Human plasmacytoid-derived dendritic cells and the induction of T-regulatory cells

Human Plasmacytoid-Derived Dendritic Cells and the Induction of T-Regulatory Cells Michel Gilliet and Yong-Jun Liu ABSTRACT: Suppression by T-regulatory (Tr) cells is essential for the induction of T-cell tolerance and the prevention of autoimmune diseases, organ rejection, and graft-versus-host disease. Increasing attention has been devoted to understand the role of dendritic cells (DC) in the control of Tr-cell differentiation. Here we review the recent evidence that cluster designation (CD)40-ligand activated plasmacytoid-derived DCs (DC2) have the ability to induce primary Tr-cell differentiation. We propose that in addition to the regulatory functions of immature myeloid DC, Tr-cell induction by DC2 represents a nonABBREVIATIONS Tr T regulatory cell Th T helper cell APC antigen-presenting cell DC dendritic cell MDC myeloid DC PDC plasmacytoid-derived DC IL interleukin

INTRODUCTION The induction of specific tolerance is critical for the maintenance of the immune homeostasis and the prevention of autoimmunity. There are several mechanisms by which the immune system distinguishes self and nonself or innocuous and harmful foreign antigens. In the thymus, central tolerance is a well-known mechanism, which involves deletion of self-reactive T cells [1]. In the periphery, the mechanisms involved include the induction of cell death, the development of a state of T-cell unresponsiveness called anergy [2], and active suppression by T-regulatory (Tr) cells. In recent years, increasing attention has been devoted to Tr cells, due to their unique ability to prevent autoimmune pathologies [3], From the DNAX Research Institute, Palo Alto, California. Address reprint requests to: Michel Gilliet, Department of Dermatology, Zurich University Hospital, Gloriastrasse 31, 8091Zurich, Switzerland; Tel: ⫹41(1)255-1111; Fax: ⫹41(1) 255-4549; E-mail: [email protected]. Received July 22, 2002; accepted September 27, 2002. Human Immunology 63, 1149 –1155 (2002) © American Society for Histocompatibility and Immunogenetics, 2002 Published by Elsevier Science Inc.

redundant mechanism for the safeguard of peripheral Tcell tolerance. DC2 can be used as tool to drive potent antigen specific Tr-cell differentiation and expansion in vitro and in vivo. Human Immunology 63, 1149 –1155 (2002). © American Society for Histocompatibility and Immunogenetics, 2002. Published by Elsevier Science Inc. KEYWORDS: human dendritic cells; T regulatory cells; immunosuppression; IL-10

IFN TGF TLR TCR MHC ILT

interferon tumor growth factor toll-like receptor T cell receptor major histocompatibility antigen immunoglobuline-like transcripts

intestinal inflammation [4], and allograft rejection [5]. Further understanding of the biology of Tr cells and the mechanisms that control their differentiation may be of key importance for the development of new therapeutic strategies. Here we summarize the recent progress in the characterization of human Tr cell subsets and review the new evidence on the Tr-cell generation by mature plasmacytoid-derived dendritic cells (PDCs) activated in the presence of cluster designation (CD)40-ligand. In addition, we provide a comparison with the current state of knowledge of the immunoregulatory properties of immature myeloid dendritic cells (MDCs) and propose that the induction of Tr cells by PDC and MDC lineages may represent nonredundant mechanisms in peripheral tolerance. Human T-Regulatory Cell Subsets T-regulatory or suppressor cells were first described in the early 1970s [6]. However, the failure to characterize these cells and to understand their mechanism of sup0198-8859/02/$–see front matter PII S0198-8859(02)00753-X

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TABLE 1 Similarities and differences between subsets of human Tr cells

Characteristics of Tr Anergic Production of IL-10 Production of TGF-␤ Mechanism of suppression Contact-dependent Cytokine-dependent Direct on T cells Indirect via APC

CD25⫹CD4⫹Tr

Tr1

Th3

CD28⫺CD8⫹Tr

⫹ ⫹ ⫹

⫹ ⫹ ⫹

? ? ⫹

⫺ ⫺ ⫺

⫹ ⫺

⫺ ⫹ IL-10/TGF-␤ ⫹ ⫹

⫺ ⫹ TGF-␤ ⫹ ⫹

⫹ ⫺

⫹ ⫺

pression led to the demise of the field in the mid 1980s [7]. More recently, distinct subsets of T-regulatory cells were identified, either by their surface phenotype or by their cytokine secretion profile (Table 1), finally providing a cellular basis for this immunologic paradigm. One of the best-characterized subsets are the CD4⫹ Tr cells with constitutive expression of CD25 [8], which in humans represent 10%–15% of total CD4⫹ T cells [9 –11]. CD25⫹ Tr cells with high affinity to self-peptides are generated in the thymus, where the cortical epithelium has been identified as critical antigen-presenting cell (APC)[12]. CD25⫹ Tr cells have the ability to suppress T-cell immune responses in a contact-dependent manner not through the modulation of APC functions, but based on an antigen (Ag) nonspecific T to T-cell interaction [13]. One hypothesis suggests that after T-cell receptor (TCR)-mediated activation, CD25⫹ Tr cells express cell surface molecules, which mediate suppression by binding to a counter receptor on CD25⫺ cells expressed after TCR ligation. Another subset of CD4⫹ Tr cells named type 1 Tr (Tr1) has been first identified by expanding human T cells in the presence of interleukin (IL)-10 [14]. Tr1 cells suppress T-cell responses through the production of IL-10 and transforming growth factor (TGF)-␤, either by direct modulation of T-cell function or by downregulating the costimulatory capacity of APC [14, 15]. Similarly, there is now evidence that within the human CD8 subset there are Tr cells, which suppress T-cell reactivity by producing IL-10 [16]. Studies of oral tolerance have identified a subset of CD4 T cells exerting regulatory functions through the production of TGF-␤. These cells named T-helper (Th) 3 were induced in patients suffering from multiple sclerosis following oral treatment with myelin basic protein (MBP) and associated with disease remissions [17]. The key role for TGF-␤ in the regulatory activity of Th3 cells was indicated by the suppression of disease induction in murine encephalomyelitis (EAE) model [18]. At present, it is not clear whether Th3 cells induced during oral tolerance and Tr1 cells represent similar or different Tr. Models of

⫺ ⫹

allotranslantation and xenotranslantation have pointed out a suppressive role of CD8⫹ T cells lacking CD28 expression [19]. These CD8⫹CD28- Tr cells are generated in vitro after multiple rounds of antigenic stimulation [20] and may not represent a distinct Tr subset, but rather a terminal differentiation state of CD8 effector T cells [21]. Suppression of primary bystander antigenspecific CD4 T-cell responses is mediated by the inhibition of CD40-ligand mediated upregulation of costimulatory molecules on APC [19]. Recently, it was illustrated that this inhibition was associated with the upregulation of inhibitory receptors ILT3 and ILT4 on APC, induced by CD8⫹CD28⫺ Tr cells [22]. Generation of T-Regulatory Cells by CD40-Ligand Activated PDC Plasmacytoid dendritic cell precursors activated in the presence of IL-3 and CD40-ligand into mature dendritic cells (named DC2), express high levels of MHC products and costimulatory molecules and exhibit potent T-cell stimulatory capacity [23]. A single stimulation with DC2 induces naı¨ve CD4 and CD8 T-cell differentiation into Th2 cells [24] and IL-10-producing CD8 T cells [16], respectively. A more detailed analysis of the effector functions of IL-10-producing CD8 T cells induced by DC2 revealed their inability to proliferate in response to further Ag-specific restimulations, as well as an inability to efficiently lyse Ag-bearing target cells [16]. This state of anergy is induced by autocrine or paracrine T-cellderived IL-10 produced during the primary stimulation (Figure 1A). Importantly, upon Ag-specific restimulation, IL-10 producing CD8 T cells suppress primary T-cell activation through the secretion of IL-10 [16] (Figure 1B). Suppression of bystander T cells may occur regardless of their Ag-specificity, provided that these CD8 Tr cells are restimulated nearby to produce IL-10 (a phenomenon named antigen-driven bystander suppression). Suppression by IL-10 occurs both through inhibition of antigen presentation and through direct effects on T cells [15]. Therefore, Tr cells induced by CD40-ligand

PDCs and Induction of Tr Cells

FIGURE 1 Cluster designation (CD)8 T regulatory (Tr) cells induced by CD40-ligand-activated plasmacytoid-derived dendritic cells (PDC). (A) Priming of anergic IL-10-producing CD8 T cells by CD40-ligand-activated PDC (DC2). DC2 express high levels of costimulatory-molecules and MHC products (A), strongly activate naive CD8 T cell proliferation, and induce a population of 10% to 20% CD8 T cells which produce IL-10. The induction of IL-10 producing CD8 T cells does not occur as default due to the lack of IL-12 but its mechanism is currently unknown (B). Anergy and poor cytolytic ability of DC2-primed CD8 T cells are induced by T cell-derived IL-10 in an autocrine (C) or paracrine (D) fashion and are Ag-specific since unstimulated T cells bearing other Ag-specificities are fully able to mount a primary immune response (E). (B) Suppressor function of CD8 T regulatory cells. Following Ag-specific restimulation (F) CD8 Tr release IL-10 into the microenvironment. IL-10 suppresses the induction of a primary T cell respone acting either directly on the naive T cells (G) or on the APC (H). CD8 Tr may also mediate bystander suppression of T cells bearing other Ag-specificities if activated at the same time in the immediate vicinity (I).

activated DC2 share many similarities with Tr1 cells: (i) they are both anergic, (ii) their generation depends on IL-10, and (iii) they both suppress primary T-cell responses through IL-10. It remains to be established whether the DC2-primed CD4 T cells exhibiting a Th2 cytokine secretion profile [24] contain IL-10-producing Tr1 cells with suppressor activity.

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Generation of T-Regulatory Cells by Immature MDC For years, immature DC, defined by their low expression of costimulatory molecules and lack of IL-12-production, were regarded as inactive in the initiation of T-cell responses [25]. However, recent evidence indicated that repetitive stimulation of naı¨ve CD4 T cells with immature monocyte-derived MDCs resulted in the differentiation of anergic T cells, producing IL-10 and exerting regulatory activity [26]. Although the exact mechanism remains unclear, suppression of T responses is independent of IL-10, requires direct cell contact between Tr cells and responder T cells, and does not occur through downregulation of the costimulatory capacity of APC [26]. The in vivo confirmation was presented by Dhodapkar et al. indicating that subcutaneous injection of immature monocyte-derived MDCs pulsed with MHC class I-restricted influenza matrix peptides induced IL-10producing CD8 Tr cells, which inhibited the Ag-specific effector function of peptide-specific T cells [27, 28]. Similarly to CD4 Tr induced in vitro, suppression by CD8 Tr was contact dependent, therefore largely independent of IL-10 [28]. Thus, immature MDCs induce the differentiation of Tr-cell population with functional properties similar to the described properties of CD25⫹Tr cells (Table 1). Jonuleit et al. suggested that Tr cells differentiated by immature MDCs may represent an activated state of thymic derived CD25⫹ Tr cells [29]. Lineage-Dependent Functional Plasticity of the DC Network Whereas the ability of MDCs to induce Tr-cell differentiation is strictly dependent on their immature phenotype, PDCs can drive Tr-cell differentiation as fully mature cells, if activation is driven by CD40-ligand stimulation (Figure 2). PDC precursors may also promote T-cell tolerance by the induction of IL-10-producing anergic T cells [30], however, their suppressor potential has not been analyzed. Input of danger signals (products of microbial invasion) induces the differentiation of immature MDCs and PDC precursors into mature immunogenic DCs. Pathogens trigger pattern recognition receptors toll-like receptor (TLR)2 and TLR4, but also CD40-ligand stimulation induce IL-12-producing MDC1, which drive Th1 differentiation and strong cytolytic T lymphocytes (CTL) responses [24]. In contrast, pathogens triggering TLR7 and TLR9, but not CD40ligand stimulation, induce interferon (IFN)-␣ production and maturation into immunogenic PDCs, which promote IFN-␥-producing T cells [31–33]. Thus, the balance between immunity and tolerance is determined (I) by a lineage-restricted responsiveness of MDC and the PDC to particular pathogens, which determines whether

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FIGURE 2 Lineage dependent functional plasticity of the dendritic cell (DC) network. Myeloid DC lineage induces T-regulatory (Tr) cells at immature DC differentiation stages, such as resting DCs or DCs activated in the presence of immunomodulatory compounds. Activation by microbial products but also cluster designation (CD)40-ligand induces immunogenic DCs which produce interleukin (IL)-12. Plasmacytoid DC activated by CD40-ligand differentiate into mature DC, which are able to induce Tr differentiation. Activation by microbial products induces immunogenic IFN-␣producing DCs.

maturation is induced and (ii) by the ability of the PDC lineage to develop into a mature but quiescent stage, which promotes Tr-mediated T-cell tolerance [34, 35]. Peripheral Tolerance and Human DC Lineages: Evidence and Speculations Given that immature MDC are disseminated throughout the peripheral tissues and capture Ag from dying cells during normal tissue turnover, their steady-state migration to the draining lymph nodes may promote self-tolerance in vivo [36, 37]. In the absence of activating stimuli, migrating DCs retain their immature phenotype and may induce self-Ag-specific anergic T cells and Tr cells. Two key studies provide the experimental support for this model: (i) Huang et al. illustrated that a subpopulation of rat intestinal DCs constitutively transport apoptotic epithelial cells to T-cell areas of mesenteric lymph nodes [38] and (ii) Hawiger

M. Gilliet and Y.-J. Liu

et al. demonstrated that in vivo targeted immature murine DCs in the absence of activation induced T-cell unresponsiveness [39]. The ability of the PDC lineage to develop into mature tolerogenic DCs, driven by endogenous factors including cytokines and T-cell-derived signals, may represent an important safeguard of self-tolerance in tissue cytokine microenvironments, which could lead to the activation of MDC. A major step forward in the understanding of T-cell tolerance will come from the identification of the in vivo counterpart of CD40ligand activated PDCs (DC2). In transplantation tolerance, PDC precursors may capture host alloantigen, mature into DC2 following activation by CD40-ligand expressing allogeneic T cells, and present the host alloantigen to tolerize the donor T cell by induction of IL-10-producing Tr cells. In contrast, CD40-ligand activated MDCs (DC1) may induce IFN-␥ producing alloantigen-specific Th1 and CTL responses, which will trigger and accelerate graftversus-host disease (GVHD) and allograft rejection. Indeed, a role of DC2 in transplantation tolerance has been provided indirectly by a study illustrating that transplantation of granulocyte colony stimulating factor (G-CSF) mobilized blood cells, which contain increased numbers of PDC precursors, induced less severe GVHD [40]. A key role of IL-10 produced by human Tr1 and CD8 Tr has been characterized in long-term acceptors of human leukocyte antigen (HLA)-mismatched bone marrow graft and kidney transplant [41, 42].

PDCs and Induction of Tr Cells

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TABLE 2 Characteristics of human Tr cells induced by immature MDC versus CD40-ligand-activated PDC Induction by

Immature MDC

CD40L-activated PDC

Phenotype Cytokine profile Primary proliferative activity Responsiveness to restimulation Mechanism of suppression Ag-specificity Relation to known Tr subsets

CD4, CD8 IL10⫹⫹, IL4⫺, IFN␥⫹/⫺ low low Contact-dependent no CD25⫹CD4⫹

CD8, (CD4?) IL10⫹⫹, IL4⫺, IFN␥⫹ strong low Soluble IL-10 Ag-driven bystander suppression Tr1

Are Tr Cells Induced by the Myeloid and the Plasmacytoid Lineage Distinct? Although their state of anergy and the production of IL-10 seems to be a common feature of both Tr cells induced by CD40-ligand activated DDCs (DC2) or immature MDCs, there are important differences in their mechanism of suppression (Table 2). Similar to Tr1, suppression by DC2-primed Tr cells is mediated by the Ag-driven production of IL-10. In contrast, suppression by Tr primed with immature MDCs was completely independent of Ag and required cell contact, therefore resembling CD25⫹ Tr cells. These data suggest that the induction of Tr cells by the PDC and the MDC lineages may represent nonredundant mechanisms in peripheral T-cell tolerance. Further studies are needed to elucidate the relations between Tr1 and CD25⫹Tr cells or DC2primed and immature MDC-primed Tr cells. The molecular characterization of these Tr subsets and the ability to track them in vivo will ultimately tell whether these are distinct subsets with specialized functions in T-cell tolerance [43].

PDC activated in vitro by CD40-ligand (DC2) may have two major advantages: (i) whereas cells mature, DC2 reveal limited plasticity to further stimuli and may therefore retain their tolerogenic potential and (ii) DC2 induce stronger and more rapid expansion and differentiation of Tr cells. Current limitation for vaccination strategies involving DC2 are the lack of protocols to generate sufficient numbers of PDCs for in vivo transfer. Future challenges include the identification of molecules and mechanism employed by DCs to induce Tr cells. A role of costimulation through Serrate1/Notch- [49], CD58-leukocyte function antigen(LFA)-3/CD2⫺ [50], and inducible T-cell costimulator (ICOS)-ligand/ICOS- [51] signaling has been described. Further understanding of these pathways may ultimately lead to the design of new strategies to generate tolerogenic DCs in vitro or to manipulate DCs directly in vivo with the goal to induce Tr cells, which are capable of inhibiting Ag-specific pathogenic T cells with a precision level that only the immune system can achieve.

DCs as Tools to Induce Tr-Mediated T-Cell Tolerance: Future Perspectives The generation of tolerogenic DCs with the ability to induce Tr-cell differentiation opens new therapeutic perspectives for the induction of Ag-specific T-cell tolerance to treat pathologies including autoimmune diseases, GVHD, and organ– graft rejection. In vitro pulsing of tolerogenic DCs with self-antigens or alloantigens followed by in vivo injections may be suitable for the induction of Tr cells capable of downregulating selfreactivity or alloreactivity. The validity of such an approach has been illustrated by a vaccination study using immature MDC [27]. However, caution must exerted, given the inherent plasticity of generated immature MDCs and their propensity to mature. Pharmacologic inhibition of maturation by anti-inflammatory compounds such a corticosteroids, vitamin D3, and IL-10 may prove helpful [44 – 46]. However, concomitant inhibition of DC differention from precursor cells may be counterproductive [46 – 48]. Vaccination strategies using

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PDCs and Induction of Tr Cells

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