Table Of ContentTheJournalofImmunology
A Novel ICOS-Independent, but CD28- and SAP-Dependent,
Pathway of T Cell-Dependent, Polysaccharide-Specific
Humoral Immunity in Response to Intact Streptococcus
pneumoniae versus Pneumococcal Conjugate Vaccine1
Quanyi Chen,* Jennifer L.Cannons,† JamesC.Paton,‡ HisayaAkiba,§
PamelaL.Schwartzberg,† and Clifford M.Snapper2*
Polysaccharide(PS)-andprotein-specificmurineIgGresponsestointactStreptococcuspneumoniae(Pn)arebothdependenton
CD4(cid:1)Tcellhelp,B7-dependentcostimulation,andCD40/CD40ligandinteractions.However,theprimaryPS-specific,relativeto
protein-specific,IgGresponseterminatesmorerapidly,requiresashorterperiodofTcellhelpandB7-dependentcostimulation,
and fails to generate memory. In light of the critical role for ICOS/ICOS ligand interactions in sustaining T cell-dependent Ig
responses and promoting germinal center reactions, we hypothesized that this interaction was nonessential for PS-specific IgG
responsestoPn.WenowdemonstratethatICOS(cid:2)/(cid:2),relativetowild-type,miceelicitanormalPS-specificIgGisotyperesponse
to Pn, despite marked inhibition of both the primary and secondary IgG anti-protein (i.e., PspA, PspC, and PsaA) response. A
blockinganti-ICOSligandmAbinjectedduringprimaryPnimmunizationinhibitsboththeprimaryanti-proteinresponseandthe
generationofprotein-specificmemory,buthasnoeffectwheninjectedduringsecondaryimmunization.IncontrasttoPn,bothPS-
andprotein-specificIgGresponsestoapneumococcalconjugatevaccineareinhibitedinICOS(cid:2)/(cid:2)mice.ICOS(cid:2)/(cid:2)miceimmunized
withintactPnorconjugateexhibitnearlycompleteabrogationingerminalcenterformation.Finally,althoughmicethatlackthe
adaptor molecule SAP (SLAM-associated protein) resemble ICOS(cid:2)/(cid:2) mice (and can exhibit decreased ICOS expression), we
observethatthePS-specific,aswellasprotein-specific,IgGresponsestobothPnandconjugatearemarkedlydefectiveinSAP(cid:2)/(cid:2)
mice. These data define a novel T cell-, SAP-, and B7-dependent, but ICOS-independent, extrafollicular pathway of Ig
induction. TheJournalofImmunology,2008,181:8258–8266.
S
ystemic immunization with intact Streptococcus pneu- (PC)ofthecellwallC-polysaccharide(C-PS,teichoicacid).The
moniae,capsulartype14(Pn14)3elicitsanIgGresponse protein- and PS-specific IgG responses to intact Streptococcus
specificforanumberofpneumococcalproteins,including pneumoniae(Pn)areeachdependentonCD4(cid:1)Tcells,B7/CD28-
pneumococcal surface protein A (PspA), as well as the capsular dependent costimulation, and CD40-CD40 ligand interactions,
polysaccharide (PPS14) and the phosphorylcholine determinant whereas the PS-specific IgM response is T cell-independent (TI)
(1–3). Nevertheless, relative to the protein-specific response, the
primaryIgGanti-PSresponsetointactPnpeaksearlier,requiresa
*DepartmentofPathology,UniformedServicesUniversityoftheHealthSciences, shorterperiodofTcellhelpandB7-dependentcostimulation,and
and†NationalHumanGenomeResearchInstitute,NationalInstitutesofHealth,Be-
in contrast to the anti-protein response, fails to elicit a boost in
thesda, MD 20814; ‡School of Molecular and Biomedical Science, University of
Adelaide, Adelaide, South Australia, Australia; and §Department of Immunology, serum PPS14-specific IgG titers upon secondary immunization.
JuntendoUniversitySchoolofMedicine,Tokyo,Japan PPS14expressedbyintactPn14behavesasadistinctimmunogen
ReceivedforpublicationMay30,2008.AcceptedforpublicationOctober15,2008. relative to either isolated, soluble PPS14 or PPS14 covalently
Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage linked to PspA (PPS14-PspA) (soluble conjugate). Thus, the IgG
charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordance
response to isolated PPS14 is TI, whereas immunization with
with18U.S.C.Section1734solelytoindicatethisfact.
PPS14-PspA induces CD4(cid:1) T cell-dependent, PPS14-specific
1ThisstudywassupportedbyNationalInstitutesofHealthGrants1R01AI49192(to
C.M.S.)andtheUniformedServicesUniversityoftheHealthSciencesDean’sRe- memory,inadditiontoPspA-specificmemory(4,5)Themecha-
searchandEducationEndowmentFund(toC.M.S.)andbyfundingfromtheintra- nismunderlyingthedivergentimmunologicbehaviorbetweenin-
muralprogramoftheNationalHumanGenomeResearchInstitute(toP.L.S.).
tactPN14andsolublepneumococcalconjugateisunresolved.
OnoptintoiobnescaonndstarsuseedrtaiosnosfficcoinatlaoinrerdeflheecrteiningathreetvhieewprsivoaftteheonDeespoafrttmheenatuothfoDrsefaenndseaorer ICOSisamemberoftheCD28familythatisinducedonCD4(cid:1)
theUniformedServicesUniversityoftheHealthSciences. T cells upon TCR cross-linking and CD28-mediated signaling,
2AddresscorrespondenceandreprintrequeststoDr.CliffordM.Snapper,Depart- whereas CD28 is constitutively expressed (6, 7). The respective
mentofPathology,UniformedServicesUniversityoftheHealthSciences,4301Jones
cognate ligands, ICOSL and B7-1/B7-2, expressed on APC are
BridgeRoad,Bethesda,MD20814.E-mailaddress:[email protected]
constitutivelyexpressedbutcanbeup-regulatedbyinflammatory
3Abbreviationsusedinthispaper:Pn14,intactStreptococcuspneumoniae,capsular
type 14; CpG-ODN, CpG-containing oligodeoxynucleotide; FB, follicular B; GC, stimuli. In this regard, CD28 is critical for initiation of CD4(cid:1) T
germinalcenter;ICOSL,ICOSligand;KLH,keyholelimpethemocyanin;MZB,mar- cellactivation(8–10),whereasICOSplaysakeyroleinthesub-
ginalzoneB;NP,(4-hydroxy-3-nitrophenyl)acetyl;PC,phosphorylcholinedetermi-
sequent T cell effector response (11, 12). Genetic disruption or
nantofteichoicorlipoteichoicacid;Pn,intactStreptococcuspneumoniae;PPS14,
capsularpolysaccharide,serotype14;PS,polysaccharide;PsaA,pneumococcalsur- blockadeoftheB7/CD28orICOS/ICOSLpathwaysinhibitsboth
faceadhesinA;PspA,pneumococcalsurfaceproteinA;PspC,pneumococcalsurface type 1 and type 2 CD4(cid:1) T cell-dependent humoral immune re-
proteinC;SAP,SLAM-associatedprotein;TD,Tcelldependent;TI,Tcellindepen-
dent;WT,wildtype. sponses, although secondary, relative to primary, responses are
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A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of
T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in 5b. GRANT NUMBER
Response to Intact Streptococcus pneumoniae versus Pneumococcal
Conjugate Vaccine 5c. PROGRAM ELEMENT NUMBER
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TheJournalofImmunology 8259
impacted to a greater degree by loss of ICOS signaling (13–15). Materials and Methods
ICOS-mediatedcostimulationiscriticalforthedevelopmentofthe Mice
germinal center (GC) reaction (16), in part through induction of
SAP(cid:2)/(cid:2)mice(30)werebackcrossedtoC57BL/6micefor8–10genera-
CD40L on the CD4(cid:1) T cell (13, 14) and the development of tions.CD28(cid:2)/(cid:2)mice(15)(C57BL/6background;B6.129S2-Cd28tm1Mak/J,
CD4(cid:1)CXCR5(cid:1)follicularhelperTcells(17,18),andisthusisa catalog no. 002666) and ICOS(cid:2)/(cid:2) mice (13) (C57BL/6 background;
key regulator of immunologic memory. Genetic deficiency in B6129P2-ICOStm1Mak/J, catalog no. 004859) were purchased from The
JacksonLaboratory.C57BL/6andBALB/cmicewerepurchasedfromthe
ICOSinhumansleadstothecommonvariableimmunodeficiency
NationalCancerInstitute.Femalemicewereusedbetween7and10wkof
syndrome, characterized by hypogammaglobulinemia and an al-
age. These studies were conducted in accordance with the principles set
mostcompletelackofmemoryBcells(19). forthintheGuideforCareandUseofLaboratoryAnimals(Instituteof
CD4(cid:1)Tcells,aswellasCD8(cid:1)andNKTcells,NKcells,and LaboratoryAnimalResources,NationalResearchCouncil,revised1996),
and they were approved by the Uniformed Services University of the
someBcells,expressacytoplasmicadaptorprotein,SAP(SLAM-
HealthSciencesInstitutionalAnimalCareandUseCommittee.
associatedprotein),whichisencodedbythegeneSH2D1Aandis
criticalforcellsignalingviaSLAMfamilyproteinsexpressedon Reagents
thecellsurface(20–22).GeneticdeficiencyofSAPinbothmice Recombinant PspA was expressed in Saccharomyces cerevisiae BJ3505
and humans (X-linked lymphoproliferative syndrome) results in andpurifiedaspreviouslydescribed(31).Recombinantpneumococcalsur-
immunologic phenotypes, some of which are similar to loss of faceadhesinA(PsaA)andpneumococcalsurfaceproteinC(PspC;CbpA)
ICOS/ICOSLinteractions(18,23).Thus,althoughSAP(cid:2)/(cid:2)CD4(cid:1) wereexpressedasN-terminalHis6fusionproteinsinrecombinantEsche-
richiacoliandpurifiedbyNi-NTAaffinitychromatography,aspreviously
T cells can undergo initial proliferation and activation similar to described(32).PurifiedPPS14waspurchasedfromAmericanTypeCul-
wild-type (WT) CD4(cid:1) T cells, they can exhibit decreased ICOS tureCollection.PC-KLHwassynthesizedaspreviouslydescribed(1).The
expression,andinvivotheyshowmarkedlyimpairedinductionof resultingconjugatehadasubstitutionratioof19PC/KLHmolecule.Sol-
uble conjugates comprising PspA covalently linked to either C-polysac-
GC and memory B cells (23–26). In this regard, the induction of
charide(C-PS-PspA)orPPS14(PPS14-PspA)weresynthesizedasprevi-
GC and memory B cells is inhibited in SAP(cid:2)/(cid:2) mice. Notably, ouslydescribed(3).AblockingmAbagainstmouseB7RP-1(ICOSligand;
immunization of SAP(cid:2)/(cid:2) mice with T cell-dependent (TD) Ags cloneHK5.3,ratIgG2a)wasgeneratedaspreviouslydescribed(33).The
HK5.3 mAb and a control rat IgG2a anti-E. coli (cid:1)-galactosidase mAb
(soluble(4-hydroxy-3-nitrophenyl)acetyl(NP)-keyholelimpethe-
(clone GL117) were purified from culture supernatant using protein G
mocyanin (KLH) or SRBC) resulted in dramatic defects in GC
chromatography.
formation, primary and secondary Ag-specific IgG production
PreparationofPn14
(IgG1mostdramaticallyreduced,thenIgG2b/IgG2a,thenIgG3),
butnotIgMproduction,anddramaticdefectsinthegenerationof A frozen stock of Pn14 was thawed and subcultured on BBL premade
long-livedplasmacellsinthebonemarrow(26).Inadoptivetrans- blood agar plates (VWR International). Isolated colonies on blood agar
ferstudiesbothTh1andTh2SAP(cid:2)/(cid:2)Tcellsfailedtopromotean were grown in Todd Hewitt broth (BD Biosciences) to mid-log phase,
collected, and heat killed by incubation at 60°C for 1 h. Sterility was
Igresponse.PatientswithX-linkedlymphoproliferativesyndrome confirmedbysubcultureonbloodagarplates.Afterextensivewashings,
likewiseexhibithypogammaglobulinemiaandamarkedreduction the bacterial suspension was adjusted with PBS to give an absorbance
in memory (CD27(cid:1)) B cells associated with reduced T cell ex- readingat650nmof0.6whichcorrespondedto109CFU/ml.Bacteriawere
thenaliquotedat1010CFU/mlandfrozenat(cid:2)80°CuntiltheiruseasAg
pression of ICOS (24, 27). Collectively, these data raise the pos-
formouseimmunizations.ThePn14stockwastestedforendotoxinusing
sibility that the failure of SAP(cid:2)/(cid:2) CD4(cid:1) T cells to up-regulate theLimulusamebocytelysateassay(QCL-1000)fromBioWhittaker.This
ICOS might contribute to the defective humoral immunity in the assaydemonstratedthatmiceinjectedwith2(cid:4)108CFUequivalentsof
SAP-deficient host. However, more recent data demonstrate that heat-killedPn14receive(cid:5)20pgofendotoxin.
SAP(cid:2)/(cid:2) CD4(cid:1) T cells can up-regulate ICOS, but fail to provide Immunizations
criticalsignalstoBcellsduetoaselectivedefectinTcell-Bcell
Mice(n(cid:6)7/group)wereimmunizedi.p.with2(cid:4)108CFUheat-killed
adhesion(28). Pn14insalineori.p.with1(cid:2)g(weightofpolysaccharide)eachofPPS14-
Not surprisingly, deficiency in either SAP or ICOS/ICOSL is PspA (cid:1) C-PS-PspA adsorbed on 13 (cid:2)g of alum (Allhydrogel, 2%;
associatedwithdefectiveTDhumoralimmunityandhostprotec- BrenntagBiosector)mixedwith25(cid:2)gofastimulatory30-merCpG-con-
tainingoligodeoxynucleotide(CpG-ODN)(34)andsimilarlyboosted.Se-
tion against a number of microbial pathogens. As mentioned
rum samples for measurement of anti-PPS14, anti-PC, anti-PspA, anti-
above,thePS-specificTDhumoralresponsetointactPn14isun- PsaA,andanti-PspCIgisotypetiterswerepreparedfrombloodobtained
usualinthattheprimaryIgGresponseandrequirementforCD4(cid:1) throughthetailvein.
TcellsandB7-dependentcostimulationarerelativelyshort-lived.
MeasurementofserumAg-specificIgisotypetiters
This is associated with a failure to generate PS-specific memory,
despite a critical role for B7/CD28 and CD40/CD40L costimula- Immulon 4 ELISA plates (Dynex Technologies) were coated (50 (cid:2)l/
well)withPC-KLH(5(cid:2)g/ml),PPS14(5(cid:2)g/ml),PspA,PsaA,orPspC
tionfortheprimaryresponse(1–3).SinceICOSinductionfollows (5(cid:2)g/ml)inPBSovernightat4°C.Plateswerewashedthreetimeswith
initialTCR-andCD28-mediatedCD4(cid:1)Tcellactivation,peaking PBS(cid:1)0.1%Tween20andwereblockedwithPBS(cid:1)1%BSAfor1h
(cid:3)48hfollowingimmunization(12,29),wehypothesizedthatthe at37°C.Three-folddilutionsofserumsamples,startingata1/50serum
PS-specific, but not protein-specific, IgG response to intact Pn14 dilution, in PBS (cid:1) 1% BSA were then added overnight at 4°C and
plateswerewashedthreetimeswithPBS(cid:1)0.1%Tween20.Alkaline
would be ICOS-independent, whereas both responses would be
phosphatase-conjugated polyclonal goat anti-mouse IgM, IgG, IgG3,
ICOS- and SAP-dependent in response to pneumococcal conju- IgG1,IgG2b,orIgG2aAbs(200ng/mlfinalconcentration)inPBS(cid:1)
gate.However,itremainsunclearhowSAPwouldaffectthePS- 1%BSAwerethenadded,andplateswereincubatedat37°Cfor1h.
PlateswerewashedfivetimeswithPBS(cid:1)0.1%Tween20.Substrate
specific responses to Pn14. In this report we demonstrate that,
(p-nitrophenylphosphate,disodium;Sigma-Aldrich)at1mg/mlinTM
indeed, the PS-specific IgG response to intact Pn14 represents a buffer(1MTris(cid:1)0.3mMMgCl (pH9.8))wasthenaddedforcolor
2
novel B7-dependent, ICOS-independent TD response. However, development.Colorwasreadatanabsorbanceof405nmonaMulti-
despite its ICOS independence, the PS-specific IgG response to skan Ascent ELISA reader (Labsystems). Serum Ig titers were calcu-
Pn14inmarkedlyinhibitedinSAP(cid:2)/(cid:2)mice,stronglysuggesting latedasfollows:Astandardcurvewasgeneratedusing3-folddilutions
ofapositiveserumsample,startingwithaninitial1/50serumdilution.
that SAP plays a key role at the earliest stages of T cell help for
The signal (in fluorescent units) from the most dilute sample of the
humoralimmuneresponses,independentofICOS. standardcurvethatwasstillabovebackgroundwasrandomlyassigned
8260 ICOS-INDEPENDENTPATHWAYOFTCELL-DEPENDENTHUMORALIMMUNITY
FIGURE 1. The IgG anti-protein and anti-poly-
saccharide response to Pn14 is CD28-dependent. WT
(C57BL/6)andCD28(cid:2)/(cid:2)mice(n(cid:6)7/group)wereim-
munizedi.p.andthenboostedonday14witheither(A)
Pn14insalineor(B)PPS14-PspA(cid:1)C-PS-PspAinal-
um/CpG-ODN. Serum titers of Ag-specific IgM and
IgGareillustratedforday0(pre-immune)andday21
(immune).(cid:1),p(cid:5)0.05betweenWTandCD28(cid:2)/(cid:2)mice.
a titer of “1,” and all signals from consecutively less dilute samples Results
wereassignedthenumbers3,9,27,andsoforth.Foreachexperimental
TheIgGanti-proteinandanti-polysaccharideresponsetoPn14
sample,adilutionwaschosenthatgeneratedasignalwithinthelinear
isCD28-dependent
part of the standard curve. The value extrapolated from the standard
curvewasthenmultipliedbytheinverseofthatdilutiontogeneratethe Immunizationi.p.withintact,heat-killedPn14inducesanIgM
finalinversetiter.
response specific for PPS14 and the PC determinant of C-PS
DetectionofGCs (teichoicacid)and/ormembranelipoteichoicacidthatisTcell-
independent (1, 3). In contrast, the IgG anti-PPS14 and IgG
Mice were injected i.p. with either Pn14 or conjugate. Spleens were
removed12daysafterimmunizationandincubatedatleast6hin15ml anti-PC responses are dependent on CD4(cid:1) T cells and CD40/
ofPLPbuffer(0.05MPBScontaining0.1ML-lysine(pH7.4),2mg/ml CD40Linteractions.Inapreviousstudywealsodemonstrated,
NaIO4, and 10 mg/ml paraformaldehyde). The fixed samples were using the unencapsulated variant of Pn2 (R36A), that the IgG
washed in PBS and dehydrated in 30% sucrose in PBS. Tissues were
snap-frozeninTissue-Tek(VWR).Twenty-to30-(cid:2)m-thickfrozensec- anti-PC response was dependent on B7-2 and CD28 (2). Pn14
tions were cut and stained with anti-mouse B220-Pacific Blue (clone also elicits IgG anti-protein responses (e.g., PspA, PsaA, and
RA36B2,catalogno.558108),anti-mouseCD3-PE(clone17A2,cata- PspC)(35).Inparticular,detailedanalysisoftheIgGanti-PspA
log no. 555275) (BD Biosciences), or peanut agglutinin-Alexa Fluor protein response to Pn14 and/or R36A also demonstrated de-
488 from Arachis hypogaea (catalog no. L21409, Invitrogen). Immu- pendence on CD4(cid:1) T cells, B7-2/CD28 costimulation, and
nofluorescenceimagingwasperformedwithaZeissPascallaserscan-
ningconfocalmicroscope.Separateimageswerecollectedforeachflu- CD40/CD40Linteractions.InlightofourcurrentuseofPn14in
orochrome and overlaid to obtain a multicolor image. Final image thisstudytodetermineapotentialroleofICOScostimulationin
processingwasperformedwithImageJsoftware(NationalInstitutesof mediating Ig responses, we first wanted to determine whether
Health)andAdobePhotoshop.Spleensfromfourmicepergroup(four
the TD IgG anti-PPS14 as well as IgG anti-PC and anti-PspA
sectionsperspleen)wereanalyzedforGCnumberandarea,foratotal
of(cid:3)12mm2ofsplenicareapergroup. responses to Pn14 were dependent on CD28-dependent co-
stimulation. Thus, CD28(cid:2)/(cid:2) and WT mice were immunized
Flowcytometricanalysis
withPn14andboosted14dayslater.SerumtitersofAg-specific
SplenicandperitonealcellsfromWTandknockoutmice(threemiceper IgM and IgG were measured on day 21. As illustrated in Fig.
group,WT,SAP(cid:2)/(cid:2),andICOS(cid:2)/(cid:2);fourCD28(cid:2)/(cid:2)mice)wereharvested 1A, the IgG anti-PspA response to Pn14 was completely abro-
acyntdomBectreyllassufboslelotswfsr:osmpleenaicchminardgiivniadluzaolnseplBee(nMwZeBre;CenDu2m1heirgahCteDd2b3ylowfl/o(cid:2)w), gatedinCD28(cid:2)/(cid:2)mice,similartowhatwepreviouslyobserved
follicularB(FB;CD21intCD23high),B-1(B220intCD5(cid:1)),peritonealB-1a usingR36A(2).WhereastheTIIgManti-PPS14andIgManti-
(B220(cid:1)CD11b(cid:1)CD5(cid:1)), B-1b (B220(cid:1)CD11b(cid:1)CD5(cid:2)), and B-2 (B220(cid:1) PC responses were unaffected in CD28(cid:2)/(cid:2) mice, both the TD
CD11b(cid:2)CD5(cid:2)). The following mAbs purchased from BD Pharmingen IgG anti-PPS14 responses and the TD IgG anti-PC responses
were used: FITC-rat IgG2b,(cid:3)anti-mouse CD21/35 (clone 7G6), PE-rat
were significantly, though partially, reduced relative to WT
IgG2a,(cid:3)anti-mouse CD23 (clone B3B4), FITC-rat IgG2a,(cid:3)anti-mouse
CD45R/B220(cloneRA3-6B2),biotin-ratIgG2a,(cid:3)anti-mouseCD5(Ly-1) mice (Fig. 1A). The partial reduction in the IgG anti-PC re-
(clone 53-7.3) (cid:1) streptavidin-PE-Texas Red, and PE-rat IgG2b,(cid:3)anti- sponse to Pn14 in CD28(cid:2)/(cid:2) mice was similar to what we pre-
mouseCD11b(cloneM1/70).CellswereanalyzedonaBDLSRIIflow viouslyobservedusingR36A(2).Similarresultswereobserved
cytometer(BDBiosciences)using488-and635-nmlasers.
from sera obtained on day 7 and/or day 14 (data not shown).
Statistics Covalent linkage of an immunogenic carrier protein to a
polysaccharide Ag (conjugate) converts the IgG anti-PS re-
SerumIgisotypetiterswereexpressedasgeometricmeans(cid:7)SEMofthe
sponse from TI-2 to TD, with the latter now dependent on
individual serum Ig isotype titers. Splenic and peritoneal B cell subset
numbersandpercentagesweredeterminedbyflowcytometryandareex- CD4(cid:1) T cells, B7-dependent costimulation, and CD40/CD40L
pressed as arithmetic means of the numbers obtained from individual interactions (36, 37), similar to what we observed for intact
mice(cid:7)SEM.SignificanceinbothcaseswasdeterminedbytheStudentt
Pn14(1).Ofinterest,whereastheIgManti-PSresponsetoPn14
test.p-valuesof(cid:5)0.05wereconsideredstatisticallysignificant.Allexper-
isTI,theIgM,aswellastheIgG,anti-PSresponsetoconjugate
iments were performed twice, except for flow cytometry, which was
performedonce. is partially TD. In contrast to a pneumococcal conjugate (i.e.,
TheJournalofImmunology 8261
FIGURE 2. The IgG anti-PPS14
andanti-PCresponsetoPn14,incon-
trast to pneumococcal conjugate, is
ICOS-independent. WT (C57BL/6)
and ICOS(cid:2)/(cid:2) mice (n (cid:6) 7/group)
wereimmunizedi.p.andthenboosted
onday42witheither(A)Pn14insa-
line or (B) PPS14-PspA (cid:1) C-PS-
PspA in alum/CpG-ODN. C, WT
(C57BL/6) and ICOS(cid:2)/(cid:2) mice (n (cid:6)
7/group) were immunized i.p. with
Pn14 in saline and then boosted on
days14and21.SerumtitersofAg-
specific IgM and IgG are illustrated.
(cid:1), p (cid:5) 0.05 between WT and
ICOS(cid:2)/(cid:2)mice.
PPS14-PspA (cid:1) C-PS-PspA in alum/CpG-ODN), the primary were also abrogated in ICOS(cid:2)/(cid:2) mice (Fig. 2C). However, in
IgG anti-PPS14 and IgG anti-PC response to Pn14 develops marked contrast to the IgG anti-protein response, ICOS(cid:2)/(cid:2) mice
with more abbreviated kinetics and does not generate PS-spe- elicited IgM and IgG anti-polysaccharide (PPS14 and PC) re-
cific immunologic memory. We thus wanted to further deter- sponsestoPn14thatwereessentiallysimilartothoseobservedin
mine the CD28 dependence of the conjugate-induced Ig re- WTmice(Fig.2A).
sponse.AsillustratedinFig.1B,theIgGanti-PspAresponseto WenextwantedtodeterminewhetherinductionofthePPS14-
conjugatewascompletelyabrogatedinCD28(cid:2)/(cid:2)mice,similar and PC-specific responses, as well as the PspA-specific Ig re-
to that observed with Pn14 immunization. In contrast to Pn14, sponses, are inhibited in ICOS(cid:2)/(cid:2) mice upon immunization with
both the IgM and IgG anti-PPS14 and anti-PC responses to conjugate. Similar to Pn14, the primary and secondary IgG anti-
conjugate were partially, but not completely, abrogated in PspA response to conjugate was markedly inhibited in ICOS(cid:2)/(cid:2)
CD28(cid:2)/(cid:2) mice. Of interest, the IgG anti-PPS14 and IgG anti- mice, although modest boosting (p (cid:5) 0.05) was observed in
PC responses to conjugate were more strikingly reduced in ICOS(cid:2)/(cid:2)micefollowingsecondaryimmunization(Fig.2B).Ad-
CD28(cid:2)/(cid:2) mice than the same responses to Pn14 (p (cid:5) 0.05). ditionally, the primary IgG anti-PPS14 was essentially abrogated
Thesedatathusdemonstrateabsoluteandrelativedifferencesin in ICOS(cid:2)/(cid:2) mice, whereas a partial, although significant, reduc-
theCD28dependenceoftheIgresponsetoPn14vsconjugate. tion in the IgG anti-PC response was also observed (Fig. 2B).
Similar to the IgG anti-PspA response, the secondary IgG anti-
TheIgGanti-PPS14andanti-PCresponsetoPn14,incontrast PPS14responsewasalsosignificantlylowerthanthatobservedin
topneumococcalconjugate,isICOS-independent WT mice, although significant boosting was observed in both
Numerous studies have documented the critical role of ICOS/ strains. No significant boosting of the IgG anti-PC response was
ICOSL interactions in mediating TD IgG responses (11–14). In observedineitherWTorICOS(cid:2)/(cid:2)mice.TheIgManti-PPS14and
light of the CD28 dependence of both the IgG anti-PPS14 and anti-PC responses to conjugate were largely ICOS-independent
anti-PCresponsestoPn14,previouslyshowntobealsodependent (Fig.2B),althoughtheseresponsesarealsopartiallyTD(datanot
onCD4(cid:1)TcellsandCD40/CD40Linteractions(1,3),wewanted shown).Thus,theIgGanti-polysaccharideresponsestoPn14and
to determine whether they were also ICOS-dependent. Mice ge- pneumococcal conjugate exhibit different requirements for ICOS
neticallydeficientinICOS(ICOS(cid:2)/(cid:2))andWTmicewereimmu- costimulationdespitebothbeingdependentonCD4(cid:1)Tcells,B7-
nized i.p. with Pn14 and boosted 6 wk later. WT mice elicited a dependentcostimulation,andCD40/CD40Linteractions.
detectableprimaryIgGanti-PspAresponseand,followingsecond-
aryimmunization,substantialboostinginPspA-specificIgGserum
ICOScostimulationforinductionoftheIgGanti-PspAresponse
titers(Fig.2A).Asreportedpreviously(35),serumtitersofPspA-
toPn14iscriticalduringprimary,butnotsecondary,
specificIgG3,IgG1,IgG2b,andIgG2awereallinducedduringthe
immunization
primaryimmunizationandboosteduponsecondaryimmunization
(datanotshown).Incontrast,boththeprimaryandsecondaryIgG To confirm our observations in ICOS(cid:2)/(cid:2) mice and further deter-
anti-PspAresponseswereessentiallyabrogatedinICOS(cid:2)/(cid:2)mice minewhen(i.e.,duringprimaryand/orsecondaryresponse)ICOS
(Fig. 2A), reflected by a lack of induction of all 4 PspA-specific costimulation was necessary for the IgG anti-PspA response, we
IgG isotypes (data not shown). In addition to the IgG anti-PspA injectedmicewithaneutralizinganti-ICOSLmAb(HK5.3)either
response to Pn14, the primary and secondary IgG responses spe- at the time of the primary and/or secondary immunization with
cificfortwoadditionalPn14-expressedproteins,PsaAandPspC, Pn14.AsillustratedinFig.3,injectionofanti-ICOSLmAbonly
8262 ICOS-INDEPENDENTPATHWAYOFTCELL-DEPENDENTHUMORALIMMUNITY
FIGURE 3. ICOScostimulationforinductionoftheIgGanti-PspAresponsetoPn14iscriticalduringprimary,butnotsecondaryimmunization.BALB/c
mice(n(cid:6)7/group)wereimmunizedi.p.withPn14insalineandboostedonday42.Anti-ICOSLmAb(cloneHK5.3)oracontrolratIgG2aanti-E.coli
(cid:1)-galactosidasemAb(cloneGL117)(1mg/mouse)wereinjectedonday0and/orday42.SerumtitersofAg-specificIgMandIgGareillustrated.(cid:1),p(cid:5)
0.05betweencontrolmAbandanti-ICOSLmAb.
atthetimeofprimaryimmunizationwithPn14completelyinhib- and thus are likely to represent a completely TD extrafollicular
itedtheprimaryIgGanti-PspAresponse.Followingsecondaryim- response in WT mice, consistent with the absence of PS-specific
munizationwithPn14,6wklaterintheabsenceofanti-ICOSL,a memory generation. In contrast, the inhibition of both the IgG
partial although significant reduction in the secondary IgG anti- anti-PS response and GC formation in response to conjugate in
PspAresponsewasalsoobserved,indicatingthatICOSwasnec- ICOS(cid:2)/(cid:2)miceareconsistentwiththisresponsebeingfollicularin
essary both for the primary response and for the generation of natureinWTmice,associatedwiththegenerationofPS-specific
memory.Ofnote,noeffectontheprimaryIgGanti-PspAresponse memory.
wasobservedwhenPn14wasfirstinjected6wkafterinjectionof
anti-ICOSLalone,indicatingthatthemAbwaseffectivelycleared Protein-andpolysaccharide-specificIgGresponsestoboth
after 6 wk (data not shown). Similar results were observed when Pn14andconjugatearedefectiveinSAP(cid:2)/(cid:2)mice,whereasthe
anti-ICOSLwasinjectedbothatthetimeofprimaryandsecondary IgManti-polysaccharideresponseisdifferentiallyregulatedin
immunizationwithPn14(Fig.3).However,whenmicewerefirst responsetothetwoimmunogens
primedwithPn14intheabsenceofanti-ICOSLandthenboosted TheadaptorproteinSAPhaspreviouslybeenshowntobecritical
in the presence of anti-ICOSL, no effect of the mAb on the sec- for a CD4(cid:1) T cell-dependent humoral immune response. In par-
ondaryIgGanti-PspAresponsewasobserved(Fig.3).Similarto ticular, both the primary and secondary IgG, but not IgM, re-
what was observed in ICOS(cid:2)/(cid:2) mice, anti-ICOSL had no signif- sponses to a number of TD Ags are strongly reduced in SAP(cid:2)/(cid:2)
icanteffectontheIgMorIgGanti-PPS14oranti-PCresponsesto mice, associated with a defect in the formation of GCs (26). In
Pn14(Fig.3).Thus,ICOScostimulationappearstobecriticalfor lightofdatademonstratingdecreasedICOSexpressiononSAP(cid:2)/(cid:2)
induction of both the primary IgG anti-protein response and the CD4(cid:1)Tcells(24,26),ithasbeenpostulatedthatdecreasedICOS
generation of memory, but not for the elicitation of the memory costimulation could lead at least in part to the defective IgG re-
responsefollowingsecondaryimmunization.Thepartial,although sponses.Iftrue,wehypothesizedthattheIgGanti-PSresponseto
substantial,inhibitionoftheIgGPspA-specificsecondaryresponse
using anti-ICOSL at the time of the primary vs the complete in-
hibition observed in ICOS(cid:2)/(cid:2) mice might reflect incomplete or
relatively short-lived neutralization of ICOSL following a single
injectionofmAb.
GerminalcenterformationinICOS(cid:2)/(cid:2)miceislargely
abrogatedinresponsetoeitherPn14orconjugate
ICOScostimulationofCD4(cid:1)Tcellshasbeenshowntobecritical
forinductionofGCandthusthegenerationofmemory(16).Al-
thoughtheIgGanti-PPS14andIgGanti-PCresponsestoPn14are
dependent on CD4(cid:1) T cells, CD28 costimulation, and CD40/
CD40L interactions, they fail to generate memory, in contrast to
the induction of PS-specific memory to conjugate (4, 5). These
data suggest the possibility that the TD IgG anti-PS responses to
Pn14andconjugateareextrafollicularandfollicular,respectively.
Toexplorethisfurther,wemeasuredthenumberandsizeofGCs
in WT and ICOS(cid:2)/(cid:2) mice following immunization with either
Pn14orconjugate(Fig.4).NaiveWTandICOS(cid:2)/(cid:2)micehaveno
detectableGCsinanareaof12mm2.WhereasGCsarestrongly
FIGURE 4. GCformationinICOS(cid:2)/(cid:2)miceislargelyabrogatedinre-
induced in WT mice, 10 days following primary immunization
with either Pn14 (GCs (cid:6) 44/12 mm2, GC total area (cid:6) 0.57 sponse to either Pn14 or conjugate. WT (C57BL/6) and ICOS(cid:2)/(cid:2) mice
(n (cid:6) 4 mice/group) were immunized i.p. with either Pn14 in saline or
mm2/12mm2)orconjugate(GCs(cid:6)29/12mm2,GCtotalarea(cid:6)
PS14-PspA(cid:1)C-PS-PspAinalum/CpG-ODN.Spleenswereremovedon
0.47 mm2/12 mm2), they are essentially abrogated in response to
day10andstainedwithpeanutagglutinin-AlexaFlour488(green),anti-
both immunizations in ICOS(cid:2)/(cid:2) mice. These data thus indicate B220-PacificBlue(blue),andanti-CD4-PE(red).Foursectionsfromeach
that the IgG anti-PS responses to Pn14, which are normal in offourspleenspergroupwereanalyzedforGCnumberandareaforatotal
ICOS(cid:2)/(cid:2)mice,canindeedoccurintheabsenceofGCformation, spleenareaof12mm2(seeResults).
TheJournalofImmunology 8263
FIGURE 5. Protein-andPS-specificIgGresponsestobothPn14andconjugatearedefectiveinSAP(cid:2)/(cid:2)mice,whereastheIgManti-polysaccharide
responseisdifferentiallyregulatedinresponsetothetwoimmunogens.WT(C57BL/6)andSAP(cid:2)/(cid:2)mice(n(cid:6)7/group)wereimmunizedi.p.witheither
(A)Pn14insaline(boostedonday21)or(B)PPS14-PspA(cid:1)C-PS-PspAinalum/CpG-ODN(boostedondays21and35).SerumtitersofAg-specificIgM
andIgGareillustrated.(cid:1),p(cid:5)0.05betweenWTandSAP(cid:2)/(cid:2)mice.
Pn14, which is ICOS-independent, would also be SAP-indepen- peakedearlierinSAP(cid:2)/(cid:2)micebutthendeclinedbelowWTlevels,
dent,whereasSAPwouldplayanimportantroleintheIgresponse whereasSAP(cid:2)/(cid:2)miceexhibitedhighersecondaryandtertiarytiters
to conjugate. However, other data argue that SAP(cid:2)/(cid:2) CD4(cid:1) T following boosting. In contrast to Pn14, immunization of SAP(cid:2)/(cid:2)
cellscanexpressnormallevelsofcostimulatorymoleculesinclud- micewithconjugateresultedinstrikingreductionsinbothIgMand
ingICOS,butfailtointeractproperlywithBcells,suggestingthat IgG anti-PPS14 and anti-PC responses, even greater than those ob-
SAP would be required for all TD responses, including the IgG servedinICOS(cid:2)/(cid:2)mice(Fig.5B).Thesedatathusdemonstratethat
anti-PPS14responsetoPn14(28).Toevaluatethesepossibilities, SAPplaysaroleinCD4(cid:1)TDIgresponsesthatisatleastpartiallyor
SAP(cid:2)/(cid:2) mice were immunized and boosted with either Pn14 or completelyindependentofICOS.
conjugate. As illustrated in Fig. 5, as expected, both the primary
AnalysisofsplenicandperitonealBcellsubsetsinWT,
and secondary IgG anti-PspA responses to Pn14 and conjugate
were markedly inhibited in SAP(cid:2)/(cid:2) mice, as they were in both SAP(cid:2)/(cid:2),ICOS(cid:2)/(cid:2),andCD28(cid:2)/(cid:2)mice
CD28(cid:2)/(cid:2)andICOS(cid:2)/(cid:2)mice,althoughthesemicedidelicitavery Splenic MZB and B-1 cells and peritoneal B-1a and B-1b cells
weak secondary response to conjugate. Surprisingly, althoughthe havebeenimplicatedinPS-specificIgisotyperesponses,whereas
IgGanti-PPS14andIgGanti-PCresponsestoPn14werenormalin splenic FB cells are thought to be responsible for most Ig re-
ICOS(cid:2)/(cid:2)mice,theywereeithercompletelyabrogated(anti-PPS14) sponsestoblood-borneproteinAgs(38,39).Thus,inafinalsetof
orsignificantlyreduced(anti-PC)inSAP(cid:2)/(cid:2)mice(Fig.5A).Incon- studies,wewantedtodeterminewhetherchangesinBcellsubset
trast,theprimaryIgManti-PPS14andIgManti-PCresponsestoPn14 developmentcouldaccount,atleastinpart,forthedifferencesin
TableI. QuantitationofsplenicandperitonealBcellsubsetsinWT,SAP(cid:2)/(cid:2),ICOS(cid:2)/(cid:2),andCD28(cid:2)/(cid:2)micea
%(No.(cid:4)106)ofCellsinSubset
TotalCells((cid:4)106) FB MZB B-1
Spleen
WT 69.16(cid:7)1.02 27.48(cid:7)0.03(19.00(cid:7)0.02) 3.78(cid:7)0.15(2.61(cid:7)0.10) 3.43(cid:7)0.05(2.37(cid:7)0.03)
SAP(cid:2)/(cid:2) 65.89(cid:7)8.90 32.55(cid:7)1.06*(21.45(cid:7)0.70*) 3.75(cid:7)0.04(2.47(cid:7)0.02) 3.45(cid:7)0.04(2.27(cid:7)0.02*)
ICOS(cid:2)/(cid:2) 75.04(cid:7)14.29 37.70(cid:7)0.20*(28.29(cid:7)0.15*) 4.16(cid:7)0.01*(3.12(cid:7)0.01*) 3.57(cid:7)0.05(2.68(cid:7)0.04*)
CD28(cid:2)/(cid:2) 69.98(cid:7)1.18 26.83(cid:7)1.15(18.78(cid:7)0.80) 3.72(cid:7)0.25(2.59(cid:7)0.17) 3.80(cid:7)0.18(2.66(cid:7)0.13)
%(No.(cid:4)106)ofCellsinSubset
B-1a B-1b B-2
Peritoneum
WT 5.31(cid:7)0.41 3.19(cid:7)0.63(0.17(cid:7)0.03) 3.30(cid:7)0.85(0.18(cid:7)0.05) 3.25(cid:7)0.75(0.17(cid:7)0.04)
SAP(cid:2)/(cid:2) 6.26(cid:7)3.86 2.71(cid:7)1.33(0.17(cid:7)0.08) 6.60(cid:7)0.66*(0.41(cid:7)0.04*) 6.39(cid:7)1.17*(0.40(cid:7)0.07*)
ICOS(cid:2)/(cid:2) 2.62(cid:7)0.41 3.40(cid:7)1.17(0.09(cid:7)0.03) 2.75(cid:7)0.51(0.07(cid:7)0.01*) 9.00(cid:7)1.17*(0.24(cid:7)0.03)
CD28(cid:2)/(cid:2) 5.44(cid:7)0.96 6.78(cid:7)0.88*(0.37(cid:7)0.05*) 5.22(cid:7)0.71(0.28(cid:7)0.04) 3.85(cid:7)0.07(0.21(cid:7)0.01)
aSpleenandperitonealcellsfromindividualmice(WT,SAP(cid:2)/(cid:2),andICOS(cid:2)/(cid:2)mice(n(cid:6)3/group);CD28(cid:2)/(cid:2)mice(n(cid:6)4))wereanalyzedbyflowcytometryasindicated
inMaterialsandMethods.Dataareexpressedasarithmeticmean(cid:7)SEM.(cid:1),p(cid:5)0.05relativetoWTmice(bytheStudentttest).
8264 ICOS-INDEPENDENTPATHWAYOFTCELL-DEPENDENTHUMORALIMMUNITY
TableII. Summaryofresultsa TD.AlthoughitmightthusbeassumedthatPSandproteinAgsin
aconjugatevaccinebehaveinanimmunologicallysimilarmanner,
CD28(cid:2)/(cid:2) ICOS(cid:2)/(cid:2) SAP(cid:2)/(cid:2) thisalsomaynotbeentirelytrue.Thus,PS,incontrasttoprotein
Ags,containsrepeatingidenticalantigenicepitopescapableofme-
Pn14
diating multivalent BCR cross-linking, suggesting a differential
IgGanti-protein 2 2 2
IgManti-PPS14 (cid:3) (cid:3) 1 requirement for BCR signaling in response to these two types of
IgGanti-PPS14 2 (cid:3) 2 linkedimmunogen.Inthisregard,wepreviouslydemonstratedthat
IgManti-PC (cid:3) (cid:3) 1? anti-PS responses to conjugate, as well as intact Pn, are more
IgGanti-PC 2 (cid:3) 2
heavily dependent on BCR-mediated signaling via Bruton’s ty-
Conjugate
rosinekinase(Btk)relativetotheanti-proteinresponse(5).More-
IgGanti-protein 2 2 2
IgManti-PPS14 2 (cid:3) 2 over,recent,unpublisheddatafromourlaboratorysuggestthatthe
IgGanti-PPS14 2 2 2 natureoftheassociationofthePSwiththeimmunogenicproteins
IgManti-PC 2 (cid:3) 2 expressedbytheintactbacteriavstheconjugatevaccinemaybea
IgGanti-PC 2 2 2
criticalfactorindeterminingtheabilityoftheimmunogentogen-
aKnockoutrelativetoWTmice((cid:6),nooveralldifferencesbetweenknockoutand erateaPS-specificIgGmemoryresponse.
WTmice).
Inthisstudywehaveextendedourobservationsontherelative
immunologic properties of PS Ags associated with intact Pn vs
conjugate vaccine by investigating the further role of ICOS co-
Ag-specific Ig isotype induction in SAP(cid:2)/(cid:2), ICOS(cid:2)/(cid:2), and/or stimulation and the signaling adaptor protein, SAP, shown to be
CD28(cid:2)/(cid:2)relativetoWTmice.AsillustratedinTableI,although criticalforCD4(cid:1)TcellhelpforGCformation(23,24).Wedem-
some significant (p (cid:5) 0.05) changes in the percentages and/or onstrate that the TD IgG anti-PS responses to intact Pn and con-
numbers of certain B cell subsets are observed in SAP(cid:2)/(cid:2), jugate vaccine are ICOS-independent and ICOS-dependent, re-
ICOS(cid:2)/(cid:2), and CD28(cid:2)/(cid:2) mice relative to WT mice, these differ- spectively, despite both responses being CD28-dependent. In
encesaremodest,andtheyfailtoexplainthealterationsobserved contrast, the anti-protein responses to both Pn and conjugate are
inIgisotypeinduction,whicharesummarizedinTableII. strongly ICOS-dependent. For the generation and elicitation of
protein-specific memory in response to Pn, ICOS was required
Discussion onlyatthetimeoftheprimary,butnotsecondary,immunization
Most studies on the regulation of PS-specific Ig responses have withPn,whereitalsowasfoundtobenecessaryfortheinduction
utilizedisolatedPSAgs,withorwithouthaptenation(40).Onthe of the primary IgG response. These data are in contrast to our
basis of these studies, the concept arose of PS Ags being either earlier observation of a requirement for B7-dependent costimula-
TI-1orTI-2,dependingonthepresenceorabsenceofanassoci- tion for the IgG anti-PspA response during both the primary and
ated TLR ligand, respectively. Although nonzwitterionic PS Ags secondary immunizations to Pn, although the dependence on B7
fail to associate with MHC-II Ags and thus are unable to recruit costimulationforthesecondaryresponsewasofmuchbrieferdu-
classical,cognateCD4(cid:1)Tcellhelp(41–43),itwasdemonstrated ration (2). Of note, ICOS(cid:2)/(cid:2) mice failed to generate a GC re-
thatthecovalentattachmentofanimmunogenicproteintothePS sponse to either Pn or conjugate, indicating that the IgG anti-PS
Ag(i.e.,conjugatevaccine)couldconvertthePSAgfromaTI-2 responsetoPncouldoccurnormallyintheabsenceofGCdevel-
toanapparentlyclassicalTDAg(36,37,44).Thus,PS-specificB opment. Collectively, these observations and those in SAP(cid:2)/(cid:2)
cells internalizing conjugate could present peptide from the PS- mice,discussedbelow,cannotbeaccountedforbythemodestand
linked protein to CD4(cid:1) T cells to receive cognate help for Ig selective alterations in B cell subset percentages and/or absolute
induction. Although studies of isolated PS Ags have been highly numbersintheseknockoutstrains.
informative, minimal attention has been paid to how anti-PS re- Previous studies have demonstrated that CD4(cid:1) T cells geneti-
sponses are regulated when PS Ags are presented to the immune cally deficient in SAP fail to provide help for GC formation and
system in a physiological context, that is, when expressed by an show impaired ICOS induction (17, 18, 23, 24). However, we
intactpathogen.Theexpressionbythepathogenofimmunogenic showthatalthoughtheIgGanti-PSresponsetoPnisICOS-inde-
proteins, TLR and NOD ligands, scavenger receptor ligands, and pendentandcanoccurnormallyintheabsenceofGCformation,
the particulate nature of the pathogen itself might confer unique it is still markedly inhibited in SAP(cid:2)/(cid:2) mice. Furthermore, IgM
immunologicpropertiesupontheassociatedPSAg.Inparticular, and IgG anti-PS responses to conjugate were significantly more
we hypothesized that perhaps pathogen-associated PS Ags were defective in SAP(cid:2)/(cid:2) as opposed to ICOS(cid:2)/(cid:2) mice. SAP is ex-
indeed classical TD Ags, similar to conjugate vaccines, specifi- pressedbycelltypesotherthanCD4(cid:1)Tcells,suchasCD8(cid:1)and
callyonthebasisoftheirassociationwithproteinAgs. NKTcells,NKcells,andsomeBcells(20–22),andthusitmight
Our previous studies have refuted this hypothesis, at least re- beinfluencingtheIgGanti-PSresponsetoPnthroughoneormore
gardingtheregulationoftheanti-PSresponsetotheintact,Gram- oftheselattercelltypes.However,theoverallenhancementofthe
positive extracellular bacterium S. pneumoniae (Pn). Thus, we TIIgManti-PSresponsetoPninSAP(cid:2)/(cid:2)micefavorsaspecific
demonstrated that PS Ags associated with intact Pn are unique role for SAP in CD4(cid:1) T cells in this model system. This idea is
immunologically, in that they exhibit both TD and TI properties. furthersupportedbythemarkedlydecreasedIgMandIgGanti-PS
Thus, similar to conjugate vaccine, the anti-PS response to Pn is responsestoconjugate,bothofwhicharedependentonCD4(cid:1) T
CD4(cid:1) T cell-, B7/CD28-, and CD40/CD40L-dependent, and it cells,inSAP(cid:2)/(cid:2)mice.Consistentwithourdata,itwaspreviously
generatesIgGcontainingallfourisotypes,inadditiontoIgM(i.e., demonstrated that immunization of SAP(cid:2)/(cid:2) mice with TD Ags
TDproperties).However,theanti-PSresponsetoPn,incontrastto (soluble NP-KLH or SRBC) resulted in dramatic defects in GC
conjugatevaccine,exhibitsmoreabbreviatedprimarykineticsand formationandprimaryandsecondaryAg-specificIgG(IgG1most
a failure to generate PS-specific memory (i.e., TI properties), as- dramaticallyreduced,thenIgG2b/IgG2a,thenIgG3),althoughnot
sociated with a shortened period of dependence on CD4(cid:1) T cell IgM,production(26).Thebasisforourcontrastingobservationof
helpandB7-dependentcostimulation(1–5).Thus,theanti-PSre- decreasedPS-specificIgMproductioninresponsetoconjugatein
sponse to Pn, in contrast to conjugate vaccine, is not classically SAP(cid:2)/(cid:2)miceisunknown,butitcouldreflectintrinsicdifferences
TheJournalofImmunology 8265
betweenPSandproteinAgs.TheimportanceofSAPforthePS- in Pn, but not conjugate, become disassociated before eventual
specificIgresponsetoPnsuggeststhatthereisacriticalrequire- contactwithPS-specificBcells,thuslimitingtheextentofspecific
ment for SAP and the associated SLAM family receptors in reg- cognate CD4(cid:1) T cell help for the anti-PS response. Third, the
ulating the earliest stages of T cell help for B cells before, or at degreeofBCRcross-linkinginresponsetoAg,whichlikelydif-
least partially independent of, ICOS signals. While these conclu- fers between protein and PS Ags, could influence whether the Ig
sionsappeartodifferfromthoseobtainedinarecentstudy,which response is of the predominantly extrafollicular plasma cell type
concluded that SAP expression in T cells was required only late (higher BCR strength) or alternatively progresses through a GC
(after5days)inresponsetoaTDproteinAg(45),thedifferences reaction (lower BCR strength) (65) Finally, the nature of the as-
are likely to result from the distinct types of Ags examined. In- sociation of capsular PS with Gram-positive vs Gram-negative
deed, the data herein support the recent demonstration that bacteria is distinct and could impart different immunologic prop-
SAP(cid:2)/(cid:2) CD4(cid:1) T cells have a selective defect in adhesion to B ertiestoPSAgsexpressedbythesetwoclassesofpathogen.
cells (28), which would be required for the early interactions be-
tweenTcellsandBcellsmediatingthePS-specificIgGresponse Disclosures
toPn(2,3,46). Theauthorshavenofinancialconflictsofinterest.
Collectively, these data strongly suggest that the IgG anti-PS
responsetoPninWTmiceisextrafollicular,mostlikelyleading References
toapredominantlyrapidplasmacellresponsetotheexclusionof
1. Wu,Z.Q.,Q.Vos,Y.Shen,A.Lees,S.R.Wilson,D.E.Briles,W.C.Gause,
memory B cell generation, which is nevertheless dependent on J.J.Mond,andC.M.Snapper.1999.Invivopolysaccharide-specificIgGisotype
CD4(cid:1) T cell help and CD40/CD40L interactions for its optimal responsestointactStreptococcuspneumoniaeareTcelldependentandrequire
CD40-andB7-ligandinteractions.J.Immunol.163:659–667.
induction (47–50). This would then account for the abbreviated 2. Wu,Z.Q.,A.Q.Khan,Y.Shen,J.Schartman,R.Peach,A.Lees,J.J.Mond,
primary kinetics, the shorter duration of CD4(cid:1) T cell help and W.C.Gause,andC.M.Snapper.2000.B7requirementsforprimaryandsec-
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B7-dependentcostimulation,andthelackofdependenceonICOS
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vaccineislikelytolargelyreflectafollicularresponseassociated anti-capsular polysaccharide and antiprotein responses to intact Streptococcus
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Plasmacellsgeneratedintheextrafollicularresponseundergoex- pneumococcalconjugatevaccinecombinedwithanunmethylatedCpG-contain-
ingoligodeoxynucleotiderestoresdefectiveimmunoglobulinGantipolysaccha-
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