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Antigen delivery by dendritic cells
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International Journal of Medical Microbiology 294 (2004) 337–344 www.elsevier.de/ijmm
REVIEW
Antigen delivery by dendritic cells Heidrun Moll* Institute for Molecular Biology of Infectious Diseases, University of Wurzburg, Rontgenring 11, D-97070 Wurzburg, Germany . . . Received 10 March 2004; accepted 15 March 2004
Abstract Dendritic cells (DC) link the innate and adaptive arms of the immune system and thus orchestrate the immune response to pathogens. A novel immune intervention strategy to control infectious diseases is based on the use of the potent immunostimulatory properties of DC for vaccination and immunotherapy. Recent advances in our understanding of DC biology and the molecular mechanisms by which DC instruct the development of an appropriate immune response to microorganisms provide means for DC-based approaches to manipulate the immune system. In experimental systems, DC vaccination has been documented to mediate protection against a wide spectrum of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens. The protocols for the generation, stimulation and antigen loading of DC are being optimized, and methods for DC targeting in situ are likely to become available soon, thus paving the way for clinical applications of DC-based vaccines. r 2004 Elsevier GmbH. All rights reserved. Keywords: Vaccination; Dendritic cells; T cells; Cytokines; Microbial pathogens
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Pathogen recognition by dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Pathogens trigger the maturation of dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Dendritic cells control the immune response to infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 The role of dendritic cells in classical vaccine approaches and DNA vaccination . . . . . . . . . . . . . . . . . . . . . . . 340 Novel immune intervention strategies based on dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 Immunological parameters determining the efficiency of dendritic cell-based vaccination . . . . . . . . . . . . . . . . . . . 341 Approaches to enhance the efficacy of dendritic cell-based vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
*Tel.: +49-931-31-2627; fax: +49-931-31-2578. E-mail address: [email protected] (H. Moll). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.03.003
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Introduction Effective and durable immunity against the diverse types of microbial pathogens that enter our body relies on the co-ordinated action of components of the innate and the adaptive immune system. The specific immune response is driven by antigen-presenting cells that are located in peripheral tissues and thus link the innate arm with the cellular and humoral arms of the immune system (Moll, 2003). Both macrophages and dendritic cells (DC) can process and present microbial antigens to T cells, but have distinct and complementary functions in the regulation of the resulting immune response and the resolution of infection. Macrophages are professional phagocytes and, after appropriate activation, the most important effector cells for pathogen clearance. DC, on the other hand, express high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules and have the unique ability to stimulate resting T cells. Moreover, DC instruct the development of pathogen-specific CD4+ T helper (Th) cell subsets. This Th cell polarization into Th1 and Th2 cells is critical for balancing the immune response to microorganisms and must be controlled tightly. A novel approach to vaccination against infectious diseases and therapeutic immune intervention is to exploit DC as ‘nature’s adjuvant’ in order to induce pathogen-specific T cells directly in vivo. Such a strategy may be particularly useful in cases of infectious diseases for which conventional therapies have failed or are still not available. This review article focuses on recent findings elucidating the instructive role of DC in the development of an appropriate immune response and highlights the emerging reports on DC-based vaccination against infections that offer new perspectives for manipulations of the immune system for clinical benefit.
The different DC subpopulations may vary in their abilities to sense pathogens because they express distinct sets of receptors for the detection of conserved molecular structures of microorganisms, known as pathogen-associated molecular patterns (PAMPs) (Janeway and Medzhitov, 2002). These microbial structures are recognized by members of the mammalian Toll-like receptor (TLR) family, homologues of Drosophila Toll, and by C-type lectins expressed on the surface of DC. Several different TLR have been identified that discriminate between characteristic microbial cell wall components, such as lipoprotein, flagellin, doublestranded RNA, lipopolysaccharide (LPS) or bacterial DNA (Takeda et al., 2003). The engagement of TLR on DC provides information about the invading pathogen that is conveyed by intracellular signaling cascades and leads to the activation of DC and induction of inflammatory cytokines. On the other hand, C-type lectins bind to specific carbohydrate structures on microorganisms, and this interaction leads to internalization of pathogens for degradation in lysosomal compartments and enhancement of antigen processing and presentation by DC (Figdor et al., 2002). DC subpopulations differ in their expression of TLR and C-type lectins and are thus specialized to respond to distinct pathogens, depending on their tissue localization and activation status. High levels of C-type lectins are expressed by immature DC in the skin or mucosal tissues, whereas their expression is often reduced after DC maturation. There are also variations in the TLR expression profiles of myeloid and plasmacytoid DC. Human plasmacytoid DC express TLR7 and TLR9, and respond to CpG-containing oligonucleotides, but lack TLR4 and do not respond to LPS. On the other hand, human myeloid DC express TLR2, TLR4 and TLR6. Murine myeloid DC also express TLR9.
Pathogens trigger the maturation of dendritic cells Pathogen recognition by dendritic cells The DC system comprises a network of different subpopulations that are phenotypically and functionally heterogeneous (Shortman and Liu, 2002). The functional properties of a given DC subset do not appear to be fixed, but can be modified by signals from the microenvironment, thus permitting a striking plasticity in response to different microbes (de Jong et al., 2002; Manickasingham et al., 2003). In humans, myeloid DC and plasmacytoid DC have been described. Murine DC express the CD11c integrin and can be distinguished on the basis of CD8a expression. A third subset of murine CD11clow DC has plasmacytoid morphology.
In the course of an infection, pathogen encounter causes the maturation of DC not only directly, but also indirectly via the broad spectrum of inflammationassociated factors released in the affected tissues (Josien et al., 2000). Furthermore, DC can be activated by necrotic cells (Gallucci et al., 1999). The DC maturation program is associated with profound changes in the phenotypic and functional characteristics of the cells. Immature DC in peripheral tissues, such as the skin and the mucosal linings of the gastrointestinal, respiratory and urogenital tract, are highly specialized for antigen uptake (Blank et al., 1993; Wick, 2002). During maturation, they lose this property,
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but up-regulate MHC class II, co-stimulatory and adhesion molecules to allow for optimal antigen presentation (Flohe! et al., 1997; The! ry and Amigorena, 2001). Interestingly, in a process referred to as cross-presentation, DC are capable of targeting exogenous antigens simultaneously to MHC class I and class II processing pathways for the induction of responses mediated by CD8+ and CD4+ T cells (Melief, 2003). The expression of inflammatory chemokine receptors, such as CCR1, CCR2, CCR5 and CCR6, and the responsiveness to their ligands is down-regulated during DC maturation. At the same time, DC up-regulate surface expression of CCR7 which is essential for the migration of DC from peripheral tissues to the T cell areas of draining lymph nodes (McColl, 2002). As CCR7 also mediates the homing of T cells to lymphoid organs, this chemokine receptor is critical for the encounter of antigen-delivering DC and specific T cells. Formation of the immunological synapse, a specialized area of contact between DC and T cells, results in sustained T cell receptor signaling and triggers T cell proliferation and differentiation into effector cells (Lanzavecchia and Sallusto, 2001).
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Dendritic cells control the immune response to infections DC integrate microbial and inflammatory stimuli and direct the selective development of polarized Th cell responses (Fig. 1). Elucidating the mechanisms by which DC instruct the appropriate type of Th cell response is a major focus of the present research on DC immunobiology. It has been suggested that different DC subsets have an intrinsic tendency to promote either a Th1 or a Th2 response (Moser and Murphy, 2000). However, this rather simplistic concept has been challenged by a number of recent reports indicating that there is a high degree of flexibility in the ability of a given DC subpopulation to respond to different microbes (Kapsenberg, 2003; Sher et al., 2003). The findings suggest that the type of DC stimulus is a critical factor leading to DC-mediated polarization of the Th cell response. This notion is supported by the observation that certain products of bacteria and helminths typically triggering Th1 or Th2 responses in vivo stimulate DC to induce the development of Th1 or Th2 cells, respectively (de Jong et al., 2002; Whelan et al., 2000). An essential aspect of the ability of DC to tailor the antimicrobial immune response is their potential to
Fig. 1. Th cell polarization by DC is influenced by the type of signals that are recognized. DC express TLR and C-type lectins to sense pathogens or their components (P), as well as receptors for inflammatory factors (Ri) released in the infected tissue. Internalization of microbial molecules results in antigen processing and presentation by MHC class II molecules to Th cells (signal 1). The co-stimulatory signal 2, mainly mediated by the interaction of CD80 and CD86 on DC with CD28 on T cells, and the expression of cell adhesion molecules and CCR by DC is triggered by recognition of PAMPs and inflammatory signals. In addition, microbial and inflammatory stimuli trigger the release of cytokines by DC that promote the development of either Th1 or Th2 cells.
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release different cytokines depending on the type of microbial signal that is recognized. For example, components from Toxoplasma and Mycobacterium were shown to induce IL-12 release by DC, whereas zymosan led to production of IL-10 (Edwards et al., 2002). IL-12 plays a dominant role in the development of Th1 cells, and the Th2 cell effector choice may be a default that occurs in the absence of IL-12. The production of IL-10 by DC may lead to tolerance in several ways: it has a suppressive effect on T cells and can induce the differentiation of regulatory T cells. Moreover, IL-10 can decrease the functional activities of DC or make them tolerogenic. There is evidence that DC also have the potential to discriminate between different forms of the same pathogen, resulting in the induction of different Th cell subsets. The yeast form of Candida albicans as well as Aspergillus fumigatus conidia elicit a protective Th1 response via stimulation of IL-12 secretion by DC, whereas the hyphal forms of both fungi inhibit IL-12 production, or induce IL-4 and IL-10 expression by DC, and prime a non-protective Th2 response (Bozza et al., 2002; d’Ostiani et al., 2000). Moreover, LPS molecules from different strains of bacteria mediate different Th cell-polarizing signals: LPS from Escherichia coli was shown to induce IL-12 production by DC and Th1 development, while LPS from Porphyromonas gingivalis did not (Pulendran et al., 2001). The recent advances in our understanding of the mechanisms by which DC translate information from the innate to the adaptive immune system and shape the resulting response to pathogens may be of great value for the development of novel immune intervention strategies. Valuable proofs of concepts of such DCbased vaccination protocols have already been obtained in the immunotherapy of cancer patients (Schuler et al., 2003).
The role of dendritic cells in classical vaccine approaches and DNA vaccination DC are highly specialized in antigen presentation via the MHC class I pathway, which is required for the activation of CD8+ cytotoxic T lymphocytes, and the MHC class II pathway, which forms structures to be recognized by CD4+ Th cells (Guermonprez et al., 2002). In addition, DC express members of the nonpolymorphic CD1 family, which are able to present microbial lipid and glycolipid antigens, such as mycobacterial cell wall components, to conventional T cells, a subset of gdT cells and natural killer T cells. Selectively delivering antigens to DC in situ, combined with administration of a DC activation stimulus, increases the efficiency in T cell activation by several orders of
magnitude (Hawiger et al., 2001). Thus, the induction of immunity by DC requires the uptake and processing of antigens as well as signals that promote DC maturation. Some existing vaccine approaches may profit from the excellent adjuvant properties of DC. Attenuated organisms in the smallpox and measles vaccines infect DC and give rise to dead cells that can be captured and processed by other DC. The uptake of necrotic cells induces DC maturation, with the release of heat shock proteins being a relevant stimulus. Moreover, there is clear evidence that DC play a key role in the induction of immune responses after DNA vaccination (Gurunathan et al., 2000). The mechanisms involve direct transfection of small numbers of DC with the DNA construct and stimulation of additional DC via TLR9, which recognizes bacterial oligonucleotide motifs.
Novel immune intervention strategies based on dendritic cells DC are strategically located at epithelial barriers where pathogens gain access to their host. Upon internalization of microorganisms and their transport to secondary lymphoid organs, matured DC present a profile of processed antigens of the pathogen to naive T cells, thus initiating the specific immune response. Furthermore, mature DC are able to retain microbial antigens in immunogenic form for prolonged periods, due to the increased stability of MHC–peptide complexes, and may thus allow the sustained stimulation of a small pool of specific T cells that maintain immunity (Moll et al., 1995). DC have also been demonstrated to induce T cell-dependent immunoglobulin secretion and isotype switching in response to bacterial protein and polysaccharide antigens (Colino et al., 2002). Thus, DC carrying microbial antigens are remarkably efficient in inducing both humoral and cell-mediated immune responses, and there is growing literature indicating that DC can be exploited for the elicitation of protective immunity against infections. DC-based vaccination protocols were shown to mediate protection against a wide spectrum of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens (Table 1). For example, DC vaccines induce protective immunity against bacterial infections caused by Borrelia burgdorferi (Mbow et al., 1997), Chlamydia trachomatis (Su et al., 1998), Mycobacterium tuberculosis (Demangel et al., 1999), or Bordetella pertussis (GeorgeChandy et al., 2001), against viral infections caused by lymphocytic choriomeningitis virus (Ludewig et al., ! 1998), influenza virus (Lopez et al., 2000) or genital . et al., 2001) and against herpes simplex virus (Schon fungal and parasitic infections caused by C. albicans (d’Ostiani et al., 2000), Toxoplasma gondii (Bourguin
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Table 1. Dendritic cell-based vaccination mediates protection against various infectious agents Pathogen
Antigen used for DC pulsing
Type of protective immune response
References
Borrelia burgdorferi Chlamydia trachomatis Mycobacterium tuberculosis Bordetella pertussis Pseudomonas aeruginosa Listeria monocytogenes Leishmania major
Live spirochetes Heat-killed organisms Live M. bovis BCG Heat-killed organisms Live or heat-killed organisms Listeriolysin peptide Lysate or recombinant antigen Sonicate Heat-killed organisms Live yeasts LCMV peptide Live organisms Killed virus or HSV peptides Chemically inactivated SIV
IgG and IgM production Th1 cells CD8+ T cells, Th1 cells IgG and IgA production CD4+ T cells CD8+ T cells Th1 cells
Mbow et al. (1997) Su et al. (1998) Demangel et al. (1999) George-Chandy et al. (2001) Worgall et al. (2001) Hamilton and Harty (2002) Floh!e et al. (1998), Berberich et al. (2003) Bourguin et al. (1998) Ahuja et al. (1999) d’Ostiani et al. (2000) Ludewig et al. (1998) ! Lopez et al. (2000) . et al. (2001) Schon Lu et al. (2003)
Toxoplasma gondii Leishmania donovani Candida albicans LCMV Influenza virus Herpes simplex virus SIV
IgG production, Th1 cells Th1 cells Th1 cells CD8+ T cells CD8+ T cells, Th1 cells Th1 cells CD8+ T cells, Ig production
DC=dendritic cells, HSV=herpes simplex virus, LCMV=lymphocytic choriomeningitis virus, and SIV=simian immunodeficiency virus.
et al., 1998), Leishmania major (Flohe! et al., 1998; Berberich et al., 2003), or Leishmania donovani (Ahuja et al., 1999). Notably, DC vaccines can also promote the control of disease in already infected hosts. Therapeutic vaccination with DC pulsed with inactivated simian immunodeficiency virus (SIV) elicits effective cellular and humoral immune responses against SIV, allowing the control of SIV replication and the reduction of viral DNA and RNA levels in SIV-infected macaques (Lu et al., 2003).
Immunological parameters determining the efficiency of dendritic cell-based vaccination The immunological mechanisms underlying the ability of DC to induce protective immune responses in vivo have been studied in several infectious disease models. Exposure to microbial antigen in vitro induces high levels of IL-12 production by DC, and this feature may play a major role in the development of protective immunity associated with Th1 cells (Berberich et al., 2003; Bourguin et al., 1998; d’Ostiani et al., 2000; Flohe! ! et al., 1998; Lopez et al., 2000; Su et al., 1998). Furthermore, the expression of chemokines and chemokine receptors, mediating DC migration to lymphatic tissues and the recruitment of T cells, has been demonstrated to correlate with the protective immunizing capabilities of antigen-pulsed DC (Shaw et al., 2001). The preferential homing of DC to a particular lymphoid organ strongly influences the quality of the resulting immune response. Thus, the efficacy of DCbased vaccination also depends on the route of immunization. For example, i.v. administration of
antigen-pulsed DC was shown to be required for the induction of protection against B. pertussis, L. major and herpes simplex virus (George-Chandy et al., 2001; . et al., 2001). Flohe! et al., 1998; Schon
Approaches to enhance the efficacy of dendritic cell-based vaccination DC may represent the key to the development of novel vaccination approaches that mimic the course of natural immune responses or trigger de novo responses that have been ignored or suppressed. To optimize DC vaccination, it will be important to design strategies for appropriate activation of DC, improved antigen delivery to DC and in situ targeting of DC. There is increasing evidence for the profound influence of microbial components and inflammatory factors on DC phenotype and function, suggesting that the type of DC stimulus determines the capacity of DC to instruct an appropriate immune response. In fact, capture of microbial antigen by DC in the absence of concurrent activation may silence immunity in an antigen-specific manner (Hawiger et al., 2001). Using the model of murine leishmaniasis, it has been demonstrated that immunization of susceptible mice with a single dose of antigen-pulsed DC that additionally had been activated ex vivo with CpG motifs, a TLR9 ligand, mediated complete and durable protection against Leishmania parasites (Ram!ırez-Pineda et al., 2004). The cytokine interferon-a has also been used to promote DC activation (Le Bon et al., 2001). The efficacy of DCbased immune intervention may also be increased by genetic engineering of DC to overexpress the
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Th1-inducing cytokine IL-12 (Ahuja et al., 1999) or to down-regulate the expression of IL-10 which is known to have a negative effect on the development of Th1 responses (Igietseme et al., 2000). Notably, IL-12engineered DC pulsed with antigen were also effective in treating an established infection, thus providing a valuable tool for immunotherapy of infectious diseases. Another important variable that impacts on DCmediated T cell priming is the migration of mature DC from the injection site to the draining lymph nodes. It has been demonstrated that DC migration can be increased by tissue conditioning with inflammatory stimuli (Mart!ın-Fontecha et al., 2003). The choice of antigen is of substantial importance for the efficacy of vaccination. The antigen preparation should be molecularly defined and it should be possible to manufacture it in a safe and reproducible manner. DC pulsed with recombinant Leishmania peptides were able to mediate high levels of protection against leishmaniasis, demonstrating that the development of a DC-based subunit vaccine is feasible (Berberich et al., 2003). Transfection of DC with DNA plasmids or RNA derived from pathogens may represent a useful strategy to increase the availability of protective antigens and enhance the in vivo priming capacity of DC. This approach has been used successfully for the induction of antitumor immunity and has also been reported to confer resistance against fungal infection: DC transfected with RNA from C. albicans yeast, but not hyphae, were able to mediate protection against Candida (Bacci et al., 2002). It is not feasible to use ex vivo antigen-loaded DC for first-line prophylactic vaccination of large numbers of humans. Therefore, DC-based therapeutic vaccination will be more useful. It will probably be restricted to cases in which conventional therapies have failed and represents a new tool for enhancing or restoring the antimicrobial immune response in immunocompromised patients, such as bone marrow transplant recipients (Grazziutti et al., 2001). Ideally, antigen should be targeted directly to DC in situ. The approaches currently being explored employ DC-specific surface molecules, such as DEC-205 (CD205) (Hawiger et al., 2001) or C-type lectins (Figdor et al., 2002), DC-specific promoters (Ross et al., 2003), synthetic TLR ligands that specifically interact with DC subsets (Maurer et al., 2002) and bacterial non-live vaccine delivery systems (Haicheur et al., 2003). Another strategy involves the use of exosomes, small vesicles of endosomal origin which are secreted by DC and may be useful to induce or modulate immune responses, as they can be loaded with antigens or additional stimulatory molecules (The! ry et al., 2002). The properties of exosomes derived from DC reflect the maturation state and subtype of DC with regard to the expression of MHC products, signal transduction molecules, lectin-
binding proteins, T cell-stimulatory molecules and chaperons, such as heat-shock proteins. Passive vaccine delivery by transcutaneous immunization, using a patch or other topical formulations, may be another useful approach to target antigen and adjuvant to DC in the skin. It has been demonstrated that application of a patch containing heat-labile enterotoxin from E. coli results in systemic antibody responses via activation of epidermal Langerhans cells (Glenn et al., 2000).
Concluding remarks During the last decade, considerable insights have been gained into the mechanisms by which DC induce and regulate the fate of immune responses to microbial pathogens. The development of protocols for generating large numbers of DC in vitro from precursor cells has opened new perspectives for DC-based immune intervention strategies. Data from infectious disease models are highly encouraging and results from completed clinical trials with cancer patients demonstrate the safety of DC vaccines. Several important issues have to be addressed. The selection of microbial antigens that are suitable for DC targeting needs careful consideration. Genetic engineering of immunogenic recombinant proteins may enhance DC-mediated antigen processing and presentation to the appropriate T cell populations. It remains to be established whether DC loading with proteins or RNA, or transfection with DNA, will be the most efficient strategy to target microbial antigens to DC and enable the activation and maintenance of T cell responses. Another critical parameter is the choice of activation signal enabling DC to induce a polarized T cell response that is appropriate to control infection with a given pathogen. Moreover, the type of DC population to be generated for vaccination or to be targeted in tissues must be taken into account. Finally, it should be considered that DC must reach secondary lymphoid organs to elicit an effective T cell response. To this end, it will be important to define cytokines and chemokines that may be used for conditioning the injection sites.
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ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC. J. Immunol. 172, 6281–6289. Ross, R., Sudowe, S., Beisner, J., Ross, X.L., LudwigPortugall, I., Steitz, J., Tuting, T., Knop, J., Reske-Kunz, A.B., 2003. Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin. Gene Ther. 10, 1035–1040. . . Schon, E., Harandi, A.M., Nordstrom, I., Holmgren, J., Eriksson, K., 2001. Dendritic cell vaccination protects mice against lethality caused by genital herpes simplex virus type 2 infection. J. Reprod. Immunol. 50, 87–104. Schuler, G., Schuler-Thurner, B., Steinman, R.M., 2003. The use of dendritic cells in cancer immunotherapy. Curr. Opin. Immunol. 15, 138–147. Shaw, J.H., Grund, V.R., Durling, L., Caldwell, H.D., 2001. Expression of genes encoding Th1 cell-activating cytokines and lymphoid homing chemokines by Chlamydia-pulsed dendritic cells correlates with protective immunizing efficacy. Infect. Immun. 69, 4667–4672. Sher, A., Pearce, E., Kaye, P., 2003. Shaping the immune response to parasites: role of dendritic cells. Curr. Opin. Immunol. 15, 421–429. Shortman, K., Liu, Y.J., 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2, 151–161. Su, H., Messer, R., Whitmire, W., Fischer, E., Portis, J.C., Caldwell, H.D., 1998. Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable chlamydiae. J. Exp. Med. 188, 809–818. Takeda, K., Kaisho, T., Akira, S., 2003. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376. Th!ery, C., Amigorena, S., 2001. The cell biology of antigen presentation in dendritic cells. Curr. Opin. Immunol. 13, 45–51. Th!ery, C., Zitvogel, L., Amigorena, S., 2002. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579. Whelan, M., Harnett, M.M., Houston, K.M., Patel, V., Harnett, W., Rigley, K.P., 2000. A filarial nematodesecreted product signals dendritic cells to acquire a phenotype that drives development of Th2 cells. J. Immunol. 164, 6453–6460. Wick, M.J., 2002. The role of dendritic cells during Salmonella infection. Curr. Opin. Immunol. 14, 437–443. Worgall, S., Kikuchi, T., Singh, R., Martushova, K., Lande, L., Crystal, R.G., 2001. Protection against pulmonary infection with Pseudomonas aeruginosa following immunization with P. aeruginosa-pulsed dendritic cells. Infect. Immun. 69, 4521–4527.
International Journal of Medical Microbiology 294 (2004) 337–344 www.elsevier.de/ijmm
REVIEW
Antigen delivery by dendritic cells Heidrun Moll* Institute for Molecular Biology of Infectious Diseases, University of Wurzburg, Rontgenring 11, D-97070 Wurzburg, Germany . . . Received 10 March 2004; accepted 15 March 2004
Abstract Dendritic cells (DC) link the innate and adaptive arms of the immune system and thus orchestrate the immune response to pathogens. A novel immune intervention strategy to control infectious diseases is based on the use of the potent immunostimulatory properties of DC for vaccination and immunotherapy. Recent advances in our understanding of DC biology and the molecular mechanisms by which DC instruct the development of an appropriate immune response to microorganisms provide means for DC-based approaches to manipulate the immune system. In experimental systems, DC vaccination has been documented to mediate protection against a wide spectrum of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens. The protocols for the generation, stimulation and antigen loading of DC are being optimized, and methods for DC targeting in situ are likely to become available soon, thus paving the way for clinical applications of DC-based vaccines. r 2004 Elsevier GmbH. All rights reserved. Keywords: Vaccination; Dendritic cells; T cells; Cytokines; Microbial pathogens
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Pathogen recognition by dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Pathogens trigger the maturation of dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Dendritic cells control the immune response to infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 The role of dendritic cells in classical vaccine approaches and DNA vaccination . . . . . . . . . . . . . . . . . . . . . . . 340 Novel immune intervention strategies based on dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 Immunological parameters determining the efficiency of dendritic cell-based vaccination . . . . . . . . . . . . . . . . . . . 341 Approaches to enhance the efficacy of dendritic cell-based vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
*Tel.: +49-931-31-2627; fax: +49-931-31-2578. E-mail address: [email protected] (H. Moll). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.03.003
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Introduction Effective and durable immunity against the diverse types of microbial pathogens that enter our body relies on the co-ordinated action of components of the innate and the adaptive immune system. The specific immune response is driven by antigen-presenting cells that are located in peripheral tissues and thus link the innate arm with the cellular and humoral arms of the immune system (Moll, 2003). Both macrophages and dendritic cells (DC) can process and present microbial antigens to T cells, but have distinct and complementary functions in the regulation of the resulting immune response and the resolution of infection. Macrophages are professional phagocytes and, after appropriate activation, the most important effector cells for pathogen clearance. DC, on the other hand, express high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules and have the unique ability to stimulate resting T cells. Moreover, DC instruct the development of pathogen-specific CD4+ T helper (Th) cell subsets. This Th cell polarization into Th1 and Th2 cells is critical for balancing the immune response to microorganisms and must be controlled tightly. A novel approach to vaccination against infectious diseases and therapeutic immune intervention is to exploit DC as ‘nature’s adjuvant’ in order to induce pathogen-specific T cells directly in vivo. Such a strategy may be particularly useful in cases of infectious diseases for which conventional therapies have failed or are still not available. This review article focuses on recent findings elucidating the instructive role of DC in the development of an appropriate immune response and highlights the emerging reports on DC-based vaccination against infections that offer new perspectives for manipulations of the immune system for clinical benefit.
The different DC subpopulations may vary in their abilities to sense pathogens because they express distinct sets of receptors for the detection of conserved molecular structures of microorganisms, known as pathogen-associated molecular patterns (PAMPs) (Janeway and Medzhitov, 2002). These microbial structures are recognized by members of the mammalian Toll-like receptor (TLR) family, homologues of Drosophila Toll, and by C-type lectins expressed on the surface of DC. Several different TLR have been identified that discriminate between characteristic microbial cell wall components, such as lipoprotein, flagellin, doublestranded RNA, lipopolysaccharide (LPS) or bacterial DNA (Takeda et al., 2003). The engagement of TLR on DC provides information about the invading pathogen that is conveyed by intracellular signaling cascades and leads to the activation of DC and induction of inflammatory cytokines. On the other hand, C-type lectins bind to specific carbohydrate structures on microorganisms, and this interaction leads to internalization of pathogens for degradation in lysosomal compartments and enhancement of antigen processing and presentation by DC (Figdor et al., 2002). DC subpopulations differ in their expression of TLR and C-type lectins and are thus specialized to respond to distinct pathogens, depending on their tissue localization and activation status. High levels of C-type lectins are expressed by immature DC in the skin or mucosal tissues, whereas their expression is often reduced after DC maturation. There are also variations in the TLR expression profiles of myeloid and plasmacytoid DC. Human plasmacytoid DC express TLR7 and TLR9, and respond to CpG-containing oligonucleotides, but lack TLR4 and do not respond to LPS. On the other hand, human myeloid DC express TLR2, TLR4 and TLR6. Murine myeloid DC also express TLR9.
Pathogens trigger the maturation of dendritic cells Pathogen recognition by dendritic cells The DC system comprises a network of different subpopulations that are phenotypically and functionally heterogeneous (Shortman and Liu, 2002). The functional properties of a given DC subset do not appear to be fixed, but can be modified by signals from the microenvironment, thus permitting a striking plasticity in response to different microbes (de Jong et al., 2002; Manickasingham et al., 2003). In humans, myeloid DC and plasmacytoid DC have been described. Murine DC express the CD11c integrin and can be distinguished on the basis of CD8a expression. A third subset of murine CD11clow DC has plasmacytoid morphology.
In the course of an infection, pathogen encounter causes the maturation of DC not only directly, but also indirectly via the broad spectrum of inflammationassociated factors released in the affected tissues (Josien et al., 2000). Furthermore, DC can be activated by necrotic cells (Gallucci et al., 1999). The DC maturation program is associated with profound changes in the phenotypic and functional characteristics of the cells. Immature DC in peripheral tissues, such as the skin and the mucosal linings of the gastrointestinal, respiratory and urogenital tract, are highly specialized for antigen uptake (Blank et al., 1993; Wick, 2002). During maturation, they lose this property,
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but up-regulate MHC class II, co-stimulatory and adhesion molecules to allow for optimal antigen presentation (Flohe! et al., 1997; The! ry and Amigorena, 2001). Interestingly, in a process referred to as cross-presentation, DC are capable of targeting exogenous antigens simultaneously to MHC class I and class II processing pathways for the induction of responses mediated by CD8+ and CD4+ T cells (Melief, 2003). The expression of inflammatory chemokine receptors, such as CCR1, CCR2, CCR5 and CCR6, and the responsiveness to their ligands is down-regulated during DC maturation. At the same time, DC up-regulate surface expression of CCR7 which is essential for the migration of DC from peripheral tissues to the T cell areas of draining lymph nodes (McColl, 2002). As CCR7 also mediates the homing of T cells to lymphoid organs, this chemokine receptor is critical for the encounter of antigen-delivering DC and specific T cells. Formation of the immunological synapse, a specialized area of contact between DC and T cells, results in sustained T cell receptor signaling and triggers T cell proliferation and differentiation into effector cells (Lanzavecchia and Sallusto, 2001).
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Dendritic cells control the immune response to infections DC integrate microbial and inflammatory stimuli and direct the selective development of polarized Th cell responses (Fig. 1). Elucidating the mechanisms by which DC instruct the appropriate type of Th cell response is a major focus of the present research on DC immunobiology. It has been suggested that different DC subsets have an intrinsic tendency to promote either a Th1 or a Th2 response (Moser and Murphy, 2000). However, this rather simplistic concept has been challenged by a number of recent reports indicating that there is a high degree of flexibility in the ability of a given DC subpopulation to respond to different microbes (Kapsenberg, 2003; Sher et al., 2003). The findings suggest that the type of DC stimulus is a critical factor leading to DC-mediated polarization of the Th cell response. This notion is supported by the observation that certain products of bacteria and helminths typically triggering Th1 or Th2 responses in vivo stimulate DC to induce the development of Th1 or Th2 cells, respectively (de Jong et al., 2002; Whelan et al., 2000). An essential aspect of the ability of DC to tailor the antimicrobial immune response is their potential to
Fig. 1. Th cell polarization by DC is influenced by the type of signals that are recognized. DC express TLR and C-type lectins to sense pathogens or their components (P), as well as receptors for inflammatory factors (Ri) released in the infected tissue. Internalization of microbial molecules results in antigen processing and presentation by MHC class II molecules to Th cells (signal 1). The co-stimulatory signal 2, mainly mediated by the interaction of CD80 and CD86 on DC with CD28 on T cells, and the expression of cell adhesion molecules and CCR by DC is triggered by recognition of PAMPs and inflammatory signals. In addition, microbial and inflammatory stimuli trigger the release of cytokines by DC that promote the development of either Th1 or Th2 cells.
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release different cytokines depending on the type of microbial signal that is recognized. For example, components from Toxoplasma and Mycobacterium were shown to induce IL-12 release by DC, whereas zymosan led to production of IL-10 (Edwards et al., 2002). IL-12 plays a dominant role in the development of Th1 cells, and the Th2 cell effector choice may be a default that occurs in the absence of IL-12. The production of IL-10 by DC may lead to tolerance in several ways: it has a suppressive effect on T cells and can induce the differentiation of regulatory T cells. Moreover, IL-10 can decrease the functional activities of DC or make them tolerogenic. There is evidence that DC also have the potential to discriminate between different forms of the same pathogen, resulting in the induction of different Th cell subsets. The yeast form of Candida albicans as well as Aspergillus fumigatus conidia elicit a protective Th1 response via stimulation of IL-12 secretion by DC, whereas the hyphal forms of both fungi inhibit IL-12 production, or induce IL-4 and IL-10 expression by DC, and prime a non-protective Th2 response (Bozza et al., 2002; d’Ostiani et al., 2000). Moreover, LPS molecules from different strains of bacteria mediate different Th cell-polarizing signals: LPS from Escherichia coli was shown to induce IL-12 production by DC and Th1 development, while LPS from Porphyromonas gingivalis did not (Pulendran et al., 2001). The recent advances in our understanding of the mechanisms by which DC translate information from the innate to the adaptive immune system and shape the resulting response to pathogens may be of great value for the development of novel immune intervention strategies. Valuable proofs of concepts of such DCbased vaccination protocols have already been obtained in the immunotherapy of cancer patients (Schuler et al., 2003).
The role of dendritic cells in classical vaccine approaches and DNA vaccination DC are highly specialized in antigen presentation via the MHC class I pathway, which is required for the activation of CD8+ cytotoxic T lymphocytes, and the MHC class II pathway, which forms structures to be recognized by CD4+ Th cells (Guermonprez et al., 2002). In addition, DC express members of the nonpolymorphic CD1 family, which are able to present microbial lipid and glycolipid antigens, such as mycobacterial cell wall components, to conventional T cells, a subset of gdT cells and natural killer T cells. Selectively delivering antigens to DC in situ, combined with administration of a DC activation stimulus, increases the efficiency in T cell activation by several orders of
magnitude (Hawiger et al., 2001). Thus, the induction of immunity by DC requires the uptake and processing of antigens as well as signals that promote DC maturation. Some existing vaccine approaches may profit from the excellent adjuvant properties of DC. Attenuated organisms in the smallpox and measles vaccines infect DC and give rise to dead cells that can be captured and processed by other DC. The uptake of necrotic cells induces DC maturation, with the release of heat shock proteins being a relevant stimulus. Moreover, there is clear evidence that DC play a key role in the induction of immune responses after DNA vaccination (Gurunathan et al., 2000). The mechanisms involve direct transfection of small numbers of DC with the DNA construct and stimulation of additional DC via TLR9, which recognizes bacterial oligonucleotide motifs.
Novel immune intervention strategies based on dendritic cells DC are strategically located at epithelial barriers where pathogens gain access to their host. Upon internalization of microorganisms and their transport to secondary lymphoid organs, matured DC present a profile of processed antigens of the pathogen to naive T cells, thus initiating the specific immune response. Furthermore, mature DC are able to retain microbial antigens in immunogenic form for prolonged periods, due to the increased stability of MHC–peptide complexes, and may thus allow the sustained stimulation of a small pool of specific T cells that maintain immunity (Moll et al., 1995). DC have also been demonstrated to induce T cell-dependent immunoglobulin secretion and isotype switching in response to bacterial protein and polysaccharide antigens (Colino et al., 2002). Thus, DC carrying microbial antigens are remarkably efficient in inducing both humoral and cell-mediated immune responses, and there is growing literature indicating that DC can be exploited for the elicitation of protective immunity against infections. DC-based vaccination protocols were shown to mediate protection against a wide spectrum of infectious diseases caused by viral, bacterial, parasitic and fungal pathogens (Table 1). For example, DC vaccines induce protective immunity against bacterial infections caused by Borrelia burgdorferi (Mbow et al., 1997), Chlamydia trachomatis (Su et al., 1998), Mycobacterium tuberculosis (Demangel et al., 1999), or Bordetella pertussis (GeorgeChandy et al., 2001), against viral infections caused by lymphocytic choriomeningitis virus (Ludewig et al., ! 1998), influenza virus (Lopez et al., 2000) or genital . et al., 2001) and against herpes simplex virus (Schon fungal and parasitic infections caused by C. albicans (d’Ostiani et al., 2000), Toxoplasma gondii (Bourguin
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Table 1. Dendritic cell-based vaccination mediates protection against various infectious agents Pathogen
Antigen used for DC pulsing
Type of protective immune response
References
Borrelia burgdorferi Chlamydia trachomatis Mycobacterium tuberculosis Bordetella pertussis Pseudomonas aeruginosa Listeria monocytogenes Leishmania major
Live spirochetes Heat-killed organisms Live M. bovis BCG Heat-killed organisms Live or heat-killed organisms Listeriolysin peptide Lysate or recombinant antigen Sonicate Heat-killed organisms Live yeasts LCMV peptide Live organisms Killed virus or HSV peptides Chemically inactivated SIV
IgG and IgM production Th1 cells CD8+ T cells, Th1 cells IgG and IgA production CD4+ T cells CD8+ T cells Th1 cells
Mbow et al. (1997) Su et al. (1998) Demangel et al. (1999) George-Chandy et al. (2001) Worgall et al. (2001) Hamilton and Harty (2002) Floh!e et al. (1998), Berberich et al. (2003) Bourguin et al. (1998) Ahuja et al. (1999) d’Ostiani et al. (2000) Ludewig et al. (1998) ! Lopez et al. (2000) . et al. (2001) Schon Lu et al. (2003)
Toxoplasma gondii Leishmania donovani Candida albicans LCMV Influenza virus Herpes simplex virus SIV
IgG production, Th1 cells Th1 cells Th1 cells CD8+ T cells CD8+ T cells, Th1 cells Th1 cells CD8+ T cells, Ig production
DC=dendritic cells, HSV=herpes simplex virus, LCMV=lymphocytic choriomeningitis virus, and SIV=simian immunodeficiency virus.
et al., 1998), Leishmania major (Flohe! et al., 1998; Berberich et al., 2003), or Leishmania donovani (Ahuja et al., 1999). Notably, DC vaccines can also promote the control of disease in already infected hosts. Therapeutic vaccination with DC pulsed with inactivated simian immunodeficiency virus (SIV) elicits effective cellular and humoral immune responses against SIV, allowing the control of SIV replication and the reduction of viral DNA and RNA levels in SIV-infected macaques (Lu et al., 2003).
Immunological parameters determining the efficiency of dendritic cell-based vaccination The immunological mechanisms underlying the ability of DC to induce protective immune responses in vivo have been studied in several infectious disease models. Exposure to microbial antigen in vitro induces high levels of IL-12 production by DC, and this feature may play a major role in the development of protective immunity associated with Th1 cells (Berberich et al., 2003; Bourguin et al., 1998; d’Ostiani et al., 2000; Flohe! ! et al., 1998; Lopez et al., 2000; Su et al., 1998). Furthermore, the expression of chemokines and chemokine receptors, mediating DC migration to lymphatic tissues and the recruitment of T cells, has been demonstrated to correlate with the protective immunizing capabilities of antigen-pulsed DC (Shaw et al., 2001). The preferential homing of DC to a particular lymphoid organ strongly influences the quality of the resulting immune response. Thus, the efficacy of DCbased vaccination also depends on the route of immunization. For example, i.v. administration of
antigen-pulsed DC was shown to be required for the induction of protection against B. pertussis, L. major and herpes simplex virus (George-Chandy et al., 2001; . et al., 2001). Flohe! et al., 1998; Schon
Approaches to enhance the efficacy of dendritic cell-based vaccination DC may represent the key to the development of novel vaccination approaches that mimic the course of natural immune responses or trigger de novo responses that have been ignored or suppressed. To optimize DC vaccination, it will be important to design strategies for appropriate activation of DC, improved antigen delivery to DC and in situ targeting of DC. There is increasing evidence for the profound influence of microbial components and inflammatory factors on DC phenotype and function, suggesting that the type of DC stimulus determines the capacity of DC to instruct an appropriate immune response. In fact, capture of microbial antigen by DC in the absence of concurrent activation may silence immunity in an antigen-specific manner (Hawiger et al., 2001). Using the model of murine leishmaniasis, it has been demonstrated that immunization of susceptible mice with a single dose of antigen-pulsed DC that additionally had been activated ex vivo with CpG motifs, a TLR9 ligand, mediated complete and durable protection against Leishmania parasites (Ram!ırez-Pineda et al., 2004). The cytokine interferon-a has also been used to promote DC activation (Le Bon et al., 2001). The efficacy of DCbased immune intervention may also be increased by genetic engineering of DC to overexpress the
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Th1-inducing cytokine IL-12 (Ahuja et al., 1999) or to down-regulate the expression of IL-10 which is known to have a negative effect on the development of Th1 responses (Igietseme et al., 2000). Notably, IL-12engineered DC pulsed with antigen were also effective in treating an established infection, thus providing a valuable tool for immunotherapy of infectious diseases. Another important variable that impacts on DCmediated T cell priming is the migration of mature DC from the injection site to the draining lymph nodes. It has been demonstrated that DC migration can be increased by tissue conditioning with inflammatory stimuli (Mart!ın-Fontecha et al., 2003). The choice of antigen is of substantial importance for the efficacy of vaccination. The antigen preparation should be molecularly defined and it should be possible to manufacture it in a safe and reproducible manner. DC pulsed with recombinant Leishmania peptides were able to mediate high levels of protection against leishmaniasis, demonstrating that the development of a DC-based subunit vaccine is feasible (Berberich et al., 2003). Transfection of DC with DNA plasmids or RNA derived from pathogens may represent a useful strategy to increase the availability of protective antigens and enhance the in vivo priming capacity of DC. This approach has been used successfully for the induction of antitumor immunity and has also been reported to confer resistance against fungal infection: DC transfected with RNA from C. albicans yeast, but not hyphae, were able to mediate protection against Candida (Bacci et al., 2002). It is not feasible to use ex vivo antigen-loaded DC for first-line prophylactic vaccination of large numbers of humans. Therefore, DC-based therapeutic vaccination will be more useful. It will probably be restricted to cases in which conventional therapies have failed and represents a new tool for enhancing or restoring the antimicrobial immune response in immunocompromised patients, such as bone marrow transplant recipients (Grazziutti et al., 2001). Ideally, antigen should be targeted directly to DC in situ. The approaches currently being explored employ DC-specific surface molecules, such as DEC-205 (CD205) (Hawiger et al., 2001) or C-type lectins (Figdor et al., 2002), DC-specific promoters (Ross et al., 2003), synthetic TLR ligands that specifically interact with DC subsets (Maurer et al., 2002) and bacterial non-live vaccine delivery systems (Haicheur et al., 2003). Another strategy involves the use of exosomes, small vesicles of endosomal origin which are secreted by DC and may be useful to induce or modulate immune responses, as they can be loaded with antigens or additional stimulatory molecules (The! ry et al., 2002). The properties of exosomes derived from DC reflect the maturation state and subtype of DC with regard to the expression of MHC products, signal transduction molecules, lectin-
binding proteins, T cell-stimulatory molecules and chaperons, such as heat-shock proteins. Passive vaccine delivery by transcutaneous immunization, using a patch or other topical formulations, may be another useful approach to target antigen and adjuvant to DC in the skin. It has been demonstrated that application of a patch containing heat-labile enterotoxin from E. coli results in systemic antibody responses via activation of epidermal Langerhans cells (Glenn et al., 2000).
Concluding remarks During the last decade, considerable insights have been gained into the mechanisms by which DC induce and regulate the fate of immune responses to microbial pathogens. The development of protocols for generating large numbers of DC in vitro from precursor cells has opened new perspectives for DC-based immune intervention strategies. Data from infectious disease models are highly encouraging and results from completed clinical trials with cancer patients demonstrate the safety of DC vaccines. Several important issues have to be addressed. The selection of microbial antigens that are suitable for DC targeting needs careful consideration. Genetic engineering of immunogenic recombinant proteins may enhance DC-mediated antigen processing and presentation to the appropriate T cell populations. It remains to be established whether DC loading with proteins or RNA, or transfection with DNA, will be the most efficient strategy to target microbial antigens to DC and enable the activation and maintenance of T cell responses. Another critical parameter is the choice of activation signal enabling DC to induce a polarized T cell response that is appropriate to control infection with a given pathogen. Moreover, the type of DC population to be generated for vaccination or to be targeted in tissues must be taken into account. Finally, it should be considered that DC must reach secondary lymphoid organs to elicit an effective T cell response. To this end, it will be important to define cytokines and chemokines that may be used for conditioning the injection sites.
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