Cellular & Molecular Immunology www.nature.com/cmi REVIEW ARTICLE OPEN Paving the way towards an effective treatment for multiple sclerosis: advances in cell therapy M. J. Mansilla1,2, S. Presas-Rodríguez3,4, A. Teniente-Serra1,2, I. González-Larreategui1,2, B. Quirant-Sánchez1,2, F. Fondelli1,2, N. Djedovic5, D. Iwaszkiewicz-Grześ6,7, K. Chwojnicki8, Đ. Miljković5, P. Trzonkowski6,7, C. Ramo-Tello3,4 and E. M. Martínez-Cáceres1,2 Multiple sclerosis (MS) is a leading cause of chronic neurological disability in young to middle-aged adults, affecting ~2.5 million people worldwide. Currently, most therapeutics for MS are systemic immunosuppressive or immunomodulatory drugs, but these drugs are unable to halt or reverse the disease and have the potential to cause serious adverse events. Hence, there is an urgent need for the development of next-generation treatments that, alone or in combination, stop the undesired autoimmune response and contribute to the restoration of homeostasis. This review analyzes current MS treatments as well as different cell-based therapies that have been proposed to restore homeostasis in MS patients (tolerogenic dendritic cells, regulatory T cells, mesenchymal stem cells, and vaccination with T cells). Data collected from preclinical studies performed in the experimental autoimmune encephalomyelitis (EAE) model of MS in animals, in vitro cultures of cells from MS patients and the initial results of phase I/II clinical trials are analyzed to better understand which parameters are relevant for obtaining an efficient cell-based therapy for MS. 1234567890();,: Keywords: multiple sclerosis; cell-based therapy; tolerance; neuroprotection; autoimmunity Cellular & Molecular Immunology (2021) 18:1353–1374; https://doi.org/10.1038/s41423-020-00618-z MULTIPLE SCLEROSIS (CSF)) is used. The most recent diagnostic criteria are the 2017 Multiple sclerosis (MS) is a chronic inflammatory and demyeli- McDonald criteria.3 nating disease that affects the central nervous system (CNS) and is characterized by inflammation, multifocal demyelination, MS pathogenesis axonal loss, and gliosis in both the white and gray matter. MS Currently, the cause of MS remains unknown. In experimental is a complex disease with considerable clinical and radiological autoimmune encephalomyelitis (EAE), an animal model of MS, heterogeneity. It was initially classified into four different pheno- myelin-specific T cells are believed to play a crucial role in its types: relapsing-remitting MS (RRMS), secondary-progressive MS pathogenesis.4 In fact, the presence of circulating myelin-reactive (SPMS), primary-progressive MS (PPMS), and relapsing-progressive T cells in MS patients has been extensively reported. However, the MS (RPMS).1 RRMS (85%) is characterized by acute relapses (acute or specific mechanisms that cause the activation and entrance of these subacute episodes of new or increasing neurologic dysfunction in cells into the CNS are still unknown. It has been postulated that a the absence of fever or infection) followed by remission with full or complex interaction between multiple genetic and environmental partial recovery. SPMS is defined as progressive clinical worsening factors contributes to the dysregulation of peripheral immune over time after an initial relapsing course, with or without acute homeostasis and the activation of autoreactive T cells.5 exacerbations during the progressive course. PPMS, accounting for Several environmental factors, including infectious agents ~15% of MS, is characterized by clinical progression without relapse (mainly viruses), tobacco, diet (long-chain fatty acids, salt), gut from disease onset. The term RPMS is used to describe the microbiota, stress, sex hormones, and vitamin D deficiency, have progressive accumulation of disability from onset with occasional been shown to be related to the triggering and development of relapses. This subtype is rarely diagnosed since it overlaps with other disease.5 The incidence of MS is higher in women than in men. MS phenotypes in terms of its features. symptoms often improve during late pregnancy, coinciding with In addition, since 2014, these phenotypes have also included high levels of estriol.6 In contrast, men are more prone to develop the concept of “disease activity” based on clinical and MRI criteria PPMS later in life, correlating with the physiological decline in in an effort to achieve better patient classification.2 For MS testosterone with age.7 diagnosis, a combination of clinical, radiological, and laboratory Extensive studies have been performed to understand the criteria (presence of oligoclonal bands in the cerebrospinal fluid genetic contribution to MS, and more than 200 loci that promote a 1 Division of Immunology, LCMN, Germans Trias i Pujol University Hospital and Research Institute, Barcelona, Spain; 2Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain; 3Multiple Sclerosis Unit, Department of Neurosciences, Germans Trias i Pujol University Hospital, Barcelona, Spain; 4 Department of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain; 5Department of Immunology, Institute for Biological Research “Siniša Stanković”- National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia; 6Department of Medical Immunology, Medical University of Gdańsk, Gdańsk, Poland; 7Poltreg S.A., Gdańsk, Poland and 8Department of Anaesthesiology & Intensive Care, Medical University of Gdańsk, Gdańsk, Poland Correspondence: M. J. Mansilla (mjmansilla@igtp.cat) or E. M. Martínez-Cáceres (emmartinez.germanstrias@gencat.cat) Received: 13 July 2020 Accepted: 3 December 2020 Published online: 6 May 2021 © The Author(s) 2021 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1354 predisposition to MS have been identified, suggesting a complex dissection.28 Up to a quarter of CD8+ T cells in the active lesions of disease etiology. Nearly all the gene regions identified so far MS patients can produce IL-17 and are thought to be mucosa- contain genes involved in immune mechanisms. The major HLA- associated invariant T (MAIT) cells. It has been suggested that DRB1∗1501 locus accounts for 30% of the overall risk.8,9 Moreover, these CD8+ cells, characterized by the expression of a semi- emerging evidence indicates that the DNA methylome actively invariant T cell receptor (a dimer of Vα7.2 with Jα12, Jα20, or Jα33), participates in gene × environment interactions, and several play an important role in disease pathogenesis.29 studies have shown an aberrant DNA methylome profile develops In recent years, the pathogenic role of B cells in MS has been in MS.10 highlighted.30 Clonally expanded B cells can be found in the Factors such as the presence of dysfunctional regulatory T cells meninges, parenchyma and CSF. B cells produce antibodies (Tregs)11 or dendritic cells (DCs)12 and alterations in cytokine intrathecally with an oligoclonal pattern (which can be observed production may facilitate the entry of proinflammatory myelin- by comparison with serum samples from the same patient). specific autoreactive T cells into the CNS (reviewed in13,14). In this Ectopic lymphoid follicles, which sequester antigens and facilitate context, IFN-γ-producing Th17.1 (CCR6+CXCR3+CCR4–) cells have B and T cell activation, have been observed proximal to cortical been identified as relevant in disrupting the permeability of the demyelinating lesions in the meninges of MS patients, and BBB in MS.15 Memory B cell precursors and IFN-γ-producing CD8+ their frequency correlates with disease severity.31–33 The T cells also express high levels of CCR6, as they can enter the CNS. presence of tertiary lymph follicles suggests that B cell maturation In addition to chemokine receptors and proinflammatory cyto- is sustained locally, contributing to the intrathecal synthesis of kines, adhesion molecules such as integrin α4β1 (VLA-4), which immunoglobulins.31,32 In addition to their roles in antigen presenta- induces firm adhesion to vascular cell adhesion protein 1 (VCAM- tion to Th1 and Th17 cells and antibody production, B cells secrete 1) on brain endothelial cells, and activated leukocyte cell adhesion proinflammatory cytokines (e.g., IL-6, TNF, granulocyte-macrophage molecule enhance the transmigration of pathogenic B and T cell colony-stimulating factor (GM-CSF)), promoting CNS inflammation subsets.16,17 and demyelination.34 As an alternative to the immune-mediated cause of MS, it has There is increasing evidence that innate immune cells (NK, DCs, been postulated that CNS-intrinsic events (for example, CNS viral macrophages, mast cells, and innate lymphoid cells (ILCs)), normal infection or processes leading to primary neurodegeneration) may residents of the meninges, are involved in the pathogenesis of MS, trigger disease development, with the infiltration of autoreactive affecting both its initiation and progression (reviewed by Brown lymphocytes occurring as a secondary phenomenon.18,19 et al.33). Mast cell transcripts that encode mast cell-associated The brain and spinal cord are surrounded by the meninges, molecules, such as tryptase histamine or FcεR1, have been which provide first-line protection to the CNS. They are observed in the demyelinating lesions of MS patients.35 They constituted by three layers: the dura mater, located directly under can increase BBB permeability, contributing to the initiation of the skull or vertebral column; arachnoid mater; and pia mater, in chronic inflammation.36 Moreover, interactions between resident close contact with the CNS parenchyma. The CSF drains through mast cells and autoreactive T cells in the meninges induce the subarachnoid space, an anatomical gap between the caspase-1-dependent IL-1β production by mast cells, activating arachnoid mater and pia mater (both known as the leptome- GM-CSF production by T cells.37,38 GM-CSF is an essential growth ninges). Interestingly, the existence of a rudiment of lymphatic factor for T cell encephalitogenicity, inducing the recruitment of vessels within the meninges was recently described, supporting CCR2+ inflammatory monocytes into the CNS.39–42 Additionally, the existence of a physical connection between the fluids, TNF expression by mast cells is essential for the early recruitment immune cells, and macromolecules of the CNS and the deep of neutrophils to the meninges and CNS.23,36,43 Neutrophil cervical lymph nodes, establishing physical contact between the products, i.e., proteolytic enzymes, including matrix metallopro- CNS and the immune system.19,20 teinase 9, ROS and structures composed of DNA and proteins Most knowledge of MS pathogenesis has been obtained in the called neutrophil extracellular traps, damage the BBB, thus EAE model, the animal model of MS. This model is typically supporting a role for neutrophils in MS pathogenesis.43–45 induced by either active immunization with myelin-derived CD45+Lin−IL-7Rα+ ILCs constitute a heterogeneous group of proteins or peptides in adjuvant or by passive transfer of activated innate immune cells that have more recently been related to MS myelin-specific CD4+ T lymphocytes and reproduces most of the pathogenesis: group 1 (ILC1), group 2 (ILC2), and group 3 (ILC3) main clinical and histopathological characteristics of MS. Although cells, analogous to Th1, Th2, and Th17 cells, respectively. These this model has some limitations, as it is an induced model, it is a cells mostly remain in tissues and exert their effects locally.46 powerful tool to investigate MS pathogenesis and potential Similar to Th cells, ILCs can also exhibit considerable phenotypic therapeutic strategies.21,22 In EAE, several days before inflamma- plasticity.46 Each group of ILCs plays a different role in EAE-related tory cells are detected in the CNS, an influx of peripherally derived inflammatory responses. ILC2s express ST2, the heterodimeric IL- immune cells within the meninges occurs.23 Myelin antigens that 33 receptor. Upon activation, these cells produce the Th2 drain from the CNS to the meninges via the CSF are presented by cytokines IL4, IL-5, IL-9, and IL-13.47 IL-33 induces ST2+ ILC2s to infiltrating or resident antigen-presenting cells (APCs) (perivascu- produce IL-13 and other Th2-polarizing cytokines, which in turn lar macrophages or resident APCs, e.g., microglial cells).24–26 This promote a nonpathogenic Th2-dominated response. In contrast, causes myelin-specific T cell reactivation. Following myelin ILC1s and ILC3s produce IFN-γ, IL-17, GM-CSF, and other cytokines recognition by autoreactive pathogenic T cells, a complex immune that have been linked to EAE pathogenesis. Interestingly, the response is produced, facilitated by the entrance of other cell lymphoid tissue inducer subset of ILC3s drives ectopic lymphoid types (B cells, NK cells, macrophages, and innate immune cells) follicle formation.48 and the production of proinflammatory cytokines and reactive Natural killer (NK) cells have been widely studied in the context oxygen and nitrogen species (ROS and RNS, respectively); this of MS and EAE and assigned both pathogenic and protective roles results in disruption of the blood–brain barrier (BBB) and the (reviewed in49,50). CD56dim cells, the major cytotoxic NK popula- entrance of cells into the CNS parenchyma, leading to perivascular tion, can kill oligodendrocytes, astrocytes, and microglia in vitro. In inflammation, demyelination, and neuronal damage.27 contrast, regulatory CD56hi NK cells produce neurotrophic factors Interestingly, in contrast to EAE, CD8+ T cells are found more such as brain-derived neurotrophic factor (BDNF) and neurotro- frequently than CD4+ cells in acute and chronic MS lesions. CD8+ phin-3, consistent with a role in neural repair. T cells directly attack oligodendrocytes (via the secretion of Additionally, CNS-resident cells (mainly microglia) are highly granzymes and perforin), causing apoptosis and damaging sensitive to homeostatic disturbances and can produce neurotoxic neurons via the release of cytolytic granules, leading to axonal inflammatory mediators (cytokines, chemokines, and ROS), Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1355 promoting and sustaining neurodegeneration (reviewed in13). processes in the CNS, followed by pronounced progressive Importantly, astrocytes are key players recruiting lymphocytes and neurodegenerative decline (reviewed in13). inducing the innate response during the early stage of white matter lesion formation. In contrast, astrocytes can also restrict inflammation through scar formation and trigger neuroprotection CELL-BASED TOLEROGENIC THERAPIES and tissue repair.51 Regulatory B cells (Bregs), T cells (CD4+FoxP3+ Targeting the fundamental cause of autoimmunity, i.e., the loss of (Tregs), CD4+Tr1+ and CD8 Tregs), tolerogenic DCs (tolDCs) and tolerance to self-antigens, will provide the next steps forward to regulatory CD56hi NK cells can regulate effector T cells in the avoid the general immunosuppression induced by current periphery or CNS through different mechanisms. It has been treatments. Accumulating knowledge on the mechanisms of postulated that either a defect in the regulatory function of these immune tolerance and activation has led to the development of cells or the increased resistance of effector T cells to suppressive tolerance-inducing cellular therapies with the specific objective of mechanisms contributes to the pathogenic function of autoreac- limited unwanted immune reactions over the long term (reviewed tive T and B cells in MS. Alternatively, the dysfunction of peripheral in54). Phase I clinical trials using Tregs, mesenchymal stem cells regulatory cells could be indirectly driven by the dysregulation of (MSCs), or tolerogenic antigen-presenting cells, such as tolDCs and tolerogenic APCs (reviewed in13). regulatory macrophages, for the treatment of autoimmune diseases (ADs) and prevention of transplant rejection have Neuropathology of MS expanded in recent years. The results have confirmed so far that In contrast to the increased inflammation of relapsing forms of MS, these cellular therapies are safe, with no relevant side effects, and neurodegenerative events are more severe in progressive MS. well tolerated by patients.54 In this context, several cellular Nevertheless, there is no clear distinction between progressive products have been developed for MS (Fig. 2), and phase I clinical and relapsing MS; both pathobiologies can occur during the trials are currently ongoing or finished. To advance the use of disease. these therapies in the clinic, we should analyze the results and Comparison of PPMS and SPMS reveals some quantitative address remaining challenges, such as the optimal dose, admin- differences in the presence of focal and active classical white istration route, frequency of administration, antigen specificity, matter lesions and in the global degree of inflammation, which is and biomarkers of clinical and biological response, to design the lower in PPMS than in SPMS.52 These differences can be explained next generation of clinical trials. by the two types of inflammation in MS patients (Fig. 1). During disease progression, patients can exhibit both types of inflamma- Tolerogenic dendritic cells tion, although the composition can be altered during relapses and DCs are key players in controlling the immune response. They are progressive periods. highly efficient APCs that are able to activate the immunogenic T During the acute and relapsing phases of the disease, in which cell response and suppress it by inducing regulatory mechan- leakage of the BBB occurs, the major players are T (CD4+ and isms.55 TolDCs are defined as semimature DCs with an inter- CD8+) and B lymphocytes that attack myelin, leading to mediate phenotype between the phenotypes of immature (iDCs) demyelination. CD8+ cells have the phenotype of tissue-resident and mature DCs (mDCs). It is not clear whether tolDCs constitute a memory cells. CD8+ T cells proliferate focally and show signs of different DC subset by themselves. TolDCs are characterized by activation or clonal expansion, indicating local antigen recogni- one or more of these features: the expression of low levels of tion. The pathogenic role of B cells is inferred by the beneficial costimulatory molecules (i.e., CD80, CD86, and CD40) and MHC effects observed after anti-CD20 therapies. However, B cells are class II, a reduced capacity to produce proinflammatory cytokines, thought to play different roles depending on their stage of upregulated expression of inhibitory molecules such as PDL1, ILT3 differentiation or the activity stage of the lesions. Plasmablasts and ILT4 and secretion of immunoregulatory cytokines and and plasma cells within MS lesions express high levels of IL-10,53 mediators (IL-10, TGF-β, IDO, heme oxygenase-1 or FasL).56,57 suggesting that they may ameliorate inflammation. Because of the semimature or mature resistance phenotype of Inflammatory infiltrates may lead to focal areas of primary tolDCs, the proper activation of T cells by costimulatory molecules demyelination with variable axonal injury, which is mainly carried and proinflammatory cytokines following antigen recognition is out by activated microglia and macrophages. Antibodies that limited. Under these conditions, instead of inducing the activation recognize oligodendrocytes or astroglia may contribute to MS and clonal expansion of T cells, tolDCs promote T cell pathogenesis at this stage. After the initial autoimmune attack, hyporesponsiveness that is mainly mediated by the induction of lymphoid cells in the parenchyma undergo apoptosis, and T cell anergy, T cell depletion triggered by apoptosis induction or macrophages and microglia can switch to an anti-inflammatory/ the induction of Treg differentiation (reviewed in58,59). reparative phenotype. Importantly, tolDCs can be generated ex vivo from autologous In the progressive phases of MS, the second pattern of human peripheral blood monocytes. Indeed, over the last 20 inflammation predominates. Leakage of the BBB is less pro- years, a wide variety of protocols describing the induction of nounced, and T and B lymphocytes slowly accumulate in the tolDCs with several stimuli, such as anti-inflammatory cytokines connective tissue spaces of the brain and spinal cord, affecting the (IL-10 and TGF-β), pharmacological agents and immunosuppres- meninges and periventricular spaces. Infiltrating cells form focal sant compounds (rapamycin, different corticosteroids, vitamin D3, aggregates resembling tertiary lymph follicles. CD8+ cells have the aspirin, mitomycin C, and the NF-κB inhibitor BAY11-7082), and phenotype of tissue-resident memory cells with focal activation. other strategies, such as genetic engineering for the selective Most cells of the B cell lineage in chronic lesions are plasmablasts repression or induction of key molecules and pathways, among and plasma cells. Tissue injury may at least be partly mediated by many others, have emerged (reviewed in54,58). Most of these microglia and macrophage activation, oxidative injury and protocols share several features, such as the differentiation of mitochondrial damage. This inflammation is associated with the monocytes in the presence of GM-CSF and IL-4, as well as the expansion of pre-existing lesions, as well as diffuse neurodegen- addition of a maturation stimulus (which usually consists of eration in normal-appearing white or gray matter. Interestingly, it different combinations of LPS, monophosphoryl lipid A, TNF, IL-1β, has been found that this second type of inflammation is already prostaglandin E2 (PGE2) and IL-6), to maintain tolDCs in an present in the early stages of MS, after which it increases gradually activation-resistant state, as this is an absolute requirement for with disease duration and patient age.52 The inflammation tolDC therapy. induced by peripheral immune cell infiltration and CNS-resident Analysis of preclinical data for the evaluation of tolDC treatment innate immune cells may contribute to acceleration of the aging in EAE, as well as in vitro studies on peripheral blood leukocytes Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1356 Fig. 1 Multiple sclerosis pathogenesis in both relapsing and progressive disease. Scheme representing the major cells and molecules that play a role in the two different stages of MS. The dashed line allows comparison of the differences between relapsing and progressive MS. Arrows indicate release, while inhibitors indicate inhibition. The black arrowhead on a dotted line indicates transmigration. CSF cerebrospinal fluid, DC dendritic cells, GM-CSF granulocyte-macrophage colony-stimulating factor, IFN interferon, IL interleukin, ILCs innate lymphoid cells, MAIT cells mucosal-associated invariant T cells, MS multiple sclerosis, RNS reactive nitrogen species, ROS reactive oxygen species, SAS subarachnoid space, Th T helper from MS patients (Table 1), is crucial to better understand the autoreactive MOG, PLP, and MBP peptides seems to be a better requirements and characteristics for the design of efficient tolDC strategy.72,73 In fact, the three phase I clinical trials of peptide- products for MS patients. loaded tolDCs in MS have employed a similar approach using a pool of myelin peptides (NCT02283671, NCT02903537, and Antigen specificity. Several studies in the EAE model of MS have NCT02618902) (Table 2). demonstrated that clinical efficacy of tolDCs is achieved only when the cells are loaded with disease-related autoantigens60–71 Dose, timing, and frequency. Although studies in animal models (Table 1). Although the etiology of MS is unknown, it is widely are extremely useful, extrapolation of the dose, timing, and accepted that it is an autoimmune-mediated disease directed frequency of administration to patients is not straightforward. against several myelin proteins, such as MBP, proteolipid protein From publications using different tolDC types to treat EAE, i.v. (PLP), and myelin oligodendrocyte glycoprotein (MOG). Conse- administration of a million antigen-specific tolDCs has demon- quently, when thinking about the translation of tolDC therapy to strated promising results in reducing EAE severity (Table 1). humans, the loading of a single antigen on tolDCs is unlikely to be However, extrapolation of the dose from these studies is not sufficiently effective. Moreover, due to the epitope spreading feasible. The monocyte-derived tolDCs recovered after the in vitro phenomenon, the same patient may also exhibit extended differentiation process number a maximum of several million, reactivity to additional myelin epitopes following disease progres- depending on the patient. Consequently, thus far, direct extra- sion. Therefore, in tolDC phase I clinical trials for MS patients, polation of tolDC doses from EAE studies is not feasible. In fact, a cocktail of myelin peptides containing the most relevant in a phase I trial of i.v. dexamethasone-tolDC (dexa-tolDC) Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1357 Fig. 2 Proposed mechanisms of action of approved treatments for multiple sclerosis and cell-based therapies. Representation of the mechanisms of action of current treatments (black boxes) and cell-based therapies (gray boxes). Arrows indicate induction, while inhibition symbols indicate inhibition. Breg regulatory B cells, CNS central nervous system, DC dendritic cells, MAIT cell mucosal-associated invariant T cells, NK natural killer cells, Th T helper, Treg regulatory T cells administration, a technical limitation was reported related to would require several rounds of leukapheresis and in vitro reaching 300 × 106 cells.74 Since cells administered intravenously differentiation under good manufacturing practice conditions). exhibit a wide biodistribution of cells (meaning that a large Hence, the use of cryopreserved cells is a feasible option.66,75 In number of tolDCs would be necessary to reach the secondary fact, at least for VitD3-tolDCs, both human and murine cryopre- lymphoid organs), other routes of administration are currently served cells retain phenotypical and functional tolerogenic being tested by our group and others (Table 2). This issue will be characteristics. Importantly, therapeutic administration of cryopre- discussed below. served MOG40–55-VitD3-tolDCs in EAE mice showed clinical efficacy, As shown in Table 1, many of the EAE studies analyzed the reducing T cell autoreactivity and triggering the generation of efficacy of tolDCs before the onset of clinical symptoms FoxP3+ Tregs in vivo.66 All these data were comparable with those (prophylactic or late prophylactic administration). In contrast, other obtained using fresh cells.67 To go one step further, we explored authors have attempted to reproduce the real-world situation of the effect of long-term treatment with cryopreserved MOG40–55- MS patients for therapeutic administration.61,66,67,70 From those VitD3-tolDCs in EAE. Following 3 therapeutic administrations of studies, antigen-specific VitD3-tolDCs demonstrated a remarkable MOG40–55-tolDCs every 4 days, additional doses were administered therapeutic effect.61,66,67 when the mean clinical score of the group increased. Strikingly, we Focusing on therapeutic studies in EAE, the administration of at observed that periods of long-lasting clinical stability increased least 3 doses of antigen-specific tolDCs treated with VitD3 or progressively. Immunological examination of mice revealed BD750 (a JAK3/STAT5 inhibitor) in the early stage of disease increased proportions of Bregs and activated NKT cells as well as abrogated disease progression.61,66,67,70 Considering translation to a reduction in immunogenic NK cells in the spleens of treated MS, the use of multiple tolDC injections seems optimal. However, mice, which might explain (at least in part) the beneficial effect of this would imply repeated production of tolDC batches (which MOG40–55-tolDCs.66 Cellular & Molecular Immunology (2021) 18:1353 – 1374 1358 Table 1. Tolerogenic DCs therapies in EAE and in vitro studies using MS patients tolDCs Tolerogenic DC therapies in EAEa Tolerogenic agent EAE model Groups of Number of administrations Type of Route Number Clinical outcome Mechanism of action Observations Ref treatment treatmentb of tolDCs 60 K313 C57BL/6 MOG-K313-DC, 3 administrations (every 3 days) Early i.v. 1 × 106 cells Clinical symptoms Strong reduction of BMDCs treated with K313 mice unpulsed K313- at days therapeutic amelioration (similar to leukocyte infiltration and inhibit antigen-specific CD4 immunized DC and MOG- 10, 13, and 16 p.i. MOG-VitD3-tolDCs group) demyelination + T cells with VitD3-tolDCs Reduction of Th1 and Th17 MOG35–55 (control group) cells and increase of Treg compared to untreated group 61 VitD3 C57BL/6 MOG mRNA 3 administrations (every 4 days) Therapeutic i.v. 1 × 106 cells Reduced disease severity Decrease of number of mice electroporated- at days with both electroporated spinal cord lesions (MRI) immunized VitD3-tolDCs, 13, 17, and 21 p.i. and pulsed antigen-specific Decreased MOG-reactive with MOG-VitD3- VitD3-tolDCs T cells in the spleen and MOG35–55 tolDCs, lymph nodes and reduction unpulsed VitD3- of proinflammatory tolDCs and PBS cytokines secretion (IL-17, IFN-γ, TNF-α, GM-CSF) 62 C57BL/6 MOG-VitD3- 3 administrations (every 3 days) Early i.v. 0.8 × 106 cells Delay of disease onset Decreased Th1 and Th17 No maturated DC mice tolDCs, at days therapeutic Reduction of EAE severity cell infiltration into the CNS immunized MOG-DC 10, 13, and 16 p.i. Enhanced % of Treg, CD4+ with and PBS IL10+ T cells and Breg in MOG35–55 spleen and/or lymph nodes 63 C57BL/6 MOG and 1 single administration at day Early i.v. 1 × 106 cells Reduced disease severity Less inflammatory foci and DC engineered to mice unpulsed 10 p.i. therapeutic (1 dose) with antigen- demyelination overexpress 25- immunized engineered or s.c. specific manner Increase of Th2, Tr1, and hydroxyvitamin D 1α- MJ Mansilla et al. Paving the way towards an effective treatment for multiple sclerosis:. . . with tolDCs 3 administrations (every 7 days) (3 doses) FoxP3+ Treg cells hydroxylase (self-tolerized) MOG35–55 overespressing at days Higher secretion of IL-4 and and release 1,25- VitD3 and 10, 17, and 24 p.i. IL-10 compared to dihydoxyvitamin D untreated EAE control groups Clinical amelioration related with FoxP3+ Treg induction 64 C57BL/6 Cryopreserved For short-term treatment Therapeutic i.v. 1 × 106 cells Reduced disease severity Short-term treatment: The long term-treatment mice MOG-VitD3- (30 days), 3 administrations following short and long- Inhibition of MOG40–55 T cell with cryopreserved tolDCs- immunized tolDCs, (every 4 days: 15, 19, and 23 p.i.) term antigen-specific reactivity and increased VitD3-MOG was well with unpulsed VitD3- For long-term treatment treatment with antigen- proportion of FoxP3+ Treg tolerated, highly effective MOG40–55 tolDCs and PBS (74 days), 3 initial administrations specific VitD3-tolDCs Long-term treatment: and exhibited a prolonged (every 4 days: 14, 18, and 22 p.i.) Inhibition of MOG40–55 T cell clinical benefit after each + extra-administrations when the reactivity; increase of Breg administration. mean clinical score of the MOG- and activated NKT cells; and VitD3-tolDCs group increased decreased proportion and activation of NK cells 65 C57BL/6 MOG-VitD3- 2 administrations for prophylactic Prophylactic, i.v. 1 × 106 cells Antigen-specific VitD3- Induction of FoxP3+ Treg Biodistribution analysis mice tolDCs, (−2, 5 p.i.) and late-prophylactic late- tolDCs showed reduced and IL-10 secretion showed that although immunized unpulsed VitD3- (5, 9 p.i.) approaches and prophylactic EAE incidence (prophylactic Inhibition of MOG40-55- tolDCs reached CNS with tolDCs and PBS 3 administrations (every 4 days: and treatment) and reduced specific T cells reactivity following i.v. injection, MOG40–55 15, 19, and 23 p.i.) for therapeutic therapeutic disease severity (late- tolDCs accumulated in prophylactic and spleen after 48 h and therapeutic treatment) remained there at day 14 p.i. 66 Chloroquine C57BL/6 MOG-CQ-BMDC, 1 single administration at day Late- i.v. 1.5 × 106 cells Reduction of EAE severity Inhibition of T cell STAT1−/− mice were also mice MOG-PBS-BMDC 10 p.i. prophylactic Suppression of Th17 proliferation and reduction used in the experiments immunized and infiltration into the CNS of differentiation and and treated with MOG-CQ- with untreated EAE infiltration of Th17 cells BMDC or MOG35–55 mediated by MOG-PBS-BMDC STAT1 signaling Increase of IL10 secretion and reduction of IFN- γ levels Induction of FoxP3+ Treg 67 Tofacitinib i.v. 1 × 106 cells Cellular & Molecular Immunology (2021) 18:1353 – 1374 Table 1. continued Tolerogenic DC therapies in EAEa Tolerogenic agent EAE model Groups of Number of administrations Type of Route Number Clinical outcome Mechanism of action Observations Ref treatment treatmentb of tolDCs C57BL/6 MOG-Tofa-DC, 3 administrations (every 4 days) Early Reduction of EAE severity Reduced leukocyte Frequencies of Th17 and mice unpulsed-Tofa- at days therapeutic in mice receiving antigen- infiltration and less Th1 cells correlated immunized DC PBS, and 7, 11, and 15 p.i. specific Tofa-DC extensive demyelination positively with the clinical with non-immunized into the CNS score and correlated MOG35–55 Decreased % of Th1 and negatively with the Th17 cells and enhanced % frequency of Treg of CD25 + FoxP3+ Treg in the spleen 68 BD750 C57BL/6 MOG-BD750- 3 administrations (every 4 days) Prophylactic, i.v. 1 × 106 cells Early therapeutic Early therapeutic: Early therapeutic treatment mice tolDCs, Prophylactic approach: days −2, early treatment: Delay on disease Reduced inflammatory using only 1 or 2 immunized npulsed BD750- 2, and 6 p.i. therapeutic onset and reduced EAE infiltrates and administrations of MOG- with tolDCs, PBS and Early therapeutic approach: days and late- severity with MOG-BD750- demyelination in the CNS BD750-tolDCs (at day 7 p.i. MOG35–55 non-immunized 7, 11 and 15 p.i. therapeutic tolDCs Reduced frequency of or days 7 and 11 p.i.) Late-therapeutic approach: days Late-therapeutic treatment: Th1 and Th17 cells and showed no clinical efficacy 19, 23, and 27 p.i. no clinical improvement increased % of FoxP3+ Treg in the spleen Cellular & Molecular Immunology (2021) 18:1353 – 1374 69 IL-35 C57BL/6 MOG loaded 1 single administration 1 day Prophylactic i.v. 2.5 × 106 cells Reduction of EAE Impairment of T cell receiving and unpulsed after passive EAE induction and Late- symptoms activation and proliferation 5·106 tumor DC cell (prophylactic) and 2 prophylactic MOG35–55- line modified to administrations at day 6 and 8 specific express IL-35 post passive EAE induction (late- T cells prophylactic) (Passive EAE) 70 LPS C57BL/6 MOG-LPS-DC, 3 administrations (every 4 days) Early i.v. 0.3 × 106 cells Antigen-specific abrogation LPS-treated BMDC increase mice MOG-DC and at days therapeutic of EAE development the frequency of Treg (CD4 immunized unpulsed DC 11, 14, and 17 p.i. + CD25+ FoxP3+ GITR+) with that are CD127+ 3G11− MOG35–55 and inhibit those CD127 + 3G11 + Treg 71 Apoptotic C57BL/6 Splenic DC 3 administrations (every 4 days) Early i.v. 0.3 × 106 cells Antigen-specific abrogation Inhibition of central and Immune tolerance induced thymocytes mice primed with at days therapeutic of EAE development effector memory CD4+ T by apoptotic T cell-treated immunized irradiated 11, 14, and 17 p.i. cell development DC is mainly related to CD4 with (apoptotic) or Reduction of IFNγ + effector memory T cells MJ Mansilla et al. MOG35–55 fresh T cells and production reduction pulsed with or without MOG In vitro studies using tolDCs from MS patients Tolerogenic agent Phenotype Functionality Stability Comparison with HD tolDCs Mechanism of action Observations Ref 82 VitD3 Reduced expression of CD86, CD83 and Reduced ability to stimulate T Stable phenotype and Equal efficiency to generate VitD3- Not provided Set up of myelin HLA-DR molecules cell allogeneic proliferation functionality tolDCs from MS patients and HD peptide-tolDCs loading Production of higher levels of IL-10 Decrease secretion of TNF-α, LPS stimulation did not Low level expression of CD83 in mDC (although not statistically significant) and IL-6, and IFN-γ induce IL-23 secretion from MS patients compared with HD low levels of IL12p70 compared to mDC Induction of antigen-specific but small amounts of T cell hyporesponsiveness IL-10 were still detectable 75 Decreased expression of CD86, CD80, Reduced allogeneic and No tested on fresh VitD3-tolDCs and mDC from MS patients Unknown Cryopreserved VitD3- CD83, and HLA-DR molecules antigen-specific T cell VitD3-tolDCs secreted higher levels of IL-1β, IL-6 and No induction of neither tolDCs were studied: proliferation Cryopreserved VitD3- TNF-α compared to HD counterparts FoxP3+ Treg nor IL-10 and/or They retained Impaired secretion of tolDCs remained TGF-β producing cells phenotypical and proinflammatory cytokines phenotypically and functional functionally stables characteristics and following LPS remain stable stimulation following Paving the way towards an effective treatment for multiple sclerosis:. . . proinflammatory stimulus 1359 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1360 Ref The effect of tolDCs in the chronic phase of EAE has been CNS central nervous system, CQ chloroquine, DC dendritic cells, EP ethyl pyruvate, HD healthy donors, i.v. intravenous, MAP7 microtubule-associated protein 7, MOG myelin oligodendrocyte glycoprotein, MRI Prophylactic treatment: treatment before or at the moment of EAE induction; Late-prophylactic treatment: treatment after EAE induction but prior to clinical onset; early therapeutic treatment: initiation of treatment when mice showed first clinical symptoms; therapeutic treatment: first dose administrated to mice with established clinical signs; late-therapeutic: treatment in mice with high degree of paralysis 87 86 analyzed using MOG35–55-BD750-tolDCs. Unfortunately, no clinical reported as biomarkers MUCL1 and MAP7 were Similar phenotype and patients VitD3-tolDCs benefits were observed even though 3 doses of MOG35–55-tolDCs of both HD and MS functionality of EP- were administered every 4 days.70 Although more studies are tolDCs and VitD3- needed, these results suggest that tolDC therapy should be used in Observations the first stages of the disease. tolDCs Route of administration. The safety of i.v., intraperitoneal (i.p.), intradermal (i.d.), intranodal (i.n.) and even intra-articular routes of human tolDC administration has been demonstrated in different phase I clinical trials.74,76–79 The route of administration is crucial due to two important issues. On the one hand, the treatment must Mechanism of action promote tolerogenic in vivo effects. In this context, i.v. adminis- tration is considered the most tolerogenic route of administration, showing tolerogenic effects superior to those of i.d. administration No provided No differences in neither phenotype nor No provided in nonhuman primates.80 On the other hand, the selected route of administration must allow tolDCs to reach the draining lymph nodes or inflamed tissues. In this regard, either i.d. administration near the draining lymph nodes or direct i.n. tolDC injection could allogenic proliferation inhibition of HD be an alternative to i.v. administration. Interestingly, two VitD3-tolDCs from HD also exhibited coordinated phase I clinical trials using VitD3-tolDCs will compare these two routes of administration81 (Table 2). Comparison with HD tolDCs increase secretion of TGF-β Mechanism of action. The mechanism of action of several types of tolDCs has been analyzed.54 Most tolDCs impair T cell alloproliferation in in vitro cultures. Moreover, they also act and MS patients through different pathways. Dexa-tolDCs produce IL-10, tolDCs generated in the presence of exogenous IL-10 (DC-10) induce IL- 10-producing Tregs (Tr1 cells), and VitD3-tolDCs induce T cell hyporesponsiveness in an antigen-specific manner without affecting the ability of other T cells to respond to unrelated antigens.75,82 In addition, a transcriptomic analysis of autologous CD4+ T-cells primed with antigen-specific VitD3-tolDCs revealed magnetic resonance imaging, MS multiple sclerosis, MUCL1 mucin-like 1, Tofa tofacitinib, VitD3 vitamin D3 profound genetic downregulation, mainly affecting factors related to Non evaluated Non evaluated the cell cycle and proinflammatory immune response processes.83 Dexa+VitD3-tolDCs regulate CD4+ T cell cytokine production in RA patients in a TGF‐β1‐dependent manner.84 Additionally, TGF-β Stability secreted by tolDCs is an important immunoregulatory mediator involved in the induction of Tregs. Interestingly, tolDCs differentiated in the presence of low doses of GM-CSF and in the absence of IL-4, Ethyl pyruvate (EP) Decreased expression of CD86, CD83, and Inhibition of allogenic T cell proliferation with reduction No increase of IL-10 levels proliferation and increase referred to as autologous tolerogenic dendritic cells (ATDCs), have the capacity to reduce T cell proliferation via a novel mechanism Inhibition of allogenic involving lactate secretion.85 Lactate secreted by ATDCs exerts its secretion of IL-10 immunosuppressive effect by downregulating T cell glycolysis. was observed Functionality Currently, the specific mechanisms triggered by different tolDCs in vivo remain elusive. Inhibition of antigen-specific T cell of IFN-γ. proliferation, increases in FoxP3+ Treg numbers, decreases in proinflammatory Th17 and Th1 cell numbers in both the periphery (spleen and lymph nodes) and CNS, and increased levels of IL-10 Reduced expression of CD83, CD86, and have been described60–65,67–70,75,82,84,86,87 (Table 1). Interestingly, an increased frequency of Bregs was found after MOG-VitD3-tolDC therapy in EAE,64,66 similar to the results of the first phase I clinical trial with genetically modified tolDCs conducted by Giannoukakis et al. in patients with type I diabetes.77 In MS, an increase in IL-10 In vitro studies using tolDCs from MS patients production by PBMCs isolated from treated patients was described.74 In addition, tolDCs can induce the secretion of HLA-DR molecules HLA-DR molecules indoleamine 2,3-dioxygenase (IDO), a potent regulatory enzyme that catalyzes the degradation of tryptophan required for T cell proliferation.88 Altogether, these data indicate that tolDC therapy Phenotype can trigger a complex tolerogenic immune cascade, with anergy or elimination of pathogenic Th1/Th17 cells and induction of Table 1. continued regulatory cells (FoxP3+ Tregs, Tr1 cells, and Bregs), that can reduce From 2015 to 2020 Tolerogenic agent EAE severity, even in mice with established clinical signs of paralysis. Phenotype, function, and stability. Currently, no common bio- markers of tolerogenic function in different types of monocyte- derived tolDCs have been identified.89 Thus, their phenotypic, as b a well as functional, characterization requires comparison with Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1361 mDCs generated in parallel to certify their correct production advantage of the polyclonal preparation of tTregs used in cell before administration to patients. In addition, the stability of therapy. The fact that pTregs arise in the periphery during tolDCs is crucial, and stability can be analyzed in vitro in tolDCs immune responses, mainly from naïve CD4+ T cells, implies that exposed to a proinflammatory milieu to ensure that there is no they are specific to the antigen that triggers the immune conversion of tolerogenicity to immunogenicity.90 No data response. The main regulatory mechanism of pTregs involves regarding the stability of these cells in vivo after their adminis- the secretion of suppressive factors such as IL-10, produced tration to patients have been obtained so far. In this context, it is mainly by Tr1 cells, or TGF-β, produced by Th3 cells.92,94 Currently, expected that the use of cell trackers will provide relevant tTregs are the main subset used in the clinic, but a limited number information to better understand the in vivo mechanism of action of trials using Tr1 cells have also been conducted. of tolDCs.91 Phenotype, function, and stability. Expression of the transcription Clinical trials in MS. To the best of our knowledge, a total of three factor FoxP3 is currently the main marker of Tregs.93 CD127 (the phase I clinical trials using tolDCs for the treatment of MS patients IL7 receptor) is negatively correlated with FoxP3.11,95 Importantly, are ongoing or have recently finished (Table 2). In one dose- mutations that render FoxP3 inactive in humans are responsible escalation phase Ib clinical trial, patients with MS (n = 8) and for immune dysregulation, polyendocrinopathy, enteropathy, and neuromyelitis optica (n = 4) received 3 i.v. injections (50, 100, 150, X-linked inheritance (IPEX). Nevertheless, the phenotype should or 300 × 106 tolDCs in total) as three independent doses always be confirmed with the suppressive function. Throughout administered every 2 weeks. However, the last group did not their development from the progenitor stage to the immune receive the planned doses due to a technical limitation in response, tTregs undergo many changes that affect their activity. obtaining the required number of cells. Clinically, the treatment For example, there is a developmental link between Th17 cells and was safe and well tolerated.74 tTregs, which implies the plasticity of cells of these phenotypes.96 Two coordinated phase I clinical trials in MS patients treated This is important for the stability of Tregs not only during in vitro with autologous VitD3-tolDCs loaded with myelin peptides are manufacturing but also when tTregs are expected to function in currently ongoing simultaneously in Belgium and Spain in the the body. The stability of Tregs is also substantially affected by context of the European H2020 framework.81 Both studies tested epigenetic changes in mature cells. For example, methylation of the safety and tolerability of autologous peptide-loaded VitD3- the Treg-specific demethylated region within the foxp3 gene tolDCs in a dose-escalation study using 5, 10, and 15 × 106 VitD3- significantly impairs suppressive function.97 Additionally, the tolDCs/administration, with the first four of six independent doses inflammatory cytokine milieu can counteract the suppressive administered every 2 weeks and the last two administered every effects of tTregs, which should be considered when therapies with 4 weeks. In addition, exhaustive immunomonitoring is being tTregs are designed. For example, TNF secreted by inflammatory performed (Table 2). cells can abrogate the suppressive effects of tTregs.98 This fragile phenotype should be taken into account during the manufactur- Regulatory T cells ing of Tregs for clinical applications, since trivial factors such as the Since Treg induction is one of the most relevant and consistent time or temperature of expansion might affect the final activity of mechanisms to achieve immunoregulation, cell therapy adminis- the cellular product.99,100 tering Treg cells is one of the most promising strategies that has been extensively investigated worldwide.92 Therapy with Tregs in EAE. While animal studies in many cases assume very early intervention, when there is little or no damage Mechanism of action. Tregs in the body comprise the naturally to the CNS, therapy in humans usually starts when the pathology occurring/thymic (tTreg) and induced/peripheral (pTreg) compart- is very advanced, as the first symptoms often occur only then. ments. The latter compartment is further divided into several Likewise, the burden of inflammation and the location of the subsets, with Tr1 cells and Th3 cells being the main representa- inflammatory process may be different between an animal model tives. While tTregs are specifically designed to regulate the and humans in a trial, and this needs to be considered when immune response from the progenitor stage in the thymus, translating the results into the clinic. One variable to consider is pTregs are generated via the conversion of conventional CD4+ the time when the treatment is administered. In this context, T cells in the periphery during the immune response.93 Both analysis of different studies on Tregs shows that their administra- subsets are efficient regulators of the immune response, but their tion in EAE was performed at different times: prophylactically origins suggest somewhat different activities. Thymic Tregs are (before or with immunization), late prophylactically (before the anergized towards self-antigens in the thymus. These cells migrate first clinical signs appeared), therapeutically (with the first clinical to a site of inflammation and the local lymphoid tissue signs), and late therapeutically (5–8 days after the initial clinical surrounding it and limit the immune response when self- signs). Details of the studies are given in Table 3. Here, we antigens are sensed. In this way, tTregs protect the body from will focus on studies in which Tregs are administered therapeu- possible autoreactivity. This suppression is very precise, occurring tically, as they more closely resemble the potential application of mainly locally via cell-to-cell interactions with other cells that take Tregs in MS. part in the immune response. The main receptor of tTregs is CTLA- Stephens et al. administered Tg4 CD25+CD62Lhi MBP(Ac1-9)- 4 (cytotoxic T lymphocyte antigen 4, CD152), which links to reactive Tregs to Ac1-9 peptide-immunized B10.PL or B10.PL x SJL receptors from the B7 family on APCs and limits the presentation mice at 18 days post immunization (d.p.i.).101 This was the time of of autoantigens. Surface TGF-β and LAG-3 are involved in the remission from the first EAE relapse. While mice that were not suppression of NK cells, and PD-1/PD-L coupling is involved in the treated with Tregs developed more relapses and chronicity of EAE suppression of T cells and B cells. High expression of CD25 (IL2Rα in the subsequent days, the severity of EAE relapse was markedly subunit) allows tTregs to preferentially take up the majority of reduced in Treg-treated mice. The effect was more pronounced in available IL-2, which triggers apoptosis of overactivated effector B10.PL x SJL mice, as Treg-treated mice were nearly disease-free at T cells (conventional T cells, Tconvs) in the surrounding the end of the observation period (100 d.p.i.). These results imply microenvironment. This suppression is not limited to self-antigens, that Treg application could be efficient in MS, even at the later as the interaction with APCs can result in so-called “linked stages of the disease when the vicious cycle of autoimmune suppression”. Specifically, when APCs present tolerized autoanti- reactivity is well established. In another study, Fransson et al. used gens with some alloantigens, tTregs interacting with APCs can also Tregs derived from CD4+ T cells that had been modified with a impose tolerance towards the alloantigen. This is an important lentiviral vector system to express a chimeric antigen receptor Cellular & Molecular Immunology (2021) 18:1353 – 1374 1362 Table 2. Clinical trials using tolDCs treatment in MS patients Type of tolDCs NCT number Purpose Number of Phase Status Antigen Dose Number, route, and Observations Ref patients frequency of administrations 74 Dexamethasone NCT02283671 —Evaluation of safety and tolerability of dexa- 8 MS I Completed (2019) Pool of 7 Dose-escalation A total of 3 i.v. doses Administration of fresh TolDec-EM-NMO tolDCs treatment patients and myelin study with a total every 2 weeks (week antigen-specific tolDCs The —Analysis of changes in the immunological 4 NMO peptidesa of 50, 150 and 0, 2, and 4) treatment was safe and profile patients of MS + 300 × 106 Dexa- well tolerated Increase AQP463- tolDCs secretion of IL-10 after 76 myelin peptides for NMO stimulation at week 12 vs baseline Decrease of memory CD8 + T cells and NK cells by week 12 vs baseline Autologous NCT02903537 —To determine the safety and tolerability of 12active MS I Recruiting Pool of 7 Dose-escalation A total of 6 Administration of On going VitD3-tolDCs TOLERVIT-MS: the intranodal administration of autologous patients myelin study using 5, 10, intranodal doses cryopreserved antigen- Tolerance- tolDCs-VitD3 pulsed with myelin peptides in peptidesa and 15 × 106 VitD3- (cervical lymph specific tolDCs Induction with multiple sclerosis patients tolDCs/injection nodes): the first 4 Dendritic Cells —To select the most appropriate regime for Additional cohort: administrations Treated with the development of future therapeutic trials the highest dose every 2 weeks + the Vitamin-D3 and —To evaluate the preliminary proof of of VitD3-tolDCs last 2 administrations Loaded with concept by clinical and/or radiological activity well-tolerated + every 4 weeks (week Myelin Peptides, in and immunological markers IFN-β treatment 0, 2, 4, 6, 10, and 14) Multiple Sclerosis —To test the effect of the selected VitD3- Patients tolDCs doses combined with IFN-β (n = 3) NCT02618902 MS- —To determine the safety and tolerability of 9 active MS I Recruiting Pool of 7 Dose-escalation A total of 6 Administration of On going tolDCs: A the intranodal administration of autologous patients myelin study using 5, 10, intradermal doses (in cryopreserved antigen- MJ Mansilla et al. Paving the way towards an effective treatment for multiple sclerosis:. . . “Negative” tolDCs-VitD3 pulsed with myelin peptides in peptidesa and 15 × 106 VitD3- the subclavicular specific tolDCs Dendritic Cell- multiple sclerosis patients tolDCs/injection region): the first 4 based Vaccine for —To select the most appropriate regime for administrations the Treatment of the development of future therapeutic trials every 2 weeks + the Multiple Sclerosis: —To evaluate the preliminary proof of last 2 administrations a First-in-human concept by clinical and/or radiological activity every 4 weeks (week Clinical Trial and immunological markers 0, 2, 4, 6, 10, and 14) AQP aquaporine, Dexa dexamethasone, i.v. intravenous, NK natural killer, NMO neuromyelitis optica, VitD3 vitamin D3 a MOG1–20, MOG35–55, MBP13–32, MBP83–99, MBP111–129, MBP146–170, and PLP139–154100,101 Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1363 Ref (CAR) targeting MOG in trans with the FoxP3 gene. They were able Prophylactic treatment: treatment before or at the moment of EAE induction; late-prophylactic treatment: treatment after EAE induction but prior to clinical onset and therapeutic treatment: first dose ATRA all-trans retinoic acid, CAR chimeric antigen receptor, CRP C-reactive protein, d.b.i. days before immunization, d.p.i. days post immunization, inh. inhibition, n.a. not analyzed, NF-M neurofilament-medium, 107 109 110 102 108 103 104 106 111 112 105 to produce Tregs with a strong affinity for MOG and persistent FoxP3 expression.102 Tregs were applied to C57BL/6 mice with Effect on EAE Amelioration Amelioration Amelioration Amelioration Amelioration Amelioration MOG35–55-induced EAE on day 15, i.e., at the time of the EAE peak. Prevention Prevention Prevention Prevention Prevention Prevention The treatment reduced the severity of EAE in the chronic phase, and the treated mice were symptom-free at the end of the observation period (30 d.p.i.). Importantly, Treg-treated mice were reimmunized with MOG35–55 at 30 d.p.i., and only one mouse LAP+, CTLA4+, GITR+, ICOS+, PD1+, OX40+, PLP139–151 (5 µM), TGF- CD62Linter CD103high CD73+ CTLA4+ GITR+ developed any clinical manifestations of EAE. This effect on the Helios−Eos−CD103+ GITR+NRP-1+ CD62L+ clinical score occurred in parallel with inhibition of IL-12 and IFN-γ expression in the CNS. Interestingly, Tregs were applied to mice intranasally (i.n.), and the authors were able to demonstrate their migratory capacity (presumably via olfactory pathways), as they detected the Tregs within the CNS. Niedbala et al. used nitric oxide (NO)-induced Tregs in EAE.103 CD103+, Tim3+ TGF-β Helios, GARP, and LAP They applied Tregs in C57BL/6 mice with MOG35–55-induced EAE at day 10, i.e., when the initial clinical signs appeared. The treatment led to a reduction in the severity of chronic EAE CTLA4 + IL-10 CTLA4 IL-10 throughout the observation period, which ended at 30 d.p.i. This Phenotype CD62Lhigh effect occurred in parallel with the limitation of immune cell CD62Lhi + infiltration into the CNS and a specific reduction in Th17 cell IL-10 n.a. n.a. n.a. numbers within the CNS. Malviya et al. generated transgenic T cells expressing a TCR specific for MOG and neurofilament medium (NF-MT) and used these T cells to treat C57BL/6 mice with EAE induced by MOG35–55 IL-2, rapamycin or PLP178–191.104 These engineered Tregs reduced the severity of MBP85–99, IL-2 PLP 30 µg/ml Propagation IL-2, TGF-β1 EAE when applied at 9 d.p.i. (when clinical symptoms were β, RA, IL-2 evident), and their effect increased towards the peak of EAE. Importantly, these Tregs containing TCRs specific for MOG were IL-2 administrated to mice with the first clinical signs and late-therapeutic: treatment 5–8 days after the initial clinical signs No No No No No equally efficient in EAE induced by MOG35–55 and EAE induced by an unrelated CNS antigen, PLP178–191. Such efficacy in restricting Prophylactic i.v. 1 d.b.i. Therapeutic MOG/NF-M TCR CRP-MBP mice autoimmune reactivity against unrelated CNS antigens, if extra- GFP-Foxp3 x Modification 5B6 TCR Tg polated to humans, would be a beneficial therapeutic property in CAR-MOG 5B6 Tregs Tg4 Tbet- NO-Tregs MS. The engineered Tregs were detected in the CNS of the treated Ob2F3 Tg4 Tg4 mice, and the authors suggested that the therapeutic effect of the No Tregs was achieved within the CNS. To summarize, at least in the EAE model of MS, a single application of Tregs to mice when the disease is well established is Prophylactic i.v. 0 d.p.i. Late- Late-therapeutic i.n. 15 d.p.i. successful and has persistent effects. The cells can be applied Late-prohylactic i.v. 7 d.p.i Prophylactic i.p. 2–3 d.b.i. systemically (i.v. or i.p.)101,103–111 or locally (i.n.).102 Nevertheless, the Therapeutic i.p. 10 d.p.i. Prophylactic i.p. 0 d.p.i. Prophylactic i.v. 1 d.b.i. Prophylactic i.v. 2 d.b.i. Prophylactic i.v. 0 d.b.i. Prophylactic i.v. 1 d.b.i. therapeutic i.v. 18 d.p.i number of cells used for i.n. application was 10 times lower than that used for systemic application, which could be of interest when Administrationa thinking about translation to humans. However, this difference in the number of Tregs required for efficient application could also be i.v. 9 d.p.i a consequence of different backgrounds and preparations. In conclusion, the results obtained in EAE clearly imply that the application of Tregs is a promising approach for the treatment of ongoing CNS autoimmunity, as observed in MS; therefore, clinical 1 × 105 3 × 105 4–5 × trials with Tregs in MS are expected to yield promising results. CD4+CD25+FoxP3+ Tregs in EAE treatment Clinical trials in MS patients. Tregs, mainly tTregs, have been No of Tregs 105 1 × 106 extensively tested in clinical trials for the treatment of not only AD 6 1 × 105 1 × 105 2 × 105 1 × 105 1 × 106 5 × 106 1 × 106 2 × 106 1 × 106 1 × 10 and graft-versus-host disease after bone marrow transplantation as a prophylaxis for solid organ rejection but also for unexpected indications such as thalassemia, muscle dystrophies or amyo- trophic lateral sclerosis.92 We recently accomplished a phase I/IIa PLP178–191 SJL/J MBP87–99 SJL/J MOG35–55 MBP Ac1-9 B10.PL PLP139–151 (B10.PL x trial of autologous CD4+CD25hiCD127-FoxP3+ Tregs administered to RRMS patients, either i.v. or intrathecally (i.t.) (trial registration: MBP Ac1-9 (C57BL/6 × B10.PL)F1 MOG35–55 or PLP178–191 C57BL/6 EudraCT: 2014-004320-22.94 The therapy proved to be safe. MOG35–55 (Tg(HLA-DR15)#Lfug) Although very preliminary, the results also suggested that intrathecal administration was more effective than intravenous o.e. over-expression administration. Experiments with adoptive transfer of Tregs (SJL/J x C57BL/6) F1 MBP Ac1-9 C57BL/6 C57BL/6 MOG35–55 C57BL/6 MOG35–55 suggested the good safety profile of Tregs administered via both PLP139–151 SJL/J MBP Ac1-9 Tg4 PLP139-151 SJL/J tested routes.94 Table 3. Dose, timing, and route of administration. To translate a treat- SJL) F1 ment from early animal use into late human pathology, the dose EAE a and route of administration of the cells should be adjusted to Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1364 address the level of inflammation and follow the progression of Mesenchymal stem cells the disease. We tried to address these issues with the use of two MSCs are nonhematopoietic multipotent and self-renewing routes of Treg administration. Patients treated i.v. received 40 × progenitor cells with the potential to differentiate into different 106 Tregs/kg b.w., which in our experience is a relatively high lineages under specific conditions. They were described for the dose. Within this arm of the trial, we tried to address the first time in 1968 by Friedenstein et al. as an adherent fibroblast- hypothesis that systemic dysregulation between Tconvs and Tregs like population in the bone marrow that was able to differentiate triggers the disease and relapse.112 The results suggest that we into adipocytes, chondrocytes and osteocytes.120 In 1991, Caplan were, at least partially, ‘too late’, as half of the treated patients et al. named these cells “mesenchymal stem cells”.121 The authors experienced relapse and progression of disease, as confirmed with demonstrated that MSCs are involved in bone and cartilage MRI. This somewhat confirms that the clinical onset of disease may turnover and examined how surrounding conditions play a crucial occur very late in the pathogenesis of MS, when the core of the role in their differentiation. Therefore, MSCs were postulated to be process has already moved from the periphery to the CNS. The a novel therapeutic strategy for self-cell repair.121 initiation of RRMS occurs somewhere in the peripheral lymphoid MSCs can be isolated from various tissues, including the bone system with the presentation of myelin peptides and the marrow, adipose tissue, placenta, umbilical cord, fetal liver, generation of autoaggressive Tconvs, as in EAE, which is not muscle, and lung. Among these tissues, bone marrow and adequately controlled by Tregs.113 However, Tconvs very quickly adipose tissue are the most widely used sources of MSCs for traffic to the CNS, destroy the BBB, attack myelin sheaths, and therapeutic purposes. According to the Mesenchymal and Tissue cause the development of lesions. Hence, the systemic adminis- Stem Cell Committee of the International Society for Cellular tration of drugs is of limited value when symptoms have already Therapies,122 the cells must be plastic adherent and able to occurred. The results of immunophenotyping in our trial seem to differentiate into osteoblasts, adipocytes and chondroblasts, and confirm such overactivity of Tconvs, which were mainly of an their phenotype must be characterized by flow cytometry as ‘experienced’ memory phenotype in all patients. At the same time, CD105+ CD73+ CD90+ CD45− CD34− CD14−/CD11b− CD79a−/ the majority of Tregs were naïve, confirming their relative CD19− HLA-class II−. MSCs can be easily cultured and expanded inactivity. Surprisingly, MS patients exhibited an extraordinarily ex vivo and have several properties, such as functions in tissue repair high percentage of peripheral Helios−FoxP3+ Tregs (20–30% of all and homeostasis maintenance, immunomodulatory properties and FoxP3+ Tregs). These cells arise during the immune response, low immunogenicity. which suggests a history of long/massive immune activation in the Due to their potential as an immunomodulatory and regen- periphery, with ineffective regulation of this process in MS. There erative therapy, MSCs have been considered an optimal candidate have been reports that Tregs in RRMS patients follow autoreactive cellular therapy for inflammatory and neurodegenerative diseases Tconvs and, attracted by inflammation, move quickly to the CNS, of the CNS, such as MS. accumulating in the CSF. Moreover, remission occurs only when Tregs have accumulated in the CSF.114 Preclinical studies. MSC-based cell therapy is the most investi- This finding justifies the second arm of our trial, in which gated and has been examined in numerous in vitro and in vivo patients received Tregs i.t. The dose could be lower (1.0 × 106 studies (EAE). In vivo treatment of EAE mice with bone marrow- Tregs) in these patients, as 100% of the cells were delivered derived MSCs (BM-MSCs) using several routes of administration behind the BBB. The patients did not experience relapse, and MRI (i.v., intraventricular (i.v.t.), and i.p.) has shown clinical amelioration confirmed stable nonprogressing lesions in the CNS of these of EAE severity, with reduced inflammatory infiltration, demyelina- patients, which proved that this approach should be further tested tion, and axonal damage. Of note, this beneficial effect was not in future trials.94 found when BM-MSCs were infused during the chronic phase of the disease.123 Novel Treg therapies. The approach based on engineered Tregs It has been widely reported that MSC therapy in EAE exerts an came from cancer studies in which the receptor specific for a important immunomodulatory effect and, to a lesser extent, a particular molecule expressed on cancer cells was inserted into neuroprotective effect that results in axonal and neuronal Tconvs using a vector (chimeric antigen receptor T (CAR-T) cells). protection through the release of antiapoptotic, antioxidant, and This approach allows CAR-T cells to identify and kill cancer neurotrophic factors (systematically reviewed in124). Induction of cells in a very specific and efficient way. Several drugs based on Tregs, TGF-β1, and IL-10 mRNA in the spleen and lymph nodes of this therapy, i.e., axicabtagene ciloleucel (Yescarta, Gilead) and treated mice was the main immunological mechanism involved in tisagenlecleucel (Kymriah, Novartis), are already routinely used. the induction of peripheral tolerance following MSC transplanta- The first CAR-Tregs were constructed with specificity towards allo- tion in EAE mice. HLA to quench the possible rejection of an allotransplant.112,115,116 The challenge is much higher in ADs such as MS, in which the Mechanisms of action of MSCs. The specific mechanisms that target antigens are not as obvious. In the majority of such mediate the clinical benefits of BM-MSCs likely involve a diseases, the complete list of autoantigens is not known.117 combination of peripheral autoimmune modulation and the Moreover, it is possible that the target antigens evolve with the induction of CNS tissue protection. MSCs have four main progression of the disease due to epitope spread.114 The properties: (1) migration capacity, (2) immunomodulation, (3) complexity of the response is also caused by the fact that the differentiation and neuroregeneration, and (4) secretion of soluble same epitopes can trigger responses in both Tregs and Tconvs, factors. and the final outcome depends on which subset prevails.118 After their systemic administration, MSCs can migrate to and Nevertheless, there have been attempts to create Tregs with engraft in inflamed locations, exerting a local effect. Injured cells engineered TCRs to direct them to particular sites and protect and immune cells involved in the immune response regulate MSC particular organs from autoimmune attack.119 These attempts for migration through the secretion of a broad range of signals, such MS are at the EAE stage. There have been reports on CAR-T cells as growth factors and chemokines. In vitro studies proved that with specificity towards MOG manufactured from Tconvs and MSC migration is regulated by receptors such as platelet-derived directed toward a regulatory function through foxP3 gene growth factor and insulin-like growth factor 1 and chemokine delivery. This cellular product trafficked to the brain and exerted receptors such as CCR2, CCR3, CCR4, and CCL5.125 Studies in suppressive activity.102 More recently, human Tregs that had a animal models have shown that MSCs can roll and tether to the transgenic TCR specific for MBP and had proven immunosuppres- endothelium, crossing the BBB through the VLA-4/VCAM-1 sive activity were described.111 interaction. Moreover, MMPs play an important role in the transit Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1365 of MSCs through the endothelial membrane. Interestingly, it is differentiation into neurons and oligodendrocytes.139–141 These important to keep in mind that culture conditions during MSC promising results encourage the use of MSCs in MS. ex vivo expansion can affect the expression of some receptors, such as VLA-4 and MMP, thus altering the migration capacity of Clinical trials in MS. The number of clinical trials investigating MSCs.125–127 MSC treatment for RRMS and progressive MS patients has The immunomodulatory properties of MSCs can be exerted via increased quickly over the past two decades. Currently, more cell-to-cell interactions or paracrine effects. On T cells, MSCs inhibit than twenty clinical trials are registered at ClinicalTrials.gov (see T cell proliferation via a mechanism independent of apoptosis Table 4). Thus far, the results have shown that MSC therapy in MS induction.128 After coculture with T cells, MSCs decreased the Th1 patients is safe, with no relevant side effects.142–151 Due to response and induced a switch towards the Th2 response (a variability in the protocols used (different types of MSCs, routes of decrease in IFN-γ secretion and an increase in IL-4 secretion).129 In administration, and doses) and the limited number of patients addition, MSCs induce the expansion of Treg subsets, increasing enrolled in each trial, most studies have been unable to draw Foxp3 and CD25 expression. These Treg cells express TGF-β1 and conclusions about the efficacy of these treatments. Nevertheless, PGE2.129,130 some trials have reported beneficial effects of MSCs through a Regarding B cells, it has been reported that PDL1 inhibits their decrease in relapse rate or disability.143,146,147 proliferation in murine cells and arrests the cell cycle in human To establish a consensus protocol for the use of MSCs for cells.131,132 Chemotactic properties are also affected by soluble treating MS patients, in 2010, a group of experts created the factors secreted by MSCs. A decrease in the expression of some International Mesenchymal Stem Cell Transplantation Study Group chemokine receptors, such as CXCR4, CXCR5, and CCR7, was (IMSCTSG). As a result, a large multicenter randomized, double- observed in B cells, together with a decrease in CXCL12 (a CXCR4 blind, crossover phase I/II clinical trial was initiated to analyze the ligand) and CXCL13 (a CXCR5 ligand) expression in MSCs. In safety and efficacy of a single i.v. dose of autologous BM-derived contrast, B cell costimulatory molecule expression and cytokine MSCs (Mesenchymal StEm cells for Multiple Sclerosis: MESEMS production were unaltered.132 Furthermore, suppression of B cell trial; NCT02403947).142 The results of this trial have not yet been terminal differentiation by soluble factors secreted by MSCs, such published. as MCP-1 or IL-6, was also reported in C57BL/6 mice.133 Recently, Sarkar et al. reported relevant data about the MSC MSCs induced the inhibition of NK cell proliferation when secretome that must be considered in the context of treatment cocultured in IL-15-supplemented medium via both cell-to-cell with MSCs. These authors found that chronic inflammatory stress contact and soluble factors, such as TGF-β1 and PGE2. Moreover, in MS patients limits MSC functionality by altering the MSC under these conditions, a decrease in the production of IFN-γ and secretome.152 Moreover, other factors, such as aging and the IL-10, a decrease in the surface expression of CD56 in NK cells in vitro expansion of MSCs, also exert detrimental effects on their (although no changes were observed in the ratio of the CD56dim functionality.152 Therefore, an in-depth analysis of the autologous and CD56bright subsets), and lower cytotoxicity against HLA class I MSC secretome should be considered as a quality control measure targets were found.129,134,135 before administrating MSC therapy. MSCs can inhibit the maturation of DCs, resulting in a decrease Regarding the effect of MSC treatment on the immune in their capacity to activate alloreactive T cells. Furthermore, MSCs response, only a few clinical trials have performed immune can inhibit TFN-α release by DCs, resulting in a tolerogenic state. monitoring to elucidate the mechanism of action of this therapy. Moreover, it has been postulated that PGE2 secreted by MSCs Llufriu et al. reported a decrease (although it was not statistically plays an important role in promoting the Th2 cell response, acting significant) in Th1 and Th17 cells and an increase in Breg cells, as against Th1 cells, during the DC-induced Th cell response.129,136 analyzed by the expression of IFN-γ, IL-17, and IL-10, respectively, As previously mentioned, MSCs release multiple soluble and using intracellular cytokine staining.143 Karussis et al. described an encapsulated molecules, such as growth factors, cytokines, and increase in Treg cells (identified as CD4+CD25+), together with a chemokines (secretome), which can exert a biological effect in decrease in the expression of CD83 and CD86 in DCs and CD40 in tissues. The conditioned medium of MSCs was shown to have a activated cells. Moreover, they performed functional analysis of beneficial effect in damaged tissue in the liver and myocardium. the T cell response, which showed a decrease in proliferation in Among these molecules are PGE2, which has anti-inflammatory and response to phytohemagglutinin (PHA).147 Finally, a clinical trial of antiproliferative effects; IL-10 and IL-1 receptor antagonist (which the administration of autologous BM-MSCs is currently ongoing in have anti-inflammatory effects); and TGF-β1 and hepatocyte growth Jordan and will analyze the levels of IgG, IgA, and IgM and factor (which suppress T cell proliferation).126,127 Moreover, it was complement factors C3 and C4 in treated patients (NCT03069170). recently reported that exosomes released by MSCs can cross the BBB due to their small size and transfer bioactive molecules. In this Hematopoietic stem-cell transplantation regard, MSC-derived exosomes polarized microglial cells mainly into Hematopoietic stem-cell transplantation (HSCT) was established the M2 anti-inflammatory phenotype, promoting a shift to an anti- for the treatment of hematological malignancies such as multiple inflammatory profile and, consequently, reducing the clinical myeloma and leukemias due to the capacity of hematopoietic symptoms of EAE in rats.137 stem cells to differentiate into all hematopoietic cell types.153 One important characteristic of MSCs is their ability to Surprisingly, treated patients who had concomitant ADs experi- differentiate towards both mesenchymal and ectodermal cells, such enced amelioration of their clinical symptoms following HSCT.154 as neurons, astrocytes, and oligodendrocytes. When analyzing the As a result, high-dose immunosuppression followed by auto- neuroprotective and regenerative functions of MSCs in MS, different logous HSCT (aHSCT) has been investigated for patients with experiments demonstrated that after i.v. administration of MSCs, severe MS, with the rationale of this therapy based on a “reset” of they spread and homed preferentially to inflamed tissues, to the the immune repertoire to eliminate autoreactive T and B cells; CNS in the case of EAE, to induce neuronal axon protection and the thus, subsequent aHSCT would allow reconstitution with the hope regeneration of damaged areas.138,139 Unfortunately, MSCs have not that a new and more self-tolerant immune system is developed.155 been demonstrated to transdifferentiate into neuronal cells. Their HSCT is carried out through different steps: mobilization, protective function is likely related to the secretion of antiapoptotic, harvesting, ablative conditioning, and transplantation of aHSCs.156 anti-inflammatory, and neurotropic factors (through the activation of First, HSCs are collected from the peripheral blood of patients after astroglial cells to secrete neurotrophins such as BDNF, glial cell- the mobilization of HSCs from bone marrow using treatment with derived neurotrophic factor, and nerve growth factor),139 as well as granulocyte colony-stimulating factor (G-CSF) or GM-CSF with the probable recruitment of local progenitor cells for subsequent cyclophosphamide to prevent possible MS relapse or worsening of Cellular & Molecular Immunology (2021) 18:1353 – 1374 1366 Table 4. Clinical trials using mesenchymal stem cells in MS patients Source Years Country Phase NCT number Type of study MS type n EDSS Route of Dose of MSCss Number of Immunological findings Ref of cells administ administrations 142 aMSCs 2013 Italy I/II NCT01854957 Randomized, Active 185 2.5–6.5 i.v. A dose of 1–2 × Single Unreported aMSCs 2013–2016 UK I/II NCT01606215 double-blind, PPMS, 106 MSCs/kg placebo- SPMS, RRMS body weight aMSCs 2014–2018 Canada II NCT02239393 compared aBM-MSCs 2015–2018 France I/II NCT02403947 aBM-MSCs 2013–2016 Spain I/II NCT02035514 aADMSCs 2012–2015 Sweden I/II NCT01730547 aMSCs 2011–2018 Iran I/II NCT01377870 Randomized, RRMS, 22 3–6.5 i.v. Unreported Single Unreported Unpublished double-blind, SPMS, RRMS placebo- compared 143 aMSCs 2010–2014 Spain II NCT01228266 Randomized, RRMS 9 3–6.5 i.v A dose of 1–2 × Single Non significative decrease of Th1 double-blind, 106 MSCs/kg and Th17 cells; increase of Breg placebo- body weight controlled, crossover 144 aMSCs 2011–2016 USA I NCT00813969 Open-label RRMS, SPMS 24 3–6.5 i.v. A dose of 2 × 106 Single Unreported MSCs/kg body weight aBM-MSCs 2014–2016 Israel II NCT02166021 Randomized, PPMS, SPMS 48 3.5–6.5 i.t. and i.v. 10 × 106/mL to Double Unreported Unpublished double-blind, 15 × 106/mL placebo- compared 145,146 MJ Mansilla et al. Paving the way towards an effective treatment for multiple sclerosis:. . . aBM-MSCs 2006–2011 UK I/II NCT00395200 Open-label SPMS 10 2–6.5 i.v. A dose of 2 × 106 Single Unreported MSCs/kg body weight aBM-MSCs 2013–2017 Jordan II NCT01895439 Open-label MS 13 ≤6 i.t. Unreported Unreported Unreported Unpublished aBM-MSCs 2006–2009 Israel I/II NCT00781872 Open-safety Active MS 15 MS 4–8 i.t. and i.v. 60 × 106 i.t. (n = Single Increase of Treg (CD4+ CD25+) and 147 clinical trial and ALS and 19 ALS 34) and 25 × 106 decrease of CD83+ and CD86+ on i.v. (n = 14) DC and activated CD40+ cells. Decrease of in vitro lymphocytes proliferation to PHA aBM-MSCs 2017 Jordan I NCT03069170 Open-label RRMS 50 3–6.5 i.t. and i.v. Unreported Single Recruiting IgG, IgA, and IgM levels Unpublished and complement factors C3 and C4 will be analysed aBM-MSCs 2015–2017 India I/II NCT02418351 Open-label RRMS 69 3.5–6.5 i.t. and i.v. 50 × 106 BM-MNC Single Unreported Unpublished (i.v.) and 100 × 106 BM-MNCs i.t. (when associated with CCSVI) alloUCMSCs 2015–2017 Trinidad I/II NCT02418325 Open-label RRMS 69 3.5–6.5 i.t. and i.v. 50 × 106 UC-MSCs Single Unreported Unpublished and Tobago (i.v.) and 100 × 106 UC-MSCs i.t. (when associated with CCSVI) aBM-MSCs 2015–2018 Spain I/II NCT02495766 Quadruple- RRMS 8 <6.5 i.v. Unreported Single Unreported Unpublished blind, placebo- and SPMS controlled study 148 aBM-MSCs 2014–2018 UK II NCT01815632 Double-blind, SPMS 80 4–6 i.v. 500–600 mL single Unreported placebo- and PPMS bone marrow controlled study 149 aBM-MSCs 2013–2018 UK II NCT01932593 Double-blind, SPMS 6 4–6 i.v. 500–600 mL Reinfusion Unreported placebo- and PPMS bone marrow (extension of controlled study NCT01815632) Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1367 Unpublished Unpublished Unpublished Unpublished clinical symptoms as a result of G-CSF or GM-CSF administra- aADMSCs autologous adipose-derived MSCs, aASVF autologous adipose stromal vascular fraction, aBM-MSCs autologous bone marrow-derived MSCs, alloUCMSCs allogenic umbilical cord-derived MSCs, ALS amyotrophic lateral sclerosis, aMSCs autologous MSCs, i.v. intravenous, i.t. intrathecal, MS multiple sclerosis, MSCs mesenchymal stem cells, PPMS primary-progressive MS, RRMS relapsing-remitting MS, SPMS tion.156 After 4 or 5 days, cells are harvested by leukapheresis and cryopreserved. Additionally, an HSC purification step is performed Ref 150 151 by CD34 positive selection to eliminate possible autoreactive lymphocytes. Then, the patient receives ablative conditioning to eradicate autoreactive cells. Different regimens of immune ablative conditioning are used based on the intensity of ablation using different chemotherapeutics and immunosuppressive drugs. High-intensity ablative regimens involve a high i.v. dose Immunological findings of immunosuppressive therapy and are associated with high toxicity and, in a small percentage of patients, mortality.156,157 In contrast, low-intensity regimens are nonmyeloablative and Unreported Unreported Unreported Unreported Unreported Unreported produce fewer adverse effects but may be associated with the early reappearance of MS disease activity post infusion. Hence, intermediate-intensity ablative regimens, referred to as BEAM (BCNU (or carmustine), etoposide, cytosine arabinoside, mel- phalan) or modified BEAM, are becoming more accepted.157 administrations Following ablative conditioning, cryopreserved HSCs are thawed Unreported Unreported Number of and reinfused into the patient. 7 doses Low-dose (1 × 106 Single Single Single Despite the promising positive results obtained using aHSCT (see below), some aHSCT-related risks must be considered: as stated above, the risk of transplant-related mortality, which is dose (4 × 106 cells/ cells/kg) or high- Infusions of 20 × most severe in the first 100 days after transplantation, and the Dose of MSCss 150 × 106 cells 106 UCMSCs increased susceptibility to infection as a result of the accompany- over 7 days Unreported Unreported Unreported Unreported Unreported Unreported Unreported ing chemotherapeutic immunosuppressive regimen. In addition, long-term side effects include the development of secondary kg) autoimmune problems and/or fertility issues.158 Interestingly, aHSCT has dramatically improved over the years, showing a 0.3% treatment-related mortality rate since 2005.157 Route of administ i.v. Unreported i.v. i.v. i.t. Clinical trials in MS. Since the end of the 1990s, several clinical trials evaluating the safety and efficacy of aHSCT using different conditioning regimens have been performed. Published results have shown that aHSCT can inhibit MS disease activity for 4–5 5.5–9 2–5.5 EDSS ≤7 years in 70–80% of patients. Interestingly, this rate is higher than that achieved with any other therapy for MS. The results placebo, 10 and 9 high- low-dose were better in young patients with inflammatory-active RRMS 30 [11 (reviewed in157). dose] MS patients 221 MS patients 20 20 MS patients 60 To go one step further in examining aHSCT, currently, a phase n MS patients 2 III randomized clinical trial in RRMS patients with significant Progressive and NMO inflammatory disease activity is being conducted to compare MS type the efficacy of aHSCT using a nonablative conditioning regimen SPMS MS with that of alemtuzumab (anti-CD52), which is considered the most effective available drug for RRMS (NCT03477500). Impor- controlled study tantly, if the results indicate improved efficacy of aHSCT over Type of study NCT03326505 Randomized, NCT01056471 Triple-blind, single-blind alemtuzumab, aHSCT will likely be approved as a part of the NCT02326935 Open-label NCT02157064 Open-label NCT02034188 Open-label NCT01364246 Open-label placebo- current standard treatment recommendations for a significant proportion of RRMS patients (except in the case of Sweden, where aHSCT has been already approved). In the same way, another phase III clinical trial to analyze the efficacy of aHSCT Phase NCT number (using high-dose myeloablative conditioning) in comparison with the best available therapy (BAT) in treatment-resistant RRMS is currently ongoing (BEAT-MS trial, NCT04047628). This multicenter, randomized, blinded study was conducted in a total of 156 RRMS patients distributed to each treatment arm at a I/II I/II I/II I/II II ratio of 1:1. Treatments included in the BAT arm are natalizumab I (anti-CD49d), alemtuzumab (anti-CD52), ocrelizumab (anti- CD20) and rituximab (anti-CD20). Relapse-free survival up to 3 Country 2014–2018 German alloUCMSCs 2014–2017 Panama Jordan alloUCMSCs 2010–2014 China 2010–2015 Spain years will be determined and used to compare the efficacy of 2014–2018 USA secondary-progressive MS aHSCT with that of the other treatments. Table 4. continued T cell vaccination Years alloUCMSCs 2017 Autologous T cell vaccination (TCV) involves collecting and expanding myelin-reactive T cells from MS patients and reinfusing them after their attenuation by irradiation. The rationale is that, as aADMSCs aADMSCs a result of this process, the immune system will attack pathogenic of cells Source aASVF myelin-specific T cells, causing their deletion or inactivation while maintaining protective immunity. Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1368 The first study using attenuated MBP-reactive T lymphocytes kidney transplantation in which different types of regulatory cells, was conducted in Lewis rats in 1981.159 The adoptive transfer of including tolDCs, were combined with conventional immunosup- MBP-reactive T cells in rats induced EAE disease onset. Interest- pressive drugs reducing the proliferative response of lymphocytes ingly, administration of the same MBP-T cells attenuated by by inhibiting IL-2 signaling (basiliximab and tacrolimus), blocking irradiation before adoptive induction resulted in disease preven- de novo synthesis of guanosine nucleotides (mycophenolate tion. Increased interest in TCV led to the initiation of multiple trials mofetil) or using a feedback mechanism to control inflammation in MS patients in the late 1990s. The results from phase I pilot and the immune response to steroids. Data from the trial showed studies demonstrated that TCV treatment was safe and well the safety and feasibility of combined therapy. Interestingly, a tolerated160–165 (Table 5) and depleted MBP-reactive T cells after decrease in infectious complications was reported in the group only 2 administrations.160 Interestingly, a correlation between administered combined therapy.100 In this context and to move MBP-reactive T cell depletion and a 40% reduction in the relapse one step forward in the clinical application of tolDCs in MS, our rate was found in RRMS patients. Nevertheless, no relevant group investigated the effect of tolDCs combined with IFN-β. We reduction in EDSS score was observed in the RRMS patients. In found that the anti-inflammatory and immunomodulatory proper- contrast, a slight increase in EDSS score after 2 years was reported ties of IFN-β combined with VitD3-tolDCs induced a reduction in in SPMS patients166 (Table 5). Th1 and Th17 cells, favoring a more potent antigen-specific To improve TCV outcomes in MS patients, T lymphocytes isolated regulatory effect of VitD3-tolDCs both in vivo (EAE model) and from CSF were used to develop CSF-derived T cell lines against in vitro in cultures of peripheral blood cells from MS patients myelin peptides, since CSF was thought to contain infiltrating (Quirant-Sánchez et al. unpublished data). pathogenic lymphocytes relevant to the disease process due to the proximity of these lymphocytes to the CNS. Data from two pilot Tregs with immunomodulatory/immunosuppressive drugs studies indicated that the administration of attenuated autoreactive The relationship between Tregs and IL-2 is probably the most CSF-derived T cell clones was feasible and safe, and no adverse explored area in MS research, with the longest history of clinical effects were observed. Phase II studies with a large number of MS trials.172 Tregs have high expression of IL-2R and are highly patients are required to evaluate the clinical and radiological efficacy dependent on IL-2; therefore, this cytokine or its muteins have of CSF-derived TCV.167,168 been used alone or in combination with Tregs in many conditions, To improve clinical remission, a new strategy using multivalent such as GvHD, transplantation or T1D.96,97 Other approaches TCV was developed using immune-dominant epitope sequences of include the administration of rapamycin as an immunosuppressive MBP, MOG and PLP. A phase I clinical trial was conducted by Achiron agent able to induce Tregs and tolDCs98,99 or adjuvant therapy et al. using attenuated T cell lines specific for different MBP and/or with Tregs added to standard immunosuppression in organ MOG peptides.169 The results of the trial showed clinical as well as transplantation.100 Our team has been testing Tregs combined radiological benefits with no adverse effects in RRMS patients who with anti-CD20 antibody in T1D.173 did not respond to disease-modifying treatments.169 In addition, multivalent TCV allows personalized therapy following prescreening TolDCs in combination with Tregs for myelin reactivity. In this regard, Tcelna (Imilecleucel-T, previously The combination of tolDC and Treg cell therapy has been known as Tovaxin), a TCV composed of autologous preselected proposed for the treatment of ADs since these cells can interact T cells reactive against up to six immunodominant peptides derived to stabilize, maintain and potentiate their tolerogenic effects.174 from MBP, MOG and PLP, was shown to be safe and well tolerated in Although coadministration or serial administration of antigen- a phase II clinical trial; however, its clinical and radiological efficacy specific tolDCs and Tregs a priori does not seem to be feasible, a was not demonstrated (TERMS trial: Tovaxin for Early Relapsing single leukapheresis could provide enough monocytes to Multiple Sclerosis, NCT00245622,170). An extension study (OLTERMS generate tolDCs and lymphocytes for autologous Treg expan- trial: Open label extension of TERMS study, NCT00595920) sion.174 However, to date, no studies investigating the efficacy of was initiated to further evaluate the clinical efficacy of Tcelna/ this combined therapy have been reported. Tovaxin. However, the study was terminated because it did not accomplish the predefined primary endpoint of reducing brain TolDCs/Tregs in combination with MSCs atrophy or the secondary endpoint of decreasing disability A different combination therapy approach for ADs is the progression (NCT00595920). administration of antigen-specific cells, such as tolDCs or Tregs, with MSCs. In this context, synergistic suppression of autoimmune arthritis was reported when collagen-induced arthritis (CIA) mice COMBINED THERAPIES were treated with RelB-silenced tolDCs and MSCs. Combined Evidence from phase I clinical trials conducted in type 1 diabetes, therapy was able to inhibit disease progression, decrease the rheumatoid arthritis, Crohn’s disease and MS patients has clinical symptoms of CIA and reduce joint damage. Immunological demonstrated that tolDCs, Tregs, TCV, MSCs, and HSCT are safe studies revealed inhibition of the collagen T cell response and a and well tolerated. However, due to the different cell types and shift towards an anti-inflammatory profile, although the most mechanisms involved in the maintenance of immune tolerance potent synergistic effect elicited by RelB-silenced tolDC and MSC and the difficulty of establishing an optimal dose, route, and therapy was a strong reduction in Th17 cells.175 frequency of administration, it has been postulated that a Another example was published by Lee et al., who used a combined therapy of antigen-specific cells with conventional murine model of acute GVD (aGVHD) to determine the efficacy immunomodulatory drugs is most likely necessary to potentiate of combined Treg and MSC therapy.176 Researchers have shown their beneficial effects and restore immune homeostasis.54 that the adoptive transfer of donor-derived MSCs and Tregs reduces the severity of aGVHD by controlling Th1 and Th17 TolDCs with immunomodulatory/immunosuppressive drugs responses (related to the function of Tregs), accompanied by A single infusion of donor-derived VitD3+IL-10-tolDCs before increased long-term survival of transferred Tregs and induction transplantation in combination with CD28 costimulatory signal of endogenous Treg repopulation in target organs (related to blockade using the fusion protein CTLA-4 Ig (abatacept) and MSC function).176 rapamycin significantly prolonged allograft survival in nonhuman In conclusion, all these data suggest that combination therapies primates by attenuating donor‐reactive memory T cells.171 have the advantage of increasing the possible clinical effective- More recently, the results of the ONE Study, an analysis of seven ness of antigen-specific cell-based tolerogenic therapies and will non-randomized, single-arm, phase I/IIa trials in living donor contribute to their optimal application in the future. Cellular & Molecular Immunology (2021) 18:1353 – 1374 Table 5. Clinical trials using T cell vaccination in MS patients Vaccination type Vaccine composition NCT number MS patients Number of Phase Status Study design Dose and number of Route of Clinical outcome TCV Center Ref MS injections administration patients 160 T cell Autologous MBP- Unregistered RRMS (n = 3) 6 Phase I Completed Non-randomized, A total of 3 injections of s.c. Decreased frequency of MBP- LUC, vaccination reactive T cell clones PPMS (n = 1) open label 2–3 irradiated MBP- reactive T cells after the 2nd Diepenbeek, (predominant SPMS (n = 2) specific T cell clones administration Remained Belgium reactivity pattern to (1 × 107 to 1.5 × 107 undetectable in 5 of 6 MBP 84–102 and cells/clone) patients at the end of 143–168 peptides) the study 161 T cell Autologous MBP- Unregistered RRMS (n = 5) 8 Phase I Completed Non-randomized, A total of 3 injections of s.c. Moderate clinical LUC, vaccination reactive T cell clones PPMS (n = 1) open label 2–4 irradiated MBP- improvement by reduction of Diepenbeek, SPMS (n = 2) specific T cell clones relapse rate, and brain lesions Belgium (15 × 106 cells/clone) compared to paired controls every 2–4 months 166 T cell Autologous MBP- Unregistered RRMS (n = 28) 54 Phase II Completed Non-randomized, A total of 3 injections s.c. Reduction of 40% in ARR MRI Baylor College vaccination reactive T cell clones SPMS (n = 26) open label every 2 months of 2–4 lesions and EDDS remained of Medicine, both whole MBP and irradiated MBP-T cell stable. Houston, MBP peptides clones (30 × 106–60 × 106 TX, USA (83–99 and 151–170) cells/clone) 169 T cell Autologous specific NCT00220428 RRMS with 20 Phase I Phase II Completed Randomized, A total of 3 injections in 6- s.c. Treatment was safe and no Sheba Medical Cellular & Molecular Immunology (2021) 18:1353 – 1374 vaccination T-cell lines against aggressive open label to 8-week intervals of up serious adverse events were Center Ramat MBP((83–99, 87–110, disease course to 1.5 × 107 cells of each observed. Decreased ARR, Gan, Israel 151–170) and/or and failure to myelin-reactive T cell line number and volume of active MOG(G respond to DMT (not exceeding 6 × 107 MRI lesions, as well as (6–26, 34–56) total cells) reduction in T2 lesion burden (quantitative MRI analysis) 165 T cell Autologous myelin- NCT00228228 Patients with 80 Phase I Phase II Unknown Randomized, Dose: 1–1.5 × 107 cells/ Unknown To evaluate the treatment Sheba Medical vaccination reactive T-cell lines probable MS double-blind line, up to 5 lines Multiple effects at the onset of disease, Center Ramat against MBP, PLP, within up to injections: a total of 5 in patients with probable MS Gan, Israel and MOG peptides 3 months after injections, 3 every within up to 3 months after the first 6 weeks and 2 boosts on the first clinical attack, in clinical attack weeks 24 and 28 order to evaluate whether early treatment can prevent the second attack (conversion to definite MS). 162 T cell Whole bovine Unregistered SPMS (n = 4) Phase I, Phase I Phase II Phase I Non-randomized, A total of 3 s.c. TCV using autologous USC, Los vaccination myelin selected with EDSS > = n=4 completed open label administrations every irradiated bovine myelin- Angeles, USA T cells 6.5 SPMS (n = Phase II, 3 months of 40 × 106 reactive T cells depletes 80) n = 80 irradiated T cells circulating MBP-specific T cells MJ Mansilla et al. in patients 168 CSF-derived T CSF-derived T cell Unregistered PPMS (n = 4) 4 Phase I Completed Non-randomized, Single, twice or 3 s.c. Administration of attenuated Harvard Medical cell vaccination lines reactive to open label administrations of 4 × 106 autoreactive CSF-derived T School, myelin peptides irradiated CSF-derived T cell clones was feasible, and Boston, USA cell clones no adverse effects were observed 167 CSF-derived T CD4+ T-cells derived Unregistered RRMS (n = 4) 5 Phase I Completed Non-randomized, A total of 3 s.c. Vaccinations were well LUC, cell vaccination from CSF SPMS (n = 1) open label administrations every tolerated; no adverse effects Diepenbeek, 2 months of 10 × 106 were found. Preliminary Belgium irradiated CSF-derived efficacy data exhibited clinical T cells vaccines stability of patients and reduced EDSS with no relapses during or after treatment 163 T cell T cells lines reactive NCT01448252 Relapsing- 26 MS Phase I Phase II Completed Randomized Dose: 10–30 × 106 s.c. Results demonstrate the Dept of vaccination against 9 myelin progressive MS patients: double-blind, irradiated autologous T- feasibility and safety of the Neurology, peptides of MBP patients with n = 19 placebo-controlled cells Multiple procedure and showed Hadassah Ein- (84–102, 143–168), EDSS from patients administrations: 4 clinical improvements Kerem MOG (1–22, 34–56, 3.0 to 7.0 treated vaccinations on days 1, (decrease of EDSS and high Jerusalem, Israel 64–86, 74–96) and with TCV 30, 90, and 180 frequency of relapse-free PLP (41–58, 184–199, and n = 7 patients during the year of the 190–209) patients study in TCV treated patients treated compared to placebo group) with MRI parameters did not placebo change significantly Paving the way towards an effective treatment for multiple sclerosis:. . . 1369 1370 Table 5. continued Vaccination type Vaccine composition NCT number MS patients Number of Phase Status Study design Dose and number of Route of Clinical outcome TCV Center Ref MS injections administration patients 164 T cell Autologous T cells NCT00587691 RRMS (n = 9) 16 Phase I Phase II Completed Non-randomized, Multiple administrations s.c. Tovaxin® was safe and well Opexa vaccination against 6 SPMS (n = 7) open label, dose- of 3 doses were tested. tolerated, and appeared to be Therapeutics, Tcelna® immunodominant escalation study Low dose: 6–9 × 106, mid associated with clinical Inc. Texas, (Imilecleucel-T, peptides derived dose: 30–45 × 106 and benefit (reduction of ARR) The United States previously from MOG high dose: 60–90 × 106 administration of 30–45 × 106 known as (MOG1–17, irradiated autologous irradiated myelin-reactive Tovaxin®) MOG19–39), PLP myelin-reactive T cells, T cells was the most effective (PLP30–49, administrated on weeks 0, dose during the PLP180–199) and 4, 12, and 20 52 weeks study MBP (MBP83–99, MBP151–170) 170 T cell Autologous T cells NCT00245622 RRMS (n = 95) 150 Phase IIb Completed Randomized, Multiple administrations: s.c. Tovaxin treatment had a Opexa vaccination against up to 6 (TERMS study: CIS (n = 5) (Tovaxin, double-blind, 5 injections of 30–45 × safety profile but efficacy Therapeutics, Tcelna® immunodominant Tovaxin for n = 100; placebo-controlled. 106 irradiated autologous results were not clear likely Inc. Texas, (Imilecleucel-T, peptides derived Early placebo, Personalized myelin-reactive T cells on due to the presence of United States previously from MBP, MOG, Relapsing n = 50) therapy as a result weeks 0, 4, 8, 12, and 24 patients with prior treatment known as and PLP Multiple of prescreening with DMT therapies Tovaxin®) Sclerosis) reactivity against peptide libraries covering MBP, MOG and PLP T cell Autologous T cells NCT00595920 RRMS CIS with 116 Phase II Terminated Multicenter, Multiple administrations: s.c. Since only 38 patients Opexa – vaccination against up to 6 OLTERMS positive myelin- (extension study) due to randomized, 5 injections of 30–45 × received the treatment and 32 Therapeutics, Tcelna® immunodominant study: Open reactive T cells financial double-blind, 106 irradiated autologous of them did not complete the Inc. Texas, (Imilecleucel-T, peptides derived label (MRTC) in their constraints placebo-controlled. myelin-reactive T cells on 5 administrations, it was not United States previously from MBP, MOG, extension of blood during Personalized weeks 0, 4, 8, 12, and 24. possible to evaluate the known as and PLP TERMS study the previous therapy using yearly as required efficacy MJ Mansilla et al. Paving the way towards an effective treatment for multiple sclerosis:. . . Tovaxin®) TERMS study prescreening reactivity test T cell Autologous pool of NCT01684761 SPMS Presence 183 Phase II Completed Multicenter, Multiple administrations: s.c. Favorable safety and Opexa – vaccination myelin-reactive T- Abili-T study of myelin- randomized, 5 injections of 30–45 × tolerability profile in SPMS Therapeutics, Tcelna® cells against reactive T-cells double-blind, 106 irradiated autologous patients No changes on Inc. Texas, (Imilecleucel-T, immunodominant EDSS score placebo-controlled myelin-reactive T cells on disease progression rate nor United States previously epitopes selected 3.0–6.0, weeks 0, 4, 8, 12, and 24. reduction of brain atrophy known as derived from MBP, inclusively was observed Tovaxin®) MOG and PLP on a per subject basis ARR annualized relapse rate, DMT disease-modifying treatments, i.d. intradermal IFA, incomplete Freund’s adjuvant, MBP myelin basic protein, MS multiple sclerosis, MOG myelin oligodendrocyte glycoprotein, PLP proteolipid protein, PPMS primary-progressive MS, RRMS relapsing-remitting MS, s.c. subcutaneous, SPMS secondary-progressive MS, TCV T cell vaccination, WBC white blood cell counts Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1371 LEGAL RULES FOR CELLULAR PRODUCTS us to approach this disease from various fronts and, without a Cellular therapies are classified as either transplants, in which doubt, increase the possibility of obtaining a suitable therapy for unmodified cells are immediately administered, or drugs, in which each patient at the right time to ensure the highest quality of life substantial laboratory modification of the cells and/or nonhomo- possible because each patient is unique, as is their disease. logous use of the cells occurs. In Europe, the use of these two kinds of products is regulated by directive 2004/23/EC, which sets standards of quality and safety for the donation, procurement, ACKNOWLEDGEMENTS testing, processing, preservation, storage and distribution of This review was possible thanks to contributions and funding from the European Union’s human tissues and cells, and directive 1394/2007, which is Horizon 2020 research and innovation program under grant agreement 779316 focused on advanced therapy medicinal products (ATMPs). Clinical (RESTORE); the support of project PI17/01521 and PI20/01313, integrated in the Plan Nacional de I + D + I and cosupported by the ISCIII-Subdirección General de Evaluación trials to test new investigational medicines are also centrally and the Fondo Europeo de Desarrollo Regional (FEDER); and the support of National regulated by regulation 536/2014. In general, these directives Centre for Research and Development, Poland (grant n° STRATEGMED1/233368/1/NCBR/ implement the guidelines established by the International Council 2014). MJ.M. is beneficiary of a Sara Borrell contract from the ISCIII and the FEDER. N.D. for Harmonization of Technical Requirements for Pharmaceuticals and Đ.M. are supported by MPNTR Republika Srbija, grant n° 451-03-68/2020-14/200007. for Human Use (ICH), a common worldwide effort of national F.F. holds a PhD fellowship from the INsTRuCT Consortium focused on developing regulatory authorities and the pharmaceutical industry to discuss innovative myeloid regulatory cell (MRC)-based immunotherapies, which receive an scientific and technical aspects of pharmaceuticals. Similar acts Innovative Training Network subsidy from the European Union Horizon 2020 Programme. based on ICH rules are also in place in other parts of the world and describe the path of new medicines from discovery to routine use. This path for cellular drugs is similar to that for other drugs. It ADDITIONAL INFORMATION starts from preclinical assessment, which establishes the most Competing interests: The authors declare no competing interests. important toxic, pharmacokinetic and pharmacokinetic features of the investigational drug. This step in humans consists of three trials: a first-in-man phase I trial to establish safety and dose, a REFERENCES phase II trial to test efficacy and a phase III trial to confirm safety 1. Lublin, F. D. & Reingold, S. C. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 46, 907–911 (1996). and efficacy in a larger population. The last study is usually pivotal 2. Lublin, F. D. et al. Defining the clinical course of multiple sclerosis: the 2013 and the basis for marketing authorization. Drugs are also revisions. Neurology 83, 278–286 (2014). continuously surveyed post authorization, which is phase IV. In 3. Thompson, A. J. et al. Diagnosis of multiple sclerosis: 2017 revisions of the Europe, the authorization of cellular drugs is centralized, as it McDonald criteria. Lancet Neurol. 17, 162–173 (2018). occurs in one step for all EU countries and is granted by the EMA. 4. Bielekova, B., Sung, M.-H., Kadom, N., Simon, R., McFarland, H., & Martin, R. Some possibilities for early access to cellular products, such as Expansion and functional relevance of high-avidity myelin-specific CD4 + T cells hospital exemption rules, exist, but these are very limited, and in multiple sclerosis. J. Immunol. 172, 3893–3904 (2004). there is pressure to provide standardized, equal treatment to all 5. Olsson, T., Barcellos, L. F. & Alfredsson, L. Interactions between genetic, lifestyle citizens of the EU. The importance of cellular drugs and high and environmental risk factors for multiple sclerosis. Nat. Rev. Neurol. 13, 26–36 (2016). expectations that these cellular drugs will be a ‘game changer’ for 6. Papenfuss, T. L. et al. Estriol generates tolerogenic dendritic cells in vivo that many unmet medical needs are highlighted by the fact that there protect against autoimmunity. J. Immunol. 186, 3346–3355 (2011). is a dedicated legal board within the EMA, the Committee for 7. Kurth, F. et al. Neuroprotective effects of testosterone treatment in men with Advanced Therapies (CAT), which addresses only regulations on multiple sclerosis. NeuroImage Clin. 4, 454–460 (2014). cellular therapy. There are also academic initiatives to regulate the 8. Jelcic, I. et al. Memory B cells activate brain-homing, autoreactive CD4 + T cells way cellular drugs are manufactured. For example, academic in multiple sclerosis. Cell 175, 85–100.e23 (2018). guidelines for tolerance-inducing cellular products based on the 9. Patsopoulos, N. A. et al. Multiple sclerosis genomic map implicates peripheral minimal information model exist.177,178 immune cells and microglia in susceptibility. Science. 365, eaav7188 (2019). 10. Celarain, N. & Tomas-Roig, J. Aberrant DNA methylation profile exacerbates inflammation and neurodegeneration in multiple sclerosis patients. J. Neuroin- flammation 17, 21 (2020). CONCLUDING REMARKS 11. Haas, J. et al. Reduced suppressive effect of CD4 + CD25high regulatory T cells Over the past 20 years, an extraordinary effort has been made to on the T cell immune response against myelin oligodendrocyte glycoprotein in develop treatments that can halt the natural evolution of MS. Thanks patients with multiple sclerosis. Eur. J. Immunol. 35, 3343–3352 (2005). to this effort, we have a variety of drugs that, due to their powerful 12. Nuyts, A. H., Lee, W. P., Bashir-Dar, R., Berneman, Z. N. & Cools, N. Dendritic cells anti-inflammatory/immunosuppressive effects, decrease the rate of in multiple sclerosis: key players in the immunopathogenesis, key players for relapse and radiological activity, thus slowing the onset of disability. new cellular immunotherapies? Mult. Scler. J. 19, 995–1002 (2013). However, by acting in an immunologically nonspecific manner and 13. Dendrou, C. A., Fugger, L. & Friese, M. A. Immunopathology of multiple sclerosis. suppressing one or more branches of the immune response, these Nat. Rev. Immunol. 15, 545–558 (2015). 14. van Langelaar, J., Rijvers, L., Smolders, J. & van Luijn, M. M. B and T cells driving treatments have the potential to cause serious adverse effects. In multiple sclerosis: identity, mechanisms and potential triggers. Front. Immunol. this context, cell therapy appears to be a promising strategy. 11, 760 (2020). Phase I clinical trials with tolDCs, MSCs and Tregs in MS patients 15. Rahman, M. T. et al. IFN-γ IL-17A, or zonulin rapidly increase the permeability of have shown these therapies to be safe and well tolerated, with no the blood-brain and small intestinal epithelial barriers: relevance for neuro- relevant adverse effects. Among cell-based strategies, MSCs have inflammatory diseases. Biochem Biophys. Res. Commun. 507, 274–279 (2018). a potent immunomodulatory effect. TolDCs loaded with self- 16. Cayrol, R. et al. Activated leukocyte cell adhesion molecule promotes leukocyte antigens against which tolerance is induced, Tregs, and more trafficking into the central nervous system. Nat. Immunol. 9, 137–145 (2008). recently CAR-Tregs have the potential to specifically act against 17. Michel, L. et al. Activated leukocyte cell adhesion molecule regulates B the cause of the disease, i.e., the autoimmune response to CNS lymphocyte migration across central nervous system barriers. Sci. Transl. Med. 11, eaaw0475 (2019). myelin, while maintaining protective immunity. In addition, 18. Ransohoff, R. M. & Engelhardt, B. The anatomical and cellular basis of immune although less explored, some cell therapies show neuroprotective surveillance in the central nervous system. Nat. Rev. Immunol. 12, 623–635 and neurorepair potential. These cells have shown promising (2012). results in experimental models, and some, such as hfNSCs, MSC- 19. Louveau, A. et al. Structural and functional features of central nervous system NPs and hESCs, are already being tested in patients. lymphatic vessels. Nature 523, 337–341 (2015). All these developments paint a very hopeful picture for the next 20. Aspelund, A. et al. A dural lymphatic vascular system that drains brain interstitial few years. The personalized combination of treatments will allow fluid and macromolecules. J. Exp. Med. 212, 991–999 (2015). Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1372 21. Lassmann H., Bradl M. Multiple sclerosis: experimental models and reality. Acta 49. Gross, C. C. et al. Regulatory functions of natural killer cells in multiple sclerosis. Neuropathologica. 133, 223–244 (2017). Front. Immunol. 7, 606 (2016). 22. Robinson, A. P., Harp, C. T., Noronha, A. & Miller, S. D. The experimental auto- 50. Lünemann, J. D. & Münz, C. Do natural killer cells accelerate or prevent auto- immune encephalomyelitis (EAE) model of MS. utility for understanding disease immunity in multiple sclerosis? Brain 131, 1681–1683 (2008). pathophysiology and treatment. Handb. Clin. Neurol. 2014, 173–189 (2014). 51. Ponath, G., Park, C. & Pitt, D. The role of astrocytes in multiple sclerosis. Front. 23. Walker-Caulfield, M. E., Hatfield, J. K. & Brown, M. A. Dynamic changes in Immunol. 9, 217 (2018). meningeal inflammation correspond to clinical exacerbations in a murine model 52. Lassmann, H. Pathogenic mechanisms associated with different clinical courses of relapsing-remitting multiple sclerosis. J. Neuroimmunol. 278, 112–122 (2015). of multiple sclerosis. Front. Immunol. 9, 3116 (2019). 24. Kivisakk, P. et al. Localizing central nervous system immune surveillance: 53. Machado-Santos, J. et al. The compartmentalized inflammatory response in the meningeal antigen-presenting cells activate T cells during experimental auto- multiple sclerosis brain is composed of tissue-resident CD8 + T lymphocytes immune encephalomyelitis. Ann. Neurol. 65, 457–469 (2009). and B cells. Brain 141, 2066–2082 (2018). 25. Bartholomäus, I. et al. Effector T cell interactions with meningeal vascular 54. ten Brinke, A. et al. Ways forward for tolerance-inducing cellular therapies—an structures in nascent autoimmune CNS lesions. Nature 462, 94–98 (2009). afactt perspective. Front. Immunol. 10, 181 (2019). 26. Brown, D. A. & Sawchenko, P. E. Time course and distribution of inflammatory 55. Nutt, S. L. & Chopin, M. Transcriptional networks driving dendritic cell differ- and neurodegenerative events suggest structural bases for the pathogenesis of entiation and function. Immunity 52, 942–956 (2020). experimental autoimmune encephalomyelitis. J. Comp. Neurol. 502, 236–260 56. Morelli, A. E. & Thomson, A. W. Tolerogenic dendritic cells and the quest for (2007). transplant tolerance. Nat. Rev. Immunol. 7, 610–621 (2007). 27. Kammona O., Kiparissides C. Recent advances in antigen-specific immu- 57. Suwandi, J. S., Nikolic, T. & Roep, B. O. Translating mechanism of regulatory notherapies for the treatment of multiple sclerosis. Brain Sci. 10, (2020). action of tolerogenic dendritic cells to monitoring endpoints in clinical trials. 28. Baecher-Allan C., Kaskow B. J., Weiner H. L. Multiple sclerosis: mechanisms and Front. Immunol. 8, 22 (2017). immunotherapy. Neuron 97, 742–768 (2018) 58. Kim, S. H., Jung, H. H. & Lee, C. K. Generation, characteristics and clinical trials of 29. Abrahamsson, S. V. et al. Non-myeloablative autologous haematopoietic stem ex vivo generated tolerogenic dendritic cells. Yonsei Med. J. Yonsei Univ. Coll. cell transplantation expands regulatory cells and depletes IL-17 producing Med. 59, 807–815 (2018). mucosal-associated invariant T cells in multiple sclerosis. Brain 136, 2888–2903 59. Van Brussel, I. et al. Tolerogenic dendritic cell vaccines to treat autoimmune (2013). diseases: Can the unattainable dream turn into reality? Autoimmun. Rev. 13, 30. \Sospedra, M. B cells in multiple sclerosis. Curr. Opin. Neurol. 31, 256–262 138–150 (2014). (2018). 60. Zhou, Y. et al. Regulatory dendritic cells induced by K313 display anti-inflam- 31. Magliozzi, R. et al. Meningeal B-cell follicles in secondary progressive multiple matory properties and ameliorate experimental autoimmune encephalitis in sclerosis associate with early onset of disease and severe cortical pathology. mice. Front. Pharmacol. 10, 1579 (2020). Brain 130, 1089–1104 (2007). 61. Derdelinckx, J. et al. Clinical and immunological control of experimental auto- 32. Serafini, B., Rosicarelli, B., Magliozzi, R. & Stigliano, E. A. F. Detection of ectopic B- immune encephalomyelitis by tolerogenic dendritic cells loaded with MOG- cell follicles with germinal centers in the meninges of patients with secondary encoding mRNA. J. Neuroinflammation. 16, 167 (2019). progressive multiple sclerosis. Brain Pathol. 14, 164–174 (2004). 62. Zhou, F., Zhang, G. X. & Rostami, A. LPS-treated bone marrow-derived dendritic 33. Brown, M. A. & Weinberg, R. B. Mast cells and innate lymphoid cells: under- cells induce immune tolerance through modulating differentiation of CD4 + appreciated players in CNS autoimmune demyelinating disease. Front. Immunol. regulatory T cell subpopulations mediated by 3G11 and CD127. Immunol. Res. 9, 514 (2018). 65, 630–638 (2017). 34. Greenfield, A. L. & Hauser, S. L. B-cell therapy for multiple sclerosis: entering an 63. Zhou, F., Zhang, G. X. & Rostami, A. Apoptotic cell-treated dendritic cells induce era. Ann. Neurol. 83, 13–26 (2018). immune tolerance by specifically inhibiting development of CD4 + effector 35. Lock, C. et al. Gene-microarray analysis of multiple sclerosis lesions yields new memory T cells. Immunol. Res. 64, 73–81 (2016). targets validated in autoimmune encephalomyelitis. Nat. Med. 8, 500–508 64. Xie, Z. et al. 1,25-dihydroxyvitamin D3-induced dendritic cells suppress experi- (2002). mental autoimmune encephalomyelitis by increasing proportions of the reg- 36. Christy, A. L., Walker, M. E., Hessner, M. J. & Brown, M. A. Mast cell activation and ulatory lymphocytes and reducing T helper type 1 and type 17 cells. neutrophil recruitment promotes early and robust inflammation in the menin- Immunology 152, 414–424 (2017). ges in EAE. J. Autoimmun. 42, 50–61 (2013). 65. Li, C. H. et al. Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 37. Karussis, D. & Vaknin-Dembinsky, A. Hematopoietic stem cell transplantation in 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific multiple sclerosis: a review of the clinical experience and a report of an inter- suppression of ongoing experimental allergic encephalomyelitis. FASEB J. 31, national meeting. Expert Rev. Clin. Immunol. 6, 347–352 (2010). 2996–3006 (2017). 38. Russi, A. E., Walker-Caulfield, M. E., Guo, Y., Lucchinetti, C. F. & Brown, M. A. 66. Mansilla, M. J. et al. Cryopreserved vitamin D3-tolerogenic dendritic cells pulsed Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity. J. with autoantigens as a potential therapy for multiple sclerosis patients. J Neu- Autoimmun. 73, 100–110 (2016). roinflammation. 13, 113 (2016). 39. El-Behi, M. et al. The encephalitogenicity of TH 17 cells is dependent on IL-1- 67. Mansilla, M. J. et al. Beneficial effect of tolerogenic dendritic cells pulsed with and IL-23-induced production of the cytokine GM-CSF. Nat. Immunol. 12, MOG autoantigen in experimental autoimmune encephalomyelitis. CNS Neu- 568–575 (2011). rosci. Ther. 21, 222–230 (2015). 40. Ponomarev, E. D. et al. GM-CSF production by autoreactive T cells is required for 68. Thome, R. et al. Chloroquine-treated dendritic cells require STAT1 signaling for the activation of microglial cells and the onset of experimental autoimmune their tolerogenic activity. Eur. J. Immunol. 48, 1228–1234 (2018). encephalomyelitis. J. Immunol. 178, 39–48 (2007). 69. Zhou, Y. et al. Tolerogenic dendritic cells generated with tofacitinib ameliorate 41. Croxford, A. L. et al. The cytokine GM-CSF drives the inflammatory signature of experimental autoimmune encephalomyelitis through modulation of Th17/Treg CCR2 + monocytes and licenses autoimmunity. Immunity 43, 502–514 (2015). balance. J. Immunol. Res. 2016, 5021537 (2016). 42. Codarri, L. et al. RORγ3t drives production of the cytokine GM-CSF in helper 70. Zhou, Y. et al. Tolerogenic dendritic cells induced by BD750 ameliorate proin- T cells, which is essential for the effector phase of autoimmune neuroin- flammatory T cell responses and experimental autoimmune encephalitis in flammation. Nat. Immunol. 12, 560–567 (2011). mice. Mol. Med. 23, 204–214 (2017). 43. Rumble, J. M. et al. Neutrophil-related factors as biomarkers in EAE and MS. J. 71. Haller, S. et al. Interleukin-35-producing CD8α + dendritic cells acquire a tol- Exp. Med. 212, 23–35 (2015). erogenic state and regulate T cell function. Front Immunol. 8, 98 (2017). 44. Pierson, E. R., Wagner, C. A. & Goverman, J. M. The contribution of neutrophils to 72. Grau-López, L. et al. Specific T-cell proliferation to myelin peptides in relapsing- CNS autoimmunity. Clin. Immunol. 189, 23–28 (2018). remitting multiple sclerosis. Eur. J. Neurol. 18, 1101–1104 (2011). 45. Manda-Handzlik, A. & Demkow, U. The brain entangled: the contribution of 73. Lutterotti, A. et al. Antigen-specific tolerance by autologous myelin peptide- neutrophil extracellular traps to the diseases of the central nervous system. Cells coupled cells: A phase 1 trial in multiple sclerosis. Sci. Transl. Med. 5, 188ra75 8, 1477 (2019). (2013). 46. Klose, C. S. N. & Artis, D. Innate lymphoid cells as regulators of immunity, 74. Zubizarreta, I. et al. Immune tolerance in multiple sclerosis and neuromyelitis inflammation and tissue homeostasis. Nat. Immunol. 17, 765–774 (2016). optica with peptide-loaded tolerogenic dendritic cells in a phase 1b trial. Proc. 47. Huang, Y. et al. S1P-dependent interorgan trafficking of group 2 innate lym- Natl Acad. Sci. USA. 116, 8463–8470 (2019). phoid cells supports host defense. Science 359, 114–119 (2018). 75. Lee, W. P. et al. Immunomodulatory effects of 1,25-dihydroxyvitamin D3 on 48. Weyand, C. M., Kurtin, P. J. & Goronzy, J. J. Ectopic lymphoid organogenesis: a dendritic cells promote induction of T cell hyporesponsiveness to myelin- fast track for autoimmunity. Am. J. Pathol. 159, 787–793 (2001). derived antigens. J. Immunol. Res. 2016, 5392623 (2016). Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1373 76. Jauregui-Amezaga, A. et al. Intraperitoneal administration of autologous tol- 104. Malviya, M., Saoudi, A., Bauer, J., Fillatreau, S. & Liblau, R. Treatment of experi- erogenic dendritic cells for refractory Crohn’s disease: a phase I study. J. Crohns mental autoimmune encephalomyelitis with engineered bi-specific Foxp3+ Colitis 9, 1071–1078 (2015). regulatory CD4 + T cells. J. Autoimmun. 108, 102401 (2020). 77. Giannoukakis, N., Phillips, B., Finegold, D., Harnaha, J. & Trucco, M. Phase I 105. Verhagen, J., Gabryšová, L., Shepard, E. R. & Wraith, D. C. CTLA-4 modulates the (safety) study of autologous tolerogenic dendritic cells in type 1 diabetic differentiation of inducible foxp3+treg cells but IL-10 mediates their function in patients. Diabetes Care. 34, 2026–2032 (2011). experimental autoimmune encephalomyelitis. PLoS ONE 9, e108023 (2014). 78. Benham, H. et al. Citrullinated peptide dendritic cell immunotherapy in HLA risk 106. Yu, P. et al. Specific T regulatory cells display broad suppressive functions genotype-positive rheumatoid arthritis patients. Sci. Transl. Med. 7, 290ra87 (2015). against experimental allergic encephalomyelitis upon activation with cognate 79. Bell, G. M. et al. Autologous tolerogenic dendritic cells for rheumatoid and antigen. J. Immunol. 174, 6772–6780 (2005). inflammatory arthritis. Ann. Rheum. Dis. 76, 227–234 (2017). 107. Zhang, H., Podojil, J. R., Chang, J., Luo, X. & Miller, S. D. TGF-β–induced myelin 80. Moreau, A. et al. Generation and in vivo evaluation of IL10-treated dendritic cells peptide-specific regulatory T cells mediate antigen-specific suppression of induction in a nonhuman primate model of AAV-based gene transfer. Mol. Ther. Methods of experimental autoimmune encephalomyelitis. J. Immunol. 184, 6629–6636 (2010). Clin. Dev. 1, 14028 (2014). 108. Chen, M.-L., Yan, B.-S., Bando, Y., Kuchroo, V. K. & Weiner, H. L. Latency- 81. Willekens, B. et al. Tolerogenic dendritic cell-based treatment for multiple sclerosis associated peptide identifies a novel CD4 + CD25 + regulatory T cell subset (MS): a harmonised study protocol for two phase I clinical trials comparing intra- with TGFβ-mediated function and enhanced suppression of experimental dermal and intranodal cell administration. BMJ Open. 9, e030309 (2019). autoimmune encephalomyelitis. J. Immunol. 180, 7327–7337 (2008). 82. Raïch-Regué, D. et al. Stable antigen-specific T-cell hyporesponsiveness induced 109. Lüth, S. et al. Ectopic expression of neural autoantigen in mouse liver suppresses by tolerogenic dendritic cells from multiple sclerosis patients. Eur. J. Immunol. experimental autoimmune neuroinflammation by inducing antigen-specific 42, 771–782 (2012). Tregs. J. Clin. Investig. 118, 3403–3410 (2008). 83. Navarro-Barriuso, J. et al. Vitamin D3-induced tolerogenic dendritic cells mod- 110. McPherson, R. C., Turner, D. G., Mair, I., O’Connor, R. A. & Anderton, S. M. T-bet ulate the transcriptomic profile of T CD4 + cells towards a functional hypor- expression by Foxp3 + T regulatory cells is not essential for their suppressive esponsiveness. Front. Immunol. 11, 599623 (2020). function in CNS autoimmune disease or colitis. Front. Immunol. 6, 69 (2015). 84. Anderson, A. E. et al. Tolerogenic dendritic cells generated with dexamethasone 111. Kim, Y. C. et al. Engineered MBP-specific human Tregs ameliorate MOG-induced EAE and vitamin D3 regulate rheumatoid arthritis CD4 + T cells partly via trans- through IL-2-triggered inhibition of effector T cells. J. Autoimmun. 92, 77–86 (2018). forming growth factor-β1. Clin. Exp. Immunol. 187, 113–123 (2017). 112. MacDonald, K. G. et al. Alloantigen-specific regulatory T cells generated with a 85. Marín, E., Cuturi, M. C. & Moreau, A. Tolerogenic dendritic cells in solid organ chimeric antigen receptor. J. Clin. Investig. 126, 1413–1424 (2016). transplantation: Where do we stand? Front. Immunol. 9, 274 (2018). 113. Paweł, Ł., Trzonkowski, P. & Chwojnicki, K. Multiple sclerosis—new therapeutic 86. Djedovic, N. et al. Ethyl pyruvate induces tolerogenic dendritic cells. Front. directions. Eur. J. Transl. Clin. Med. 2, 7–22 (2017). Immunol. 10, 157 (2019). 114. Riedhammer, C. & Weissert, R. Antigen presentation, autoantigens, and immune 87. Navarro-Barriuso, J. et al. MAP7 and MUCL1 are biomarkers of Vitamin D3- regulation in multiple sclerosis and other autoimmune diseases. Front. Immunol. induced tolerogenic dendritic cells in multiple sclerosis patients. Front. Immunol. 6, 322 (2015). 10, 1251 (2019). 115. Boardman, D. A. et al. Expression of a chimeric antigen receptor specific for 88. Chung, D. J. et al. Indoleamine 2,3-dioxygenase-expressing mature human donor HLA class I enhances the potency of human regulatory T cells in pre- monocyte-derived dendritic cells expand potent autologous regulatory T cells. venting human skin transplant rejection. Am. J. Transplant. 17, 931–943 (2017). Blood 114, 555–563 (2009). 116. Noyan, F. et al. Prevention of allograft rejection by use of regulatory T cells with an 89. Navarro-Barriuso, J., Mansilla, M. J. & Martínez-Cáceres, E. M. Searching for the MHC-specific chimeric antigen receptor. Am. J. Transplant. 17, 917–930 (2017). Transcriptomic Signature of Immune Tolerance Induction-Biomarkers of Safety 117. Trzonkowski, P. et al. Hurdles in therapy with regulatory T cells. Sci. Transl. Med. and Functionality for Tolerogenic Dendritic Cells and Regulatory Macrophages. 7, 304ps18 (2015). Front Immunol. 9, 2062 (2018). 118. Gliwiński, M. et al. Proinsulin-specific T regulatory cells may control immune 90. Voigtländer, C. et al. Dendritic cells matured with TNF can be further activated responses in type 1 diabetes: Implications for adoptive therapy. BMJ Open in vitro and after subcutaneous injection in vivo which converts their tolero- Diabetes Res Care. 8, e000873 (2020). genicity into immunogenicity. J. Immunother. 29, 407–415 (2006). 119. Hull, C. M. et al. Generation of human islet-specific regulatory T cells by TCR 91. Lee, H. W., Gangadaran, P., Kalimuthu, S. & Ahn, B. C. Advances in molecular gene transfer. J. Autoimmun. 79, 63–73 (2017). imaging strategies for in vivo tracking of immune cells. BioMed Res. Int. 2016, 120. Friedenstein, A. J., Petrakova, K. V. & Kurolesova, A. I. F. G. Heterotopic of bone 1946585 (2016). marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. 92. Gliwiński, M., Iwaszkiewicz-Grześ, D. & Trzonkowski, P. Cell-based therapies with Transplantation 6, 230–247 (1968). T regulatory cells. BioDrugs 31, 335–347 (2017). 121. Caplan, A. I. Mesenchymal stem cells. J Orthop Res. 9, 641–650 (1991). 93. Zhang, X. et al. IL-10 is involved in the suppression of experimental autoimmune 122. Dominici, M. et al. Minimal criteria for defining multipotent mesenchymal encephalomyelitis by CD25 + CD4 + regulatory T cells. Int Immunol. 16, stromal cells. The International Society for Cellular Therapy position statement. 249–256 (2004). Cytotherapy 8, 315–317 (2006). 94. Chwojnicki, K. et al. Intrathecal administration of CD4+ CD25 high CD127− foxp3+ 123. Zappia, E. et al. Mesenchymal stem cells ameliorate experimental autoimmune regulatory T cells slows progression of multiple sclerosis—Results of phase I study. encephalomyelitis inducing T-cell anergy. Blood 106, 1755–1761 (2005). Biodrugs. 35, 47–60 (2021). 124. Yanwu, Y., Meiling, G., Yunxia, Z., Qiukui, H. & Birong, D. Mesenchymal stem cells 95. Feger, U. et al. Increased frequency of CD4 + CD25 + regulatory T cells in the in experimental autoimmune encephalomyelitis model of multiple sclerosis: a cerebrospinal fluid but not in the blood of multiple sclerosis patients. Clin. Exp. systematic review and meta-analysis. Mult. Scler. Relat. Disord 44, 102200 (2020). Immunol. 147, 412–418 (2007). 125. Mansoor, H. et al. Current trends and future perspective of mesenchymal stem 96. Sharabi, A. et al. Regulatory T cells in the treatment of disease. Nat. Rev. Drug cells and exosomes in corneal diseases. Int. J. Mol. Sci. 20, 2853 (2019). Discov. 17, 823–844 (2018). 126. Wang, S., Qu, X. & Zhao, R. C. Clinical applications of mesenchymal stem cells. J. 97. Marek-Trzonkowska, N. et al. Factors affecting long-term efficacy of T regulatory Hematol. Oncol. 5, 19 (2012). cell-based therapy in type 1 diabetes. J. Transl. Med. 14, 332 (2016). 127. Li, L. et al. Exosomes derived from mesenchymal stem cells ameliorate renal 98. Long, S. A. et al. Rapamycin/IL-2 combination therapy in patients with type 1 ischemic-reperfusion injury through inhibiting inflammation and cell apoptosis. diabetes augments Tregs yet transiently impairs β-cell function. Diabetes 61, Front. Med. 6, 269 (2019). 2340–2348 (2012). 128. Di Nicola, M. et al. Human bone marrow stromal cells suppress T-lymphocyte 99. Schmitz, R. et al. Kidney Transplantation using Alemtuzumab, Belatacept and proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99, Sirolimus—Five‐Year Follow‐up. Am. J. Transplant. 20, 3609–3619 (2020). 3838–3843 (2002). 100. Sawitzki, B. et al. Regulatory cell therapy in kidney transplantation (The ONE 129. Aggarwal, S. & Pittenger, M. F. Human mesenchymal stem cells modulate Study): a harmonised design and analysis of seven non-randomised, single-arm, allogeneic immune cell responses. Blood 105, 1815–1822 (2005). phase 1/2A trials. Lancet. 395, 1627–1639 (2020). 130. English, K. et al. Cell contact, prostaglandin E2 and transforming growth factor 101. Stephens, L. A., Malpass, K. H., Anderton, S. M. & Curing, C. N. S. autoimmune beta 1 play non-redundant roles in human mesenchymal stem cell induction of disease with myelin-reactive Foxp3+ Treg. Eur. J. Immunol 39, 1108–1117 CD4 + CD25Highforkhead box P3 + regulatory T cells. Clin. Exp. Immunol 156, (2009). 149–160 (2009). 102. Fransson, M. et al. CAR/FoxP3-engineered T regulatory cells target the CNS and 131. Augello, A. et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte suppress EAE upon intranasal delivery. J. Neuroinflamm 9, 112 (2012). proliferation by activation of the programmed death 1 pathway. Eur. J. Immunol. 103. Niedbala, W. et al. Nitric Oxide–Induced Regulatory T Cells Inhibit Th17 but Not 35, 1482–1490 (2005). Th1 Cell Differentiation and Function. J. Immunol. 191, 164–170 (2013). Cellular & Molecular Immunology (2021) 18:1353 – 1374 Paving the way towards an effective treatment for multiple sclerosis:. . . MJ Mansilla et al. 1374 132. Corcione, A. et al. Human mesenchymal stem cells modulate B-cell functions. 158. Pluchino, S., Smith, J. A. & Peruzzotti-Jametti, L. Promises and limitations of Blood 107, 367–372 (2006). neural stem cell therapies for progressive multiple sclerosis. Trend. Mol. Med. 26, 133. Asari, S. et al. Mesenchymal stem cells suppress B-cell terminal differentiation. 898–912 (2020). Exp. Hematol. 37, 604–615 (2009). 159. Ben-Nun, A. & Wekerle, H. C. I. Vaccination against autoimmune encephalomyelitis 134. Sotiropoulou, P. A., Perez, S. A., Gritzapis, A. D., Baxevanis, C. N. & Papamichail, M. with T-lymphocyte line cells reactive against myelin basic protein. Nature 292, 60– Interactions between human mesenchymal stem cells and natural killer cells. 61 (1981). Stem Cells 24, 74–85 (2006). 160. Zhang, J., Medaer, R., Stinissen, P., Hafler, D. & Raus, J. MHC-restricted depletion 135. Spaggiari, G. M., Capobianco, A., Becchetti, S., Mingari, M. C. & Moretta, L. of human myelin basic protein-reactive T cells by T cell vaccination. Science. Mesenchymal stem cell-natural killer cell interactions: evidence that activated 261, 1451–1454 (1993). NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK- 161. Medaer, R., Stinissen, P., Raus, J., Zhang, J. & Truyen, L. Depletion of myelin-basic- cell proliferation. Blood 107, 1484–1490 (2006). protein autoreactive T cells by T-cell vaccination: pilot trial in multiple sclerosis. 136. Chen, L. et al. Effects of human mesenchymal stem cells on the differentiation of Lancet. 346, 807–808 (1995). dendritic cells from CD34 + cells. Stem Cells Dev 16, 719–731 (2007). 162. Correale J., et al. T cell vaccination in secondary progressive multiple sclerosis. J. 137. Li, Z. et al. Exosomes derived from mesenchymal stem cells attenuate inflam- Neuroimmunol. 107, (2000). mation and demyelination of the central nervous system in EAE rats by reg- 163. Karussis, D. et al. T cell vaccination benefits relapsing progressive multiple ulating the polarization of microglia. Int Immunopharmacol. 67, 268–280 (2019). sclerosis patients: a randomized, double-blind clinical trial. PLoS ONE. 7, e50478 138. Devine, S. M., Cobbs, C., Jennings, M., Bartholomew, A. & Hoffman, R. (2012). Mesenchymal stem cells distribute to a wide range of tissues following systemic 164. Loftus, B. et al. Autologous attenuated T-cell vaccine (Tovaxin®) dose escalation infusion into nonhuman primates. Blood 101, 2999–3001 (2003). in multiple sclerosis relapsing-remitting and secondary progressive patients 139. Li, Y. et al. Human marrow stromal cell therapy for stroke in rat: neurotrophins nonresponsive to approved immunomodulatory therapies. Clin. Immunol. 131, and functional recovery. Neurology 59, 514–523 (2002). 202–215 (2009). 140. Munoz, J. R., Stoutenger, B. R., Robinson, A. P., Spees, J. L. & Prockop, D. J. Human 165. Vandenbark, A. A. & Abulafia-Lapid, R. Autologous T-cell vaccination for multiple stem/progenitor cells from bone marrow promote neurogenesis of endogenous sclerosis: a perspective on progress. BioDrugs 22, 265–273 (2008). neural stem cells in the hippocampus of mice. Proc. Natl Acad. Sci. USA. 102, 166. Zhang, J. Z. et al. T cell vaccination in multiple sclerosis: results of a preliminary 18171–18176 (2005). study. J. Neurol. 249, 212–218 (2002). 141. Rivera, F. J. et al. Mesenchymal stem cells instruct oligodendrogenic fate deci- 167. Van Der Aa, A. et al. T cell vaccination in multiple sclerosis patients with auto- sion on adult neural stem cells. Stem Cells 24, 2209–2219 (2006). logous CSF-derived activated T cells: results from a pilot study. Clin. Exp. 142. Uccelli, A. et al. MEsenchymal StEm cells for Multiple Sclerosis (MESEMS): A ran- Immunol. 131, 155–168 (2003). domized, double blind, cross-over phase I/II clinical trial with autologous 168. Hafler, D., Iruncohen, D. A., Benjamin, D. & Weiner, H. L. T cell vaccination in multiple mesenchymal stem cells for the therapy of multiple sclerosis. Trials. 20, 263 (2019). sclerosis: a preliminary report. Clin. Immunol. Immunopathol. 62, 307–313 (1992). 143. Llufriu, S. et al. Randomized placebo-controlled phase II trial of autologous 169. Achiron, A. et al. T cell vaccination in multiple sclerosis relapsing-remitting mesenchymal stem cells in multiple sclerosis. PLoS ONE. 9, e113936 (2014). nonresponders patients. Clin. Immunol. 113, 155–160 (2004). 144. Cohen, J. A. et al. Pilot trial of intravenous autologous culture-expanded 170. Fox, E. et al. A randomized clinical trial of autologous T-cell therapy in multiple mesenchymal stem cell transplantation in multiple sclerosis. Mult. Scler. J. 24, sclerosis: Subset analysis and implications for trial design. Mult. Scler. J. 18, 843– 501–511 (2018). 852 (2012). 145. Connick, P. et al. The mesenchymal stem cells in multiple sclerosis (MSCIMS) trial 171. Ezzelarab, M. B. et al. Regulatory dendritic cell infusion prolongs kidney protocol and baseline cohort characteristics: an open-label pre-test: Post-test allograft survival in nonhuman primates. Am. J. Transplant. 13, 1989–2005 study with blinded outcome assessments. Trials. 12, 62 (2011). (2013). 146. Connick, P. et al. Autologous mesenchymal stem cells for the treatment of 172. Koreth, J. et al. Interleukin-2 and regulatory T cells in graft-versus-host disease secondary progressive multiple sclerosis: An open-label phase 2a proof-of- abstract from the divisions of hematologic malig-nancies. N. Engl. J. Med. 22, concept study. Lancet Neurol. 11, 150–156 (2012). 2055–2066 (2011). 147. Karussis, D. et al. Safety and immunological effects of mesenchymal stem cell 173. Zielinski, M. et al. Combined immunotherapy with T regulatory cells and anti- transplantation in patients with multiple sclerosis and amyotrophic lateral CD20 antibody prolongs survival of pancreatic islets in type 1 diabetes. Diabetes sclerosis. Arch. Neurol. 67, 1187–1194 (2010). 69, 66–LB (2020). 148. Rice, C. M. et al. Repeat infusion of autologous bone marrow cells in multiple 174. Phillips, B. E., Garciafigueroa, Y., Engman, C., Trucco, M. & Giannoukakis, N. sclerosis: Protocol for a phase i extension study (SIAMMS-II). BMJ Open. 5, Tolerogenic dendritic cells and T-regulatory cells at the clinical trials crossroad e009090 (2015). for the treatment of autoimmune disease; emphasis on type 1 diabetes therapy. 149. Rice, C. M. et al. Assessment of bone marrow-derived Cellular Therapy in pro- Front. Immunol. 10, 148 (2019). gressive Multiple Sclerosis (ACTiMuS): Study protocol for a randomised con- 175. Li, R. et al. Synergistic suppression of autoimmune arthritis through concurrent trolled trial. Trials. 16, 463 (2015). treatment with tolerogenic DC and MSC. Sci. Rep. 7, 43188 (2017). 150. Fernández, O. et al. Adipose-derived mesenchymal stem cells (AdMSC) for the 176. Lee, E. S. et al. Adoptive transfer of treg cells combined with mesenchymal stem treatment of secondary-progressive multiple sclerosis: A triple blinded, placebo cells facilitates repopulation of endogenous treg cells in a murine acute GVHD controlled, randomized phase I/II safety and feasibility study. PLoS ONE. 13, model. PLoS ONE. 10, e0138846 (2015). e0195891 (2018). 177. Lord, P. et al. Minimum information about tolerogenic antigen-presenting cells 151. Riordan, N. H. et al. Clinical feasibility of umbilical cord tissue-derived (MITAP): a first step towards reproducibility and standardisation of cellular mesenchymal stem cells in the treatment of multiple sclerosis. J. Transl. Med. 16, therapies. PeerJ. 4, e2300 (2016). 57 (2018). 178. Fuchs, A. et al. Minimum information about T regulatory cells: a step toward 152. Sarkar, P. et al. Reduced neuroprotective potential of the mesenchymal stromal reproducibility and standardization. Front. Immunol. 8, 1844 (2018). cell secretome with ex vivo expansion, age and progressive multiple sclerosis. Cytotherapy 20, 21–28 (2018). 153. Massey, J. C., Sutton, I. J., Ma, D. D. F. & Moore, J. J. Regenerating immunoto- Open Access This article is licensed under a Creative Commons lerance in multiple sclerosis with autologous hematopoietic stem cell transplant. Attribution 4.0 International License, which permits use, sharing, Front. Immunol. 9, 410 (2018). adaptation, distribution and reproduction in any medium or format, as long as you give 154. Fassas, A. et al. Peripheral blood stem cell transplantation in the treatment of appropriate credit to the original author(s) and the source, provide a link to the Creative progressive multiple sclerosis: First results of a pilot study. Bone Marrow Commons license, and indicate if changes were made. The images or other third party Transplant. 20, 631–638 (1997). material in this article are included in the article’s Creative Commons license, unless 155. Arruda, L. C. M. et al. Immunological correlates of favorable long-term clinical indicated otherwise in a credit line to the material. If material is not included in the outcome in multiple sclerosis patients after autologous hematopoietic stem cell article’s Creative Commons license and your intended use is not permitted by statutory transplantation. Clin. Immunol. 169, 47–57 (2016). regulation or exceeds the permitted use, you will need to obtain permission directly 156. Cuascut, F. X. & Hutton, G. J. Stem cell-based therapies for multiple sclerosis: from the copyright holder. To view a copy of this license, visit http://creativecommons. current perspectives. Biomedicines. 7, 26 (2019). org/licenses/by/4.0/. 157. Muraro, P. A. et al. Autologous haematopoietic stem cell transplantation for treatment of multiple sclerosis. Nat. Rev. Neurol. 13, 391–405 (2017). © The Author(s) 2021 Cellular & Molecular Immunology (2021) 18:1353 – 1374