The Dark Side of Dendritic Cells: Development and Exploitation of Tolerogenic Activity That Favor Tumor Outgrowth and Immune Escape

Dendritic cells (DC) play a central role in the regulation of the immune responses by providing the information needed to decide between tolerance, ignorance, or active responses. For this reason different therapies aim at manipulating DC to obtain the desired response, such as enhanced cell-mediated toxicity against tumor and infected cells or the induction of tolerance in autoimmunity and transplantation. In the last decade studies performed in these settings have started to identify (some) molecules/factors involved in the acquisition of a tolerogenic DC phenotype as well as the underlying mechanisms of their regulatory function on different immune cell populations.


INTRODUCTION
The immune system evolved with the difficult task of preserving the integrity of the "self," while protecting it from "non-self " and/or dangerous invaders, thus finding the right balance between aggression and tolerance. A central role in orchestrating the different immune cell subpopulations is played by dendritic cells (DC), the major professional antigen presenting cells (APC). Over the last two decades, many different DC subsets have been identified and classified into myeloid DC, which comprise all monocytederived cells and blood-resident CD1c + DC and into plasmacytoid DC (PDC).
A particularly difficult task for the immune system is to fight tumors, since they derive from the "self," but based on their high proliferative potential they are dangerous for the survival of the host. Moreover, due to the high mutation rates of tumor cells the selection pressure posed by an immune response can result in tumor immunoediting with the outgrowth of immune escape variants or the induction of a suppressive microenvironment. In line with the central role of DC in balancing response versus tolerance, many of the immune escape mechanisms displayed by cancer cells affect DC. These include alterations in the frequency and/or function of circulating and tumor-infiltrating DC in patients with tumors of different histologies. In particular, DC in cancer patients can be affected in their differentiation capacity, with either enhanced apoptosis or skewed phenotype toward immature cells with suppressive properties collectively named myeloid derived suppressor cells [MDSC; (1,2)], in their ability to process and/or present tumor-associated antigens (TAA) and in their ability to interact with effector cells, e.g., to activate and/or correctly polarize them.
Studies performed with DC differentiated in vitro in the presence of tumor cells or of their conditioned medium as well as with purified tumor-infiltrating DC identified the underlying mechanisms responsible for such alterations leading to a pro-tumorigenic phenotype. This review summarizes the known processes employed by tumor cells to subvert professional APC (summarized in Table 1) and how the increasing knowledge can not only help in fighting cancer, but also in inducing tolerance to transplanted organs and suppression of autoimmune diseases.

MYELOID DC AND CANCER
Tumor cells can influence the phenotype and function of myeloid cells at different time points of their life and with distinct mechanisms. These include the metabolic shift of tumor cells toward the anaerobic glycolytic pathway for glucose degradation resulting in increased concentrations of extracellular lactate and an acidification of the microenvironment, the so-called Warburg effect (3). Monocytes cultured in vitro in the presence of lactate and low pH have shown an impaired differentiation toward DC favoring either an expansion of MDSC (4) or of macrophages that promote a Th17 polarization (5). Despite prolonged incubation in the presence of lactate impairs DC responsiveness to lipopolysaccharide [LPS; (6)], a transient exposition promotes DC maturation and enhances their ability to induce a type 1 immune response (7). In addition to pH alterations, the tumor microenvironment is characterized by hypoxia that skews DC toward a type 2 polarization (8), reduces their ability to uptake antigens (Ag), and alters their migratory properties (9).
In addition, expression of hyaluronan (HA), a component of the extracellular matrix of the tumor stroma, correlates with tumor invasiveness and poor survival of patients with ovarian, breast, and colorectal cancer (10-13), while high HA levels correlate with more differentiated tumor phenotype and an enhanced survival in patients with oral squamous carcinoma (14). The effects of HA www.frontiersin.org Frontiers in Immunology | Tumor Immunity An other escape strategy exploited by tumor cells is the hijacking of endogenous mechanisms of tolerance induction used by immuno-privileged organs. This is mediated by the non-classical HLA-G antigen, which exhibit a tightly controlled physiologic expression restricted to cornea, thymic epithelial cells, reproductive organs, embryonal tissues, and the extravillous cytotrophoblasts at the maternal-fetal interface. Furthermore, HLA-G is often expressed in solid and hematologic tumors either as a transmembrane and/or a secreted/shed protein, thereby protecting tumor cells from the cytolytic activity of natural killer (NK) and T cells (21). In addition, HLA-G can also impair myeloid DC by binding to the inhibitory receptors ILT2 and ILT4 in humans and PIR-B in mice (22-24). Receptor triggering by HLA-G inhibits the nuclear translocation of the transcription factor NF-κB (25), which is consequently accompanied by reduced expression of costimulatory molecules and proinflammatory cytokines as well as impaired presentation of MHC class II-restricted epitopes (22). As a consequence, HLA-G treated DC lack the ability to induce NK cells activation (26)  Glycodelin (previously called placental protein 14 or PP14, α2globulin, progesterone-associated endometrial protein or zonabinding inhibitory factor) has been originally identified as the molecule responsible for the immunosuppressive activity in the decidua during early gestation (28), but is also expressed in tumors of the reproductive tract, e.g., ovarian carcinoma, where its glycosylated form glycodelin A (GdA) correlated with unfavorable prognosis (29). Furthermore, glycodelin correlate with a worse patients' prognosis in familiar, non-BRCA1/2 breast carcinoma (30) and in lung cancer (31). In vitro characterization of glycodelin function demonstrated suppressive effects on all immune cell populations (32), including DC. Treatment of DC with GdA results in lower expression levels of costimulatory molecules, a low IL-12/IL-10 ratio (33), and a reduced ability to induce a type 1 polarization of effector cells (34). Depending on the culture conditions, GdA has also been reported to induce or suppress apoptosis in monocytes [see discussion in Ref. (34)].
Transforming growth factor-β plays not only a role in MDSC development, like vascular endothelial growth factor [VEGF; (40-42)], IL-6 and/or macrophage colony stimulating factor [M-CSF; (43)], but also impairs the DC migratory capacity by altering the expression pattern of chemokine receptors (44, 45) and inducing downregulation of CD1d thus impairing DC interactions with NK-T cells (46). sCD83 was found in total blood cell cultures after stimulation and might represent a feed back mechanism to shut down an immune response (47). Indeed, enhanced serum levels of sCD83 detected in hematologic malignancies and solid tumors, like lung carcinoma correlate with shorter tumor-free survival (48-50).

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In vitro treatment of DC with recombinant sCD83 results in enhanced IDO production and induction of TGF-β producing Foxp3 + Treg (51).
An additional strategy of immune escape mechanisms exploited by tumor cells consists in the upregulation of molecules with negative effects on DC. These include alterations in the Wnt/β-catenin pathway inducing activation of MerTK receptor (c-mer proto-oncogene tyrosine kinase) in infiltrating cells like macrophages and DC that help tumor growth in vivo (52). In vitro studies have found that tumor-derived Wnt5a can impair the differentiation of monocytes toward DC (53) and inhibit the maturation response to TLR ligand by myeloid DC (54,55) as well as by PDC (56).
Mucins are expressed by many epithelial tumors and their presence during differentiation of monocytes toward DC results in less differentiated cells with increased apoptosis (57), impaired response to TLR ligand stimulation, cytokine production skewed toward the immunosuppressive IL-10, impaired ability to induce proliferation of T cells, and enhanced induction of suppressive T cells (58)(59)(60). Those effects seem to be mediated by binding to the mannose receptor, siglec-3 and -9 (57)(58)(59).
A hallmark of many tumors is the secretion of high levels of prostaglandin E 2 (PGE 2 ) due to upregulation of cyclooxygenase (COX)1/2. The consequences of PGE 2 on DC functionality are complex. While it represent a component of the "gold standard" cocktail for vaccine DC maturation (61) due to its role in promoting CCR7-mediated migration (62), and it also induces the expression of costimulatory molecules like OX40-L and CD70 promoting T cell functions (63,64), it can inhibit the synthesis of IL-12p70 (65), while favoring the secretion of IL-23 that promote Th17 immune responses (66) and tumor development (67,68). Moreover, PGE 2 enhances MDSC differentiation (69), induces expression of IDO and soluble CD25 that inhibit T cell stimulation (70) and impairs the cross talk with NK cells (71). A possible explanation for the contrasting effects can be due to the specific receptor triggered by PGE 2 (72) and/or the relative ratio between PGE 2 -treated DC and effector cells (73).
Altered and/or secreted gangliosides have also been demonstrated to affect DC differentiation and survival (74)(75)(76)(77)(78). Moreover, gangliosides impair the ability of monocytes to induce T cell proliferation due to a downregulated expression of components of the antigen processing machinery [APM; (79,80)], a suppressed costimulation and a reduced cytokine production (81,82). In vivo, a correlation between elevated levels of the ganglioside GM3 and a higher frequency of immature DC was found in non-small-cell lung cancer (83).
Furthermore, "tumor-deviated" DC/MDSC exhibit an altered phosphorylation pattern of STAT3 (84,85) that has also been linked to the inhibition of IL-12p40 transcription (86) and/or of p38 (87) that is involved in the induction of Th17 responses (88).
In addition to boost the immune suppression, tumorconditioned DC can also provide direct help to tumor cells by secreting mitogens for the tumor cells (89), by favoring the epithelial mesenchymal transition (90), by promoting their invasiveness and ability to metastasize (8,90) and by inducing angiogenesis (91,92).
Characterization of the immunosuppressive activity of PDC in vitro have highlighted their ability to induce unresponsiveness of effector cells, to promote the development of suppressive CD8 + T cells, to differentiate naïve CD4 + T cells toward Foxp3 + or IL-10 producing Treg as well as to expand pre-existing Treg (108)(109)(110)(111)(112)(113). From the molecular point of view, important roles have been identified for ICOS ligand (ICOS-L), IDO, notch ligand delta-like 4 (Dll4), and granzyme B. ICOS-L is upregulated shortly after maturation induced by CD40-L or TLR9 triggering (108), is involved in inducing IL-10 production in CD45RO + T cells (114) and in sustaining the survival and proliferation of Foxp3 + Treg (115). A role in vivo for this pathway is supported by the co-localization between ICOS + Treg and ICOS-L + PDC within breast and ovarian carcinoma (115,116). Murine and human PDC can produce IDO in vitro upon triggering of TLR9, CTLA-4, GITR, or CD200 (117)(118)(119). PDC expressing IDO have been identified in melanoma draining lymph nodes in murine models and human patients and have been correlated with the activation of naïve and mature Treg (120)(121)(122). In murine models, the constitutively expressed Dll4 allow PDC to induce Th1 cells to produce IL-10 even under type 1 polarizing conditions, thus favoring the shut down of an immune response (123). Granzyme B, whose secretion by PDC is boosted by tumor-derived IL-3 and IL-10, is involved in the downregulation of the CD3ζ chain of T effector cells, thereby resulting in their anergy or deletion by apoptosis induction (124,125).
In addition to their immunosuppressive role, PDC play a protumorigenic role by promoting angiogenesis via secretion of TNFα and IL-8 (126) and favoring metastasis dissemination into the bone (127).

IMPROVED PROTOCOLS FOR DC-BASED VACCINATION AGAINST CANCER
Two major strategies of DC-based tumor immunotherapy have been implemented. The first is based on the ex vivo production and manipulation of DC that are then injected into the patients while the second targets the DC directly in vivo (Figure 1).
The classical strategy for the first approach consists in the differentiation of CD14 + circulating monocytes or CD34 + -mobilized precursor cells into immature DC by culturing them in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and IL-4 for 7 days, after which they are loaded with TAA Frontiers in Immunology | Tumor Immunity and induced to mature before in vivo injection. Studies performed using cells from patients with different solid as well as hematologic cancer histotypes have demonstrated that precursor cells are either not irreversibly impaired and can be matured with this protocol or that is possible to rescue their differentiation into functional DC upon inhibition of STAT3, p38, and/or IL-6 (128,129). Despite the good results obtained in vitro with patient-derived DC, and the induction of immune response in treated patients demonstrated by expanded Ag-specific T cells and delayed type hypersensitivity (DTH) reactions, the first clinical trials with vaccine DC resulted in poor clinical outcome. Based on the increased knowledge of the DC biology and of tumor escape mechanisms the protocol for the ex vivo production of vaccine DC has to be optimized (Box 1).
The initial poor results of DC-based immunotherapy could be due to the immature or only partially mature phenotype of the DC, and in particular to their reduced levels of IL-12 secretion. Thus, many alternative maturation protocols have been developed, which induce DC with an enhanced IL-12 secretion and functionality in vitro, with some of them that have also reached clinical application. The "alpha type-1 polarized DC" obtained upon maturation in the presence of IL-1β, IFN-α, IFN-γ, poly IC, and TNF-α (130) have been tested in patients with recurrent glioma www.frontiersin.org

Box 1 Optimization of DC-based tumor immunotherapy.
Different anatomical and tumor derived factors pose problems to the success of DC-based therapy. Following is a list of key points that have to be optimized.

(A) Ex vivo DC preparation
• DC subset: terminally differentiated blood DC (PDC, CD1c + DC, mixed) or monocyte derived DC (GM-CSF + IL-4, GM-CSF + IFN-α, GM-CSF + IL-15; standard 7 days or shortened 2-3 days protocol). • Antigen loading: protein, DNA, or mRNA; one or multiple Ag, defined or total tumor repertoire. • Maturation:TLR-ligand(s) (poly IC, MPLA, R848) and/or immune-derived (CD40-L, IFN-γ). Is PGE2 to be added for the migratory ability? • Targeted effector cells: CD8 + T cell only; also CD4 + T helper and/or innate effector cells (NK, iNKT, γδ T cells) (B) Vaccination protocol: •  (135) and colorectal cancer (136). In parallel to the manipulation of the maturation protocol, the type of DC was also optimized. Alternative differentiation protocols for monocytes have been tested to obtain more physiologic DC types. GM-CSF has been combined with IFN-α to induce inflammatory DC, which have already been tested in patients with medullary thyroid carcinoma (137), or with IL-15 to induce Langerhans-like DC that despite the enhanced functionality in vitro did not provide higher responses in melanoma patients when compared to standard DC (138). Furthermore, terminally differentiated DC have been used both as single or mixed populations. Regarding the myeloid subset, in a preclinical trial sufficient amount of CD1c + blood DC have been purified from healthy as well as melanoma and Bowel cancer disease patients under GMP (good manufacturing practice) conditions and could be induced to secrete proinflammatory cytokines, thus opening the way for a possible clinical application (139). Two different approaches using PDC have been developed. A leukemic cell line with PDC characteristic has been isolated and, after having demonstrated functional activity in humanized murine models (140) and with melanoma PBL in vitro (141) will be evaluated in a clinical trial in HLA-matched melanoma patients (NCT01863108). In contrast, de Vries and co-workers have employed autologous, patientderived PDC in a phase I clinical trial against melanoma (142). A GMP platform has been established to purify all subtypes of circulating APC resulting in a population able to induce Agspecific CTL both from healthy donors and myeloma patients (143). The injection of a highly purified DC population does not seem to be required since the sipuleucel-T (also called APC8015 or Provenge®) vaccination approved by the Food and Drug Administration (FDA) for treatment of prostate cancer patients is based on a highly mixed population, in which the DC targeted with TAA only represent a small component (144,145).
Other optimizations have been also evaluated in order to modulate the suppressive environment that impair the in vivo ability of the vaccine DC to prime immune responses. This is mediated by rendering DC insensitive to the tumor-induced suppressive microenvironment by blocking inhibitory signaling pathways, like TGF-β (146,147), IL-6 (148), and STAT3 (129,149,150). On the other side, the costimulatory function of DC have been further improved by providing the T lymphocytes with all required positive signals and/or the absence of negative feedback regulators in order to acquire full functionality and resistance to suppressor cells. DC unable to produce IL-10 (151, 152), insensitive to CTLA-4 triggering (153), providing enhanced levels of CD70 (154)(155)(156)(157), CD80 (154), or GITR-L costimulation (153,158) have proved to induce T cells with enhanced resistance to Treg suppression, delayed induction of tolerance as well as reversion of the tolerized status. Some of those "costimulatory enhanced" DC have also started the path of clinical trials like the TriMix DC (expressing CD40L, CD70, and a constitutively active TLR4 receptor) in melanoma patients (159,160).
To provide a more general pro-stimulatory phenotype, multiple signaling pathways have also been enhanced by either inducing expression of the transcription factor T-bet (161,162) or by silencing A20, an inhibitor of signaling pathway downstream of TLR and TNF receptors (163) resulting in DC with improved functionality. Similarly, with the increased knowledge of the important role of micro RNA (miR) in the fine tuning of gene transcription and their role in the immune response and in DC functions (164), DC-specific miR have been targeted. For example, inhibition of miR-22 and miR-503, two miRNA upregulated in DC upon coculture with tumor cells, resulted in improved therapeutic activity due to enhanced DC survival (165).
The major aim of the second line of therapy is to revert the tolerized phenotype of tumor-infiltrating and/or recirculating DC in order to allow proper activation of effector cells. The most clinically advanced strategies are those focusing on PDC and using ligands of the TLR-7 and -9 to recover their IFN-α secreting capabilities. After the successful use of imiquimod and CpG-containing oligonucleotides in murine models, many clinical trials have also been performed (166), leading to the approval of imiquimod for Frontiers in Immunology | Tumor Immunity cancer immunotherapy by the European Medicines Agency and the FDA. A problem with such a strategy is the fact that in some, but not all tumors a downregulation of the two TLR in tumorinfiltrating PDC have been reported (93). At the basis of the positive outcome upon PDC in situ triggering there can be not only the activation of other immune cells and the inhibition of Treg (167), but also a direct tumoricidal activity upon upregulation of TRAIL and granzyme B (168,169). Is to be underlined that the upregulation of granzyme B by PDC can also have detrimental effect by killing T cells (124,125,169). An other reported effect of CpG injection is the differentiation of MDSC toward functional monocytes with consequent reduction in the amount of suppressive cells (170,171).
On the side of targeting myeloid DC in vivo, similar approaches to the ex vivo manipulation have been tested. Chemical inhibitors of negative signaling pathways like STAT3 (150), or positive modulators like miR-155 (172), have been injected in vivo with the aim to be uptaken by DC and to revert their tolerogenic phenotype and promote immune-mediated tumor rejection. Provision of missing IL-12 through different technical approaches have demonstrated that in addition to the stimulation of NK and T cells, also myeloid cells are positively affected with the activation of cytotoxic macrophages and reversion of MDSC with loss of their suppressive properties (173,174). Many different strategies are also aiming at the removal of MDSC acting on their differentiation and/or suppressive functions (175). For example all-trans retinoic acid (ATRA), a compound reducing MDSC number and function has been combined to DC vaccination in a phase I trial in small cell lung cancer patients (176).

EXPLOITATION OF TOLEROGENIC DC IN TRANSPLANTATION AND AUTOIMMUNITY
In the setting of autoimmunity and transplantation the aim of immunotherapy is to reduce inflammation and to induce a local and/or antigen-specific immunosuppression/tolerance in order to avoid organ rejection and reduce the disease score without increasing the risk of opportunistic infections. Like for tumor immunotherapy, DC have been manipulated ex vivo or directly targeted in situ.
Tolerogenic DC have been differentiated in vitro from human monocytes and murine bone marrow cells upon culture in the presence of different combination of IL-10 (177), TGF-β (178), vitamin D3 (179), dexamethasone (180), protein kinase C inhibitor (181), and rapamycin (182). These cells are characterized by a semi-mature phenotype, the ability to expand Treg and to preserve such properties even in an inflamed microenvironment, as mimicked by stimulation with TLR ligands (183,184). Murine models of organ transplantation as well as different autoimmune diseases have demonstrated the therapeutic applicability of such tolerogenic DC (185)(186)(187)(188) and opened the way to preclinical evaluation in multiple sclerosis (189) as well as in a phase I trial in rheumatoid arthritis [RA; (190)]. Similarly, the good results obtained with non-obese diabetic (NOD) mice injected with DC silenced in the major costimulatory molecules [i.e., CD40, CD80 and CD86; (191)] have opened the way for a phase I safety study in patients with type 1 diabetes (192). In the setting of organ transplantation, "classical" murine bone marrow-derived DC from the organ donor have been either triggered with tetramer of sHLA-G1 (25) or silenced in NF-κB (193) in order to induce a transplant-specific tolerance that allow (longer) acceptance of the graft.
Direct in situ targeting of DC has also been implemented in order to promote local and/or antigen specific tolerance. Clinical trials have been performed with apilimod (or STA5326), a specific inhibitor of IL-12 and IL-23, which are the central mediators of the Th1 and Th17 responses involved in autoimmunity. Whereas in psoriasis a reduction in inflammatory cytokine and DC infiltration of the skin lesions was observed upon apilimod (194), no robust clinical improvement was found in RA (195) and contrasting results were reported in Crohn's disease (196,197). Although still in the preclinical phase systemic or topic injection of sCD83 was able to prolong survival of grafted organs (51,198) as well as to reduce experimental autoimmune encephalomyelitis (EAE) in both prophylactic and therapeutic setting (199). Additional strategies inducing antigen specific tolerance consist in coupling the desired antigen to antibodies or ligands for specific receptors expressed by DC. Examples are DEC-205 (200,201), the human DC immunoreceptor (DCIR) (202,203), and the murine acid binding Ig-like lectin H [siglec-H; (204)] that induce specific tolerance to the antigen they have been targeted with.

CONCLUSION
Despite the ever growing knowledge on the immunologic function of DC and how tumor cells try to subvert them, a long way has still to be performed before defining the best protocol of vaccination regarding not only the maturation/resistance of the DC but also the road of injection, the number of injections, the type of antigen(s) and the loading strategy (see Box 1). Of particular interest is the recent report that in therapeutic setting a single immunization performs better alone that with a following boost, a setting that is on the contrary highly favorable in prophylactic immunization (205). Frontiers in Immunology | Tumor Immunity