Tolerogenic Dendritic Cells as a Promising Antigen-Specific Therapy in the Treatment of Multiple Sclerosis and Neuromyelitis Optica From Preclinical to Clinical Trials

The identification of activated T-lymphocytes restricted to myelin-derived immunogenic peptides in multiple sclerosis (MS) and aquaporin-4 water channel in neuromyelitis optica (NMO) in the blood of patients opened the possibility for developing highly selective and disease-specific therapeutic approaches. Antigen presenting cells and in particular dendritic cells (DCs) represent a strategy to inhibit pro-inflammatory T helper cells. DCs are located in peripheral and lymphoid tissues and are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of receptors involved in pathogen recognition confers to DCs the property to initiate immune responses. However, in the absence of danger signals different DC subsets have been revealed to induce active tolerance by inducing regulatory T cells, inhibiting pro-inflammatory T helper cells responses or both. Interestingly, several protocols to generate clinical-grade tolerogenic DC (Tol-DC) in vitro have been described, offering the possibility to restore the homeostasis to central nervous system-related antigens. In this review, we discuss about different DC subsets and their role in tolerance induction, the different protocols to generate Tol-DCs and preclinical studies in animal models as well as describe recent characterization of Tol-DCs for clinical application in autoimmune diseases and in particular in MS and NMO patients. In addition, we discuss the clinical trials ongoing based on Tol-DCs to treat different autoimmune diseases.

1 year of disease progression (retrospectively or prospectively determined) plus 2 of 3 of the following criteria: 1. evidence for DIS in the brain based on 1 or more T2 lesions in the MS-characteristic (periventricular, juxtacortical, or infratentorial) regions 2. evidence for DIS in the spinal cord based on 2 or more T2 lesions in the cord 3. positive CSF (isoelectric focusing evidence of oligoclonal bands and/or elevated IgG index) are based on their clinical phenotype (3). These subtypes are: The primaryprogressive MS (PPMS) which is a disabling subtype from the beginning, the relapsingremitting type (RRMS) that is characterized by clinical relapses without progression of disabi lity and finally, the secondaryprogressive subtype that appears about 20 years after RRMS.
The MS diagnosis is summarized in the revised 2010 Mc Donald criteria which is included in Table 1 (4). Although the cause of the immune deregulation is unknown, there are evidences that implicate Th1 and Th17 lymphocytes in the pathophysiology of MS (5-10). Furthermore, it was supported by studies performed in experimental models of MS either knocking out or blocking using monoclonal antibodies for IL17 or IL23 resulted in a suppression of the activity of this disease (11,12). Other authors have described that memory Tcells are activated in the periphery by different processes that can be promoted by environmental or genetic factors. These activated cells cross the blood-brain bar rier, penetrate to CNS where they are locally reactivated (9,13).
Firstline therapies for MS include injectable treatments such as IFNβ, and glatiramer as well as oral therapies such as terif lunomide and dimethylfumarate. Secondline therapies include fingolimod, and the intravenous natalizumab, which present higher levels of efficacy in reducing the relapse rate; however, it has poten tial severe side effects. Moreover, Alentuzumab, Cladribine, and Ocrelizumab were recently added as approved therapies, and they are in progress of being defined in the pyramid of the MS therapy. All these mentioned treatments are systemic immunomodulatory or immunosuppressive treatments with risks of adverse events.
Neuromyelitis optica (NMO) is an inflammatory disease affec ting the CNS (14) with similar physiopathology as MS, but is considered an autoimmune astrocytopathy. NMO is a rare disease which presents with incidence between 0.05 and 0.4/100,000 (15,16). About 70% of the patients diagnosed with NMO shows the presence of antiaquaporin4 (AQP4) antibody as well as specific Tlymphocytes in the bloodstream or CSF which suggest the proinflammatory role of these cells (17). Importantly, the detection of antiAQP4 antibodies is related with more severe disease (14). Recently, among seronegative patients, anti(MOG) antibodies have been described as the pathological antibody (18). This disease has its own international consensus diagnostic criteria (19), defining the NMO spectrum disorder (NMOSD) concept ( Table 2). Different MS drugs such as natalizumab or finolimob have been evaluated in NMO resulting in exacerbation of relapses (20). Immunomodulatory or immunossuppressant therapies are used for label in NMOSD (e.g., azathioprine, mycophenolate, cyclophosphamide, or rituximab) (21). Furthermore, several monoclonal antibodies are in clinical trials to evaluate their efficacy and safety, as tocilizumab, satralizumab, eculizumab, or aquapuromab for example (22). Based in the unmet need of achieving higher levels of efficacy and/or better safety profile, antigenspecific therapies are being considered as a potential treatment for MS and NMO (19).

DeNDRiTiC CellS (DCs)
Dendritic cells act as a link between innate and adaptive immune responses. Their main function is to capture and process exog enous antigens in the peripheral tissues to present them to Tcells after migration to the draining lymph nodes. In addition, they polarize immune responses by promoting both pro and anti inflammatory immune responses depending on the presence of danger signals associated to the antigens (Figure 1) (24,25).  Dendritic cells are located in peripheral tissues (skin and mucosa) and remain in an immature state (iDCs) until they interact with the antigens. After cells activation, DCs initiate a maturation process in which mature DCs (mDCs) lose capacities for antigen uptake in favor of acquiring stimulatory properties for the activa tion of naïve Tcells and the development of effector Tcells (27). Maturation process involves different processes and physiological changes in DCs, which are illustrated in Figure 2 (28).
Due to their immunological functions and the availability of clinicalgrade reagents, immunogenic DCs have been safely used in clinical trials to potentiate immune response against tumors or infectious diseases (30). However, only a few studies recently published have taken advantage of their specific tolerogenic properties to treat Type 1 diabetes, rheumatoid arthritis (RA) and Crohn's disease patients (25,31,32).

HUMAN DCs SUbSeTS
Dendritic cells can be subclassified based on anatomical location, origin, and function. In humans, different DC subsets have been identified in blood, spleen and skin and in nonlymphoid tissues.
Each DC subset presents different specialization in Tcell priming and induction of immune responses, although their functions can partially overlap (33).
In peripheral blood, DCs that express Human Leukocyte Antigen-antigen D Related (HLADR) and lineage negative fraction are divided into two main groups: conventional myeloid DCs (cDCs) and nonconventional plasmacytoid DCs (pDC). Within myeloid DCs two main subsets have been identified based on their surface marker expression: CD1c/BDCA1 cDCs and CD141/BDCA3 cDCs. However, recently new DC subset classification has been described (CD16 and DC5) (23). Circu lating DCs represent a little fraction of total circulating peripheral blood mononuclear cells (PBMCs) as they account for less than 1% of PBMCs (24,34).
In the skin two different subsets of DCs can be found. Langerhans cells (LCs) which contributes to immune surveillance and CD14 DCs, which are involved in tolerance induction (35,36).
From all the different DC subsets above mentioned, the BDCA1, pDCs, LCs, and CD14 have been described to gener ate both immunogenic and suppressive functions (Figure 3). BDCA1 have the capacity to produce IL10 in response to E. coli and potentially contribute to suppress immune responses. Recently, a particular subset of BDCA1 (BDCA1CD14 + ) has been shown to act as immunosuppressive cells in certain types of tumor environment and may hamper anticancer DCs vaccines (37,38) Moreover, in an steady state, pDCs are able to induce tolerogenic immune responses by inducing Tcell anergy and promoting Treg cells development. They have been found to be infiltrated in tumors activating Tr1 cells (33,39,40). LCs, apart from respond to intracellular pathogens and viruses under inflam matory conditions are in charge to maintain epidermal health and tolerance to commensals from the skin, while retaining the  ability to respond to selected pathogens (40)(41)(42). Finally CD14 DCs also have the ability to generate Tregs through the elevated IL10 production (43,44).
To sum up, BDCA1, pDCs, LCs, and CD14 have been shown to present immunoregulatory effects. However, deeper charac terization of this tolerogenic profile and mechanisms needs to be performed.

TOleROgeNiC DCs (Tol-DCs) AND MeCHANiSMS OF TOleRANCe iNDUCTiON
As described in the previous section, DCs play a crucial role in the initiation of immune responses and also in maintaining the immune tolerance. DCs present both foreign antigens as well as endogenous antigens derived from tissues. For this reason, the immune system is able to distinguish between innocuous and harmful antigens to avoid autoimmune or undesired immune responses (45). Several studies point that a key factor for DCs to initiate immunity or tolerance is the maturation stage of DCs (25). It is generally accepted that in absence of danger signals provided by infection or inflammation, DCs remain in an imma ture state which will induce tolerance by deleting or inducing apoptosis of selfantigenspecific Tcells (25,46). However, other several mechanisms to explain how DCs induce tolerance have been proposed. Some authors have reported that low expression of MHC molecules and costimulatory receptors on DC surface fail to stimulate Tcells sufficiently, thus resulting in Tcell anergy (47)(48)(49). Currently, it has been demonstrated that the expression  of single immunoglobulin IL1 related receptor, which is lower in iDCs, has a role in maintain low levels of costimulatory mole cules and in the regulation of Treg cell expansion (50). Furthermore, it is well established that the expression of certain molecules such as PDL1 rather than promote activation signals to Tcells, they induce Tcell anergy (28,51,52). Moreover, some authors dem onstrated that suboptimal antigen presentation, together with indoleamine 2,3dioxygenase (IDO) or FasL (CD95L) expres sion by DCs leads to inhibition of Tcell proliferation and Tcell deletion, respectively. Finally but not the least, the production of the potent antiinflammatory cytokine IL10 by DCs is crucial for peripheral tolerance induction. IL10 acts on a wide variety of immune cells and it has been clearly involved in Treg as well as Tr1 induction (38). In the steady state, peripheral Treg cells rise from peripheral CD4 + CD25 − FOXP3 − T cells that are exposed to antigen in the presence of transforming growth factorβ as well as IL10 without IL6 or IL1β, which promotes the upregulation of FOXP3 (17) (Figure 4). Recent developments carried on by Agrawal et al., have shown that Clectin receptor (CLEC2) upre gulation in DCs, is associated with Treg induction. Moreover, they have also described that platelet growth factor is able to induce IL10 production by DCs and in consequence Treg cell induction (53).
In consequence, major efforts have been focused on in vitro generation of TolDCs. In this regard, different immunosup pressive drugs, such as corticosteroids, cyclosporine, tacrolimus, rapamycin, deoxyspergualin, vitamin D3 (vitD3), mycophenolate mofetil, and sanglifehrin A, have been successfully used to modu late DCs differentiation and function. Thus, several protocols that include the generation of monocytederived DCs in the presence of corticosteroids and a defined maturation cytokine cocktail (including TNFα, IL1β, IL6, and PGE2) or lipopolysaccharide (LPS) activation in order to boost their tolerogenic properties, have been described to generate TolDCs in vitro (54,55).
Tolerogenic DCs present an intermediate phenotype between iDCs and mDCs regarding costimulatory molecules, a pronounced shift toward antiinflammatory versus proinflammatory cyto kine production (high amounts of IL10 versus low levels of IL12p70 and IL23) and a reduced capacity to stimulate Tcells response. In addition TolDCs present an increment of IL10 production upon Gramnegative bacterial interaction which rep resents a relevant factor to induce tolerance due to the potent anti inflammatory role of IL10 (Figure 5) (56)(57)(58).
The role of in vitro generated TolDCs as potential immuno modulatory and immunosuppressive agents have been evaluated by different groups (44,60,61). The first experimental data to objectify the potential of human TolDC to induce tolerance in MS, was the induction of Tcell hyporesponsiveness by TolDC from MS patients. The results obtained shown that only TolDCs (vitD3) derived from RRMS patients, induced hyporesponsive ness in autologous antigenspecific Tcells restricted to myelin derived peptides and produced higher levels of IL10 and reduced levels of TNFα compared to healthy controls, making the tolero genic potential of these autologous TolDCs may be an effective tool to reestablish tolerance in RRMS patients and set up the basis for the ongoing clinical trials (62). In addition, a critical consideration for TolDC application in immunotherapy is the phenotype stability once the cells are injected into the patients. It has been demonstrated that in vitro generated TolDCs have a stable tolerogenic profile after LPS stimulation as they produce higher amounts of IL10 and as well as they are able to induce antigenspecific Tcell hyporesponsiveness (58,63). In summary, TolDCs generated ex vivo using immunosup pressive agents, induced T regulatory cells through different mechanism such as lower expression of co stimulatory molecules, expression of inhibitory receptors and IL10 production.

Tol-DC Therapy in the Animal Model of MS
Animal models are the first step in the development of new therapies, and antigenspecific therapies are not an exception to this rule. Over the past several decades animal models have been used to understand different aspects of human MS. There are three different animal models of MS that are the most com monly used: (1) the experimental autoimmune encephalomyelitis (EAE), (2) viral induced models, and (3) toxininduced models of demyelination (6).
In addition to the in vitro demonstration of the capacity of TolDC to induce immune tolerance, the role of TolDCs has been evaluated in the EAE model. The critical role of mDCs and pDCs in the chronic pathogenesis of EAE in Lewis rats described by Miller and colleagues makes this model extremely relevant to study positive and negative regulatory pathways involved in MS and other chronic autoimmune diseases (64). Wang et al. demonstrated the involvement of CD11b + and CD11c + DCs in the generation of both Tregs and Tr1 cells, by depleting DCs they observed that tolerance effect disappeared (65). In consequence, the induction of DCs with a regulatory profile is a key mechanism underlying auto antigeninduced tolerance (64). It is interesting to highlight that studies performed in EAE induced in Lewis rats demonstrated that the maturation state as well as the route of administration influence on the induction of tolerance by these DCs which is in concordance with the in vitro performed studies (65,66). Moreover, different authors have described that the administration of TolDCs generated with different immunosuppressive agents such as vitD3 or estriol induced a decrease of the incidence of the disease as well as they promoted the induction of regulatory Tcells though higher levels of IL10 production (63,67).
In addition, other authors have performed comparisons regar ding the use of immunosuppressive oral drugs such as vitD3 and (for 20 days after EAE induction) or pretreating DCs before EAE induction. The results obtained were similar in both cases: significant improvement of clinical severity and an increase of regulatory CD4 + Foxp3 + cells and increased IL10 levels in lymph nodes from treated animals suggesting that DCs are the main target of tolerogenic effect of vitamin D. Some studies pointed out that in the absence of DCs during the priming process of autoreactive Tcells leads to a unidirectional deficiency of cell generation which results in a fulminant attack against CNS (65,66,68). Different studies using DCs to induce tolerance have been performed in EAE animal models of mice and rats and they are summarized in Table 3.
In addition, TolDCs have also been generated for another disease models such as type I diabetes T1D by using a combina tion of both dexamethasone and vitD3. This generated TolDCs presented a stable phenotype and a high capacity to induce Treg cells (73). Moreover, other protocols, such as DC treatment with CD40, CD80, and CD86 antisense oligo nucleotides or even low doses of GMCSF has also been reported although in some cases partial loss of tolerance have been reported.
The critical part is that after being culture, all generated TolDCs have to present different characteristics: (a) low levels of co stimu latory molecules, (b) stability when challenges with maturation stimuli and also produce IL10, (c) lower activation of Tcells (73).
Overall, different protocols for TolDCs in preclinical studies has been shown to be beneficious to treat different autoimmune  (65,67,69,70 Therapeutic Application of Tol-DCs in Type i Diabetes, RA and Crohn's Disease Following the encouraging results obtained from different in vitro and preclinical studies in animal models, TolDCs are revealed as a promising therapy for autoimmune diseases and transplanta tion (32). Consequently, in 2011, the first phase I clinical trial with TolDCs was conducted at the University of Pittsburgh. The trial enrolled 10 insulindependent diabetic patients, and admin istrated control DCs to three patients and immunosuppressive DC (iRNA for CD40, CD80, and for CD86) to seven patients. The treatment was safe and well tolerated. There were no changes in insulin requirements, hematology assessments or blood immune cell population levels in both groups, showing a slight increase of CD4 + CD25 +++ FoxP3 + T cells in immunosuppressive DC group. All treated patients had normal immune responses to vaccination and alloantigen stimulation in vitro (74). Thus, a doubleblinded, placebocontrolled crossover phase II trial is planned to start in Diabetes mellitus type 1 in 24 patients with a recent onset of the disease, inducing tolerability of DC with antisense DNA targeting CD40, CD80, and CD86 (NCT02354911). Among autoimmune arthritis, two trials have been published recently. In the first one, a unique intradermal administration of "Rheumavax" (autologous DCs modified with a nuclear factor κb inhibitor exposed to 4citrullinated peptide antigens), was studied in a phase I clinical trial of RA patients. They observed a significant increased ratio of regulatory to effector T cells and a reduction of IL15, which is a relevant proinflammatory cytokine. Moreover, in a more clinical level they found a decrease of DAS28 which is a clinical scale for RA severity together with no disease flares (75). Furthermore, in 2017, results from AUTODECRA trial (NCT01352858) came out resulting a safe and well tolerated therapy with no target knee flares, but with no significant clinical and immunomodulatory changes in serum (76).
Importantly, other clinical trials have been recently reported in other autoimmune diseases such as Crohn's disease. In Crohn's disease, our institution conducted a phase I clinical trial to dem onstrate the safety of intraperitoneal administration of autologous TolDCs in refractory patients. The immune monitoring stud ies showed an increase of circulating Tregs and a decrease of IFNγ production after Tcell activation (31). Regarding organ transplantation, two trials are ongoing. A phase I clinical trial, openlabel and noncontrolled, in liver transplantation is aimed to assess the safety of TolDCs therapy in this type of patients (NCT03164265). The ONEatDC study, aims to assess if Tol DC administration before renal transplantation is beneficial to reduce immunosuppression needs (NCT02252055).
Overall, the encouraging results obtained in above mentioned clinical trials, of an increase immunosuppressive activity, drawn TolDCs as a potential tool to modulate autoinflammatory dis eases in the coming years.

ANTigeN-SPeCiFiC THeRAPieS iN MS AND NMO
In the recent years, several strategies to modulate antigen specific Tcells have been evaluated in therapeutic clinical trials for patients with MS and NMO. Among the advantages to use antigenspecific therapies, they lack of general immunosuppres sion and its side effects as infections and cancer, as well as the lack of metabolic activity that activates selfreactive T cells, the induction of tolerance to a specific antigen without changing the general immunity (77). The use of DC to induce immune toler ance is also pursuit in patients with MS and NMO. In this sense, a phase I trial to assess the safety of TolDC in MS and NMO patients in an ascending dose of intravenous administration of the DCs (NCT02283671) has been performed at our institution and the results are under evaluation. In addition, two more cli nical trials are ongoing (NCT02618902) and (NCT02903537), which will provide precious information about safety, modulation of immune response and clinical efficacy.
Several approaches to induce antigenspecific tolerization have been evaluated as DNA vaccination of myelin protein, pep tides inoculation, altered peptide ligand (APL) administration to modify TCR recognition, autologous myelinreactive T cells administration, HLA/MOG recombinant construct administra tion and autologous PBMCs coupled with myelinpeptides administration, TolDCs with myelinpeptides administration (78). Specifically, myelinpeptides approaches are based in a myelin relevant immunodominant peptide administration, like administration of the synthetic peptide itself like MBP, MOG, or PLP, administration of APL or the administration of a region of TCRpeptide complex.
Antigenspecific therapeutic approaches have been dem onstrated in the majority of the phase I clinical trials to be safe and well tolerated. However, a trial conducted at NIH with APL induced disease exacerbation and the trial was stopped due to safety issues (79). The concept of APL is based in the administra tion of modified peptides by introducing some amino acids in substitution in specific positions relevant to link with the TCR, but without changing the MHC binding part. This strategy is aimed to inhibit the inflammatory T cell response, as acts as partial agonist or as antagonist. A phase II trial using MBP83-99 was inter rupted as three out of eight participants presented relapses during the clinical trial, that were considered as inflammatory activation as MRI controls showed disease worsening, and this was corre lated with MBP specific T cell expansion in blood and CSF samples (80). Two more trials with APLs were done afterward, without objectifying exacerbations of the disease activity (81).
DNA vaccination aims to induce tolerance using heterotopic expression of some antigens, for example using whole human MBP protein. The BHT3009 molecule is a union of the whole MBP molecule, a human cytomegalovirus promoter and an altered plasmid. In two clinical trials it was demonstrated safe and gadoliniumenhancing lesions were fewer in the treated groups comparing with placebo groups; although, there were significant improvement in clinical outcomes. Immunologically, a decrease in IFNγ production and T cell proliferation by MBP, PLP, and MOG specific Tcells was observed (82). In another trial, reduction of autoreactive T cells was demonstrated with this approach, creating a proof of concept of the possible efficacy of DNA vaccination (80).
The vaccination with Tcell consists in the administration of activated and irradiated MPBspecific Tcell lines and clones (attenuated autologous Tcells). Phase I and phase II clinical trials have been done, with no relevant side effects, but without significant clinical improvement in treated group comparing with placebo group (83).
Other antigenspecific tolerization approach studied in MS was the antigencoupled cell tolerance, based on inactivated auto logous PBMCs chemically linked with myelin relevant peptides. After proving reduction of onset and severity as well as preventing epitope spreading in EAE, this approach was evaluated in humans. In 2013, a phase I clinical trial (ETIMS trial) was published where antigenspecific tolerance induced with inactivated PBMCs cou pled with six immunodominant myelinpeptides was safe, with some immunological promising results to objectify clinical significance (78). Significant advantage of this approach is that the tolerization to several myelin relevant peptides derived from three different antigens (MBP, MOG, and PLP) simultaneously is aimed to prevent the epitope spreading situation.
To synthetize, there are different antigenspecific therapies that have been asses in MS patients. The majority has been presented as safe and well tolerated with encouraging data regarding the clinical benefits.

CONClUSiON AND FUTURe PeRSPeCTiveS
Antigenspecific tolerance in autoimmune diseases is a therapeu tic approach that is currently been evaluated in MS and NMO as well as in other autoimmune diseases. Different reports have demonstrated that DCs are powerful therapeutic tools to modify the immune response and restore the immune tolerance in ani mal models and in preclinical data. Most importantly, the use of TolDCs in clinical trials is being safe in several phase I clinical trials (type I Diabetes, RA and Crohn's disease) showing in some of the studies promising clinical and immunomodulatory results.
In MS several reports have revealed the therapeutic effect of TolDCs in ameliorating EAE in animal model. These results highlight the importance of DCs in the homeostasis control and open new avenues for an innovative therapeutic indication for human patients. A major challenge is to translate all these results obtained in animal models to humans. For that reason, it will be crucial to correlate clinical efficacy with modulation of immuno logical parameters and also to define the optimal administration route, dose of cells, tolerogenic treatments and the potential tolerogenic effect of circulating DCs.
From the studies conducted so far, several important consider ations have been raised, application of TolDCs in humans is safe and well tolerated without remarkable side effects and showing promising immunological and clinical results. However, phase II and/or III clinical trials including control (placebo) group will bring some light about the clinical efficacy of this therapy in MS/ NMO patients. In addition, more studies are needed to evaluate the real effectiveness and the possibility to use TolDC as a real treatment for autoimmune diseases.

AUTHOR CONTRibUTiONS
GFG, IZ, and RC wrote the manuscript and designed figures. PV and DBR revised the manuscript.