Mouse CD103+ DCs as well as both human and mouse pDCs were shown to mediate oral tolerance through an IDO-dependent mechanism (49). In human blood, DC-10 induce and expand Tr1 cells through the release of IL-10 and TGF-β (31, 75). DC-10 identified as CD11b+CD11c+CD14+CD16+CD83+HLA-DR+ cells that do not express CD1a and CD1c. Furthermore, DC-10 expresses the inhibitory receptors ILT2, ILT3, ILT4, and HLA-G. Despite concomitant high surface levels of costimulatory molecules (CD40, CD80, and CD86), DC-10 exhibit potent tolerogenic activity (31). Another DC population characterized by CD1c+CD14+CD16− expression was found in the blood of melanoma patients and exhibited immunosuppressive functions by suppressing T-cell proliferation in an antigen-specific manner. It was suggested that this DC subtype modulated T-cell responses through the expression of PD-L1 (18). A similar phenotype was described in minced lung tissues. The frequency of the CD1c+ subset (including the CD1c+CD14+ fraction) was increased in patients with chronic obstructive pulmonary disease, suggesting that these cells may be involved in the enhanced susceptibility of these patients to infections. Indeed, this subset favored the generation of IL-10-secreting CD4+ T cells and mediated immunosuppression through IL-10, IL-27, and ICOS-L (76). In skin dermis, resident CD141+CD14+ DCs were shown to produce large amounts of IL-10 and were able to induce Treg (50).

Besides their role in controlling peripheral immune responses, DCs play a role in the maintenance of central tolerance, as trafficking peripheral DCs can home to the thymus and promote negative selection of antigen-reactive T cells, thus contributing to a safe peripheral T-cell repertoire (77, 78). By presenting antigens directly within the thymus, DCs and particularly resident pDCs influence the generation of natural Foxp3+ Treg, a process mediated by the IL-7-related molecule, thymic stromal lymphopoietin (TSLP), which is secreted in the thymic medulla (79–81).

### GENERATION OF tolDCs

Dendritic cell-based therapeutic approaches are being explored with the aim to reestablish self-tolerance in autoimmune diseases, and to promote alloimmune tolerance after SOT. Several strategies for the generation of tolDCs are being explored (**Figure 1**). These include treatment with pharmacologic agents or cocktails of immunomodulatory cytokines, genetic engineering, and exposure to apoptotic cells. Research groups have developed protocols to generate and expand antigen-specific tolDCs *in vitro*. Most of these *in vitro* conditioning regimens aim to stabilize the immature state of DCs, even in the presence of strong inflammatory challenges [e.g., lipopolysaccharide (LPS)]. The resulting tolDCs also express and/or secrete immunomodulatory molecules that favor the development and expansion of Treg (82, 83). After adoptive transfer *in vivo*, maturation-resistant tolDCs may, therefore, promote peripheral tolerance mainly by inducing antigenspecific T-cell hyporesponsiveness and an immuno-regulatory microenvironment.

### Pharmacologic Interventions for tolDC Induction

Various pharmacological agents have been used to generate tolDCs, including immunosuppressive drugs, cyclic AMP inducers, chemicals, cytokines, and growth factors (**Table 2**) (84, 85). Many of these agents were primarily studied for their inhibitory

(in the case of autoimmune diseases or transplantation) and can be further pulsed *in vitro* with specific antigens (peptides, donor cell lysates, apoptotic cells). tolDCs can regulate Teff responses by various mechanisms: 1. Fas/FasL pathway-mediated deletion; 2. Production of IDO which degrades the essential amino acid tryptophan through kynurenine pathway, causing starvation of Teff. The production of IDO is favored by reversed signaling via interaction between CD80/CD86 on DCs and CTLA-4 on regulatory T cell (Treg). 3. Surface expression of inhibitory molecules and secretion of regulatory mediators. Abbreviations: CTLA-4, cytotoxic T-lymphocyte antigen-4; IDO, indoleamine 2,3-dioxygenase; iTreg, induced regulatory T cell; PBMC, peripheral blood mononuclear cells; tolDC, tolerogenic dendritic cell; Teff, effector T cell; Tmem, memory T cell; Tnaive, naïve T cell; tTreg, thymic-derived regulatory T cell.

Table 2 | Pharmacologic interventions to induce tolerogenic DCs (tolDCs).

*AMP, adenosine monophosphate; DSG, 15-deoxyspergualine; GM-CSF, granulocytemacrophage colony-stimulating factor; HLA, human leukocyte antigen; IL, interleukin; TGF, transforming growth factor-beta.*

effects on T-cell activation and proliferation. As such, some of them are currently used in the clinical treatment of autoimmune diseases and in the prevention of allograft rejection after SOT.

Dexamethasone, a potent immunosuppressant, blocks the differentiation and maturation of DCs and enhances their death by apoptosis (92, 109). Using rat bone marrow-derived DCs and human GM-CSF/IL-4-induced ModDCs, we demonstrated that pretreatment with dexamethasone-induced selective expansion of Treg and T-cell alloantigen-specific hyporesponsiveness in re-challenge experiments (110). Dexamethasone was reported to have synergistic effects with other drugs to induce tolDC, in particular 1,25-dihydroxyvitamin D3 (VitD3) (111). While traditionally known for its role in the regulation of calcium and bone homeostasis, VitD3 and its receptor were also described to regulate innate and adaptive immune responses. Exposure to VitD3 inhibited the expression of MHC class II, CD80, and CD86 on DCs with a high ratio of PD-L1/CD86, while reducing the production of pro-inflammatory cytokines, such as IL-12 and IL-23, and increasing that of TGF-β and IL-10. Moreover, VitD3 favored Treg development and blocked B-cell proliferation and differentiation toward antibody-producing plasma cells (112).

Inhibitors of the mammalian target of rapamycine (mTOR) pathway engage FK506-binding protein 12 forming a complex that blocks mTOR, but not calcineurin, resulting in non-specific inhibition of cell cycle progression and, therefore, of T- and B-cell proliferation. *In vitro* assays, together with experimental and clinical data, suggest that immunosuppression based on mTORinhibitors may favor the induction of peripheral tolerance. In rodent models, the adoptive transfer of rapamycin-conditioned alloantigen-pulsed DCs resulted in prolonged cardiac and skin allograft survival (87, 113). Moreover, rapamycin was shown to facilitate peripheral deletion of alloreactive Teff by promoting activation-induced cell death in experimental transplantation models, while selectively expanding human Foxp3+ Treg and Tr1 cells both *in vitro* and *in vivo* (114–117).

Various other immunosuppressive drugs and biologic agents were described to generate tolDCs *in vitro* including mycophenolic acid (MPA) formulations (90, 118), deoxyspergualin (DSG) and its analogs (93), aspirin (98, 99), retinoic acid (117), and prostaglandin E2 (119). These substances mainly interfere with NF-κB pathway-mediated DCs maturation and the capacity of DCs to produce IL-12p70 (84, 85).

Bone marrow-derived DCs exposed *in vitro* to the immunomodulatory cytokines IL-10, TGF-β, or low-dose GM-CSF in the absence of IL-4 exhibit low expression levels of costimulatory molecules and pro-inflammatory cytokines with minor changes in MHC class-I and -II molecules. These conditioned DCs were less immunogenic when co-cultured with CD4+ and CD8+ T cells, while promoting the expansion of Treg (both natural Treg and iTreg) and antigen-specific T-cell hyporesponsiveness *in vivo* upon re-challenge (108, 120).

Some of these agents also directly promote the differentiation of tolDCs *in vivo*. For example, local or systemic presence of cytokines such as IL-10, TGF-β, and even IFN-γ-induced differentiation of monocytes into tolDCs and promoted FoxP3+ Treg (121). The eye is a known locally immune-privileged site and the aqueous humor constitutively contains molecules that maintain DCs in an immature state, such as TGF-β2, IDO, the neuropeptide α-melanocyte stimulating hormone (α-MSH) and FasL (122). The epithelium also plays a critical role in dampening inflammation through the release of epithelial-derived factors, including prostaglandin E2, TSLP, retinoic acid, and TGF-β, which are able to promote tolDC (119, 123).

Cobalt protoporphyrin is an inducer of heme oxygenase (HO)-1, an intracellular enzyme that catalyzes the degradation of heme, resulting in the production of biliverdin and carbon monoxide. HO-1 expression is induced by local oxidative stress. Experimentally, HO-1 upregulation was protective in the context of inflammatory processes and after allogeneic SOT (124). Besides maintaining HO-1 expression on human DC, cobalt protoporphyrin prevents their maturation and promotes the secretion of regulatory cytokines (124, 125). Interestingly, HO-1 is also able to inhibit the activation of T, B, and NK cells (126).

### Genetic Engineering of DCs

We and others have used gene transfer technology to generate tolDCs *via* increased expression of immunomodulatory molecules such as IL-10, TGF-β, CTLA-4, IDO, PD-L1, or ligands for receptors resulting in T-cell deletion such as CD95/Fas (TNF-family related death receptor) and TNF-related apoptosis-inducing ligand (127–129). Using gene therapy approaches, recombinant adenovirus vectors typically achieve great transfection efficiencies but are limited by pro-inflammatory effects leading to DCs maturation (130). Methods using recombinant retrovirus vectors and, recently, non-viral gene transfer methods such as nucleoporation may induce lower degrees of DCs maturation (131). As an example, genetically engineered DCs over-expressing IDO regulated T-cell alloresponses *in vitro*, while IDO adenovirus-mediated gene transfer into the donor heart attenuated acute rejection of MHC-mismatched cardiac allografts in rats (132). Compared to pharmacological conditioning, genetic engineering of DCs using retroviral or lentiviral vectors offers the advantage of a potentially more stable cell phenotype and function *in vivo*. Another interesting approach that was described is the *in vivo* transfer of antigen-encoding bone marrow progenitor cells which, at steadystate, prevent antigen-specific sensitization and promote T-cell tolerance mostly by deletional mechanisms (133–135).

### DC Exposure to Apoptotic Cells

Exposure of DCs to early apoptotic cells down-modulates their stimulatory functions (136). DC internalization of apoptoticcells-associated molecular patterns selectively leads to decreased production of pro-inflammatory cytokines (e.g., IL-1, TNF-α, IL-6, and IL-12), while enhancing the secretion of IL-10 and TGF-β (137). This process also limits upregulation of MHC class II and costimulatory molecules such as CD40, CD80 and CD86, hence maintaining DCs in an immature state (138). This mechanism contributes to self-tolerance and was exploited to induce tolerance to alloantigens in SOT. Following intravenous injection, donor allogeneic apoptotic cells were rapidly internalized in the spleen by red pulp macrophages and marginal zone DCs and were able to prolong cardiac allograft survival in rodents (139). Interestingly, T-cell depleting therapies used in clinical SOT, such as anti-CD3 or anti-CD52 monoclonal antibodies, were shown to induce T-cell apoptosis *in vivo.* This effect was associated with TGF-β secretion by DCs and subsequent expansion of Treg (140). Thus, administration of apoptotic cells or direct induction of apoptosis *in vivo* could be used to promote the generation of tolDCs *in vivo*. Of note, the induction of apoptosis *in vivo* must be carefully controlled to prevent simultaneous activation of the necrotic cell death mechanism and subsequent release of necrotic cell-associated antigens. These antigens were demonstrated to be able to stimulate CD141+CD14− DCs through the Clec9A receptor favoring antigen cross-presentation and, hence, CD8+ T-cell responses (8).

### THERAPEUTIC USE OF tolDCs IN AUTOIMMUNE DISEASES

In Europe and North America, 5% of adults, of whom two-thirds are females, suffer from autoimmune diseases, the most prevalent pathologies being type 1 diabetes (T1D), psoriasis, RA, inflammatory bowel disease (IBD), and multiple sclerosis (MS).

### Type 1 Diabetes

Type 1 diabetes is due to a breakdown of self-tolerance and is mainly orchestrated by CD4+ and CD8+ autoreactive T cells that activate B cells, resulting in the production of autoantibodies specific for pancreatic islets β-cell antigens with progressive immune-mediated destruction of the β-cell mass and insulin insufficiency (141). Insulin treatment increases the life expectancy of T1D patients; however, it often fails to prevent T1D-associated cardiovascular and renal complications with increased morbidity and mortality. This opens the way to immune-based interventions aiming at restoring immune tolerance and preventing early T1D either by targeting autoreactive T cells (142) or by modifying the immunogenicity of DC. As T1D is a progressive and not a relapsing-remitting autoimmune disease, there is only a window of opportunity treatment period at the onset of the disease, in order to reinstitute self-tolerance and preserve the function of the existing β-cells mass.

In non-obese diabetic (NOD) mice, GM-CSF treatment prevented the development of diabetes primarily by inducing tolDCs and Treg (143). Another therapeutic option for T1D consists of transplantation of pancreatic islets or whole pancreas; however, such approaches require long-term immunosuppression to prevent allograft rejection and reoccurrence of autoimmunity. In an experimental model of syngeneic pancreatic islets transplantation in NOD mice, both transient TGF-β expression within islets and transplantation of islets grafts containing TGF-β-conditioned tolDCs reduced the activation of islet autoantigen-specific T cells in graft-draining LNs, resulting in prolonged graft survival (144). These results support the notion that TGF-β-induced tolDCs could be an effective strategy to restore peripheral tolerance within the context of an established autoimmune disease. Aside from generating tolDC, TGF-β promoted the survival of thymicderived Treg and the differentiation of iTreg (64, 145, 146).

Other strategies using tolDCs for the prevention or treatment of T1D currently are being explored in experimental models. Antisense oligonucleotides have been used to specifically downregulate costimulatory molecules, resulting in DCs with an immature phenotype. A single injection of bone marrow-derived DCs engineered *ex vivo* with a mixture of antisense oligonucleotides targeting the CD40, CD80, and CD86 primary transcripts significantly delayed the onset of diabetes in syngeneic NOD recipients. The beneficial effect of these tolDCs was partly mediated by an increase in Treg (147). Based on these encouraging experimental data, efforts have been made at translating the use of autologous tolDCs in patients with new-onset T1D, aiming to prevent disease progression, or even to revert established disease (148, 149). A phase I clinical study showed that intradermal injection of autologous monocyte-derived costimulationimpaired tolDCs (10 × 106 cells every 2 weeks for a total of 4 administrations), treated *ex vivo* with antisense oligonucleotides, was safe and well tolerated in patients with established T1D (150). A phase II follow-up clinical trial (ClinicalTrials.gov identifier NCT02354911) using DCs isolated from patients with recent-onset T1D is ongoing. Potential therapeutic success will be evaluated through the improvement of the glycemic control as evidence for a preserved β-cell mass. A similar clinical study using costimulation-impaired tolDCs is currently registered (identifier NCT01947569). This clinical trial includes a sequential open-label, phase-IB safety assessment and a randomized, double-blind, phase-IIA efficacy trial aiming at maintaining and improving residual β-cell function in new-onset T1D patients.

### Psoriasis

Psoriasis is a chronic inflammatory skin disease mainly characterized by abnormal keratinocyte proliferation and differentiation causing thickening of the epidermis (151). The psoriatic skin shows a prominent infiltration of neutrophils in the epidermis, together with macrophages, DCs, and T cells in the dermis. The successful use of cyclosporine A, a drug that inhibits early T-cell activation by blocking the TCR-downstream calcium–calcineurin pathway, highlights the role of T cells in the pathogenesis of the disease (152). Psoriasis has been associated with impaired Treg suppressive functions, resulting in overproduction of pro-inflammatory cytokines such as TNF-α and IFN-γ, as well as IL-17 and IL-22 produced by Th1 and Th17 effector cells, respectively (151, 153–155). The neuropeptide α-MSH is a well known mediator of skin pigmentation and has recently been shown to exert anti-inflammatory and immunomodulatory activities (106). Treatment with α-MSH ameliorated psoriasislike skin inflammation, in part by suppressing the proliferation and effector function of Th17 cells. The beneficial effect of α-MSH was shown to be mediated by tolDCs and functional iTreg (107).

### Rheumatoid Arthritis

Rheumatoid arthritis, an autoimmune disease associated with chronic joint inflammation and destruction, is characterized by infiltration of innate immune cells (neutrophils, monocytes, NK cells and DCs) as well as T and B cells in the synovial compartment (156). The current treatment of RA includes immunosuppressive drugs such as corticosteroids, cytokine antagonists (anti-TNF-α), costimulation blockade (CTLA-4 Ig), and B-cell depleting monoclonal antibodies. More recently, the potential of DC-mediated immunomodulation for the treatment of RA was investigated. Clinical-grade tolDCs have been generated from monocytes of patients with RA by conditioning them *ex vivo* with VitD3 and dexamethasone. The resulting tolDCs exhibited reduced costimulatory molecules expression, low production of pro-inflammatory cytokines, and impaired capacity to stimulate antigen-specific T cells. Importantly, the phenotype and functional characteristics of tolDCs generated from RA patients were comparable to those generated from healthy individuals. These tolDCs remained stable in the absence of immunosuppressive drugs even after further challenge with pro-inflammatory mediators (157). Interestingly, the tolDCs exhibited high cell-surface expression of TLR2 compared to mature immunogenic DC. The use of this marker should be considered in future immunotherapeutic protocols to assess the quality and stability of tolDCs produced *ex vivo*. An ongoing registered phase I randomized, placebo-controlled trial (identifier NCT01352858, AutoDECRA) aims to generate autologous tolDCs to be injected (single dose) into the knee joint of patients suffering from RA. This clinical study will evaluate the effect of injected tolDCs on both the local and the systemic disease activity.

### Inflammatory Bowel Disease

The gut mucosa is constantly exposed to food antigens, pathogens, and commensal microorganisms, and holds the largest mass of lymphoid tissues in the body. The interplay between the intestinal epithelium and the local innate and adaptive immune system is crucial to the maintenance of immune homeostasis and oral tolerance. IBD (Crohn's disease and ulcerative colitis) is mainly a consequence of loss of peripheral tolerance to otherwise harmless bacterial flora with dysregulated T-cell function in response to local intestinal antigens (158). Current treatments include corticosteroids, azathioprine, and 6-mercaptopurine, as well as anti-TNF-α and anti-α4β7 integrin therapies in severe cases. T-cell activation and effector function is dependent on the microenvironment in which antigen presentation occurs; hence, tolDCs may have a potential for reestablishing intestinal immune regulation (159). CD103+ DCs are found in the human gut under normal conditions (160). These cells express low levels of CD40, TLR2 and TLR4, secrete IL-10 but not IL-12, and produce retinoic acid and IDO that promote the differentiation of iTreg and local immune regulation (49, 161). Peripheral tissue-resident pDCs exert a pivotal role in oral and mucosal tolerance (15, 16). An aberrant pDC distribution and effector function was described in the mesenteric LNs and inflamed mucosa of patients with Crohn's disease compared to healthy individuals (162). Moreover, immature peripheral blood pDCs and cDCs were reduced during flares in IBD patients (163). A phase I randomized clinical study (identifier NCT02622763 TolDecCDintra) currently evaluates the safety and clinical efficacy of autologous tolerogenic ModDCs injected into the intestinal lesions identified by endoscopy in patients with refractory Crohn's disease.

G-CSF therapy has been shown to be beneficial in Crohn's disease patients. The benefit was associated with increased numbers of pDCs in the gut mucosa and induction of IL-10 production (164). IL-10 is a crucial immunoregulatory cytokine in the gut, as documented by severe spontaneous IBD in IL-10-knockout mice (165). Protocols are under development to produce tolDCs under clinical-grade conditions for IBD patients by conditioning ModDCs with IL-10, together with a cocktail of other cytokines and prostaglandin E2 (166). The generated tolDCs display a semi-mature phenotype (intermediate expression of CD80 and CD86, MHC class II low), produce IL-10 with low levels of IL-12p70, IL-23, and TNF-α, and remain stable even in proinflammatory conditions. These data suggest that the strategy based on using autologous DCs (derived from patients with autoimmune diseases) may indeed be feasible for future immune therapies. Although the initial monocyte population is selected from patients with an overt inflammatory disease, the cells can be conditioned to acquire beneficial tolerogenic properties *ex vivo*.

### Multiple Sclerosis

Multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS), is predominantly a T-cell-mediated autoimmune disease characterized by leukocyte infiltration into the CNS, demyelination, and axonal loss. Besides current strategies targeting T and/or B cells, tolDCs may represent a potential therapeutic approach. Myelin peptide-loaded tolDCs were generated *ex vivo*, exhibiting a stable semi-mature phenotype and an anti-inflammatory cytokine profile. These tolDCs induced antigen-specific hyporesponsiveness in myelin-reactive T cells isolated from relapsing-remitting MS patients (167). IFN-β is an immunomodulatory agent used in the treatment of MS (168). Using healthy donors as well as MS patients PBMCs, it was shown that *in vitro* treatment with IFN-β enhanced PD-L1 expression on monocytes and DCs, inhibited antigen-specific CD4+ T-cell activation, and increased Treg numbers. In addition, serial *in vivo* measurements in MS patients before and 6 months after initiation of IFN-β therapy revealed a significant increase in PD-L1 mRNA (169). Sex hormones such as estrogens can modulate immune responses, contributing to the observed difference in the incidence of autoimmune diseases between males and females. Although the overall incidence of autoimmune diseases is higher in women compared with men, estrogens were protective in the experimental autoimmune encephalomyelitis (EAE) animal model of MS, even during pregnancy (170, 171). *In vivo* exposure to estriol (E3), a pregnancy-specific estrogen, induced tolDCs (E3 tolDC) characterized by increased expression of the inhibitory molecules PD-L1, PD-L2, B7-H3, and B7-H4, as well as mediators such as IL-10 and TGF-β, along with decreased expression of IL-12, IL-23, and IL-6. The transfer of E3 tolDCs to mice prior to active induction of EAE prevented the development of the disease. The protective effect was associated with immune deviation from pathogenic Th1/Th17 cells to a Th2 response (172). Two phase I clinical trials are currently registered on the ClinicalTrials. gov website, assessing the feasibility and safety of tolDCs loaded with myelin peptide in patients with MS (identifier NCT02618902 and NCT02903537). The TOLERVIT-MS (NCT02903537) trial involves VitD3-induced tolDCs used at increasing doses, starting from 5 × 106 cells. Interestingly, different routes of administration will be explored for optimal efficacy, such as intravenous, intradermal or direct intranodal cell injection into cervical LNs.

### THERAPEUTIC POTENTIAL OF tolDCs IN SOT

In the absence of adequate immunosuppression after SOT, the recognition of donor alloantigens by recipient T cells initiates a strong immune response leading to alloimmunization and allograft rejection. As T cells play a central role in alloresponses, immunosuppressive regimens have been historically developed to target T cells, whereas recent attention has also been devoted to the roles of B cells and alloantibodies (173). The development of immunosuppressive drugs has led to decreased rates of acute rejection after SOT, but their long-term administration is associated with side-effects including cardiovascular and renal toxicities, as well as infections and tumors (174, 175). Moreover, current regimens have limited effects on T- and B-cell memory responses and may interfere with the induction and expansion of donor-specific Treg (83, 176). Therefore, inducing sustained donor-specific tolerance with minimal drug exposure remains an important goal to improve long-term outcomes in transplantation medicine.

Compared to immune responses to autoantigens or pathogens, SOT constitutes a unique situation whereby DCs present antigens to alloreactive T cells through three different mechanisms: the direct, indirect and semi-direct pathways (177). Recipient T cells can be activated by donor antigens either as intact allogeneic MHC:peptide complexes presented by donor mature DCs that have migrated out of the allograft (direct allorecognition) or as donor-derived MHC:peptide complexes that have been processed and presented by recipient DCs (indirect allorecognition) (178, 179). The semi-direct pathway involves the presentation by recipient DCs of intact donor MHC:peptide complexes that have been captured from donor cell membranes or exosomes (177). Therefore, DCs from either donor or recipient origin can be considered for the development of immunotherapeutic protocols in SOT.

### Donor-Derived tolDC

Experimental models have illustrated the potential of donorderived tolDCs to promote peripheral transplantation tolerance through the induction of donor-specific T-cell hyporesponsiveness, deletion, and/or regulation of alloreactive T cells (180–182). Costimulation-deficient tolDCs generated from donor bone marrow in the presence of GM-CSF, without IL-4, significantly prolonged the survival of MHC-mismatched heart allografts in mice when delivered (2 × 106 cells intravenously) 1 week before transplantation. The effect was only partially antigen-specific, as third-party tolDCs also prolonged graft survival, albeit to a lesser extent (median graft survival time 22 vs. 16.5 days, respectively; vs. 9.5 days in control non-treated mice) (180). However, *in vivo* maturation of the injected tolDC occurred, as evidenced by upregulation of CD80 and CD86, partially explaining the limited efficacy of tolDCs *in vivo*. Based on these results, a newer approach evaluated donor-derived tolDCs generated in the presence of GM-CSF and TGF-β, delivered in conjunction with CD40–CD40L costimulation blockade 1 week before transplantation. This strategy resulted in extended allograft survival (181). In a preclinical non-human primate (NHP) model, the infusion of donor-derived tolerogenic ModDCs (3.5–10 × 106 cells/kg) in combination with B7-CD28 costimulation blockade (CTLA-4 Ig given at day −7 and up to 8 weeks after transplantation) and rapamycin (started on day −2 and tapered over 6 months) significantly prolonged renal allograft survival (median graft survival time 113.5 vs. 39.5 days in controls) (86). In this study, tolDCs generated *in vitro* with VitD3 and IL-10 expressed low MHC class II and costimulatory molecules, high levels of PD-L1, and were resistant to inflammation-induced maturation. *Ex vivo* immune monitoring demonstrated regulation of donor-reactive memory T cells in tolDC-treated NHP compared to controls. Importantly, no adverse events, and particularly no significant allosensitization, were observed in the recipients.

Donor-derived tolDCs could also contribute to the induction of donor-specific central tolerance after SOT. A recent study evaluated the thymus-homing ability of DCs and their tolerogenic function. FMS-related tyrosine kinase 3 ligand (Flt3L) is a cytokine that synergizes with other growth factors to stimulate the proliferation and differentiation of hematopoietic progenitor cells. Flt3L-induced bone marrow-derived DCs (FLDCs), but not GM-CSF-induced DCs, were able to traffic to recipient thymus after systemic injection. Infusion of allogeneic donor-derived FLDCs induced clonal deletion of both CD4 and CD8 single-positive alloreactive thymocytes, leading to donorspecific central tolerance and prolonged survival of donor skin grafts (183).

### Recipient-Derived tolDCs

It should be emphasized that the use of recipient autologous DCs appears to be more feasible than that of donor DCs, as tolDCs can be prepared from peripheral blood of the recipient before SOT. Recipient tolDCs could be generated and stored while the patient is on the waiting list, and loaded with donor-derived antigens (HLA peptides, donor cell lysates) at the time of transplantation. Taking advantage of linked suppression mechanisms, it could be sufficient to generate recipient tolDCs expressing some but not all donor alloantigens (184). Importantly, the use of recipient DCs allows for the generation of tolDCs with the potential to induce indirect pathway CD4+ T-cell hyporesponsiveness and donor-specific Treg, while also controlling anti-donor humoral responses, which could have protective effects against chronic rejection (185–188). Recipient DCs pulsed with donor allopeptides and injected into the thymus in combination with one dose of anti-lymphocyte serum 7 days before transplantation induced donor-specific tolerance to cardiac (189) and pancreatic islets (190) allografts in rats. In an experimental model, the infusion of recipient DCs expressing donor intact MHC class I antigens selectively prevented indirect pathway alloreactive CD4+ T cells activation and the generation of alloantibodies, resulting in long-term survival of MHCmismatched skin allografts (191). Interestingly, recipient bone marrow-derived tolDCs (generated in the presence of low-dose GM-CSF) infused one day before transplantation significantly prolonged cardiac allograft survival, even in the absence of prior *in vitro* pulsing with donor antigens. Although recipients showed reduced anti-donor cellular and humoral responses *ex vivo,* the effect of these tolDCs was not antigen-specific (192). This study suggests that, while alloantigen-specific immune regulation is desirable in the setting of SOT, recipient autologous tolDCs may also modulate the microenvironment in a way that favors allograft survival. The ONE study consortium aims at promoting clinical tolerance in living-donor renal transplant recipients (193). Within this consortium, a multicenter phase I/II safety/efficacy trial (ClinicalTrials.gov identifier NCT02252055) is currently evaluating the administration of autologous tolDCs on top of the standard immunosuppressive regimen (tacrolimus-MPA-corticosteroids).

### FEASIBILITY AND SAFETY OF DC-BASED IMMUNOTHERAPY

### Phenotype and Function of Generated tolDCs

In recent years, an improved understanding of the mechanisms that govern central and peripheral immune tolerance, along with a more precise characterization of DC subsets, has opened the door to their therapeutic use in autoimmune diseases and SOT (**Table 3**). There are, however, some caveats that need to be addressed in order to safely translate and evaluate DC-based immunotherapy to the clinical arena. One major hurdle is the generation of high amounts of tolDCs of reproducible quality using clinical-grade procedures. Cells isolation, culture, and preconditioning protocols need to be optimized to generate tolDCs with stable phenotypes and functions. A better identification of subset-specific markers of human tolDCs would be valuable for safe delivery to patients. Another major concern is the stability of *ex vivo* generated tolDCs in a pro-inflammatory environment *in vivo*. While many studies support a maturationresistant state of human tolDCs produced *in vitro* using various conditioning regimens, the *in vivo* environment may still induce maturation toward undesired immunogenic DCs (180, 194, 195). In the context of SOT, infused donor tolDCs have been shown to be short-lived, being eliminated mainly by recipient NK cells. Autologous recipient DCs could then process and present donorderived MHC:peptide alloantigens in an immunogenic context, leading to allosensitization and accelerated allograft rejection (196). However, this aspect remains controversial as there is evidence suggesting that, while donor tolDCs indeed rapidly die after infusion and are re-processed by splenic recipient DC,

*NP, not provided; T1DM, type 1 diabetes mellitus; NIAID, National Institute of Allergy and Infectious Diseases; NIDDK, National Institute of Diabetes and Digestive and Kidney Diseases.*

this may not lead to allograft rejection because alloantigens are delivered in the context of tolDCs (197). Nonetheless, tolDCbased "negative vaccination" most likely will need to be combined with immunomodulatory drugs *in vivo* to harness strong immune activation, as demonstrated in NHP transplantation models (86, 198). Overall, additional preclinical and clinical trials are now needed to demonstrate the feasibility and safety of tolDC-based immunotherapy in humans. For instance, in a clinical study antigen-loaded autologous tolDCs were injected intradermic in healthy volunteers. While the treatment was well tolerated, it also resulted in antigen-specific regulation of Teff (199, 200).

### Source of DCs and Administration Protocols

The source of DCs is a central aspect of tolDC-based strategies*.* In the context of autoimmunity or end-stage organ disease, a possible effect of the disease on DCs population and function needs to be addressed. Specifically, it must be demonstrated that autologous tolDCs from patients are as stable as tolDCs derived from healthy individuals (157). The number of cells to be delivered, the most appropriate timing of injection, and the route of administration also need to be carefully evaluated. Antigen-specificity of DC-based immunomodulation is another open issue with respect to clinical applications. Targeted regulation of antigen-specific T-cell responses would avoid generalized immunosuppression and impaired immune surveillance leading to infections or the development of malignancies. However, specific autoantigens that are responsible for T-cell priming have not been identified in some autoimmune diseases such as IBD, and peripheral DCs used for immunotherapy may not present some tissue-specific antigens. It also should be considered that, in deceased donor SOT, donor alloantigens are not known until the day of transplantation.

In an attempt to limit the workload and costs of *ex vivo* generation of tolDCs, while also circumventing the uncertainty regarding their stability, these cells could be directly generated *in vivo*. As discussed, the administration of apoptotic cells or *in vivo* induction of apoptosis could be a first option in this regard (136, 201). Another possibility may involve the administration of specific immunomodulatory cytokines or drugs. Infliximab, a chimeric monoclonal antibody that neutralizes both soluble and membrane-bound TNF-α, is an effective treatment in autoimmune diseases such as psoriasis, RA and IBD, which can lead to long-term remission. Besides its inhibitory effects on T-cell activation and homing, infliximab was shown to impair the differentiation and antigen-presenting capacity of ModDCs (202) and to restore the suppressive function of previously compromised Treg in patients with RA (203). CTLA-4 Ig, a fusion protein that blocks B7-CD28 costimulation and is currently used in RA patients as well as in SOT, can also promote tolDC (204, 205). The combination of adoptive transfer of tolDCs and CTLA-4 Ig resulted in extended survival of MHC-mismatched heart allografts in mice (206). In an experimental model, CTLA-4 Ig suppressed collagen-induced arthritis by inducing tolDCs and expanding Treg. This effect was abrogated by anti-TGF-β treatment (207). Other drugs that can be used to modulate DCs functions are listed in **Table 2**. Finally, antigens could be directly delivered to steady-state quiescent DCs *in vivo* by specifically targeting DC-restricted endocytic receptors (DEC-205) with monoclonal antibodies (208), aiming at inducing antigen-specific T-cell hyporesponsiveness. Altogether, it will be important to take advantage of some currently used drugs or biological agents in order to promote an environment that favors tolDCs.

### Overcoming Memory Responses

Based on their central role in immune activation, it is very tempting to consider immunotherapy using tolDCs for tolerance induction to specific antigens. Indeed, the type of DC and the cytokine microenvironment at the time of antigen presentation are major components in the regulation of T-cell responsiveness. While many *in vitro* and *in vivo* experimental data support the potential of tolDCs in regulating the priming of naïve T cells, less evidence exist regarding memory T cells. This is an important issue as the human immune repertoire harbors antigen-specific as well as cross-reactive long-lived memory T cells and antibodies which could represent an obstacle for clinical translational of tolDCs-based immunotherapy in chronic autoimmune diseases as well as in SOT (209, 210). There are, however, some experimental and preclinical studies that illustrate the potential of tolDCs in controlling memory T cells (108, 133, 134), suggesting that tolDCs would be advantageous compared to Treg-based immunotherapy or more conventional immunosuppression that are less efficient in the presence of preexisting memory responses (211). In an experimental allergic airway disease model, tolDCs were shown to inhibit allergenspecific memory Th2 responses and airway inflammation in sensitized hosts (135). In a clinically relevant NHP model of MHC-mismatched renal transplantation with minimal immunosuppression, infusion of donor-derived tolDCs 1 week before transplantation prolonged allograft survival, with no evidence of host sensitization. This therapeutic effect was associated with selective attenuation of donor-reactive memory T-cell responses (86, 198).

### Migration of tolDCs

Although significant progress has been made in the generation of tolDCs, the capacity of DCs to access LNs and encounter T cells, as well as the survival of treated DCs remains a critical concern. It is known that immature DCs traffic poorly to LNs. Different approaches have been made to overcome this issue. Genetically engineered DCs that express CCR7 and IL-10 showed improved migration ability to T-cell zones of secondary lymphoid organs and promoted prolonged heart allograft survival in a mouse model (212). Interestingly, in the setting of cancer vaccines development, the direct intra-lymphatic delivery approach is being evaluated in a clinical setting. DCs injected into a lymphatic vessel showed a prolonged half-life compared to DCs injected intravenously and were rapidly detected in LNs where they elicited a strong T-cell response (213, 214). The technique proved to be feasible and safe for short-term delivery of DCs. However, research is still needed to assess the efficacy of this route and the clinical relevance in the context of tolerance induction.

### CONCLUSION AND PERSPECTIVES

The field of immune regulation has become increasingly complex with the identification of both lymphoid and non-lymphoid cells that are involved in the induction and maintenance of immune tolerance. DCs are at the cornerstone of adaptive immune responses and, therefore, represent an appealing tool for immunoregulatory therapies in autoimmune diseases as well as in SOT. While extensive data have been generated in animal models, questions remain regarding the distribution and function of human DCs subsets *in vivo*. Clinical protocols need to be optimized for the safe generation of tolDCs, ensuring stable phenotypes and immunomodulatory functions. Interestingly, although both exogenously transferred or *in vivo* induced tolDCs may have a short half-life, they were shown to induce a regulatory environment (regulatory cell-subsets and cytokines) that could promote a more prolonged maintenance of peripheral tolerance (215). Finally, tolDCs immunotherapy has a unique potential for inducing antigen-specific central tolerance.

Whether or not tolDCs can maintain their function and regulate memory T-cell and B-cell responses in a strong inflammatory environment still remains to be demonstrated in more stringent preclinical models (209, 216, 217). In the clinical setting,

### REFERENCES

tolDC-based therapy, therefore, may not be sufficient *per se* to promote tolerance and may need to be used in conjunction with immunomodulatory drugs. While further preclinical and clinical studies are needed before tolDC-based immunotherapy can be successfully translated to the clinic, the quest for modalities to induce immune tolerance has clearly improved our understanding of human DC biology. The production and use of tolDCs in the upcoming years will continue to represent a challenging and exciting road, which, ultimately, may improve various immunemediated clinical pathologies.

### AUTHOR CONTRIBUTIONS

All authors contributed to writing the article. The final version of the manuscript was reviewed and approved by all authors.

### ACKNOWLEDGMENTS

This work was supported by the Fondation Pierre Mercier pour la Science, the Fondation Medi-CAL Futur, the Fondation Lausannoise pour la Transplantation d'Organes (to DG), the Fondation Vaudoise de Cardiologie (to GV), as well as an unrestricted grant from Astellas (to DG and MP).

and the inflamed setting. *Annu Rev Immunol* (2013) 31:563–604. doi:10.1146/ annurev-immunol-020711-074950

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Obregon, Kumar, Pascual, Vassalli and Golshayan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Regulating Immunogenicity and Tolerogenicity of Bone Marrow-Derived Dendritic Cells through Modulation of Cell Surface Glycosylation by Dexamethasone Treatment

*Kevin Lynch1 , Oliver Treacy1 , Jared Q. Gerlach1,2, Heidi Annuk 2 , Paul Lohan1 , Joana Cabral1 , Lokesh Joshi 2 , Aideen E. Ryan1,3 and Thomas Ritter1 \**

#### *Edited by:*

*Catharien Hilkens, Newcastle University, United Kingdom*

#### *Reviewed by:*

*Lenka Palová Jelínková, Charles University, Czechia Maja Wallberg, University of Cambridge, United Kingdom*

*\*Correspondence: Thomas Ritter<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology*

*Received: 15 September 2017 Accepted: 13 October 2017 Published: 30 October 2017*

#### *Citation:*

*Lynch K, Treacy O, Gerlach JQ, Annuk H, Lohan P, Cabral J, Joshi L, Ryan AE and Ritter T (2017) Regulating Immunogenicity and Tolerogenicity of Bone Marrow-Derived Dendritic Cells through Modulation of Cell Surface Glycosylation by Dexamethasone Treatment. Front. Immunol. 8:1427. doi: 10.3389/fimmu.2017.01427*

*1School of Medicine, Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Galway, Ireland, 2Glycoscience Group, NCBES National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland, 3Discipline of Pharmacology and Therapeutics, School of Medicine, National University of Ireland, Galway, Ireland*

Dendritic cellular therapies and dendritic cell vaccines show promise for the treatment of autoimmune diseases, the prolongation of graft survival in transplantation, and in educating the immune system to fight cancers. Cell surface glycosylation plays a crucial role in the cell–cell interaction, uptake of antigens, migration, and homing of DCs. Glycosylation is known to change with environment and the functional state of DCs. Tolerogenic DCs (tDCs) are commonly generated using corticosteroids including dexamethasone, however, to date, little is known on how corticosteroid treatment alters glycosylation and what functional consequences this may have. Here, we present a comprehensive profile of rat bone marrow-derived dendritic cells, examining their cell surface glycosylation profile before and after Dexa treatment as resolved by both lectin microarrays and lectin-coupled flow cytometry. We further examine the functional consequences of altering cell surface glycosylation on immunogenicity and tolerogenicity of DCs. Dexa treatment of rat DCs leads to profoundly reduced expression of markers of immunogenicity (MHC I/II, CD80, CD86) and pro-inflammatory molecules (IL-6, IL-12p40, inducible nitric oxide synthase) indicating a tolerogenic phenotype. Moreover, by comprehensive lectin microarray profiling and flow cytometry analysis, we show that sialic acid (Sia) is significantly upregulated on tDCs after Dexa treatment, and that this may play a vital role in the therapeutic attributes of these cells. Interestingly, removal of Sia by neuraminidase treatment increases the immunogenicity of immature DCs and also leads to increased expression of pro-inflammatory cytokines while tDCs

**Abbreviations:** iDC, immature bone marrow-derived dendritic cell; tDC, tolerogenic bone marrow-derived dendritic cell; niDC, neuraminidase-treated bone marrow-derived immature dendritic cell; ntDC, neuraminidase-treated bone marrowderived tolerogenic dendritic cell; mDC, mature bone marrow-derived dendritic cell (LPS-treated); MLR, mixed lymphocyte reactions; Sia, sialic acid; Dexa, dexamethasone.

are moderately protected from this increase in immunogenicity. These findings may have important implications in strategies aimed at increasing tolerogenicity where it is advantageous to reduce immune activation over prolonged periods. These findings are also relevant in therapeutic strategies aimed at increasing the immunogenicity of cells, for example, in the context of tumor specific immunotherapies.

Keywords: tolerogenic dendritic cells, glycosylation, dexamethasone, immunogenicity, tolerogenicity, sialic acid, autoimmunity, cell therapy

### INTRODUCTION

Dendritic cells are professional antigen-presenting cells, which are a component of the innate immune system which induce adaptive immune responses (1). Dendritic cells (DCs) were first described by Steinman and Cohn in 1973 (2) and were subsequently identified to be potent activators of the immune system when employed in mixed lymphocyte reactions (MLRs) (3). DCs are a heterogeneous population classified in different subsets dependent on the origin (4). DCs have been extensively investigated for potential use as a cellular therapy due to their ability to maintain peripheral tolerance, which is of importance in the field of transplantation and autoimmunity. Since mature DCs are potent activators of the T-cell responses, pharmacological approaches have been used to maintain DCs in a maturation resistant state (5–7). The glucocorticoid dexamethasone (Dexa) has been widely used in this context (8–11). Glucocorticoids are potent immunosuppressive drugs that are used in clinical regimens to treat both Th1- and Th2-mediated inflammatory diseases including allograft rejection (12). Dexa is known to exert potent effects on many immune cells including DCs (8, 13). It has been consistently described in the literature that Dexa has inhibitory effects on the development of immature DCs (iDCs) (5, 8, 12, 14), and that it also impairs lipopolysaccharide (LPS) (TLR4) stimulation of DCs, which would otherwise lead to their maturation (mDCs) (15–17). In addition to this, Dexa-treated DCs have a reduced capacity to activate naïve T lymphocytes by interfering with Signals 1–3 important for T-cell activation (17).

In the context of transplantation, preclinical experiments suggested the potential therapeutic use of both donor and recipientderived tolerogenic DCs to prevent organ graft rejection (18). In a rat model, we have recently shown that pretreatment of donor DCs with Dexa *ex vivo* prevents the maturation of DCs and prolongs rat corneal allograft survival upon injection in corneal transplant recipients (13). However, the mechanisms of how tolerogenic DCs engage with other immune cells and exert their immunomodulatory effects are not completely understood. Despite this, tolerogenic DCs have been already tested in humans suffering from various diseases. As of this writing, there are currently eight tolerogenic DC cell therapies listed in Phase I/ II clinical trials for treatment of autoimmune disease and graft rejection (https://clinicaltrials.gov. September 2017, search for key words tolerogenic DCs), which highlights the importance and urgency of understanding the mechanisms associated with the therapeutic effect.

Glycosylation is one of the most vital and frequent forms of posttranslational modification and is involved in the function of many immune associated molecules. Some of the functions of glycosylation include, but are not limited to, protein folding and molecular trafficking to the cell surface (19–23). Glycosylation has also been implicated in the stability of proteins and protection from proteolysis (24). All immune cells are coated by a glycocalyx composed of a complex assortment of oligosaccharides (glycans), of which one frequent terminal component is sialic acid (Sia). Sias are a broad family of negatively charged, 9-carbon monosaccharides that are exposed to the cellular microenvironment and are involved in communication and in cellular defense (25). It has been reported that a typical somatic cell surface presents millions of Sia molecules (26) and also that they have long been noted to be important in immune cell behavior (27). It has been suggested that Sias can play important roles in both acting as a recognizable molecule for cellular interactions but also as a biological shield preventing receptors on cells recognizing their ligands (28). Large amounts of Sias on the cell surface of immune cells will result in an overall negative charge, which can have biophysical effects, such as the repulsion of cells from each other and subsequently disrupting cellular interactions (29).

Since immune cell interactions form the basis of immune responses, glycosylation is, therefore, likely to play a major role in dictating these responses. However, there is a significant knowledge gap as to how glycosylation modulates immune responses. Currently, little information exists on how DC glycosylation patterns change after Dexa treatment. Here, we present a comprehensive profile of bone marrow-derived DCs (BMDCs), examining their cell surface glycosylation before and after Dexa treatment as resolved by both lectin microarrays and lectincoupled flow cytometry.

In this work, the composition of the glycocalyx of both iDCs and tolerogenic DCs (tDCs) was altered using neuraminidase (sialidase) treatment and the functional consequences in immunogenicity and inhibition of T-cell proliferation were observed. We show that Sia is upregulated on tDCs contributing to the tolerogenic state of tDCs. However, removal of Sia leads to increased stimulatory activity of iDCs leading to enhanced T-cell activation and proliferation. These findings have important implications in strategies aimed at increasing tolerogenicity where it is advantageous to reduce immune activation over prolonged periods. These findings are also relevant in therapeutic strategies aimed at increasing the immunogenicity of cells, for example, in the context tumor specific immunotherapies.

## MATERIALS AND METHODS

### Animals

All animals used in experiments were accommodated in an accredited animal housing facility under a license granted by the Department of Health, Ireland, and were approved by the Animals Ethics Committee of the National University of Ireland, Galway. Bone marrow used in the generation of BMDCs was isolated from male Dark Agouti (DA, RT-1avl) rats at 8–14 weeks of age. For the allogeneic MLRs, male Lewis (LEW, RT-1l ) rats served as a source of lymphocytes, isolated from both the cervical and mesenteric lymph nodes and spleen. DA and LEW rats were obtained from Harlan Laboratories (Bicester, UK).

### Isolation and Generation of iDCs and tDCs

Immature DCs were generated using an adapted version of the protocol, which has been previously described (13) (Figure S1 in Supplementary Material). Briefly, on day 0, male DA rats of the specified age were sacrificed and the tibia and femur were surgically removed postmortem. The epiphyses were cut and the bone marrow was flushed from the long bones with a syringe/needle combination. The erythrocytes were removed from the suspension by lysis using a standard red blood cell lysis buffer (Sigma-Aldrich, Dublin, Ireland). After erythrocyte lysis, the cells were washed in RPMI-1640 (Gibco, Grand Island, NY, USA) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L l-glutamine, 100 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 55 µmol/L 2-β-mercaptoethanol (2β-ME) (Gibco). Cells were resuspended at a concentration of 1.5 × 106 /mL and plated at a concentration of 4.5 × 106 per well of a 6-well plate. The culture medium was supplemented with 5 ng/mL rat granulocyte-macrophage colony-stimulating factor (GM-CSF) (Invitrogen, Paisley, UK) and 5 ng/mL rat IL-4 (Peprotech EC, London, UK). Cells were incubated under standard cell culture conditions (37°C at 5% CO2) and, on the third day of culture, half of the medium from each well was harvested and cells were resuspended in fresh medium supplemented with rat GM-CSF and IL-4 and added back into the culture. On the fifth day, the supernatant was exchanged with fresh supplemented growth medium to remove dead granulocytes and lymphocytes. In experiments requiring tDCs, Dexa (Sigma-Aldrich) was added to the culture at 10−6 mol/L at this point. On the seventh day of culture, half of the medium was again removed and replaced with fresh supplemented medium (Dexa was added as required). To generate mDCs, LPS (1 µg/mL; Sigma-Aldrich) was added 24 h before the cells were cultured. Cultures were maintained until day 10 and then gently pipetted off the bottom of the wells for the *in vitro* assays.

### Neuraminidase Treatment

To produce neuraminidase-treated iDCs and tDCs (niDCs and ntDCs), BMDCs were harvested on day 10 of culture and 2 × 105 /mL were treated with 400 U/mL of recombinant *Clostridium perfringens* neuraminidase (P0720S, New England Biolabs, Ipswitch, MA, USA) in phosphate buffered saline (PBS) supplemented with 1 mM MgCl2 (Sigma-Aldrich), 1 mM CaCl2 (Sigma-Aldrich), and 1% bovine serum albumin (Sigma) for 90 min at 37°C.

### RNA-Isolation and RT-PCR

RNA was exacted from iDCs, tDCs, mDCs, niDCs, and ntDCs on day 10 using Bioline Isolate II RNA mini kits according to manufacturer's protocols. All cDNA was produced using RevertAidTM H Minus Reverse Transcriptase (Thermo Fisher Scientific, MA, USA) with random primers. For primer sequences of GAPDH, TNF-α, IL-12p40, inducible nitric oxide synthase (iNOS) IL-10, IDO, IL-6, and IL-1β, see Table S1 in Supplementary Material. All samples were normalized to expression of the house-keeping gene GAPDH and made relative to iDCs. All quantitative realtime PCR was performed according to the standard program using a real-time PCR system (StepOne Plus, Applied Biosystems, Thermo Fisher Scientific).

### Flow Cytometry

Cells were characterized by flow cytometry using the monoclonal antibodies (mAbs) CD11b/c-APC, CD80-PE, CD86-PE, MHCI-FITC, and MHCII-PE (BioLegend, San Diego, CA, USA). For analysis of the glycocalyx, lectins from *Maackia amurensis* (MAL II, indicating α2-3 Sia) and *Sambucus nigra* (SNA-I, indicating α2-6 Sia) were used (Vector Labs). Lectins were biotin conjugated. PE-streptavidin was used for detection. Negative controls for non-specific fluorescence were used, these consisted of PE-streptavidin staining solutions in the absence of the lectin conjugated to biotin. Lectins were prepared in lectin staining buffer (PBS containing 1% FBS, 1 mmol/L CaCl2, and 2 mmol/L MgCl2) and resuspended in FACS buffer (PBS containing 2% fetal calf serum and 0.01% NaN3, all from Sigma-Aldrich) before analysis using a FACS Canto II (BD Biosciences, Oxford, UK).

For analysis of the assays involving lymphocytes from the lymph nodes and spleen, the following mAbs were used CD3/PE, CD8/PE-Cy7, CD4/APC (BioLegend), and CD25/FITC (eBioscience, San Diego, CA, USA). Prior to staining, cells were washed with FACS buffer. mAbs were diluted in 50 µL FACS buffer, added to the cells, and incubated for 15 min at 4°C. To remove any unbound antibodies, the cells were washed three times with FACS buffer. The cells were then filtered through a nylon mesh (40 µm) before analysis in the cytometer.

### Mixed Lymphocyte Reaction/T Cell Proliferation Assays

Lymphocytes were isolated from the spleen and lymph nodes of LEW rats. T cells were washed with phosphate-buffered saline and stained in prewarmed (37°C) CellTrace™ Violet (CTV) phosphate-buffered saline staining solution (Invitrogen, Carlsbad, CA, USA) as per manufacturer's instructions. 2 × 105 CTV-stained T cells were stimulated at a 1:1 ratio with antirCD3/anti-rCD28-labeled beads in supplemented RPMI 1640 media. Assays were incubated at various BMDC: T-cell ratios in a humidified incubator for 4/5 days at 37°C following which T-cell proliferation and CD4 and CD8 expression were assayed by flow cytometry (mAbs CD4-APC and CD8α-PE-Cy7; Biolegend). T-cell proliferation, activation, and differentiation were analyzed using a FACS Canto II.

## Membrane Protein Extraction and Labeling

Membrane proteins were extracted from iDCs, tDCs, niDCs, and ntDCs using a commercial protein extraction kit (Mem-Per®, Thermo Fisher Scientific). Proteins recovered from 106 cells were labeled with 100 µg (10 mg/mL in DMSO) Alexa Fluor® succinimidyl ester 555 dye (Thermo Fisher Scientific) as per the manufacturer's instructions. Labeled protein was separated from unconjugated dye with Bio-Gel® P6 (Bio-Rad Laboratories, Dublin, Ireland).

### Lectin Microarray Construction and Sample Interrogation

Lectin microarrays were constructed essentially as described previously in Ref. (30). Forty-four lectins (Table S2 in Supplementary Material) sourced from multiple vendors were diluted to 0.5 mg/mL in PBS supplemented with 1 mM of respective haptenic sugar to maintain binding site integrity (see Table S2 in Supplementary Material) and printed on Nexterion® H (Schott, Mainz, Germany) functionalized glass substrates using a sciFLEXARRAYER S3 non-contact spotter (Scienion, Berlin, Germany). During printing, relative humidity and temperature were maintained at 62% (±2%) and 20°C, respectively. Following printing, slides were incubated in a humidity chamber overnight at 20°C to ensure completion of covalent conjugation. Unoccupied functional groups were deactivated by 1 h incubation with 100 mM ethanolamine in 50 mM sodium borate, pH 8. Finished slides were washed with PBS with 0.05% Tween-20 (PBS-T) three times for 3 min and once with PBS for 3 min, centrifuged dry (450 × *g*, 5 min), and stored at 4°C with desiccant until use.

Labeled cellular proteins were incubated with finished microarrays following extensive optimization as described in Ref. (30). All processes were carried out with limited light exposure. Samples were applied to microarrays using an 8-well gasket slide and incubation cassette system (Agilent Technologies, Cork, Ireland). 70 µL of each labeled glycoprotein at 0.5 mg/mL, in incubation buffer [TBS-T; Tris-buffered saline (TBS; 20 mM Tris– HCl, 100 mM NaCl, pH 7.2, supplemented with 1 mM CaCl2, and 1 mM MgCl2) with 0.05% Tween®-20], was applied to each well of the gasket. A total of 18 technical replicates were carried out for iDC and tDC profiling (encompassing samples of five biological replicates). Each microarray slide was loaded into a cassette with an accompanying gasket slide and placed in a rotating incubation oven (23°C, approximately 4 rpm) for 1 h. Incubation cassettes were disassembled under TBS-T, and microarrays were washed in a Coplin jar twice in TBS-T for 2 min each and once with TBS for 2 min. Microarrays were dried by centrifugation (450 × *g*) and imaged immediately using an Agilent G2505B microarray scanner at 5 µm resolution (532 nm laser, 100% laser power, 90% PMT).

### Microarray Data Extraction and Analysis

Data extraction and analysis was performed essentially as previously described (30, 31). In brief, raw intensity values were extracted from high-resolution \*.tif files using GenePix Pro v<IP_ADDRESS> (Molecular Devices, Berkshire, UK) and a proprietary \*.gal file (containing feature spot addresses and identities) using adaptive diameter (70–130%) circular alignment based on 230 mm features. Numerical data were exported as text to Excel (Version 2010, Microsoft, Dublin, Ireland). Local backgroundcorrected median feature intensity data (F543median-B543) was analyzed. The median value, derived from data from six replicate spots per subarray, was handled as a single data point for graphical and statistical analyses.

Lectin microarray intensity values were normalized to the median total intensity value for all features across all subarrays. The significance of difference between relative intensity data (\**p*< 0.05, \*\**p*< 0.01, \*\*\**p*< 0.001, \*\*\*\**p*< 0.0001) was evaluated for each set of replicates on a lectin-by-lectin basis using a standard Student's *t*-test (two-tailed, two sample unequal variance). Unsupervised, hierarchical clustering of lectin-binding data was performed with Hierarchical Clustering Explorer v3.0 (http:// www.cs.umd.edu/hcil/hce/hce3.html). For clustering analysis, previously, normalized data were imported directly and clustered with the following parameters: no pre-filtering, complete linkage, Euclidean distance. Principal component analysis (PrCA) of previously normalized and pre-filtered data (those lectins which demonstrated *p* < 0.01 or better in the above *t*-tests, 15 in total) was performed using Minitab version 16.1.1 (Minitab, Inc., State College, PA, USA).

### Statistical Analysis

Data were analyzed using the software package FlowJo v10 (Tree Star, Ashland, OR, USA). All data were analyzed with Graphpad Prism V6 software (Graphpad Software, CA, USA) and are expressed as mean ± SEM unless otherwise indicated. Comparisons among multiple groups were made with one-way ANOVAs followed by Tukey's multiple comparisons test. Data sets with two groups were analyzed using an unpaired *t*-test. Differences were considered statistically significant when *p-*value was <0.05.

### RESULTS

### Dexamethasone Treatment of BMDC Induces a Tolerogenic Phenotype

Dexamethasone treatment of DCs has been reported to generate tolerogenic DCs (tDCs) (32). To generate iDCs, bone marrow was flushed from the long bones of the tibia and femur of DA rats and cultured in medium supplemented with GM-CSF, IL-4, and Dexa (for tDCs) as required (Figure S1A in Supplementary Material). Following isolation, cell surface characterization was performed using flow cytometry by gating on the CD11b/c population (**Figure 1A**). tDC generation did not result in any significant changes in cell size (**Figure 1B**, i) but the number of cells harvested from wells that were treated with Dexa was significantly lower than that of wells that were Dexa-free (**Figure 1B**, ii). This may be due to Dexa-induced apoptosis of the DCs, which has been reported by other groups (33). While lower numbers of cells were obtained from tDC wells, after harvesting and washing of the

#### Figure 1 | Continued

Isolation, generation, and characterization of immature DCs (iDCs), tolerogenic DCs (tDCs), and stimulated DCs (mDCs). Bone marrow was flushed from the femur and tibia of 8- to 14-week-old DA rats and cultured in IL-4 and GM-CSF cultured media for 10 days (Figure S1 in Supplementary Material). (A) Representative gating strategy. Cells were selected according to size and granularity (i) followed by live/dead discrimination based on Sytox blue negative cells (live) (ii). After single cell selection (iii), cells were selected by CD11b/c (APC) positivity (iv). (B) Changes in cell size (*n* = 3) (i), the number of cells harvested (*n* = 8) (ii), and viability of iDCs to tDCs (*n* = 4) (iii) was compared.  Representative histograms and bar charts displaying relative fluorescence intensity (RFI) for flow cytometric analysis of DC cell surface. Median fluorescence intensities were established relative to iDCs. (D) The mRNA expression of interleukin 6 (IL-6), Indoleamine 2,3-dioxygenase (IDO), interleukin 1 beta (IL-1β), inducible nitric oxide synthase (iNOS), and IL-12p40 was analyzed in iDCs and tDCs. Normalized to GAPDH and fold change relative to iDCs. Error bars: mean ± SEM \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 one-way ANOVA, Tukey's multiple comparisons test.

cells, no significant changes in viability was noted (**Figure 1B**, iii). We also analyzed the expression levels of the costimulatory molecules CD80/CD86 and the major histocompatibility complex class I and II molecules (MHCI/II) as an indicator of the maturation status of generated iDCs and tDCs (**Figure 1C**). The expression levels of CD80, CD86, MHC I, and MHC II indicate that the iDCs display a semi-mature phenotype. However, when the cells were treated with Dexa, a significant reduction in the expression level of MHC II was observed with no changes in MHC I (**Figure 1C**). To mature iDCs or tDC *in vitro*, LPS was added to the cultures (1 µg/mL) for 24 h. A significant increase in both CD80/CD86, MHC I and MHC II was noted. However, tDCs following LPS treatment showed significantly reduced expression levels of CD80/CD86 and MHC I/II molecules compared to stimulated iDCs indicating a phenotype that is maturation resistant. iDC and tDC populations were also assessed for expression of pro- and anti-inflammatory markers with and without Dexa-treatment by qRT-PCR (**Figure 1D**). Results indicate that LPS stimulation of iDCs leads to an increase in mRNA expression of pro-inflammatory molecules such IL-6, IL-12p40, and iNOS. In contrast, tDCs are less sensitive to TLR4 stimulation compared to mDCs, indicated by no observed increases in IL-6, IL-12-p40, and iNOS after LPS treatment. Higher levels of IDO mRNA, which is known as a marker in tolerogenic cells, is present in LPS-treated tDCs when compared to mDCs. Interestingly, IL-1β mRNA expression does not seem to be regulated by Dexa, as LPS stimulation leads to a profound increase, which cannot be blocked by Dexa. All together these data indicate that Dexa treatment of iDCs leads to the generation of a tolerogenic DC phenotype with reduced expression of markers of immunogenicity and reduced expression of pro-inflammatory molecules but increases in immunoregulatory molecules.

### tDC Generation Modulates the Glycocalyx by Significantly Increasing Levels of **α**2-6-Linked Sia

Changes in DC glycocalyx after induction of tolerogenic phenotype have not been investigated. To address this knowledge gap, lectin microarray profiling of proteins extracted from the membranes of iDCs and tDCs and lectin-coupled flow cytometry of intact iDCs and tDCs was undertaken.

Comparisons of all lectin microarray replicate profiles were made by unsupervised hierarchical clustering. This clustering approach revealed two major clusters with separation at 53% minimum similarity (**Figure 2A**). With the complete linkage method employed, two untreated iDC replicates were placed into the tDC group while only three of the iDC replicates, two from biological set 2 and one from set 5 (**Figure 2A**), showed outlier behavior and were excluded from the major cluster containing the balance of the iDC replicate data. However, the well-defined separation of the vast majority of the iDC and tDC replicates into two groups (**Figure 2A**, Group 1 and 2) supports the solidity of the subtle profile differences and also the high level of reproducibility for the lectin profiling method in distinguishing membrane glycoprotein samples from iDCs and tDCs.

Median values obtained from normalized lectin microarray profile data (*n* = 18) for iDCs and tDCs were broadly similar with only small, but significant, differences in intensities noted at a subset of the lectin panel (**Figure 2B**). The general profiles of tDC glycoproteins remained similar to those of iDCs across lectin features. Furthermore, the lectin profiles displayed no obvious signs of cell stress as evidenced by a lack of elevation of signals suggesting increased endoplasmic reticulum- and proximal Golgi-associated glycan structures (i.e., increased evidence of high mannose structures). However, SNA-I showed a consistent intensity increase with tDC surface glycoproteins (*p* = 2 × 10−10) relative to iDCs, which is in line with previous findings from our group (13). PrCA performed using the 15 lectins, which demonstrated *p* < 0.01 (SNA-II, BPA, PNA, DSA, LEL, SNA-I, RCA-I, CPA, ECA, LTA, UEA-I, EEA, GS-I-B4, MPA, and VRA) revealed a division of replicate lectin profiles dominated by distinct groups containing iDCs or tDCs with minimal overlap and further reinforced the ability of these lectins to distinguish untreated iDCs from tDCs (**Figure 2C**). In short, these lectin microarray profiles demonstrate that the glycocalyxes of the iDC and tDCs are distinct. These changes were validated using lectin-coupled flow cytometry. The increase in SNA-I binding suggests an increase in quantity or better accessibility to α2-6-linked with no significant change suggested for α2-3-linked Sia (MAL-II) confirmed lectin microarray findings (**Figure 2D**).

### Neuraminidase Treatment of iDCs and tDCs Modulates Levels of **α**2-6-Linked Sia and Alters Expression Levels of Immunogenicity Markers

Sia has long been reported to be important in DC biology (28). Considering the dramatic increase observed after Dexa treatment confirmed by both flow cytometry and lectin microarray (**Figures 2B–D**), we cleaved Sia using neuraminidase to study phenotypical and functional changes upon removal. iDCs and tDCs were treated with neuraminidase (designated niDC and ntDC, respectively) and lectin binding profiles for SNA-I and MAL-II were analyzed using flow cytometry. Both niDCs and ntDCs showed a significant reduction in SNA-I binding intensities and trend decreases MAL-II binding intensities suggesting the successful removal of α2-6-linked and α2-3-linked Sia, respectively (**Figure 3A**, i–iv). Based on these results, we further investigated if the removal of Sia resulted in a detectable increase of the expression of MHC I, MHC II, CD80, and CD86 immunogenicity markers after treatment with neuraminidase. niDCs (**Figure 3B**, i) had small but significant increases in MHC II and CD86 expression when compared to iDCs. MHC I showed

\*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 one-way ANOVA, Tukey's multiple comparisons test.

a trend increase in expression on niDCs compared to iDCs, and there was no change in CD80 expression after treatment with neuraminidase. ntDCs (**Figure 3B**, ii) displayed a significant increase in both MHC I and MHC II with no changes in CD80 and a trend increase in CD86 after neuraminidase treatment. niDC and ntDC populations were also assessed for expression of pro- and anti-inflammatory markers by qRT-PCR (**Figure 3C**). Although there was some sample-to-sample variation, our data indicate that neuraminidase treatment of iDCs leads to dramatic increases in pro-inflammatory mRNA expression of IL-6, IL-1β, iNOS, TNF-α, and IL-12-p40. However, ntDCs are protected from this strong increase in pro-inflammatory cytokine expression in the case of iNOS and IL-12-p40, but mRNA levels of IL-6, IL-1β, and TNF-α are increased. Interestingly, levels of anti-inflammatory IL-10 are lost after neuraminidase treatment in both iDCs and tDCs. In summary, these results indicate that neuraminidase treatment reduces Sia on the cell surface of both iDCs and tDCs and leads to the stimulation of pro-inflammatory cytokine mRNA expression, which can be largely inhibited by Dexa treatment.

### Neuraminidase Treatment Alters Immunomodulatory Properties of iDCs and tDCs

Considering that the removal of Sia altered the immunogenic phenotype of both iDCs and tDCs, we further analyzed the effects of neuraminidase treatment on iDCs and tDCs through *in vitro* allogeneic coculture experiments. iDCs or tDCs from DA rats were treated with neuraminidase and cocultured with allogeneic lymphocytes. The immunogenic potential or the ability of niDCs and ntDCs to induce the proliferation and/or the activation of allogeneic lymphocytes was analyzed by T-cell proliferation assays (**Figure 4A**). Responder LEW rat T cells were analyzed based on their co-expression of CD3+CD4+ or CD3+CD8+ (**Figure 4B**). Proliferation of lymphocytes was measured using CellTraceTM Violet (CTV) and activation of lymphocytes was measured using CD25 as an activation marker. DA iDCs (**Figure 4C**, i) and tDCs (**Figure 4C**, ii) did not induce an allogeneic response as indicated by a lack of changes in LEW CD3+CD4+ or CD3+CD8+ T cell proliferation when compared to unstimulated lymphocytes alone. Additionally, we observed no significant changes in CD3+CD4+CD25 or CD3+CD8+CD25 expression (data not shown) supporting our data on reduced immunogenicity of iDCs and tDCs. However, niDCs (**Figure 4C**, i) significantly stimulated both CD3+CD4+ and CD3+CD8+ T cell proliferation when compared to both unstimulated lymphocyte controls and iDCs. This indicates the importance of Sia in the maintenance of an iDCs phenotype. While ntDCs (**Figure 4C**, ii) show a trend increase to stimulate CD3+CD8+ T cells, there were no significant changes noted (**Figure 4C**). To eliminate the possibility of cell death as a potential cause of this increase in proliferation, we assessed cell death using Sytox Blue. We observed that iDCs have less cell death after neuraminidase treatment than tDCs (Figure S2 in Supplementary Material) enabling us to exclude this possibility. Finally, we investigated if niDCs and ntDCs can regulate the proliferation of stimulated T cells. LEW T cells were labeled with CTV, stimulated with CD3/CD28 labeled beads, and cocultured with niDCs and ntDCs (**Figure 5A**) and CD3+CD4+ and CD3+CD8+ proliferation was measured by flow

#### Figure 3 | Continued

Neuraminidase treatment of immature DCs (iDCs) and tolerogenic DCs (tDCs) decreases levels of α2-6-linked Sia significantly and alters phenotype. iDCs or tDCs were treated with neuraminidase for 90 min at 37°C to cleave Sia residues on the surface. (A) α2-6-linked Sia (SNA-I) (i) and α2-3-linked Sia (MAL-II) (ii) was measured on iDCs and niDCs after neuraminidase treatment (*n* = 3). α2-6-linked Sia (SNA-I) (iii) and α2-3-linked Sia (MAL-II) (iv) was measured on tDCs and ntDCs after neuraminidase treatment (*n* = 3). (B) Both iDCs (i) and tDCs (ii) were analyzed by flow cytometry for their expression of MHC I (FITC), MHC II (PE), CD 80 (PE), and CD 86 (PE) after neuraminidase treatments. (C) The mRNA expression of interleukin 6 (IL-6), interleukin 1 beta (IL-1β), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin subunit beta (IL-12p40), and interleukin 10 (IL-10) was analyzed in iDCs, niDCs, tDCs, and ntDCs. Normalized to GAPDH and fold change relative to iDCs. Representative histograms and bar charts displaying relative fluorescence intensity (RFI) for flow cytometric analysis of DC cell surface. Median fluorescence intensities were established relative to iDCs in the case of niDCs and tDCs in the case of ntDCs. Error bars: mean ± SEM \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 one-way ANOVA, Tukey's multiple comparisons test. Data sets with two groups were analyzed using an unpaired *t*-test.

cytometry. Neuraminidase treatment completely abrogates the T cell inhibitory effect of iDCs leading to full restoration of T cell proliferation (**Figure 5A**, i). Interestingly, Dexa treatment is not sufficient to enable iDCs to inhibit the proliferation of activated T cells as no differences were observed between tDCs and ntDCs (**Figure 5B**, ii). In summary, these data indicate that the removal of Sia from iDCs increases the immunogenicity by its ability to stimulate CD4 and CD8 T cell proliferation, which can be prevented by Dexa treatment. In contrast, neuraminidase treatment completely restores the proliferation of polyclonally activated T cells, which cannot be prevented by Dexa treatment.

## DISCUSSION

Organ transplantation is often considered as the only therapeutic option for patients with life-threatening organ disease and is now performed on a routine basis. Due to incompatibilities between donor and recipient MHC-molecules, patients are required to take immunosuppressive drugs to prevent the destruction of the transplanted organ by the recipient's immune system. Immunosuppressive drug regimens are associated with severe side effects long term (34, 35). As a result, alternative immunosuppressive treatment strategies have been researched and developed including the use of therapeutic DCs in the treatment of autoimmune diseases and in the prevention of allograft rejection. DCs promote central and peripheral tolerance through various mechanisms, such as T cell anergy, inhibition of memory T cell responses, and clonal deletion amongst others (36). These characteristics form the basis of the use of DCs in the induction of tolerance. iDCs even have displayed the ability to convert naïve conventional T cells to regulatory T cells (Tregs) both *in vitro* (37, 38) and *in vivo* (39)*.* As shown here, and as shown by others, iDCs in non-inflammatory conditions display a poor immunogenic phenotype. One of the major barriers for use of iDCs in cellular therapies is that they respond to inflammatory stimuli, exemplified here by TLR4 (LPS) stimulation. In the context of autoimmunity and transplantation, iDCs are bound to encounter inflammatory environments if employed in therapeutic regiments. A potential solution to overcome this is the use of tDCs, which are maturation resistant.

Using tDC cellular therapies for the treatment of organ transplantation looks promising (18). tDCs are now routinely generated using different induction protocols, including the use of corticosteroids such as Dexa (11, 14, 15, 17, 40) and, in fact, we have recently shown in a rat model of corneal transplantation that Dexa generated tDCs significantly prolonged allograft survival without the need for additional immunosuppression (13). In this manuscript, we generate tDCs using Dexa and we characterize their maturation resistant phenotype by analyzing the expression of the immunogenicity markers MHCI, MHCII, CD80, and CD86 before and after TLR4 stimulation. We also analyze the expression of several immunomodulatory cytokine mRNAs. Dexa generated, maturation resistant, tDC have been well characterized by us (13, 32) and by other groups (17). However, to our knowledge, little is known on how Dexa induction of tDCs may affect the glycosylation profile of these cells and what functional consequences this may have. Glycosylation changes are not routinely assayed, but are likely to play crucial roles in iDC and tDC biology.

We describe here for the first time, using both lectin microarray and flow cytometry, that generation of tDCs by Dexa treatment leads to significant alterations in the cell surface glycosylation profile when compared to iDCs. We noted highly significant changes in lectin binding for α2-6-linked Sia (SNA-I) with no significant changes in lectin binding for α2-3-linked Sia (MAL-II). Interestingly, Jenner et al. (41) when comparing human iDCs with iDCs matured with a cytokine cocktail (IL-6, IL-1β, TNF-α, and prostaglandin E2) noted decreased α2-6-linked Sia with no changes in α2-3-linked Sia on the more immunogenic DC. This study also showed that Tregs have higher levels of α2-6-linked Sia when compared to activated conventional T cells. This suggests a possible link between α2-6-linked Sia content and tolerogenicity, where the increased α2-6-linked Sia may potentially serve as ligands for inhibitory sialic acid-binding proteins (Siglecs) on the surface of effector cells (41). In fact, hyper-sialylated antigens loaded onto DCs were recently shown to impose a regulatory program in the DCs. This resulted in the inducement of Tregs *via* Siglec-E and the inhibition of effector T cells (42).

Looking more closely at the lectin microarray analysis, other differences in lectin profiles observed here also hint at significant changes in the total abundance or potential branching alterations of underlying oligosaccharide structures, particularly N-acetyllactosamine (LacNAc), which may have occurred because of Dexa treatment. The relationship of responses among the 15 lectins (SNA-II, BPA, PNA, DSA, LEL, SNA-I, RCA-I, CPA, ECA, LTA, UEA-I, EEA, GS-I-B4, MPA, and VRA) which demonstrated the most significant differences between untreated iDCs and tDCs may hold further clues as to the nature of these variations in the glycocalyx, and it is possible that a portion of

#### Figure 4 | Continued

Neuraminidase treatment alters immunogenic properties of immature DCs (iDCs) and tolerogenic DCs (tDCs). To test the immunomodulatory properties of iDCs, niDCs, tDCs, and ntDCs, they were placed into MLRs for 5 days. (A) Schematic representation of experimental design. DA iDCs, niDCs, tDCs, and ntDCs were placed in cocultures for 5 days with allogeneic LEW lymphocytes isolated from the spleen and lymph nodes. (B) Representative gating strategy. Cells were selected according to size and granularity (i) followed by live/dead discrimination based on Sytox AADvancedTM negative cells (live) (ii). After single cell selection (iii) cells were selected by CD3 (PE) positivity (iv). Further selected by CD4 (APC) and CD8 (PE-CY7) and proliferation was measured by successive generations of CellTraceTM Violet positive cells. (C) The ability of iDCs, niDCs, tDCs, and ntDCs to stimulate allogeneic LEW T-cells was analyzed using unstimulated splenocytes/lymphocytes as a negative control (*n* = 3). (i) Representative histograms and bar charts displaying CD4+ and CD8+ T cell proliferation following a 5-day coculture with iDCs and niDCs. (ii) Representative histograms and bar charts displaying CD4+ and CD8+ T cell proliferation following a 5-day coculture with tDCs and ntDCs. Error bars: mean ± SEM \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 one-way ANOVA, Tukey's multiple comparisons test.

such variations exist among the membrane glycolipid structures as well as membrane proteins, which were analyzed here. With extracted glycoproteins, only one of the three lectins on the microarray, which has been reported to be indicative of Sia presence, SNA-I, demonstrated a significant intensity increase for tDCs. This was also demonstrated by lectin coupled flow cytometry showing how highly regulated Sia metabolism is in DCs. However, responses at lectins, which bind to structures, which are the most frequent attachment points for sialylation, those which bind to galactose (Gal) or *N*-acetylgalactosamine (GalNAc) (SNA-II, BPA, PNA), and those which bind to the associated disaccharide Type II LacNAc (RCA-I, CPA, ECA) or poly-LacNAc (LEL), are particularly interesting because the expected relationship of higher SNA-I binding and simultaneously lower Gal/GalNAc and LacNAc lectin binding did not consistently hold true across the lectin microarray profiles for DCs. The binding profiles and behavior of SNA-I and MAL-II in these experiments strongly infer quantitative differences between iDC and tDC surface Sia content; however, absolute quantitation will ultimately require chromatographic (e.g., HPLC) or chromatography-conjugated mass spectrometric analysis (LC-MS).

Because of the reported importance of Sias in DC pattern recognition (41, 43), endocytosis/phagocytosis (44–47), antigen presentation (48), migration (28, 49–52), and T cell interactions (28, 53). But also, considering that α2-6-linked Sia was the most significantly increased change after tDC generation by Dexa, we choose to investigate Sia's importance in iDC and tDC immunogenicity in an allogeneic setting, which would have potential implications in iDC and tDC cellular therapies.

For this, we removed Sia from the surface of the cells by enzymatic digestion using neuraminidase (sialidase). These experiments showed that Sia is involved in maintaining the tolerogenic phenotype of both iDCs and tDCs, as removal of Sia resulted in an increase in immunogenicity markers and increases in proinflammatory TH1 mRNA transcripts notably IL-6, IL-1β, iNOS (iDCs only), TNF-α, and IL-12p40 (iDCs only) with significant decreases in anti-inflammatory or tolerogenic IL-10. In experiments where neuraminidase-treated human monocyte derived DCs were cultured with ovalbumin (45) or *Escherichia coli* (44), there were reported increases in immunogenicity markers and cytokine gene expression also. Here, we show that even after Dexa treatment and tDC generation the removal of Sia from the cell surface results in increases in both cell surface immunogenicity markers and TH1 pro-inflammatory cytokine gene expression, underpinning the importance of Sia in a non-immunogenic phenotype.

In the context of allogeneic cell therapy for the treatment of autoimmune diseases and in the prevention of allograft rejection, it is important that the cell therapy itself does not elicit a deleterious immune response. In unstimulated allogeneic co-cultures using LEW responder lymphocytes, we show that iDCs and tDCs are non-immunogentic and do not elicit either CD3+CD4+ nor CD3+CD8+ proliferation. This attribute makes them ideal candidates in DC cellular therapies. We show that removal of Sia from iDCs is sufficient enough to stimulate the allogeneic responders, again showing the importance of Sia in a non-immunogenic phenotype. This may indicate that the removal of Sia uncaps underlying structures, which are then recognized as a signal for T-cell proliferation or that the Sias may act as ligands for inhibitory Siglecs on the surface of effector cells and once removed, this inhibitory effect is lost. Sia removal of tDCs did not induce CD3+CD4+ proliferation, but we noted a trend increase in CD3+CD8+ proliferation. Interestingly, this indicates that, despite the increase of immunogenicity markers and the transcript increase in several pro-inflammatory mRNAs, Dexa treatment of iDCs was sufficient to keep the cells, at least partially, in a non-immunogenic state.

In CD3/CD28 stimulated (hyper stimulated) allogeneic co-cultures using LEW responder lymphocytes, we show that iDCs had an impressive ability to supress stimulated allogeneic lymphocytes. Sia is critical in maintaining this suppressive ability as when it was absent we observed complete restoration of T cell proliferation for both CD3+CD4+ and CD3+CD8+ populations. These results are supported by the fact that Crespo et al. (45) showed increased T-lymphocyte proliferation in autologous mixed lymphocyte cultures using human monocyte-derived DCs where the lymphocytes were stimulated with tetanus toxoid, inactivated with mitomycin C, and cocultured with neuraminidase monocyte-derived DCs. Interestingly, we showed that tDCs do not have the ability to suppress hyperstimulated allogeneic lymphocytes to the same extent as iDCs. Sia removal had little effect on tDCs suppressive ability and did not exaggerate proliferation. Together, these experiments highlight that the tolerogenic properties between iDCs and tDCs are not inherently the same and understanding these characteristics and limitations will inform us on how to optimize therapy strategies.

The findings outlined here could also have numerous implications for our understanding of DC phenotype and function in the tumor microenvironment. Efficient induction of antitumor

Figure 5 | Neuraminidase treatment alters T-cell suppression properties of immature DCs (iDCs) and tolerogenic DCs (tDCs). To test the T-cell suppression properties of iDCs, niDCs, tDCs, and ntDCs, they were placed into stimulated MLR cultures for 4 days. Splenocytes/lymphocytes were stimulated with CD3/CD28 beads. (A) Schematic representation of experimental design. DA iDCs, niDCs, tDCs, and ntDCs were placed in cocultures for 4 days with CD3/CD28-stimulated allogeneic LEW lymphocytes isolated from the spleen and lymph nodes. (B) The ability of iDCs, niDCs, tDCs, and ntDCs to suppress CD3/CD28 stimulated allogeneic T-cells was analyzed using stimulated splenocytes/lymphocytes as a positive control. Error bars: mean ± SEM \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001 one-way ANOVA, Tukey's multiple comparisons test. (i) Representative histograms and bar charts displaying stimulated CD4+ and CD8+ T cell proliferation following a 4-day coculture with iDCs and niDCs. (ii) Representative histograms and bar charts displaying stimulated CD4+ and CD8+ T cell proliferation following a 4-day coculture with tDCs and ntDCs.

responses requires that DCs in the tumor undergo proper maturation and activation (54). Understanding DC activation is important both in terms of their role in regulating immune responses locally in the tumor microenvironment (55), and also their use in *ex vivo* cellular and vaccination strategies to induce tumor specific immune responses.

In the context of tumor vaccination strategies using DCs, the required response is to induce tumor-specific effector T cells that can eliminate tumor cells specifically and that can induce immunological memory to control tumor relapse. Our findings suggest that Dexa, a common component of chemotherapy regimens, could suppress DC maturation and activation, their ability to present antigen (56), as well as their ability to induce T cell proliferation and activation. Interestingly, our data indicate that these potent Dexa-induced effects could be somewhat reversed in the presence of a neuraminidase, suggesting a key role for sialylation in Dexa generated tDCs. Removal of sialic acid has also previously been shown to increase tumor antigen-specific T cell responses (48). Our data also show that as well as a more potent ability to induce CD8+ T cell activation. In terms of modulating the tumor microenvironment directly, local delivery targeted approaches using sialyltransferase inhibitors delivered either to the tumor or the local lymph nodes could be exploited. In terms of *ex vivo* generated DCs for either cellular therapy or in vaccination strategies, treatment of DCs with sialyltransferase inhibitors could be sufficient to allow efficient priming of T cells systemically. As DCs provide an essential link between innate and adaptive immunity, these findings could have important implications in our understanding of the suppressive mechanisms within the tumor microenvironment that hinder adaptive antitumor immune responses and potential mechanisms by which they could be overcome.

Together, these results highlight the importance of Sia's in DC biology, especially in the context of iDC allogeneic cellular therapy. While the precise implications of increased or decreased Sia expression on iDCs and tDCs remain to be elucidated *in vivo*, we show here strong evidence that supports a function of Sia in the therapeutic aspects of DC cellular therapies. Identification of the molecular mechanisms and factors, which are regulated by Sia's are important to exploit this phenomenon in the clinic. This study points toward the potential of DC surface sialylation as a therapeutic target to improve and diversify DC-based therapies and treatments. In the context of disease, cell glyco-engineering could have positive implications in the treatment of autoimmunity, DC-based vaccines, the tumor microenvironment, and transplant biology.

### ETHICS STATEMENT

All animals used in experiments were accommodated in an accredited animal housing facility under a license granted by the Department of Health, Ireland, and were approved by the Animal Ethics Committee of the National University of Ireland, Galway.

### REFERENCES

### AUTHOR CONTRIBUTIONS

Substantial contributions to the conception or design of the work (KL, TR, LJ); or the acquisition (KL, OT, HA, PL), analysis (KL, OT, HA, JG, JC), or interpretation of data for the work (KL, JG, AR, TR); and drafting the work or revising it critically for important intellectual content (KL, JG, TR, AR); and final approval of the version to be published (KL, OT, JG, HA, PL, JC, LJ, AR, TR); and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved (KL, OT, JG, HA, PL, JC, LJ, AR, TR).

### ACKNOWLEDGMENTS

National University of Ireland flow cytometry core facilitated by Dr. Shirley Hanley. Functional genomics core facilitated by Dr. Enda O'Connell.

### FUNDING

This work is supported by Science Foundation Ireland (12/TIDA/ B2370 and 12/IA/1624) and European Cooperation in Science and Technology (COST) for the AFACTT project (Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies; BM1305).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01427/ full#supplementary-material.

Table S1 | Table listing forward primers, reverse primers, and probes for interleukin 6 (IL-6), indoleamine 2,3-dioxygenase (IDO), interleukin 1 beta (IL-1β), inducible nitric oxide synthase (iNOS), and IL-12p40, tumor necrosis factor alpha (TNF-α), GAPDH and interleukin 10 (IL-10).

Table S2 | Lectin names and common major binding ligands. Table listing the abbreviations, the kingdom, species, common name, and major binding ligand of the lectins used in lectin micro array profiling.

Figure S1 | Isolation and generation of immature DCs and tolerogenic DCs.

Figure S2 | Resultant cell death due to neuraminidase treatment of immature DCs (iDCs) and tDCs. Both iDCs and tDCs were treated with neuraminidase for 90 min. These cells were then washed and placed into culture for 48 h. Cells were prepared for flow cytometry as previously described and stained with the live/dead indicator sytox blue (n = 1, technical replicate of 2).

doi:10.1002/1521-4141(200007)30:7<1807::AID-IMMU1807>3.0. CO;2-N

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past co-authorship with one of the authors TR.

*Copyright © 2017 Lynch, Treacy, Gerlach, Annuk, Lohan, Cabral, Joshi, Ryan and Ritter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Dexamethasone and Monophosphoryl lipid a induce a Distinctive Profile on Monocyte-Derived Dendritic cells through Transcriptional Modulation of genes associated With essential Processes of the immune response

*Paulina A. García-González 1,2†, Katina Schinnerling1,2†, Alejandro Sepúlveda-Gutiérrez 3 , Jaxaira Maggi 1,2, Ahmed M. Mehdi4 , Hendrik J. Nel 4 , Bárbara Pesce1 , Milton L. Larrondo5 , Octavio Aravena1 , María C. Molina1 , Diego Catalán1,2, Ranjeny Thomas 4 , Ricardo A. Verdugo3 \* and Juan C. Aguillón1,2\**

*1Programa Disciplinario de Inmunología, Facultad de Medicina, Instituto de Ciencias Biomédicas (ICBM), Universidad de Chile, Santiago, Chile, 2Millennium Institute on Immunology and Immunotherapy, Santiago, Chile, 3Programa de Genética Humana, Facultad de Medicina, Instituto de Ciencias Biomédicas (ICBM), Universidad de Chile, Santiago, Chile, 4 Translational Research Institute, University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia, 5Banco de Sangre, Hospital Clínico de la Universidad de Chile, Santiago, Chile*

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/ inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation.

Keywords: tolerogenic dendritic cells, immune regulation, dexamethasone, transcriptome, tolerance induction

#### *Edited by:*

*Piotr Trzonkowski, Gdan´ sk Medical University, Poland*

#### *Reviewed by:*

*Lenka Palová Jelínková, Second Faculty of Medicine, Charles University, Czechia Nick Giannoukakis, Allegheny Health Network, United States*

#### *\*Correspondence:*

*Ricardo A. Verdugo<EMAIL_ADDRESS>Juan C. Aguillón<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology*

*Received: 06 September 2017 Accepted: 03 October 2017 Published: 23 October 2017*

#### *Citation:*

*García-González PA, Schinnerling K, Sepúlveda-Gutiérrez A, Maggi J, Mehdi AM, Nel HJ, Pesce B, Larrondo ML, Aravena O, Molina MC, Catalán D, Thomas R, Verdugo RA and Aguillón JC (2017) Dexamethasone and Monophosphoryl Lipid A Induce a Distinctive Profile on Monocyte-Derived Dendritic Cells through Transcriptional Modulation of Genes Associated With Essential Processes of the Immune Response. Front. Immunol. 8:1350. doi: 10.3389/fimmu.2017.01350*

## INTRODUCTION

Dendritic cells (DCs) are a heterogeneous group of specialized antigen-presenting cells with the capacity to orchestrate specific immune responses according to the antigen they encounter and the environmental signals derived from the local milieu (1). After antigen capture and processing, DCs undergo a complex process of differentiation to mature DCs, which express high levels of surface peptide–HLA complexes and co-stimulatory molecules. Mature DCs also produce inflammatory cytokines and T-cellattracting chemokines enabling the induction of Th1, Th2 or Th17 responses (2, 3). DCs can also differentiate into tolerogenic DCs (tolDCs), capable of inducing anergy or deletion of effector T cells, and/or differentiation and proliferation of regulatory T-cell (Treg) subsets. Regulation may result from various processes, including deficient antigen presentation, reduced co-stimulatory molecules, expression of inhibitory molecules, and/or secretion of anti-inflammatory cytokines such as IL-10 and TGF-β (4, 5). The ability of DCs to modulate T-cell responses has made them an interesting target of study for the immunotherapy of autoimmune diseases, since these cells are supposed to induce and maintain immune tolerance to harmless or self-antigens (6, 7).

Differentiation of DCs from peripheral blood monocytes using GM-CSF and IL-4 is a useful approach to obtain large numbers of DCs *in vitro* to study their function and biology. This approach is also used to generate tolDCs *in vitro* by adding immune modulators such as immunosuppressant drugs [dexamethasone (Dex), rapamycin, aspirine, rosiglitazone, tacrolimus] (8–12); anti-inflammatory cytokines (IL-10 and TGF-β) (13–15); natural compounds (vitamin D3, retinoic acid, and curcumin) (8, 16, 17); the JAK inhibitor tofacinib (18); and the NF-kB inhibitor BAY11- 7082 (19). All strategies lead to DCs with regulatory capacities, although some features may vary between protocols. Our group has described a protocol for tolDC generation from peripheral blood monocytes of healthy controls (20) and rheumatoid arthritis (RA) patients (21) using Dex to induce a tolerogenic phenotype and subsequent activation with the non-toxic lipopolysaccharide (LPS) analog monophosphoryl lipid A (MPLA) to confer lymph node homing capacity and stability. These cells, herein termed Dex-modulated and MPLA-activated DCs (DM-DCs), expressed low levels of CD83, CD86, and CD40, secreted high levels of IL-10 and TGF-β and low levels of IL-12, and stimulated T-cell proliferation and cytokine production at low levels in allogeneic and autologous cultures (20, 22).

While we generally understand the cellular mechanisms by which tolDCs modulate T-cell responses and induce tolerance, the molecular switches determining tolDC differentiation and function are still poorly known. The knowledge of molecular regulators and pathways could be of great benefit for searching targets for effective cellular therapies. However, only few studies have attempted to identify specific molecules or processes involved in tolerogenic functions of monocyte-derived DCs (moDCs) using whole-genome transcriptomic or proteomic analyses (23, 24). Most studies focus on vitamin D3-modulated moDCs (25, 26). Studies of Dex-treated moDCs comprised only proteomic approaches and focused on the identification of potential tolDC markers (27, 28).

We recently compared tolDCs derived from monocytes of healthy controls and RA patients at phenotypic, functional, and transcriptional levels (21) and demonstrated that Dex and MPLA treatments removed disease-associated features of moDCs to yield a uniform signature. Here, we describe a genome-wide differential expression study of tolDCs derived from monocytes of healthy controls in which we elucidate molecular processes that drive DC differentiation toward a tolerogenic profile in response to Dex and MPLA treatments. We found that DM-DCs exhibit a transcriptional profile that distinguishes them from other moDC subsets, characterized by the upregulation of several genes related to immunoregulatory functions and biological processes that could be involved in tolerance induction.

### MATERIALS AND METHODS

### Blood Samples

A total of 10 buffy coat samples from healthy controls were used for microarray analysis and phenotypic and functional studies. An additional 10 buffy coat samples were used to confirm differential expression of genes by qRT-PCR and flow cytometry. All subjects signed an informed written consent and all procedures were approved by the Ethics Committees for Research in Human Beings from the Faculty of Medicine and from the Clinical Hospital of the University of Chile. Demographic characterization of healthy controls is detailed in Table S1 in Supplementary Material.

### Generation of moDC Subsets

Human moDCs were generated from monocytes as previously described (20). Monocytes were isolated from peripheral blood of 10 healthy individuals by negative selection using RosetteSep Human Monocytes enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer's instructions. Monocytes were cultured at 2 × 106 cells/ml in serum-free AIM-V medium (Gibco BLR, Grand Island, NY, USA), supplemented with 500 U/ml of recombinant human GM-CSF and IL-4 (eBioscience, San Diego, CA, USA) for 5 days at 37°C and 5% CO2. At day 3, culture medium was replenished and cells were incubated with Dex (Sigma-Aldrich, St. Louis, CO, USA) at a final concentration of 1 µM [Dex-modulated DCs (D-DCs)]. At day 4, cells were stimulated with 1 µg/ml of cGMP-grade MPLA (Avanti Polar Lipids Inc., Alabaster, AL, USA) (DM-DCs). Unstimulated cells (DCs) and MPLA-matured DCs (M-DCs) generated in the absence of Dex were used as controls of immature and mature DCs, respectively. On day 5, cells were harvested and characterized by flow cytometry.

### Flow Cytometry

Antibodies used for analysis were anti-human CD80 FITC (clone 2D10.4), CD83 FITC (clone HB15e), CD40 PE (clone 5C3), CD86 PE (clone IT2.2), IDO1 PECy7 (clone eyedio), CD4 PECy7 (clone OKT4), IFN-γ APC (clone 4S.B3) (eBioscience); CD11c BUV395 (clone B-ly6), CD83 BUV737 (clone HB15e) (BD Biosciences); CD86 BV650 (IT2.2), CD163 BV605 (clone GHI/61), CD1c BV510 (clone L161), MERTK BV421 (clone 590H11G1E3), CD32 APC (clone FUN-2), ZBTB16/PLZF PE (clone Mags.21F7) (BioLegend) and TLR2 Alexa Fluor 700 (clone 383936), JAG1 Fluorescein (clone 188331), and FPR2 APC (clone 304405) (R&D Systems). Prior to antibody staining, cells were labeled with fixable viability dye eFluor 780 (eBioscience). Cells were resuspended in PBS supplemented with 10% of fetal bovine serum (FBS) (HyClone Thermo Scientific, Logan, UT, USA), stained with specific antibodies, fixed with IC fixation buffer (eBioscience), and resuspended in FACSFlow buffer (Becton Dickinson, San Diego, CA, USA) for subsequent analysis. Data were acquired on a FACSAria III with FACSDiva v6.1.3 software (both Becton Dickinson) and analyzed by FlowJo software (Treestar, USA).

### Cytokine Production

A total of 1 × 105 DCs were incubated for 24 hours with or without CD40L-transfected irradiated NIH3T3 cells at 1:1 ratio in AIM-V medium, in 96-well U bottom plates (BRAND, Wertheim, Germany). Supernatants of cocultures with NIH3T3 cells or T cells were recovered and stored at −80°C until quantification of IL-10, IL-12p70, and IFN-γ by ELISA (eBioscience).

### CD4**+** T Cell-Stimulatory Capacity of DCs

CD4+ T cells were isolated by negative selection using RosetteSep Human T-cell enrichment cocktail (Stemcell Technologies) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE).

For the assessment of antigen-specific CD4+ T-cell activation, DCs were loaded with 1 µg/ml tuberculin purified protein derivative (Staten Serum Institute, Copenhagen, Denmark) 4 hours prior to activation with MPLA and co-cultured with autologous CD4+ T cells at a DC:T-cell ratio of 1:2 in RPMI medium (HyClone Thermo Scientific) with 10% FBS in 96-well U bottom plates for 6 days (20). CD4+ T cells alone and stimulated with anti-human CD3 mAb (clone OKT3; 0.65 μg) (eBioscience) were used as negative and positive controls, respectively. Supernatants were collected to assess cytokine secretion. For intracellular IFN-γ detection, 50 ng/ml phorbol-12-myriastate-13-acetate (Sigma-Aldrich), 1 µg/ml ionomycin (Sigma-Aldrich), and 1 µg/ml brefeldin-A (eBioscience) were added for the last 5 hours of culture. Proliferation and IFN-γ production of CD4+ T cells were analyzed by flow cytometry.

### RNA Isolation and Microarray Analysis

RNA was isolated from 5 × 105 DCs on day 5 using total RNA isolation RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Yield and quality of RNA samples were evaluated with NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity (RIN score) was analyzed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) or LabChip GX/ GX II (Caliper LifeSciences, Hopkinton, MA, USA). A total of 40 samples, corresponding to 10 healthy donors under four experimental conditions were considered for microarray analysis (Figure S1 in Supplementary Material). All RNA samples used for microarrays showed A260/A280 values between 1.8 and 2.2, and RIN scores >7. RNA samples were reverse transcribed, amplified, and labeled using an Illumina® TotalPrep™ RNA Amplification Kit, and cDNA was hybridized onto Illumina Human HT-12 v4 BeadChips (Illumina, San Diego, CA, USA), covering the whole human genome. Expression data were extracted with GenomeStudio Project Software from Illumina.

### Confirmation of Gene Expression by qRT-PCR

cDNA was prepared from moDCs RNA samples using the Superscript II Reverse Transcriptase kit (Thermo Fisher Scientific). Quantitative RT-PCR was performed in Stratagene Mx300P, using Brilliant II SYBR Green QPCR Master Mix (Agilent Genomics) with primer sets from IDT. The housekeeping genes *GAPDH* and *r18S* were used as internal controls and target gene expression was normalized to untreated DCs. Primer sequences for each target gene are described in Table S2 in Supplementary Material.

### Data Exploration and Statistical Analyses

For flow cytometry and qPCR data, Friedman repeated measures test and Dunn's *post hoc* test were used for data comparison between moDC culture conditions. Analyses were performed using Prism 5.01 software (Graphpad, San Diego, CA, USA).

Microarray data were log transformed followed by quantile normalization using the preprocess Core package v1.28.0 from Bioconductor. Differentially expressed (DE) genes in modulated DCs relative to unstimulated DCs were identified with the Maanova package v1.36.0 *t*-test for gene pairwise comparisons (29), and *p*-values were adjusted using false discovery rate (FDR) method. Genes with adjusted *p*-value ≤0.05 were considered differentially expressed and reported. K-means clustering of residual values of DE genes between DM-DCs and DCs was performed using the cluster package and a *K* value of 6 to maximize cluster distance and minimize distance between clustered genes.

Overrepresentation of pathways and biological functions was assessed using Ingenuity Pathway Analysis (Ingenuity Systems, Qiagen, Hilden, Germany).

## RESULTS

### Modulation of moDCs with Dex and MPLA Induces a Distinctive Transcriptional Profile

First, we confirmed that treatment with Dex and MPLA during moDC differentiation induces a tolerogenic phenotype on these cells. As previously described (20), DM-DCs expressed low levels of CD86, CD80, CD40, and CD83 and produced low levels of IL-12 and high levels of IL-10 relative to M-DCs. T-cell proliferation and IFN-γ production in response to antigen-exposed DM-DCs were significantly reduced when compared with T cells stimulated with DCs or M-DCs (Figure S2 in Supplementary Material). Subsequently, we used RNA from the same moDC preparations to conduct whole-genome analysis through microarray technology, using a total of 40 moDC samples, corresponding to four different DC subsets, i.e., unstimulated (DC), M-DCs, D-DCs, and Dexmodulated/MPLA-activated DCs (DM-DC), differentiated from monocytes from 10 healthy individuals. We defined statistically significant differences between samples by a FDR value of 0.05 or lower. Since all four differentiation protocols led to moDCs with different phenotypic characteristics, we tested whether these differences could also be found at transcriptional level. A principal component analysis was used to explore the data by projecting samples onto the major orthogonal components or genes expression. The first two dimensions separated moDCs samples by their differentiation state (unstimulated, MPLA, Dex, or Dex plus MPLA) (**Figure 1**). The first component (*X*-axis) naturally clustered samples as M-DCs < DCs < (D-DCs:DM-DCs). D-DCs and DM-DCs, which together could be associated with a tolerogenic potential, could not be distinguished on component 1. The second component (*Y*-axis) clustered samples by their grade of activation, with both moDC subtypes treated with MPLA projecting toward positive values, i.e., DCs < D-DCs < (M-DCs:DM-DCs). Thus, each protocol induces a unique transcriptional profile in moDCs that distinguishes them from other moDC subtypes.

### Dex and MPLA Treatment Modulates Genes Associated With Cell Movement, Signaling, and Metabolism

Given their tolerogenic phenotype, we next focused on the transcriptional effects of combined treatment with Dex and MPLA treatment on moDCs. Considering *p*-values (corrected using FDR) 0.05 or lower to define statistically significant differences in gene expression, we identified 259 DE transcripts in DM-DCs compared to the unstimulated control (DCs). The scavenger receptor CD163, several MT1 (metallothionein 1) isoforms and MT2A, C1QTNF1, ADORA3, S100A9, and both isoforms of Fc receptor for IgG FCGR2A/CD32 and FCGR2B/CD32B (**Table 1**; Table S3 in Supplementary Material) were among the genes most upregulated by Dex and MPLA treatments; while CD1b, FCER1A (high affinity I Fc fragment of IgE receptor subunit alpha polypeptide), and MMP12 (Matrix metallopeptidase 12) were the most downregulated genes (**Table 1**; Table S3 in Supplementary Material).

A pathway enrichment analysis of the DE genes found on DM-DCs done with ingenuity pathway analysis (IPA) identified cell movement, cell signaling, and metabolism as main functions modulated by Dex and MPLA (**Figure 2A**; Table S4 in Supplementary Material), assembling regulated genes into 11 networks representing significantly enriched biological functions, mostly associated with cellular movement, growth, immune cell trafficking, and metabolism (**Figure 2A**; Table S4 in Supplementary Material). The main regulated genes in DM-DCs such as CD163, ADORA3, FCGR2A, CD1c, CD1b, and MMP12 belong to these networks. In terms of canonical pathways and consistent with functional enrichment analysis, Dex and MPLA treatments affected cell adhesion and diapedesis (*p*-value 8.52e−3) (**Figure 2B**), modulating the expression of 10 genes, including the upregulation of chemokines such as CCL8, CCL18, CCL23, and CXCL5. Interferon signaling (*p*-value 7.49e−3) was predicted to be an active process, with a positive *z*-score (2.449) and high expression levels of the IFN-inducible genes IFI6, IFITM1, IFITM2, and IFITM3 (Table S4 in Supplementary Material). Processes associated with DC maturation and activation were inhibited, and several molecules associated with these processes such as CD80, CD83, CD1c, ACTA2, ACTG1, TMBS10, AP-1, and RAP1GAP were downregulated (Table S4 in Supplementary Material). Complement system pathway was highly modulated, with increased expression of C1QA, C1QB, C1QC, and CFB. IL-10 signaling (*p*-value 2.93e−3), along with TLR (*p*-value 4.11e−2), iNOS (*p*-value 4.65e−2), and p38MAPK signaling (*p*-value 1.31e−2), which also signal through IRAK and STAT1, was enriched processes on DM-DCs according to

IPA knowledge database (**Figure 2B**; Table S4 in Supplementary Material). Upstream regulator analysis revealed GILZ, STAT1, FOXO3, STAT3, SMARCA4 and CEBPD to be amongst the main transcriptional regulators of many genes from this dataset (Table S4 in Supplementary Material).

### The Transcriptional Program of DM-DCs Is Regulated by an Interplay between Dex and MPLA

The 259 DE genes in DM-DCs *versus* untreated DCs were further grouped into six clusters according to their expression pattern using K-means clustering of residual values (clusters C1–C6; **Figure 3**). Clusters 1 and 3 contain 61 DE genes (Cluster 1 = 20 genes; Cluster 3 = 41 genes) that correspond mainly to DC differentiation and maturation, including the DC markers CD1c, DC-SCRIPT/ZNF366, co-stimulatory molecule CD80, and other molecules involved in DC maturation and inflammation such as CD83, RAP1GAP, NDRG2, CD1b, FCER1A, CCL22, and MMP12 (**Figure 3**; Table S3 in Supplementary Material). Dex treatment alone or combined with MPLA leads to downregulation of these genes (**Figure 3**). Clusters 2 and 5 comprise 79 genes upregulated by MPLA (Cluster 2 = 44 genes; Cluster 5 = 35 genes), regardless of the presence of Dex. These clusters contain most genes related to IFN signaling and granulocyte and agranulocyte adhesion and diapedesis, in addition to IL-1B, STAT1, and IDO1. Cluster 4 contains 75 genes, which are upregulated by Dex irrespective of MPLA addition. This cluster includes many genes associated to inhibition of DC activation and maturation such as FCGR2B, C1Q, and MAFB, as well as genes related to anti-inflammatory responses of DCs leading to IL-10 production (TSC22D3/GILZ and MAP3K8/TPL-2) and modulation of T-cell activation and/ or expansion of Treg-cell populations such as JAG1 (Jagged 1), MERTK (receptor tyrosine kinase Mer), TBXAS1, and SEMA4A, thus contributing to the tolerogenic profile of DM-DCs (**Figure 3**; Table S3 in Supplementary Material). Additionally, Cluster 4 is characterized by the expression of membrane receptors (MERTK, FCGR2A, FCGR2B, and SEMA4A), signaling proteins (MAP3K8, S100A9, and JAG1) and transcriptional regulators (TSC22D3/GILZ) as well as molecules related to the complement system (C1QA, C1QB, C1QC, and CFH) and cell adhesion and migration (CCL13, CCL18, CCL23, SH3PXD2B, and ADORA3) (**Figure 3**; Table S3 in Supplementary Material). IL-10 signaling and complement system are among the pathways overrepresented in this group.

Finally, Cluster 6 contains 44 genes, which were regulated in response to a synergistic effect between Dex and MPLA. Metallothioneins, proteins involved in stress response, heavy metal scavenging, and immunosuppression were highly represented in this group, including several MT1 isoforms (MT1A, MTE, MT1F, MT1G, MT1H, MT1M, and MT1X) and MT2A. Enrichment of several molecules involved in cell adhesion and diapedesis (CXCL5 and MMP19), T-cell migration (THBS1 and TNFRSF6B), and production of reactive oxygen species (ROS) (FPR1, FPR2, NCF1, and SLAMF1) was also found in this cluster as well as molecules associated with a low inflammatory state (C1QTNF1, SLC39A8/ZIP8 and IRAK3), suggesting that genes in this cluster also contribute to the regulatory features of DM-DCs in addition to the genes of Cluster 4. Functional and pathways analysis of genes from this cluster showed that cellular movement and metabolic processes, particularly ROS metabolism is highly represented (Table S5 in Supplementary Material).

### Dex and MPLA Treatment Promotes the Upregulation of Genes Related to the Modulation of Biological Processes That Control Immune Responses

Biological functions related to cellular movement, growth, and proliferation, cell-to-cell signaling, and free radical scavenging were found to be upregulated. Canonical pathway analysis also provided agranulocyte/granulocyte adhesion and diapedesis, T helper differentiation, and DC maturation as main pathways regulated in our dataset (**Figure 2**; Tables S4 and S5 in Supplementary Material). Since these functions were highly enriched in the two clusters that are potentially involved in DM-DCs tolerogenic features (Clusters 4 and 6), we further analyzed interactions between genes contained in these clusters. Functional annotations involving activation and proliferation of T lymphocytes were highly represented in several networks and predicted to be inhibited in DM-DC, while chemotaxis of T lymphocytes and synthesis of ROS, also highly represented, were predicted to be activated in these cells (**Figure 4**). Most downregulated genes in DM-DCs, which are associated with T-cell activation and proliferation, are also involved in DC maturation and activation, while upregulated genes interacting in these networks encode inhibitory membrane receptors or transcriptional regulators that lead to inhibition of effector T-cell activation and

promote differentiation of Tregs (FCGR2B, MT1, IDO1, CCL18, JAG1, and MERTK). This is further supported by upregulation of genes involved in the activation of ROS production (FPR1 and NCF1 and NAMPT) and recruitment of naive and Tregs (CCL18, CCL23, and SAA1). Many of the genes that were upregulated in response to Dex and MPLA together (Cluster 6) also participate in the main metabolic processes in DM-DCs, mainly ROS production and zinc homeostasis (Table S5 in Supplementary Material), contributing to the particular metabolic profile of these tolDCs.

The differential expression of several genes of interest that are modulated on DM-DCs such as CD163, JAG1, IDO1, MERTK, MT1F, FPR1, and CD32, which might participate in the main processes enriched on DM-DCs, was confirmed through realtime PCR (**Figure 5**) and were shown to correlate with protein levels determined by flow cytometry (**Figure 6**).

### DISCUSSION

Here, we demonstrate, by whole-genome transcriptomic analysis of different moDC subtypes, that DM-DCs exhibit a distinctive transcriptional program that potentially endows them with regulatory functions through modulation of chemotactic responses, cell-to-cell signaling and interaction, and metabolic processes.

Glucocorticoids, including Dex, have been widely used for tolDC generation and have been demonstrated to inhibit DC maturation and inflammation (30, 31). Since activation of tolDCs has been shown to increase lymph node homing, antigen presentation, and stability against other inflammatory modulators (32), we activated Dex-treated DCs with MPLA (DM-DCs), a non-toxic clinical grade analog of LPS, which also signals via toll-like receptor 4 and exhibits potent immune-stimulatory capacity (33, 34). Glucocorticoid receptors are transcription factors that suppress the pro-inflammatory program induced by TLRs and in turn potentiate TLR-mediated anti-inflammatory responses such as IL-10 secretion (35, 36). Our group has previously shown that Dex and MPLA induce a tolerogenic profile in moDCs from both healthy controls and RA patients (21, 22). The resulting DM-DCs exhibit characteristic tolDCs features, with low expression of co-stimulatory and maturation markers, high production of anti-inflammatory cytokines, and reduced

capability to promote effector T-cell responses. Consistent with their phenotypic properties, we found that modulation of moDCs with either stimulus alone or a combination of both also induces a distinctive transcriptional profile, which allows separation of moDCs samples according to differentially expressed genes.

Several methallothioneins (MTs) (MT1G, MT1H, MT1E, MT2A, MT1M, MT1A, and MT1X) appear in this study as some of the most upregulated genes within the 259 DE transcripts of DM-DCs. These are small cysteine-rich metal-binding proteins involved in the regulation of homeostasis of zinc and other heavy metals at cytoplasmic level (37). MTs can also affect different cellular processes such as gene expression, apoptosis, proliferation, and differentiation (38) and have been described to suppress collagen-induced arthritis via induction of TGF-β and reduction of pro-inflammatory modulators such as TNF (39) and to promote the expansion of Treg (40, 41). Besides MTs, another molecule involved in zinc transport, the zinc importer SLC39A8/ ZIP8, is also highly upregulated in DM-DCs. Zinc is known to act as a modulator of immune responses through its availability, and zinc deficiency affects the immune system leading to increased inflammation and inflammatory diseases such as RA (38). In DCs, zinc supplementation has been shown to interfere with maturation, by inhibiting the upregulation of MHCII and co-stimulatory molecules (42), as well as to induce the expression of the tolerogenic markers PD-L1, IDO1, and CD103 (43). Thus, a tight regulation of zinc concentration is required and zinc may contribute to the immunoregulatory functions of DM-DCs, since several regulators of intracellular zinc concentration are overexpressed in these cells.