Dendritic Cell Biology

Dendritic Cell Biology Francesca Granucci, Maria Foti, and Paola Ricciardi‐Castagnoli Department of Biotechnology and Bioscience, University of Milano‐Bicocca, Piazza della Scienza 2, 20126 Milan, Italy

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Abstract............................................................................................................. Introduction ....................................................................................................... DC Subtypes ...................................................................................................... Deciphering DC Biology with Genomic Approaches .................................................. DC Interactions with the Microbial World ............................................................... DC Functions..................................................................................................... Conclusions........................................................................................................ References .........................................................................................................

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Abstract Dendritic cells (DCs) are a special type of leukocytes able to alert the immune system to the presence of infections. They play a central role in the initiation of both innate and adaptive immune responses. This particular DC feature is regulated by the activation of specific receptors at the cell surface called Toll‐ like receptors (TLRs) that bind a number of microbial products collectively referred to as microbial-associated molecular patterns (MAMP). TLRs initiate a cascade of events, which together define the process of DC maturation. This phenomenon allows DCs to progressively acquire varying specific functions. DC maturation depends on the nature of the perturbation and permits unique and efficient immune responses for each pathogen. In this review the discussion is focused on DCs in the context of interactions with pathogens and DC‐specific functions are highlighted. 1. Introduction During millions of years of coevolution, microorganisms have evolved mechanisms to infect the host by subverting the organism’s first lines of defense, the mucosal barrier and the innate response. The innate immune system is as old as the emergence of multicellular organisms and has been selected over evolutionary time rather than in individual cells. Indeed the receptors for innate immunity are found in all multicellular organisms, whereas adaptive immunity receptors are found uniquely in vertebrates. Because it is phylogenetically more ancient, the innate response has been regarded as a response broadly directed to the clearance of microorganisms. However, the discovery of a new class of receptors, involved in the

193 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88006-X

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recognition of defined groups of microorganisms has more recently readdressed the role of innate immunity as a sophisticated discriminating system that uses a broad innate receptor repertoire to sense the nature of the environmental perturbation (Medzhitov and Janeway, 1997). Indeed, the induction of different types of effector responses seems to be dictated and conditioned by the type of innate response that follows microbial recognition. It was first suggested that the innate immune responses could be activated via receptors called pattern recognition receptors (PRRs) that are able to recognize microbial associated molecular patterns (Janeway, 1992). The identification of the Toll‐like receptor (TLR) family members capable of binding specific components of a wide variety of pathogens has provided evidence to this new theory (Medzhitov et al., 1997). DCs were first described as the ‘natural adjuvants’ inducing adaptive immune responses (Banchereau and Steinman, 1998; Ibrahim et al., 1995; Steinman, 1991). They represent a special type of leukocytes able to alert the immune system to the presence of infections and responsible for activation and control of both innate and adaptive immune responses (Steinman, 2001; Zitvogel, 2002). DCs are especially distributed in tissues that interface the external environment, such as the skin, the gut, and the lungs (Nelson et al., 1994; Nestle et al., 1993; Sertl et al., 1986), where they can perform a sentinel function for incoming pathogens, and have the capacity to recruit and activate cells of the innate immune system (Fernandez et al., 1999; Foti et al., 1999; Rescigno et al., 1998). After antigen uptake has occurred, DCs efficiently process antigens for their presentation in association with major histocompatibility complex (MHC) molecules. However, before DCs can prime the adaptive immune response they must complete a full maturation process that is initiated by direct exposure to TLR ligands or to other receptors of the innate receptor repertoire. Interaction with pathogens results in a DC activation state that leads to their migration to the T‐cell area of lymph nodes where antigen‐specific cells of the adaptive immune response can be primed. Given the high plasticity of DCs, the signals that determine a particular DC function, and consequently, the type of adaptive immune response, depend mostly on the local microenvironment and on the interaction between the DCs and the microbial signals. These interactions are complex and very different from one pathogen to the next. In this review the discussion is focused on DCs in the context of interactions with pathogens and DC‐specific functions are highlighted. 2. DC Subtypes DCs originate from hematopoietic precursors within the bone marrow. Different subtypes have been described according to the surface expression of

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particular markers and tissue distribution (Ardavin, 2003; Ardavin et al., 2001; Shortman and Liu, 2002). In mice, immature DCs are characterized by the expression of CD11c (the integrin a‐chain), low levels of the costimulatory molecules CD80 and CD86, and low levels of MHC class II (MHC II); these molecules can be upregulated at the cell surface upon activation (Henri et al., 2001). Interestingly, DCs can also express the T‐cell markers CD4 and CD8. This last molecule is expressed as aa‐homodimer, whereas in T cells are expressed as ab‐heterodimer. Another molecule that has been used to identify mouse DC subtypes is the CD11b; using CD4, CD8, and CD11b markers five distinct DC classes have been identified. Three DC classes have been observed in the spleen, CD4þCD8–, CD4–CD8þ, and CD4–CD8–. The CD8– DC reside mostly in the marginal zone while the CD8þ are mainly located in the T‐cell area (Shortman and Liu, 2002). CD8– DCs can migrate to T‐cell areas following stimulation with microbial stimuli (De Smedt et al., 1998). Another DC population has been identified in all lymph nodes. These lymph node DCs are CD4–CD8–CD11bþ and also express moderate levels of the scavenger receptor CD205 (Anjuere et al., 1999). Finally, in skin‐draining lymph nodes an additional DC subtype has been found. It expresses high levels of Langerin, a molecule typically produced by Langerhans cells (LCs), an immature population of DCs located in the skin (Henri et al., 2001). In humans, DC subtypes are less characterized. Human DCs do not express CD8 and in spleen and tonsils, DCs differentially positive for CD11b, CD11c, and CD4 have been identified (Shortman and Liu, 2002). Most of the information on human DC subtypes and their possible origin derive from in vitro studies. Blood monocytes are the most commonly used precursors to generate DC in culture. In the presence of granulocyte macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, monocytes can differentiate to immature nonproliferating DCs, expressing low levels of CD86 and MHC II (Sallusto and Lanzavecchia, 1994). Following incubation with inflammatory products these DCs can reach the mature phenotype showing high levels of MHC II and costimulatory molecules (Sallusto and Lanzavecchia, 1994). A second human DC subtype, called interferon (IFN)‐ producing plasmacytoid DC was found in blood and many lymphoid tissues (Kadowaki and Liu, 2002). They are phenotypically characterized as CD11c–CD45RAþCD123þ. Recently the corresponding population of IFN‐ producing plasmacytoid DCs has been also identified in mouse blood and lymph nodes (Asselin‐Paturel et al., 2001). In this review, the discussion will focus on the functions of conventional myeloid CD8þCD11cþ, CD8–CD11cþCD11bþ mouse DCs, and human monocyte‐derived DCs.

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3. Deciphering DC Biology with Genomic Approaches The immune response involves a complex network of dynamic interactions of a wide array of tissues, cells, and molecules. Each single cell of the immune system recruited in response to a perturbation induced by an invading microorganism undergoes a sophisticated activation process. This process transforms it from a quiescent cell to an effector functional cell able to communicate with other effectors. The final goal is to eliminate the perturbation and restore resting conditions. All these processes are extremely complex and their interpretation requires integrated approaches that, together with the classical reductionistic studies, help to reconstruct the complexity of immunological phenomena. Traditional approaches are based on analysis of a single parameter at a time and, though they provide detailed knowledge of a particular molecular entity or a particular isolated phenomenon, they cannot provide an exhaustive interpretation regarding how the immune system fights a particular pathogen, maintains self‐tolerance, or remembers past infections. The completion of draft sequences of the human and mouse genomes offers many opportunities for gene discovery in the field of immunology through the application of methods of computational genomics. In concert with emerging genomic and proteomic technologies, the biology of the immune system can be defined. Initiation and regulation of the immune response is complicated and occurs on many levels. Multicellular organisms have been obliged to develop multifaceted innate and adaptive immune systems to cope with the challenges to survival originating from microorganisms and their products. The diversity of innate immune mechanisms is in large part conserved in all multicellular organisms (Mushegian and Medzhitov, 2001). Some basic principles of microbial recognition and response are emerging, and recently, the application of computational genomics has played an important role in extending such observations from model organisms, such as Drosophila, to higher vertebrates, including humans (Burley and Bonanno, 2002). Analysis of gene expression in tissues, cells, and biological systems has evolved in the last decade from analysis of a selected set of genes to an efficient high‐throughput whole‐genome screening approach of potentially all genes expressed in a tissue or cell sample. Development of sophisticated methodologies such as microarray technology for gene expression studies allows an open‐ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and define the samples’ unique biology. This discovery‐based research provides the opportunity to characterize either new genes with unknown function or genes not previously known to be involved in a biological process, and can lead to unpredictable and unexpected results, which may advance new insights in immunology.

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Large‐scale gene expression analysis is of great relevance in the field of immunology to generate a global view of how the immune system integrates its resources to maintain the integrity of the organism. To this purpose, the study of host–pathogen interactions is instrumental in understanding how to control infectious diseases. Host eukaryotes are constantly exposed to attacks by microbes seeking to colonize and propagate in host cells. To counteract them, host cells utilize a whole battery of defense systems to combat microbes. However, in turn, successful microbes evolved sophisticated systems to evade host defense. As such, interactions between hosts and pathogens are perceived as evolutionary arms races between genes of the respective organisms (Bergelson et al., 2001; Kahn et al., 2002; Woolhouse et al., 2002). Any interaction between a host and its pathogen involves alterations in cell signaling cascades in both partners that may be mediated by transcriptional or posttranslational changes. Transcriptomics is one of the methodologies that can be used to select important genes to study in detail from among thousands of genes encoded in the genome and can help in defining complex gene networks. Many genomic studies have been performed to interpret how human and mouse DCs respond to microbial and nonmicrobial inflammatory stimuli. In kinetic experiments, gene expression profiles of immature in vitro‐derived mouse or human DCs have been compared with gene expression patterns of activated DCs at different times after challenge with the activation stimulus (Tang and Saltzman, 2004). Analysis of entire kinetic data sets has revealed that DCs undergo a profound reorganization of gene expression in the first few hours after activation and then they progress versus a new resting state that is clearly distinct from the original immature DC state (Aebischer et al., 2005; Granucci et al., 2001a); the process of DC maturation is almost complete 24 hours after activation (Granucci et al., 2001a). Moreover, the number of genes expressed at different stages of DC ontogeny remains similar, indicating that the same number of genes are induced and suppressed at different time‐points after activation. The diversity of transcripts expressed in immature, transitional, and mature DCs is, thus, similar in magnitude, as has already been suggested for resting and activated T cells (Marrack et al., 2000). Comparing the response of DCs to inflammatory microbial and nonmicrobial stimuli has shown clear differences in the activation of the DC maturation program induced by these two types of stimuli. In particular, when lipopolysaccharide (LPS) and Tumor Necrosis Factor (TNF)a have been compared, LPS has stimulated a more potent and more rapid stimulus for DC activation (Granucci et al., 2001b). The expression of many genes was modulated by LPS but not TNF, and most of the genes that are commonly modulated were upregulated or downregulated 2–5 times more with LPS than with TNF. Genes involved in the activation and control of inflammatory processes, such

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as the complement component C1q, IL‐1b, IL‐1 receptor antagonist (IL‐ 1RA), and IL‐6, were markedly affected by DC exposure to LPS, but not to TNF. From this analysis, TNF emerged as a weak stimulus that could not drive DCs to a stage of maturation necessary for optimal immune‐response activation. This interpretation has been confirmed in a recent study showing that TNFa‐exposed DCs, although able to upregulate MHC II and costimulatory molecules, remain weak producers of proinflammatory cytokines and reach a semimature state that is tolerogenic rather than immunogenic (Lutz and Schuler, 2002; Menges et al., 2002). The tolerogenic activity of semimature DCs would be due to their capacity to induce IL‐10‐producing antigen‐ specific T cells (Menges et al., 2002). Besides in vitro‐derived DCs, the inefficacy of inflammatory nonmicrobial stimuli to induce full DC activation has also been demonstrated in vivo for naturally developing DCs. Indirect activation of DCs by inflammatory mediators generated antigen‐presenting DCs able to support T‐cell expansion but not the production of effector CD4þ T lymphocytes. In contrast, DC exposure to microbial stimuli resulted in generation of fully activated DCs able to sustain the differentiation of helper T‐cell responses (Sporri and Reise Sousa, 2005). These observations imply that DCs are extremely plastic and can adjust their responses depending on the nature of the stimulus they encounter. Therefore, DCs can distinguish between the actual presence of an infection and an inflammatory process mediated only by cytokines (Sporri and Reis e Sousa, 2005). This is particularly relevant for the activation of the appropriate responses. Genomic approaches for the study of DC maturation have emphasized this aspect revealing that DCs are able to activate distinct genetic reprogramming depending on the nature of the stimulus, even if the diverse stimuli are all of microbial origin. Thus, for distinct microorganisms it is always possible to identify a core and a pathogen‐specific DC response (Huang et al., 2001). An important aspect of using genomic approaches is the identification of molecules that regulate biological functions and, in this particular context, DC functions. One of the most unanticipated finding of global gene expression analysis applying to the study of mouse DC maturation was that they produce IL‐2 transiently at early time points after microbial encounter. The first observation was made for DCs stimulated with Gram‐negative bacteria (Granucci et al., 2001a). Afterward, it was shown that many different microbial stimuli are able to induce IL‐2 production by DCs while none of the inflammatory cytokine tested was able to elicit IL‐2 production by DCs (Granucci et al., 2003a), confirming the hypothesis that DCs are extremely plastic and can modulate their responses depending on the nature of the stimulus. DC‐ derived IL‐2 has been associated with regulatory functions in both innate and adaptive immune responses in mice (Granucci et al., 2001a; 2004).

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4. DC Interactions with the Microbial World 4.1. DC Receptor Repertoire 4.1.1. Toll‐like Receptor Family The receptor repertoire of DCs include a broad family of receptors that recognize microbial components which, whether pathogenic or not, serve as ligands to alert the immune response. One of the fastest growing families, collectively named TLRs, senses a distinct repertoire of conserved molecules expressed by fungi, viruses, bacteria, and protozoa. The TLR field was initiated thanks to the observation that Toll‐mutant flies were highly susceptible to fungal infection (Lemaitre et al., 1996; Poltorak et al., 1998). This study revealed for the first time that the innate immune system had a sophisticated means of detecting invasion by pathogens, such as Aspergillus fumigatus. Mammalian homologues were identified initially on the basis of their homology to the Drosophila Toll protein, the developmental protein required for antifungal immune responses in adult flies (Medzhitov et al., 1997). In 1998 one mammalian homologue named TLR4 was positionally identified as the LPS receptor, encoded by the Lps locus, known to be required in mice to respond to Gram‐negative bacteria (Poltorak et al., 1998). Since then, many other mammalian TLR have been identified: 10 in humans and 12 in mice, named TLR‐1 to TLR‐12 (Beutler, 2004). Their discovery is as fundamental as the discovery of antigen receptors on lymphocytes. Understanding TLR signaling pathways may allow us to dissect the complexity of immune responses in the balance between immunopathology and protective immunology. TLRs are expressed and modulated in myeloid mouse DCs and have a more selective distribution on human blood DCs (Degli‐Esposti and Smyth, 2005). Not all TLRs are expressed on the cell surface of DCs. TLR1, TLR2, and TLR4 are located at the cell surface; in contrast, TLR3, TLR7, and TLR9, all of which are involved in the recognition of microbial nucleic‐acid‐like structures, are not present on the cell surface (Ahmad‐Nejad et al., 2002; Heil et al., 2003; Matsumoto et al., 2003), but rather in the endoplasmic reticulum (ER). These receptors can be recruited from the ER to the endosomal compartments by microbial nucleic acid ligands (Latz et al., 2004). The TLR molecules are characterized by an extracellular domain with a leucine‐rich repeats (LRR) and a cytoplasmic domain (TIR domain) similar to that of the IL‐1R family (Akira and Takeda, 2004; Takeda et al., 2003). Functional analysis of each mammalian TLR member has revealed that they recognize different microbial ligands, such as LPS, lipoproteins, peptidoglycan, bacterial CpG DNA, single‐ and double‐stranded RNA, and bacterial flagellin, which are mostly conserved among pathogens and are not found in

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mammals. In particular, the microbial compounds able to activate cellular responses mediated by TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 have been identified (Bendelac and Medzhitov, 2002; Diebold et al., 2004). In general, TLR2 is mainly involved in responses to microbial products from Gram‐positive bacteria (peptidoglycans or lipoproteins) as well as from yeast (Hirschfeld et al., 1999; Underhill et al., 1999; Yoshimura et al., 1999). By contrast, TLR4 mediates the interaction with Gram‐negative bacteria, by transducing the signals deriving from LPS. The activation of TLR signaling pathways originates from the cytoplasmic TIR domains. Mutations of the TIR domain of the tlr4 gene in C57BL/10ScCr or C3H/HeJ mice impede LPS signal transduction so that these mutant mice become resistant to endotoxin and highly susceptible to Gram‐negative infection (Poltorak et al., 1998). Downstream of the TIR domain, the adaptor protein Myeloid Differentiation Factor 88 (MyD88) has been shown to recruit the IL‐ 1 receptor‐associated kinase (IRAK) when activated. IRAK is then activated by phosphorylation and its association with TRAF6 (tumor‐necrosis‐factor‐ receptor‐associated‐factor 6) leads to the activation and the nuclear translocation of NF‐kB transcription factor or to the activation of c‐Jun N‐terminal kinase (JNK) (Takeda and Akira, 2004). In addition to the adaptor protein MyD88, other relevant adaptors have been identified: TRIF, TRAM, and TIRAP (Beutler, 2004). A MyD88‐independent pathway, typical of the TLR3 and TLR4 signaling pathways, has also been described (Akira et al., 2001; Takeuchi et al., 1999). This pathway activates IFN‐regulatory factor (IRF3) (Taniguchi and Takaoka, 2002) leading to the production of IFNb and the expression of a number of IFN‐inducible genes such as IRG1 (immunoresponsive gene1), GARG16 (glucocorticoid‐attenuated response gene 16), or the IFN‐inducible protein (IP)10 chemokine. Thus, myeloid DC activation can lead to type I IFN production. In particular, DC secretion of type I IFNs has been first shown after viral infections (Diebold et al., 2003). It was demonstrated that dsRNA, produced by most viruses, can elicit type I IFN production in MyD88‐ independent manner. Subsequently, it has been observed that type I IFN are rapidly produced by DCs also following exposure to many other infectious agents, including bacteria (Granucci et al., 2004), and play a key role in the control of immune responses. Interestingly, in myeloid DCs, it has been shown that helminths, such as the Schistosoma mansonii, trigger the MyD88‐ independent pathway leading to IFNb secretion (Trottein et al., 2004). Surprisingly, it has been found that Schistosoma eggs contain a TLR3 ligand (Aksoy et al., 2005). Thus, the engagement of TLRs in DCs leads to the production of type I IFNs (IFNa and b) that are responsible for the subsequent expression of chemokine genes in DCs and DC maturation

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(Luft et al., 1998). Type I IFNs produced by TLR‐activated DCs have been shown to elicit specific Ig production of all isotypes (Le Bon et al., 2001). There is an increased interest in studying the role of TLR signaling in host defense against live pathogens. Notably, MyD88‐deficient mice still generate an immune response against viruses and intracellular bacteria such as Mycobacterium tuberculosis or Listeria monocytogenes, and TLR‐independent mechanisms for their recognition have been proposed (Inohara and Nunez, 2003). TLRs recognize microbial ligands in the extracellular space, but many pathogens reach the cell cytoplasm so that hosts have had to evolve mechanisms to detect intracellular pathogens. For viral infections, a good example of this coevolution is the detection of dsRNA in the cell cytoplasm by the activated endogenous protein kinase R (PKR, IFN‐inducible dsRNA‐dependent protein kinase). The relevance of this host recognition is exemplified by the presence of the influenza virus protein NS1 that can mask the viral dsRNA genome and prevent PKR activation (Diebold et al., 2003). Intracellular receptors are also NOD1 and NOD2 (nucleotide‐binding oligomerization domain) that bind in the cytosol, the core structures of bacterial peptidoglycans. The NOD protein family has been shown to act as intracellular receptors of bacterial lipids (Girardin et al., 2003) and to play a major role in shaping the cell response to cytoplasmic invasion by L. monocytogenes (McCaffrey et al., 2004). NOD proteins may have evolved to complement the detection of pathogens through intracellular recognition also in DCs. DC maturation induced by TLRs seems to direct polarization of the effector cells of the adaptive response. In this regard, the so‐called ‘dirty trick of immunologists’ to mix the protein antigen with Freund adjuvants to achieve immunization, now has a molecular basis. The active component of the Freund adjuvant, heat‐killed M. tuberculosis, has several ligands for TLRs. It has been demonstrated that antigen mixed with adjuvant failed to trigger T‐cell responses in MyD88‐deficient mice, indicating that DC engagement of TLRs in vivo is necessary to induce T‐cell priming (Schnare et al., 2001). In addition to the Freund adjuvant, several other microbial components have potent immune‐stimulatory activity on DCs. CpG DNA, which is recognized by TLR9, is a potent adjuvant eliciting Th1 responses (Krieg, 2000; Lipford et al., 1998) and the outer membrane protein (the porin) of Neisseria, which is recognized by TLR2 is also an adjuvant with potent immunogenicity (Massari et al., 2002). The cell‐wall skeletal fraction (CWS) of M. bovis Calmette‐ Guerin strain (BCG) recognized by TLR2 and TLR4 and used as an adjuvant in anticancer therapy also has a potent immunostimulatory effect on DCs (Azuma and Seya, 2001; Tsuji et al., 2000).

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TLR engagement promotes uptake of bacteria through upregulation of expression of scavenger receptors (SR), such as SR‐1 and the Mannose Receptor (MMR) (Doyle et al., 2004) and through maturation of the phagosome (Blander and Medzhitov, 2004). In DCs, TLR signaling induces delivery of degraded antigen to MHC II loading compartments and interestingly, through the upregulation of the expression of the MARCO receptor, it induces a cytoskeletal rearrangement (Granucci et al., 2003b). Comparing the effects of the ligation of different TLRs in DCs has been a difficult task as the signals from the TLRs can be not only qualitative, but also quantitative and additive. For example, ligation of TLR4 on DCs can upregulate the expression of TLR2, TLR4, and TLR9 (An et al., 2002; Nilsen et al., 2004; Visintin et al., 2001), resulting in signaling amplification. Furthermore, when molecularly defined ligands are substituted with live pathogens that possess multiple TLR‐activating ligands, the conditioning of DCs responses might become very complex because the adaptation of pathogens might have evolved to circumvent effector immune responses and therefore TLR‐mediated signaling. Recent studies analyzing the association between polymorphism in TLR sequences and disease susceptibility have demonstrated the protective role of TLRs in vivo. For example, polymorphisms in the TLR4 gene have been associated with increased susceptibility to respiratory syncytial virus (RSV) and Gram‐negative bacterial infections (Agnese et al., 2002; Tal et al., 2004). Similarly, a role for TLR5 has been demonstrated by the increase in Legionella pneumophila infection in individuals with a common polymorphism that introduces a stop codon into the TLR5 gene (Hawn et al., 2003). In mice, infection with Yersinia enterocolitica, a pathogen that can activate TLR2, is cleared more efficiently in the absence of TLR2 (Sing et al., 2002), but no protective effect of TLR2 was observed in Borrelia burgdorferi and Staphylococcus aureus infections (Takeuchi et al., 2000; Wooten et al., 2002). It has also been suggested that pathogens could exploit TLRs to generate nonresolving Th2‐biased responses. 4.1.2. C‐Type Lectin Family: Their Targeting by Infectious Agents Might Support Pathogen Spreading This family of receptors recognizes a wide range of carbohydrate structures. These receptors include the MMR, Dectin‐1, DEC‐205, DC‐SIGN, BDCA‐2, and Langerin. They all possess at least one carbohydrate recognition domain and bind sugars in a variety of secondary and tertiary structures (Geijtenbeek et al., 2004). Most of these molecules are involved in receptor‐mediated phagocytosis or endocytosis of microbes but some of them, such as the

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DC‐SIGN, actually support pathogen spreading. Thus, the adaptation of pathogens to target DC‐SIGN has assured longer pathogen survival. The MMR is preferentially expressed on myeloid cells and in DC. Increasing evidence shows that the MMR is involved in the silent clearance of inflammatory molecules and in maintenance of homeostasis. The MMR is an endocytic and phagocytic receptor that binds carbohydrate moieties on several pathogens, such as bacteria, fungi, parasites, and viruses, in particular Influenza virus (Reading et al., 2000) and HSV‐1 (Milone and Fitzgerald‐ Bocarsly, 1998; Rong et al., 2003). The high mannan content in the bacterial oligosaccharide cell envelope in both Gram‐positive and Gram‐negative bacteria assures efficient molecular recognition. In addition, MMR also binds endogenous molecules (Allavena et al., 2004). DC‐SIGN, the nonintegrin DC‐specific intercellular adhesion molecule 3 (ICAM‐3) is a type II transmembrane protein expressed on DCs with a C‐type lectin extracellular domain, capable of binding endogenous ligands such as ICAM‐2 and ICAM‐3 on resting T cells (Geijtenbeek et al., 2000b), but also capable of recognizing bacteria, fungi, (Geijtenbeek et al., 2004) and viruses. In particular, DC‐SIGN has been shown to bind the Human Immunodeficiency Virus (HIV‐1) envelope glycoprotein gp120 but does not function as a receptor for viral entry. Instead, DC‐SIGN allows mucosal DCs to carry HIV‐1 through the lymphatics in a ‘Trojan horse’ fashion, where it is eventually delivered to the T cells. Thus, the period of infectivity of HIV‐1 is increased by several days as a result of DC‐SIGN‐gp120 binding (van Kooyk and Geijtenbeek, 2003). DCs that express DC‐SIGN are mostly located close to the mucosal barriers, such as lamina propria DCs and dermal DCs. In contrast, LCs of the skin do not express DC‐SIGN but rather the C‐type lectin, Langerin. The discovery that in the early phase of infection DCs are among the first target cells of HIV at mucosal sites and from there the virus is shuttled to the lymph nodes and transmitted to the T cells has led to a reexamination of the mechanisms underlying interactions between DCs and T cells and the pathogenesis of HIV‐1 infection. Other pathogens, such as M. tuberculosis, have been also shown to target the DC‐SIGN. Notably, these pathogens misuse DC‐SIGN by distinct mechanisms that either circumvent antigen processing or alter TLR‐mediated signaling. This implies that adaptation of pathogens to target DC‐SIGN might support pathogen survival. Recently, the signaling events downstream of C‐type lectin receptors have attracted attention. It has been shown that Dectin‐1 and TLR2 can cooperate to increase zymosan‐induced signaling and cytokine secretion (Gantner et al., 2003). Cross‐linking the MMR, despite inducing upregulation of costimulatory molecules, induces secretion of IL‐10 (Chieppa et al., 2003; Nigou et al., 2001).

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Finally, the pentraxin protein, PTX3, which is produced by activated DCs and plays an important role in the clearance of Aspergillus infections, is associated with various TLRs and may be a host mechanism to expand the repertoire of microbial structures that are recognized (Doni et al., 2003; Garlanda et al., 2002). The receptor repertoire of DCs to sense microbes is, in fact, much broader and also includes phagocytic opsonic receptors such as the Fc receptors and the complement receptors. DCs express a moderate level of Fc receptors that are not modulated during maturation. DCs also express the Mac‐1 molecule (CD11b/CD18; aMb2 integrin), which is the CR3 complement receptor used for phagocytosis of complement‐coated bacteria. As with FcR, the surface expression of Mac‐1 molecules is not changed during activation of DCs. This is in contrast to monocytes and neutrophils that strongly upregulate Mac‐1 expression during differentiation, and in the presence of inflammatory stimuli. In addition to its role in receptor‐mediated internalization, the Mac‐1 molecule also mediates adhesion and chemotaxis (Anderson et al., 1986). Studies have shown that Mac‐1 is stored in intracellular vesicles, which are rapidly mobilized to the cell surface in response to chemoattractants (Miller et al., 1987). 4.2. DC Interaction with Bacteria Despite several early reports on the uptake of particulate material and cells by DCs (Austyn, 1996), the phagocytic capacity of DCs has long been denied. One reason for this was the technical difficulty of growing DCs in their immature state. Only in the past decade has it become possible to grow and maintain in vitro homogeneous immature DCs as long‐term growth‐factor‐ dependent lines (Winzler et al., 1997). This has allowed the investigation of DC biology in the absence of other contaminating cell populations. DCs have phagocytic activity that decreases with maturation. Indeed, several studies have shown that DCs can internalize Latex and zymosan beads (Austyn, 1996; Inaba et al., 1993; Matsuno et al., 1996; Reis e Sousa et al., 1993), but also apoptotic bodies (Parr et al., 1991), as well as microbes such as BCG (Henderson et al., 1997; Inaba et al., 1993), Saccaromyces cerevisiae, Corynebacterium parvum, S. aureus (Reis e Sousa et al., 1993), Leishmania spp. (Blank et al., 1993), and B. burgdorferi (Filgueira et al., 1996). The ability of DCs to phagocytose particulates or bacteria is greatest in immature DCs, whereas this capacity is reduced, but not eliminated, in mature DCs (Henderson et al., 1997). DCs are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial

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replication and producing cytokines and chemokines that activate and recruit additional cells. In the lamina propria, DCs have been shown to take up bacteria across the mucosal epithelium (Rescigno et al., 2001). Recently, this process has been elucidated: DCs open the tight junctions between epithelial cells, send dendrites outside of the epithelium, and sample bacteria directly in the gut lumen. The molecular mechanism that allows it to preserve the integrity of the epithelial barrier is based on expression and modulation by DCs of tight junction proteins, such as occludin, claudin 1, Zonula occludens 1, and junctional adhesion molecule (JAM) (Rescigno et al., 2001). Upon attachment, DCs engulf the bacterium by actively surrounding it with pseudopodia. This process is facilitated by Fc‐type and complement‐type receptor‐mediated endocytosis. The movement of the pseudopodia in activated DCs involves actin‐binding proteins, and it can be blocked by the drug cytochalasin D, which stops the polymerization of actin and inhibits phagocytosis. The rearrangement of the cytoskeleton, associated with DC motility, involves depolymerization of the actin (Winzler et al., 1997). Once a bacterium has been fully internalized into the phagosome, fusion of the phagosome with other intracellular vacuoles or granules takes place. Processing bacterial molecules for antigen presentation occurs in lysosomes following their fusion with phagosomes. This process may take several hours, as antigen presentation of bacterial antigens is not observed earlier than 6 h following infection (Rescigno et al., 1998). Some bacteria prevent the normal maturation and trafficking of the phagosome and impair its normal bacteriolytic activities. Other bacteria escape from the vacuole and can replicate in the cytosol. After infection with live bacteria, DCs sense the intruders by reprogramming the transcription of about 1000 genes (Granucci et al., 2001a). Cytokine and chemokine genes as well as IFN‐inducible genes are differentially expressed in the first few hours, whereas at later time‐points, apoptosis and antiapoptosis genes as well as genes involved in T‐cell activation are induced. Model antigens expressed in recombinant Gram‐positive and Gram‐ negative bacteria are processed and can be directly presented on both MHC I and II molecules (Corinti et al., 1999; Rescigno et al., 1998; Svensson et al., 1997). Unlike macrophages, this exogenous pathway of MHC I presentation is transporter associated with antigen presentation (TAP)‐dependent (Rescigno et al., 1998). This implies that exogenous bacterial antigens introduced by phagocytosis are directed into the classical pathway of MHC I presentation. Indeed, transport of whole bacterial proteins from phagolysosome to the cytosol takes place after phagocytosis of bacteria as also shown with immune‐ complex internalization (Rodriguez et al., 1999). The capacity of DCs to

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present bacterial antigens with very high efficiency on both MHC I and II molecules can be exploited to induce strong and long‐lasting immunity to bacteria as well as nominal antigens of interest. Several examples of partial protective immune responses achieved by injecting in vivo DCs loaded with microbes have been described against Chlamydiae trachomatis (Su et al., 1998), B. burgdorferi (Mbow et al., 1997) or M. tuberculosis (Demangel et al., 1999). A few hours after bacterial infection, DCs synthesize a number of cytokines and chemokines (Rescigno et al., 1998). TNFa and IL‐6 are readily detected in DCs infected with either Gram‐positive or Gram‐negative bacteria. Interestingly, activation of DCs is achieved when bacteria are alive. Indeed, heat‐inactivated bacteria, although able to induce maturation of DCs, fail to induce inflammatory cytokines (Rescigno et al., 2002). Biologically active p70 IL‐12 is produced by myeloid mouse DCs only in a very small amount after bacterial encounter, as compared to human monocyte‐derived DCs (Corinti et al., 1999). TNFa production is rapidly induced following infection. It is likely that the phenotypic and the functional maturation, which occurs in DCs within 24 h of bacterial uptake is the result of cytokine amplification during this response. Indeed, DC activation by TNFa alone mimics the phenotypic maturation observed after bacterial infection, although the addition of anti‐TNFa antibodies only partly inhibits this process. This is consistent with the finding that the pattern of genes induced after activation of DC by TNFa and LPS is very different (Granucci et al., 2001b). Moreover, DC maturation obtained by whole bacteria is quantitatively and qualitatively more pronounced indicating the induction of several transducing pathways, likely via receptors that recognize distinct bacterial components. Treatment of DCs with bacteria results in a clear modification of cell‐ surface DC‐activation markers. Consistent with acquisition of costimulatory activity during maturation is the upregulation of CD86 and CD40 molecules. Upregulation of CD86 and CD40 molecules has also been observed with BCG (Thurnher et al., 1997) and M. tuberculosis (Henderson et al., 1997), but it was not observed following the use of inert Latex beads of various sizes. Upregulation of the costimulatory molecules and the coordinated translocation of MHC molecules at the cell surface are essential molecular events for the subsequent antigen presentation and activation of both CD4þ and CD8þ T cells. The internalization of bacteria is also associated with increased stability of MHC class I‐ and class II‐peptide complexes. Indeed, the half‐lives of MHC class II‐ and class I‐peptide complexes change from 10 to 20 h and from 3 to 9 h, respectively. This has important consequences for T‐cell induction because it increases the chances of DCs to encounter antigen‐specific T cells in the draining lymph nodes (Rescigno et al., 1998).

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4.3. DC Interaction with Viruses Mucosal surfaces represent the main sites of interaction of DC with viruses. The ability of DCs in inducing a strong antiviral immunity suggests that it would be an evolutionary advantage for viruses to subvert this particular cell. In fact DCs often act as a portal for virus invasion and viruses have indeed evolved to trigger epithelial responses, via chemokine production that result in the recruitment of DCs at the mucosal sites (Reinhart, 2003). Viruses that can target DC receptors are also able to evade the host’s immune system and acquire a selective advantage. An interesting example is provided by a‐dystroglycan (a‐DG), a DC receptor for lymphocytic choriomeningitis virus (LCMV), and several other Arenaviruses. By selection, variants of LCMV that bind a‐DG with high affinity replicate in the majority of DC, causing a generalized immune‐suppression, and establish a persistent infection. In contrast, viral strains that bind with low affinity to a‐DG display minimal replication in DCs and generate a robust anti‐LCMV cytotoxic T‐lymphocyte response that clears the virus infection. Hence, receptor–virus interaction on DCs in vivo is an essential step in the initiation of virus‐induced immune‐suppression and viral persistence (Sevilla et al., 2003). The best example is represented by HIV‐1 which has, in fact, evolved mechanisms to subvert DC immune function. DCs promote replication of HIV by capture and infection of the cells themselves followed by transmission of the virions to T cells. HIV‐1 infection of DCs can occur either via CD4 or chemokine receptors, leading to full viral replication, or via binding to C‐type lectin receptors resulting in transfer and replication of the virus to T cells. DCs respond to this invasion by processing viral proteins through MHC class I and II pathways and undergoing a maturation that enhances their presentation of antigen to T cells for induction of adaptive antiviral immunity (Wilflingseder et al., 2005). In contrast to acute HIV‐1 infection, individuals in the chronic phase regularly show impaired HIV‐1‐specific CD4 helper responses and very low or no specific neutralizing antibodies. In addition to HIV‐1, DCs are likely to be responsible for virus‐induced immune‐suppression that has been observed with measles (MV) infections or with members of the herpes virus family, such as herpes simplex virus (HSV) or human cytomegalovirus (HCMV). HSV‐1 infects the epidermis, where the predominant cell type it encounters is keratinocytes. These cells allow efficient replication (Mikloska et al., 1996). In the epidermis, LCs can be infected at early stages, whereas dermal DCs seem to be unaffected by the virus and no detectable viruses are found in the draining lymph nodes (Mueller et al., 2002). Following HSV‐1 infection, LC functions are impaired allowing the virus sufficient time to replicate and infect

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a second round of keratinocytes. This increases the local virus titre and favors an efficient infection of nerve endings (van Lint et al., 2004) that are in close anatomical association with LCs (Hosoi et al., 1993); retrograde migration of the virus to the dorsal root ganglions (DRG) allows the establishment of latency (Jones, 2003). It is still unclear whether a lytic lesion breaks through the epidermal‐dermal barrier. Infection of LCs/DCs with herpes viruses such as HSV‐1, HSV‐2, HCMV, VZV (varicella‐zoster virus), or HSV‐6 results in an impaired capacity to stimulate T‐cell proliferation (Kakimoto et al., 2002; Morrow et al., 2003; Moutaftsi et al., 2002). The ability of HCMV to bind DC‐SIGN may aid HCMV infection of T cells in trans, similarly to that seen with HIV‐1 (Geijtenbeek et al., 2000a; Halary et al., 2002). In addition, Epstein‐Barr virus (EBV) and HCMV also prevent the differentiation of DCs from their precursors (Gredmark and Soderberg‐Naucler, 2003; Guerreiro‐Cacais et al., 2004; Li et al., 2002) providing the establishment of latency, a characteristic of all herpes viruses. In fact it has been shown that HCMV reservoirs are myeloid DC progenitors (Hahn et al., 1998). Herpes viruses also modify the expression of relevant molecules in the DC–T cell interaction. HSV‐1‐ and HCMV‐infected DCs are unable to upregulate costimulatory molecule expression during maturation (Moutaftsi et al., 2002; Salio et al., 1999) and CD83 downregulation has been observed (Morrow et al., 2003). Herpes viruses may have evolved to specifically target DC–T cell interaction. In fact, HSV‐1 and HCMV infection of DCs induces upregulation of TRAIL and Fas, resulting in bystander killing of antigen‐ specific T cells (Muller et al., 2004). Other viruses such as Parainfluenza virus (PIV) and RSV also impair infected‐DC ability to activate T cells (Bartz et al., 2003; Plotnicky‐Gilquin et al., 2001; Vidalain et al., 2000) or to secrete cytokines, including IL‐2 (Andrews et al., 2001; Fugier‐Vivier et al., 1997). The downregulation of IL‐2 production by DCs likely accounts for the profound immune‐suppression observed in infected individuals (Klagge et al., 2000; Marie et al., 2001). Other microbes may avoid immunity by altering migration of DCs; examples include Poxvirus that mediates the release of chemokines antagonists. Some viruses also act on several signal transduction pathways known to modulate DC functions. For example, Vaccinia viruses express intracellular proteins that interfere with signal transduction from TLR or cytokine receptors (Bowie et al., 2000; Harte et al., 2003) whereas Paramyxoviruses target the CD40 signaling pathway (Fugier‐Vivier et al., 1997). Thus, viruses have evolved several strategies to evade the immune response. This could have resulted in uncontrolled microbial replication, which would have been deleterious for the host. One may imagine that to counteract such strategy the

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mammalian host has developed strategies to control the infection with minimal damage to self‐tissues that consist primarily in the coordination of the innate and the adaptive immune responses through DC activation and TLR signal transduction. 5. DC Functions 5.1. Role of DCs in Innate Immunity As highlighted before, innate immunity is the most ancient line of defense against microbial infection. It is present and conserved in all animals throughout evolution (Janeway and Medzhitov, 2002). Among the cells that are involved in innate responses, an important role is held by natural killer (NK) cells. NK cells are lymphocytes of the innate immune system that potently contribute to infection eradication (Moretta, 2002). NK cells comprise about 15% of circulating lymphocytes and are also found in peripheral tissues. Recently, the presence of NK cells in resting human lymph nodes and in lymph nodes of mice injected with mature DCs has also been described (Ferlazzo et al., 2004; Martin‐Fontecha et al., 2004). NK cells exert their activity by producing high amounts of IFNg, which activates a strong inflammatory response, and by having direct cytotoxic function (Moretta, 2002). The functions of NK cells are regulated by a balance of activating and inhibiting signals. These signals are transmitted by inhibitory receptors, which bind MHC I molecules, and activating receptors, which bind ligands on tumors and pathogen‐infected cells (Smyth et al., 2002). Other than surface receptors, cytokines such as IL‐2, IL‐12, IL‐18, and type I IFNs have been shown to promote NK cell priming (Smyth et al., 2002). Recent studies have focused on the function of DCs during the early phases of the immune response, and a predominant role for DCs in activation of NK cells has been described both in mice and in humans (Ferlazzo et al., 2002; Fernandez et al., 1999; Gerosa et al., 2002; Piccioli et al., 2002). The first place of contact between NK cells and DCs could be the site of infection where both resident and recruited DCs would be able to activate NK cells (Moretta, 2002). Activated DCs can migrate to the draining lymph nodes where they can probably stimulate resident and newly recruited NK cells. Indeed, in the T‐cell area of human lymph nodes NK cells have been described as colocalizing with DCs (Ferlazzo et al., 2004). Moreover, it has been found that a subpopulation of NK cells enriched in lymph nodes (CD16–CD56hi) is able to respond to DC‐derived stimuli, such as IL‐12, that elicits IFNg production by NK cells, and membrane‐bound IL‐15, which induces NK cell proliferation (Ferlazzo et al., 2004).

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Maturing DCs can be recruited at the draining lymph nodes via the upregulation of the expression of the CC‐chemokine receptor CCR7 (Weninger and von Andrian, 2003). Concerning NK cell recruitment, it has been proposed that inflammatory stimuli can induce NK cell migration at the draining lymph nodes with a process partially dependent on the expression of CXCR3 (Martin‐Fontecha et al., 2004). Thus following activation with inflammatory microbial stimuli, DCs would produce CXCR3 ligands (CXCR3Ls) that would favor the recruitment of NK cells (Martin‐Fontecha et al., 2004). In accordance with this hypothesis, we have found that DCs, activated with microbial stimuli (LPS and CpG) able to guarantee NK‐cell accumulation at the draining lymph nodes, produce large amounts of two CXCR3Ls, CXCL9 and CXCL10. This suggests that the accumulation of NK cell at the draining lymph nodes might actually be due to DC‐mediated CXCR3‐dependent NK cell recruitment (Zanoni et al., 2005). Two pathways for DC‐mediated NK cell activation have been described in mice: one dependent on IL‐4 and the other dependent on microbial stimuli (Granucci et al., 2004; Terme et al., 2004). Therefore, an appropriate cytokine milieu containing IL‐4 renders DCs competent for NK‐cell activation independent of microbial stimuli, although the presence of microbial stimuli increases the efficiency of NK‐cell activation (Ferlazzo et al., 2003); alternatively, following a microbial encounter, DCs become capable of efficiently activating NK cells (Granucci et al., 2004). DCs differentiated in the presence of IL‐4 are strong producers of IL‐12 following activation with inflammatory stimuli (Macatonia et al., 1995). This cytokine has been shown to be required to obtain optimal IFNg production by NK cells (Smyth et al., 2002). In the context of viral infections, another DC‐derived cytokine able to promote efficient IFNg production by NK cells is IL‐18 (Andrews et al., 2003). In the absence of DC exposure to IL‐4 and in response to bacterial challenges or to bacterial cell products, DC‐derived IL‐2 plays a major role in eliciting IFNg production by NK cells (Granucci et al., 2004). The biological relevance of NK‐cell activation mediated by DCs during bacterial infections resides mainly in the secretion of IFNg (Ferlazzo et al., 2003), which represents the principal phagocyte‐activating factor (Boehm et al., 1997; Ferlazzo et al., 2003). The role of DC‐derived IL‐2 in inducing IFNg production by NK cells has been studied in the mouse system. Nevertheless, human monocyte‐ derived DCs cultured in vitro in the presence of IL‐15 and not in the presence of IL‐4 can produce IL‐2 (Feau et al., 2005); thus, it would be interesting to investigate whether, also in humans, IL‐2 could play a role in stimulating NK cells in a context in which IL‐15 is present. Bacterially activated DCs can also induce NK‐cell cytotoxic function. This phenomenon is type I IFN‐dependent and IL‐2‐independent. As stated

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previously, the production of type I IFNs by myeloid DCs has been shown in the context of viral infections (Diebold et al., 2003). In support of this observation, it has been found that myeloid DCs can also produce type I IFNs following bacterial activation (Granucci et al., 2004). The precise contribution of myeloid and plasmacytoid DCs in the production of type I IFNs in vivo in response to different types of infection remains to be determined. Results suggest that production of type I IFNs may represent a poorly recognized response of myeloid DCs to several infectious agents, including bacteria. The upregulation of NK‐cell cytotoxic function by bacterially activated DCs may not be directly related to NK‐cell antibacterial effects. The biological relevance of this response may reside in the fact that cytotoxicity could contribute to controlling the late phases of the immune response by limiting inflammation and restoring homeostatic balance after infection (Ferlazzo et al., 2003; Zitvogel, 2002). DCs are susceptible to NK cell‐mediated lysis, therefore the ability to elicit NK‐cell cytotoxicity may be a means for DCs to limit their own activity. Moreover, given the ability of NK cells to acquire strong cytolytic function following interaction with bacterially activated DCs, and given the fact that NK cell antitumor functions can be upregulated following contact with activated DCs (Fernandez et al., 1999; Van Den Broeke et al., 2003), it has been proposed that bacterial infections may contribute to maintaining a basal level of alert against tumors. It is well established that DC‐mediated NK‐cell activation requires cell–cell contact, although the relevant molecules have not been defined. In humans, the induction of IFNg production by NK cells occurs through the formation of an organized supramolecular structure called immunological synapse between DCs and NK cells (Borg et al., 2004; Grakoui et al., 1999). The formation of DC–NK cell contacts depends on cytoskeleton reorganization and lipid raft mobilization. Synapse formation allows the polarization and concentration of preformed IL‐12 at the contact site, a process required to elicit IFNg production (Borg et al., 2004). The outcome of DC–NK cell interaction is not univocal. Under appropriate conditions, NK cells can contribute to DC activation. Indeed, IL‐2‐activated NK cells can promote DC maturation measured in terms of upregulation of costimulatory and MHC molecules and production of inflammatory cytokines. As previously mentioned for DC‐mediated NK cell activation, NK‐mediated DC activation requires cell–cell contact and soluble mediators produced by NK cells and DCs, such as TNFa(Gerosa et al., 2002). 5.2. Role of DCs in Acquired Immunity Activated antigen‐loaded DCs migrate to the T‐cell zone of secondary lymphoid organs to prime T‐cell responses. The requirement of DCs for T‐cell

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activation has been demonstrated in vivo for CD8þ T lymphocytes. Mice temporarily deprived of CD11cþ DCs are impaired in their ability to mount specific CD8þ T‐cell responses to infections with the intracellular bacterium Listeria monocytogenes, the parasite Plasmodium yoelii, the LCMV, or following antigen immunization (Jung et al., 2002; Probst and van den Broek, 2005). DCs can enter the lymph node either through blood or lymph (Cavanagh and Von Andrian, 2002). Initially they are concentrated in close vicinity to high endothelial venules (HEV), then progressively they change their intranodal distribution and, 1 day after reaching the lymph node, DCs are predominantly distributed throughout the T‐cell area (Mempel et al., 2004). The interaction of DCs with CD8þ T cells has been followed in vivo over a 48‐h period after T‐cell entry in the lymph node (Mempel et al., 2004). Experimental animals were injected in the footpad with immature DCs either pulsed or not with the peptide. To allow DC migration to draining lymph nodes, mice were also treated with LPS. Antigen‐specific T cells were then injected intravenously 18 h after DC administration. DC–T cell interactions were analyzed over time using intravital multiphoton microscopy (Sumen et al., 2004). It has been observed that during the initial 8 h after entering the lymph node, T cells scan many different DCs rapidly and establish only short interactions lasting no more than a few minutes. Subsequently, after 6–8 h, the motility of T cells decrease and the contact they form with DCs last longer than 1 h. Long‐lasting contacts between DCs and T cells continue for the first day after T‐cell entry in the lymph node and until they start to proliferate (Mempel et al., 2004). Stable DC–T cell interactions are necessary to induce T‐cell priming (Hugues et al., 2004). In vitro stable DC–T cell contacts are associated with the formation of an immunological synapse (Grakoui et al., 1999). Three‐dimensional (3D) analysis of the binding site between DCs and T cells by confocal microscopy shows that this specialized contact zone is a concentric structure with a central supramolecular activation cluster (cSMAC) enriched in TCR and CD28, which interact with peptideþMHC complexes and CD80 or CD86, respectively (Bromley et al., 2001), surrounded by a peripheral ring (pSMAC) enriched in lymphocyte function‐associated antigen (LFA‐1). The synapse depends on T‐cell cytoskeletal rearrangements that are required for receptor clustering (Friedl and Storim, 2004). The capacity of DCs to prime T‐cell responses has not been attributed to one particular DC‐specific surface or secreted molecule, but to a combination of factors such as the high level of expression of cell membrane costimulatory proteins, the secretion of specific cytokines, and the efficient antigen‐processing machinery, properties acquired by DCs during maturation (Steinman, 2000). Stimuli that induce DC activation and maturation increase the efficiency of antigen processing for both class I and class II pathways and the half‐life

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of peptideþMHC complexes at the cell surface that otherwise are rapidly internalized and recycled (Cella et al., 1997; Pierre et al., 1997; Rescigno et al., 1998). Regarding the class II pathway, DCs contain a large number of multivesicular bodies (MVBs), that store a presynthesized pool of MHC II molecules. The vesicles storing MHC II proteins are formed by invagination of the limiting MVB membrane. In MVBs the accessory molecules H2‐DM, required for MHC loading with peptides, are physically segregated into the MVB‐limiting membrane and cannot come in contact with class II molecules. This segregation prevents class II loading in immature DCs. Following DC activation by microbial or microbial product encounter, an extensive fusion of the MVB internal vesicles occurs, resulting in the formation of long tubular compartments where MHC II molecules and H2‐DM molecules become closely associated. This MVB reorganization process favors an efficient peptide‐ loading of MHC II molecules and allows massive export of peptide‐MHC complexes at the cell surface (Kleijmeer et al., 2001). In contrast with class II molecules, class I molecules are not stored inside immature DCs but are newly synthesized after activation stimuli encounter. The kinetics of new MHC I molecule biosynthesis, has a peak at 18 h after bacterial or LPS stimulation (Rescigno et al., 1998). Interestingly, DCs can delay the processing of internalized antigens by antigen retention in a storage compartment with a mildly acidic pH content (Lutz et al., 1997). In these vesicles, the internalized antigens are not immediately degraded and fusion with the lysosomes is delayed. This mechanism is apparently coordinated with the generation of newly synthesized MHC I molecules (Rescigno et al., 1998). Exclusively in DCs, class I molecules can cross‐present exogenous antigens. Thus, antigen retention could represent a good way to maximize the efficiency of class I presentation during DC maturation. The mechanism by which exogenous antigens internalized in phagosomes can access the MHC I loading compartment has been reported (Guermonprez et al., 2003). During or immediately after their formation, phagosomes can fuse with the endoplasmic reticulum. This permits a release of antigens in the cytosol and the consequent degradation in the proteasome. After degradation, peptides are transported into the lumen of the same phagosome (Guermonprez et al., 2003). In adaptive immunity DCs play the important role of directing the adequate type of immune response depending on the invading microorganism. DCs plasticity consists in their ability to express distinct polarizing signals on the basis of the type of PRR that is activated at the cell surface. The precise mechanisms that regulate this phenomenon have not been defined yet. In general, in response to most TLR stimuli, DCs skew T‐cell responses toward the Th1 type (Schnare et al., 2001). An exception is represented by TLR2 that, when stimulated on DCs by Pam3Cys, can mediate Th2 responses (Agrawal

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et al., 2003). The activation of Th1 responses by DCs has been often associated with the production of cytokines of the IL‐12 family (Trinchieri et al., 2003). Surprisingly, the role of IL‐12 has been recently questioned and it seems to be redundant in many human infectious diseases (Fieschi and Casanova, 2003) and in many mouse models (Oxenius et al., 1999; Zhang et al., 2003). To this regard, a more important role for IL‐23 and IL‐27 has been proposed (Cua et al., 2003; Murphy et al., 2003). Concerning Th2 responses, although some DC‐expressed molecular mediators, such as monocyte chemoattractant protein (MCP‐1) (Chensue et al., 1996; Matsukawa et al., 2000) and OX40L (Akiba et al., 2000), have been proposed in particular infection models, crucial molecules still need to be identified. As will be discussed, DCs may also produce molecules involved in the peripheral differentiation of regulatory T cells (Lutz and Schuler, 2002). Besides their well‐established role in priming T lymphocytes, DC have the capacity to regulate B‐cell responses directly (Banchereau et al., 2000). Human tonsillar interdigitating DCs have been shown to interact with B cells in situ (Bjorck et al., 1997). Similarly, it has also been observed that, in rats, DCs can form T cell‐independent short‐lived clusters with B cells with a mechanism that depends on the LFA‐1 integrin (Kushnir et al., 1998). In vivo DCs have been shown to uptake the antigen, retain it unprocessed, and transfer it to naı¨ve B cells to initiate an antigen‐specific response (Wykes et al., 1998). The migration of DCs to the B‐cell zone in lymph nodes would be controlled by CXCR5 (Wu and Hwang, 2002). In vitro DCs are able to induce proliferation and IgM secretion of B cells activated through CD40 (Dubois et al., 1997). After 20 days of coculture approximately 20% of B cells differentiate to IgM‐secreting plasma cells with a mechanism that involve IL‐12 production by DCs (Dubois et al., 1998). DCs are also able to promote the differentiation of CD40‐activated memory B cells to Ig‐secreting cells (Dubois et al., 1997). This process is IL‐12 independent and requires secretion of soluble IL‐6R, a chain which can bind IL‐6 to form a complex that can interact with high affinity with the IL‐6R transducing chain and increase the biological activity of IL‐6 (Dubois et al., 1998). DCs are also involved in the induction of B‐cell class switch (Dubois et al., 1999). Upon exposure to different molecular mediators, such as IFNg, CD40 ligand, LPS, or IFNa, marginal zone DCs upregulate the expression of members of the TNF family, such as B lymphocyte stimulator protein (BlyS) and proliferation‐inducing ligand (APRIL). In the presence of IL‐4, IL‐10, or transforming growth factor‐b (TGFb activated DCs, expressing BlyS and APRIL, can induce B‐cell class switch recombination to Cg, Ca, and C in a CD40‐independent manner (Litinskiy et al., 2002). To acquire the ability to secrete immunoglobulins, BlyS‐ and APRIL‐stimulated B cells also require

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cross‐linking of the surface B‐cell receptor and exposure to IL‐15. Synergism of BlyS and APRIL with IL‐15 is needed for activation of transcription factors, including NF‐kB, that lead to efficient B‐cell proliferation and antibody production (Litinskiy et al., 2002). 5.3. Role of DCs in Central Tolerance The immune system of vertebrate animals has evolved to protect against perturbations provoked by incoming pathogens. Given the variety of pathogens, it is necessary that the T‐cell repertoire be extremely diverse to allow elimination of all possible invading microorganisms. However, the generation of an immense T‐cell repertoire increases the possibility of developing autoreactive T cells. To limit self‐tissue damages while maintaining T‐cell diversity, the immune system has therefore developed mechanisms for eliminating or rendering nonfunctional autoreactive T cells. Tolerance to tissue antigens is achieved through a combination of thymic and peripheral events that eliminate or inactivate potentially dangerous T cells (Stockinger, 1999). The thymus provides a very important initial step in eliminating potentially dangerous self‐specific T cells (Liston et al., 2003). Positive and negative selection of T lymphocytes are considered qualitatively distinct processes that depend on thymic compartmentalization and on the cellular context of TCR‐MHC interaction (Laufer et al., 1999). Different thymic cell types can give qualitatively different signals to T cells so that positive and negative selections can occur sequentially following T‐cell interaction with thymic stromal cells (Laufer et al., 1999). Three different stromal cell types are present in the thymus: cortical epithelial cells; medullary epithelial cells, and bone marrow‐derived cells that comprehend DCs, macrophages, and B cells. By developing mouse models in which the expression of MHC‐peptide complexes was limited to particular thymic cell types, it has been shown that cortical epithelium has the exclusive capacity to induce positive selection of both autoreactive and nonautoreactive thymocytes (Capone et al., 2001; Laufer et al., 1996). Negative selection is, then, consequent to positive selection, and mainly occurs in the corticomedullary junction and within the thymic medulla (Murphy et al., 1990; Surh and Sprent, 1994). In these regions, negative selection is mediated by medullary epithelial cells (Kyewski and Derbinski, 2004) and antigen presenting cells (APCs) (Marrack et al., 1988). The capacity of medullary epithelial cells to negatively select has been attributed to the fact that they are able to express tissue‐specific genes (Gotter and Kyewski, 2004). It has been proposed that the promiscuous gene expression of thymic epithelial cells is regulated by expression of a particular transcription factor called transcriptional regulator autoimmune regulator (Aire) (Gotter and Kyewski, 2004). Expression of

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many tissue‐specific genes is, indeed, highly reduced in Aire‐deficient mice (Anderson et al., 2002) who develop tissue‐specific antibodies and lymphoid cell infiltration in many peripheral organs (Anderson et al., 2002). Moreover, the deletion of CD4þ T lymphocytes specific for a self‐antigen expressed under the rat insulin promoter is abrogated in Aire‐deficient mice (Liston et al., 2003). The function of DCs in central tolerance has been investigated in different experimental settings. DCs loaded with low doses of C5, the fifth component of the complement, or DCs spontaneously presenting the C5 are able, together with thymic epithelial cells, to delete C5‐specific T lymphocytes in culture (Volkmann et al., 1997; Zal et al., 1994). Moreover, in an in vivo model in which MHC II was selectively expressed by DCs under the CD11c promoter, efficient negative selection of I‐E reactive Vb5þ and Vb11þ CD4þ T cells has been described (Brocker et al., 1997). Recently, using a similar model in which MHC I was expressed exclusively by DCs the tolerizing role of these cells has also been shown in the context of CD8þ T cells (Cannarile et al., 2004). Thus, at present, it seems that thymic DCs are specialized in tolerance induction and cannot positively select either CD4þ or CD8þ T cells. 5.4. Role of DCs in Peripheral Tolerance Many tissue proteins are not expressed in the thymus at sufficient levels to induce clonal deletion or tolerization (Avery et al., 1995). For this reason, several mechanisms of peripheral T‐cell tolerization have evolved and it has been shown that autoreactive T cells that actively recognize the antigen in the periphery can undergo anergy (Schwartz, 2003), deletion (Burkly et al., 1990), or downregulation of T‐cell receptors (TCRs) (Schonrich et al., 1991) or coreceptors (Robbins and McMichael, 1991). Several models have been proposed to explain the induction of tolerance in peripheral autoreactive T cells. The first model (Bretscher and Cohn, 1970) hypothesized that the immune system is capable of responding only to nonself‐antigens, not to self‐antigens, and that antigen‐specific cells make the discrimination between self and nonself and make the decision to respond. According to this model, self is an invariant property of the individual and antigenic exposure early in ontogeny is tolerogenic because of the low frequency of effector T‐helper lymphocytes (Bretscher, 1999). Nevertheless, as emphasized previously, evidence accumulated in the last 20 years (Mueller et al., 1989) have led to a different assumption that the decision to initiate an adaptive immune response is not made by the antigen‐specific cells, but by the APCs. According to the infectious nonself and noninfectious self (INS) model, APCs are maintained in a resting state until they encounter microbes or microbial cell products that

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activate them and induce the upregulation of costimulatory molecules (signal two) necessary for peripheral T‐cell activation (Medzhitov et al., 1997). Thus, distinction between self and nonself in the periphery is made by the APCs. The INS model does not deal with the problem of peripheral tolerance and claims that self‐peptides presented by APC in the periphery are not recognized as nonself by T cells because self‐reactive T cells are eliminated during negative selection in the thymus (Medzhitov and Janeway, 2000). In contrast, the role of peripheral T‐cell tolerance is strongly emphasized in the Danger model (Matzinger, 1994). Like the INS model, the Danger theory proposes that T‐cell activation is mediated by activated APCs that deliver signals one and two to naı¨ve T cells, but that activation of APCs is mediated by danger signals derived from injured cells such as those exposed to pathogens or other possible stress signals. In the presence of danger, APCs are activated, expressing signal one and two and are capable of activating T cells; in the absence of danger, APCs are not activated and T cells (antigen‐experienced or naı¨ve) that interact with resting APCs die for lack of costimulation (Matzinger, 2002). Recently, the Danger model has been experimentally tested on antigen‐presenting DCs and it has been proposed that the activation state of DCs is relevant for the decision to suppress or activate an immune response (Steinman et al., 2003). In particular, immature or CD40‐activated DCs expressing the scavenger receptor CD205 have been targeted with the antigen in vivo and the fate of CD4þ and CD8þ antigen‐specific T cells followed over time (Bonifaz et al., 2002; Hawiger et al., 2001). T‐lymphocyte tolerization is observed when they encounter immature antigen‐loaded DCs, whereas T‐cell activation is described when they encounter activated antigen‐presenting DCs. The immature state of DCs, characterized by the absence of sufficient costimulation and sufficient signal two, has been interpreted as responsible for the tolerization process. A second evidence of the capacity of immature DCs to induce peripheral T‐cell tolerance relies on their ability to cross‐present peripheral tissue antigens and induce abortive T‐cell activation (Hernandez et al., 2001). Finally, a system that allows inducible antigen presentation by resting or activated DCs has been described. In this model, three distinct LCMV‐ derived CTL epitopes could be presented by 5% of the total DC population following induction (Probst et al., 2003). Presentation of LCMV‐derived CTL epitopes by resting DCs resulted in antigen‐specific tolerance, which could not be broken by subsequent infection with LCMV. Conversely, antigen presentation by activated DC-induced CTL activation and protective memory (Probst et al., 2003). The tolerization process depended on the synergistic effect of the PD‐1 and CTLA‐4 molecules (Probst et al., 2005). Although other explanations concerning all these observations may be possible, such as antigen persistence, they have been interpreted in the context of

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the Danger model with a fundamental role exerted by immature DCs in maintaining peripheral tolerance. DCs have also been implicated in the functional control of regulatory T cells (Treg). These particular lymphocytes are involved in maintaining tolerance to self‐antigens by inhibiting responses mediated by effector CD4þ and CD8þ T cells (Sakaguchi, 2000). Some Treg cell populations [those expressing CD4 and IL‐2 receptor a chain (CD25)], originate in the thymus, while other Treg subtypes (those identified as IL‐10 producers) differentiate in the periphery (Roncarolo et al., 2003; Roncarolo and Levings, 2000). A role of DCs in the peripheral differentiation of Treg cells has been proposed. In particular, it has been observed that repetitive stimulation of naı¨ve T cells with allogeneic immature DCs may result in the generation of IL‐10‐producing anergic T cells able to suppress effector T‐cell functions (Jonuleit et al., 2000). The generation of Treg cell require production of IL‐10 by immature DC (Levings et al., 2005). Similar results have also been obtained in vivo in humans; injection of immature DCs pulsed with the influenza peptide induced the differentiation of peptide‐specific IL‐10‐producing T cells, and the disappearance of influenza‐specific effector CD8þ T cells. In contrast, a single injection of peptide‐pulsed mature DCs led to rapid expansion of specific T lymphocytes (Dhodapkar et al., 2001). Moreover, as mentioned previously, there are semimaturation stimuli, such as TNFa, that can induce a particular population of semimature DCs, able to sustain the peripheral differentiation of Treg cells. Repetitive injections of bone‐marrow‐derived TNFa‐activated DCs prevent the development of experimental autoimmune encephalomyelitis (EAE) by inducing IL‐10‐producing Treg cells (Menges et al., 2002). Thus, T‐cell interaction with antigen‐presenting immature or semimature DC could induce peripheral differentiation of Treg lymphocytes. Besides a role in peripheral Treg‐cell differentiation, DCs have been shown capable of influencing the function of thymus‐derived CD4þCD25þ Treg cells (Guiducci et al., 2005; Steinman et al., 2003). Mature DCs are, indeed, able to induce expansion of CD4þCD25þ Treg cells both in vitro and in vitro in the presence of a specific antigen or in the presence of IL‐2 (Yamazaki et al., 2003). Once expanded, following the DC encounter, the Treg cells show more of an increased suppressive activity in vitro than their ex vivo counterpart (Tarbell et al., 2004). Concerning the mechanism by which DCs would be able to control Treg‐ cell functions, it has been hypothesized that DC‐derived IL‐2 could play an important role (Malek and Bayer, 2004) because it has been observed that IL‐2 is necessary for Treg‐cell functionality. In this experiment, IL‐2‐ or CD25‐ deficient T cells were transferred in mice who harbor a monoclonal myelin basic protein (MBP)‐specific ab T‐cell repertoire and spontaneously develop

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EAE (Lafaille et al., 1994). Only IL‐2‐deficient T cells protected from the disease, whereas CD25‐negative lymphocytes did not, indicating that IL‐2 is not required for the generation of Treg cells in the thymus but is necessary for Treg cells in the periphery to exert their functions. Finally, DCs have been shown to be involved in the maintenance of CD4þCD25þ cell homeostasis through a mechanism involving cell–cell contact, CD40–CD40L interaction, and IL‐2 production (Guiducci et al., 2005). 6. Conclusions DCs are key regulators of immune responses. They react to infectious agents undergoing a complex reprogramming of their functions. On one hand, DCs are programmed to react to different microorganisms modulating a constant core of genes. On the other hand, they show a functional plasticity in their maturation process, which depends on the nature of the perturbation and which permits to arise unique and efficient immune responses for each pathogen. Different DC functions are segregated in time and in space, allowing these cells to control both innate and adaptive immune responses. Acknowledgments This work was supported by grants from AIRC (Italian Association Against Cancer), The European Commission 6th Framework Program (contracts DC VACC LSHB‐CT‐2003–503037, DC THERA LSHB‐CT‐2004–512074, and MICROBAN MRTN‐CT‐2003–504227), The Italian Ministry of Education and Research (Programs FIRB and COFIN), The Italian Ministry of Health, and The Foundation Cariplo.

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