Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets

Induction of Anergic and Regulatory T Cells by Plasmacytoid Dendritic Cells and Other Dendritic Cell Subsets Masataka Kuwana ABSTRACT: The induction of antigen-specific tolerance is critical for maintaining immune homeostasis and preventing autoimmunity. Because the central tolerance that eliminates potentially harmful autoreactive T cells is incomplete, peripheral mechanisms for suppressing selfreactive T cells play an important role. Dendritic cells (DCs) are professional antigen-presenting cells, which have an extraordinary capacity to stimulate naı¨ve T cells and initiate primary immune responses. Recent accumulating evidence indicates that several subsets of human DCs also play a critical role in the induction of peripheral tolerance by anergizing effector CD4⫹ and CD8⫹ T cells or by inducing the differentiation of naı¨ve T cells into T-regulatory cells, which produce interleukin (IL)-10. Human DC subsets with the property of suppressing an Abbreviations APC antigen-presenting cell CTL cytotoxic T lymphocyte DC dendritic cell G-CSF granulocyte colony-stimulating factor GVHD graft-versus-host disease IFN interferon

INTRODUCTION Dendritic cells (DCs) are professional antigen-presenting cells (APCs), which have an extraordinary capacity to stimulate naı¨ve T cells and initiate primary immune responses [1]. DCs also play critical roles in the immune system, including the induction of peripheral tolerance and regulation of the types of T-cell responses and they function as effector cells in innate immunity against microbes [1–3]. These diverse functions of DCs in immune regulation are now recognized to depend on the diversity of DC subsets, lineages, and maturation stages From the Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan Address reprint requests to: Masataka Kuwana, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 Japan; Phone: ⫹81(3)5363-3778; Fax: ⫹81 (3) 5362-9259; E-mail: [email protected]. Received July 22, 2002; accepted September 27, 2002. Human Immunology 63, 1156 –1163 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.

antigen-specific T-cell response include plasmacytoid DCs, which are either in an immature state or in a mature state induced by CD40 ligand stimulation, and monocyte-derived DCs, which are either in an immature state or have had their state modulated by treatment with IL-10 or CD8⫹CD28⫺ T cells. These “tolerogenic” DCs may be relevant to therapeutic applications for autoimmune and allergic diseases as well as organ transplant rejection. Human Immunology 63, 1156 –1163 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: anergy; autoimmunity; dendritic cell; Tregulatory cell; tolerance

ILT MHC PDC TCR TGF-␤ Tr cell

immunoglobulin-like transcript major histocompatibility complex plasmacytoid dendritic cell T-cell-receptor transforming growth factor-␤ T-regulatory cell

[4]. Precursors for DCs are present in human peripheral blood and comprise at least four phenotypically and functionally different subsets; three are cluster designation (CD)11c⫹ subsets derived from common myeloid progenitors and the other is a CD11c⫺ subset derived from common lymphoid progenitors. CD11c⫹ myeloid DC precursors include CD1a⫹ epidermal Langerhans cell precursors, CD1a⫺ interstitial DC precursors, and CD14⫹ monocytes (pre-DC1). These myeloid DC precursors migrate into the skin epidermis and other tissues and become immature DCs [5]. Upon appropriate stimulation, such as by lipopolysaccharide, tumor necrosis factor (TNF)-␣, or CD40 ligand, immature myeloid DCs mature into fully competent DCs, produce a large amount of interleukin (IL)-12, and preferentially induce T-helper 1 cell (Th1) development and strong cytotoxic T-lymphocyte (CTL) responses [6]. On the other hand, 0198-8859/02/$–see front matter PII S0198-8859(02)00754-1

Tolerogenic Dendritic Cell Subsets

CD11c⫺ plasmacytoid DCs (PDCs) or precursors for DC2 reveal a plasma cell-like morphology and lack myeloid markers CD13, CD14, and CD33, but they express CD123 (the IL-3 receptor) at high levels as well as transcripts specific for lymphocytes, such as the pre-Tcell receptor ␣, immunoglobin (Ig)␭-like 14.1, and Spi-B [5–7]. Immature PDCs that are activated by IL-3 and CD40-ligand stimulation preferentially promote Th2 differentiation [8] and, in some instances, Th1 responses [9]. Recently, several human DC subsets have been found to induce antigen-specific suppression of the immune response and to be involved in the maintenance of peripheral tolerance by inducing anergic or regulatory T cells [10]. This review summarizes these “tolerogenic” DC subsets and discusses their potential role in tolerance maintenance and their application to immunotherapy aimed at suppressing harmful T-cell responses in various pathologic conditions. PDCs Induce an Anergic State in Effector CD4ⴙ T Cells Plasmacytoid dendritic cells are mainly present in the peripheral blood and tonsils, but cells with a similar morphology were originally identified as “plasmacytoid T cells,” which are clustered around the high endothelial venules of inflamed lymph nodes [11]. PDCs are identical to natural type I interferon (IFN)-producing cells, which rapidly produce enormous amounts of IFN-␣/␤ in response to viruses [12, 13]. Immature PDCs undergo maturation upon recognizing viral components via tolllike receptors on the cell surfaces [14]. PDCs that are activated by virus prime naı¨ve T cells to produce IFN-␥ and IL-10 and promote Th1 differentiation, although PDCs activated by IL-3 and CD40 ligand stimulation preferentially promote Th2 differentiation [8, 13]. The differentiation of immature PDCs into a mature form during viral infection does not require exogenous cytokines, but is mediated by type I IFN produced in an autocrine manner [13]. These findings indicate that PDCs play an important role in both innate and adaptive immunity and provide a physical link between the two arms of immunity. We recently demonstrated that immature PDCs freshly enriched from human peripheral blood can induce an anergic state in human antigen-specific CD4⫹ T-cell lines [15]. Human CD4⫹ T-cell lines specific to tetanus toxoid, a foreign recall antigen, incubated with tetanus toxoid-pulsed autologous PDCs, fail to proliferate in secondary cultures with optimal antigen stimulation. Autoreactive CD4⫹ T-cell clones specific for topoisomerase I, derived from a patient with scleroderma, as well as clones specific for ␤2-glycoprotein I, derived from patients with antiphospholipid syndrome, are also rendered anergic after coculture with autologous PDCs

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pulsed with the corresponding autoantigens, resulting in their failure to proliferate or to provide help to B cells to produce autoantibodies [15 and unpublished observations]. The PDC-induced unresponsiveness is associated with a loss of IL-2 production and is completely or partially reversible in the presence of a high concentration of exogenous IL-2 in the secondary cultures. The cellular state of these unresponsive T cells is consistent with T-cell anergy, a cellular state where T cells fail to proliferate when optimally restimulated with functional APCs and antigen [16]. CD4⫹ T cells treated with antigen-pulsed PDCs fail to upregulate CD40 ligand expression upon recognizing an antigenic peptide by the T-cell receptor (TCR). Th1 clones treated with antigenpulsed PDCs produce a trace amount of IFN-␥, but no IL-2, upon mitogenic stimulation, whereas Th0 clones, which can secrete IL-10 produce more after treatment with antigen-pulsed PDCs. Like T-regulatory (Tr) cells, these IL-10-producing anergic CD4⫹ T cells suppress in vitro autoantibody production mediated by autoantigenspecific effector CD4⫹ T-cell clones by producing IL-10 (unpublished observations). These results suggest that immature PDCs in circulation have the function of anergizing effector CD4⫹ T cells, which are responsive to foreign and self-antigens. T-cell anergy induction by immature PDCs requires cognate contact through the engagement of the TCR with the antigenic peptide, which is presented on major histocompatibility complex (MHC) molecules [15], but important details concerning the interaction between PDCs and CD4⫹ T cells, which induce T-cell anergy, remain unclear. T-cell activation requires two signals: TCR occupancy by an antigenic peptide presented on MHC molecules and the engagement of costimulatory molecules on the APC with their ligands on the T cell [16]. TCR occupancy in the absence of costimulation leads to T-cell anergy [16, 17]. According to this “twosignal” hypothesis of T-cell activation, the mechanism for T-cell anergy induction could simply be explained by TCR-MHC engagement without costimulatory signals through CD80 and CD86, because freshly isolated PDCs express no amount or a minimal amount of CD80 and CD86 [5, 18]. However, Albert et al. recently developed an in vitro human cell culture system for testing the “two-signal” hypothesis for the regulation of CD8⫹ Tcell priming versus tolerance and found that monocytederived mature DCs expressing CD80 and CD86 were able to tolerize CD8⫹ T cells [19]. This finding suggests that refinement of the current “two-signal” model is necessary. What, then, is the mechanism for the T-cell anergy induction mediated by PDCs? One of the key events in the induction of an anergic state in T cells during interaction with antigen-pulsed PDCs is a failure in CD40

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ligand upregulation [15], which is presumed to be mediated by inhibitory signals from PDCs, either via cellsurface molecules or soluble factors. Culture supernatants from PDCs, whether or not they are co-cultured with T cells and antigen, do not have any effect on the status of T cells cultured with antigen-bearing functional APCs [15]. Moreover, monoclonal antibodies to IL-10, IFN-␣, or transforming growth factor-␤ (TGF-␤) fail to inhibit the T-cell anergy induction, which is mediated by antigen-pulsed PDCs (unpublished observations). These findings strongly suggest that soluble factors alone, in addition to the TCR-MHC engagement, are not sufficient to induce an anergic state and cell surface factors, which transmit negative signals to T cells may play a more important role. Two such candidate molecules are the inhibitory receptors immunoglobulin-like transcript (ILT) 3 and ILT4, which are expressed on some subsets of DCs and monocytes [20, 21], including immature PDCs [22]. These receptors belong to a family of Ig-like inhibitory receptors, which are structurally and functionally related to killer cell inhibitory receptors and display a long cytoplasmic tail containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ILT3 and ILT4 mediate inhibition of cell activation by recruiting the tyrosine phosphatase SHP-1, and interfere with CD40 – CD40 ligand-mediated signaling. Although the ligand for ILT3 is currently unknown, ILT4 has been found to bind HLA-A, B, C, and G [23]. Tolerogenic DC Subsets In addition to immature PDCs, which are freshly isolated from peripheral blood, several other human DC subsets can suppress immune responses by anergizing effector T cells or inducing the differentiation of naı¨ve T cells into T-regulatory (Tr) cells in vitro. These tolerogenic DC subsets are reported across lineages (myeloid vs lymphoid DCs) and maturation status (immature vs mature DCs), as described below. IL-10-Modulated Immature Myeloid DCs IL-10 is known as an immunosuppressive cytokine, which reduces the upregulation of expression of MHC class II molecules and several costimulatory and adhesion molecules, as well as the DC-specific activation marker CD83 [24, 25]. Steinbrink et al. have reported that immature monocyte-derived DCs treated with IL-10 induce an antigen-specific anergy in alloreactive CD4⫹ and CD8⫹ T cells and an influenza hemagglutinin-specific CD4⫹ T-cell clone [25]. Moreover, IL-10-modulated monocyte-derived DCs induce an anergic state in melanoma antigen-specific CD4⫹ and CD8⫹ T cells and the anergic CD8⫹ cells lose their capacity to lyse target tumor cells [26]. This anergic state is characterized by

M. Kuwana

impaired T-cell proliferation and markedly reduced production of IL-2 and IFN-␥. The induction of anergy requires direct contact between effector T cells and DCs, but, after incubation with IL-10-modulated DCs, anergic T cells do not produce the immunoregulatory cytokine IL-10 or TGF-␤. Unlike other tolerogenic DC subsets, IL-10-modulated DCs are resistant to maturation signals [25, 26]. This is potentially beneficial when this tolerogenic DC subset is used in a DC-based vaccine for suppressing a specific T-cell response. DCs with properties similar to the IL-10-modulated DCs generated in vitro can be detected in vivo in IL-10-producing tumors [27] and ultraviolet-irradiated skin [28]. CD8ⴙCD28ⴚ T-Cell-Treated Immature Myeloid DCs Human CD8⫹CD28⫺ T cells have been reported as a distinct Tr-cell population, which suppresses antigenspecific CD4⫹ T-cell responses by inhibiting their capacity to produce IL-2 and upregulate CD40 ligand expression [29]. CD8⫹CD28⫺ Tr cells can be generated in vitro after human peripheral blood mononuclear cells are stimulated with multiple rounds of allogeneic- or xenogeneic-donor APCs [30]. Chang et al. recently found that human CD8⫹CD28⫺ T cells can modulate the function of immature monocyte-derived DCs. These modulated DCs lack CD80 and CD86 and are able to anergize alloreactive memory CD4⫹ T cells [31]. This suppressive effect by CD8⫹CD28⫺ T-cell-treated DCs is MHC-restricted and antigen-specific. The induction of CD4⫹ T-cell anergy is not caused by a suppressor effect mediated by soluble factors, but requires direct interactions between effector CD4⫹ T cells and DCs. The mechanism for generating tolerogenic DCs has been extensively analyzed in this system. Exposure of immature DCs to CD8⫹CD28⫺ T cells in an antigen-specific manner results in interference with CD40 –CD40 ligandmediated signaling, which normally induces functional maturation of DCs and high expression of CD80 and CD86. Moreover, the tolerogenic influence of CD8⫹CD28⫺ T cells is associated with the induction of the inhibitory molecules ILT3 and ILT4 on the DC surface [31]. Interestingly, CD8⫹CD28⫺ T cells, which can upregulate ILT3 and ILT4 in donor APCs, are present in the circulation of human heart transplant recipients, especially in rejection-free patients [31]. Immature Myeloid DCs Jonuleit and associates have reported that repeated stimulation of naı¨ve cord blood-derived CD4⫹ T cells with allogeneic immature monocyte-derived DCs results in their differentiation to IL-10-producing CD4⫹ Tr cells in vitro [32]. These CD4⫹ Tr cells depict an inhibited proliferation, which cannot be restored by restimulation

Contact-dependent MHC-specific Antigen-specific Naı¨ve CD8⫹ T cells

Contact-dependent MHC-specific Antigen-specific Naı¨ve CD4⫹ T cells Naı¨ve CD8⫹ T cells Low IFN-␥, high IL-10 Low INF-␥(Th1) Low INF-␥, high IL10 (Th0)

Target T cells

Mechanisms for T cell suppression

Contact-dependent MHC-specific Antigen-specific Effector CD4⫹ T cells Effector CD8⫹ T cells Low IFN-␥,IL-2 Cytokine profiles of T cells after interaction with DCs

?

High High ? — Low Low ? Sensitive to TNF-␣, LPS, other stimulators

Low Absent or very low ILT3⫹,ILT4? Sensitive to virus and CD40 ligand stimulation Contact-dependent MHC-specific Antigen-specific Effector CD4⫹ T cells MHC expression Expression of CD80/CD86 Expression of ILT3/ILT4 Maturation sensitive/resistant

Low Low ? Resistant

IL-10 treatment of immature myeloid DCs Freshly isolated from peripheral blood Preparation

Contact-dependent MHC-specific Antigen-specific Effector CD4⫹ T cells

CD40 activation of PDCs

IL-10-modulated myeloid DCs

Immature myeloid DCs cultured with allogeneic CD8⫹CD28⫺ T cells Low Absent ILT3⫹,ILT4⫹ ?

Incubation of monocytes with GMCSF and IL-4

Low IFN-␥, high IL-10

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Freshly isolated Immature PDCs

TABLE 1 The features of human tolerogenic DC subsets

CD8⫹CD28⫺ T cell-treated myeloid DCs

Immature myeloid DCs

CD40-activated mature PDCs

Tolerogenic Dendritic Cell Subsets

with functional mature DCs or the addition of exogenous IL-2. These IL-10-producing CD4⫹ Tr cells indicate an early upregulation of cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4) and inhibit the antigen-induced proliferation of Th1 cells in a contact- and dose-dependent, but antigen-nonspecific, manner. These features appear to be similar to the CD4⫹CD25⫹ Tr cells, a well-characterized Tr cell subset in mice [33] and humans [34]. In addition, injection of influenza matrix peptide-pulsed immature monocyte-derived DCs in humans results in the induction of influenza matrix-specific CD8⫹ T cells, which produce IL-10, but are defective in IFN-␥ secretion and killer function [35]. In this human in vivo study, immature DCs also dampened the function of preexisting antigen-specific effector CD4⫹ T cells [35]. CD40 Ligand-Activated PDCs It was reported recently that naı¨ve CD8⫹ T cells primed with allogeneic CD40 ligand-activated PDCs differentiate into CD8⫹ T cells, which display poor secondary proliferative and cytolytic responses, but produce a large amount of IL-10 and low IFN-␥ upon restimulation [36]. This indicates that the induction of tolerance versus immunity may not simply be determined by the maturity of the DCs. These IL-10-producing CD8⫹ Tr cells suppress bystander proliferation of CD8⫹ T cells, which is mediated through the production of IL-10, but not through the production of TGF-␤. These CD8⫹ Tr cells seem to be the same as the CD8⫹ suppressor T cells, which were first described in the 1970s, but were discounted in the 1980s [37]. These CD8⫹ Tr cells differ from CD8⫹CD28⫺ Tr cells in several aspects. IL-10producing CD8⫹ Tr cells can be directly generated from naı¨ve CD8⫹ T cells by one round of stimulation with mature PDCs, but CD8⫹CD28⫺ Tr cells appear to differentiate into end-stage CTL cells after repeated stimulations [38]. Moreover, IL-10-producing CD8⫹ Tr cells directly inhibit primary T-cell responses through IL-10 secretion, whereas CD8⫹CD28⫺ Tr cells exert immunosuppressive functions by inducing the differentiation of immature myeloid DCs into tolerogenic DCs as described above [31]. Rather, these CD8⫹ Tr cells share many similarities with CD4⫹ Tr cells [39], including the properties induced by repeated stimulation with immature myeloid DCs [32], in that they proliferate poorly when stimulated via the TCR and suppress immune responses via a mechanism dependent on the production of IL-10. Features of Tolerogenic DC Subsets As summarized in Table 1, tolerogenic DC subsets have common features, such as low MHC expression and little or no expression of CD80 and CD86, except for the

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CD40-activated mature PDCs. The induction of anergic or nonproliferating T cells requires direct cell– cell contact between T cells and DCs and suppression of T cells is MHC-restricted and antigen-specific. These tolerogenic DCs can be divided into two categories: those that directly anergize effector T cells, including PDCs, IL10-modulated DCs, and CD8⫹CD28⫺ T-cell-treated DCs and those that promote the differentiation of naı¨ve T cells into IL-10-producing Tr cells, including immature myeloid DCs and CD40-activated PDCs. Regarding this point, freshly isolated PDCs are unique because they can induce the differentiation of effector Th0 cells into IL-10-producing cells with a possible regulatory function [15]. The mechanisms that lead to the suppression of T-cell function as a result of the interaction with DCs remain unclear. The absence or low expression of CD80 and CD86 on the DC surfaces may be one of the mechanisms, but additional mechanisms are definitely required. In this regard, the inhibitory molecules ILT3 and ILT4, which have been reported to be upregulated on CD8⫹CD28⫺ T-cell-treated DCs [31], may be associated with the tolerogenic property of these DC subsets. Because a suppressive effect has been seen in vitro in all tolerogenic DC subsets except the immature monocytederived DCs [35], in vivo experiments are necessary to confirm their tolerogenic function, although humans and mice are different in terms of the phenotypic and functional classification of DC subsets [6]. Several murine DC subsets appear to be counterparts of human tolerogenic DC subsets. Specifically, freshly enriched murine Langerhans cells pretreated with IL-10 inhibit induction of antigenspecific Th1 responses [40]. In addition, injection of bone marrow-derived MHC class II⫹CD80dimCD86⫺ immature myeloid DCs induce donor-specific T-cell anergy and prolong cardiac allograft survival in recipient mice without the use of immunosuppressants [41]. In contrast, CD8⫹CD28⫺ Tr cells have not been reported in mice and a murine counterpart of human PDCs has not been evident despite a long search. A recently identified murine cell population, which resembles human PDCs, possesses key features, including the production of large amounts of type I IFN upon viral challenge and differentiation into mature DCs upon CD40 stimulation [42, 43]. However, in contrast to human PDCs, the murine cells express CD11c and produce IL-12. Nevertheless, this murine cell population is potentially useful in analyzing the in vivo tolerogenic function of immature PDCs and CD40-activated mature PDCs. Control of Peripheral T Cell Tolerance by DC Subsets T-cell tolerance is generally maintained by eliminating self-reactive T cells in the thymus, but a significant

M. Kuwana

number of self-reactive T cells escape the central thymic tolerance and are released into the circulation [44, 45]. Therefore, the prevention of harmful autoimmune responses is largely dependent on peripheral mechanisms and the DC lineages may have evolved to maintain peripheral tolerance in several distinct pathways. Immature myeloid DCs, including Langerhans cells and interstitial DCs, are located at sites of pathogen entry, such as the skin and mucosal tissues [46]. Under steady-state physiologic conditions, immature myeloid DCs constantly sample self-antigens and apoptotic cells. In the absence of inflammation, these DCs remain immature, but still migrate to regional lymph nodes, where naı¨ve T cells may encounter antigen on immature DCs and differentiate into Tr cells rather than effector T cells. Therefore, the primary function of immature myeloid DCs in vivo would be to prime Tr cells and generate tolerance to self-antigens in various tissues and immature myeloid DCs may function as “the police” of the immune system, which actively maintain tolerance to self-antigens [47]. In contrast, because CD8⫹CD28⫺ T cells are generated by multiple rounds of CTL stimulation [38], CD8⫹CD28⫺ T-cell-treated DCs mainly play a role in terminating Th1 inflammatory responses, rather than in the maintenance of tolerance. In contrast to immature myeloid DCs, immature PDCs are mainly located in the circulation and T-cell areas of lymphoid tissues, but are absent at sites where most pathogens access the body [2], suggesting that PDCs are not the first to respond to microbial invasion via the body surface and may be specialized to recognize blood-borne pathogens and self-antigens. Therefore, it is likely that immature PDCs sample circulating self-antigens and dead cells and present self-peptides to autoreactive T cells to prevent autoimmunity under a physiologic state. On the other hand, PDCs are known to play a central role in antiviral immunity by rapidly producing type I IFN and subsequently inducing strong Th1 responses [4]. Paradoxically, PDCs can suppress Th1 responses independent of their maturation status; immature PDCs directly induce an anergic state in effector Th1 cells [15] and CD40 ligand-activated mature PDCs promote the differentiation of Th2 [8] and IL-10-producing CD8⫹ Tr cells from naı¨ve CD4⫹ and CD8⫹ T cells, respectively [36]. Because CD40-mediated PDC activation is delayed until antigen-specific T cells are activated to express the CD40 ligand in the inflamed lymph nodes, it is possible that the PDC lineage functions as a “shut-off” system, which suppresses excessive Th1 immune responses induced by virus-activated mature PDCs and mature myeloid DCs (Figure 1). In this regard, during bone marrow transplantation, donor DCs are activated by allogeneic T cells and mature myeloid DCs trigger and accelerate graft rejection and graft-

Tolerogenic Dendritic Cell Subsets

FIGURE 1 Suppression of T-helper 1 cell (Th1) responses by plasmacytoid dendritic (PDC) lineage cells. The development of Th1 and cytotoxic T-lymphocyte responses is mediated by mature myeloid dendritic cells (DCs) through interleukin (IL)-12 secretion and the development of virusactivated mature PDCs is mediated through interferon (IFN)-␣ secretion. Immature PDCs supplied from the bone marrow directly anergize antigen-specific effector Th1 cells. Mature PDCs activated by CD40 ligand stimulation promote the differentiation of naı¨ve CD4⫹ and CD8⫹ T cells into Th2 and IL-10-producing CD8⫹ T-regulatory (Tr) cells, respectively. Soluble factors IL-4 and L-10 secreted by Th2 and IL-10-producing CD8⫹ Tr cells suppress Th1 responses.

versus-host disease (GVHD), whereas immature and mature PDCs may function to inhibit these Th1-mediated pathogenic conditions. Specifically, transplanted donor PDCs may capture host allo-antigens and anergize activated donor alloreactive T cells. In addition, PDCs that have captured allo-antigens differentiate into a mature form and tolerize donor Th1 cells and CTLs by inducing Th2 and CD8⫹ Tr cell responses. Therefore, the fate of immune responses in bone marrow transplant recipients is dependent on the balance between allospecific Th1 responses mediated by myeloid DCs and Th1-suppressing responses mediated by PDCs. Because PDCs are mobilized by the administration of granulocyte-colony stimulating factor (G-CSF) in vivo in humans [48, 49], this hypothesis is supported by a recent study illustrating that transplantation of G-CSF-mobilized blood cells, which contain a large number of donor PDCs, reduces the severity of GVHD [48]. Therefore, the PDC lineage is likely to play a key role in the suppression of Th1mediated pathogenic conditions, such as transplant rejection, GVHD, and several autoimmune diseases, such as multiple sclerosis. Therapeutic Applications of Tolerogenic DCs to Suppress Pathogenic T-Cell Responses Because the ability of DCs to present antigenic peptides and initiate T-cell-dependent immune responses is far greater than that of other types of professional APCs,

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various strategies utilizing mature myeloid DCs have already been adapted to augment tumor immunity for cancer immunotherapy [50]. Because of the ability of several DC populations to induce antigen-specific T-cell anergy or generate Tr cells, manipulation of tolerogenic DCs is a potential strategy to suppress harmful T-cell responses in patients with autoimmune and allergic diseases as well as those with transplant rejection. There are several reports proposing the idea of using DC subsets in tolerizing allo- and auto-antigen-specific T cells in experimental models in mice [51]. However, human studies using tolerogenic DCs have not been tried yet, although there is one study, which illustrates that injecting immature monocyte-derived DCs pulsed with an influenza matrix peptide into healthy subjects leads to the appearance of IL-10-producing specific CD8⫹ T cells [35]. A possible protocol for DC-based immunotherapy to selectively suppress a harmful T-cell response in autoimmune patients includes the isolation and generation of autologous tolerogenic DCs, incubation of the DCs with a target antigen, and reinfusion of the DCs. This DC-based strategy has a potential advantage compared with peptide-based immunotherapy, because immature DCs can capture and process soluble protein antigens; therefore, it is unnecessary to identify T-cell epitopes on the target antigens and restricted HLA alleles. In addition, preparation of a large quantity of tolerogenic DCs is feasible: immature myeloid DCs can be induced from peripheral blood CD14⫹ monocytes and circulating PDCs can be mobilized by administering fms-like tyrosine kinase 3 (FLT3) ligand or G-CSF in vivo [48, 49]. CONCLUSION The immune regulatory properties of the human DC system are currently under active clinical investigation. The suppressive properties of several DC subsets described in this review should be pursued for antigenspecific inhibition of T-cell function in the setting of autoimmune diseases and organ transplantation. ACKNOWLEDGMENTS

The study was supported by grants from the Keio University Medical Fund and the Ichiro Kanehara Foundation.

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