequeststoDr.CliffordM.Snapper,Depart-
tointactheat-killedPn(13).
mentofPathology,UniformedServicesUniversityoftheHealthSciences,4301Jones
BridgeRoad,Bethesda,MD20814.E-mailaddress:[email protected] Using various preparations of isolated pneumococcal polysac-
4Abbreviationsusedinthispaper:PS,polysaccharide;(cid:1)(cid:2)-dex,multipleanti-IgDAbs charides, including the commercial pneumococcal PS (PPS) vac-
conjugatedtodextran;DOC,deoxycholate;KLH,keyholelimpethemocyanin;LTA, cine, Pneumovax-23 (14), we now demonstrate that the polysac-
lipoteichoicacid;mIg,membraneIg;PBA,polyclonalBcellactivator;PC,phos-
charideAgitselfisineffectiveatinducingdetectableIgGand,toa
phorylcholine;Pn,Streptococcuspneumoniae;PPS,capsularPS;SN,supernatant;
TEA,triethanolamine;TI-2,Tcellindependenttype2. lesser extent, IgM responses in vivo in the absence of a distinct
Copyright©2005byTheAmericanAssociationofImmunologists,Inc. 0022-1767/05/$02.00
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
In Vivo Humoral Immune Responses to Isolated Pneumococcal
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Polysaccharides Are Dependent on the Presence of Associated TLR
Ligands 5c. PROGRAM ELEMENT NUMBER
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TheJournalofImmunology 3085
secondsignal,inthiscaseprovidedbytheconcomitantandhith- 0.5%DOC.Water-saturatedphenol(0.5ml)wasaddedandvortexedin-
ertounappreciatedpresenceofTLRligandswithinthePPSprep- termittently for 5 min. The solution remained undisturbed for 5 min at
roomtemperatureforphaseseparation,thenwasplacedoniceforanad-
arations.WealsoshowthatthepresenceofaTLR2ligand(s)inthe
ditional 5 min. The material underwent centrifugation for 2 min at
commercialPPSconjugatevaccine,Prevnar(15),up-regulatesthe 10,000(cid:2)gat4°C.Theupper,aqueouslayerwastransferredtoanother
inducedIgGanti-PPSresponse.Thesedataprovideamechanism tube. The phenol layer was re-extracted with 0.5 ml of water-saturated
underlyingtheinductionofspecificIgGanti-PSresponsesinvivo phenol containing 0.2% TEA and 0.5% DOC. The aqueous layers were
that is consistent with earlier predictions made using the in vitro pooled and re-extracted with 1 ml of water-saturated phenol containing
polyclonal (cid:1)(cid:2)-dex model system (2) and highlight the potential 0.2%TEAand0.5%DOC.Pooledaqueouslayerswereadjustedto75%
ethanolcontaining30mMsodiumacetateandstoredfor1hat(cid:1)20°Cfor
importanceofpreviouslyunappreciated,copurifiedand/orcontam- precipitation. The material then underwent centrifugation for 10 min at
inatingTLRligandsinPPSvaccinepreparations. 10,000 (cid:2) g at 4°C. The precipitate was washed with 1 ml of ice-cold
absoluteethanolandthenair-dried.ThePSprecipitatewaslaterdissolved
Materials and Methods in0.5mlofsaline.
Mice Isolationandactivationofperitonealmacrophages
TLR2(cid:1)/(cid:1)(16)andMyD88(cid:1)/(cid:1)mice(17)wereobtainedoriginallyfromDr. RPMI1640(10ml)wasinjectedintotheperitonealcavitythroughthefat
S.Akira(OsakaUniversity,Osaka,Japan)andwerebredinourfacility. pad using a 23-gauge needle. After 3 min, lavage fluid containing the
ControlB6129SF2/J(forTLR2(cid:1)/(cid:1))andC57BL/6(forMyD88(cid:1)/(cid:1))mice peritonealcellswaswithdrawnusingan18-gaugeneedle.Cellswerepel-
wereobtainedfromTheJacksonLaboratoryandtheNationalCancerIn- letedbycentrifugationat400(cid:2)gfor10minatroomtemperature.Thecell
stitute, respectively. TLR2(cid:1)/(cid:1) (C3H/HeN background) and TLR2(cid:1)/(cid:1) (cid:2) pelletwassuspendedinculturemedium(RPMI1640supplementedwith
C3H/HeJmicewereobtainedfromDr.C.J.Kirschning(TechnicalUni- 10%FBS,2mML-glutamine,0.05mM2-ME,50(cid:4)g/mlpenicillin,and50
versityofMunich,Munich,Germany).C3H/HeJmice(18)wereobtained (cid:4)g/mlstreptomycin)andadjustedto1(cid:2)106totalcells/ml,and1mlofcell
fromTheJacksonLaboratory,andC3H/HeNmicewereobtainedfromthe suspensionwasaddedtoeachwellofa24-wellplateandincubatedfor3h
NationalCancerInstitute.Micewereusedbetween6and10wkofage. at 37°C in a 5% CO -containing incubator. Plates were then washed to
2
Thesestudieswereconductedinaccordancewiththeprinciplessetforthin removeunboundcells.Freshmedium(0.5ml)containingisolatedPSwas
theGuideforCareandUseofLaboratoryAnimals,InstituteofLaboratory addedtotheculturesofboundcellsandincubatedfor48hat37°C,fol-
Animal Resources, National Research Council, revised 1996, and were lowedbyplatecentrifugationandcollectionofculturesupernatant(SN)for
approved by the Uniformed Services University of the Health Sciences determinationofIL-6andTNF-(cid:1)concentrationsbyELISA.
institutionalanimaluseandcarecommittee.
Measurementofcytokineconcentrationsinperitoneal
Mousegenotyping macrophagecultureSN
AllTLR2(cid:1)/(cid:1)mice(backgroundB6129SF2)andMyD88(cid:1)/(cid:1)mice(back- TheconcentrationsofIL-6andTNF-(cid:1)releasedintothemediaofstimu-
ground C57BL/6) used in these experiments were first genotyped in our latedperitonealmacrophageculturesweremeasuredusingoptimizedstan-
laboratory as previously described (13). The following mice were also dardsandwichELISA.Recombinantcytokinesusedasstandardsaswellas
genotypedinourlaboratory:1)TLR2(cid:1)/(cid:1)(backgroundC3H/HeN):primer thecapturemAbs,biotinylatedmAbsusedfordetection,andavidin-HRP
A,5(cid:3)-GGGCCAGCTCATTCCTCCCACTCAT-3(cid:3);B,5(cid:3)-CTTCCT werepurchasedfromBDPharmingen.Afterincubationwithavidin-HRP,
GAATTTGTCCAGTACAGGG-3(cid:3);C,5(cid:3)-TCGACCTCGATCAAC plates were washed and developed using SureBlue tetramethylbenzidine
AGGAGAAGGG-3(cid:3);PCR,200ngofgenomicDNA;94°Cfor5min, microwell peroxidase substrate (Kirkegaard & Perry Laboratories). The
94°Cfor1min,59°Cfor30s,70°Cfor1min(30cycles),then72°Cfor reactionwasstoppedusingtetramethylbenzidinestopsolution,andplates
2min;wide-typeproduct,499bp;TLR2(cid:1)/(cid:1)product,334bp;and2)C3H/ werereadusinga450-nmELISAreader.
HeJ (TLR4-defective, background C3H/HeN): primer A, 5(cid:3)-TGT CAG
TGG TCA GTG TGA TTG-3(cid:3); B, 5(cid:3)-TCA GGT CCA AGT TGC CGT ImmunizationofmiceanddeterminationofserumtitersofIgM
TTC-3(cid:3);PCR,200ngofgenomicDNA;94°Cfor5min,94°Cfor1min, andIgGisotypeanti-PPStiters
59°C for 30 s, 70°C for 1 min (30 cycles), then 72°C for 2 min; PCR
products,BstNIdigestfor4hat60°C;separateproductson3%NuSieve Micewereimmunizedi.p.with1(cid:4)gofPPS3,PPS14,orC-PSdissolved
GTGagarosegel;HeNproducts,twobandsbetween300and350bp;HeJ insterilePBS.Serawerepreparedfrombloodobtainedfromthetailvein
products,twobandsbetween300and400bp. onday0(beforebleeding),day7,and/orday14.SerumtitersofIgMand
IgG anti-PPS or anti-PC (for C-PS) Abs were determined by ELISA as
Reagents previouslydescribed(13).Fortheanti-PPSELISAs,5(cid:4)g/mlC-PSand10
(cid:4)g/ml PPS22F were added to the serum dilution buffer to block PPS-
Phosphorylcholine (PC)-keyhole limpet hemocyanin (KLH) was synthe- nonspecific binding. For measurement of serum titers of IgM and IgG
sizedasdescribedpreviously(19).Theresultingconjugatehadasubsti- anti-PPSinducedafterPneumovax-23immunizations,ELISAplateswere
tutiondegreeof19PC-KLHmolecules.PurifiedS.pneumoniaecapsular coatedwith23(cid:4)g/mlPneumovax-23,representing1(cid:4)gofeachPPS/ml
polysaccharides(PPS)types3,4,6B,9V,14,18C,19F,22F,and23Fwere (50 (cid:4)l/well). For measurement of serum titers of IgM and IgG isotypes
allvaccinegrade,producedbyMerck,andpurchasedfromAmericanType inducedafterPrevnarimmunizations,ELISAplateswerecoatedwith5(cid:4)g/ml
CultureCollection.PurifiedS.pneumoniaecellwallC-PSwaspurchased eachofPPS4,6B,9V,14,18C,19F,and23F(50(cid:4)l/well).SerumtitersofIgM
fromStatensSerumInstitute.Pneumovax-23waspurchasedfromMerck andIgGanti-PPSAbswerecalculatedasreportedpreviously(21).
andconsistedofamixtureofhighlypurifiedcapsularpolysaccharidesfrom
the23mostprevalentorinvasivepneumococcaltypesofS.pneumoniae Statistics
(14).Each0.5-mldoseofvaccinecontained25(cid:4)gofeachPStypedis-
Serum titers of Ig were expressed as the arithmetic mean of individual
solvedinisotonicsalinesolutioncontaining0.25%phenolasapreserva-
serumsamplesfromfivetosevenmice(cid:5)SEM.Concentrationsofcyto-
tive.PrevnarwaspurchasedfromWyethPharmaceuticalsandconsistedof
kines in peritoneal macrophage culture SN were expressed as the arith-
seven PPS serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) conjugated to
metic mean of duplicate cultures from macrophages pooled from five
diphtheriaCRM197protein(15).Each0.5mlofPrevnarcontained0.125
mgofaluminumphosphateadjuvant,atotalof20(cid:4)gofCRM197,and2 mice(cid:5)SD.Levelsofsignificanceofthedifferencesbetweengroupswere
(cid:4)gofeachPPS,exceptforPPS6B(4(cid:4)g).ThedoseofPrevnarstatedin determinedbyStudent’sttest.Avalueofp(cid:5)0.05wasconsideredsta-
tisticallysignificant.
thetextreferredtothetotalamountofPPS.AllPPS-containingprepara-
tionsweretestedforthepresenceofendotoxinbytheLimulusamebocyte
lysateassay(LALtest;BioWhittaker)andwerefoundtobe(cid:4)0.13U/10 Results
(cid:4)gPPS/ml. IgGresponsestoisolatedPPSareabrogatedinTLR2(cid:1)/(cid:1)mice
PhenolextractionofPPS14 PPSs are T cell-independent type 2 (TI-2) Ags capable of stimu-
latingspecificIgMandIgG(mostlyIgG3)responsesinvivo,but
PhenolextractionofPPS14wasconductedasdescribedbyHirschfeldetal.
themechanismbywhichtheypromotedifferentiationandswitch-
(20) with minor modifications. Specifically, triethanolamine (TEA) was
addedto0.5mlofPPS14(5mg/ml)tomakea0.2%finalconcentrationof ing of PPS-specific B cells is poorly understood. In light of the
TEA. Deoxycholate (DOC) was added to make a final concentration of importantroleofTLR2ininnateandadaptiveimmunitytointact
3086 TLRSIGNALINGANDANTI-PSIgRESPONSE
S. pneumoniae (13), we wanted to determine whether TLR2 was
alsoimportantforpromotingPPS-specificIgMandIgGresponses
to various isolated PPS preparations. Thus, wild-type and
TLR2(cid:1)/(cid:1) mice were immunized with isolated PPS3, PPS14, or
C-PS. All PPS preparations induced serum titers of PPS-specific
IgMandIgGinwild-typemice,withpeaktitersobservedbyday
7 after immunization (Fig. 1). In contrast, IgG anti-PPS3, anti-
PPS14, and anti-PC (C-PS) responses were essentially abrogated
inTLR2(cid:1)/(cid:1)mice(Fig.1).ThePPS-specificIgGconsistedmostly
ofIgG3,withbarelydetectableandvariableamountsofIgG1(data
notshown).IgManti-PPS3,butnotIgManti-PPS14orIgManti-
PC,wasalsosignificantlyreduced,butnoteliminatedinTLR2(cid:1)/(cid:1)
mice(Fig.1).NofurtherincreasesinserumtitersofPPS-specific
IgMorIgGwereobservedfromdays7to14ineitherwild-typeor
TLR2(cid:1)/(cid:1)mice(datanotshown).
Thecytokine-inducingactivityofPPS3,PPS14,andC-PSisdue
tothepresenceofaTLR2ligand(s)
ThedatapresentedabovesuggestedthattheIg-inducingcapacity
ofthePPSsinvivomightbecriticallydependentonthepresence
ofaTLR2ligand(s).Todeterminethis,weisolatedmacrophages
FIGURE 2. IsolatedPPSpreparationscontainaTLR2ligand(s).Peri-
from both wild-type and TLR2(cid:1)/(cid:1) mice and measured IL-6 and toneal macrophages from wild-type and TLR2(cid:1)/(cid:1) mice were incubated
TNF-(cid:1)secretion48hafterinitiationofculturewithPPS3,PPS14, at 37°C for 48 h with PPS3, PPS14, C-PS, (each at 10 (cid:4)g/ml), LTA (2
or C-PS (Fig. 2). Wild-type, but not TLR2(cid:1)/(cid:1), macrophages se- (cid:4)g/ml), or LPS (1 (cid:4)g/ml), and SNs were collected for measurement of
creted IL-6 (Fig. 2) and TNF-(cid:1)(data not shown) in response to cytokine concentrations by ELISA. This experiment is representative of
PPS3, PPS14, or C-PS, indicating that the cytokine-inducing ac- twoindependentexperimentsperformed.
tivitiesinthesepreparationswereindeedsecondarytothepresence
ofaTLR2ligand(s).TLR2(cid:1)/(cid:1)macrophagesfailedtosecreteIL-6
(Fig. 2) and TNF-(cid:1)(data not shown) in response to the TLR2
ligand, LTA, whereas LPS (a TLR4 ligand) induced comparable detectableinductionofIL-6orTNF-(cid:1)wasobservedinresponseto
cytokineresponsesinwild-typeandTLR2(cid:1)/(cid:1)macrophages(Fig. PPS2, 6B, 7F, 9V, 18C, 19F, 22F, or 33F (our unpublished ob-
2).ThesedatademonstrateacriticalroleforaTLR2ligand(s)in servations).AllPPSswereobtainedfromAmericanTypeCulture
stimulating IgG and, to a lesser and more variable extent, IgM Collection. The implications of these latter findings for Ig induc-
anti-PS Ig in vivo in response to isolated PS preparations. Re- tioninvivoarecurrentlyunderinvestigation.
cently,weobservedthatPPS4andPPS23FalsoinducedIL-6and
TLR2activityinPPSpreparationsisdistinctfromPPS3,
TNF-(cid:1)frommurineperitonealmacrophagesinvitro,whereasno
PPS14,orC-PS
AlthoughisolatedPPSpreparationsareknowntocontain(cid:5)5%of
various non-PPS, pneumococcal contaminants, the precise nature
andfunctionalrelevanceofthesearelargelyunknown.Inthisre-
gard,itwasunclearwhetherthereportedcytokine-inducingactiv-
itiesofsomePPSs(3–6)wereduetothesenon-PPScontaminants
orwereanintrinsicpropertyofthePPSitself.Todeterminethis,
we phenol-extracted all PPSs in an attempt to remove these po-
tentially distinct cytokine-inducing activities. Peritoneal macro-
phagesfromwild-typemicewereisolatedandculturedinthepres-
ence of varying amounts of stock or phenol-extracted PPS3,
PPS14,orC-PSfor48h,followedbymeasurementofconcentra-
tionsofsecretedIL-6andTNF-(cid:1).StockPPS3,PPS14,andC-PS
allinducedbothIL-6(Fig.3A)andTNF-(cid:1)(datanotshown)from
culturedperitonealmacrophagesinaverysimilardose-dependent
manner.Thisactivitycouldbemostlyabrogated,ineachcase,by
prior phenol extraction. Previous incubation of PPS14 and C-PS,
althoughnotPPS3,at80°Cfor1halsoledtothelossofcytokine-
inducingactivity(datanotshown).
To further determine whether the removal of this cytokine-in-
ducing activity affected the subsequent Ig response to the PPS in
FIGURE 1. InvivoIgGanti-PPSresponsestoisolatedPPSpreparations
vivo,weimmunizedwild-typemicewithstockorphenol-extracted
aredependentonTLRsignaling.Wild-type(B6129SF2/J)andTLR2(cid:1)/(cid:1)
PPS14. Phenol-extracted PPS14 induced a significantly reduced
mice(fivepergroup)wereimmunizedi.p.withPPS3,PPS14,andC-PS
IgG,althoughnotIgM-anti-PPS14,responsecomparedwithstock
dissolvedinsterilePBS.Ineachcase,1(cid:4)gofeachPPSwasinjected.Sera
PPS14 (Fig. 3B), suggesting that this cytokine-inducing activity
were prepared from blood obtained from the tail vein on day 0 (before
immunization),day7,and/orday14(datanotshown).SerumtitersofIgM was important for IgG induction in vivo. A similar reduction in
andIgGanti-PPSweredeterminedbyELISA.Thisexperimentisrepre- serumIgG,butnotIgM,anti-PPS14titerswasobserveduponim-
sentativeoftwoindependentexperimentsperformed. munization with heat-inactivated (80°C for 1 h) vs stock PPS14
TheJournalofImmunology 3087
FIGURE 3. TheTLR2ligand(s)isdistinctfromPPS3,PPS14,orC-PS.A,Peritonealmacrophagesfromwild-typemicewereincubatedat37°Cfor48h
with the indicated concentrations of either untreated (stock) or phenol-extracted PPS3, PPS14, or C-PS. Supernatants were collected 48 h later for
measurementofcytokineconcentrationsbyELISA.Thisexperimentisrepresentativeoftwoindependentexperimentsperformed.B,Wild-typemice(seven
pergroup)wereimmunizedi.p.with1(cid:4)g/mouseofeitherstockorphenol-extractedPPS14.Serawerepreparedfrombloodobtainedfromthetailvein
onday0(beforeimmunization)andday7.SerumtitersofIgMandIgGanti-PPS14weredeterminedbyELISA.Thisexperimentisrepresentativeoftwo
independentexperimentsperformed.
(data not shown). Coating the ELISA plates with stock, phenol- elicited serum titers of IgG anti-PPS Ab between TLR2(cid:1)/(cid:1) and
extracted, or heat-inactivated PPS14 had no significant effect on MyD88(cid:1)/(cid:1)micecomparedwiththeirwild-typecontrolssuggested
theserumIgGanti-PPS14titersinanyoftheimmunizationgroups thepresenceinthePneumovax-23preparationofaligand(s)fora
(data not shown), strongly suggesting that neither phenol extrac- TLR(s)inadditiontoTLR2.
tionnorheatinactivationsignificantlyalteredPPS14epitopes. Pneumovax-23 induced IL-6 (Fig. 4B) and TNF-(cid:1)(data not
shown) secretion in wild-type peritoneal macrophages in a dose-
Pneumovax-23containsbothTLR2andTLR4ligandsthatare dependent manner. Macrophages from wild-type and TLR2(cid:1)/(cid:1)
collectivelycriticalforinductionofIgGand,toalesserextent,
mice showed equivalent cytokine induction in response to Pneu-
IgManti-PPSresponsesinvivo
movax-23(Fig.4C).Incontrast,C3H/HeJ(TLR4-defective)mac-
We next determined the potential clinical relevance of possible rophages were largely, although not completely, defective,
contamination of commercial PPS vaccines with TLR ligands. whereas no detectable cytokine release was observed in macro-
Pneumovax-23isacommercialvaccineconsistingofamixtureof phages from TLR2(cid:1)/(cid:1) (cid:2) HeJ mice. Although heat pretreatment
highlypurifiedcapsularpolysaccharidesinisotonicsalinefromthe had no effect on Pneumovax-23-mediated cytokine induction in
23mostprevalentorinvasivepneumococcaltypesofStreptococ- wild-type and TLR2(cid:1)/(cid:1) macrophages, it eliminated the residual
cus pneumoniae (14). This vaccine is administered to adults, es- cytokine secretion observed in macrophages from C3H/HeJ mice
pecially the elderly, who are at increased risk for pneumococcal (Fig.4C).Thesedatastronglysuggestthepresenceofbothaheat-
infections,andhasshownmoderatesuccessinhostprotectionvia sensitive TLR2 and a heat-resistant TLR4 ligand(s) in the Pneu-
inductionofIgGanti-PPSAb.Wefirstdeterminedapotentialrole movax-23preparationthatappearstobecriticalforIgGand,toa
for TLR signaling in the ability of Pneumovax-23 to induce IgM lesser extent, IgM anti-PPS responses in vivo. Analysis of endo-
andIgGanti-PPSAbinvivo.TLR2(cid:1)/(cid:1)orMyD88(cid:1)/(cid:1)micewere toxin content in Pneumovax-23 strongly suggests that this TLR4
immunizedi.p.withPneumovax-23,andserawereobtained7and ligand(s)isnotLPS(seeMaterialsandMethods).
14 days later for measurement of IgM and IgG titers specific for
the whole PPS vaccine. TLR2(cid:1)/(cid:1) mice showed a significant, al- PrevnarcontainsaTLR2ligand(s)thatenhanceselicitedIgG
anti-PPSresponsesinvivo
thoughpartial,reductioninbothelicitedIgMandIgGanti-PPSAb
titerscomparedwithwild-typemiceonbothday7(Fig.4A),when Prevnarisacommercialpneumococcalconjugatevaccineconsist-
the peak of the response occurred, and day 14 (data not shown) ingofsevenPPSserotypesconjugatedtodiphtheriaCRM197pro-
after immunization. More strikingly, MyD88(cid:1)/(cid:1) mice failed to tein and suspended in aluminum phosphate adjuvant (15). The
makeanydetectableIgGanti-PPSAbafterPneumovax-23immu- linkageoftheCRM197proteintoPPSconvertsthevaccinefrom
nization compared with wild-type mice (Fig. 4A). Similar to aTI-2toaTDAgwithresultanthigherPPS-specificIgtitersand
TLR2(cid:1)/(cid:1)mice,MyD88(cid:1)/(cid:1)miceexhibitedasignificant,although abroaderIgGisotypeprofile.Moreimportantly,althoughinfants
partial,reductioninIgManti-PPSAbtiters.Thedifferenceinthe make poor anti-PPS responses to isolated PPS Ags, they elicit a
3088 TLRSIGNALINGANDANTI-PSIgRESPONSE
FIGURE 4. Pneumovax-23containsTLR2andTLR4
ligands critical for IgG induction in vivo. A, Wild-type
(B6129SF2/J) and TLR2(cid:1)/(cid:1) mice (seven per group) or
wild-type (C57BL/6) and MyD88(cid:1)/(cid:1) mice (seven per
group)wereimmunizedi.p.withPneumovax-23(1(cid:4)gof
eachPPS).Serawereobtainedondays0(beforeimmu-
nization)andday7.SerumtitersofIgMandIgGspecific
forallPPSscontainedwithinPneumovax-23weredeter-
minedbyELISA.Thisexperimentisrepresentativeoftwo
independentexperimentsperformed.B,Peritonealmacro-
phages from wild-type (B6129SF2/J) mice were incu-
batedat37°Cfor48hwiththeindicatedconcentrationsof
Pneumovax-23,andSNswerecollectedformeasurement
ofcytokineconcentrationsbyELISA.Thisexperimentis
representativeoftwoindependentexperimentsperformed.
C, Peritoneal macrophages from wild-type (C3H/HeN),
TLR2(cid:1)/(cid:1) (C3H/HeN background), C3H/HeJ, and
TLR2(cid:1)/(cid:1)(cid:2)C3H/HeJmicewereincubatedat37°Cfor
48hwith10(cid:4)g/mlPneumovax-23(withorwithoutpre-
viousincubationat80°Cfor1h),andSNswerecollected
for measurement of cytokine concentrations by ELISA.
Oneexperimentwasperformed.
robust,protectiveanti-PPSresponsetoPrevnar.AswithPneumo- long been known that isolated PS Ag preparations alone induce
vax-23,wewantedtodeterminewhetherPrevnarcontainsaTLR specific IgM and IgG responses in vivo, although the underlying
ligand(s),andwhetherthisinfluencestheelicitedIgMand/orIgG mechanismforthisinductionhasremainedobscure.Thereported
anti-PPS responses in vivo. Prevnar induced both IL-6 (Fig. 5A) abilityofisolatedcapsularPSpreparationstoinducecytokinesin
and TNF-(cid:1)(data not shown) production in wild-type peritoneal vitro (3–6) suggested the presence of an activity that might co-
macrophages in a dose-dependent manner. In contrast, this cyto- stimulatePS-specificBcellstosecreteIginvivo,butthenatureof
kine response was markedly attenuated in macrophages from this activity has not been elucidated. Although specific amplifier
TLR2(cid:1)/(cid:1)mice(Fig.5A).Onthebasisofthesedata,wild-typeand andsuppressorTcellscanregulateIgresponsestoisolatedPSAgs
TLR2(cid:1)/(cid:1) mice were immunized i.p. with either 1.0 or 4.0 (cid:4)g of (22), such Ig responses can still occur normally in the complete
Prevnar and boosted i.p. on day 42 with the same corresponding absenceofTcells,suggestingthepresenceofanon-Tcellsecond
dose.Serawerecollectedonthedaysindicatedformeasurementof signal for Ig induction (23). Recently, a role for endogenous
serum titers of IgM and IgG isotypes specific for all seven PPSs CD40-CD40Linteractionsinpromotinganti-PSresponsestoiso-
containedwithinthevaccine(seeMaterialsandMethods).Prevnar latedPSAgshasbeendemonstrated,althoughthecellularbasisfor
elicitedIgMandIgGofallisotypesinwild-typemice,withsub- thisremainsunknown(24).Inthisregard,previousinvitrostudies
stantial boosting of IgG, although not IgM, upon secondary im- have demonstrated that combined mIg plus CD40 signaling is
munization (Fig. 5B). IgM and IgG anti-PPS titers were higher markedlysynergisticforBcellproliferation(25),butdoesnotlead
(4.0- to 7.5-fold for IgM and 5.5- to 21-fold for IgG) in mice todifferentiationorIgclassswitchingintheabsenceofadditional
receiving 4.0 (cid:4)g of Prevnar compared with those given 1.0 (cid:4)g stimuli (26). PS Ags may also activate complement in vivo (27),
(Fig.5B).Wild-typeandTLR2(cid:1)/(cid:1)miceelicitedverysimilarIgM resulting in cocross-linking of mIg and the type 2 complement
anti-PPS responses to either dose of Prevnar. In contrast, both receptor(CD21).AlthoughcoligationofmIgandCD21issyner-
primary and secondary IgG anti-PPS responses to 4.0 (cid:4)g of Pre- gisticforBcellactivation(28),andlinkageofC3dtoisolatedPS
vnar were substantially higher in wild-type compared with canenhanceanti-PSresponsesinvivo(29),itisunclearwhether
TLR2(cid:1)/(cid:1)mice(day7,4.4-fold;day14,3.4-fold;day42,1.9-fold BcelldifferentiationandIgclassswitchingwilloccursolelyonthe
(primary);day49,4.0-fold(secondary)).Asignificantlyhighersec- basisofmIg/CD21cocross-linking.However,complementcanac-
ondaryIgGanti-PPSresponseinwild-typecomparedwithTLR2(cid:1)/(cid:1) tivate numerous immune cell types, potentially leading to the re-
micewasalsoobservedusing1.0(cid:4)gofPrevnar(2.9-fold;Fig.5B). lease of mediators that promote B cell differentiation. Thus, the
AnalysisoftheIgGisotypeprofilesofanti-PPSAbselicitedinthe nature of the differentiative signal(s) for in vivo Ig responses to
primaryresponsedemonstratedthatthereductioninIgGinTLR2(cid:1)/(cid:1) isolatedPSAgshasremainedofinterest.
micewaslargelyduetodecreasesinIgG3andIgG2a.Incontrast,all Our observations that specific in vivo IgG and, to a lesser and
IgGisotypesinTLR2(cid:1)/(cid:1)miceweresignificantlyreducedaftersec- morevariabledegree,IgMresponsestoisolatedPPSpreparations
ondaryimmunizationcomparedwithwild-typemice,especiallywith critically depend on the presence of a distinct, copurified TLR2
thehigher,4.0-(cid:4)gdoseofPrevnar(Fig.5B).Collectively,thesedata ligand(s),suggestthatpurifiedPSAgsbythemselvesareineffec-
indicatethattheunexpectedpresenceofaTLR2ligand(s)inPrevnar tive immunogens for IgG induction in the absence of a separate
exertsasignificantenhancingeffectontheelicitationofbothprimary TLR/cytokinecostimulus.Inthisstudywemeasuredmacrophage
andsecondaryIgGanti-PPSAbs. productionofIL-6andTNF-(cid:1)inresponsetovariousPPSprepa-
rationstoassessthepresenceofTLRligands.However,thisdoes
Discussion notspecificallyimplyarolefortheseparticularcytokinesinme-
Wepreviouslydemonstrated,usingapolyclonalinvitromodelfor diating the IgG anti-PPS response. Collectively our early studies
multivalentmIgcross-linkinginresponsetoPSAgs((cid:1)(cid:2)-dex),that using (cid:1)(cid:2)-dex (2), our more recent study using intact Pn14 (13),
multivalent mIg cross-linking alone induces vigorous B cell pro- and the current study support the idea that physiologic anti-PS
liferation,butnodifferentiationorIgclassswitchingunlessvari- responsestointactbacteriaareprobablymediatedatleastinpart
ousTLRligandsorcertaincytokinesarealsopresent(1,2).Ithas byPS-inducedmIgcross-linkingofspecificBcellscombinedwith
TheJournalofImmunology 3089
FIGURE 5. PrevnarcontainsaTLR2ligand(s)thatstimulatesanti-PPSresponsesinvivo.A,Peritonealmacrophagesfromwild-type(B6129SF2/J)or
TLR2(cid:1)/(cid:1) mice were incubated at 37°C for 48 h with the indicated concentrations of Prevnar, and SNs were collected for measurement of cytokine
concentrationsbyELISA.B,Wild-type(B6129SF2/J)andTLR2(cid:1)/(cid:1)mice(sevenpergroup)wereimmunizedi.p.witheitherPrevnar(containingeither
0.12or0.5(cid:4)g(PPS)ofeachindividualconjugateexceptPPS6B(0.25or1.0(cid:4)g);totalof1.0or4.0(cid:4)g,respectively)andboostedi.p.42dayslaterwith
thesamedoseofPrevnar.SerawerecollectedontheindicateddaysformeasurementofPPS-specificIgMandIgGisotypetitersbyELISA.Thisexperiment
isrepresentativeofthreeindependentexperimentsperformed.
costimulationbyassociatedbacterialTLRligands.Additionalac- mococcalprotein,pneumolysin,likeendotoxin,isaTLR4ligand
tivationsignalsmediatedbyCD40/CD40LinteractionsandCD4(cid:6) (12),aheat-resistantTLR4activitywasobservedinPneumovax-
T cells appear to amplify this response (19). The mechanism un- 23, suggesting the presence of an additional, nonprotein, TLR4
derlying the residual IgM anti-PS response remains unresolved. ligand. TLR2 ligands expressed by Pn include LTA, peptidogly-
TheabilityofNKcellstodirectlyinducemIg-activatedBcellsto can,andlipoprotein(9–11).
secreteIgM(30)providesonepossibleTLR-independentmecha- This study is of interest in light of recent data indicating that
nism,butthisremainstobedemonstratedinvivo. commercialenterobacterialLPSpreparationshavemodestTLR2-
ThesedataalsosuggestthattheeffectivenessofPS-containing stimulatingactivityinadditiontostrongTLR4-activatingproper-
vaccinesmaycurrentlydependontheunintendedcopurificationof ties.ThisTLR2activitywasshowntobetheresultofalipoprotein
bacterial TLR ligands during the PS purification process. Thus, contaminantthatcouldberemovedbyphenolextraction(20,31).
detectable IgG anti-PPS responses to Pneumovax-23 were totally Morerecently,itwasdemonstratedthatanLPSpreparationfrom
dependentonthepresenceofassociatedTLR2andTLR4ligands. Porphyromonas gingivalis, previously shown to activate immune
Thislatterpointmayhavesubstantialrelevancewithregardtothe cells through TLR2, but not TLR4, also had a contaminating li-
specificproceduresbywhichthesePSAgsarepurified,including poproteinastheprincipalcomponentoftheTLR2activity(32).In
the immunogenic consistency of different vaccine lots, because contrast,P.gingivalislipidAexhibitedrelativelyweakTLR4ac-
IgGisconsideredthemainprotectiveIgisotype.Thenatureofthe tivity.Finally,ithasbeensuggestedthatthepresenceofcontam-
TLR ligands present within the PPS preparations is currently un- inating LPS and an undefined TLR2 ligand(s) may be important
known.Ofnote,noneofthepreparationshadsignificantendotoxin fortheTIimmunostimulatorypotentialofrecombinanthepatitisB
contamination (see Materials and Methods). Although the pneu- nucleocapsidpreparationsproducedinEscherichiacoli(33).
3090 TLRSIGNALINGANDANTI-PSIgRESPONSE
The ability of an associated TLR2 ligand in the pneumococ- 4. Soell,M.,M.Diab,G.Haan-Archipoff,A.Beretz,C.Herbelin,B.Poutrel,
cal conjugate vaccine, Prevnar, to enhance both primary and andJ.-P.Klein.1995.Capsularpolysaccharidetypes5and8ofStaphylococ-
cusaureusbindspecificallytohumanepithelial(KB)cells,endothelialcells,
secondary IgG anti-PPS responses was somewhat surprising, and monocytes and induce release of cytokines. Infect. Immun. 63:
because Prevnar is injected with aluminum phosphate adjuvant 1380–1386.
and,unlikeisolatedPPSAgs,promotesrecruitmentofclassical 5. Leiva, L. E., B. Butler, J. Hempe, A. P. Ortigas, and R. U. Sorensen. 2001.
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6. Jagger,M.P.,Z.Huo,andP.G.Riches.2002.Inflammatorycytokine(interleukin
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6andtumornecrosisfactor(cid:1))releaseinahumanwholebloodsysteminresponse
inpromotingtype1IgGisotypeproduction(i.e.,IgG3,IgG2b, toStreptococcuspneumoniaeserotype14anditscapsularpolysaccharide.Clin.
andIgG2a,butnotIgG1)inresponsetointactPn14(13).TLR Exp.Immunol.130:467–474.
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(36) demonstrated that a soluble conjugate of meningococcal
10. Koedel,U.,B.Angele,T.Rupprecht,H.Wagner,A.Roggenkamp,H.-W.Pfister,
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producingloweranti-HibPSIgGandIgMtiterscomparedwith
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wild-typemice.Theoutermembraneproteincomplex,thecar- Peptidoglycan-andlipoteichoicacid-inducedcellactivationismediatedbyToll-
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