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CD20 therapies in multiple sclerosis and experimental autoimmune encephalomyelitis – Targeting T or B cells?

      Highlights

      • Evidence indicates that therapies targeting CD20+ B cells in MS also target T cells.
      • EAE is used to identify mechanisms and therapies for MS, a uniquely human disease.
      • B cells have many function in MS other than as a source of pathogenic antibodies.
      • B cell therapies in EAE do not reflect the efficacy in people with MS.
      • The efficacy of CD20 therapy may be removal of Epstein-Barr virus.

      Abstract

      MS is widely considered to be a T cell-mediated disease although T cell immunotherapy has consistently failed, demonstrating distinct differences with experimental autoimmune encephalomyelitis (EAE), an animal model of MS in which T cell therapies are effective. Accumulating evidence has highlighted that B cells also play key role in MS pathogenesis. The high frequency of oligoclonal antibodies in the CSF, the localization of immunoglobulin in brain lesions and pathogenicity of antibodies originally pointed to the pathogenic role of B cells as autoantibody producing plasma cells. However, emerging evidence reveal that B cells also act as antigen presenting cells, T cell activators and cytokine producers suggesting that the strong efficacy of anti-CD20 antibody therapy observed in people with MS may reduce disease progression by several different mechanisms. Here we review the evidence and mechanisms by which B cells contribute to disease in MS compared to findings in the EAE model.

      Abbreviations:

      CNS (central nervous system), EAE (experimental autoimmune encephalomyelitis), MS (multiple sclerosis), PwMS (Person with MS), EBV (Epstein-Barr virus)

      Keywords

      1. Introduction

      Multiple sclerosis (MS) is a chronic immune-mediated neurodegenerative disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal loss. The most widely-used experimental animal model to study mechanisms of CNS damage, as well as for drug development for MS is experimental autoimmune encephalomyelitis (EAE) (
      • Baxter A.G.
      The origin and application of experimental autoimmune encephalomyelitis.
      ,
      • Gold R.
      • Linington C.
      • Lassmann H.
      Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research.
      ,
      • Kipp M.
      • Van der Star B.
      • Vogel Y.S.V.
      • Puentes F.
      • et al.
      Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond.
      ). EAE is a spectrum of experimentally induced immune-mediated neurological diseases of the CNS (
      • Kipp M.
      • Van der Star B.
      • Vogel Y.S.V.
      • Puentes F.
      • et al.
      Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond.
      ), induced in many species. It is well known that the mode of induction, animal species and strains of rodents heavily influence the course of disease and observed immunological alterations and pathology in the CNS (
      • Kipp M.
      • Van der Star B.
      • Vogel Y.S.V.
      • Puentes F.
      • et al.
      Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond.
      ,
      • Baker D.
      • Amor S.
      Experimental autoimmune encephalomyelitis is a good model of multiple sclerosis if used wisely.
      ). Most of the current drugs available for MS are broad-spectrum immunomodulatory agents, although more recently specific therapies using cell-depleting antibodies such as those targeting B cells have been shown to be effective (
      • Kappos L.
      • Li D.
      • Calabresi P.
      • O’Connor P.
      • et al.
      Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicenter trial.
      ,
      • Brück W.
      • Gold R.
      • Brett
      • Oreja-Guevara C.
      • et al.
      Therapeutic decision in multiple sclerosis: moving beyond efficacy.
      ). Despite the apparent efficacy of B cell depletion there is much to be learned about how B cells contribute to the regulation and pathogenesis of MS, given the balance between regulatory and effector cell functions as shown both in EAE and MS. In addition, broad B cell-targeted depletion carries risks, notably associated infections. This indicates that despite being very effective, the depletion regimen could be better targeted to remove pathogenic B cells while leaving the protective and regulatory arm intact.

      2. B cells and autoantibodies in EAE

      Until the last decade MS has been widely considered a white matter disorder, and thus EAE has been commonly induced following immunization with myelin antigen such as myelin basic protein (MBP). However, clinical and pathological features of MS are usually better represented when EAE is induced using total CNS tissues (
      • Sospedra M.
      • Martin R.
      Immunology of multiple sclerosis.
      ) suggesting that autoimmunity to other myelin components may be essential to model chronic relapsing neurological disease, demyelination and axonal damage and secondary progressive disease. Currently, the most widely used autoantigen for induction of EAE is the minor protein myelin oligodendrocyte glycoprotein (MOG), present on the outer membrane of oligodendrocytes (
      • Schluesener J.H.
      • Sobel R.A.
      • Linington C.
      • Weiner H.L.
      A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease.
      ,
      • Linington C.
      • Bradi M.
      • Lassmann H.
      • Brunner C.
      • et al.
      Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein.
      ,
      • Amor S.
      • Groome N.
      • Linington C.
      • Morris M.M.
      • et al.
      Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encepahalomyelitis in SJL and Biozzi AB/H mice.
      ). Similar to EAE induced with other autoantigens, the clinical course and pathology of MOG-induced EAE is heavily dependent on the nature of the MOG antigen for example the presence of post translational modifications, as well as the mouse strain used for immunization (
      • Smith P.A.
      • Heijmans N.
      • Ouwerling B.
      • Breij E.C.
      • Evans N.
      • van Noort J.M.
      • Plomp A.C.
      • Delarasse C.
      • 't Hart B.
      • Pham-Dinh D.
      • Amor S.
      Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice.
      ,
      • de Graaf K.L.
      • Albert M.
      • Weissert R.
      Autoantigen conformation influences both B- and T-cell responses and encephalitogenicity.
      ) (Table 1).
      Table 1Outcome of MOG-induced EAE in WT and B cell deficient mice.
      AntigenMutation or treatmentEAE CourseAntibody orBell requirementReference
      (m)recMOGBiozzi ABH miceChronic relapsing
      • Amor S.
      • Groome N.
      • Linington C.
      • Morris M.M.
      • et al.
      Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encepahalomyelitis in SJL and Biozzi AB/H mice.
      Biozzi mice+MOG antibodiesIncreased EAE severityYes
      • Morris-Downes M.M.
      • Smith P.A.
      • Rundle J.L.
      • Piddlesden S.J.
      • et al.
      Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.
      Augmented demyelination
      (m)SCHBiozzi mice+RituximabNo effect on EAENoD Baker - Unpublished
      (m)MOG35–55Biozzi ABHMonophasic chronicNo
      • Amor S.
      • Smith P.A.
      • Hart B.’
      • Baker D.
      Biozzi mice: of mice and human neurological diseases.
      (m)recMOGWT C57BL/6Chronic EAENo
      • Lyons J.A.
      • San M.
      • Happ M.P.
      • Cross A.H.
      B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide.
      μMT C57BL/6No EAE
      CD20+ cell depletionNot known
      (r)MOGWT C57BL/6Chronic EAENo
      • Oliver A.F.
      • Lyon G.M.
      • Huddle N.H.
      Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice.
      μMT C57BL/6Chronic EAE
      CD20+ cell depletionNot known
      (h)recMOGWT C57BL/6ChronicYes
      • Oliver A.F.
      • Lyon G.M.
      • Huddle N.H.
      Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice.
      μMT C57BL/6No EAE
      B cell depletionNot known
      (m)MOG35–55WT C57BL/6ChronicNo
      • Lyons J.A.
      • San M.
      • Happ M.P.
      • Cross A.H.
      B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide.
      Mouse specific CD20+cell depletionSevere or reduced EAE dependson timing of CD20 depletionYes
      • Matsushita T.
      • Yanaba K.
      • Bouazi J.D.
      • Fujimoto M.
      • et al.
      Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.
      MOG – myelin oligodendrocyte glycoprotein, m – mouse; h – human; recMOG – recombinant MOG; WT – wild type; μMT – Knock-out mice that lack functional B cells.
      Importantly, the majority of studies, including those focusing on the role of B cells, make use of the C57BL/6 mice since most transgenic or mutant mice are bred on this background. Notably, the majority of EAE studies in this mouse strain uses mouse MOG35–55 peptide which induces a monophasic in which B cells and antibodies to MOG do not play a role (
      • Oliver A.F.
      • Lyon G.M.
      • Huddle N.H.
      Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice.
      ).
      The role of pathogenic autoantibodies was first indicated following the demonstration that circulating autoantibodies to myelin in EAE induce myelin damage in vitro and in vivo (
      • Apple
      • Borstein
      The application of tissue culture to the study of experimental allergic encephalomyelitis: II serum factors responsible for demyelination.
      ). This is supported by studies showing that titers of antibodies to myelin are high during chronic stages of EAE when demyelination was most pronounced, and that some MOG antibodies, notably those that fix complement, augment EAE (
      • Linington C.
      • Lassmann H.
      • Morgan B.P.
      • Compston D.A.
      Immunohistochemical localisation of terminal complement component C9 in experimental allergic encephalomyelitis.
      ,
      • Morris-Downes M.M.
      • Smith P.A.
      • Rundle J.L.
      • Piddlesden S.J.
      • et al.
      Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.
      ,
      • Piddlesden S.J.
      • Lassmann H.
      • Zimprich F.
      • Morgan B.P.
      • et al.
      The demyelinating potential of antibodies to myelin oligodendrocyte glycoprotein is related to their ability to fix complement.
      ,
      • Menon K.K.
      • Piddlesden S.J.
      • Bernard C.C.A.
      Demyelinating antibodies to myelin oligodendrocyte glycoprotein and galactocerebroside induce degradation of myelin basic protein in isolated human myelin.
      ,
      • Mead R.J.
      • Singhroa S.K.
      • Neal J.W.
      • Lassmann H.
      • et al.
      The membrane attack complex of complement causes severe demyelination associated with acute axonal injury.
      ,
      • Hundgeburth L.C.
      • Wunsch M.
      • Rovituso D.
      • Recks M.S.
      • Addicks K.
      • Lehmann P.V.
      • Kuerten S.
      The complement system contributes to the pathology of experimental autoimmune encephalomyelitis by triggering demyelination and modifying the antigen-specific T and B cell response.
      ). In addition, MOG antibodies have functional effects on oligodendrocyte inducing stress, physiological and morphological changes (
      • Duvanel C.B.
      • Monnet-Tschudi F.
      • Braissant O.
      • Matthieu J.M.
      • Honegger P.
      Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.
      ,
      • Marta C.B.
      • Oliver A.R.
      • Sweet R.A.
      • Pfeiffer S.E.
      • et al.
      Pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology.
      ) and have recently been shown to trigger T cell activation by opsonisation of endogenous antigen (
      • Kinzel S.
      • Lehmann-Horn K.
      • Torke S.
      • Häusler D.
      • Winkler A.
      • Stadelmann C.
      • Payne N.
      • Feldmann L.
      • Saiz A.
      • Reindl M.
      • Lalive P.H.
      • Bernard C.C.
      • Brück W.
      • Weber M.S.
      Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.
      ). It is clear that pathogenic antibodies, possibly secondarily to damage, are generated during disease progression in MS (
      • Willis S.N.
      • Stathopoulos P.
      • Chastre A.
      • Compton S.D.
      • Hafler D.A.
      • O’Connor K.C.
      Investigating the antigen specificity of multiple sclerosis central nervous system-derived immunoglobulins.
      ) and can cause demyelination and pathology when injected in to animals (
      • Linington C.
      • Bradi M.
      • Lassmann H.
      • Brunner C.
      • et al.
      Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein.
      ,
      • Morris-Downes M.M.
      • Smith P.A.
      • Rundle J.L.
      • Piddlesden S.J.
      • et al.
      Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.
      ). However, it appears that autoreactive T cells are necessary for the clinical disease to develop. It was shown in animals studies that T cells initiate the blood-brain barrier disturbances that facilitate entry of B cells and immunoglobulins into the CNS. Once in the CNS autoantibodies bind to oligodendrocytes and myelin, activate complement and induce demyelination that subsequently enhances inflammation and increases EAE severity.

      3. B cell as immune modulators

      Emerging evidence using B cell-deficient mice has revealed a complex role for B cells in antigen presentation, cytokine production and immune regulation during EAE (Fig. 1). While early studies showed that depletion of IgM from birth prevented B cell maturation and subsequent EAE induction (
      • Willenborg D.O.
      • Prowse S.J.
      Immunoglobulin-deficient rats fail to develop experimental allergic encephalomyelitis.
      ), recent evidence for B cells in EAE comes from studies using the MOG EAE model in C57BL/6 following genetic ablation or B cell depletion using antibodies directed to CD20 (Table 1). These studies suggest that B cells are necessary for EAE induced with recombinant MOG protein but not MOG35–55 EAE, revealing the critical impact of the source and nature of immunogens (
      • Weber M.S.
      • Prod’homme T.
      • Patarroyo J.
      • Molnarfi N.
      • et al.
      B cell activation influences T cell polarization and outcome of anti-CD20 B cell depletion in CNS autoimmunity.
      ). This difference in EAE induction can be explained by the role of B cells to differentially present MOG protein and peptide (
      • Weber M.S.
      • Prod’homme T.
      • Patarroyo J.
      • Molnarfi N.
      • et al.
      B cell activation influences T cell polarization and outcome of anti-CD20 B cell depletion in CNS autoimmunity.
      ). While genetic ablation of MHC class II antigens on B cells abrogates antigen presentation of MOG to T cells and thus inhibits EAE induction (
      • Molnarfi N.
      • Schulze-Topphoff U.
      • Weber M.S.
      • Patarroyo J.C.
      • et al.
      MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies.
      ), antigen presentation by B cells and thus EAE induction requires dendritic cells (
      • Parker C.R.
      • Archambault A.S.
      • Sim J.
      • Ferris S.T.
      • et al.
      B cell antigen presentation is sufficient to drive neuroinflammation in an animal model of multiple sclerosis.
      ). Next to the requirement of MHC class II the expression of T cell costimulatory molecules expressed by B cells, such as CD80/CD86 and CD40, plays a crucial role in EAE (
      • Mann M.K.
      • Maresz K.
      • Shriver L.P.
      • Tan Y.
      • et al.
      B cell regulation of CD4 CD25 T regulatory cells and IL-10 Via B7 is essential for recovery from experimental autoimmune encephalomyelitis.
      ,
      • Ray A.
      • Mann M.K.
      • Basu S.
      • Dittel B.
      A case for regulatory B cells in controlling the severity of autoimmune-mediated inflammation in experimental autoimmune encephalomyelitis and multiple sclerosis.
      ). While mice (in this case B10. PL) in which B cells lack co-stimulatory CD80/CD86 molecules develop EAE, they fail to recover from acute disease due to a delay in IL-10 production and mobilization of Foxp3 regulatory T cells (
      • Mann M.K.
      • Maresz K.
      • Shriver L.P.
      • Tan Y.
      • et al.
      B cell regulation of CD4 CD25 T regulatory cells and IL-10 Via B7 is essential for recovery from experimental autoimmune encephalomyelitis.
      ). In addition, B cells produce pro-inflammatory and anti-inflammatory cytokines that impact on EAE (
      • Molnarfi N.
      • Schulze-Topphoff U.
      • Weber M.S.
      • Patarroyo J.C.
      • et al.
      MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies.
      ) (Table 2).
      Fig. 1.
      Fig. 1The multifunctional roles of B cells in EAE and MS. A) B cells act as antigen presenting cells that activate autoreactive T cells. B) B cells mature to plasma cells that produce antibodies including CNS reactive autoantigens. C) Mature B cells process antigens, and once activated produce pro- and anti-inflammatory cytokines important in D) the development of tertiary lymphoid tissue. B cells also harbor Epstein-Barr Virus (EBV) thought to a key etiological agent in MS.
      Table 2B cell production of cytokines during EAE.
      CytokineProduced viaEffect mechanismEffect on EAEReference
      LT1α2βFDCs and CXCL13Follicle formationIncreased inflammation
      • Magliozzi R.
      • Columba-Cabezas S.
      • Serafini B.
      • Aloisi F.
      Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis.
      ,
      • Magliozzi R.
      • Howell O.
      • Serafini B.
      • Nicholas R.
      • et al.
      Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.
      TNFLT1α2βIncreased IFN-γ and inflammationIncreased severity
      • Menrad L.C.
      • Minns L.A.
      • Darche S.
      • Mielcarz D.W.
      • et al.
      B cells amplify IFN-γ production by T cells via a TNF-α-mediated mechanism.
      ,
      • Matejuk A.
      • Dwyer J.
      • Ito A.
      • Bruender Z.
      • et al.
      Effects of cytokine deficiency on chemokine expression in CNS of mice with EAE.
      IL-6Innate receptorsIncreased ICAM and VCAM expression. Increased T cell infiltrationIncreased severity
      • Okuda Y.
      • Sokada S.
      • Fujimura H.
      • Saeki Y.
      • et al.
      IL-6 plays a crucial role in the induction phase of myelin oligodendrocyte glycoprotein 35–55 induced experimental autoimmune encephalomyelitis.
      ,
      • Eugster H.P.
      • Frei K.
      • Kopf M.
      • Lassman H.
      • et al.
      IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.
      IL-10CD40 and B7Increased Tregs, decreased inflammationReduced severity
      • Bettelli E.
      • Das M.P.
      • Howard E.D.
      • Weiner H.L.
      • et al.
      IL10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10-and IL-4 deficient and transgenice mice.
      ,
      • Fillatreau S.
      • Sweenie C.H.
      • McGeachy M.J.
      • Gray D.
      • et al.
      B cells regulate autoimmunity by provision of IL-10.
      • Matsushita T.
      • Yanaba K.
      • Bouazi J.D.
      • Fujimoto M.
      • et al.
      Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.
      IL-35TLR4 and CD40Decreased APC functionReduced severity
      • Shen P.
      • Roch T.
      • Lampropoulau V.
      • O’Connor R.A.
      • et al.
      IL‑35–producing B cells are critical regulators of immunity during autoimmune and infectious diseases.
      Abbreviations: APC – antigen presenting cell; IL – Interleukin; Tregs – regulatory T cells, TLR – Toll-Like receptor, FDCs – Follicular Dendritic Cells, CXCL13 – Chemokine CXC-motif Ligand 13.
      B cells secrete lymphotoxin α1-β2 (LTα1β2) essential for lymph node expansion and development of tertiary lymphoid tissue, which are sometimes present in the CNS during EAE (
      • Lassmann H.
      • Niedobitek G.
      • Aloisi F.
      • Middeldorp J.M.
      • NeuroproMiSe EBV Working Group
      Epstein-Barr virus in the multiple sclerosis brain: a controversial issue – report on a focused workshop held in the Centre for Brain Research of the Medical University of Vienna, Austria.
      ). LTα1β2 is observed in the CNS during EAE in SJL mice (
      • Magliozzi R.
      • Columba-Cabezas S.
      • Serafini B.
      • Aloisi F.
      Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis.
      ,
      • Columba-Cabezas S.
      • Griguoli M.
      • Rosicarelli B.
      • Magliozzi R.
      • et al.
      Suppression of established experimental autoimmune encephalomyelitis and formation of meningeal lymphoid follicles by lymphotoxin β receptor-Ig fusion protein.
      ) and clinical disease is prevented with antibodies to LTα1β2 receptor, due to inhibition of CXCL13 important for lymphoid follicle development (
      • Columba-Cabezas S.
      • Griguoli M.
      • Rosicarelli B.
      • Magliozzi R.
      • et al.
      Suppression of established experimental autoimmune encephalomyelitis and formation of meningeal lymphoid follicles by lymphotoxin β receptor-Ig fusion protein.
      ). Antigen activated B cells also produce TNF, a key cytokine in EAE (
      • Matejuk A.
      • Dwyer J.
      • Ito A.
      • Bruender Z.
      • et al.
      Effects of cytokine deficiency on chemokine expression in CNS of mice with EAE.
      ). Another B cell-derived cytokine is interleukin-6 (IL-6) that is increased during EAE. That IL-6 production by B cells is key for EAE induction has been shown following B cell depletion studies in mice (
      • Barr T.A.
      • Shen P.
      • Brown S.
      • Lampropulaulau V.
      • et al.
      B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells.
      ). A mechanism by which IL-6 may contribute to EAE is by regulating T cell infiltration into the CNS since expression of ICAM-1 and VCAM-1 are absent in IL-6 deficient mice (
      • Eugster H.P.
      • Frei K.
      • Kopf M.
      • Lassman H.
      • et al.
      IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.
      ). B cells also produce IL-10 as well as IL-35; cytokines that reduce EAE severity. This is supported by studies in which regulatory B cells (Bregs) extracted from mice during EAE remission, produce IL-10 in a CD40 dependent manner and, following transfer into mice with active EAE induce recovery (
      • Fillatreau S.
      • Sweenie C.H.
      • McGeachy M.J.
      • Gray D.
      • et al.
      B cells regulate autoimmunity by provision of IL-10.
      ). Thus, IL-10 producing Bregs are important for disease recovery. Furthermore, IL-10-producing B cells play an important role in EAE initiation by down-regulating the conditions leading to T cell CNS infiltration (
      • Matsushita T.
      • Yanaba K.
      • Bouazi J.D.
      • Fujimoto M.
      • et al.
      Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.
      ,
      • Matsushita T.
      • Horikawa M.
      • Iwata Y.
      • Tedder T.F.
      Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling EAE initiation and late-phase immunopathogenesis.
      ). Finally, IL-35 producing B cells also control EAE severity since mice lacking these B cells develop exacerbated EAE. IL-35 regulates APC functions of B cell that together with IL-10 induces differentiation of plasma cells. Mice in which B cells lack IL-35 have stronger antigen presentation capabilities, this is associated with stronger inflammatory responses and more severe EAE (
      • Shen P.
      • Roch T.
      • Lampropoulau V.
      • O’Connor R.A.
      • et al.
      IL‑35–producing B cells are critical regulators of immunity during autoimmune and infectious diseases.
      ).

      4. B cells and autoantibodies in MS

      B cells, plasma cells, plasmablasts and antibodies directed to oligodendrocytes, myelin and neurons are detectable in peripheral blood and the cerebrospinal fluid (CSF) of people with MS (pwMS) (
      • Olsson T.
      • Baig S.
      • Höjeberg B.
      • Link H.
      Antimyelin basic protein and antimyelin antibody-producing cells in multiple sclerosis.
      ,
      • van Noort J.M.
      • Verbeek R.
      • Meilof J.F.
      • Polman C.H.
      • Amor S.
      Autoantibodies against alpha B-crystallin, a candidate autoantigen in multiple sclerosis, are part of a normal human immune repertoire.
      ,
      • Amor S.
      • van der Star B.J.
      • Bosca I.
      • Raffel J.
      • Gnanapavan S.
      • Watchorn J.
      • Kuhle J.
      • Giovannoni G.
      • Baker D.
      • Malaspina A.
      • Puentes F.
      Neurofilament light antibodies in serum reflect response to natalizumab treatment in multiple sclerosis.
      ). In the CSF oligoclonal IgG bands (OCBs), present in 95% pwMS, are an established diagnostic tool for MS. That immunoglobulins may play a role in disease is reflected by the close association of lesions to CSF flow such as ventricular and subpial lesions, and that OCB-negative patients are characterized by less global and regional brain atrophy (
      • Ferreira D.
      • Voevodskaya O.
      • Imrell K.
      • Stawiarz L.
      • Spulber G.
      • Wahlund L.O.
      • Hillert J.
      • Westman E.
      • Karrenbauer V.D.
      Multiple sclerosis patients lacking oligoclonal bands in the cerebrospinal fluid have less global and regional brain atrophy.
      ). Further support comes from a study showing that clonally expanded plasma cells from the CSF of pwMS induce myelin damage in vitro, thus implicating intrathecal IgG in MS pathogenesis (
      • Blauth K.
      • Soltys J.
      • Matschulat A.
      • Reiter C.R.
      • Ritchie A.
      • Baird N.L.
      • Bennett J.L.
      • Owens G.P.
      Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.
      ). In addition the CNS immunoglobulins and complement deposits are present on disintegrated myelin sheath and demyelinating lesions (
      • Raine C.S.
      • Cannella B.
      • Hauser S.L.
      • Genain C.P.
      Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation.
      ,
      • Lucchinetti C.
      • Bruck W.
      • Parisi J.
      • Scheithauer B.
      • et al.
      Heterogeneity of multiple sclerosis lesions: Implications for the pathogenesis of demyelination.
      ,
      • Storch M.K.
      • Piddlesden S.
      • Haltia M.
      • Livanainen M.
      • et al.
      Multiple sclerosis: in situ evidence for antibody- and complement-mediated d demyeli.
      ), indicating that like EAE CNS reactive antibodies mediate demyelination and neuronal damage (Fig. 2); findings that have been confirmed in vitro studies (
      • Zhou D.
      • Srivastava R.
      • Nessler S.
      • Grummel V.
      • Sommer N.
      • et al.
      Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis.
      ,
      • Blauth K.
      • Soltys J.
      • Matschulat A.
      • Reiter C.R.
      • Ritchie A.
      • Baird N.L.
      • Bennett J.L.
      • Owens G.P.
      Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.
      ). In addition, the finding of B cell follicle-like structures as well as chemokines and cytokines that support B cell development all provide support for a key role of B cells, plasma cells and autoantibodies in MS pathogenesis. Like EAE, B cells extracted from pwMS and stimulation with CD40L and IL-4 have been shown to present myelin specific antigens to autoreactive T cells (
      • Harp C.
      • Lovett-Racke A.
      • Racke M.
      • Frohman E.
      • et al.
      Impact of myelin-specific antigen presenting B cells on T cell activation in multiple sclerosis.
      ) indicating that B cells also have the capacity for antigen presentation in MS.
      Fig. 2.
      Fig. 2Antibody functions in MS and EAE. The primary effect of pathogenic antibodies is induction of tissue damage. Anti-myelin antibodies produced by plasma cells (1) attach to myelin leading to opsonisation (2). Macrophages bearing FcR bind to antibody on myelin (3). Alternatively, macrophages bind immunoglobulin via FcR receptors (4) that then bind to myelin inducing antibody dependent cell mediate cytotoxicity (ADCC). Antibodies on myelin (5) trigger complement activation leading to the formation of membrane attack complex (MAC) and cell death (6).
      Moreover, B cell follicles have been reported in the meninges of people with secondary progressive MS (pwSPMS;
      • Serafini B.
      • Rosicarelli B.
      • Magliozzi R.
      • Stigliano E.
      • et al.
      Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.
      ), although this is controversial since follicles are not present in all pwSPMS and MS cohorts (
      • Peferoen L.A.
      • Lamers F.
      • Lodder L.N.
      • Gerritsen W.H.
      • Huitinga I.
      • Melief J.
      • Giovannoni G.
      • Meier U.
      • Hintzen R.Q.
      • Verjans G.M.
      • van Nierop G.P.
      • Vos W.
      • Peferoen-Baert R.M.
      • Middeldorp J.M.
      • van der Valk P.
      • Amor S.
      Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis.
      ). In pwMS in which follicles are observed, the presence of the ectopic follicles were more pronounced in younger patients with more severe disease than the group in which follicles were not observed (
      • Magliozzi R.
      • Howell O.
      • Serafini B.
      • Nicholas R.
      • et al.
      Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.
      ). The number of these B cells characterized by up regulated chemokine receptors to CXCL13 (
      • Corcione A.
      • Casazza S.
      • Ferretti E.
      • Glunti D.
      • et al.
      Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis.
      ), were significantly higher in the CSF as compared to blood. Together with LT1α2β, CXCL13 has been found in the CSF and MS brain tissue in active lesions (
      • Corcione A.
      • Casazza S.
      • Ferretti E.
      • Glunti D.
      • et al.
      Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis.
      ,
      • Krumbholz M.
      • Thiel D.
      • Cepok S.
      • Hemmer B.
      • et al.
      Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment.
      ) and may thus contribute to disease severity by aiding inflammation. In support of this, B cell follicles and meningeal infiltrates have been associated with cortical grey matter lesions widely considered to contribute to cognitive decline in MS (
      • Magliozzi R.
      • Howell O.
      • Serafini B.
      • Nicholas R.
      • et al.
      Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology.
      ). How these follicles arise is as yet unknown, although several studies suggest strong association with EBV widely considered to be an etiological agent in MS.
      There is also clinical evidence for regulatory B cell functions in MS (
      • Fillatreau S.
      • Sweenie C.H.
      • McGeachy M.J.
      • Gray D.
      • et al.
      B cells regulate autoimmunity by provision of IL-10.
      ,
      • Shen P.
      • Roch T.
      • Lampropoulau V.
      • O’Connor R.A.
      • et al.
      IL‑35–producing B cells are critical regulators of immunity during autoimmune and infectious diseases.
      ). Recently a subset of IL-10 producing B cells has been identified in blood of healthy controls and pwMS (
      • Iwata Y.
      • Matshushita T.
      • Horikawa M.
      • DiLillo D.J.
      • et al.
      Characterization of a rare IL-10–competent B-cell subset in humans that parallels mouse regulatory B10 cells.
      ) in which production is lower in pwMS and then restored or increased via therapy (
      • Özenci V.
      • Kouwenhoven M.
      • Huang Y.M.
      • Kivisäkk P.
      • et al.
      Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-α)- and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-β) treatment.
      ). As in rodents production of IL-10 in human B cells increases following treatment with CD40L, lipopolysaccharide (LPS) or CpG. Together the gathered data on the role for B cells in MS and EAE demonstrates its involvement in antigen presentation, cytokine production and T cell activation. This abundant evidence demonstrates that the role of B cells in MS pathology is not merely antibody production. Therefore targeting B cell in the treatment of MS has been a rational approach towards improving the MS pathology.

      5. B cell therapies

      Many current therapies for MS also inhibit or modify B cell function. For example the cytostatic agents mitoxantrone, cyclophosphamide preferentially kill B cells due to their high basal proliferation rate (Fox 2004), alemtuzumab depletes T and B cells (
      • Ruck T.
      • Bittner S.
      • Wiendl H.
      • Meuth S.G.
      Alemtuzumab in multiple sclerosis: mechanism of action and beyond.
      ) and glatiramer acetate reduces B cells numbers in subset of pwMS (
      • Rovituso D.M.
      • Duffy C.E.
      • Schroeter M.
      • Kaiser C.C.
      • Kleinschnitz C.
      • Bayas A.
      • Elsner R.
      • Kuerten S.
      The brain antigen-specific B cell response correlates with glatiramer acetate responsiveness in relapsing-remitting multiple sclerosis patients.
      ). Likewise, fingolimod decreases memory B cells but increases naive B cells (
      • Claes N.
      • Dhaeze T.
      • Fraussen J.
      • Broux B.
      • Van Wijmeersch B.
      • Stinissen P.
      • Hupperts R.
      • Hellings N.
      • Somers V.
      Compositional changes of B and T cell subtypes during fingolimod treatment in multiple sclerosis patients: a 12-month follow-up study.
      ) while dimethyl fumarate is reported to also reduce B cells numbers (
      • Spencer C.M.
      • Crabtree-Hartman E.C.
      • Lehmann-Horn K.
      • Cree B.A.
      • Zamvil S.S.
      Reduction of CD8(+) T lymphocytes in multiple sclerosis patients treated with dimethyl fumarate.
      ). In contrast, natalizumab increases circulating B cells while IFN-β treatment increases CD19, CD24, CD38 B cells in peripheral blood, although whether B cells in the CNS are affected has not been reported. Cladribine is a highly effective depleting agent of peripheral B cells, compared to T cells (
      • Mitosek-Szewczyk K.1
      • Tabarkiewicz J.
      • Wilczynska B.
      • Lobejko K.
      • Berbecki J.
      • Nastaj M.
      • Dworzanska E.
      • Kolodziejczyk B.
      • Stelmasiak Z.
      • Rolinski J.
      Impact of cladribine therapy on changes in circulating dendritic cell subsets, T cells and B cells in patients with multiple sclerosis.
      ) and through targeting dividing and non-dividing lymphocytes inhibits lesion formation in both relapsing MS and progressive MS (
      • Rice G.P.
      • Filippi M.
      • Comi G.
      Cladribine and progressive MS: clinical and MRI outcomes of a multicenter controlled trial. Cladribine MRI Study Group.
      ,
      • Giovannoni G.
      • Comi G.
      • Cook S.
      • Rammohan K.
      • Rieckmann P.
      • Soelberg Sørensen P.
      • Vermersch P.
      • Chang P.
      • Hamlett A.
      • Musch B.
      • Greenberg S.J.
      • CLARITY Study Group
      A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis.
      ). It is the only MS disease-modifying agent that is CNS penetrant, with an immunomodulatory action that is active in the CNS, which has the capacity to target plasma cells and can inhibit oligoclonal band protein formation (
      • Sipe J.C.
      • Romine J.S.
      • Koziol J.A.
      • McMillan R.
      • Zyroff J.
      • Beutler E.
      Cladribine in treatment of chronic progressive multiple sclerosis.
      ). Whilst efficacy in relapsing MS has been reported (
      • Giovannoni G.
      • Comi G.
      • Cook S.
      • Rammohan K.
      • Rieckmann P.
      • Soelberg Sørensen P.
      • Vermersch P.
      • Chang P.
      • Hamlett A.
      • Musch B.
      • Greenberg S.J.
      • CLARITY Study Group
      A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis.
      ), trials in progressive MS were too short to determine efficacy in progression (
      • Rice G.P.
      • Filippi M.
      • Comi G.
      Cladribine and progressive MS: clinical and MRI outcomes of a multicenter controlled trial. Cladribine MRI Study Group.
      ). Unfortunately, due to probably unfounded concerns on safety, oral cladribine was withdrawn (
      • Pakpoor J.
      • Disanto G.
      • Altmann D.R.
      • Pavitt S.
      • Turner B.P.
      • Marta M.
      • Juliusson G.
      • Baker D.
      • Chataway J.
      • Schmierer K.
      No evidence for higher risk of cancer in patients with multiple sclerosis taking cladribine.
      ). In summary, although studies clearly reveal the multifunctional roles of B cells in MS several therapeutic approaches can be applied to modulate B cell functions.

      5.1 CD20 targeted therapy

      Three anti-CD20 antibodies rituximab, a chimeric human/mouse IgG1 antibody; ocrelizumab, a humanized antibody, and ofatumumab, a full recombinant human IgG1 antibody have been used in clinical trials for MS (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • et al.
      B-cell depletion with rituximab in relapsing–remitting multiple sclerosis.
      ,
      • Kappos L.
      • Li D.
      • Calabresi P.
      • O’Connor P.
      • et al.
      Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicenter trial.
      ,
      • Sorensen P.S.
      • Lisby S.
      • Grove R.
      • Derosier F.
      • et al.
      Safety and efficacy of ofatumumab in relapsing-remitting multiple sclerosis: a phase 2 study.
      ). All antibodies deplete cells expressing CD20, a surface antigen present on maturating B cells, from pre-B cells to plasmablasts, precursors of plasma cells (Fig. 3), as well as a population of T cells. The first study of rituximab, conducted in 5 people with primary progressive MS (pwPPMS), showed that most peripheral blood B cells were depleted until 14 months (
      • Monson N.L.
      • Cravens P.D.
      • Frohman E.M.
      • Hawker K.
      • et al.
      Effect of rituximab on the peripheral blood and cerebrospinal fluid B cells in patients with primary progressive multiple sclerosis.
      ). The depletion of CSF B cells was less efficient. A phase I open study, designed to assess the drug safety and tolerability in RRMS did not reveal serious adverse effects (
      • Bar-Or A.
      • Calabresi P.A.
      • Arnold D.
      • Markowitz C.
      • et al.
      Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial.
      ). Clinical trials that followed showed that B cell depletion effectively suppressed MS disease activity (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • et al.
      B-cell depletion with rituximab in relapsing–remitting multiple sclerosis.
      ,
      • Hawker K.
      • O’Connor P.
      • Freedman M.
      • Calabresi P.
      • Antel J.
      • et al.
      Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial.
      ,
      • Kappos L.
      • Li D.
      • Calabresi P.
      • O’Connor P.
      • et al.
      Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicenter trial.
      ). In a double-blind placebo-controlled phase II trial, rituximab reduced newly formed CNS lesions and relapses in relapsing remitting MS (RRMS) (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • et al.
      B-cell depletion with rituximab in relapsing–remitting multiple sclerosis.
      ). Studies with ocrelizumab, reduced gadolinium-enhancing lesions in RRMS by 89% compared to placebo and Interferon β (
      • Kappos L.
      • Li D.
      • Calabresi P.
      • O’Connor P.
      • et al.
      Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicenter trial.
      ) and inhibited relapsing MS and lesion formation in phase III trails (
      • Hauser S.L.
      • Comi G.C.
      • Hartung H.-P.
      • Selmaj KTraboulsee A.
      • Bar-Or A.
      • Arnold D.L.
      • Klingelschmitt G.
      • Kakarieka A.
      • Lublin F.
      • Garren H.
      • Kappos L.
      • on behalf of the OPERA I and II clinical investigators
      Efficacy and safety of ocrelizumab in relapsing multiple sclerosis - results of the interferon-beta-1a-controlled, doubleblind, phase III OPERA I and II studies.
      ). Ocrelizumab has shown some positive results in PPMS in the phase III trial (
      • Montalban E.
      Efficacy and safety of Ocrelizumab in primary progressive multiple sclerosis- results of the placebo-controlled, double blind, phase III ORATORIO study.
      ). Here, the study focused on younger people with active disease and closer to progressive onset of MS, based on the responder profile of an earlier trial in PPMS with rituximab (
      • Hawker K.
      • O’Connor P.
      • Freedman M.
      • Calabresi P.
      • Antel J.
      • et al.
      Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial.
      ). Lastly, ofatumumab, an approved drug for chronic lymphocytic leukemia is expected to be far less antigenic than rituximab. This was examined in RRMS, revealing over 99% reduction in newly formed lesions in RRMS compared to controls. No increase in adverse effects was noticed in treated compared to controls (
      • Sorensen P.S.
      • Lisby S.
      • Grove R.
      • Derosier F.
      • et al.
      Safety and efficacy of ofatumumab in relapsing-remitting multiple sclerosis: a phase 2 study.
      ). While CD20 targeting therapies reduce MS lesions and clinical relapses the immunoglobulin levels are not significantly affected (
      • Hauser S.L.
      • Waubant E.
      • Arnold D.L.
      • Vollmer T.
      • et al.
      B-cell depletion with rituximab in relapsing–remitting multiple sclerosis.
      ), not surprisingly since plasma cells do not express CD20 and are therefore not depleted. This suggests that CD20+ cell depletion interferes with antigen presentation, T cell activation or cytokine production (Fig. 4). The preclinical findings in EAE showing that B cells polarize T cell differentiation were confirmed in pwRRMS treated with anti-CD20 antibody treatment, emphasizing the APC function of B cells in RRMS (
      • Bar-Or A.
      • Fawaz L.
      • Fan B.
      • Darlington P.J.
      • et al.
      Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?.
      ). The number of CSF T cells also decreased following treatment in the majority of RRMS patients (
      • Cross A.H.
      • Stark J.L.
      • Lauber J.
      • Ramsbottom M.J.
      • Lyons J.A.
      Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients.
      ).
      Fig. 3.
      Fig. 3B cell development. CD19 is highly expressed throughout B cell development but not on terminally differentiated plasma cells. CD20 is expressed on the majority of B cell subtypes but is down-regulated in plasmablasts. B cell activating factor receptor (BAFF-R) is expressed later in development from immature B cells to plasmablasts.
      Fig. 4.
      Fig. 4Effect of anti-CD20 antibody in MS. Anti-CD20 antibody binds to and depletes CD20+ cells, including CD20+ pro-inflammatory CD4 and CD8 cells, involved in CNS inflammation and demyelination. Breg – regulatory B cells, Mϕ – macrophage CTL: cytotoxic T cell.

      5.2 CD20+ T cells

      In addition to the effect on B cell function and thereby indirectly affecting T cells, emerging evidence shows that anti-CD20 antibody therapies also directly affect CD20+ T cells. Interestingly, evidence for the direct T cell effect was suggested in early rituximab studies for treatment of rheumatoid arthritis (
      • Wilk E.
      • Witte T.
      • Marquardt N.
      • Horvath T.
      • et al.
      Depletion of functionally active CD20+ T cells by rituximab treatment.
      ) who also reported CD20+ T cells in peripheral blood of healthy people and those with rheumatoid arthritis (
      • Wilk E.
      • Witte T.
      • Marquardt N.
      • Horvath T.
      • et al.
      Depletion of functionally active CD20+ T cells by rituximab treatment.
      ). CD20+ cells comprise both CD8 and CD4 T cell subtypes and are depleted with CD20+ B cells during rituximab therapy (
      • Wilk E.
      • Witte T.
      • Marquardt N.
      • Horvath T.
      • et al.
      Depletion of functionally active CD20+ T cells by rituximab treatment.
      ). Data from RRMS supports the presence of CD20+ T cells in the peripheral blood and CSF in pwMS (
      • Palanichamy A.
      • Jahn S.
      • Nickles D.
      • Derstine M.
      • et al.
      Rituximab efficiently depletes increased CD20-expressing T cells in multiple sclerosis patients.
      ) and cerebral subventricular white matter samples (
      • Holley J.E.
      • Bremer E.
      • Kendall A.C.
      • de Bruyn M.
      • et al.
      CD20+ inflammatory T cells are present in blood and brain of multiple sclerosis patients and can be selectively tarteted for apoptotic elimination.
      ). Since CD20 expression is lower on CSF T cells was less noticeable, they were characterized as CD20dim T cells that are both CD8+ and CD4+. Interestingly, CD20-specific antibodies predominantly depleted CD8+ CD20dim T cells although the overall CD4/CD8 ratio remained unchanged (
      • Palanichamy A.
      • Jahn S.
      • Nickles D.
      • Derstine M.
      • et al.
      Rituximab efficiently depletes increased CD20-expressing T cells in multiple sclerosis patients.
      ). In the CNS, particularly in active MS lesions, T cells are CD20high and express IL-17 and IFN-γ indicating a more pro-inflammatory profile (
      • Holley J.E.
      • Bremer E.
      • Kendall A.C.
      • de Bruyn M.
      • et al.
      CD20+ inflammatory T cells are present in blood and brain of multiple sclerosis patients and can be selectively tarteted for apoptotic elimination.
      ) suggesting that CD20-specific immunotherapy may thus modify disease by directly depleting inflammatory CD20high/dim T cells. Furthermore, it was demonstrated that rituximab was most effective in controlling EAE in human CD20 transgenic mice when antibody treatment induced marked T cell depletion (
      • Weber M.S.
      • Prod’homme T.
      • Patarroyo J.
      • Molnarfi N.
      • et al.
      B cell activation influences T cell polarization and outcome of anti-CD20 B cell depletion in CNS autoimmunity.
      ).

      5.3 Risks of infection

      Due to the broad B cell target, anti-CD20 antibodies also deplete B cells that regulate immune responses including those controlling viruses. Incidental cases of progressive multifocal leukoencephalopathy (PML) in people treated with rituximab with other immune disorders have been observed (
      • Clifford D.B.
      • Ances B.
      • Costello C.
      • Rosen-Schmidt S.
      • Andersson M.
      • Parks D.
      • Perry A.
      • Yerra R.
      • Schmidt R.
      • Alvarez E.
      • Tyler K.L.
      Rituximab-associated progressive multifocal leukoencephalopathy in rheumatoid arthritis.
      ), which suggests a risk of PML development in MS. Furthermore, ocrelizumab development in rheumatoid arthritis and systemic lupus erythematosus was halted because it was linked to serious life-threatening infections (
      • Tak P.P.
      • Mease P.J.
      • Genovese M.C.
      • Kremer J.
      • Haraoui B.
      • Tanaka Y.
      • Bingham 3rd, C.O.
      • Ashrafzadeh A.
      • Travers H.
      • Safa-Leathers S.
      • Kumar S.
      • Dummer W.
      Safety and efficacy of ocrelizumab in patients with rheumatoid arthritis and an inadequate response to at least one tumor necrosis factor inhibitor: results of a forty-eight–week randomized, double-blind, placebo-controlled, parallel-group phase III trial.
      ). However, development of B cell depletion has a better risk: benefit profile in MS, because of the poor prognosis of MS. Whilst it has similar levels of efficacy to alemtuzumab in relapsing MS (
      • Coles A.J.
      • Twyman C.L.
      • Arnold D.L.
      • Cohen J.A.
      • Confavreux C.
      • Fox E.J.
      • Hartung H.P.
      • Havrdova E.
      • Selmaj K.W.
      • Weiner H.L.
      • Miller T.
      • Fisher E.
      • Sandbrink R.
      • Lake S.L.
      • Margolin D.H.
      • Oyuela P.
      • Panzara M.A.
      • Compston D.A.
      CARE-MS II investigators. Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial.
      ;
      • Cohen J.A.
      • Coles A.J.
      • Arnold D.L.
      • Confavreux C.
      • Fox E.J.
      • Hartung H.P.
      • Havrdova E.
      • Selmaj K.W.
      • Weiner H.L.
      • Fisher E.
      • Brinar V.V.
      • Giovannoni G.
      • Stojanovic M.
      • Ertik B.I.
      • Lake S.L.
      • Margolin D.H.
      • Panzara M.A.
      • Compston D.A.
      CARE-MS I investigators. Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial.
      ,
      • Kappos L.
      • Li D.
      • Calabresi P.
      • O’Connor P.
      • et al.
      Ocrelizumab in relapsing–remitting multiple sclerosis: A phase 2, randomised, placebo-controlled, multicenter trial.
      ), it does not cause the high incidence of secondary B cell autoimmunities following treatment with alemtuzumab (
      • Cohen J.A.
      • Coles A.J.
      • Arnold D.L.
      • Confavreux C.
      • Fox E.J.
      • Hartung H.P.
      • Havrdova E.
      • Selmaj K.W.
      • Weiner H.L.
      • Fisher E.
      • Brinar V.V.
      • Giovannoni G.
      • Stojanovic M.
      • Ertik B.I.
      • Lake S.L.
      • Margolin D.H.
      • Panzara M.A.
      • Compston D.A.
      CARE-MS I investigators. Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial.
      ,
      • Tuohy O.
      • Costelloe L.
      • Hill-Cawthorne G.
      • Bjornson I.
      • Harding K.
      • Robertson N.
      • May K.
      • Button T.
      • Azzopardi L.
      • Kousin-Ezewu O.
      • Fahey M.T.
      • Jones J.
      • Compston D.A.
      • Coles A.
      Alemtuzumab treatment of multiple sclerosis: long-term safety and efficacy.
      ) thus making it an attractive new treatment for MS.

      6. Novel B cell targeting therapies

      Anti-CD19 therapy, under development for neuromyelitis optica has also been suggested as a new treatment for MS. This approach targets B cells including plasma blasts and short-lived plasma cells (
      • Stüve O.
      • Warnke C.
      • Deason K.
      • Stangel M.
      • et al.
      CD19 as a molecular target in CNS autoimmunity.
      ), thereby reducing the levels of pathogenic antibodies (
      • Stüve O.
      • Warnke C.
      • Deason K.
      • Stangel M.
      • et al.
      CD19 as a molecular target in CNS autoimmunity.
      ). MEDI-551, a humanized anti-CD19 IgG1 antibody has been reported to reduce lesions in RRMS (Aguis et al., 2015). The humanized antibody is able to bind and deplete a broader range of B cells but its method of action is similar to that of anti-CD20. In a humanized CD19 B cell animal model, anti-CD19 treatment could reduce antibody levels because of its ability to deplete plasma blasts (
      • Yazawa N.
      • Hamaguchi Y.
      • Poe J.C.
      • Teder T.F.
      Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease.
      ). In a CD19 and CD20 transgenic animal model, MEDI-551 depletes B cells by depleting pro-B cells, delaying B cell renewal (
      • Agius M.
      • Klodowska-Duda G.
      • Maciejowski M.
      • Potemkowski A.
      • Sweeny S.
      • Li J.
      • Yao W.
      • Patra N.
      • Ratchford J.N.
      • Katz E.
      • Flor A.
      Safety and tolerability of MEDI-551 in patients with relapsing forms of multiple sclerosis: results from a phase 1 randomised, placebo-controlled, escalating intravenous and subcutaneous dose study.
      ;
      • Herbst R.
      • Wang Y.
      • Gallagher S.
      • Mittereder N.
      • Kuta E.
      • Damschroder M.
      • Woods R.
      • Rowe D.C.
      • Cheng L.
      • Cook K.
      • Evans K.
      • Sims G.P.
      • Pfarr D.S.
      • Bowen M.A.
      • Dall'Acqua W.
      • Shlomchik M.
      • Tedder T.F.
      • Kiener P.
      • Jallal B.
      • Wu H.
      • Coyle A.J.
      B-cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody.
      ). However, such approaches might lead to more adverse events such as PML or opportunistic infections otherwise controlled by humoral immune responses in the CNS. In view of this VAY736, directed against the B-cell activating factor receptor (BAFF-R) has been designed to reduce the B cell fostering milieu in the CNS (Krumbholz et al., 2005,
      • Magliozzi R.
      • Columba-Cabezas S.
      • Serafini B.
      • Aloisi F.
      Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis.
      ). Upon binding to its receptor, BAFF aids B cell survival but it is also involved in B cell development and B cell mediated meningeal follicle formation (
      • Magliozzi R.
      • Columba-Cabezas S.
      • Serafini B.
      • Aloisi F.
      Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis.
      ). Since BAFF is elevated in MS, targeting the BAFF-R prevents its binding to take place thereby reducing B cell survival and lymphoid follicle-like structures. BAFF-R deficient mice with EAE had fewer mature B cells and increased EAE severity, suggesting a predominant effect on Breg (
      • Sasaki Y.
      • Casola S.
      • Kutok J.L.
      • Rajewsky K.
      • et al.
      TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology.
      ,
      • Kim S.S.
      • Richman D.P.
      • Zamvil S.S.
      • Agius M.A.
      • et al.
      Accelerated central nervous system autoimmunity in BAFF-receptor-deficient mice.
      ). Similarly increased disease severity was also shown with atacicept, a BAFF-immunoglobulin fusion protein that targets BAFF receptor and A ProlifeRation-Inducing ligand (APRIL). However, the study was halted after increased inflammatory activity was observed in pwRRMS (
      • Hartung H.P.
      • Kieseier B.C.
      Atacicept: targeting B cells in multiple sclerosis.
      ) demonstrating that B cell depletion is not universally a predictable and positive therapeutic effect, as was shown in initial studies in EAE, when B cell depletion inhibited, was ineffective or augmented disease (
      • Matsushita T.
      • Yanaba K.
      • Bouazi J.D.
      • Fujimoto M.
      • et al.
      Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.
      ).

      7. Future perspectives and concluding remarks

      Active RR- and PPMS is inhibited by peripheral B cell depletion with CD20-depleting antibodies, which may also be active in the CNS due to blood-brain barrier disturbances (
      • Bonnan M.
      • Ferrari S.
      • Bertandeau E.
      • Demasles S.
      • Krim E.
      • Miquel M.
      • Barroso B.
      Intrathecal rituximab therapy in multiple sclerosis: review of evidence supporting the need for future trials.
      ,
      • Hauser S.L.
      • Comi G.C.
      • Hartung H.-P.
      • Selmaj KTraboulsee A.
      • Bar-Or A.
      • Arnold D.L.
      • Klingelschmitt G.
      • Kakarieka A.
      • Lublin F.
      • Garren H.
      • Kappos L.
      • on behalf of the OPERA I and II clinical investigators
      Efficacy and safety of ocrelizumab in relapsing multiple sclerosis - results of the interferon-beta-1a-controlled, doubleblind, phase III OPERA I and II studies.
      ;
      • Montalban E.
      Efficacy and safety of Ocrelizumab in primary progressive multiple sclerosis- results of the placebo-controlled, double blind, phase III ORATORIO study.
      ). The thought that central B cell activity my drive progressive MS has led to the idea of targeting B cells via local administration (
      • Bonnan M.
      • Ferrari S.
      • Bertandeau E.
      • Demasles S.
      • Krim E.
      • Miquel M.
      • Barroso B.
      Intrathecal rituximab therapy in multiple sclerosis: review of evidence supporting the need for future trials.
      ,
      • Svenningsson A.
      • Bergman J.
      • Dring A.
      • Vågberg M.
      • Birgander R.
      • Lindqvist T.
      • Gilthorpe J.
      • Bergenheim T.
      Rapid depletion of B lymphocytes by ultra-low-dose rituximab delivered intrathecally.
      ). Yet, intrathecal administration of B cell targeting therapies fails to induce marked B cell depletion within the CNS and simply drains from the natural flow of CSF into the circulation and depletes peripheral B cells (
      • Komori M.
      • Lin Y.C.
      • Cortese I.
      • Blake A.
      • Ohayon J.
      • Cherup J.
      • Maric D.
      • Kosa P.
      • Wu T.
      • Bielekova B.
      Insufficient disease inhibition by intrathecal rituximab in progressive multiple sclerosis.
      ,
      • Topping J.
      • Dobson R.
      • Lapin S.
      • Maslyanskiy A.
      • Kropshofer H.
      • Leppert D.
      • Giovannoni G.
      • Evdoshenko E.
      The effects of intrathecal rituximab on biomarkers in multiple sclerosis.
      ). Therefore, it remains to be established whether intraventricular CD20 B cell depletion would have merit in targeting CNS-B cells or whether novel plasma cell depleting antibodies (
      • Moreau P.
      • Touzeau C.
      Elotuzumab for the treatment of multiple myeloma.
      ) will be necessary to target ectopic B cell follicles in the CNS.
      Immunodepleting agents, especially if used early after diagnosis, can induce long-term no-evidence of Disease Activity (NEDA;
      • Giovannoni G.
      • Turner B.
      • Gnanapavan S.
      • Offiah C.
      • Schmierer K.
      • Marta M.
      Is it time to target no evident disease activity (NEDA) in multiple sclerosis?.
      ) in MS (
      • Tuohy O.
      • Costelloe L.
      • Hill-Cawthorne G.
      • Bjornson I.
      • Harding K.
      • Robertson N.
      • May K.
      • Button T.
      • Azzopardi L.
      • Kousin-Ezewu O.
      • Fahey M.T.
      • Jones J.
      • Compston D.A.
      • Coles A.
      Alemtuzumab treatment of multiple sclerosis: long-term safety and efficacy.
      ). For many years MS has been thought to be a T cell mediated disease, largely due to similarities with EAE (
      • Baker D.
      • Amor S.
      Experimental autoimmune encephalomyelitis is a good model of multiple sclerosis if used wisely.
      ). With the knowledge that antibodies and B cells play a significant role in MS, B cell targeted therapies have been developed and have been found to have marked inhibitory activity on active MS although there are so far no surrogate markers to monitor efficacy. Success in treating relapsing and progressive MS by B cell therapy, has been hailed as the beginning of the end of the neurodegeneration of progressive MS (
      • Steinman L.
      • Zamvil S.S.
      Beginning of the end of two-stage theory purporting that inflammation then degeneration explains pathogenesis of progressive multiple sclerosis.
      ), however this remains to be established especially as inhibiting peripheral autoimmunity does not appear to stop EAE (
      • Al-Izki S.
      • Pryce G.
      • Jackson S.J.
      • Giovannoni G.
      • Baker D.
      Immunosuppression with FTY720 is insufficient to prevent secondary progressive neurodegeneration in experimental autoimmune encephalomyelitis.
      ) or MS following haematopoietic stem cell therapy (
      • Burt R.K.
      • Balabanov R.
      • Han X.
      • Sharrack B.
      • Morgan A.
      • Quigley K.
      • Yaung K.
      • Helenowski I.B.
      • Jovanovic B.
      • Spahovic D.
      • Arnautovic I.
      • Lee D.C.
      • Benefield B.C.
      • Futterer S.
      • Oliveira M.C.
      • Burman J.
      Association of nonmyeloablative hematopoietic stem cell transplantation with neurological disability in patients with relapsing-remitting multiple sclerosis.
      ). Even though there has been much focus on T cell autoimmunity in MS, it is clear that the highly effective agents can target B cells. Although the etiology of MS is unknown, it is clear that genetics and environmental factors contribute to susceptibility to MS. One consistent feature of MS is the potential role of EBV as a trigger of disease activity (
      • Pakpoor J.
      • Giovannoni G.
      • Ramagopalan S.V.
      Epstein-Barr virus and multiple sclerosis: association or causation?.
      ). Given that EBV readily infects B cells another mechanism by which B cell depletion may operate removal of this etiological trigger of relapsing MS (
      • Pakpoor J.
      • Giovannoni G.
      • Ramagopalan S.V.
      Epstein-Barr virus and multiple sclerosis: association or causation?.
      ).
      It is clear that broad B cell targeted depletion carries risks, notably associated with risks of infection. However, compared to drugs of similar efficacy such as natalizumab and alemtuzumab the adverse events may even be favorable. Yet, there is much to be learned about how B cells contribute to the regulation and pathogenesis of autoimmunity given the balance between B regulatory and B effector cell functions as shown EAE and MS. Irrespective of this it appears that depletion of CD20+ B cells offers promise as a disease modifying therapy of MS and it will probably gain approval for treatment of MS.

      Conflict of interests

      The authors declare no conflict of interest.

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