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MicrobesandInfection5(2003)311–319 www.elsevier.com/locate/micinf Review Dendritic cells, new tools for vaccination JesusColino,CliffordM.Snapper* DepartmentofPathology,UniformedServicesUniversityoftheHealthSciences,4301JonesBridgeRoad,Bethesda,MD20814,USA Abstract Ourrapidlyexpandingknowledgeofthebiologyofthedendriticcell(DC),amajorantigen-presentingcellconnectinginnateandadaptive immunity,suggestsnewpossibilitiesforthedevelopmentofvaccinesandtherapeuticstrategiesagainstpathogens,throughthemanipulation oftheirfunctioninvivo,ortheinjectionoftheDCitself,onceproperlyinstructedexvivo. ©2003ÉditionsscientifiquesetmédicalesElsevierSAS.Allrightsreserved. Keywords:Vaccines;Immunotherapy;Dendriticcells 1.Introduction non-opsonized pathogens by macropinocytosis, phagocyto- sis or through mannose receptors (C-type lectins), or com- Duringthelastdecade,intensiveresearchhasfocusedon plexes of antibody and microbial antigen via Fcc receptor understandingdendriticcell(DC)biology[1,2].Asaresult, types I and III. By virtue of their high phagocytic and en- wenowhaveasignificantunderstandingoftheplasticityof docyticcapacities,DCsconstitutivelyinternalizesamplesof DC function and its major role in maintaining homeostasis theirantigenicmicroenvironment,whichintheeventofin- through the induction of protective immune responses fection, will also include microbial antigens. In addition, against pathogens and tumors, and in the maintaining of DCsbecomeactivatedandmobilizedinresponsetoinfection peripheraltolerance.Inthisreview,wewilldiscussthemajor (i) by direct recognition of pathogen-associated molecular checkpoints involved in DC function (Fig. 1), focusing on patterns (PAMPS); e.g. LPS or cell-wall components, by theirroleinanti-bacterialimmunity,anddiscusshowDCsor Toll-like receptors (TLRs), or (ii) indirectly through recep- theirproductsmaybeusedoralteredtopreventandmanage torsforinflammatorycytokinesandchemokines. invasiveinfections. Immature DCs upon activation with PAMPS secrete in- flammatory cytokines (e.g. IL-1b, TNF-a, IL-6 and IL-12) and chemokines (e.g. MIP-1a and MIP-1b; RANTES and 2.OverviewofDCfunction MCP-1) which serve in part to recruit circulating DC pre- cursors and other immune cells to the site of infection and DCsshowauniquefunctionaldualityduringtheirdevel- participate in their activation (Fig. 1, point 1). Later on, opment,designedtoultimatelyprovidesecondarylymphoid anti-inflammatory cytokines, chiefly IL-10, are secreted by tissueswithusefulinformationabouttheantigeniccomposi- DCs to counterbalance the pathogenic stimulus, thus pre- tionintheperiphery.Attheimmaturestagesofdevelopment, ventingtheinductionofexaggeratedimmuneandinflamma- DCsresidentinperipheraltissuesarespecializedinantigen- toryresponses. capture, acting as sentinel cells (Fig. 1). They are strategi- callylocated,amongmacrophages,atepithelialbarriersthat Pathogen-mediated activation induces DCs to undergo oftenserveasmajorportalsofpathogenentry.Thus,inthe maturation.Thisisacomplexprocesswiththedualgoalof gastrointestinal tract, DCs located in intraepithelial pockets transformingimmatureDCsintheperipheraltissueintocells belowMcellsarewellequippedtodetectandsampleinfec- thatwillmigratetothesecondarylymphoidorgansandthen tious agents for the transport of microbial antigen to the act as professional antigen-presenting cells (APCs) for the subepithelial lymphoid tissue [3]. DCs avidly internalize primingofnaiveTcells(Fig.1,points2–4).Towardaccom- plishing the first goal, activated DCs lose their phagocytic capacityandtissueadhesivestructures,increasetheirexpres- *Correspondingauthor.Tel.:+1-301-295-3490;fax:+1-301-295-1640. E-mailaddress:[email protected](C.M.Snapper). sionofreceptorsforlymphoidchemokines(i.e.CCR7),and ©2003ÉditionsscientifiquesetmédicalesElsevierSAS.Allrightsreserved. DOI:10.1016/S1286-4579(03)00033-9 Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 3. DATES COVERED 2003 2. REPORT TYPE 00-00-2003 to 00-00-2003 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Dendritic cells, new tools for vaccination 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Uniformed Services University of the Health Sciences,Department of REPORT NUMBER Pathology,Bethesda,MD,20814 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Our rapidly expanding knowledge of the biology of the dendritic cell (DC), a major antigen-presenting cell connecting innate and adaptive immunity, suggests new possibilities for the development of vaccines and therapeutic strategies against pathogens, through the manipulation of their function in vivo, or the injection of the DC itself, once properly instructed ex vivo. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE Same as 9 unclassified unclassified unclassified Report (SAR) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 312 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 Fig.1.OverviewofDCfunctionduringaprimaryinfection.NumbersonthefigurerefertothemajorstepsofDCfunctionwhichcouldbealteredinorderto designvaccinesandimmunotherapeuticapproachesagainstpathogeninfections,asindicatedinTable1.Uponinflammation,immatureDCslocatedatthemajor portalsofpathogenentry,internalizethepathogen(3,antigen-capture),becomingactivatedbydirectrecognitionofPAMPS(2,activation).ActivatedDCs releaseinflammatorychemokinestorecruitDCprecursors(1,recruitmentofprecursors)andinflammatorycytokinesthatreinforceinflammationandbystander DCactivation(2).DCsactivatedbythepathogenundergomaturation,aprocessthatoccursduringDCmigrationfromtheperipheraltissuestothesecondary lymphoidorgans(4,migration).MaturationtransformstheDCfromanantigen-capturecell(3)intoaprofessionalAPC(5,antigen-deliveryand-presentation). MigratingDCsenteringasecondarylymphoidorgan,interactandpresenttheprocessedantigenicprofileofthepathogentoTcells,andlikelyBcells,toinitiate aprimaryimmuneresponse(5).Dependingonthepatternofcytokinessecretedduringtheinteraction,theimmuneresponsewillpolarizetowardseitheraTh1 orTh2response(6,polarization).DCsuponcompletingtheirAPCfunction,likely,diebyapoptosis.ReprocessingbyresidentDCsofapoptoticbodies containingprocessedantigencouldbeasecondary,butunlikely,sourceforantigenpresentation.Alternatively,DCscoulddeliverantigenlocallyoratadistance throughsecretedexosomes.ThephysiologicalroleofDCexosomesisamatterofdebate. J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 313 reorganizetheircytoskeletonfortheacquisitionofhighcel- strated markedly impaired polysaccharide- and protein- lular mobility [4]. Towards the second goal, DCs strongly specific IgG responses upon transfer into naive mice upregulatetheirexpressionofco-stimulatorymolecules(i.e. (submitted manuscript). Collectively, our data argue for an CD40,CD80andCD86)andupregulatesynthesisandtrans- active, direct role for the antigen-loaded migrating DCs in location to the surface of MHC molecules complexed to this system. However, we hypothesize that for anti- processedmicrobialantigens.Concomitantly,MHCclassII polysaccharideresponses,theactivereleaseofexosomesby degradationisreduced,resultinginenhancedretention(sev- antigen-loaded DCs could play an important inductive role eral days) of peptide-MHC complexes on the cell surface. boththroughantigentransferbetweenDCsthemselves,and Thus, DCs, unlike macrophages and other professional between DCs and B cells, that are in tight contacts or syn- “scavengers”,respondtothemicrobialchallengebymigrat- apse. ing to secondary lymphoid organs while generating a pro- DCsandTcellscanalsointeractthroughtheformationof cessed antigenic profile of the pathogen associated with animmunologicalsynapse[7].Inthissynapse,T-cellrecep- MHCmolecules.AnotheruniquefeatureofDCsrelativeto tors and co-stimulatory molecules congregate in a central macrophages, is the ability of DCs to attenuate their pro- areasurroundedbyaringofadhesionmolecules.Sustained teolyticpotential.Thus,DClysosomescansequesterantigen signalingviathesesynapticinteractionsisrequiredinorder forextendedperiodsandstillefficientlyusetheantigenfor fortheTcelltoenterthefirstcelldivisioncycle.Specifically, generatingpeptide-MHCclassIIcomplexes[5]. naiveCD4+Thelpercellsrequiremorethan20hofcontinu- Onceinthesecondarylymphoidorgan,matureDCs(ef- ous stimulation to become committed to cell division, fectorstate)instructTcells,andlikelyBcellsandNKcells, whereasshorterperiodsdidnotresultineffectorT-celldif- toinduceaneffectiveprimaryimmuneresponse,andestab- ferentiation.Thus, the stability and duration of the synapse lishimmunologicalmemory(Fig.1,point5).Thisabilityof will determine T-cell function. Factors disrupting the syn- DCstoinduceaprimaryimmuneresponseisalsoaunique apse,suchasDCdeath,willsignificantlyaffectT-cellfunc- propertyamongAPCs.Werecentlydemonstratedthatbone- tion.Inthisregard,werecentlyobservedastrongassociation marrow-derived dendritic cells (BMDCs) loaded in vitro betweentheprogressionofS.pneumoniaeinducedDCapo- with Streptococcus pneumoniae and then transferred into ptosisandadecreasingabilityofsuchDCstoinduceT-cell- naivemicewereabletoinduceprimaryIgisotyperesponses dependentprimaryIgisotyperesponsesinvivo.Ontheother not only for bacterial proteins, but also for capsular and hand, factors that prevented DC apoptosis, such as IL-10, cell-wall polysaccharide antigens [6]. The Ig isotype re- servedtoprolongtheAPCfunctionofDCsforinductionof sponsestothebacterialproteinsuniformlyrequiredDCex- in vivo anti-bacterial humoral immunity (submitted manu- pressionofmajorhistocompatibilitycomplexclassII,CD40 script). and B7 and the production of IL-6, strongly suggesting a DCsinduceantigen-specificTcellsnotonlytoproliferate, requirementforcognateinteractionswithTcellsandlikely but also determine the development of T-cell regulatory or with B cells. Similar requirements for anti-polysaccharide effectorfunctions,includingCD4+helperT-cellpolarization antigenswereobservedonlyfortheIgG1isotype,suggesting (i.e. Th1 vs. Th2) to secrete different patterns of cytokines that for polysaccharide-specific responses classical cognate (Fig. 1, point 6). IL-12 is the prototypic Th1-polarizing interactionsmayaffectthequalityoftheIgisotyperesponse, cytokineproducedbyDCs,whereasotherfactors,including but are not critical for DC induction of primary anti- IL-4,ortheabsenceofIL-12productionleadtoTh2polar- polysaccharideIg.Anunresolvedquestionhasbeenwhether ization.The ability of DCs to influence the pattern of cyto- DCsmigratingtothesecondarylymphoidorgansactedeither kinessecretedbyTcellsrepresentsacriticalfunctionwhich directly as APCs or transferred their captured antigens to can profoundly influence the final outcome of the immune residentDCsforantigenpresentation.Twomechanismshave responsetoapathogen.Thus,cytokinessecretedbyTh1cells beenproposedtoexplainhowDCsmighttransferantigenin (IFN-c,TNF-bandIL-2),aretypicallyconsiderednecessary anindirectpathwayofantigenpresentation(Fig.1,point5): forprotectionagainstinfectionswithvirusesandwithintra- (i) antigen-loaded DCs that migrate to the secondary lym- cellular bacteria and protozoans, whereas secreted Th2 cy- phoid organ could undergo apoptosis or necrosis and be tokines(IL-4,IL-5,IL-6andIL-13)areimportantforprotec- phagocytosed by resident DCs (ii) migrating DCs could tive responses against multicellular nematode parasites. activelyreleaseantigen-bearingvesicles(exosomes)derived Several factors appear to influence the ability of DCs to from the DC lysosomal compartment, which could then be polarizeT-cellcytokineresponses.(i)DCsubsets:thesubset captured by resident DCs. The relative importance of the ofDCusedforT-cellstimulationmayeffectabiasforeither directandindirectpathwaysinanti-microbialimmunityhas aTh1orTh2response.(ii)DynamicsofDCmigrationtothe not yet been resolved, but recent data from our laboratory lymphoid organ: early after initiation of the immune re- stronglyargueagainsttheindirectpathwayhypothesis.Thus, sponse, large numbers of recently stimulated DCs actively weshowedthatviable,butnotnecrotic,BMDCsloadedwith secretingIL-12andenteringintotheT-cellareasmaypref- wholeS.pneumoniaeinducedsignificantantigen-specificIg erentiallyinduceTh1cellpriming.Incontrast,atalatertime, isotype responses in vivo [6]. Furthermore, BMDCs under- when the DC influx decreases, and the surviving DCs in goingapoptosisinducedaftercontactwithbacteriademon- T-cell areas have downregulated their secretion of IL-12, 314 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 preferentialprimingforTh2andotherregulatoryTcellsmay Table1 occur [8]. (iii) Nature of the maturation stimuli: bacterial SummaryofthepotentialtherapeuticandprophylacticusesofDC PAMPs induce strong production of IL-12 by DCs, which StepofDC Potentialtherapeuticapproach canbepotentlyboostedbyactivatedTcellsthroughCD40L, function whereas other DC stimuli such as cholera toxin, TNF-a or Recruitmentof TreatmentofpatientswithDC-poietins:Flt3L,GM-CSF DCprecursors IncreasingDC-poietindepositionandantigenco-delivery: IL-1 do not induce IL-12 (iv) Microenvironmental factors: DC-poietinconjugatesorfusionproteinswiththe IL-12productionbyDCscanalsobeenhancedbymediators antigen released in the DC microenvironment. For example, IFN-c Encapsulation and IL-4 can enhance IL-12 production by activated DCs, ViralvectorsencodingGM-CSF whileprostaglandinE2andIL-10exertaninhibitoryeffect. TransferofautologousDCexpandedexvivowith ResultsfromourgroupindicatearoleforautocrineIL-10in DC-poietins modulatingDCIL-12productionearlyafteractivationbyS. DCactivation Treatmentwithproinflammatorycytokines:IL-1b,IFN-a pneumoniaeinordertoadjustthematurationresponsetothe andmaturation Microbial-derivedproducts: Mucosaladjuvants:Choleratoxoidmutants magnitudeofthestimuli(submittedmanuscript).Thus,inthe PAMPS:CpG-oligos,viraldsRNA absence of autocrine or paracrine IL-10, the level of IL-12 Antigen-capture DCloadedexvivowithinactivatedpathogenbefore productionwassustainedforlongperiodsoftime(>8h). transfer Thus,DCsrepresentthecriticallinkbetweeninnateand DCtransfectedexvivowithpathogenDNAplasmids adaptive immunity, upon which, appropriate concerted ac- orRNA tion is required for a successful host defense against an DCloadedwithimmunologicallyrelevantpeptides invadingpathogen. TargetingDCsinvivowithsensormolecules: LigandsofMHCandco-stimulatorymolecules(CD40) Antibodiesspecificforendocytosisreceptors(CD205) 3.Therapeuticandprophylacticusesofdendriticcells PAMPs:CpG-oligos,OmpA Heat-shockproteins SignificantprogressinourunderstandingofDCbiology Engineeredbacteriaandvirus andtheircriticalfunctioninimmunityhavepromptedmany Migrationand RouteofDCadministration:subcutaneousvs. homing intravenous toexploretheirpotentialuseinimmunotherapyandprophy- Chemokine and chemokine receptors engineered DC: laxis. Most of these pioneering strategies have been devel- CXCR5,CCR7 oped for anti-tumor therapy, and some are currently being DCsubset testedinhumantrials,withvariablesuccess.Acomprehen- Antigendelivery Migratorypathways sivelistofthesafetyrequirementsofDCpreparationsused andpresentation Exosomes forcancerimmunotherapyhasbeenpublished[9].Below,we Polarization DCsubset discuss various strategies to prevent or treat infectious dis- NatureoftheDCmaturationoractivationstimuli eases for optimizing the use of DCs for mediating their (e.g.bacterialPAMPSvs.inflammatorycytokines) DC-poietin(e.g.Flt3vs.GM-CSF) functional effects (Fig. 1) upon transfer into the host as RouteofDCadministration(e.g.i.v.vs.s.c.) summarizedinTable1. andDChoming DCtreatmentwithanti-inflammatorycytokines:IL-10 3.1.ExpandingDCnumbers One approach to optimizing DC function is to increase subsets could influence the nature of the induced response. their numbers, usually, prior to the administration of an Thus, Pulendran et al. [10] observed that pretreatment of antigen-basedvaccine.Itwillfavorandreinforcethenatural micewithFlt3LselectivelyenhancedtheIgG2aresponseto recruitment of DC precursors (Fig. 1, point 1) Two major soluble ovalbumin which resulted from the induction of a strategies have been explored to achieve this goal: (i) the polarizedTh1response,whereasGM-CSFpretreatmentin- transferofautologousDCsexpandedinvitrointhepresence creasedIgG1antigen-specificresponses,indicatingpolariza- of cytokine cocktails and (ii) the in vivo mobilization of tionofthecytokineresponsetoTh2.Therefore,theselection stem-cellprecursorsofDCsandexpansionofimmatureDCs betweenGM-CSForFlt3L,intheory,mightallowformodu- by treatment with the DC-poietins Flt3L or granulocyte- lationoftheresponsethatmayfavorhostdefense,depending macrophagecolony-stimulatingfactor(GM-CSF). onthenatureoftheinfectiousdisease. Flt3L, the ligand of the FMS-like tyrosine kinase 3, TheuseofFlt3Lforaugmentinghostresistancetopatho- strongly promotes the proliferation, differentiation and sur- gens has met so far with limited success. Thus, Flt3L pre- vival of early hematopoietic progenitor/stem cells toward treatmenthasbeenshowntopromoteresistanceagainstsec- DCs and to a lesser extent, NK cells. Noticeably, Flt3L ondary infection with Listeria monocytogenes [11] and inducesincreasesinthenumbersofallDCsubsets.Incon- partialprotectionagainstprogressivecutaneousLeishmania- trast,GM-CSFisselectiveinitsinductionofproliferationof sis[12]inmice,butinboththecases,itisnotclearthatthe cells of the monocyte-macrophage lineage leading to my- protective effect was directly due to DC expansion as op- eloidDCexpansion.TherelativeexpansionofdifferentDC posedtotheexpansionofothercelllineages.Moreover,tobe J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 315 effective, repetitive injections of high doses of Flt3L were two major advantages as adjuvants for human use: (i) low required. Further, the treatment did not resolve an ongoing toxicityand(ii)theabilitytosynthesizetheminapureand Leishmania infection. In light of these studies, the future standardizedform. applicationofFlt3Ltohumanpopulationappearsuncertain. 3.3.Improvingantigen-capture:learningtouseDCs GM-CSF is also a very poor adjuvant when delivered systemically. This drawback has been partially resolved A next level of intervention is the design of strategies to through strategies focused on effecting efficient and pro- improve the delivery of the antigen to the DCs by ex vivo longeddepositionofGM-CSFatthevaccinationsiteandits loading,orinvivotargetinginordertoreproduceandrein- co-delivery with the antigen (i) by coupling the GM-CSF force the physiologic antigen-capture step of DC function with carrier molecules such as polyethylene-glycol, (ii) as (Fig. 1, point 3).An invaluable amount of information has fusionproteinswiththeantigen,(iii)byencapsulationinside beenobtainedusingDCsloadedexvivowithbacteriaprior liposomesincombinationwiththeantigen,(iv)orencoded to transfer to the recipient animal. This experimental ap- intoviralvectors.Someofthesestrategieshaveproventobe proachprovedforthefirsttimethattransferofDCsloaded effectiveinmousemodelsofbacterialinfection.Thus,vac- with the inactivated pathogen induces protective immune cination with a fusion protein of a pneumococcal surface responsesinnaiverecipientslatersubjectedtoexperimental protein(PspA)andGM-CSFprotectedmiceagainstalethal infection. Immunity has been obtained against pathogens challenge with virulent S. pneumoniae [13]. Most recently, that have so far proved difficult to prevent using vaccine co-immunization of mice with transgenic adenoviral GM- approaches:M.tuberculosis[19],Borreliaburgdorferi[20] CSF and BCG vaccine, strongly enhanced Th1 antigen- Chlamydiatrachomatis[21]andCandidaalbicans[22].C. specificimmunity,andtheprotectiveefficacyofthevaccine albicans provides a paradigmatic example of how this ap- againstachallengewithMycobacteriumtuberculosis[14]. proachcanalsohelptodesignvaccinestrategies.DCsloaded withtheyeastform,butnotwiththehyphalform,generate 3.2.ActivatingDCs protectiveanti-fungalimmunity.Thiswasbasedontheabil- DCs that capture antigen, in the absence of concurrent ityoftheDCstodiscriminatebetweenthetwoformsofthe activation,mayinduceantigen-specifictolerance,asopposed pathogen, responding to the yeast by producing IL-12 and to immunity. Thus, when the antigen does not activate the stimulatingaprotectiveTh1response,butsecretingtheTh2 DCsbyitself,anadjuvantmightberequiredincombination cytokineIL-4inresponsetohyphae,whichwasnotprotec- withtheDC-poietininordertoinduceproperimmunity.In tive. However, suppression of IL-4 production by hyphae- thisregard,ithasbeenshownthatDCscanplayanactiverole pulsedDCsallowedforaprotectiveresponsetooccur. intheinductionofmucosaltolerancetofoodororallyadmin- The success of this approach is based on two factors. (i) istered soluble antigens when a concurrent adjuvant is not Pathogens can directly activate the DCs. (ii) A single DC present.Thus,inductionoftolerancetoorallyadministered afterpathogeninternalization,isabletocompetentlypresent soluble ovalbumin, is further enhanced in mice pretreated an array of T-cell pathogen-derived peptides covering the with Flt3L to expand DCs, but this can be reversed by complete T-cell antigenic repertoire of the bacteria, thus additionaladministrationoftheinflammatorycytokineIL-1 inducingaprotective,multi-specificimmuneresponse.This (Fig. 1, point 2), a cytokine which can activate DCs [15]. approachallowsforuseofhiddenorunknownT-cellprotec- IFN-a can also promote DC activation and hence act as an tiveepitopes,hardtodetectinscreeningtests.Furthermore, adjuvant. Thus, IFN-a markedly enhances an antigen- ourresultsdemonstratingthatDCsloadedwithinactivatedS. specific humoral response in mice when co-administered pneumoniaeareabletostimulateapolysaccharide-specific, withFlt3L[16]. aswellasprotein-specific,humoralresponsesinnaiverecipi- A second group of molecules that can activate DCs are ents[6]expandthepotentialprophylacticuseofDCs,since microbial-derived products (Fig. 1, point 2). The most protectionagainstextracellularbacteriaistypicallymediated broadly used products in experimental models are LPS a bycapsularpolysaccharide-specificantibodyresponses. systemicadjuvantandTLR4ligand,andcholeratoxin(CT), Formostinfectiousdiseases,theuseofDCspulsedwith a mucosal adjuvant.Although both are highly toxic, many thewholepathogen,whetherinactivatedorattenuated,may attempts have been made to detoxify them while retaining not represent an ideal approach for human vaccination be- theiradjuvantproperties.Onepromisingstrategyistheuseof cause of safety concerns.We thus need to determine which amutantoftheCTsubunitA,whichisnon-toxicandinduces pathogencomponentsarebothsafeandusefulwhenusedin astrongandpolarizedTh2responsewhenusedasamucosal combination with DCs. The consequent reduction of anti- adjuvant [17].Alternatively, DCs may be activated through genic variety inherent in this latter approach might have TLR9 by oligodeoxynucleotides containing unmethylated unexpected pitfalls. For instance, adoptive transfer of DCs CpG motifs or through TLR3 by viral dsRNA or polyI- pulsedwithinactivatedC.trachomatishasprovensuccessful :polyC. Stimulatory oligodeoxynucleotides in combination in experimental models for promoting anti-Chlamydial im- withFlt3Lhavebeenshowntobeeffectiveintheenhance- munity through stimulation of a Th1 response. However, a ment of humoral and cellular responses to soluble proteins similaradoptivetransferofDCspulsedwiththerecombinant andanti-tumorimmunityinmice[18].Polynucleotideshave chlamydial major outer membrane protein, which has been 316 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 regarded as a promising candidate for the development of troduced class II-restricted T-cell epitopes, as sensors conventional vaccines, did not induce protection, and this [23]. These antibodies, termed “troybodies”, strongly was associated with a polarized Th2 response [21]. One enhanced epitope delivery to the APC for efficient alternative is the use of DCs transfected ex vivo with DNA T-cellstimulation,andthuscouldbeapromisingstrat- plasmids or RNA derived from the pathogen, in order to egy for targeted peptide delivery. However, caution endogenouslyproducethemicrobialantigensaftertransfer. mustbetakenwhentargetingDCsthroughMHC,since This strategy has been used with success in experimental DCsareverysensitivetotheinductionofapoptosisvia tumors, and recently has shown promise in anti-infectious surfaceMHCcross-linking,andthelevelofexpression immunity.Thus,DCstransfectedwithRNAfromyeast,but of MHC will vary with the activation and functional not hyphal, C. albicans induced a protective immune re- maturationstageoftheDC. sponsesimilartothatobservedupontransferofDCspulsed 2.Antibodies and their fragments specific for endocytic withtheintactyeast[22]. receptors:An analogous approach involved the use of AnotherapproachistheuseofDCsloadedwithrelevant fusionproteinsofapeptideandoneantibodyspecificto immunogenic peptides in an attempt to increase the safety DEC-205(CD205),anendocyticreceptorandmember and reproducibility of the vaccine. Nevertheless, this ap- ofthemannosereceptorfamily.DEC-205expressionis proach may introduce more problems than it resolves. (i) restricted to DCs within the T-cell areas of lymphoid Loading with peptides drastically restricts the repertoire of tissuesandparticularlyexpressedbylymphoidDCs.It potentialT-cell clones stimulated, and therefore, the ability is well known that endocytosis of microbial antigens toinduceaprotectiveresponse.(ii)Itisdifficulttoenvisage through DEC-205 binding results in efficient antigen- standardizedmixesofpeptidesabletostimulateCD4,CD8 processingand-presentationtoTcells.DCstargetedin andregulatoryTcells.DCs,instead,arespecializedtopro- vivo with the fusion antibodies for DEC-205 indeed, ducethesepeptidesfromthewholepathogen.(iii)Efficient efficiently stimulated the proliferation of T cells spe- MHCclassIandclassIIpresentationoccursonlywhenthe cificforthepeptide.Nevertheless,sincetheendocyto- peptides are generated within the DC itself (iv) Only in sisofthefusionantibodiesdidnotinduceDCmatura- mature DCs are the cell surface MHC-peptide complexes tion,thetreatmentresultedintheinductionofpeptide- stable;inimmatureDCs,theyareconstantlyrenewed.This specifictolerance[24].Thisstudywasamongthefirst approachwouldrequiretheconcurrentuseofaDCactivator todemonstratethatDCshaveakeyroleinthemainte- iftheDCsareimmature,oralternatively,analreadymature nanceofperipheraltolerance,andthatthisisrelatedto DC. The use of a fully matured DC immediately prior to the state of activation/maturation of the DCs. These transfer,couldprovelesseffectivesincetheinvivohalf-life resultsstressedtheimportanceoftheuseofconcurrent of the mature DC is likely less than the activated immature DCactivationsignalsorvaccinedesignsabletoinduce DC. DC maturation in order to elicit protective immunity. Vaccinationofthegeneralhumanpopulationwithexvivo Nevertheless, this approach could be used to induce generated DCs, a procedure non-exempt of risk, costly and antigen-specifictoleranceforthetreatmentofautoim- difficulttoreproduce,doesnotappeartobeafeasiblefirst- munediseases. lineprophylacticapproach,andprobablywillberestrictedto 3.PAMPs, (e.g. CpG oligonucleotides that signal via instancesinwhichconventionaltherapiesfortreatingmalig- TLR9, or peptidoglycan and the outer membrane pro- nancyorchronicinfectionhavefailed,tofocalvaccinationor teinA of Klebsiella pneumoniae (OmpA), both utiliz- toimproveimmunityinimmunodeficientpatients(e.g.bone ing TLR2 for signaling): the use of PAMPs as sensor marrowtransplantrecipients).Amorefeasibleapproachisto moleculesallowsafurthermanipulationoftheoutcome efficientlydelivertheantigeninvivototheendogenousDC of the immune response to induce protective immune bycell-targeting.Severalapproachescurrentlybeingevalu- responses. SinceTLR molecules are differentially ex- ated employ fusion proteins or chemical complexes of the pressed on DC subsets, it is possible to selectively immunogenic antigen or the viral vector, with a “sensor targetfunctionallydistinctDCsthroughtheselectionof molecule” which bind surface receptors expressed on DCs, the PAMP. Thus, TLR9 and TLR7 are selectively ex- and in many cases, able to deliver an activation signal.The pressed by plasmacytoid human DCs and produce currentsensormoleculestestedinclude: IFN-a after TLR9 binding with CpG motifs, whereas 1.LigandsofMHCandco-stimulatorymolecules,which myeloidhumanDCsexpressallTLRsexceptTLR7and areexpressedmostlybyAPCs:manystudieshavedem- TLR9andproducehighlevelsofIL-12afterTLR2or onstrated the ability of CD40-ligand and anti-CD40 TLR4-mediatedactivationwithpeptidoglycanorLPS, antibodies to activateAPCs which selectively express respectively[25].Furthermore,OmpAandtheantigens CD40,suggestingthattheseligandswouldbeeffective coupled to OmpA, are delivered into the MHC class I carriersforantigentargetingtoandsubsequentpresen- presentation pathway after DC uptake, through cross- tation by DCs. Recently, a new approach has been presentation [26]. This ability to present exogenous tested using MHC class II, as target, and recombinant antigensforpresentationtoCD8+TcellsmakesOmpA antibodiesspecifictoMHC,containinggeneticallyin- apromisingcarriertodevelopCTL-inducervaccines. J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 317 4.Heat-shock proteins (Hsps): Although some contro- thestimulatedDCmigrates.Intradermally(i.d.)andsubcu- versyexists,hostHspsappeartoactivateDCsviaTLR2 taneously (s.c.) injected DCs preferentially home to the andTLR4inasimilarmannerasPAMPs.Thisproperty, T-cell areas of the draining lymph nodes, not to the spleen, in combination with the recognized ability of Hsp to whereas intravenously (i.v.) injected DCs home first to the bind peptides and direct them into the MHC class I lung, and then, preferentially to the spleen, and, to some presentationpathway,makesHsp,anidealsensormol- extent,tothekidneyandliver,butnottolymphnodes[28]. ecule for peptide-based vaccines against tumors and Intraperitoneal, and probably intralymphatic, injected intracellularpathogens.Indeed,Hspsappeartoplayan DCsshowanintermediatebiodistribution.DCsinjectedi.v., importantroleinautologousresistancetotumors[27]. as we observed in mice, may remain in the spleen for rela- The use of Hsp has additional advantages which are tively longer periods of time (>5 d).The specific lymphoid reminiscent of those offered by the use of ex vivo sitetowhichtheDChomescouldhaveamajorimpactonthe pulsedDCs.Duetotheirpromiscuityinpeptidebind- particularcytokinepolarizationoftheresponse,duetoarchi- ing,theHspstrategyis,intheory,applicabletoawide tectural and functional differences intrinsic to the different varietyofantigens.TheimmunizationwithHsppeptide lymphoidorgans(Fig.1,point6).Thus,thesamepreparation complexes derived from infected cells is potentially ofDCswheninjectedi.v.arepronetoinducenon-polarized directed against the whole antigenic repertoire and, responses, whereas DCs injected i.d. or s.c. are prone to therefore, the specific identification of the immuno- induceTh1polarizedresponses. genic epitopes should not be required. Thus, Hsp- The preferential homing of the DC to a particular lym- peptidecomplexvaccinescouldbe“blindly”prepared phoidorgan,basedontherouteofadministration,couldbe against a newly emergent pathogen or variant, once used not only to affect cytokine polarization, but also to isolated, without further characterization of its anti- enhancetheimmuneresponsetoantigensoflowimmunoge- genic or immunogenic profile. Furthermore, since hsp nicity,aswellasthymus-independent(TI)antigens.Itiswell are self-antigens, they should not elicit immune re- knownthatthespleenplaysamajorroleintheinductionof sponsestothemselves.BacterialHspproteins,suchas anti-capsular polysaccharide antibody responses which are mycobacterial hsp70, have similar properties as syn- criticalforhostdefenseagainstencapsulatedbacteria.Inthis genic Hsp and could be exploited for possible novel regard, we demonstrated that the i.v. injection of BMDCs properties.Thus,syngenicHspandpeptidecomplexes pulsed ex vivo with S. pneumoniae induced antibody re- induce restricted CD8+T-cell responses, but the com- sponsesspecificforthebacterialcapsularpolysaccharideand plexesofmycobacterialhsp70alsoelicitedCD4+T-cell for the phosphorylcholine determinant of the cell-wall responses, and as fusion proteins with ovalbumin, in- C-polysaccharide [6]. However, these polysaccharide- ducespecificantibodyresponses. specific antibody responses were impaired when DCs were 5.Engineeredbacteriaandvirus:Duetotheabilityofthe injectedi.p.Recently,newdataimplicatingmyeloidBMDCs DCtospecificallyrecognizeandinternalizepathogens, intheinitiationofantibodyresponsesspecificforTIantigens attenuatedvirusorbacteriacouldbeusedascarriersfor hasbeenobtained,withtheidentificationofasubsetofblood specific antigens or genetic information. Interestingly, CD11c-low immature mouse DCs in vivo, likely counter- this approach was largely explored before our under- partsoftheexvivogeneratedBMDCs,asbeingtheprimary standing that DCs would be a major target for these cells capturing and transporting hematogenous bacteria to vaccines. thespleen[29].ThisCD11c-lowDCprovidedcriticalsignals toTIantigen-specificBcellsresidentinthesplenicmarginal 3.4.ReinforcingDCantigen-deliveryandpresentation zone,allowingthemtosurvive,andpromotingtheirdifferen- DCs process the microbial antigens obtained in the pe- tiation into IgM-secreting plasmablasts.Thus, our observa- riphery, and present them to T cells in the secondary lym- tion that DCs can induce polysaccharide-specific Ig re- phoid organs.This involves the proteolysis of proteins into sponses in vivo may open a new window for designing peptidesofappropriatesizesforbindingtoMHCclassIIand polysaccharide-based vaccines against encapsulated bacte- class I molecules. To optimize T-cell priming during an ria.DChomingwillalsodependonthesubsetandactivation ongoinginfection,DCsmusthomefromtheperipherytothe stageoftheDCs,astheyexpressdifferentchemokinerecep- appropriate microenvironments within the secondary lym- torsandhavedifferentadhesionproperties.Thus,alteration phoidorgans.Therefore,anotherlevelofinterventioncould ofthesepropertiesinantigen-pulsedexvivoDCsmayrepre- betofacilitateanddirectDChoming. sentafurthermeansofoptimizingimmuneresponses.Poten- tialchemokinereceptorsofinterestinclude(i)CCR7,which 3.4.1.Routeofadministrationandmigration isnecessaryforlymphaticentryandmigrationtotheT-cell It is well known that the route of antigen administration areas of secondary lymphoid organ, (ii) CCR6, which may can affect the quantity and quality of an immune response. direct DC migration into the epithelial layer of the Peyer’s This is due, in part, to the particular subset and migration patch or possibly the marginal zone of the spleen or, (iii) pathways of the DC capturing the antigen (Fig. 1, point 4), CXCR5, whichcandirect themigrationofDCsintoB-cell andinpartduetotheparticularlymphoidorganintowhich follicles. Thus, CXCR5-specific gene transduction of BM- 318 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 DCsledtotheirredirectiontoB-cellfollicles,asopposedto [3] M.R.Neutra,N.J.Mantis,A.Frey,P.J.Giannasca,Thecomposition T-cell areas of the draining lymph node after s.c. injection, andfunctionofMcellapicalmembranes:implicationsformicrobial pathogenesis,Semin.Immunol.11(1999)171–181. with concurrent enhancement of the elicited antibody re- sponse [30].We additionally observed a strong decrease in [4] C. Winzler, P. Rovere, M. Rescigno, F. Granucci, G. Penna, L.Adorini,V.S. Zimmermann, J. Davoust, P. Ricciardi-Castagnoli, the ability of i.v. injected CXCR5-/- BMDCs to induce Maturationstagesofmousedendriticcellsingrowthfactor-dependent polysaccharide-specific antibody responses in vivo (unpub- long-termcultures,J.Exp.Med.185(1997)317–328. lisheddata). [5] S.J. Turley, K. Inaba, W.S. Garrett, M. Ebersold, J. Unternaehrer, R.M.Steinman,I.Mellman,Transportofpeptide-MHCclassIIcom- 3.4.2.Exosomes plexesindevelopingdendriticcells,Science288(2000)522–527. Idealvaccines,aswecomment,shouldbecell-free.Inthis [6] J.Colino,Y.Shen,C.M.Snapper,DendriticcellspulsedwithIntact regard, the question arises as to how we might utilize the Streptococcus pneumoniae elicit both protein- and polysaccharide- specificimmunoglobulinisotyperesponsesinvivothroughdistinct essential properties of the DC without using the DC itself. mechanisms,J.Exp.Med.195(2002)1–14. One potential solution involves the use of exosomes. 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Thus, ovsky,C.R.Maliszewski,Distinctdendriticcellsubsetsdifferentially exosomes containing loaded peptides can stimulate, by regulatetheclassofimmuneresponseinvivo,Proc.Natl.Acad.Sci. themselves,antigen-specificCD4+T-cellandCTLresponses USA96(1999)1036–1041. bothinvitroandinvivo. [11] S.H.Gregory,A.J.Sagnimeni,N.B.Zurowski,A.W.Thomson,Flt3 ligandpretreatmentpromotesprotectiveimmunitytoListeriamono- Todate,exosomeshavebeenimplicatedjustintheinduc- cytogenes,Cytokine13(2001)202–208. tionofT-cellprimingandtherefore,havebeenexploredfor [12] I.B. Kremer, M.P. Gould, K.D. Cooper, F.P. Heinzel, Pretreatment their potential use in anti-cancer therapy. The first trials in with recombinant Flt3 ligand partially protects against progressive humanpatientshavebeencarriedoutwithsomeveryprom- cutaneousleishmaniasisinsusceptibleBALB/cmice,Infect.Immun. ising success seen in the regression of tumors at skin and 69(2001)673–680. lymphnodesites[31]. 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MicrobesandInfection5(2003)311–319 www.elsevier.com/locate/micinf Review Dendritic cells, new tools for vaccination JesusColino,CliffordM.Snapper* DepartmentofPathology,UniformedServicesUniversityoftheHealthSciences,4301JonesBridgeRoad,Bethesda,MD20814,USA Abstract Ourrapidlyexpandingknowledgeofthebiologyofthedendriticcell(DC),amajorantigen-presentingcellconnectinginnateandadaptive immunity,suggestsnewpossibilitiesforthedevelopmentofvaccinesandtherapeuticstrategiesagainstpathogens,throughthemanipulation oftheirfunctioninvivo,ortheinjectionoftheDCitself,onceproperlyinstructedexvivo. ©2003ÉditionsscientifiquesetmédicalesElsevierSAS.Allrightsreserved. Keywords:Vaccines;Immunotherapy;Dendriticcells 1.Introduction non-opsonized pathogens by macropinocytosis, phagocyto- sis or through mannose receptors (C-type lectins), or com- Duringthelastdecade,intensiveresearchhasfocusedon plexes of antibody and microbial antigen via Fcc receptor understandingdendriticcell(DC)biology[1,2].Asaresult, types I and III. By virtue of their high phagocytic and en- wenowhaveasignificantunderstandingoftheplasticityof docyticcapacities,DCsconstitutivelyinternalizesamplesof DC function and its major role in maintaining homeostasis theirantigenicmicroenvironment,whichintheeventofin- through the induction of protective immune responses fection, will also include microbial antigens. In addition, against pathogens and tumors, and in the maintaining of DCsbecomeactivatedandmobilizedinresponsetoinfection peripheraltolerance.Inthisreview,wewilldiscussthemajor (i) by direct recognition of pathogen-associated molecular checkpoints involved in DC function (Fig. 1), focusing on patterns (PAMPS); e.g. LPS or cell-wall components, by theirroleinanti-bacterialimmunity,anddiscusshowDCsor Toll-like receptors (TLRs), or (ii) indirectly through recep- theirproductsmaybeusedoralteredtopreventandmanage torsforinflammatorycytokinesandchemokines. invasiveinfections. Immature DCs upon activation with PAMPS secrete in- flammatory cytokines (e.g. IL-1b, TNF-a, IL-6 and IL-12) and chemokines (e.g. MIP-1a and MIP-1b; RANTES and 2.OverviewofDCfunction MCP-1) which serve in part to recruit circulating DC pre- cursors and other immune cells to the site of infection and DCsshowauniquefunctionaldualityduringtheirdevel- participate in their activation (Fig. 1, point 1). Later on, opment,designedtoultimatelyprovidesecondarylymphoid anti-inflammatory cytokines, chiefly IL-10, are secreted by tissueswithusefulinformationabouttheantigeniccomposi- DCs to counterbalance the pathogenic stimulus, thus pre- tionintheperiphery.Attheimmaturestagesofdevelopment, ventingtheinductionofexaggeratedimmuneandinflamma- DCsresidentinperipheraltissuesarespecializedinantigen- toryresponses. capture, acting as sentinel cells (Fig. 1). They are strategi- callylocated,amongmacrophages,atepithelialbarriersthat Pathogen-mediated activation induces DCs to undergo oftenserveasmajorportalsofpathogenentry.Thus,inthe maturation.Thisisacomplexprocesswiththedualgoalof gastrointestinal tract, DCs located in intraepithelial pockets transformingimmatureDCsintheperipheraltissueintocells belowMcellsarewellequippedtodetectandsampleinfec- thatwillmigratetothesecondarylymphoidorgansandthen tious agents for the transport of microbial antigen to the act as professional antigen-presenting cells (APCs) for the subepithelial lymphoid tissue [3]. DCs avidly internalize primingofnaiveTcells(Fig.1,points2–4).Towardaccom- plishing the first goal, activated DCs lose their phagocytic capacityandtissueadhesivestructures,increasetheirexpres- *Correspondingauthor.Tel.:+1-301-295-3490;fax:+1-301-295-1640. E-mailaddress:[email protected](C.M.Snapper). sionofreceptorsforlymphoidchemokines(i.e.CCR7),and ©2003ÉditionsscientifiquesetmédicalesElsevierSAS.Allrightsreserved. DOI:10.1016/S1286-4579(03)00033-9 Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 3. DATES COVERED 2003 2. REPORT TYPE 00-00-2003 to 00-00-2003 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Dendritic cells, new tools for vaccination 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Uniformed Services University of the Health Sciences,Department of REPORT NUMBER Pathology,Bethesda,MD,20814 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Our rapidly expanding knowledge of the biology of the dendritic cell (DC), a major antigen-presenting cell connecting innate and adaptive immunity, suggests new possibilities for the development of vaccines and therapeutic strategies against pathogens, through the manipulation of their function in vivo, or the injection of the DC itself, once properly instructed ex vivo. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE Same as 9 unclassified unclassified unclassified Report (SAR) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 312 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 Fig.1.OverviewofDCfunctionduringaprimaryinfection.NumbersonthefigurerefertothemajorstepsofDCfunctionwhichcouldbealteredinorderto designvaccinesandimmunotherapeuticapproachesagainstpathogeninfections,asindicatedinTable1.Uponinflammation,immatureDCslocatedatthemajor portalsofpathogenentry,internalizethepathogen(3,antigen-capture),becomingactivatedbydirectrecognitionofPAMPS(2,activation).ActivatedDCs releaseinflammatorychemokinestorecruitDCprecursors(1,recruitmentofprecursors)andinflammatorycytokinesthatreinforceinflammationandbystander DCactivation(2).DCsactivatedbythepathogenundergomaturation,aprocessthatoccursduringDCmigrationfromtheperipheraltissuestothesecondary lymphoidorgans(4,migration).MaturationtransformstheDCfromanantigen-capturecell(3)intoaprofessionalAPC(5,antigen-deliveryand-presentation). MigratingDCsenteringasecondarylymphoidorgan,interactandpresenttheprocessedantigenicprofileofthepathogentoTcells,andlikelyBcells,toinitiate aprimaryimmuneresponse(5).Dependingonthepatternofcytokinessecretedduringtheinteraction,theimmuneresponsewillpolarizetowardseitheraTh1 orTh2response(6,polarization).DCsuponcompletingtheirAPCfunction,likely,diebyapoptosis.ReprocessingbyresidentDCsofapoptoticbodies containingprocessedantigencouldbeasecondary,butunlikely,sourceforantigenpresentation.Alternatively,DCscoulddeliverantigenlocallyoratadistance throughsecretedexosomes.ThephysiologicalroleofDCexosomesisamatterofdebate. J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 313 reorganizetheircytoskeletonfortheacquisitionofhighcel- strated markedly impaired polysaccharide- and protein- lular mobility [4]. Towards the second goal, DCs strongly specific IgG responses upon transfer into naive mice upregulatetheirexpressionofco-stimulatorymolecules(i.e. (submitted manuscript). Collectively, our data argue for an CD40,CD80andCD86)andupregulatesynthesisandtrans- active, direct role for the antigen-loaded migrating DCs in location to the surface of MHC molecules complexed to this system. However, we hypothesize that for anti- processedmicrobialantigens.Concomitantly,MHCclassII polysaccharideresponses,theactivereleaseofexosomesby degradationisreduced,resultinginenhancedretention(sev- antigen-loaded DCs could play an important inductive role eral days) of peptide-MHC complexes on the cell surface. boththroughantigentransferbetweenDCsthemselves,and Thus, DCs, unlike macrophages and other professional between DCs and B cells, that are in tight contacts or syn- “scavengers”,respondtothemicrobialchallengebymigrat- apse. ing to secondary lymphoid organs while generating a pro- DCsandTcellscanalsointeractthroughtheformationof cessed antigenic profile of the pathogen associated with animmunologicalsynapse[7].Inthissynapse,T-cellrecep- MHCmolecules.AnotheruniquefeatureofDCsrelativeto tors and co-stimulatory molecules congregate in a central macrophages, is the ability of DCs to attenuate their pro- areasurroundedbyaringofadhesionmolecules.Sustained teolyticpotential.Thus,DClysosomescansequesterantigen signalingviathesesynapticinteractionsisrequiredinorder forextendedperiodsandstillefficientlyusetheantigenfor fortheTcelltoenterthefirstcelldivisioncycle.Specifically, generatingpeptide-MHCclassIIcomplexes[5]. naiveCD4+Thelpercellsrequiremorethan20hofcontinu- Onceinthesecondarylymphoidorgan,matureDCs(ef- ous stimulation to become committed to cell division, fectorstate)instructTcells,andlikelyBcellsandNKcells, whereasshorterperiodsdidnotresultineffectorT-celldif- toinduceaneffectiveprimaryimmuneresponse,andestab- ferentiation.Thus, the stability and duration of the synapse lishimmunologicalmemory(Fig.1,point5).Thisabilityof will determine T-cell function. Factors disrupting the syn- DCstoinduceaprimaryimmuneresponseisalsoaunique apse,suchasDCdeath,willsignificantlyaffectT-cellfunc- propertyamongAPCs.Werecentlydemonstratedthatbone- tion.Inthisregard,werecentlyobservedastrongassociation marrow-derived dendritic cells (BMDCs) loaded in vitro betweentheprogressionofS.pneumoniaeinducedDCapo- with Streptococcus pneumoniae and then transferred into ptosisandadecreasingabilityofsuchDCstoinduceT-cell- naivemicewereabletoinduceprimaryIgisotyperesponses dependentprimaryIgisotyperesponsesinvivo.Ontheother not only for bacterial proteins, but also for capsular and hand, factors that prevented DC apoptosis, such as IL-10, cell-wall polysaccharide antigens [6]. The Ig isotype re- servedtoprolongtheAPCfunctionofDCsforinductionof sponsestothebacterialproteinsuniformlyrequiredDCex- in vivo anti-bacterial humoral immunity (submitted manu- pressionofmajorhistocompatibilitycomplexclassII,CD40 script). and B7 and the production of IL-6, strongly suggesting a DCsinduceantigen-specificTcellsnotonlytoproliferate, requirementforcognateinteractionswithTcellsandlikely but also determine the development of T-cell regulatory or with B cells. Similar requirements for anti-polysaccharide effectorfunctions,includingCD4+helperT-cellpolarization antigenswereobservedonlyfortheIgG1isotype,suggesting (i.e. Th1 vs. Th2) to secrete different patterns of cytokines that for polysaccharide-specific responses classical cognate (Fig. 1, point 6). IL-12 is the prototypic Th1-polarizing interactionsmayaffectthequalityoftheIgisotyperesponse, cytokineproducedbyDCs,whereasotherfactors,including but are not critical for DC induction of primary anti- IL-4,ortheabsenceofIL-12productionleadtoTh2polar- polysaccharideIg.Anunresolvedquestionhasbeenwhether ization.The ability of DCs to influence the pattern of cyto- DCsmigratingtothesecondarylymphoidorgansactedeither kinessecretedbyTcellsrepresentsacriticalfunctionwhich directly as APCs or transferred their captured antigens to can profoundly influence the final outcome of the immune residentDCsforantigenpresentation.Twomechanismshave responsetoapathogen.Thus,cytokinessecretedbyTh1cells beenproposedtoexplainhowDCsmighttransferantigenin (IFN-c,TNF-bandIL-2),aretypicallyconsiderednecessary anindirectpathwayofantigenpresentation(Fig.1,point5): forprotectionagainstinfectionswithvirusesandwithintra- (i) antigen-loaded DCs that migrate to the secondary lym- cellular bacteria and protozoans, whereas secreted Th2 cy- phoid organ could undergo apoptosis or necrosis and be tokines(IL-4,IL-5,IL-6andIL-13)areimportantforprotec- phagocytosed by resident DCs (ii) migrating DCs could tive responses against multicellular nematode parasites. activelyreleaseantigen-bearingvesicles(exosomes)derived Several factors appear to influence the ability of DCs to from the DC lysosomal compartment, which could then be polarizeT-cellcytokineresponses.(i)DCsubsets:thesubset captured by resident DCs. The relative importance of the ofDCusedforT-cellstimulationmayeffectabiasforeither directandindirectpathwaysinanti-microbialimmunityhas aTh1orTh2response.(ii)DynamicsofDCmigrationtothe not yet been resolved, but recent data from our laboratory lymphoid organ: early after initiation of the immune re- stronglyargueagainsttheindirectpathwayhypothesis.Thus, sponse, large numbers of recently stimulated DCs actively weshowedthatviable,butnotnecrotic,BMDCsloadedwith secretingIL-12andenteringintotheT-cellareasmaypref- wholeS.pneumoniaeinducedsignificantantigen-specificIg erentiallyinduceTh1cellpriming.Incontrast,atalatertime, isotype responses in vivo [6]. Furthermore, BMDCs under- when the DC influx decreases, and the surviving DCs in goingapoptosisinducedaftercontactwithbacteriademon- T-cell areas have downregulated their secretion of IL-12, 314 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 preferentialprimingforTh2andotherregulatoryTcellsmay Table1 occur [8]. (iii) Nature of the maturation stimuli: bacterial SummaryofthepotentialtherapeuticandprophylacticusesofDC PAMPs induce strong production of IL-12 by DCs, which StepofDC Potentialtherapeuticapproach canbepotentlyboostedbyactivatedTcellsthroughCD40L, function whereas other DC stimuli such as cholera toxin, TNF-a or Recruitmentof TreatmentofpatientswithDC-poietins:Flt3L,GM-CSF DCprecursors IncreasingDC-poietindepositionandantigenco-delivery: IL-1 do not induce IL-12 (iv) Microenvironmental factors: DC-poietinconjugatesorfusionproteinswiththe IL-12productionbyDCscanalsobeenhancedbymediators antigen released in the DC microenvironment. For example, IFN-c Encapsulation and IL-4 can enhance IL-12 production by activated DCs, ViralvectorsencodingGM-CSF whileprostaglandinE2andIL-10exertaninhibitoryeffect. TransferofautologousDCexpandedexvivowith ResultsfromourgroupindicatearoleforautocrineIL-10in DC-poietins modulatingDCIL-12productionearlyafteractivationbyS. DCactivation Treatmentwithproinflammatorycytokines:IL-1b,IFN-a pneumoniaeinordertoadjustthematurationresponsetothe andmaturation Microbial-derivedproducts: Mucosaladjuvants:Choleratoxoidmutants magnitudeofthestimuli(submittedmanuscript).Thus,inthe PAMPS:CpG-oligos,viraldsRNA absence of autocrine or paracrine IL-10, the level of IL-12 Antigen-capture DCloadedexvivowithinactivatedpathogenbefore productionwassustainedforlongperiodsoftime(>8h). transfer Thus,DCsrepresentthecriticallinkbetweeninnateand DCtransfectedexvivowithpathogenDNAplasmids adaptive immunity, upon which, appropriate concerted ac- orRNA tion is required for a successful host defense against an DCloadedwithimmunologicallyrelevantpeptides invadingpathogen. TargetingDCsinvivowithsensormolecules: LigandsofMHCandco-stimulatorymolecules(CD40) Antibodiesspecificforendocytosisreceptors(CD205) 3.Therapeuticandprophylacticusesofdendriticcells PAMPs:CpG-oligos,OmpA Heat-shockproteins SignificantprogressinourunderstandingofDCbiology Engineeredbacteriaandvirus andtheircriticalfunctioninimmunityhavepromptedmany Migrationand RouteofDCadministration:subcutaneousvs. homing intravenous toexploretheirpotentialuseinimmunotherapyandprophy- Chemokine and chemokine receptors engineered DC: laxis. Most of these pioneering strategies have been devel- CXCR5,CCR7 oped for anti-tumor therapy, and some are currently being DCsubset testedinhumantrials,withvariablesuccess.Acomprehen- Antigendelivery Migratorypathways sivelistofthesafetyrequirementsofDCpreparationsused andpresentation Exosomes forcancerimmunotherapyhasbeenpublished[9].Below,we Polarization DCsubset discuss various strategies to prevent or treat infectious dis- NatureoftheDCmaturationoractivationstimuli eases for optimizing the use of DCs for mediating their (e.g.bacterialPAMPSvs.inflammatorycytokines) DC-poietin(e.g.Flt3vs.GM-CSF) functional effects (Fig. 1) upon transfer into the host as RouteofDCadministration(e.g.i.v.vs.s.c.) summarizedinTable1. andDChoming DCtreatmentwithanti-inflammatorycytokines:IL-10 3.1.ExpandingDCnumbers One approach to optimizing DC function is to increase subsets could influence the nature of the induced response. their numbers, usually, prior to the administration of an Thus, Pulendran et al. [10] observed that pretreatment of antigen-basedvaccine.Itwillfavorandreinforcethenatural micewithFlt3LselectivelyenhancedtheIgG2aresponseto recruitment of DC precursors (Fig. 1, point 1) Two major soluble ovalbumin which resulted from the induction of a strategies have been explored to achieve this goal: (i) the polarizedTh1response,whereasGM-CSFpretreatmentin- transferofautologousDCsexpandedinvitrointhepresence creasedIgG1antigen-specificresponses,indicatingpolariza- of cytokine cocktails and (ii) the in vivo mobilization of tionofthecytokineresponsetoTh2.Therefore,theselection stem-cellprecursorsofDCsandexpansionofimmatureDCs betweenGM-CSForFlt3L,intheory,mightallowformodu- by treatment with the DC-poietins Flt3L or granulocyte- lationoftheresponsethatmayfavorhostdefense,depending macrophagecolony-stimulatingfactor(GM-CSF). onthenatureoftheinfectiousdisease. Flt3L, the ligand of the FMS-like tyrosine kinase 3, TheuseofFlt3Lforaugmentinghostresistancetopatho- strongly promotes the proliferation, differentiation and sur- gens has met so far with limited success. Thus, Flt3L pre- vival of early hematopoietic progenitor/stem cells toward treatmenthasbeenshowntopromoteresistanceagainstsec- DCs and to a lesser extent, NK cells. Noticeably, Flt3L ondary infection with Listeria monocytogenes [11] and inducesincreasesinthenumbersofallDCsubsets.Incon- partialprotectionagainstprogressivecutaneousLeishmania- trast,GM-CSFisselectiveinitsinductionofproliferationof sis[12]inmice,butinboththecases,itisnotclearthatthe cells of the monocyte-macrophage lineage leading to my- protective effect was directly due to DC expansion as op- eloidDCexpansion.TherelativeexpansionofdifferentDC posedtotheexpansionofothercelllineages.Moreover,tobe J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 315 effective, repetitive injections of high doses of Flt3L were two major advantages as adjuvants for human use: (i) low required. Further, the treatment did not resolve an ongoing toxicityand(ii)theabilitytosynthesizetheminapureand Leishmania infection. In light of these studies, the future standardizedform. applicationofFlt3Ltohumanpopulationappearsuncertain. 3.3.Improvingantigen-capture:learningtouseDCs GM-CSF is also a very poor adjuvant when delivered systemically. This drawback has been partially resolved A next level of intervention is the design of strategies to through strategies focused on effecting efficient and pro- improve the delivery of the antigen to the DCs by ex vivo longeddepositionofGM-CSFatthevaccinationsiteandits loading,orinvivotargetinginordertoreproduceandrein- co-delivery with the antigen (i) by coupling the GM-CSF force the physiologic antigen-capture step of DC function with carrier molecules such as polyethylene-glycol, (ii) as (Fig. 1, point 3).An invaluable amount of information has fusionproteinswiththeantigen,(iii)byencapsulationinside beenobtainedusingDCsloadedexvivowithbacteriaprior liposomesincombinationwiththeantigen,(iv)orencoded to transfer to the recipient animal. This experimental ap- intoviralvectors.Someofthesestrategieshaveproventobe proachprovedforthefirsttimethattransferofDCsloaded effectiveinmousemodelsofbacterialinfection.Thus,vac- with the inactivated pathogen induces protective immune cination with a fusion protein of a pneumococcal surface responsesinnaiverecipientslatersubjectedtoexperimental protein(PspA)andGM-CSFprotectedmiceagainstalethal infection. Immunity has been obtained against pathogens challenge with virulent S. pneumoniae [13]. Most recently, that have so far proved difficult to prevent using vaccine co-immunization of mice with transgenic adenoviral GM- approaches:M.tuberculosis[19],Borreliaburgdorferi[20] CSF and BCG vaccine, strongly enhanced Th1 antigen- Chlamydiatrachomatis[21]andCandidaalbicans[22].C. specificimmunity,andtheprotectiveefficacyofthevaccine albicans provides a paradigmatic example of how this ap- againstachallengewithMycobacteriumtuberculosis[14]. proachcanalsohelptodesignvaccinestrategies.DCsloaded withtheyeastform,butnotwiththehyphalform,generate 3.2.ActivatingDCs protectiveanti-fungalimmunity.Thiswasbasedontheabil- DCs that capture antigen, in the absence of concurrent ityoftheDCstodiscriminatebetweenthetwoformsofthe activation,mayinduceantigen-specifictolerance,asopposed pathogen, responding to the yeast by producing IL-12 and to immunity. Thus, when the antigen does not activate the stimulatingaprotectiveTh1response,butsecretingtheTh2 DCsbyitself,anadjuvantmightberequiredincombination cytokineIL-4inresponsetohyphae,whichwasnotprotec- withtheDC-poietininordertoinduceproperimmunity.In tive. However, suppression of IL-4 production by hyphae- thisregard,ithasbeenshownthatDCscanplayanactiverole pulsedDCsallowedforaprotectiveresponsetooccur. intheinductionofmucosaltolerancetofoodororallyadmin- The success of this approach is based on two factors. (i) istered soluble antigens when a concurrent adjuvant is not Pathogens can directly activate the DCs. (ii) A single DC present.Thus,inductionoftolerancetoorallyadministered afterpathogeninternalization,isabletocompetentlypresent soluble ovalbumin, is further enhanced in mice pretreated an array of T-cell pathogen-derived peptides covering the with Flt3L to expand DCs, but this can be reversed by complete T-cell antigenic repertoire of the bacteria, thus additionaladministrationoftheinflammatorycytokineIL-1 inducingaprotective,multi-specificimmuneresponse.This (Fig. 1, point 2), a cytokine which can activate DCs [15]. approachallowsforuseofhiddenorunknownT-cellprotec- IFN-a can also promote DC activation and hence act as an tiveepitopes,hardtodetectinscreeningtests.Furthermore, adjuvant. Thus, IFN-a markedly enhances an antigen- ourresultsdemonstratingthatDCsloadedwithinactivatedS. specific humoral response in mice when co-administered pneumoniaeareabletostimulateapolysaccharide-specific, withFlt3L[16]. aswellasprotein-specific,humoralresponsesinnaiverecipi- A second group of molecules that can activate DCs are ents[6]expandthepotentialprophylacticuseofDCs,since microbial-derived products (Fig. 1, point 2). The most protectionagainstextracellularbacteriaistypicallymediated broadly used products in experimental models are LPS a bycapsularpolysaccharide-specificantibodyresponses. systemicadjuvantandTLR4ligand,andcholeratoxin(CT), Formostinfectiousdiseases,theuseofDCspulsedwith a mucosal adjuvant.Although both are highly toxic, many thewholepathogen,whetherinactivatedorattenuated,may attempts have been made to detoxify them while retaining not represent an ideal approach for human vaccination be- theiradjuvantproperties.Onepromisingstrategyistheuseof cause of safety concerns.We thus need to determine which amutantoftheCTsubunitA,whichisnon-toxicandinduces pathogencomponentsarebothsafeandusefulwhenusedin astrongandpolarizedTh2responsewhenusedasamucosal combination with DCs. The consequent reduction of anti- adjuvant [17].Alternatively, DCs may be activated through genic variety inherent in this latter approach might have TLR9 by oligodeoxynucleotides containing unmethylated unexpected pitfalls. For instance, adoptive transfer of DCs CpG motifs or through TLR3 by viral dsRNA or polyI- pulsedwithinactivatedC.trachomatishasprovensuccessful :polyC. Stimulatory oligodeoxynucleotides in combination in experimental models for promoting anti-Chlamydial im- withFlt3Lhavebeenshowntobeeffectiveintheenhance- munity through stimulation of a Th1 response. However, a ment of humoral and cellular responses to soluble proteins similaradoptivetransferofDCspulsedwiththerecombinant andanti-tumorimmunityinmice[18].Polynucleotideshave chlamydial major outer membrane protein, which has been 316 J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 regarded as a promising candidate for the development of troduced class II-restricted T-cell epitopes, as sensors conventional vaccines, did not induce protection, and this [23]. These antibodies, termed “troybodies”, strongly was associated with a polarized Th2 response [21]. One enhanced epitope delivery to the APC for efficient alternative is the use of DCs transfected ex vivo with DNA T-cellstimulation,andthuscouldbeapromisingstrat- plasmids or RNA derived from the pathogen, in order to egy for targeted peptide delivery. However, caution endogenouslyproducethemicrobialantigensaftertransfer. mustbetakenwhentargetingDCsthroughMHC,since This strategy has been used with success in experimental DCsareverysensitivetotheinductionofapoptosisvia tumors, and recently has shown promise in anti-infectious surfaceMHCcross-linking,andthelevelofexpression immunity.Thus,DCstransfectedwithRNAfromyeast,but of MHC will vary with the activation and functional not hyphal, C. albicans induced a protective immune re- maturationstageoftheDC. sponsesimilartothatobservedupontransferofDCspulsed 2.Antibodies and their fragments specific for endocytic withtheintactyeast[22]. receptors:An analogous approach involved the use of AnotherapproachistheuseofDCsloadedwithrelevant fusionproteinsofapeptideandoneantibodyspecificto immunogenic peptides in an attempt to increase the safety DEC-205(CD205),anendocyticreceptorandmember and reproducibility of the vaccine. Nevertheless, this ap- ofthemannosereceptorfamily.DEC-205expressionis proach may introduce more problems than it resolves. (i) restricted to DCs within the T-cell areas of lymphoid Loading with peptides drastically restricts the repertoire of tissuesandparticularlyexpressedbylymphoidDCs.It potentialT-cell clones stimulated, and therefore, the ability is well known that endocytosis of microbial antigens toinduceaprotectiveresponse.(ii)Itisdifficulttoenvisage through DEC-205 binding results in efficient antigen- standardizedmixesofpeptidesabletostimulateCD4,CD8 processingand-presentationtoTcells.DCstargetedin andregulatoryTcells.DCs,instead,arespecializedtopro- vivo with the fusion antibodies for DEC-205 indeed, ducethesepeptidesfromthewholepathogen.(iii)Efficient efficiently stimulated the proliferation of T cells spe- MHCclassIandclassIIpresentationoccursonlywhenthe cificforthepeptide.Nevertheless,sincetheendocyto- peptides are generated within the DC itself (iv) Only in sisofthefusionantibodiesdidnotinduceDCmatura- mature DCs are the cell surface MHC-peptide complexes tion,thetreatmentresultedintheinductionofpeptide- stable;inimmatureDCs,theyareconstantlyrenewed.This specifictolerance[24].Thisstudywasamongthefirst approachwouldrequiretheconcurrentuseofaDCactivator todemonstratethatDCshaveakeyroleinthemainte- iftheDCsareimmature,oralternatively,analreadymature nanceofperipheraltolerance,andthatthisisrelatedto DC. The use of a fully matured DC immediately prior to the state of activation/maturation of the DCs. These transfer,couldprovelesseffectivesincetheinvivohalf-life resultsstressedtheimportanceoftheuseofconcurrent of the mature DC is likely less than the activated immature DCactivationsignalsorvaccinedesignsabletoinduce DC. DC maturation in order to elicit protective immunity. Vaccinationofthegeneralhumanpopulationwithexvivo Nevertheless, this approach could be used to induce generated DCs, a procedure non-exempt of risk, costly and antigen-specifictoleranceforthetreatmentofautoim- difficulttoreproduce,doesnotappeartobeafeasiblefirst- munediseases. lineprophylacticapproach,andprobablywillberestrictedto 3.PAMPs, (e.g. CpG oligonucleotides that signal via instancesinwhichconventionaltherapiesfortreatingmalig- TLR9, or peptidoglycan and the outer membrane pro- nancyorchronicinfectionhavefailed,tofocalvaccinationor teinA of Klebsiella pneumoniae (OmpA), both utiliz- toimproveimmunityinimmunodeficientpatients(e.g.bone ing TLR2 for signaling): the use of PAMPs as sensor marrowtransplantrecipients).Amorefeasibleapproachisto moleculesallowsafurthermanipulationoftheoutcome efficientlydelivertheantigeninvivototheendogenousDC of the immune response to induce protective immune bycell-targeting.Severalapproachescurrentlybeingevalu- responses. SinceTLR molecules are differentially ex- ated employ fusion proteins or chemical complexes of the pressed on DC subsets, it is possible to selectively immunogenic antigen or the viral vector, with a “sensor targetfunctionallydistinctDCsthroughtheselectionof molecule” which bind surface receptors expressed on DCs, the PAMP. Thus, TLR9 and TLR7 are selectively ex- and in many cases, able to deliver an activation signal.The pressed by plasmacytoid human DCs and produce currentsensormoleculestestedinclude: IFN-a after TLR9 binding with CpG motifs, whereas 1.LigandsofMHCandco-stimulatorymolecules,which myeloidhumanDCsexpressallTLRsexceptTLR7and areexpressedmostlybyAPCs:manystudieshavedem- TLR9andproducehighlevelsofIL-12afterTLR2or onstrated the ability of CD40-ligand and anti-CD40 TLR4-mediatedactivationwithpeptidoglycanorLPS, antibodies to activateAPCs which selectively express respectively[25].Furthermore,OmpAandtheantigens CD40,suggestingthattheseligandswouldbeeffective coupled to OmpA, are delivered into the MHC class I carriersforantigentargetingtoandsubsequentpresen- presentation pathway after DC uptake, through cross- tation by DCs. Recently, a new approach has been presentation [26]. This ability to present exogenous tested using MHC class II, as target, and recombinant antigensforpresentationtoCD8+TcellsmakesOmpA antibodiesspecifictoMHC,containinggeneticallyin- apromisingcarriertodevelopCTL-inducervaccines. J.Colino,C.M.Snapper/MicrobesandInfection5(2003)311–319 317 4.Heat-shock proteins (Hsps): Although some contro- thestimulatedDCmigrates.Intradermally(i.d.)andsubcu- versyexists,hostHspsappeartoactivateDCsviaTLR2 taneously (s.c.) injected DCs preferentially home to the andTLR4inasimilarmannerasPAMPs.Thisproperty, T-cell areas of the draining lymph nodes, not to the spleen, in combination with the recognized ability of Hsp to whereas intravenously (i.v.) injected DCs home first to the bind peptides and direct them into the MHC class I lung, and then, preferentially to the spleen, and, to some presentationpathway,makesHsp,anidealsensormol- extent,tothekidneyandliver,butnottolymphnodes[28]. ecule for peptide-based vaccines against tumors and Intraperitoneal, and probably intralymphatic, injected intracellularpathogens.Indeed,Hspsappeartoplayan DCsshowanintermediatebiodistribution.DCsinjectedi.v., importantroleinautologousresistancetotumors[27]. as we observed in mice, may remain in the spleen for rela- The use of Hsp has additional advantages which are tively longer periods of time (>5 d).The specific lymphoid reminiscent of those offered by the use of ex vivo sitetowhichtheDChomescouldhaveamajorimpactonthe pulsedDCs.Duetotheirpromiscuityinpeptidebind- particularcytokinepolarizationoftheresponse,duetoarchi- ing,theHspstrategyis,intheory,applicabletoawide tectural and functional differences intrinsic to the different varietyofantigens.TheimmunizationwithHsppeptide lymphoidorgans(Fig.1,point6).Thus,thesamepreparation complexes derived from infected cells is potentially ofDCswheninjectedi.v.arepronetoinducenon-polarized directed against the whole antigenic repertoire and, responses, whereas DCs injected i.d. or s.c. are prone to therefore, the specific identification of the immuno- induceTh1polarizedresponses. genic epitopes should not be required. Thus, Hsp- The preferential homing of the DC to a particular lym- peptidecomplexvaccinescouldbe“blindly”prepared phoidorgan,basedontherouteofadministration,couldbe against a newly emergent pathogen or variant, once used not only to affect cytokine polarization, but also to isolated, without further characterization of its anti- enhancetheimmuneresponsetoantigensoflowimmunoge- genic or immunogenic profile. Furthermore, since hsp nicity,aswellasthymus-independent(TI)antigens.Itiswell are self-antigens, they should not elicit immune re- knownthatthespleenplaysamajorroleintheinductionof sponsestothemselves.BacterialHspproteins,suchas anti-capsular polysaccharide antibody responses which are mycobacterial hsp70, have similar properties as syn- criticalforhostdefenseagainstencapsulatedbacteria.Inthis genic Hsp and could be exploited for possible novel regard, we demonstrated that the i.v. injection of BMDCs properties.Thus,syngenicHspandpeptidecomplexes pulsed ex vivo with S. pneumoniae induced antibody re- induce restricted CD8+T-cell responses, but the com- sponsesspecificforthebacterialcapsularpolysaccharideand plexesofmycobacterialhsp70alsoelicitedCD4+T-cell for the phosphorylcholine determinant of the cell-wall responses, and as fusion proteins with ovalbumin, in- C-polysaccharide [6]. However, these polysaccharide- ducespecificantibodyresponses. specific antibody responses were impaired when DCs were 5.Engineeredbacteriaandvirus:Duetotheabilityofthe injectedi.p.Recently,newdataimplicatingmyeloidBMDCs DCtospecificallyrecognizeandinternalizepathogens, intheinitiationofantibodyresponsesspecificforTIantigens attenuatedvirusorbacteriacouldbeusedascarriersfor hasbeenobtained,withtheidentificationofasubsetofblood specific antigens or genetic information. Interestingly, CD11c-low immature mouse DCs in vivo, likely counter- this approach was largely explored before our under- partsoftheexvivogeneratedBMDCs,asbeingtheprimary standing that DCs would be a major target for these cells capturing and transporting hematogenous bacteria to vaccines. thespleen[29].ThisCD11c-lowDCprovidedcriticalsignals toTIantigen-specificBcellsresidentinthesplenicmarginal 3.4.ReinforcingDCantigen-deliveryandpresentation zone,allowingthemtosurvive,andpromotingtheirdifferen- DCs process the microbial antigens obtained in the pe- tiation into IgM-secreting plasmablasts.Thus, our observa- riphery, and present them to T cells in the secondary lym- tion that DCs can induce polysaccharide-specific Ig re- phoid organs.This involves the proteolysis of proteins into sponses in vivo may open a new window for designing peptidesofappropriatesizesforbindingtoMHCclassIIand polysaccharide-based vaccines against encapsulated bacte- class I molecules. To optimize T-cell priming during an ria.DChomingwillalsodependonthesubsetandactivation ongoinginfection,DCsmusthomefromtheperipherytothe stageoftheDCs,astheyexpressdifferentchemokinerecep- appropriate microenvironments within the secondary lym- torsandhavedifferentadhesionproperties.Thus,alteration phoidorgans.Therefore,anotherlevelofinterventioncould ofthesepropertiesinantigen-pulsedexvivoDCsmayrepre- betofacilitateanddirectDChoming. sentafurthermeansofoptimizingimmuneresponses.Poten- tialchemokinereceptorsofinterestinclude(i)CCR7,which 3.4.1.Routeofadministrationandmigration isnecessaryforlymphaticentryandmigrationtotheT-cell It is well known that the route of antigen administration areas of secondary lymphoid organ, (ii) CCR6, which may can affect the quantity and quality of an immune response. direct DC migration into the epithelial layer of the Peyer’s This is due, in part, to the particular subset and migration patch or possibly the marginal zone of the spleen or, (iii) pathways of the DC capturing the antigen (Fig. 1, point 4), CXCR5, whichcandirect themigrationofDCsintoB-cell andinpartduetotheparticularlymphoidorganintowhich follicles. 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