Tolerogenic dendritic cells: molecular and cellular mechanisms in transplantation

Transcription

1 Review Tolerogenic dendritic cells: molecular and cellular mechanisms in transplantation Urban Švajger 1 and Primož Rožman Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia RECEIVED JUNE 19, 2013; REVISED AUGUST 22, 2013; ACCEPTED SEPTEMBER 24, DOI: /jlb ABSTRACT During the discovery of mechanisms that govern immune activation and suppression, immune tolerance always came second in the scientific timeline. This has subsequently shaped the advances in the clinical translation of DC therapy protocols used for immunostimulation or immunosuppression. With several hundred clinical trials already registered within the U.S. National Institutes of Health for the use of DCs in cancer vaccination, only a few involve TolDCs for use as negative vaccines. However, as a result of the strong scientific rationale from preclinical and clinical trials, the use of negative vaccination in organ transplantation is likely on its way to reach the extent of the use of positive cancer vaccines in the future. As the underlying mechanisms emerge, the role of DCs in the induction of transplant tolerance is recognized unambiguously as central in the bidirectional communication with various types of immune cells. This is achieved by a complex interplay of numerous tolerogenic signals involving regulatory cytokines and other surface-bound or soluble inhibitory molecules associated with corresponding inhibitory signaling cascades. A detailed understanding of these processes will accelerate the advances of clinical immunologists in translating their knowledge from bench to bedside. In this review, we present the role of TolDCs as well as the most recent findings concerning associated molecular and cellular mechanisms that shape the balance between regulatory and effector immune responses during organ transplantation. J. Leukoc. Biol. 95: 53 69; Introduction Abbreviations: AA-DC alternatively activated DC, CD40L/CD95L CD40/ CD95 ligand, cdc conventional DC, CO carbon monoxide, FasL Fas ligand, FoxP3 forkhead box P3, GVHD graft-versus-host disease, HSCT hematopoietic stem cell transplantation, ICOSL ICOS ligand, ILT Ig-like transcript, itreg inducible regulatory T cell, mdc myeloid DC, MDSC myeloid-derived suppressor cell, MI maintenance immunosuppression, MSC mesenchymal stem cell, PD programmed death, pdc plasmacytoid DC, PDL programmed death ligand, TOL operationally tolerant, TolDC tolerogenic DC, Tr1 type 1 regulatory cell, Treg regulatory T cell, TT tetanus toxoid, vit D 3, vitamin D 3 active form of vitamin D Since their discovery by Steinman and Cohn in 1973 [1], DCs have been progressively established as central players in immunity and tolerance. The term professional APCs was coined as a result of their unique ability to capture and present antigens endogenous and exogenous and present them to antigen-specific T cells. This process is enriched further by a bidirectional communication between DCs and T cells in the form of cytokines and costimulatory and inhibitory molecules. In this way, the T cell not only sees the antigen but is further instructed on how to react upon its recognition, leading to T cell polarization into various effector and regulatory types [2]. Recently, numerous advances have been made in understanding how immunological tolerance is established and maintained during various conditions including transplantation, and DCs undoubtedly play a major role in these processes. This was demonstrated by the constitutive ablation of major DC types (CD11c-cre mice carrying diphtheria toxin -chain, expressed only in DCs), namely, cdcs, pdcs, and Langerhans cells, which led to the spontaneous development of autoimmunity, neutrophilia, autoantibody formation, and high numbers of Th1 and Th17 cells in animal models [3]. Additional studies, depleting individual DC subtypes, will be necessary to determine the contribution of various DC types for immunological tolerance. This can be performed by applying novel depletion methods [4]. Nevertheless, present evidence clearly highlights the general importance of DCs in maintaining immunohomeostasis and is explained further by their functional plasticity. Whereas only some time ago, the classification of DC function was defined by its immature (tolerogenic) and mature (immunogenic) states, it is now known that DCs can adopt several distinct activation states depending on their subtype and environment. Indeed, the exposure of DCs to immunosuppressive signals triggers the development of TolDCs, which are enriched with immunosuppressive characteristics and therefore, exert functions not only of passive (lack of costimulatory signals) but also of active tolerance (presence of inhibitory signals) [5, 6]. Furthermore, several recent studies have demonstrated novel ways to increase the tolerogenic potential of DCs and their ability to induce tolerance in interacting immune cells. By having a detailed understanding of the DC lifecycle and the mechanisms leading to 1. Correspondence: Blood Transfusion Center of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Slovenia. urban.svajger@ztm.si or svajgerurban@yahoo.com /14/ Society for Leukocyte Biology Volume 95, January 2014 Journal of Leukocyte Biology 53

2 their activation, we will better understand not only which factors can be used to induce TolDCs but also how to use them in different contexts of DC types (for example, pdcs vs. cdcs) and the stages of their development. In clinical transplantation, there is a great desire to reduce the dependence on immunosuppressive drugs. These usually serve as general immunosuppressants for the prevention of GVHD and host-versus-graft disease and are associated with numerous side-effects. For this reason, the ability to achieve donor-specific tolerance remains the holy grail of transplant tolerance induction. DCs are well-characterized in the context of organ transplantation, and efforts to use them as immunosuppressive cell therapies are becoming plausible and increasingly intriguing. These efforts are encouraged by a number of successful preclinical studies, accompanied by several ongoing clinical trials using DCs as positive vaccines in cancer treatment. Furthermore, a DC-based vaccine intended for treatment of prostate cancer was registered recently by the U.S. Food and Drug Administration [7]. As a result of many advances in this field, we can expect that in the near future, a significant number of various types of TolDCs will be used with the intention of achieving a tolerance to graft-derived antigens in humans. DCs AND THEIR ROLE IN TOLERANCE INDUCTION One of the main tasks of the immune system is to clearly distinguish the antigenic information into self or nonself. In this way, our bodies are protected from invading microorganisms, whereas at the same time, the immune system does not attack our own tissues. The latter is achieved through complex processes of central and peripheral tolerance. In central tolerance, the negative and positive selection mechanisms allow the maturation of double-positive CD4 CD8 thymocytes with appropriate affinities of their TCRs for self or foreign antigens presented in the thymus. This presentation is mediated by APCs, including DCs, which migrate to the thymus from the periphery [8]. In this way, only lymphocytes with TCRs that are not likely to present the danger of an autoimmune response are selected to enter the periphery. For more in-depth information on central tolerance, the reader is directed to refs. [9 12]. Although central tolerance is a rigorous process that removes most self-reactive lymphocytes, peripheral tolerance mechanisms are crucial to maintain immune homeostasis throughout the life of an individual. Besides naturally occurring Tregs that originate in the thymus, DCs play an important role in providing tolerance induction to antigens when presented under steady-state conditions [13]. This is possible, as tissue-resident, immature DCs, which capture antigens from apoptotic cells, constantly circulate between the organs and lymphoid tissues and present self-antigens in the absence of infection and therefore, with the lack of costimulatory signals [14]. The activation state of these migratory DCs, therefore, causes T cell unresponsiveness and as recently shown, also the induction of Tregs [15, 16]. However, as always in an immune process, there is a strong, functional relationship among many interacting cell types. It has been shown in this manner that it is also the naturally occurring FoxP3 Tregs that help retain the DCs in their steady-state in vivo, in other words, preventing them from their full maturation so they can maintain a peripheral tolerance and prevent autoimmunity [17]. BASIC CHARACTERISTICS OF TolDCs It is now more than 10 years ago when we first began to realize that DCs adopt several alternative or intermediate activation states. In addition to their strongly immunogenic, fully mature and tolerogenic, immature lifecycle extremes, immunologists found that so-called partial maturation also does not induce an effector immune response but rather, a tolerogenic one [18]. This third population was called semimature and was characterized by the high expression of MHC class II and B7 costimulatory molecules but produced little or no proinflammatory cytokines, such as IL-1, IL-6, IL-12p40, and IL- 12p70 or TNF-. The process of semimaturation was shown to be induced by various conditions, including apoptotic cells [19], lactobacilli from the gut [20], the systemic delivery of antigens, such as intranasally applied OVA [21], or cytokines, such as TNF-, when used alone [22] (when used in combination with other proinflammatory cytokines, TNF- indeed causes extensive DC maturation [23, 24]). The theory of semimaturation is attractive, as it also explains, in some way, the phenomenon of steady-state, migratory DCs. It was postulated that during partial maturation, the DCs also up-regulate other molecules associated with full maturation, such as the chemokine receptor CCR7, which directs their migration from tissues to the secondary LNs [25 27]. In addition, molecules that anchor the DCs in tissues, such as E-cadherin or 6-integrins, in the case of skin DCs, are down-regulated, thereby allowing migration. As evidenced above, although semimatured DCs express induced levels of costimulatory molecules, it is the absent production of proinflammatory cytokines, i.e., the lack of signal 3, that determines their ability to induce tolerogenic immune responses. In other words, their main tolerogenic mechanisms rely on their deficit of inflammatory signals. However, we know now that true TolDCs represent a broad variety of immunosuppressive APCs that are characterized by additional elements of active tolerance, such as the production of immunosuppressive cytokines and the expression of inhibitory molecules. Various immunosuppressive environmental factors, such as IL-10; molecules expressed by Tregs, such as CTLA-4; as well as immunosuppressive drugs, can induce a more prominent tolerogenic state of DCs, superior to immature and semimature DCs. Each of these factors, alone or in combination, can induce the development of TolDCs with similar, although not identical, characteristics [5, 28]. The diversity of TolDCs becomes even greater when immunosuppressive signals are combined with maturation-inducing molecules, e.g., the treatment of DCs using a combination of corticosteroids/vit D 3 and LPS [29]. For this reason, in addition to TolDCs and semimature DCs, another term has been coined that is used most frequently when DCs are activated with maturation signals in the 54 Journal of Leukocyte Biology Volume 95, January

3 Švajger and Rožman Tolerogenic dendritic cell mechanisms in transplantation presence of immunosuppressive agents. The resulting DCs have been named AA-DCs [30, 31], which are known to express relatively low levels of costimulatory molecules CD40, CD80, and CD86, as well as MHC class II [31]. On the other hand, they display an increased production of immunosuppressive cytokines, namely IL-10, and mostly retain their expression of inhibitory molecules compared with DCs that are only treated with immunosuppressive signals (unpublished observations). A particular characteristic of AA-DCs is that they can express increased levels of the chemokine receptor CCR7. It has been shown that in this manner, CCR7 is responsive to LN-homing chemokine CCL19 [29]. One of major hallmarks of TolDCs is that they can induce tolerogenic responses to a much greater extent and in a shorter time compared with, e.g., immature DCs (Fig. 1). One of the reasons is their extensive production of immunosuppressive cytokines, such as IL-10 and/or TGF-, which directs the polarization of T cells into Tregs. In addition, these cytokines serve as a positive-feedback loop, further strengthening the immunosuppressive biology of TolDCs [21, 32 34]. Another reason is the ability of TolDCs to express increased levels of various inhibitory molecules, such as ILT-2, ILT-3, and ILT-4, HLA-G, PDL-1, FasL, ICOSL, and others [35 38]. These molecules interact with their corresponding proteins on the Figure 1. Molecular and cellular mechanisms exerted by TolDCs in transplantation. In transplantation tolerance, DCs undoubtedly play a major role and are associated with numerous immune cell types. Several immunoregulatory mechanisms are associated with DCs and involve soluble and surface-bound molecules. In this figure, major known molecular pathways involved in tolerogenic potential of various TolDCs are depicted, as well as their interactions with other immune cell types involved. Volume 95, January 2014 Journal of Leukocyte Biology 55

4 surface of T cells and trigger negative intercellular signaling, often bidirectional, which results in the immunosuppression of the cell types involved. In addition, biomolecules, such as IDO and HO-1, are induced in TolDCs and readily secreted in their environment. IDO serves as a catabolic and a signaling molecule [39]. By catabolizing tryptophan, a major energy source of activated T cells, it suppresses their function and allows for the differentiation of Tregs [40, 41]. HO-1 produces CO by degrading heme, thereby inhibiting the DC function and T cells as well [42, 43]. Differences in the nomenclature of various TolDC types can be confusing, so we outlined these distinctions in Fig. 2, as they have appeared throughout the literature. It is, however, hard to make these separations in practice as a result of the many flavors of tolerogenicity and is thus easier to regard to immunogenic DCs or TolDCs with corresponding characteristics. Nevertheless, in preclinical transplantation models, several TolDC types have been shown to benefit graft survival and promote long-term tolerance induction [44 48]. For these reasons and for the ease of understanding, we will regard, in this paper, all DCs with induced tolerogenic activation states as TolDCs. INDUCED AND NATURAL SUBSETS OF TolDCs Induced TolDCs As the notion of the tolerogenic potential of the DC became clear, there was considerable interest in discovering how its tolerogenic state is established in vitro and in vivo. Besides Figure 2. Various tolerogenic activation states of DCs. Through numerous studies during the past, various DCs with immunosuppressive properties were defined and named most frequently according to their corresponding activation states and methods of generation (immature and semimature DCs, TolDCs, and AA-DCs). DCs can be activated in different ways, which can also lead to their increased tolerogenic potential. In the present manuscruipt, we designate all mdc types with immunosuppressive properties as TolDCs, except immature DCs. However, to clarify the existing nomenclature, the differences between various TolDCs types and their major characteristics are depicted in the figure. poly I:C, Polyinosinic:polycytidylic acid. 56 Journal of Leukocyte Biology Volume 95, January

5 Švajger and Rožman Tolerogenic dendritic cell mechanisms in transplantation learning of the immunosuppressive potential of immature DCs, it was soon discovered that their exposure to ril-10, when administered at 40 ng/ml or above, greatly increased their tolerogenic potential [49]. Such cells displayed a strongly reduced capacity to stimulate allogeneic CD4 T cells and could induce a state of alloantigen-specific anergy. Furthermore, they also affected CD8 T cells, inhibiting their cytotoxic and anti-tumor function and also had the ability to generate CD4 and CD8 T cells with a suppressive capacity [50, 51]. Such characteristics are important for the potential regulation of GVHD in HSCTs, where donor T cells are considered the major cause of the disease [52]. Through the years, it has been discovered that several additional factors can induce tolerogenic characteristics in DCs, some more superior than others. Among these are endogenous biomolecules, such as the TGF-, VEGF, neuropeptides, histamine, glucosamine, glucocorticoids, and 1,25-dihydroxyvit D 3 ; immunosuppressants and anti-inflammatory drugs, such as cyclosporine, rapamycin, tacrolimus, niflumic acid, aspirin, and resveratrol; and many others. The characteristics of individual TolDCs obtained by various treatments are described thoroughly in the refs. [5, 53 62]. Natural DC subsets with tolerogenic properties mdcs. The immunostimulatory capacity of DCs is, to a large extent, dependent on their residential location in the body. In addition to steady-state, immature DCs, those present in mucosal or immune-privileged sites display induced tolerogenic characteristics [63]. In this context, DCs that were isolated from the lungs [21], the small intestine lamina propria [64, 65], or the eye [66] displayed a high production of immunosuppressive cytokines and the ability to induce Tregs. Another example is a population of tolerogenic CD11c low, CD45RB high DCs found in spleen and LNs of normal mice that produces extensive amounts of IL-10 and is able to induce Tr1 cells in vitro and in vivo [33]. As the characteristics of tolerogenic mdcs have been reviewed above, we will further outline just the recent findings. Recently, novel TolDC types have been found in vivo. A group led by M. G. Roncarolo [67] identified and characterized a subset of IL-10-producing human DCs, termed DC-10, which are present in vivo and are characterized as CD1a, CD1c, CD14, CD16, CD11c, CD11b, HLA-DR, and CD83. In addition, DC-10 expresses inhibitory molecules ILT-2, ILT-3, ILT-4, and HLA-G, as well as the costimulatory molecules CD40 and CD86, and possesses extensive tolerogenic functions. They were potent inducers of IL-10-secreting regulatory Tr1 cells, a process that required IL-10 and the interactions between ILT-4 and HLA-G [67]. Interestingly, DCs with similar characteristics could also be induced in vitro by culturing human monocytes with GM-CSF, IL-4, and IL-10. As the effects of IL-10 on DCs were studied mostly on DCs already established, this observation highlights the importance of how affecting different stages in the DC lifecycle can influence their final phenotype and function. As a result of the extensive tolerogenic characteristics of DC-10, the involvement of the HLA-G/ILT-4 pathway in transplantation (described below), and the possibility of generating them in vitro, it would be interesting to see their capacity to prolong the acceptance of allogeneic grafts or regulate GVHD in animal models. Another group found that TolDCs can be generated in vivo when influenced by estriol. Such cells have an increased expression of PDL-1 and -2 and B7-H3 and -H4 inhibitory molecules in mice [68]. These TolDCs were suppressive in vivo, preventing the onset of experimental autoimmune encephalomyelitis after transfer. pdcs with tolerogenic properties. In comparison with DCs of the myeloid lineage, pdcs display a general bias toward favoring the induction of Treg responses [69]. This is a result of several differences, mostly regarding antigen presentation, their maturation characteristics, and the subsequent expression of different costimulatory molecules. The less-effective antigen presentation is a result of differences in MHC class II synthesis, ubiquitination, and the expression of MHC class II ubiquitin ligase (membrane-associated ring finger[ch] protein I), during and after maturation compared with mdcs [70]. This durable turnover enables pdcs to present endogenous viral antigens constantly but makes them more inefficient in the presentation of exogenous antigens. The subsequent weak TCR stimulation is speculated to promote Treg induction. Additionally, pdcs can deliver much less costimulation to responding T cells as a result of a lower expression of CD80 and CD86 and higher levels of PDL-1 upon maturation [71]. The expression of PDL-1 on pdcs was shown recently to be dependent on the autocrine activity of IL-27, which induces PDL-1 expression in a STAT3-dependent manner [72]. Expression of functional IDO is another immunoregulatory mechanism of pdcs [73]. Fallarino et al. [74] demonstrated that murine pdcs display induced levels of IDO upon the ligation of CD200R, a natural receptor for cell-surface glycoprotein OX-2 (CD200). Indeed, a role for pdcs has been established for solid organ transplant tolerance. Cardiac allograft survival was prolonged by the infusion of pdcs in combination with anti-cd40l therapy [75, 76], and similar results were obtained for allogeneic HSCTs [77 79]. Furthermore, it was also shown that CD8 DCs, in contrast to their myeloid CD8 counterparts, can prolong transplant survival significantly in the absence of immunosuppressive therapy when administered before transplantation [80]. In a recent case, a subset of CD8 DCs was characterized in mice and found to be CD103, CD207. This subset was found mainly in the marginal zone instead of T cell areas and proved crucial for tolerance induction by apoptotic cell clearance [81]. Ochando et al. [82] demonstrated that pdcs, circulating through the blood, can pick up alloantigens from vascularized transplants. These alloantigen-presenting pdcs then migrate to recipient secondary LNs and induce the generation of CD4 CD25 FoxP3 CCR4 Tregs. pdcs have also been demonstrated to be essential in suppressing asthmatic reactions to harmless antigens [83]. A subset of pdcs in lymphoid tissues, characterized by the expression of the chemokine receptor CCR9 and an immature phenotype, was discovered recently to exert tolerogenic functions. These CCR9 pdcs expressed low levels of CD40, CD80, and CD86 and migrated to the CCL25, a chemokine that di- Volume 95, January 2014 Journal of Leukocyte Biology 57

6 rects DCs and T cells to the gut [84]. Their priming capacity of CD4 T cells was low in vitro and in vivo. Additionally, they induced fewer activated FoxP3 CD4 CD25 T cells than their CCR9 counterparts. Instead, they induced a predominant population of FoxP3 CD4 CD25 that phenotypically and functionally resembled Tregs. In an animal model of a bone marrow transplant, CCR9 pdcs suppressed the GVHD effect extensively, which resulted in 100% overall survival and the amelioration of clinical signs, such as diarrhea and weight loss. The cellular mechanisms of GVHD suppression were ascribed to the suppression of effector T cell responses (particularly the production of IL-17) and the de novo induction of FoxP3 Tregs [84]. In humans, correlations have been found between increased pdc blood levels and operational tolerance in many cases. This was, most often, seen in cases of liver transplantation, most likely as a result of the fact that these patients most frequently develop graft tolerance [85, 86]. MOLECULES AND MECHANISMS INVOLVED IN TolDC-DEPENDENT TRANSPLANTATION TOLERANCE Although we still have to learn many things about how T cell responses are silenced in various transplantation settings, the role of immunosuppressive cytokines and inhibitory and other tolerogenic molecules in inducing graft acceptance has been explained reasonably well. We can say with certainty that transplantation tolerance is associated with multiple immunoregulatory mechanisms that evolve over time and is associated with different cell types throughout the tolerogenic time span. Below, we outline the most recent findings concerning various immunomodulatory molecules and their mechanisms, which are summarized in Tables 1 and 2. IL-10, TGF-, and IDO In general. Contrary to immature DCs, whose tolerogenicity is based mostly on the absence of adequate costimulatory signals, TolDCs possess several elements of active tolerance. Their ability to produce extensive amounts of immunosuppressive cytokines, such as IL-10 and TGF-, is crucial in the bidirectional communication with responding T cells, as these cytokines can direct the polarization of naive T cells into Tregs. Indeed, IL-10 and TGF- can tolerize T cells in vitro or in vivo [87, 88]. IL-10, in particular, is crucial for the de novo differentiation of IL-10-secreting Tr1 [89]. It is therefore suggested that the ability of TolDCs to secrete IL-10 in the steady-state or after activation determines their effectiveness in inducing Tr1 polarization in the manner of the extent and time needed for this response to occur [90]. Another important feature of TABLE 1. Description of Soluble Molecular Factors Associated with TolDCs Molecule Description/function Cellular mechanism Relevance to transplantation Reference IL-10 TGF- Associated with: mdcs and pdcs General immunosuppressive cytokine Affects all major immune cell types Associated with: mdcs and pdcs 2 Th1 and inflammatory responses, DC maturation 1 Tr1 cells, 1 TolDCs 2 GVHD in animal models DC maturation, Renal allograft tolerance early ( 100 days post-transplant) 2 Lymphocyte activation Regulation of graft-reactive 1 FoxP3 expression T cells General immunsouppressive cytokine IFN- Associated with: mdcs 1 IDO Heart allograft, skin allograft tolerance Pleiotropic cytokine 1 ILT-4 and HLA-G Promotes GVHD in the Immunosuppressive in certain Supports FoxP3 Treg function absence of PDL-1/PD-1 conditions signaling IL-27 Associated with: mdcs 1 IL-10 secretion from Th1 cells and Experimental cardiac FoxP3 Tregs allografts cooperation Regulation of T and B cell Suppression of Th17 responses together with TGF- function with TGF- IDO Associated with: mdcs and Tryptophan starvation of effector T cells pdcs Beneficial effects in several animal models: bone marrow, lung, heart, liver, kidney, and skin transplants Catabolizes tryptohan 2 Cytotoxic CD8 function Associated with long-term Augments FoxP3 Treg function graft acceptance HO-1 Associated with: mdcs 2 DC maturation Prolongs allograft survival in animal models Catabolizes heme 1 IL-10 production by DCs Associated with long-term Regulation of DC and T cell Augments Treg function graft acceptance function 87, 94, , 96, , Journal of Leukocyte Biology Volume 95, January

7 Švajger and Rožman Tolerogenic dendritic cell mechanisms in transplantation TABLE 2. Description of Surface-Bound Molecular Factors Associated with TolDCs Molecule Description/function Cellular mechanism ILT-2, ILT-4 Associated with: mdcs Negative intercellular signaling Inhibitory molecules with 2 T cell alloproliferation intercellular ITIM motifs, 1 Generation of IL-10- HLA-G ligands secreting T cells HLA-G (both surface-bound and secreted forms) PDL-1 (B7-H1) ICOSL (B7-H2) Fas/FasL (CD95/CD95L) Associated with: mdcs MHC class I inhibitory molecule Tolerogenic function associated with pregnancy, inflammation, cancer, transplantation, etc. Associated with: mdcs and pdcs Inhibitory molecule important in regulating T cell activation/proliferation Associated with: mdcs (immature) and pdcs Costimulatory molecule expressed on immature DCs and pdcs Regulates T cell activation by interaction with ICOS Associated with: mdcs Exerts immunosuppression by signaling via ILT-2 and ILT-4 Relevance to transplantation tolerance Mechanisms associated with HLA-G Increased expression in tolerant patients 2 NK and T cell function Correlation with increased FoxP3 2 DC maturation Treg activity in tolerant patients Binds to PD-1 Induces ITIM-dependent signaling Blocks T cell proliferation and cytokine production Prevents stable DC T cell contacts Associated with increased FoxP3 Treg numbers and function Stabilizes IL-10R expression on T cells [uarrow ]Generation of Tr1 cells Activation of caspase cascade Increased PDL-1/CD86 ration in tolerant patients Neutralization with antibody inhibits GVHD suppression and Treg function Reference 67, , , Not yet documented 38, Increased expression on DCs associated with lower GVHD incidence Apoptosis-inducing molecule 1 Apoptosis of target cells Prolonged survival of Belongs to TNF superfamily cardiac alografts 47, IL-10 is that it serves as a positive feedback loop, potentiating the tolerogenic circuit in an auto/paracrine manner by influencing the DCs and other immune cells in the microenvironment, such as Th1 cells, NK cells, macrophages, and others [91 93]. As briefly mentioned above, IDO is frequently associated with the immunosuppressive mechanisms of TolDCs. The immunosuppressive effects of IDO are exerted on multiple cell types, including T cells and DCs. Its effects lead to starvation of activated T cells, thereby inhibition of proliferation and increased T cell apoptosis. On the other side, IDO contributes to induction/maintainance of Tregs and TolDCs [95]. In clinical immunology, the involvement of IDO has been documented as important for tolerance induction during pregnancy, infection, malignant diseases, autoimmunity, and transplantation [ ]. In transplantation. Tregs, induced by IL-10 or TGF-, can reduce GVHD significantly when transferred to recipients [87, 88]. One of the initial studies implementing DCs in graft tolerance was performed in cases where renal allografts of fully mismatched, nonimmunosuppressed mice were accepted spontaneously in some strain combinations. The development of DCs with tolerogenic properties was suggested as one of the main mechanisms, including the involvement of TGF- and IDO [94]. In this scenario, grafts are tolerated in a two-step time course, where early escape from rejection is associated with the regulation of graft-reactive T cells by the transient TGF- dependent mechanism. The expression of TGF- was also associated with the increased presence of FoxP3 Tregs. This is expected, as TGF- is known to convert naive CD4 CD25 T cells into CD4 CD25 FoxP3 T cells with a regulatory function [95]. However, at later time-points ( 150 days post-trans- Volume 95, January 2014 Journal of Leukocyte Biology 59

8 plant), the neutralization of TGF- with antibodies did not restore alloreactivity and concomitantly, an increased IDO expressed by TolDCs was observed in the accepted allografts [94]. This suggests that the development of TolDCs is a crucial step in the development of a long-term tolerance to allografts. Furthermore, IDO-competent TolDCs have been shown to augment the function of CD4 CD25 FoxP3 Tregs in an allograft setting, confirming a well-known synergy between both cell types [107, 108]. The role of TGF- must, however, not be looked on simply as pro-tolerogenic. Namely, TGF- is involved in development of Th17 cells, which are important inflammatory players in transplantation. It is therefore a common factor associated with the Th17 Treg axis [166]. Furthermore, Tregs are known for their plasticity and can be converted to Th17 cells in the presence of inflammatory cytokines. It is likely that the quantitative balance between TGF- and other inflammatory cytokines finally determines the fate of T cell polarization, favoring tolerance or rejection [103]. These facts must be taken into consideration when translating basic knowledge, mostly as a result of the highly inflammatory environment surrounding transplanted organs and tissues. The importance of IDO is by now well-documented in transplant-related research, and its immunosuppressive properties have been described in a variety of animal models, including bone marrow [109], lung [110, 111], heart [112], liver [113], kidney [93], and skin transplantation [114]. IDO is also strongly associated with the suppression of the cytotoxic CD8 T cell function. A recent study conducted on a rodent lung transplant model showed that graft-infiltrating effector CD8 lymphocytes occur in greater numbers than CD4 T cells and are the major effector cell for the acute rejection of lung transplants [115]. In this context, IDO, induced locally by a human transgene in the rat lung allograft, prevented CD8 T cells in the graft from attacking the allogeneic donor lung cells in vivo and allogeneic target cells in vitro [116]. Pleiotropic cytokines In general. In addition to well-known immunosuppressive cytokines, such as IL-10 and TGF-, there is growing proof that certain cytokines, such as IFN-, have paradoxical roles, supporting tolerance and inflammation, depending on the specific immunological environment and the time-frame of an immune response. IFN- is known for its pleiotropicity, supporting, for example, macrophage activation and the function of CD4 CD25 Tregs [167]. IFN- is also a major inducer of IDO and capable of inducing IDO competence in DCs [96]. IL-27 was also identified as an important product of TolDCs and is known to induce IL-10 production in T cells [104, 105]. Just recently, the IL-27/IL-10 immunoregulatory axis was explained further by a study showing that IL-27 induces a high IL-10 production from the IFN- Th1 effectors as a mechanism of self-control during infection [106]. In transplantation. IFN- has been shown to promote GVHD in the absence of PDL-1/PD-1 signaling [96]. In contrast, it has been shown in experimental rat heart allograft models that the graft tolerance induced by TolDC-based therapy correlated with a threefold increase of IFN- in the spleen [98]. The blockade of IFN- led to graft rejection with a mean survival time of 25 days. The major source of IFN- is the double-negative T cells. The induction of IFN- was achieved upon contact with TolDCs in an EBV-induced gene (EBI-3) manner, which codes for a part of the cytokinic chain of IL-27 and IL-35 [99, 100]. In a model of allogeneic skin grafts, the neutralization of IFN- simultaneously with an adoptive transfer of alloantigen-specific Tregs resulted in skin graft necrosis in all recipients [101]. In addition to these findings, we demonstrated just recently that when present in high concentrations, similar to those produced by Th1 cells, IFN- induces a specific activation program of DCs in the absence of danger signals [102]. This activation state is characterized by extensive expression of inhibitory ILT-4 and HLA-G molecules on DCs. Such -high DCs possess extremely low allostimulatory capacity and are unable to induce further Th1 polarization and production of IL- 12p70, even upon CD40 ligation. Furthermore, -high DCs are very efficient in preventing the activation of cytotoxic CD8 T cells, which are known to be crucial in acute and chronic graft rejection [115, 168, 169]. We have shown that silencing of cytotoxic T cells is clearly apparent through down-regulation of granzyme B expression and suppression of proliferation. Both effects can be reversed almost completely by disrupting the HLA-G/ILT-4/ILT-2 pathway using blocking antibodies [102]. We can speculate that similar regulatory mechanisms can occur in transplant settings, where high presence of IFN- in the environment is a frequent characteristic. The immunoregulatory role of IL-27 also extends into transplantion. In a study of experimental cardiac allografts, the effects of TGF- and IL-27 were important for the inhibition of the Th17 function, while supporting the activity of CD4 CD25 Tregs and the allograft survival. As expected, the main effect of TGF- was seen in the induction of the FoxP3 Tregs. On the other hand, IL-27 did not induce the Treg expansion but rather, their secretion of IL-10 [103]. Such studies highlight the importance of immune regulation via negative-feedback axes containing elements with well-known proinflammatory characteristics. HO-1 In general. The degradation of heme is catalyzed by an enzyme HO-1, which results in subsequent release of iron, biliverdin, and CO. HO-1 has been shown an immunosuppressive role on many occasions and is now known to mediate active tolerance by TolDCs, inhibiting T cell responses [43]. The release of CO represents a major tolerogenic mechanism that works via numerous pathways. It triggers the expression of peroxisome-proliferator-actvated receptor-, which can down-regulate the expression of proinflammatory genes induced by TLRs. Another signaling pathway subsequently mediating immunosuppression is the activation of hypoxia-inducible factor-1 [117]. DCs expressing HO-1 have reduced ability to mature properly but at the same time, are able to produce IL-10 [118]. In addition, CO, as produced via HO-1, represents an important immunosuppressive mechanism of CD4 CD25 Tregs [119]. In transplantation. HO-1 has been demonstrated to be an essential mechanism of supporting tolerance induction for 60 Journal of Leukocyte Biology Volume 95, January

9 Švajger and Rožman Tolerogenic dendritic cell mechanisms in transplantation transplanted organs. It has been implicated in induction of tolerance following donor-specific transfusion in mouse models [120]. In this context, induction of HO-1 by cobalt protoporhyrin led to induction and maintainance of tolerance for allogeneic heart transplants [120]. Expression of HO-1 was demonstrated as significant for the immunosuppressive effect of MSCs. Injection of MSCs into recipients of heart allografts prolonged graft survival significantly. The sole inhibition of HO-1 was sufficient to block their tolerogenic effect [121]. In vitro, TolDCs from nonhuman primates and other animals were generated with immunosuppressive properties linked to induced expression of HO-1. They all displayed the ability to suppress proliferation of allogeneic T cells. In rats, such TolDCs, sygeneic to the recipient, prolonged the survival of cardiac allografts. The induction of graft tolerance could be reversed by a HO-1 inhibitor [43]. HLA-G and ILT-2/ILT-4 In general. Besides immunosuppressive cytokines, inhibitory molecules, expressed on the DC surface or secreted in the environment, have been associated with their tolerogenic functions. One of those is the family of receptors called ILTs, which are structurally and functionally related to killer inhibitory receptors [170, 171]. Certain isoforms of ILTs have a long cytoplasmic tail that contains ITIMs. These receptors mediate negative intracellular signaling upon activation by recruiting tyrosine phosphatase Src homology-2-containing tyrosine phosphatase 1 [172]. DCs express several inhibitory ILT receptors with ILT-3 and ILT-4, as well as ILT-2, most frequently associated with their tolerogenicity [67, 122, 123]. They can be induced by IL-10, HLA-G, tryptophan deprivation, Tregs, and also by certain immunosuppressive pharmacological agents, namely vit D 3 and niflumic acid [61, ]. A nonclassical MHC class I inhibitory molecule, called HLA-G, is strongly associated with the function of ILT-2 and ILT-4 and has been shown to be the receptor for both ligands [173]. It is called nonclassical as a result of its low polymorphism, restricted tissue distribution, and immunosuppressive characteristics. HLA-G is readily expressed on TolDCs in a membrane-bound or soluble form and is associated with immune regulatory mechanisms involved in pregnancy, inflammatory diseases, cancer, viral infections, and transplantation [ ]. HLA-G exerts its immunosuppressive function by activating signaling through ILT-2 and ILT-4. Mechanisms of TolDCs associated with the HLA-G/ILT-2/ ILT-4 pathway are known to be involved in the inhibition of NK and T cell function, as well as the suppression of DC maturation [ ]. In transplantation. It has been demonstrated that HLA-G expression by DCs suppresses the T cell alloproliferation that depends on the engagement of ILT-2 and ILT-4 [134]. After this engagement, T cells display a lower expression of CD4 and CD8 and have a suppressive function associated with IL- 10. They have been detected in the blood of transplant patients with high HLA-G plasma concentrations [135]. In addition, DCs expressing HLA-G/ILT-2/ILT-4 and IL-10 have been shown as potent inducers of Tr1 [67]. A high expression of HLA-G has been associated frequently with positive outcomes in graft acceptance [ ]. The involvement of HLA-G was suspected in patients treated with CTLA4-Ig (Belatacept), a drug efficient in preventing acute rejection after renal transplantation. After treatment, the patients displayed elevated plasma concentrations of soluble HLA-G [141]. CTLA4-Ig acts by interacting with CD80/86 costimulatory molecules on DCs. By this interaction, it not only steals signal 2, normally delivered to responding T cells, but also induces the IDO expression in the DCs and their ability to produce HLA-G during an allogeneic challenge. This may explain the progress in graft acceptance observed in Belatacept-treated patients compared with cyclosporine [177]. As described above, HLA-G is readily recognized by ILT-2 and ILT-4 inhibitory molecules, and their interaction serves as a pathway for the inhibition of T cell alloproliferation and the generation of IL-10-secreting Tregs [67, 134]. A group by A. W. Thomson [133] analyzed the expression of HLA-G and ILT-4 on circulating mdcs and pdcs obtained from different groups of liver transplant patients. They demonstrated that in operationally TOL patients, the expression of HLA-G on mdcs, not pdcs, is significantly higher than in patients receiving MI. Additionally, TOL patients had elevated levels of CD4 CD25 high CD127 Tregs and an induced expression of FoxP3. They have found a significant correlation between HLA-G expression on mdcs and that of FoxP3, suggesting the possible role of HLA-G in immune regulation by the host FoxP3 Tregs. ICOSL In general. Structurally resembling important costimulatory molecules expressed on T cells, namely CD28 and CTLA-4, the ICOS, as the name implies, needs to be de novo-induced on the T cell surface [149]. After activation with the ICOSL, it can strongly induce the synthesis of IL-10 in T cells, which makes the ICOS/ICOSL pathway an important immunoregulatory mechanism involving IL-10-mediated negative feedback and induction of Tr1 cells [150]. As a member of the B7 family of costimulatory molecules, ICOSL (also designated as B7- H2) shares an 20% sequence identity with CD80 and CD86 [151]. The expression of ICOSL is characteristic for many immune cell types and is expressed on mdcs, particularly in their immature state [38]. This is not true in the case of pdcs, where the ICOSL expression is high, even after activation with various maturation signals [150]. In transplantation. The importance of the expression of the ICOSL on pdcs or mdcs needs to be analyzed further in the context of potential benefit for graft tolerance induction. Whereas the ICOSL-dependent negative regulation of T cell activation is well-documented, the blockade of the ICOS/ ICOSL pathway in vivo by neutralizing antibodies resulted in the inhibition of chronic rejection [152]. PDL-1 In general. Another important molecule associated with TolDCs is the PDL-1. Along with the PDL-2, it binds its receptor, the PD-1, an inhibitory molecule expressed mainly on activated T cells. The blockade of the PDL-1 and PDL-2 results in Volume 95, January 2014 Journal of Leukocyte Biology 61

10 an enhanced T cell proliferation and cytokine production [142]. The ligation of the PD-1 has been shown to activate the intracellular ITIM motif, and mice deficient in the PD-1 develop autoimmune disorders, suggesting a role for the PDL-1/ PD-1 pathway in peripheral tolerance [143, 144]. Another suppression mechanism of T cells by the PDL-1/PD-1 pathway was recently reported to rely on the blockade of the TCR-induced stop signal and thereby, to prevent stable conjugates between the T cells and the antigen-bearing DCs as a result of the increased T cell motility [145]. In transplantation. An analysis of the B7 family of stimulatory/ regulatory molecules on circulating DC subsets in liver-transplant patients found an increase in the PDL-1:CD86 ratio in TOL patients compared with those receiving MI. The induced expression of PDL-1 was, however, not found on the mdc but on the pdc population. The induced PDL-1:CD86 ratio correlated with an increased FoxP3 Treg frequency [85]. The importance of the PDL-1/PD-1 pathway for FoxP3 Tregs was highlighted further in a transplantation model, where the use of the anti-pdl-1 antibody inhibited the Treg function and the suppression of GVHD [146]. In another study of xenogeneic GVHD, human monocytederived DCs, tolerized by isolated Tregs, displayed a reduced ability to initiate GVHD and prevented death after transplantation (infusion of ex vivo-generated, Treg-conditioned DCs). Tregs, which expressed the PDL-1, induced, in turn, the expression of PDL-1 on DCs after coculture. The blockade of PDL-1 by neutralizing antibodies prevented the immunosuppressive effect of Tregs and the generation of TolDCs [147]. These results highlight the bidirectional communication between the Tregs and DCs in establishing transplantation tolerance and the importance of surface-bound inhibitory molecules to spread the tolerogenic effect to other cell types. The expression of PDL-1 is, however, not limited to DCs but can also be found on tissue cells, where it also plays an important role in controlling the activity of self-reactive effector T cells [148]. Fas/FasL pathway In general. Activation-induced cell death is one of the primary homeostatic mechanisms used by the immune system to control the responses of activated T cells [178]. The process involves the induction of apoptosis to limit the pool size of activated T cells by a Fas/FasL pathway [153, 154]. Fas, also known as CD95, is a trimeric receptor that binds to the FasL (CD95L). Upon the receptor activation on the cell surface, a signaling cascade that involves the hierarchical activation of caspases transduces the death signal and causes the apoptosis of the target cell [ ]. The FasL, as well as Fas, belongs to the TNF superfamily. It is expressed particularly on activated T cells and cells present in immune-privileged sites, such as epithelial cells in the eye [158]. Other cells of the immune system, like B cells, NK cells, and APCs, are also involved in immune regulation by the Fas/FasL pathway. It has been shown that murine splenic CD8 DCs can express high levels of the FasL and can cause apoptosis of the responding T cells [159]. In transplantation. The involvement of the Fas/FasL pathway in protection from allograft rejection has been documented on several occasions. A recent study has highlighted the potential mechanism of less-severe GVHD after the transplantation of allogeneic cord blood. Its use for hematopoitetic reconstitution is increasing lately and also associated with a lower incidence of GVHD. It has been found that the expression of the FasL on DCs isolated from cord blood is significantly higher than on DCs from peripheral blood [160, 161]. In addition, cord blood DCs augmented the apoptosis of the T cells and had a low capacity to induce the proliferation of allogeneic T cells. Previously, an animal model study of allogeneic bone marrow transplantation showed that the induction of tolerance upon transplantation is dependent on the expression of the FasL on the infused donor cells [162]. To study further the possibility of DC involvement in Fas-dependent allograft tolerance, DCs have been genetically modified to express the FasL. The transfection of DCs markedly increased their capacity to induce apoptosis of the Fas-bearing cells. A transfusion of FasL-transfected DCs significantly prolonged the survival of the fully MHC-mismatched, vascularized cardiac allografts [47]. Arginase expression as a potential mechanism Above, we have outlined TolDC mechanisms associated directly with DC tolerance in the context of transplantation. The expression of enzyme arginase by tumor-associated leukocytes is frequently associated with immune suppression and tumor evasion [179]. In liver transplantation, the activity of arginase is important to prevent ischemia/reperfusion injury as a result of hepatoprotective effects of de novo-synthesized NO [180]. To our knowledge, there is yet no direct evidence of TolDCmediated immunosuppression in graft tolerance mediated by the activity of arginase. However, some evidence exists, demonstrating that arginase might contribute to increased tolerance in certain transplantation settings. In a recent study by Highfill et al. [181], arginase activity by MDSCs, derived from bone marrow, was shown to play an important role in reducing GVHD in an allogeneic setting. The mechanism of suppression depended on L-arginine depletion by the arginase, and suppression was blocked by an arginase-specific inhibitor. Furthermore, in vivo administration of human arginase into mice also resulted in depletion of L-arginine and suppression of GVHD [181]. Whether arginase activity in DCs in the context of transplantation settings also plays a role for graft tolerance induction, remains to be defined. A tumor microenvironment favors the development of arginase-competent cell types, such as MDSCs. However, the induction of arginase competence might not be exclusive for tumors. Proinflammatory components, such as PGE2, potentially released at the inflamed transplant site, could induce arginase activity in immune cells, potentially influencing graft acceptance [182]. THE BIDIRECTIONAL INTERACTION BETWEEN T CELLS AND TolDCs FoxP3 Tregs and TolDCs DCs have a central role in the bidirectional communication with naturally occurring CD4 CD25 FoxP3 Tregs, as well as 62 Journal of Leukocyte Biology Volume 95, January