Balancing between immunity and tolerance: an interplay ... - CiteSeerX

Balancing between immunity and tolerance: an interplay between dendritic cells, regulatory T cells, and effector T cells Nathalie Cools,1 Peter Ponsaerts, Viggo F. I. Van Tendeloo, and Zwi N. Berneman Laboratory of Experimental Hematology, Faculty of Medicine, Antwerp University, Belgium, and Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Belgium

Abstract: Dendritic cells (DC), professional antigen-presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the main function of immature DC (iDC) in their in vivo steady-state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell-based therapies. J. Leukoc. Biol. 82: 1365–1374; 2007. Key Words: antigen-presenting cells stimulation 䡠 cytokines

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DC-based therapies

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INTRODUCTION Dendritic cells (DC) are a highly specialized population of white blood cells, also known as the “sentinels” of the immune system, that patrol the body to capture invading pathogens and certain malignant cells in order to induce efficient antimicrobial or antitumor T cell immune responses. However, more recently, DC are also regarded as the “police” of the immune system, that is, they actively maintain tolerance to self-antigens [1]. This review focuses on both, at first sight opposite, specialized functions of DC. 0741-5400/07/0082-1365 © Society for Leukocyte Biology

DISTINCT DC SUBSETS AND DIFFERENTIATION STAGES DC are continuously differentiated from CD34⫹ hematopoietic progenitor cells within the bone marrow [2] (Table 1). A myeloid progenitor cell can differentiate to 1) CD11c⫹CD1a⫹ immature DC that migrate into the skin epidermis and become Langerhans cells (LC) and 2) to CD11c⫹CD1a– immature DC that migrate into the skin dermis and various other tissues and become interstitial DC [3]. Interstitial DC reside in the skin dermis, airways, and intestine, in the interstitial spaces of many organs, lymphoid tissues, blood, and afferent lymphatics. Their widespread distribution underlines their sentinel function. In addition, CD14⫹ monocytes are also a source of DC precursors during physiological stress. Another subset of DC, plasmacytoid DC (pDC) originates from a lymphoid progenitor cell (CD11c-pDC precursor) in lymphoid organs. In humans, the lymphoid DC lineage shows remarkable characteristics that distinguish them from the myeloid DC lineage. Of importance, pDC lack the expression of myeloid markers CD13, CD14, and CD33, but they express high levels of CD123 [interleukin (IL)-3 receptor], CD4, and CD62 ligand (CD62L) [4, 5]. DC have a remarkable functional plasticity. Because of the unique biological function of each DC subset, it was proposed that a specific DC lineage determines the outcome of T cell contact, i.e., tolerance or immunity. To support this hypothesis, several studies in mice suggested that tolerogenic DC represent a specialized lineage. Kronin et al. [6, 7] showed that splenic CD8␣-DC (the mouse counterpart of human myeloid DC) induce strong proliferative responses in CD4⫹ T cells, while CD8␣⫹ lymphoid DC induce less strong proliferative responses. Moreover, these murine CD8␣⫹ DC induce Fasdependent apoptosis in vitro [8], indicating their tolerogenic properties. More recently, the same DC subset was also shown to induce peripheral self-tolerance to tissue antigens in vivo [9 –11]. However, the role of CD8␣⫹ DC in suppression of T cell responses has been challenged by several reports demonstrating that CD8␣⫹ DC can also produce high amounts of

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Correspondence: Laboratory of Experimental Hematology, Antwerp University Hospital (UZA), Wilrijkstraat 10, B-2650 Edegem, Belgium. E-mail: [email protected] Received March 16, 2007; revised July 12, 2007; accepted July 17, 2007. doi: 10.1189/jlb.0307166

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TABLE 1.

Different Subsets of Dendritic Cells CD34⫹ hematopoietic stem cell Myeloid progenitor cell

Phenotype CD11c CD1a CD123 Birbeck granules Factor XIIIa MHC class II Function molecules involved in antigen uptake, processing and presentation IL-10* IL-12* IFN-␣*

Lymphoid progenitor cell

Monocyte-derived DC

Langerhans cells

Interstitial DC

Plasmacytoid DC

⫹ ⫹/⫺ ⫺/low ⫹/⫺ ⫹/⫺ ⫹⫹

⫹ ⫹⫹ ⫺ ⫹⫹ ⫺ ⫹

⫹ ⫹/⫺ ⫺/low ⫹/⫺ ⫹⫹ ⫹

⫺ ⫹/⫺ ⫹⫹ ⫺ ⫺ ⫹

⫹⫹ ⫹ ⫹⫹ ⫹/⫺

⫹ ⫹ ⫺ ⫺

⫹/⫺ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫹⫹

* After application of danger signals.

IL-12 and can stimulate antiviral cytotoxic CD8⫹ T cells [12–14]. In humans, pDC are now believed to be the key effector cells in the early antiviral innate immune response by producing large amounts of type I interferon (IFN) upon viral infection [15, 16]. Therefore, pDC are sometimes referred to as IFN-producing cells (IPC) [17–19]. Moreover, production of type I IFN by pDC may be important to promote maturation of myeloid DC [16, 18]. Therefore, it is a more likely hypothesis that distinct DC developmental and activation stages play a role in the induction of tolerance vs. immunity, instead of simply the DC origin hypothesis. Both myeloid and plasmacytoid immature DC have been described as inducers of T cell tolerance [20 –23]. Immature DC are specialized in capturing antigens, that is, they efficiently take up pathogens, apoptotic cells and antigens from the environment by phagocytosis, macropinocytosis, or endocytosis, but they are considered to be unable to process and present these antigens efficiently to T cells [24]. Therefore, immature DC are believed to induce T cell anergy or regulatory T cells [25, 26]. Indeed, in a steady-state condition, DC remain immature and tissue-resident, expressing only small amounts of major histocompatibility complex (MHC) class II and costimulatory molecules, which leads to T cell anergy following T cell stimulation [27, 28]. Other studies, using direct DC targeting with antigen, also suggested that immature DC are the tolerogenic APC for self-antigens in vivo [29]. Furthermore, injection of immature DC in humans can be tolerogenic [30 – 32]. However, several reports describe the presence of both iDC and more mature DC in skin draining lymph nodes [33, 34]. Interestingly, studies on the migratory capacity of steadystate DC have demonstrated that some spontaneous maturation can occur (e.g., up-regulation of CCR7 expression [35]), allowing DC to present antigens and stimulate T cells. These studies favor the hypothesis that maturing DC, but not immature DC, might be involved in stimulating T cell tolerance [36, 37]. On the other hand, mature DC are considered to be immunogenic, mainly because of the marked up-regulation of MHC class II and of costimulatory molecules. These phenotypic changes 1366

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make DC potent inducers of T cell immunity. In contrast, stimulation of T cells by mature, antigen-presenting DC might also be required for tolerance induction as several reports have demonstrated the importance of mature DC in mediating T cell tolerance due to the induction of regulatory T cells (Treg) [38, 39]. The latter suggests that a difference in maturation stimulus results in different states of DC maturation leading to different T cell polarizing or tolerating immune effector functions. Indeed, molecules derived from bacterial or viral products such as lipopolysaccharide (LPS), CpG motifs, and double-stranded RNA (dsRNA), as well as proinflammatory cytokines [tumor necrosis factor (TNF)-␣, interferon (IFN)-␥] and T cell signals [CD40-ligand (CD40L)] will promote the production of cytokines by DC, such as IL-12p70 and IFN-␣ and therefore induce T helper 1 (Th1) responses [40, 41]. By contrast, anti-inflammatory molecules, such as IL-10, prostaglandin (PG)E2, and corticosteroids will inhibit DC maturation and cytokine production and therefore promote T helper 2 (Th2) [42– 44] or regulatory T cell (Treg) responses [45]. The role played by DC in the outcome of T cell stimulation (tolerance or immunity) is therefore more likely to depend on their activation or “licensing” status instead of their origin.

DENDRITIC CELLS INDUCING PERIPHERAL T CELL TOLERANCE Lack of MHC class II and of costimulation Antigen presentation in the absence of costimulation can lead to impaired clonal expansion and T cell anergy [27, 28, 46]. Immature DC residing in peripheral tissues, such as epidermal Langerhans cells (LC), fail to induce T cell activation because they express only moderate levels of MHC class II and no or very low levels of costimulatory molecules [47].

Peripheral deletion of autoreactive T cells There is increasing evidence that, under steady-state conditions, antigen presentation by iDC leads to T cell deletion and http://www.jleukbio.org

TABLE 2. Study (reference)

DC type

Jonuleit et al. (68) Dhodapkar et al. (30) Levings et al. (69) Yamazaki et al. (72) Yamazaki et al. (73) Verhasselt et al. (38) Lundqvist et al. (71) Banerjee et al. (74) Cools et al. (117)

DC-Expanded Regulatory T Cells

Host

Activation

iDC iDC iDC mDC mDC mDC

Human Human Human Murine Murine Human

mDC mDC iDC

Human Human Human

IL-10-producing CD4⫹ T cells IL-10-producing CD8⫹ T cells IL-10 and TGF-␤-producing Tr1 cells CD4⫹CD25⫹ Treg CD4⫹CD25⫹FOXP3⫹ Treg CD4⫹CD25⫹FOXP3⫹ Treg and IL-10 and TGF-␤-producing T cells IL-10-producing Tr1 cells CD4⫹CD25⫹FOXP3⫹ Treg IL-10 and TGF-␤-producing Treg

Antigen Allo-antigen IMP and KLH Allo-antigen OVA Allo-antigen No exogenous antigen PSA Allo-antigen or no antigen No exogenous antigen

IMP, influenza matrix protein; KLH, keyhole limpet hemocyanin; OVA, ovalbumin; PSA, prostate specific antigen.

peripheral tolerance [48]. Targeting of antigens to iDC through specific DC markers, such as 33D1 or DEC-205, can lead to T cell unresponsiveness [29, 49]. In a mouse model, Hawiger et al. [29] and others [50] reported antigen-specific deletion of T cells after exposure of responding T cells to iDC that were targeted in situ by an antibody against an endocytosis receptor, called DEC-205, coupled to the model antigen hen egg lysozyme (HEL). The treated mice became tolerant and were unable to be primed by injection of the peptide with the powerful Freund’s adjuvant. However, peripheral tolerance could be converted to immunity if the anti-DEC-205/HEL were given together with a DC activation stimulus, e.g., an activation CD40 antibody [51]. As discussed above, these results do not directly imply the induction of T cell tolerance by iDC. It cannot be excluded that DEC-205 targeting might have altered the in vivo maturation and activation status of some iDC toward a more mature DC with T cell deletion capacity, a subject that needs to be further investigated. T cell tolerance mediated by T cell deletion has also been reported in a tryptophan-depleted environment. Indoleamine 2,3-dioxygenase (IDO) is responsible for the degradation of tryptophan, an amino acid essential for cell proliferation. Moreover, tryptophan metabolites (kynurenine, 3-hydroxy-kynurenine and 3-hydroxy-anthranilinic acid) inhibit T cell proliferation by a cytotoxic action. In addition, a discrete subset of human DC that express IDO constitutively has recently been identified. A few IDO⫹ cells were detected in normal lymphoid tissues, and larger numbers were found in a proportion of tumor-draining lymph nodes, suggesting that these cells may represent a regulatory subset of APC [52, 53]. There is also some evidence that signaling trough CD95 (Fas ligation) may be involved in tolerance induction [8, 54]. Also, mDC stimulated with TNF-␣ and PGE2, can lead to tolerance induction by stimulating CD8⫹ T cell proliferation that results in T cell deletion [55]. More recently, it has been suggested that DC matured in the presence of TNF-␣ and PGE2 up-regulate the expression of IDO, possibly explaining their T cell deleting capacity [56, 57].

Inhibition of T helper type 1 cell proliferation Recently, the group of Cao et al. reported a new mechanism that may account for the regulatory properties of DC. In a mouse model, they showed that mature DC started proliferating

again when they were isolated and cultured on a monolayer of stromal cells derived from neonatal mouse spleen and further differentiated into a unique form of DC with regulatory effects on T cell responses [58]. The resulting regulatory DC (DCreg) was able to activate naive T cells, as demonstrated by the expression of the activation markers CD69 and CD25 and secretion of IFN-␥ and IL-2. In contrast, DCreg did not promote proliferation of the responder T cells. It was demonstrated that DCreg attract Th1 cells by the secretion of CXCR3 chemokine, IFN-␥ inducible protein (IP-10) [59], and that there inhibitory effects were mediated by NO production and did not involve DCreg-induced differentiation of CD4⫹ T cells into regulatory T cells. The authors claim that this might be a mechanism by which DC exert negative feedback control on the immune response in its late phase.

Induction of regulatory T cells Regulatory T cells (Treg) play an important role in controlling ongoing immune responses and silencing self-reactive T cells. Currently, various subsets of regulatory T cell populations (Table 2) have been identified and are subdivided based on their expression of cell surface markers, production of cytokines, and mechanisms of action [60]. In brief, naturally occurring thymic-derived CD4⫹CD25⫹ Treg are characterized by their constitutive expression of the transcription factor FOXP3, while antigen-induced Treg are mainly identified by their expression of immune-suppressive cytokines [IL-10 and transforming growth factor (TGF)-␤]. These inducible or adaptive Treg are also designated as Tr1. It has been documented that iDC in the steady state are able to prime Treg in order to maintain tolerance to self-antigens [24, 61, 62]. Moreover, iDC can also induce Treg specific for foreign peptides [30, 31] or CD4⫹ Treg specific for alloantigens [63, 64]. Indeed, when iDC pulsed with influenza matrix protein (IMP) and keyhole limpet hemocyanin (KLH), a general stimulator of CD4⫹ T cells, were injected, a decline in influenza-specific CD8⫹ IFN-␥-secreting T cells was observed, while peptide-specific IL-10-secreting T cells appeared [30]. In addition, Jonuleit et al. [63] demonstrated the capacity of iDC to induce alloantigen-specific Treg. Recently, a natural murine DC subset has been identified that induces the differentiation of Tr1 cells [65]. Although DC seem to trigger IL-10-producing Tr1 cells mainly at the imma-

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ture state, other reports show that phenotypically mature myeloid [66], as well as plasmacytoid [67] DC also induce T cell tolerance. Indeed, several reports indicate that mDC are also able to induce IL-10-producing Treg in humans [38, 68]. In addition, Yamazaki et al. [69, 70] reported in a mouse model that mature DC expand FOXP3⫹ CD4⫹CD25⫹ Treg. Recently, others [38, 71–73] also reported the induction of FOXP3⫹CD4⫹CD25⫹ Treg in humans after stimulation with mDC. Moreover, these FOXP3⫹-expressing Treg also expanded in myeloma patients after injection with DC matured with a cytokine-cocktail consisting of IL-1, IL-6, TNF-␣, and PGE2 [71]. Similarly, vaccination of melanoma patients with DC either loaded with synthetic peptides or with tumor lysates also induced increased frequencies of Treg [74]. In summary (Table 3), these data suggest that the former hypothesis according to which phenotypically immature DC induce T cell tolerance and phenotypically mature DC induce immunity is no longer valid. It is most likely that different maturation stimuli will result in the differentiation of phenotypically identical iDC (or maturing) and mDC but will lead to different immune effector functions, either tolerance or immunity, independent of DC phenotype but dependent on intrinsic and environmental properties of the DC (e.g., secretion of soluble factors). The latter has only recently become the subject of intensive investigation [75], and it is of great importance that this is further addressed in future experiments.

DENDRITIC CELLS INDUCING T CELL IMMUNITY Maturation stimuli for DC differ in their ability to induce mature tolerogenic or mature immunogenic DC by their capacity to induce specific cytokine production. Activated immunestimulatory DC produce large amounts of proinflammatory cy-

TABLE 3. T cell fate

DC type

T cell anergy and deletion

iDC mDC mDC

Inhibition of Th1 cell proliferation Expansion of IL-10-producing CD4⫹ and CD8⫹ Treg

iDC mDC

FOXP3⫹ Treg induction

mDC

Induction of Th1 cell responses

mDC

Induction of Th2 cell responses

mDC

Induction of Th17 cell responses

mDC

CTL priming

mDC

1368

tokines (IL-12p40/p70, TNF-␣, IL-1␤, IL-6, and type I IFN) [61]. Therefore, proinflammatory cytokine production by mature DC might be crucial to induce T cell immunity. In particular, mature immune-stimulatory DC are a critical source of IL-12, a cytokine that is involved in innate immune responses and pivotal for Th1 polarization [76]. Signals that induce this activation and cytokine production by DC are associated with microbial recognition of evolutionarily conserved pathogenassociated molecular patterns (PAMP) by pattern recognition receptors (PRR) on DC, such as the recently discovered Tolllike receptors (TLR) [77–79]. Also, specific T cell signals, among them CD40-CD40L (CD154) crosstalk of DC with helper T cells, may be involved in switching from tolerogenic DC toward immunogenic DC. However, CD40 ligation alone is unable to induce IL-12p70 production in vivo [40, 80]. Cytokine production by DC is subject to tight regulation mediated by TLR signaling and CD40 ligation. Only this allows for the development of mature DC capable of polarizing T cell responses or inducing tolerance. For a long time, the mechanisms for the induction of adequate immune responses have relied on what is designated as the two-signal system of acquired immunity (Fig. 1) [81– 83]. The proposal was that the presentation of (self-)antigens (signal one), in the absence of costimulation (signal two) induces T cell anergy or deletion. This second signal has been suggested to be necessary to activate naive T cells. At present, the “signal one” theory of tolerance seems to be oversimplified. More recently, it has clearly been shown that costimulation is required for the induction of T cell anergy [84], as well as for the generation of Treg [70, 83]. In addition, Albert et al. [55] demonstrated that DC matured with TNF-␣ and PGE2 induced CD8⫹ T cell proliferation eventually leading to tolerance due to T cell deletion. It is commonly assumed now that the appropriate generation of immunity requires DC or stimulated T cells that have received an additional “signal 3,” for instance from

Proposed DC Effector Functions/Subsets DC characteristics

Lack of MHC class II and of costimulatory molecules Expression of FasL, IDO and/or soluble CD25 Reduced expression of CD11c and MHC class II; increased expression of CD80, CD40, CD106, CD11b and NO Expression of MHC class II and of costimulatory molecules; possible migratory potential; lack of expression of cytokines (IL-6, IL-12, TNF-␣) High expression of MHC class II and costimulatory molecules; high expression of IL-10 and low expression of IL-12 Upregulated expression of MHC class II and of costimulatory molecules (e.g., CD80/CD86 high) High expression of MHC class II and costimulatory molecules; production of proinflammatory cytokines (e.g., IFN-␣, IL-6, and IL-12) and/or CD70 CD8␣-DC; moderate expression of MHC class II and of costimulatory molecules; production of IL-10, TGF-␤, and IL-6 High expression of accessory molecules and enhanced expression of TGF-␤, IL-6, and IL-23 High expression of MHC class II and costimulatory molecules; production of proinflammatory cytokines and/or CD70

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References (49, 50) (8, 52–57) (58, 59) (30, 31, 63–65, 117) (66, 68) (69, 73) (40, 41, 96, 97) (42–44) (94) (85–88, 100–102, 106–108)

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Figure 1. Overview of the signals necessary for induction of tolerance and immunity. Antigen presentation (signal 1) in the absence of costimulation (signal 2) results in T cell anergy or depletion. Besides effector T cell activation, costimulation is also important for the activation and expansion of different regulatory T cell subsets. Currently, it is assumed that (an) additional signal(s) (signal 3), such as CD40 ligation of DC and/or the production of inflammatory cytokines by DC, is necessary for induction of efficient T cell immunity.

interaction with CD4⫹ T cells through CD40 on the surface of TLR-stimulated DC (Fig. 1). CD8⫹ T cells require this third signal in vitro [85, 86], as well as in vivo [87] to undergo optimal clonal expansion, develop effector functions, and establish a memory population. CD8⫹ T cells that are activated in the absence of a third signal display limited clonal expansion and inadequate effector function (lytic activity and the ability to produce IFN-␥). Several studies now suggest that the production of a variety of proinflammatory cytokines, such as type I IFN, IL-6, and IL-12, by DC may provide for the necessary third signal for induction of efficient T cell immunity [85, 88]. Interestingly, proinflammatory cytokine production by DC has been demonstrated to reverse Treg-mediated immune suppression [89 –92]. However, caution needs to be taken as DC can also produce cytokines, other than proinflammatory cytokines, involved in tolerance induction [93]. Therefore, in our opinion, signal 3 is an important DC-dependent component, implicated in the early polarization of the immune response. Because of the expression of soluble or membranebound T cell-polarizing molecules by DC, DC exert an important function in the determination of the balance between Th1, Th2, Th17 [94] or Treg development. Upon ligation of selective pattern recognition receptors, DC display a discrete expression profile of T cell polarizing signals [93, 95]. However, the detailed mechanisms are still unclear. The knowledge is emerging that ligation of certain TLR (TLR3, TLR7, TLR8, and TLR9) on DC results in high expression of IL-12, important for the initiation of Th1 responses [96, 97]. In contrast, in certain circumstances, TLR ligation can induce expression of immunosuppressive cytokines by APC such as IL-10 [98, 99], with inhibition of the immune response as a consequence. In conclusion, it is clear that, depending on the context of their activation, DC display a different T cell polarizing effect. Thus, the creation of a particular cytokine environment by DC is critical for the determination of the appropriate type of immune response.

CLINICAL IMPLICATIONS FOR DENDRITIC CELL-BASED THERAPIES DC have become an attractive cell type for therapeutic manipulation of the immune system in order to enhance insufficient immune responses in infectious diseases and cancer or to attenuate excessive immune responses in allergy, autoimmunity and transplantation. Under optimal DC activation, e.g., induced by so-called danger signals, it might be possible to break established T cell tolerance in cancer patients and to generate effective antitumor responses. The success of immunotherapeutic vaccination with DC loaded with tumor-associated antigens (TAA) for induction of protective antitumor responses has been demonstrated in several animal models [100 –102]. Currently, multiple clinical trials are ongoing in cancer patients. A number of these trials, using TAA-loaded DC, demonstrated some clinical and immunological responses [as evidenced by T cell proliferation, IFN-␥ ELISPOT, and delayed type of hypersensitivity (DTH)] without any significant toxicity [103–105]. However, despite the presence of expanded antigen-specific T cells in patients after vaccination, only a minor population of these patients showed a beneficial clinical response, that is, tumor regression and increased disease-free survival [106 –110]. Until now, clinical trials using DC have only shown moderate, if any, success. Since DC possess the exceptional capacity to stimulate the patient’s own immune system against cancer, the reasons for the failure to eliminate tumor burden in a majority of patients need to be discussed and carefully examined in future experimental settings. Novel strategies to improve DC-induced tumor immune responses are already under investigation in our laboratory. One possible way is to exploit the capacity of DC to present peptides from phagocytosed dead tumor cells on both MHC class I and class II molecules [111, 112]. In this context, we have shown that the immunogenicity of tumor cells can be improved

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after transfection with Toll-like receptor (TLR) ligands, and these TLR ligand-transfected tumor cells were able to activate and mature DC as compared with nontransfected tumor cells [113].These results again suggest the need for strong maturation stimuli for ex vivo treatment of DC in order to generate stable fully activated DC capable of inducing tumor-specific cytotoxic T lymphocytes (CTL) with limited or no induction of Treg. For this, research is now focusing on the use of pathogenderived molecules for DC maturation [41, 114, 115]. The use of TLR ligands in tumor vaccination strategies allows DC to sense “danger” leading to the differentiation of a fully mature immunostimulatory DC. In this context, TLR3 ligation by polyinosinic polycytidylic acid [poly(I:C)] has proven to be very useful in previous research [116] and in our current work. We have recently demonstrated that TLR3 stimulation of DC by poly(I:C) seems to be able to break the tolerogenic function of DC [117]. This is a strategy that is currently under intensive investigation in several research models and in different laboratories, including ours. Other immune effector cells need to be considered as well, including CD4⫹ T cells, NKT, and NK cells, as well as B cells. Previously, it was shown by our group that B cells can act as efficient antigen-presenting cells to induce CMV- and HIVspecific CD8⫹, as well as CD4⫹ T cell responses [118, 119]. In addition, several reports demonstrated the helper function of NK cells in inducing Th1-mediated immune responses [120, 121]. Tumor-induced NK cells activate DC to produce IL-12, which is important in stimulating Th1 responses. Last but not least, CD4⫹ T cells play an important role in the regulation of immune responses [122, 123]. Helper CD4⫹ T cells upregulate CD40 ligand (CD40L) following activation. CD40L is an important costimulatory signal as CD40 ligation of DC will stimulate their consecutive production of IL-12 [124]. In conclusion, novel DC-based vaccination strategies will most likely be focused on a multitargeted approach to attain the goal of effective immunotherapy in cancer. Therefore, procedures such as depletion of Treg [125, 126], neutralization of inhibitory factors [e.g., administration of 1-methyl tryptophan (1-MT), a powerful antagonist of IDO [53]] and gene targeting of tumors and cancer draining lymph nodes with DC chemoattractants are currently considered as adjuvant therapies prior to vaccination. Regarding immune tolerance, immunization with iDC or maturing DC might become an important clinical strategy for dampening or inhibition of destructive immune responses in immune-mediated pathologies. Currently, antigen-nonspecific agents (such as cyclosporine A), which suppress the function of all T cells, are used as standard therapeutic approach to inhibit destructive immune responses. In the future, vaccination with immature or maturing DC might open the possibility to decrease autoimmune responses in patients or transplant recipients in an antigen-specific manner. The possibility for this has already been demonstrated in several animal models for autoimmunity, as well as in humans [30, 32]. However, one should remain cautious, as it is unlikely that iDC will stay immature in vivo after circulation to inflamed tissues. Therefore, several strategies in which DC are treated with biological (e.g., cytokines such as IL-10 and TGF-␤) or pharmacological immunesuppressive agents or transduced with vectors encoding tolero1370

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genic molecules (IL-10, TGF-␤, CTLA-4, or Notch ligands) have been proposed in order to obtain stable tolerogenic DC. IL-10-treated DC actively 1) down-regulate costimulatory molecules, 2) inhibit IFN-␥ secretion by activated T cells, and 3) reduce the production of antibodies by activated B cells. Interestingly, all of these effects could only be observed when using IL-10-treated iDC, but not using IL-10-treated mDC. The latter down-regulate the IL-10-receptor and subsequently become insensitive to IL-10 treatment [127]. In addition, IL-10treated iDC induce activation and proliferation of CD4⫹ regulatory and CD8⫹ suppressor T cells [45, 128, 129]. Moreover, such IL-10-treated DC have been successfully applied in animal models for immune-mediated pathologies [130 –132]. Also TNF-␣ acts as a stimulus to induce tolerogenic murine bone marrow-derived DC. Despite the fact that these DC have a mature phenotype, as judged by surface marker expression of MHC class II and costimulatory molecules, they failed to secrete IL-1␤, IL-6, TNF-␣, and IL-12. Moreover, these tolerogenic DC were able to reverse autoimmunity in an experimental autoimmune encephalomyelitis (EAE) mouse model, and their suppressive effect was attributed to the induction of IL-10-producing Treg [133]. Interestingly, these TNF-␣treated DC probably represent an intermediate differentiation state, as they still can produce proinflammatory cytokines upon a second stimulation with LPS and CD40 ligation in vitro and after subcutaneous injection in vivo [26, 134]. Again, these data demonstrate that intrinsic properties (e.g., cytokine profile) of cultured DC seem to be more important than phenotypic properties (e.g., expression of membrane molecules) with regard to their functional properties (e.g., T cell stimulation or tolerance induction). DC function can also be influenced by pharmacological agents such as corticosteroids, estrogen, rapamycin, chloroquine, ATP, and vitamin D3 [135]. All of these pharmacological agents share some common properties: 1) inhibition of differentiation and of maturation of DC, 2) reduced expression of costimulatory molecules by DC, and 3) reduced IL-12 production by DC. Treatment of DC with vitamin D3 (or some of its analogs) results in the up-regulation of the inhibitory receptor immune globulin-like transcript (ILT)3 and increased secretion of IL10, while the expression of costimulatory molecules and the production of IL-12 is down-regulated both in vitro and in vivo [136, 137]. Moreover, treatment of DC with vitamin D3 results in the induction of CD4⫹CD25⫹ Treg, which can mediate tolerance in experimental models of transplantation and autoimmunity [138, 139]. Triptolide, an active compound identified in a traditional Chinese herb, has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. In addition, Liu et al. have demonstrated that triptolide can prevent DC migration into tissues and secondary lymphoid organs by down-regulation of cyclooxygenase-2 (COX-2) and CCR7 expression [140]. Moreover, triptolide-treated DC display an impaired production of chemokines attracting neutrophils and T cells (e.g., MIP-1␣, MIP-1␤ and RANTES) [141]. Alternatively, tolerogenic DC might also be used for the ex vivo expansion of Treg for adoptive cellular therapy against immune-mediated pathologies. Regarding the latter, it has http://www.jleukbio.org

been shown in humans that increased numbers of Treg persist at least 3 years following heart transplantation in patients without chronic rejection [142, 143], suggesting the role that Treg might play in future immunotherapies against excessive inflammation. Finally, several studies also suggested that the route of injection of antigen-presenting DC can determine tolerance vs. immunity. It has been demonstrated that the intravenous route is tolerogenic, whereas the subcutaneous route is immunogenic [144, 145]. However, more research, especially in the clinical setting is needed to examine the full potential of DC as cellular vaccines and to standardize DC vaccination for a number of variables, including DC subset, mode of maturation, frequency of vaccination, and route of administration [146].

CONCLUSIONS Although it is well established that both developmental and activation state of DC determines the polarization of T cell immune responses, recently performed clinical trials aiming to stimulate the immune system of cancer patients did not yet fulfill their promise. One of the main problems might have been the maturation stimuli used for obtaining mature immunostimulatory DC. Currently, much attention is given to TLR ligands for additional DC maturation, as this might result in improved stable DC maturation needed for optimal induction of T cell immunity. On the other hand, more insight will be needed to unravel the mechanisms underlying induction and maintenance of T cell tolerance by different DC subsets and interfering factors. Eventually, vaccination with tolerogenic DC in combination with regulatory T cells might gain clinical interest in the treatment of autoimmune disorders, allergy, and transplant rejection.

ACKNOWLEDGMENTS This work was supported by grants G.0456.03, G.0313.01, and WO.012.02 from the Fund for Scientific Research, Flanders, Belgium (FWO-Vlaanderen), by grants from the Fortis Bank Verzekeringen (financed cancer research), by research grants from the Foundation against Cancer (Belgische Federatie tegen Kanker, now Stichting tegen Kanker), by grant 802 of the Antwerp University Concerted Research Action, and by an interuniversity attraction pole grant of the Belgian Science Policy. V. F. I. V. T. and P. P. are postdoctoral fellows of the FWO-Vlaanderen. N. C. held a Ph.D. fellowship of the Flemish Institute for Science and Technology.

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