Biol. Chem., Vol. 389, pp. 469–485, May 2008 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.054

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Review

Dendritic cell subtypes as primary targets of vaccines:the emerging role and cross-talk of pattern recognitionreceptors

Szilvia Benko, Zoltan Magyarics, Attila Szabo´´and Eva Rajnavolgyi*

Institute of Immunology, Medical and Health ScienceCentre, University of Debrecen, H-4032 Debrecen,Hungary

* Corresponding authore-mail: evaraj@dote.hu

Abstract

Preventive vaccination is the most successful approachagainst infectious diseases and has a great impact onworld health. Vaccines operate through the activation ofinnate immunity that helps to stimulate antigen-specificT- and B-lymphocytes. These events are orchestrated bydendritic cells (DCs) that are able to sample foreign struc-tures and concomitantly sense ‘danger signals’. Thus,DCs provide a functional link between innate andacquired immunity, and due to their regulatory potentialare referred to as natural adjuvants. Human conventionaland plasmacytoid DCs express different sets of well-characterized Toll-like membrane receptors (TLRs) thatrecognize a broad range of conserved molecular patternsof pathogens. The recently discovered cytosolic Nod-likereceptors (NLRs) and RIG-like helicases (RLHs) alsoturned out to participate in pathogen recognition andmodulation of immune responses through interacting sig-naling pathways. As a result of their collaboration, theTLR, NLR and RLH recognition systems induce thesecretion of different combinations of cytokines that playa fundamental role in T-cell activation and instruction.Ligands of the innate recognition systems emerge asnew adjuvants for vaccine design, whereas manipulationof the signaling pathways mediated by these receptorsoffers new avenues for fine tuning immune responsesand optimizing immunotherapies.

Keywords: cross-priming; cytokines;immunomodulation; innate immunity; signaling;T-cell polarization.

Introduction

Preventive vaccination is the most successful approachagainst infectious diseases and has had a significantimpact on world health over the last 50 years (Hilleman,2000; Arvin and Greenberg, 2006). Furthermore, betterunderstanding of immune regulatory pathways facilitated

the development of new therapeutic vaccines againstpersistent infections and cancer (Berzofsky et al., 2004).Traditional prophylactic vaccines contain whole inacti-vated or attenuated live pathogens that express immu-nostimulatory components and in most cases inducerobust protective immune responses. The new genera-tion of vaccines, however, is composed of purified, syn-thetic or recombinant subunit antigens with weakimmunogenicity due to the lack of immunomodulatorypotential. Thus, the key elements of subunit vaccinesinvolve (i) the target antigen(s), (ii) immunostimulatoryadjuvants that induce rapid activation of innate immunity,and (iii) delivery systems that target both the antigen andthe adjuvant to the most relevant cell types of the innateimmune system. Innate immunity plays a crucial role inpriming highly specific adaptive immune responses,whereas delivery systems ensure optimal stimulation ofimmune cells and the development of long-term immu-nologic memory (Pashine et al., 2005). Various vaccineformulations containing adjuvants and delivery systemsact through various mechanisms that involve (i) theincreased stability and delayed release of the antigen, (ii)the efficient uptake and transport of antigen by phago-cytic cells, and (iii) overriding immunological tolerance bythe induction of immunostimulatory and/or immunomo-dulatory cytokines that facilitate and polarize immuneresponses to either inflammatory helper T type 1 (Th1),Th17, or anti-inflammatory Th2 antigen-specific re-sponses together with the production of typical antigen-specific antibody isotypes (Steinman and Banchereau,2007). All these effects directly or indirectly operatethrough innate immune responses, which are orchestrat-ed by professional antigen presenting cells (APCs). Den-dritic cells (DCs) are major regulators of innate immunity,possess the unique capability to prime adaptive immuneresponses, and are the most efficient APCs. Thus, DCsare also referred to as natural adjuvants of the immunesystem.

Differentiation, subsets and functional activityof dendritic cells

Based on their origin, anatomical localization and func-tional activities, human DCs are classified into two majorlineages: conventional DCs (cDCs) and plasmacytoidDCs (pDCs). The majority of human DCs derive frommyeloid precursors and give rise to various subsets, suchas Langerhans cells (LCs), interstitial (dermal or tissue)DCs, and monocyte-derived DCs (moDCs) (Shortman

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and Liu, 2002). pDCs represent a homogeneous popu-lation of cells, but their origin is still controversial, as theymay derive from both myeloid and lymphoid progenitorsexpressing the FMS-like receptor tyrosine kinase-3 (Flt-3), suggesting that a DC developmental program can beinduced in both precursors due to the substantial flexi-bility of DC generation (Chicha et al., 2004). In contrastto cDCs that reside to peripheral tissues, circulating pre-pDCs enter lymphoid tissues and acquire a typical DCmorphology after activation.

Circulating monocytes are considered as precursors ofmigratory DCs, LCs originate from inflammatory CD14high

monocytes (Schaerli et al., 2005), whereas interstitial DCsdevelop from non-inflammatory CD16q monocytes (Qu etal., 2004; Gordon and Taylor, 2005). Granulocyte-mac-rophage colony stimulatory factor (GM-CSF)-inducedmobilization and transition of monocytes to DCs wassuggested to model inflammatory DC development, andFlt-3 ligand was shown to be crucial for the steady-statedevelopment of both pDC and cDC (Shortman and Naik,2007). Conventional DCs are present in all peripheraltissues and are specialized for sampling their micro-environment (Steinman, 1991). Saturation of cDCs withphysiological tissue-derived material induces steady-state migration to draining lymph nodes (LNs) where theyinteract with T-lymphocytes. This cell-to-cell communi-cation is translated to tolerance induction mediated bynegative regulators of the immune response that causeinhibition of signaling pathways and cell communication.

Pathogenic invasion or inflammation induce the acti-vation of resting DCs and result in the transition to amature cell type with the altered expression of chemo-kine receptors that ensure the rapid migration of activat-ed DCs through the lymphatics to the draining LNs. Hereactivated DCs, carrying their accumulated antigenic con-tent, become highly potent APCs and instruct various T-lymphocytes to effector and memory cell differentiation.Microbial compounds are highly potent activators of rest-ing DCs and mediate inflammatory stimulatory signalsthrough conserved pattern recognition receptors (PRRs).

Conventional dendritic cells as targets ofvaccine delivery systems

Novel approaches of vaccine design focus on the tar-geted delivery of antigens to appropriate DC subtypes incombination with controlled stimulatory signals thatresult in prompt activation of innate immunity (Pashine etal., 2005). DCs are equipped with a wide array of inter-nalizing receptors that mediate the continuous uptake ofsoluble and particulate material from exogenous sources(Figdor et al., 2002; Gogolak et al., 2003). Due to theirhigh phagocytic and internalizing capacity, immature DCsare primary targets of vaccine delivery systems aimed atthe targeted and prolonged delivery of antigens to thesecells.

Targeted antigen delivery

Ex vivo targeting strategies use DCs or their precursorsisolated from the peripheral blood of patients. After exvivo manipulations (differentiation, antigen loading and

activation to mature antigen presenting cells) autologousDCs are reintroduced to the patient to exert immuno-modulatory functions. This approach is utilized by novelDC-based cancer immunotherapies where DCs aremanipulated to carry tumor antigens (Wang and Wang,2002; Tuyaerts et al., 2007).

In vivo DC targeting uses free antigens, fusion proteinsor viral gene therapy (Bonifaz et al., 2004) introduced tothe patients by various delivery systems. Antibody-basedtargeting strategies have been proved to be highly effi-cient as they are able to deliver the antigen to definedDC subsets resident at various tissue sites. One possiblestrategy is to design genetically engineered antibodiesthat carry microbial proteins and are specific for selectedreceptors of DCs, such as subtype-specific C-type lec-tins (Boscardin et al., 2006; Trumpfheller et al., 2006).Antibody-mediated targeting of antigen to DEC-205, anabundant type I membrane protein with multiple extra-cellular lectin domains expressed by skin and lymphnode DCs, results in a dramatic increase of antigen pres-entation by both MHC class I and class II molecules(Soares et al., 2007). Another approach uses antibodiesto target tumor cells to stimulatory Fcg receptors that notonly support antigen uptake to major histocompatibilitycomplex class II (MHC II) compartments but also aug-ment antigen processing and result in DC triggering(Dhodapkar et al., 2002; Kalergis and Ravetch, 2002).

DC subsets possess unique mechanisms optimized forantigen processing and presentation (Trombetta andMellman, 2005; Dudziak et al., 2007). Particulate anti-gens, such as microbes, apoptotic cells or designedbeads or other particulate formulates used for vaccina-tion (Table 1), can be sorted to distinct intracellularcompartments of DCs based on their co-expression ofToll-like membrane receptor (TLR) ligands (Blander andMedzhitov, 2006). Microbes and beads carrying bothantigenic structures and TLR ligands induce phagosomematuration and the presentation of antigenic peptides inthe context of MHC class II proteins at the cell surface.However, in the absence of coupled TLR signals peptidesof phagocytosed non-infected apoptotic cells internal-ized even together with microbes are not immunogenicdue to inappropriate phagosome maturation and antigenprocessing (Blander and Medzhitov, 2004).

The mode and robustness of DC activation may dra-matically influence the functional activity of other innateand adaptive cells through direct cell-to-cell interactionsand/or by the cross-talk of secreted cytokines. Thus,modulation of DC activity is able to regulate the effectorfunctions of T-lymphocytes, the magnitude and isotypedistribution of antibody responses and the induction oflong-term immunologic memory.

Action of traditional and new adjuvants

Antigen delivery systems and immune potentiatingagents altogether referred to as adjuvants can increaseimmunogenicity, speed up and sustain immuneresponses, modulate the qualitative features of antibody(avidity, isotype distribution) and cellular (T-cell subtypes,cytokine secretion) immunity, induce local mucosal orsystemic responses and decrease to dose of antigenrequired to elicit protective immune responses (Singh

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Table 1 Type, mode of action and DC target of various adjuvants grouped on the basis of their source,composition and physicochemical properties (mineral, microbial, emulsion, particulate) (based on Vogel, 2000;Pink et al., 2004; Pashine et al., 2005).

Type Mode of action DC type

MineralAluminum salts* Ag1 delivery, uptake, presentation cDCHydroxide/phosphate Co-stimulation, IL-1b, IL-18Alum Th2 response, Ab2 production

EmulsionFreund’s adjuvant (FA) Delayed Ag release, depo effect cDCwater-to-oil emulsion CFA contains heat killedComplete (CFA) mycobacteriaIncomplete (IFA) MDP-NOD2/Nalp3 ligand

MF59* oil-to-water, montanide* Delayed Ag release, depo effect cDCMicro-fluidized detergent stabilized emulsions Ag delivery to APC

ParticulateImmunostimulatory complexes Targeting to APC and LN, co- cDCSaponin3, cholesterol, phospholipid stimulation, reduced Ag doseISCOM, ISCOPREP�703 Cytotoxic CD8q T-cell response30–80 nm, amphipathic protein Ag on surface IL-6, IL-1b

Liposomes (w or w/o saponin) Protect Ag from degradation cDC100–1000 nm, Ag internal Delayed Ag release

Biodegradable macrospheres (w or w/o saponin) Enhanced, prolonged immune cDCPolylactide polyglycolide PLG responses)1000 nm, Ag internal Intracellular release of Ag

Cationic microparticles4 Targeting to APC, concentrate, cDCdisplay and co-localize Ag withimmunostimulators

Microbial products NaturalNucleic acids5: intermediers of virus infection TLR ligands – co-stimulation cDC/pDCIngested through infected or dying cells RLH ligands – co-stimulation cDC

Peptidoglycan cell wall component of Gram-/q NOD2 ligand – IL-6, cDCbacteria, muramyl-dipeptide (MDP) NOD2 and Nalp3 ligand – IL-1b

Endotoxin: bacterial lipopolysaccharide (LPS) TLR ligand – co-stimulation cDC

Exotoxin: cholera/pertussis toxin, E. coli Co-stimulation, mucosal immunity cDC/pDCheat labile toxin

Microbial products SyntheticTLR ligands/imidazoquinolines TLR7, TLR8, Nalp3 cDC/pDCR848, R837 IL-12, IL-1b, IL-18, IFN-a

NLR ligands/MDP analogs NOD2, Nalp3 ligands cDCIL-1b, IL-18

Oligonucleotides/CpG ODN/ISS1018 TLR9 ligand, IFN-a, TNF-a pDCTLR9 ligand, IFN-a

1Ag, antigen; 2Ab, antibody; 3source of saponins (triterpene glycosides) is Quillaia saponaria Molina tree andinvolves various purified fractions (Sjolander et al., 1998); 4O’Hagan et al., 2004; 5see Table 2; *licensed forhuman use.

and O’Hagan, 2003). The traditional aluminum-basedadjuvants (aluminum phosphate or hydroxide, alum)(Hem and White, 1995) have been used for more than60 years as a delivery system for human and veterinaryvaccines (Table 1). Alum is safe and provides long-termprotective immunity, but is unable to induce Th1 immuneresponses required for the induction of protective immu-nity against many intracellular pathogens. DCs take upalum-adsorbed antigens more efficiently than the solubleones (Morefield et al., 2005) and acquire enhanced anti-gen presenting potential, augmented expression of co-

stimulatory molecules and the secretion of interleukin(IL)-1b and IL-18 cytokines (Sokolovska et al., 2007).However, in the absence of IL-12, these cytokines incombination with IL-4 and IL-13 predominantly stimulateIL-4- and IL-5-secreting Th2 cells, whereas IL-6, IL-1 andIL-23 support the differentiation of human Th17 cells thatconfer protection against extracellular bacteria and fungi(Chen and O’Shea, 2007). As DCs constitutively expresspro-IL-1b and alum does not increase IL-1 b mRNA, itwas suggested that alum induces caspase-1-dependentcleavage of IL-1b and IL-18 (Matsue et al., 1992).

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Although the precise mechanism of Th2 polarizationby IL-1b is not known, absence of IL-1b results in in-complete Th2 polarization both in vivo and in vitro (Helm-by and Grencis, 2004). In a murine model systemGr-1hiCCR2q monocytes, recruited by CCL20 in a CCR6-dependent manner, were identified as the precursor ofDCs recruited to epithelial surfaces after skin or mucosaladministration of antigen administered with alum. Thesecells were able to mediate cross-priming of antigen toCD8q T-lymphocytes (Le Borgne et al., 2006). This uniquecell type also activated naive B-cells and facilitated thedifferentiation of immunoglobulin secreting cells (Jordanet al., 2004).

The other traditional adjuvant, complete Freund’sadjuvant (CFA) (Table 1) remains a preferred water-to-oiladjuvant to increase the efficacy of experimental immu-nization. It contains heat killed mycobacteria and is ableto induce cell mediated immune responses, as well as toenhance humoral immunity against various antigens. Theminimal essential component of CFA has been identifiedas muramyl dipeptide (MDP), a degradation product ofthe peptidoglycan (PGN) cell wall component of Gram-negative and Gram-positive bacteria. Muropeptides arereleased during bacterial growth, or as a result of the hostresponse mediated by lysozyme or induced by antibiotictreatment. MDP alone elicits only a very weak responseas compared to TLR-induced activation; however, itseffect synergizes with TLR agonists in inducing cytokineresponses.

Recently developed delivery and adjuvant systemsinvolve liposomes, vesicles, micelles, cationic peptidesor cationic microparticles, and also biomaterial drug vehi-cles that offer new avenues for targeting DCs to improvevaccine efficiency (Table 1). Polymer particles and lipo-somes are able to protect the antigen from degradation,and after internalization by DCs, they enable the intra-cellular release of antigen that favors antigen presenta-tion by both MHC class I and class II molecules. Largemicroparticles are phagocytosed, small microparticlesare taken up by macropinocytosis, while biomaterialscarrying targeting ligands to internalizing receptors facil-itate receptor-mediated uptake. Particle size is a crucialfactor for lymphatic uptake, and synthetic adjuvants cou-pled to nanoparticles were shown to help robust DC acti-vation (Pashine et al., 2005). As thymic, splenic, tissueresident and approximately half of LN-resident DCs arein immature state (Wilson and Villadangos, 2004), bothtissue and lymphoid organ resident DCs can be targetedfor the accumulation or transport of large amounts ofantigens. However, the most potent DC subset for vac-cine targeting has not yet been identified.

Intramuscular injection or immunization through theskin is traditional routes of vaccine introduction. Themajor professional APCs of the skin are epidermal CD1aq

LCs that are replenished from resident precursors(Romani et al., 2003), whereas circulating blood-borneLC precursors are recruited to the skin only as a con-sequence of dramatic LC loss induced by UV-irradiation(Merad et al., 2002). Dermal CD1a- DCs resemble tissueresident DCs, whereas moDCs also generate CD1a- andCD1aq subsets. All these subtypes belong to migratoryDCs and deliver their antigenic cargo to draining LNs ifactivated by appropriate stimuli. We have shown that the

CD1a- and CD1aq DC subsets – detectable in peripheraltissues and also in LNs – differ significantly in their capa-bility to secrete inflammatory cytokines (Gogolak et al.,2007). We also defined a lipoprotein and lipid-mediatedregulatory pathway of CD1a dichotomy mediated by thenuclear hormone receptor peroxisome-proliferator acti-vated receptor-g (Szatmari et al., 2004).

DNA vaccination has emerged as a new approach toinduce protective immunity against non-cultivatable orhighly pathogenic microbes (Fraser et al., 2007) and usu-ally include eukaryotic expression plasmids encodingselected pathogenic antigens under the transcriptionalcontrol of strong viral promoters. The gene gun methodintroduces the vaccine to the upper layer of the skin thatresults in direct transfection of DCs for endogenous anti-gen presentation by both MHC class I and class II mol-ecules, and cross-presentation of antigen through theuptake of transfected tissue cells (Rock and Shen, 2005).Recent studies revealed that human LCs are dispensablefor the induction of immune responses to DNA-coatedmicroparticles introduced by the gene gun technique andsuggested that cross-presentation is confined to migra-tory dermal DCs characterized by strong T-lymphocytestimulating potential (Stoecklinger et al., 2007). Charac-terization of human dermal APCs revealed the presenceof a myeloid, highly immunostimulatory CD11cqBDCA1q

myeloid subtype (Zaba et al., 2007).

Ligands of pattern recognition receptors asvaccine adjuvants

Professional APCs, such as DCs, B-cells and macro-phages, display the broadest repertoire and express thehighest levels of TLRs. However, the two major DC line-ages, cDCs and pDCs, express a characteristic combi-nation of TLRs ensuring the recognition of a wide arrayof pathogenic or damaged self structures (Figure 1). Asmany other body cells express at least one or a subsetof TLRs, these conserved receptors may have evolvedto control physiological functions while responding topathological ‘danger signals’ (Yamamoto et al., 2004).TLR-mediated signaling results in the activation of nucle-ar factor k B (NF-kB), but TLR4 ligands may also induceinterferon regulatory factor 3 (IRF3). TLR3 activation iscoupled to both IRF3 and IRF7; however, the expressionlevel of IRF3 in most cells is far higher than that of IRF7,and thus the primary target of TLR3-mediated activationis IRF3 (Mamane et al., 1999), whereas TLR7, TLR8 andTLR9 ligands induce IRF7 activation (Figure 2). Signalingthrough TLRs triggers the secretion of proinflammatorycytokines and chemokines and results in the recruitmentof other inflammatory cells, such as granulocytes andnatural killer (NK) cells. Thus, the coordinated activationand contact of various cell types, as well as the cross-talk mediated by cytokines, create a local environmentthat favors the regulated activation of adaptive immunity(Iwasaki and Medzhitov, 2004). As inducers of this cas-cade of events, natural or synthetic ligands of TLRs actas adjuvants and are able to induce robust antigen-specific immune responses and support long-termimmunologic memory.

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Figure 1 Expression and specificity of Toll-like receptors (TLRs) by conventional and plasmacytoid dendritic cells (DCs).Conventional and plasmacytoid DCs express different sets of TLRs localized to the cell or intracellular membranes. NLRs and RLHsare expressed in DCs, but the expression pattern and functional activity of the individual members of these intracellular sensors havenot yet been studied systematically in various DC subtypes. Activation of the two major lineages of DCs by TLRs and presumablyby NLRs and RLHs results in the secretion of different combinations of inflammatory cytokines.

In addition to TLRs that are expressed in cellular orvesicular membranes, PRRs also involve conservedintracellular sensors, such as NACHT-LRRs (Martinonand Tschopp, 2005) or Nod-like receptors (NLRs) (Petrilliet al., 2007a,b), retinoic acid induced gene (RIG)-like heli-cases (RLH), and intracellular DNA-binding sensors(Creagh and O’Neill, 2006; Ishii and Akira, 2006) that alsorecognize pathogen associated molecular patterns(PAMPs). As all these molecular sensors are expressedin different DC subsets (Figure 1), the combination ofextracellular, membrane and cytoplasmic stimuli, accu-mulated and coordinated by DCs, will determine themagnitude and quality of the immune response.

Nod-like receptors as molecular targets ofadjuvants

The field of vaccinology has been beneficially fueled bystudies focusing on the understanding of the mode ofadjuvant actions (Table 1). The discovery of PRRs thatmediate innate and adaptive immune responses uponrecognition of PAMPs has had a huge impact on ourknowledge on immunoregulation and enables the target-ed design of adjuvants. Recent studies demonstratedthat TLRs mediate a critical link between innate andadaptive immunity. This link, which is normally activatedby the collaboration of adjuvants with TLRs in triggeringadaptive immunity, has been the subject of numerousrecent studies. In this review, we focus on the possiblerole and collaboration of TLRs with the newly identifiedNLRs and RLHs as potential new targets of vaccinedesign. As adjuvants are able to alert the host’s immune

system through mechanisms similar to that of an infec-tion by pathogens, and the highly conserved PRRs areexpressed in DCs and mediate cell activation uponappropriate stimulation, it is reasonable to assume thatsome agents having DCs activating potential may actthrough NLRs and thus are able to exert adjuvant effects(Bendelac and Medzhitov, 2002; Kaisho and Akira, 2002).

NLRs are intracellular PRRs that are specialized forrecognition of PAMPs present within the cell and areimplicated in host defense (Martinon and Tschopp, 2005).The expression of most NLRs is widespread, but typicallyoccurs in cell types that mediate defense function, suchas leukocytes and epithelial cells. However, the expres-sion of certain NLR proteins, such as NACHT domain-,leucine-rich repeat- and pyrin (PYD)-containing protein-4(Nalp4), Nalp5, Nalp9 and Nalp14 is restricted to cellsthat are involved in gametogenesis, folliculogenesis andearly embryonic development. Although more than 20members of the NLR family have been identified, the bio-logical function of most of them has not been clarifiedyet. Based on the type of their N-terminal domain,NLR family members are grouped into subfamilies, suchas the PYD-domain containing Nalps and the caspase-associated recruitment-domain (CARD)-containingnucleotide-binding oligomerization domain proteins(Nods). As they possess a leucine-rich repeat (LRR) sim-ilar to TLRs and plant R (resistance) proteins, they areassumed to function as sensors of pathogens anddanger signals (Tschopp et al., 2003). Although somespecific NLR-activating ligands have already beendescribed, most members of the NLR family still exist as‘orphan sensors’ without known or characterized ligands.

NLRs are thought to be synthesized in an inactive formand their activation is triggered by ligands binding to the

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Figure 2 Cross-talk of TLR-, NLR- and RLH-mediated signaling pathways.Pathway 1: TLRs expressed in cell- or intracellular membranes recognize various sets of pathogenic structures and transduce signalsthrough the NF-kB pathway, which results in the production of IL-6 and TNF-a. The TLR3- and TLR4-mediated signaling pathwaysare independent of MyD88, interleukin-1 receptor-associated kinase-1 (IRAK1) and IRAK4, whereas TLR7 and TLR9 signaling occursthrough the MyD88 pathway. TRAF6 has a cardinal role in both pathways, as stimulation of TLRs results in the translocation of TRAF6and transforming growth factor-b-activated kinase-1 (TAK1/MAP3K7) to the cytosol where TAK1 is phosphorylated and activated,leading to the activation of IkB kinase and NF-kB. SOCS-1, which specifically interferes with JAK dimerization, also controls thecollaboration of MyD88 and NF-kB.Pathway 2: Intracellular NLR proteins form complexes that consist of recognition receptors, adaptors and enzymes. The Nod-sig-nalosome, that regulates the NF-kB pathway, is formed by the Nod1 or Nod2 sensors and the receptor-interacting protein-2 kinase(Rip2). The Nalp1-inflammasome consists of the Nalp1 sensor, the ASC adaptor and the caspase-1 and caspase-5 enzymes, whereasthe Nalp3-inflammasome consists of the Nalp3 sensor, ASC and Cardinal as adaptors, and the caspase-1 enzyme. Binding of ligandsthrough the leucine-rich domains of Nod and Nalp proteins results in a conformational change of these molecules that enables thebinding of caspases through the adaptors. As a result of complex formation, caspase-1 is activated and cleaves pro-IL-1b and pro-IL-18 to secrete biologically active cytokines. The Rip2 kinase represents a mediator of cross-talk among complexes. It is able tobind to TRAF6 and TAK1 to regulate NF-kB, but also interacts with caspase-1 of the inflammasome. Besides stimulatory NLRs, somemembers of the NLR family, such as Nalp2, Nalp4 and Nalp12, may act as inhibitors by modifying the NF-kB signaling pathway.CARD-only proteins (COPs) are able to participate in homophylic interactions with CARD-containing members of the Nod-signalosomeand the Nalp-inflammasome thus inhibiting complex formation through competing with other CARD-containing molecules.Pathway 3: The interaction of dsRNA – a replication intermediate of RNA viruses – with the helicase domain of RIG-I or MDA5induces association to the CARD domain of RIG-I or MDA5 and the CARD-like domain of the adaptor protein CARDIF localized tothe mitochondrial membrane. This receptor-adaptor interaction results in the activation of TBK1 through TANK binding. ActivatedTBK1 induces the phosphorylation of IRF3 and IRF7 on specific serine residues, resulting in their homodimerization. These dimersthen translocate to the nucleus and activate the transcription of type I IFN-induced genes. The expression of IRF3, IRF7, RIG-I andMDA5 is coordinately upregulated by type I IFN-mediated signaling acting as an amplification process. This pathway is implicatedto be connected to the NF-kB activation pathway through the interaction of FADD (FAS-associated via death domain), RIP1 andTRAF6 together with CARDIF that results in the induction of proinflammatory cytokine genes, such as IL-1b, IL-6 and TNF-a.

LRR domain. This interaction leads to a conformationalchange that enables the binding of the adaptor proteinsapoptosis-associated speck-like protein containing aCARD (ASC) and CARD inhibitor of NF-kB-activatingligands (Cardinal) and the interaction with the enzymescaspase-1 and caspase-5 or the receptor-interactingprotein-2 kinase (Rip2). These interactions result in theformation of protein complexes forming the Nalp-inflam-masome and the Nod-signalosome. The association ofthe subunits within a complex is mediated by homophylicinteractions of CARD-CARD or PYD-PYD domains of theindividual members. The activation of Nod-signalosomeleads to the regulation of the NF-kB, the mitogen-acti-

vated kinase and/or the activation protein-1 pathways(Kufer et al., 2005; Fritz et al., 2006), whereas the assem-bly of the Nalp-inflammasome complex results in thecleavage of pro-IL-1b and pro-IL-18 to generate func-tional cytokines by the activated caspase-1. It has beenshown that Nalp1, Nalp3 and Nalp6, co-expressed withthe ASC adaptor, are capable to activate the NF-kB path-way (Martinon et al., 2002) that leads to the expressionof a variety of cytokines, chemokines and cell surfacemolecules that are involved in pro- and anti-inflammatoryresponses. These mechanisms, together with the actionof IL-1b and IL-18, are able to modulate the outcome ofDC activation and the polarization of T-cells.

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Synergistic effect of TLR and Nod1 ligandrecognition in dendritic cells

It has been shown that Nod1 and Nod2 are involved inthe antimicrobial response to a variety of different bac-teria through the recognition of distinct PGN motifs.Nod1 was shown to have an important role in the in vivopriming of antigen-specific responses. It is able to rec-ognize structures (mesoDAP or TriDAP) specific forGram-negative bacteria and associate with Rip2 kinaseto form the Nod-signalosome and to activate the NF-kBpathway (Girardin et al., 2003a). It was also reported thatNod1 activation alone enhances pro-IL-1b processing(Yoo et al., 2002) and elicits the priming of antigen-spe-cific immunity with predominant Th2 cell polarization.However, efficient boosting of the immune responserequires mycobacterial cell-wall constituents of CFA.Recognition of PGN by Nod1 synergizes with TLR sig-naling and results in the NF-kB-mediated production ofIL-6, and the secretion of IL-12 and IL-23 that results inTh1 and Th17 polarization (Fritz et al., 2007). Thus, abroad range of inflammatory cytokine secretion requiresthe synergistic effect of Nod1 and several TLR ligands(van Heel et al., 2005).

Recognition of a common ligand by Nod2 and Nalp3expressed in dendritic cells

MDP is a common PAMP of both Gram-negative andGram-positive bacteria. MDP was first identified as aspecific ligand of Nod2 that forms the Nod-signalosomewith the contribution of the Rip2 kinase, and the complexwas shown to activate the NF-kB pathway (Girardin etal., 2003b; Inohara et al., 2003). MDP alone induces weaktumor necrosis factor a (TNF-a) or IL-1b production;however, co-stimulation of DCs with TLR agonists resultsin IL-12 production and predominant Th1 polarization.MDP was also reported to have the capability to induceIL-1b secretion via the activation of the Nalp3-inflam-masome (Akira et al., 2006). It was recently found thatthe MDP-induced IL-1b release requires the presence ofboth Nod2 and Nalp3 together with their appropriateadaptor proteins, whereas the sole presence of Nod2 andRip2 is sufficient for MDP-induced IL-6 secretion. Inter-estingly, MDP was also able to enhance the induction ofIL-23 and IL-1 in DCs primed by bacteria and promotedIL-17 expression in T-cells (van Beelen et al., 2007). Thus,the secretion of a wide repertoire of cytokines dependson both Nod2 and Nalp3 that have non-redundant func-tions in DCs (Pan et al., 2007).

Activation of the Nalp3-inflammasome by variousstimuli

Similar to Nalp1, Nalp3 is involved in IL-1b and IL-18processing through presenting a molecular platform forinflammasome formation. Besides sensing MDP, Nalp3-inflammasome can be activated by signals that mimicendogenous danger signals, such as adenosine triphos-phate, uric acid or hypotonic stress (Kanneganti et al.,2006; Mariathasan et al., 2006; Martinon et al., 2006).Moreover, Nalp3 was shown to sense several stimuli thatindicate bacterial presence, i.e., anthrax toxin or bacterial

RNA that may derive from extracellular lysates and/orfrom phagocytosed bacteria transported to the cytosolof the DC. Bacterial RNA or its synthetic imidazoquinolinemimics R837 or R848 are recognized by human TLR7and TLR8 and/or by Nalp3. TLR7/8-mediated activationof DCs by R837 results in the secretion of IL-12 (Koskiet al., 2004), the critical cytokine for Th1 polarization.However, as a result of Nalp3-inflammasome activationthe release of IL-1b and IL-18 was also shown. As dem-onstrated recently, DCs stimulated by R848 express thechemokine receptor CCR7, mediating DC migration toLN, and exhibit strong CD8q cytotoxic T-lymphocyte acti-vating potential (Lehner et al., 2007). R848 and R837have been successfully used as immune response mod-ifiers of anti-tumor and antiviral immunity (Tomai et al.,1995; Craft et al., 2005). Their beneficial effect can beinterpreted as a synergizing adjuvant effect mediated bya unique combination of TLR and NLR signals.

Cross-talk of Toll-like and Nod-like receptorspotentiates adjuvant activity

Microbial interactions with the innate immune systeminvolve triggering of multiple PRR, including NLRs andTLRs, by multiple antigens simultaneously presented bythe whole pathogen. The cross-talk between TLRs andNLRs is likely to be a crucial collaboration to maintain abalance of immune effectors and enhance the innateimmune response against microorganisms. In DCs andmacrophages, the activation of TLRs by their specificligands results in the induction of the NF-kB-pathwaywhich results in the expression of pro-IL-1b. However,the conversion of inactive pro-IL-1b to fully mature, bio-logically active secreted IL-1b requires the cleavage bycaspase-1, which is a component of the Nalp-inflam-masome and is activated as part of the properly formedcomplex upon sensing pathogenic or self danger signals.

The cross-talk of TLRs and NLRs can occur at differentmolecular levels within DCs (Kufer and Sansonetti, 2007).This may involve the transcriptional up- or downregula-tion of NLR proteins or other proteins involved in theirsignaling pathways. Upregulation of Nod1 and Nod2gene expression was reported upon TNF-a, interferon(IFN)-g or TLR agonist treatment (Rosenstiel et al., 2003;Takahashi et al., 2006). Furthermore, MDP was shown toincrease myeloid differentiation primary response gene88 (MyD88) expression, which is a critical adaptor mol-ecule of several TLR signaling pathways (Takada et al.,2002). Recently, the inducability of Nod2 expressionby flagellin, a TLR5 agonist was reported in intestinalepithelial cells (Begue et al., 2006).

Another level of cross-talk is represented by the directprotein-protein interaction of NLRs and the proteins ofdownstream signaling pathways. One good example ofthis interaction is Rip2 that, besides binding to Nod1 orNod2 in the Nod-signalosome, is able to interact withcaspase-1 of the inflammasome through CARD-CARDinteraction (Sarkar et al., 2006). Rip2 was also shown tobind TNF receptor associated factor 6 (TRAF6) that inturn is involved in NF-kB pathway regulation (Meylan andTschopp, 2005) (Figure 2). Thus, Rip2 seems to balance

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Table 2 Expression of nucleotide recognizing TLRs and intracellular sensors in dendritic cells.

Receptor Recognized Expression Recognized pathogens Recognizednucleotides on APC host structures

TLR7 ssRNA pDC Negative-sense ssRNA viruses Dead or dyingwvesicular stomatitis virus cells, immune(VSV) and influenza virusx complexes of

small nuclearribonucleoproteins(snRNP) and IgG

TLR8 cDC Influenza virus Dead or dyingcells

TLR3 dsRNA cDC dsRNA, positive- and Necrotic cells,negative-sense ssRNA viruses virus infected(herpes simplex virus, dying cellsinfluenza virus, respiratorysyncytial virus)

RLH (RIG-like Negative- and positive sense Not knownhelicases) ssRNA viruses

(VSV and Sendai virus)

TLR9 dsDNA, pDC Mycobacterium tuberculosis, Dead or dyinghemozoin Neisseria meningitidis, cells, immune

Candida albicans, herpes complexes ofsimplex virus, Trypanosoma host DNA andcruzi IgG

DLM- B-DNA, Not known Not known Not known1/ZBP1/DAI Z-DNA

Different DC subtypes express both common and unique pattern recognition receptors that respond to awide variety of pathogens, as well as recognize certain host structures.

the formation of inflammasome or Nod-signalosome at amolecular level. Another good example is the cross-talkof the TLR2 and Nod2 signaling pathways. It has beenshown that the activation of TLR2 by PGN or Nod2 byMDP results in the activation of NF-kB and the secretionof IL-12. However, PGN-induced IL-12 production isdownregulated by the concomitant stimulation of Nod2through MDP (Watanabe et al., 2004, 2006). This sug-gests that activation of the Nod2 pathway may exert anegative effect on the TLR2-mediated response. If so,this may explain the susceptibility of individuals withNod2 mutations to Crohn’s disease, caused by the lossof Nod2-mediated control of TLR2 signaling. Separatesignaling pathways with different kinetics may also beinvolved in the dual effects of Nod2 (Yang et al., 2007).

The significance of intracellular nucleotidesensors in vaccination

RIG-I and melanoma-differentiation associated gene-5(MDA5) are newly identified intracellular PRRs. RIG-I wasfirst described in promyelocytic leukemia cells, the struc-turally homologous MDA5 is induced during differentia-tion of a melanoma cell line (Kang et al., 2002). Bothproteins exhibit RNA helicase activity and their expres-sion are triggered by double-stranded RNA (dsRNA) orby synthetic RNA-analogs. RIG-I- and MDA5-mediatedsignaling is coordinated by the CARD domain-containingadaptor CARDIF (CARD adaptor inducing interferon-b,IFN-b) alternatively referred to as IPS1 (IFNB-promoter

stimulator-1), MAVS (mitochondrial antiviral signaling pro-tein) or VISA (virus-induced signaling adaptor) (Figure 2).Interestingly, this adaptor protein is localized to the mito-chondrial membrane thus linking innate immunity andanti-viral responses to an organelle with evolutionary ori-gin of aerobic bacteria (McWhirter et al., 2005). As mito-chondria play a determining role in regulating apoptosis,and silencing of MAVS was shown to increase apoptosis,this pathway may also be involved in protecting virus-infected cells from programmed cell death (Seth et al.,2005). Furthermore, the collaboration of RLHs, and theconsequent secretion of type I interferons and NF-kB-dependent cytokine production can be highly efficientagainst viral infection through multiple pathways.

After viral infections, rapid innate defense mechanismsare activated that are also crucial for the development ofadaptive immunity. Both major DC types play essentialroles in the priming and orchestrating humoral and cel-lular immunity and long-term immunologic memory (Ban-chereau and Steinman, 1998). Extra- or intracellularmodified nucleotides, released from damaged cells orgenerated in infected cells, are recognized by highly spe-cific TLRs (Table 2). TLR3 is specific for dsRNA, TLR7and TLR8 for single-stranded RNA (ssRNA), TLR9 fordsDNA. TLR3 signals via a MyD88-independent pathwaythrough the alternate Toll/interleukin-1 receptor (TIR)domain containing adaptor inducing IFN-b (TRIF) (Denget al., 2000). TRIF interacts with the TNF receptor (TNFR)associated factor-3 (TRAF3) to activate TRAF-family-member-associated NF-kB activator (TANK)-bindingkinase-1 (TBK1), and the non-canonical inhibitor of NF-

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kB (IkB) kinase (IKKi). These kinases directly phospho-rylate IRF3. Phosphorylated IRF3 forms dimers andtranslocates to the nucleus and induces the expressionof IFN-b. TRIF is also able to interact with RIP1 andTRAF6 to induce a late phase proinflammatory cytokineresponse (Takaoka et al., 2005). Conventional DCs arerich in TLR3 and are able to mediate dsRNA-mediatedsignaling; however, the interplay of this pathway with thatinitiated through RLHs is unknown, but may have animpact on vaccine design.

Recently, various attenuated strains of vaccinia virus,NYVAC and MVA have been tested to find new vectorsfor vaccination (Beattie et al., 1996; Moss, 1996; Hor-nemann et al., 2003; Vanderplasschen and Pastoret,2003). NYVAC was constructed from a Copenhagen vac-cine strain by the deletion of 18 open reading framesfrom the viral genome (Tartaglia et al., 1992). Currently,NYVAC is a frequently used virus vector for recombinantvaccines against pathogens and/or tumors (Kanesa-tha-san et al., 2000; Franchini et al., 2004). Structural genesof the MVA strain remained unaltered, but lost a smallpart of the original genome to eliminate genes which areinvolved in the evasion of the immune response (Meyeret al., 1991; Antoine et al., 1998; Wyatt et al., 1998). MVAis among the candidate strains for vaccines used in trialsagainst HIV and smallpox (Belyakov et al., 2003; Cebereet al., 2006). Taken the pivotal regulatory role of DCs inimmune responses, Guerra et al. (2007) studied the char-acteristics of the NYVAC- and MVA-based poxvirus vec-tors by gene expression profiling of human moDCsloaded by these constructs. The results showed thatMVA infection upregulates immunomodulatory genesmore effectively than the infection by NYVAC. The levelof IFN-a, TNF-a and IL-6 cytokines was higher in MVA-infected DCs than in the NYVAC-infected ones. In linewith these findings, an MVA-dependent upregulation ofRIG-I and MDA5, and consequently, the production oftype I interferons was also shown. These findings sug-gest that viral vectors with the capacity to stimulate theRLH system may be more potent in vaccination againstcertain pathogens due to their capability to switch oncollaborating signaling pathways.

Antigens acquired from body fluids or dead cells canbe cross-presented by APCs to cytotoxic T-lymphocytes(Rock et al., 1993). Cross-presentation is the only path-way by which the immune system is able to detect andrespond to viral infections that occur exclusively in epi-thelial or parenchymal cells rather than in bone marrow-derived APCs. This pathway depends on the highlyefficient uptake of exogenous material by phago- ormacropinocytosis and allows DCs and certain macro-phage types to initiate CD8q T-cell responses againstantigens that are not synthesized within the APCs. Theinternalized material can be processed through differentroutes. (i) Exogenous antigens internalized by phagocy-tosis may escape to the cytosol, degraded by the pro-teasome and reach the endoplasmic reticulum (ER)lumen in a transporter associated with antigen presen-tation (TAP) and tapasin-dependent manner (Rodriguezet al., 1999; Arrode et al., 2000). (ii) The cytoplasmic tailof MHC class I molecules with a conserved tyrosine sort-

ing motif targets unfolded MHC class I molecules to theendosomal/lysosomal compartment, where peptide load-ing occurs in a TAP-independent manner ensured by theexchange of bound endogenous peptides onto recyclingMHC class I molecules (Heath and Carbone, 2001). (iii)Active phagocytosis of particles may result in the fusionof the phagolysosome with the ER membrane by whichthe phagolysosome acquires the entire MHC class I load-ing complex, including newly synthesized MHC class Imolecules, TAP and tapasin (Ackerman and Cresswell,2003; Guermonprez et al., 2003; Houde et al., 2003). (iv)An alternative pathway of cross-presentation is mediatedby the passive transfer of short linear peptides throughgap junctions formed between adjacent cells. This path-way ensures the direct cytoplasmic contact of profes-sional APCs with neighboring infected cells and resultsin the cross-presentation of the acquired peptides andthe rapid priming of CD8q cytotoxic T-lymphocytes(Neijssen et al., 2005).

Depending on the nature of the antigen, one or a com-bination of these pathways may contribute to cross-pres-entation in vivo, and the outcome of cross-presentationcan be either tolerance or immunity (Bennett et al., 1998;Liu et al., 2002). This decision highly depends on con-comitant activation signals that promote the develop-ment of the immune response against the acquiredantigens. Induction of cross-presentation requires CD40/CD40 ligand-mediated signaling conferred by helper T-cells (Schoenberger et al., 1998), but bacterial (Hamiltonet al., 2001), viral or host cell-derived stimuli (Cho et al.,2000) are also able to license DCs for cross-priming(Ridge et al., 1998; Heath and Carbone, 2001). In additionto the critical role of cross-presentation in normalimmune physiology, it has considerable potential fordeveloping subunit vaccines that elicit both CD4q andCD8q T-cell immunity (Rock and Shen, 2005). The effi-ciency of exogenous antigen presentation by MHC classI and class II molecules is differentially regulated duringDC maturation (Delamarre et al., 2003). Soluble proteinsinternalized by immature DCs are stored intracellularlyuntil activated by an appropriate cross-presentation sig-nal, while increased expression of MHC class II mole-cules in the cell surface could be induced by variousactivation signals.

Secretion of type I interferons by infected host cells isa sensitive and common indicator of virus infections.Besides their adjuvant effects on CD4q helper cells andantibody production (Le Bon et al., 2001), IFN-a/b areable to prime DCs directly and systematically for cross-presentation and increase the induction of CD8q T-lym-phocyte responses (Le Bon et al., 2003; Lapenta et al.,2006). As the RLH system is closely linked to the pro-duction of type I interferons, intracellular nucleotide-bind-ing proteins may be involved in facilitating indirectantigen presentation and the efficacy of cross-priming.

The possibility that the collaboration of intracellularsensor systems – similar to the concerted action of var-ious TLR-mediated signaling – may converge to betteradjuvant activity has not been elucidated. However, thefinding that the anti-viral effect of IFN-a is dramaticallyenhanced by the co-administration of Murabutide, a bio-

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logically active but non-toxic derivate of MDP, suggeststhat such synergisms may exist and would have greatimpact on vaccine design (Pouillart et al., 1996).

Targeting signaling cascades initiated byvarious molecular sensors to modify dendriticcell functions – new approaches

PRR ligands act on the maturation of DCs, whereas themodulation of this process can be used for fine tuningDC-mediated functions. An important regulatory pathwayinduced by TLR activation of DCs is associated withregulatory T-cell (Treg) function that is suppressed byTLR-induced IL-6 secretion (Schnare et al., 2001).Furthermore, natural inhibitors or regulators of thesignaling pathways associated with PRRs are potentialtargets of vaccine design. For example, splice variants ofTLR2, TLR4 and TLR3 may act as dominant-negativemolecules by interfering with TLR-mediated signaling(Mbow and Sarisky, 2005). Lipopolysaccharide-bindingprotein or soluble CD14 may facilitate TLR4-mediatedsignaling (Miyake, 2004), or the flagellin-induced humanTLR5 response can be amplified by a soluble TLR5ortholog of fish acting as an adjuvant (Tsujita et al., 2006).As membrane and cytosolic PRRs signal through differ-ent adaptor molecules and initiate well-defined signalingpathways, overexpression of adaptors may promote(Sasai et al., 2006), whereas inhibition of receptor-adap-tor interactions by small synthetic molecules can blockTLR-associated functions (Ii et al., 2006). Besides theactivity of stimulatory NLRs, some members of the NLRfamily, such as Nalp2, Nalp4 and Nalp12, may act asinhibitors by modulating the NF-kB-mediated signalingpathway. CARD-only proteins (COPs) are partners ofhomophylic interactions with CARD-containing membersof the Nod-signalosome and the Nalp-inflammasome,but they can inhibit complex formation through compet-ing with other CARD-containing molecules (Lee et al.,2001) (Figure 2).

As discussed before, the secretion and activity of IL-1b and IL-18 is controlled by the regulated expression ofcaspase-1 that is involved in the enzymatic cleavage ofpro-cytokines to generate biologically active secretedcytokines. This process is under check through the nat-ural inhibitor of caspase-1, the P9 serpin (Young et al.,2000). Furthermore, soluble IL-1 receptor (IL-1Ra) andthe endogenous IL-18 binding protein (IL-18BP) are ableto inhibit ligand-receptor interactions suggesting multiplelevels of controlling cytokine secretion and signaling.

A novel and broad approach to increase the adjuvanteffects of PRR-mediated signaling is based on targetingthe suppressor of cytokine signaling (SOCS) proteins thatregulate Janus kinases, and probably by Mal degradation– the NF-kB-mediated pathway, both transmitting signalsfrom TLRs and cytokine receptors to the nucleus (Man-sell et al., 2006). Mal is a key mediator of TLR2 and TLR4signaling that is able to trans-activate NF-kB by thephosphorylation of p65 and modulate inflammatoryresponses. The results of Mansell et al. showed that Malinteracts with SOCS-1 and this interaction first leads tophosphorylation and than Mal poly-ubiquitination and

degradation. This control mechanism may have a role inlimiting proinflammatory responses. Inhibition of SOCS1by small interfering RNA resulted in augmented anti-HIVimmunity mediated by increased cytokine secretion ofboth CD4q and CD8q T-cells and prolonged andincreased antibody and anti-viral cytotoxic T-lymphocyteresponses (Song et al., 2006).

In the RLH system, LGP2 lacking the CARD domainwas shown to inhibit type I interferons induction throughblocking the multimerization of RIG-I and by competingwith the protein kinase IKK´ for the same interaction siteon MAVS (Figure 2).

Modulation of plasmacytoid pre-dendritic cellfunction by vaccination

Plasmacytoid pre-dendritic cells are key players of theimmune system by linking innate and adaptive immuneresponses, and also bearing effector function through theproduction of type I interferons upon viral infection (Cellaet al., 1999; Siegal et al., 1999). Since their discovery in1958 (Lennert and Remmele, 1958), the proper name ofthis special cell type has remained controversial due tothe unclarified link between pDCs and the previouslyidentified professional type I interferon-producing cells(IPCs) (Perussia et al., 1985). IPCs have later beendescribed as a differentiation state of pDCs (Siegal et al.,1999) that represent their resting state referred to as pDCprecursors (Liu, 2005). They are capable to respond toRNA and DNA viruses as they express a special set ofTLRs, namely TLR7 and TLR9 (Kadowaki et al., 2001;Krug et al., 2001a), and they are 10–100-fold more effi-cient producers of type I interferons than any other celltype, including cDCs. TLR7 recognizes viral ssRNA, butseveral synthetic compounds, such as loxoribine, resi-quimod (R848) and imiquimod (R837), also bind to thisreceptor (Hemmi et al., 2002). TLR9 interacts with natu-rally occurring hypo- or unmethylated DNA sequencesand synthetic CpG-oligonucleotides (Rothenfusser et al.,2002). The resulting type I interferon response of pDC isdependent on MyD88, TRAF6 and the IKK complex, andthe signaling events initiated by TLR7 and TLR9 engage-ment are similar (Akira and Takeda, 2004). Although pDCalso express intracellular helicases (Colonna, 2007),pDCs use the TLR system rather than helicases for viraldetection (Kato et al., 2005). It was also proposed thatpDCs sense ssRNA viruses after endosomal uptake, thusinvasion of the cytoplasm by the virus is not necessary(Kato et al., 2005). This assumption was confirmed byusing HIV-1 (Beignon et al., 2005) or infectious and UV-irradiated herpes simplex virus (Lund et al., 2003; Kruget al., 2004), supporting the notion that an important fea-ture of this type of recognition is that pDCs initiate thetype I interferon response, even against inactivated ornon-replicating viruses, thus allowing effective immuni-zation without viral replication. Recently, this view waschallenged by showing the necessity of replication inter-mediates and autophagy dependency of the recognitionof certain ssRNA viruses by pDCs (Lee et al., 2007).Based upon these observations, the study of recognition

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strategies used by pDCs for different virus types is ofgreat importance.

As many viruses have developed effective mecha-nisms for blocking type I interferon responses early afterinfection (Hengel et al., 2005), the contribution of pDC toantiviral immunity is crucial and their existence is bene-ficial for the host, as they can alarm the immune systemwithout being infected. Upon activation with specific TLRligands, pDCs differentiate to professional antigen-pre-senting cells and acquire a ‘dendritic’ morphology, similarto cDC, while also upregulating costimulatory molecules.Freshly isolated pDC do not express high levels of MHCclass II and CD80/CD86 co-stimulatory molecules andthus are inefficient to induce antigen-specific T-cell acti-vation and proliferation. Resting pDCs are able to drivenaive CD4q T-lymphocytes into IL-10 producing regula-tory T-cells (Tr1) (Moseman et al., 2004). Thus, pDC playan essential role in maintaining peripheral tolerance andin preventing asthmatic reaction to inhaled antigens inde-pendent on the activation state (de Heer et al., 2004).Plasmacytoid pre-dendritic cells activated by IL-3 andCD40 ligand maintain their tolerogenic potential, or dif-ferentiate to a form with high surface expression of OX40ligand and the potential to polarize CD4q T-lymphocytesto produce IL-4, IL-5 and IL-10, or CD8q T-cells tosecrete IL-10 (Gilliet and Liu, 2002). Virus infection or sig-naling through TLR7 and/or TLR9 triggered by specificligands results in IFN-a and TNF-a secretion and inducesCD4q T-cells with the capacity to secrete IFN-g and IL-10 (Kadowaki et al., 2000). It has also been shown thatinfluenza virus infection induces pDC maturation, primingof virus-specific T-lymphocytes and activation of memoryT-cells (Salio et al., 2004). Type I interferons produced bypDCs act on both innate and adaptive immune cells. IFN-a has a strong adjuvant effect on antibody production(Ito et al., 2001), activates gd T-lymphocytes and increas-es the cytotoxic activity of NK cells (Liang et al., 2003).

Beside type I interferons, cross-presentation is also akey element of antiviral immune responses, as it allowspresentation of antigens acquired from autologous cells,making DCs capable to stimulate naive CD8q T-cells(Rock and Shen, 2005). The role of type I IFNs in licens-ing cross-presentation by cDCs, as discussed earlier,strongly suggested a functional link to pDCs that are notclassical cross-presenting APC. However, it was shownthat human pDCs effectively present exogenous peptidesthrough MHC class I molecules and activate anti-gen-specific CD4q and CD8q memory T-cell responses(Fonteneau et al., 2004). As murine pDCs were still con-sidered as unable to induce cross-priming in vivo (Salioet al., 2003), the cross-presenting capacity of pDCremained a controversial issue. A recent study showedthat human pDCs are capable to cross-present vaccineformulates consisting of lipopeptides and HIV-1 antigensand this activity was comparable to that of cDCs (Hoeffelet al., 2007). Cross-presentation by pDC was proved tobe amplified after influenza virus infection stimulatingpDCs through TLR9. Thus, natural and synthetic TLR9ligands became promising vaccine adjuvants with pre-dictable potential to modulate the beneficial cross-pre-senting activity of pDC.

CpG dinucleotides target both B-cells and pDCs asthese are the only human subsets expressing TLR9. pDC

subsets respond differently to CpGs and are able tomodulate the outcome of T-cell responses (Chen et al.,2006; Fallarino and Puccetti, 2006; Ito et al., 2007). A-type CpGs induce efficient production of type I interfer-ons by pDC, but B-types do not, and the C-type groupwas distinguished by an intermediate efficacy on type Iinterferon production (Vollmer et al., 2004). The back-ground of differential interferon induction remained con-troversial until the study of Honda et al. (2005), showingthat spatiotemporal regulation of MyD88-dependent IRF7signaling is a decisive factor of this function. They alsoshowed that interferon induction by type B CpG can beenhanced by cationic lipids, as it helps the polymeriza-tion of CpG. A recent study showed that the effect ofCpGs can be enhanced by protamine nanoparticlesresulting in a 20-fold increase of the type I interferonresponse of pDCs as compared to CpG alone and cati-onic gelatin nanoparticles have the same effect on pDCs(Zwiorek et al., 2007). These results showed that not onlythe quantity and the type of CpG but also other proper-ties of CpG, including polymerization and binding tocarrier structures, are important factors to modify theoutcome of pDC activation.

Based on earlier observations showing that TLR9ligands activate pDCs (Krug et al., 2001b), and CpG issuperior to the TLR7-agonistic imidazoquinoline com-pounds in the induction of humoral and cell mediatedimmune responses (Weeratna et al., 2005), unmethylatedCpG-oligonucleotides became widely used as vaccineadjuvants. Several clinical trials started between 2002and 2005 against melanoma, non-Hodgkin’s lymphoma,and renal cell carcinoma using CpG-7909 as adjuvant(Paul, 2003). Another CpG, ISS1018, was introduced asadjuvant in clinical trials against non-Hodgkin’s lympho-ma (Friedberg et al., 2005) and allergic rhinitis. The latterstudy with a therapeutic vaccine against allergic rhinitisshowed that the ragweed Amb a1 antigen conjugated tothe immunostimulatory DNA sequence ISS1018 wasbeneficial for allergic rhinitis patients (Creticos et al.,2006), suggesting that CpG sequences induce long-termimmune modulation, although the precise mechanism isnot completely understood and the exact role of pDCs isstill questionable. CpG are under evaluation as an adju-vant for Engerix-B, the Hepatitis B vaccine introducedearlier with other adjuvants (Halperin et al., 2003; Cooperet al., 2004). In these studies, the anti-HbsAg-specificantibody response appeared earlier and resulted in high-er titers as compared to the treatment with the vaccinealone. A randomized, double-blind, controlled trial in HIV-infected patients showed enhanced responses againstHbsAg using CpG-7909 as adjuvant for Engerix-B vac-cine (Cooper et al., 2005). The clinical trial using CpG-7909 as adjuvant in a therapeutic anti-cancer vaccineagainst non-small cell lung carcinoma entered phase III(Murad et al., 2007). An interesting aspect of the adjuvanteffect of CpG is that it has wider effects (Henry et al.,1997; Levin, 1999) and also side effects, even lethal‘cytokine storm’ (Sparwasser et al., 1997) in rodents,used as a model. This may be due to the differentexpression pattern of TLR9 in humans and rodents andindicates the necessity of primate experiments.

Recent developments of pDC biology suggest that theadjuvant effect of TLR9 is not restricted to interferon

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induction by pDC, but further studies are needed toreveal the underlying mechanisms of pDC activation asa result of vaccination combined with TLR9 ligand as anadjuvant. These studies may improve our understandingof the TLR system in pDCs and work out the approachof using TLR9 in therapeutic vaccinations.

Based on their distinct functional properties in termsof tissue localization at various differentiation states, anti-gen uptake, processing and presentation, expression ofvarious TLRs and cytokine secretion (Figure 1, Tables 1and 2), cDCs and pDCs require distinct strategies for tar-geting antigen uptake or activation for directed T-cellpolarization and cytokine secretion. Targeted antigenuptake combined with directed activation of variousDC subsets through the simultaneous stimulation ofmembrane and intracellular PRRs seem to provide a newtool to promote the collaboration of innate and adaptiveimmunity and result in the optimal activation of theimmune system against vaccines.

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