Endurance Exercise Attenuates Established Progressive Experimental Autoimmune Encephalomyelitis and Is Associated with an Amelioration of Innate Immune Responses in NOD Mice.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease causing axonal degeneration and demyelination. Exercise in mice with active monophasic experimental autoimmune encephalomyelitis (EAE) attenuates disease severity associated with diverse impacts on T cell-mediated immunity. However, studies have so far focused on preventive approaches. In this study, we investigated the impact of endurance exercise on established EAE disease in a model of secondary progressive MS. When the exercise program on motorized running wheels was started at disease manifestation, the disease course was significantly ameliorated. This was associated with a significant decrease in B cell, dendritic cell, and neutrophil cell counts in the central nervous system (CNS). Furthermore, we observed an increased expression of major histocompatibility complex class II (MHC-II) as well as alterations in costimulatory molecule expression in CNS B cells and dendritic cells. In contrast, T cell responses were not altered in the CNS or periphery. Thus, exercise training is capable of attenuating the disease course even in established secondary progressive EAE, potentially via modulation of the innate immune compartment. Further studies are warranted to corroborate our findings and assess the potential of this lifestyle intervention as a complementary therapeutic strategy in secondary progressive MS patients.

Enhanced pathogenicity of Th17 cells due to natalizumab treatment: Implications for MS disease rebound.

After natalizumab (NAT) cessation, some multiple sclerosis (MS) patients experience a severe disease rebound. The rebound pathophysiology is still unclear; however, it has been linked to interleukin-17-producing T-helper (Th17) cells. We demonstrate that during NAT treatment, MCAM+CCR6+Th17 cells gradually acquire a pathogenic profile, including proinflammatory cytokine production, pathogenic transcriptional signatures, brain endothelial barrier impairment, and oligodendrocyte damage via induction of apoptotic pathways. This is accompanied by an increase in Th17 cell frequencies in the cerebrospinal fluid of NAT-treated patients. Notably, Th17 cells derived from NAT-treated patients, who later developed a disease rebound upon treatment cessation, displayed a distinct transcriptional pathogenicity profile associated with altered migratory properties. Accordingly, increased brain infiltration of patient Th17 cells was illustrated in a humanized mouse model and brain histology from a rebound patient. Therefore, peripheral blood-accumulated MCAM+CCR6+Th17 cells might be involved in rebound pathophysiology, and monitoring of changes in Th17 cell pathogenicity in patients before/during NAT treatment cessation might enable rebound risk assessment in the future.

CNS Pericytes Modulate Local T Cell Infiltration in EAE.

Pericytes at the blood-brain barrier (BBB) are located between the tight endothelial cell layer of the blood vessels and astrocytic endfeet. They contribute to central nervous system (CNS) homeostasis by regulating BBB development and maintenance. Loss of pericytes results in increased numbers of infiltrating immune cells in the CNS in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS). However, little is known about their competence to modulate immune cell activation or function in CNS autoimmunity. To evaluate the capacity of pericytes to directly interact with T cells in an antigen-specific fashion and potentially (re)shape their function, we depleted major histocompatibility complex (MHC) class II from pericytes in a cell type-specific fashion and performed T cell-pericyte cocultures and EAE experiments. We found that pericytes present antigen in vitro to induce T cell activation and proliferation. In an adoptive transfer EAE experiment, pericyte-specific MHC II KO resulted in locally enhanced T cell infiltration in the CNS; even though, overall disease course of mice was not affected. Thus, pericytes may serve as non-professional antigen-presenting cells affecting states of T cell activation, thereby locally shaping lesion formation in CNS inflammation but without modulating disease severity.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

Polarizing receptor activation dissociates fibroblast growth factor 2 mediated inhibition of myelination from its neuroprotective potential.

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.

Teriflunomide treatment for multiple sclerosis modulates T cell mitochondrial respiration with affinity-dependent effects.

Interference with immune cell proliferation represents a successful treatment strategy in T cell-mediated autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (MS). One prominent example is pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), which mediates de novo pyrimidine synthesis in actively proliferating T and B lymphocytes. Within the TERIDYNAMIC clinical study, we observed that the DHODH inhibitor teriflunomide caused selective changes in T cell subset composition and T cell receptor repertoire diversity in patients with relapsing-remitting MS (RRMS). In a preclinical antigen-specific setup, DHODH inhibition preferentially suppressed the proliferation of high-affinity T cells. Mechanistically, DHODH inhibition interferes with oxidative phosphorylation (OXPHOS) and aerobic glycolysis in activated T cells via functional inhibition of complex III of the respiratory chain. The affinity-dependent effects of DHODH inhibition were closely linked to differences in T cell metabolism. High-affinity T cells preferentially use OXPHOS during early activation, which explains their increased susceptibility toward DHODH inhibition. In a mouse model of MS, DHODH inhibitory treatment resulted in preferential inhibition of high-affinity autoreactive T cell clones. Compared to T cells from healthy controls, T cells from patients with RRMS exhibited increased OXPHOS and glycolysis, which were reduced with teriflunomide treatment. Together, these data point to a mechanism of action where DHODH inhibition corrects metabolic disturbances in T cells, which primarily affects profoundly metabolically active high-affinity T cell clones. Hence, DHODH inhibition may promote recovery of an altered T cell receptor repertoire in autoimmunity.

Vitiligo after alemtuzumab treatment: Secondary autoimmunity is not all about B cells.

OBJECTIVE: To report 3 patients with relapsing-remitting multiple sclerosis (RRMS) showing vitiligo after treatment with alemtuzumab. METHODS: Retrospective case series including flow cytometric analyses and T-cell receptor (TCR) sequencing of peripheral blood mononuclear cells. RESULTS: We describe 3 cases of alemtuzumab-treated patients with RRMS developing vitiligo 52, 18, and 14 months after alemtuzumab initiation. Histopathology shows loss of epidermal pigmentation with absence of melanocytes and interface dermatitis with CD8+ T-cell infiltration. Also compatible with pathophysiologic concepts of vitiligo, peripheral blood mononuclear cells of one patient showed high proportions of CD8+ T cells with an activated (human leukocyte antigen-DR+), memory (CD45RO+), and type 1 cytokine (interferon-γ + tumor necrosis factor-α) phenotype at vitiligo onset compared to a control cohort of alemtuzumab-treated patients with RRMS (n = 30). Of note, analysis of CD8 TCR repertoire in this patient revealed a highly increased clonality and reduced repertoire diversity compared to healthy controls and treatment-naive patients with RRMS. We observed a predominance of single clones at baseline in this patient and alemtuzumab treatment did not substantially affect the proportions of most abundant clones over time. CONCLUSION: The 3 cases represent a detailed description of vitiligo as a T-cell-mediated secondary autoimmune disease following alemtuzumab treatment. The prevailing concept of unleashed B-cell responses might therefore not cover all facets of alemtuzumab-related secondary autoimmunity. Mechanistic studies, especially on TCR repertoire, might help clarify the underlying mechanisms.

Mechanisms underlying lesion development and lesion distribution in CNS autoimmunity.

It is widely accepted that development of autoimmunity in the central nervous system (CNS) is triggered by autoreactive T cells, that are activated in the periphery and gain the capacity to migrate through endothelial cells at the blood-brain barrier (BBB) into the CNS. Upon local reactivation, an inflammatory cascade is initiated, that subsequently leads to a recruitment of additional immune cells ultimately causing demyelination and axonal damage. Even though the interaction of immune cells with the BBB has been in the focus of research for many years, the exact mechanisms of how immune cells enter and exit the CNS remains poorly understood. In this line, the factors deciding immune cell entry routes, lesion formation, cellular composition as well as distribution within the CNS have also not been elucidated. The following factors have been proposed to represent key determinants for lesion evaluation and distribution: (i) presence and density of (auto) antigens in the CNS, (ii) local immune milieu at sites of lesion development and resolution, (iii) trafficking routes and specific trafficking requirements, especially at the BBB and (iv) characteristics and phenotypes of CNS infiltrating cells and cell subsets (e.g. features of T helper subtypes or CD8 cells). The heterogeneity of lesion development within inflammatory demyelinating diseases remains poorly understood until today, but here especially orphan inflammatory CNS disorders such as neuromyelitis optica spectrum disorder (NMOSD), Rasmussen encephalitis or SUSAC syndrome might give important insights in critical determinants of lesion topography. Finally, investigating the interaction of T cells with the BBB using in vitro approaches or tracking of T cells in vivo in animals or even human patients, as well as the discovery of lymphatic vasculature in the CNS are teaching us new aspects during the development of CNS autoimmunity. In this review, we discuss recent findings which help to unravel mechanisms underlying lesion topography and might lead to new diagnostic or therapeutic approaches in neuroinflammatory disorders including multiple sclerosis (MS).

B7-H1 shapes T-cell-mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity.

Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood-brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4+ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity.

The farnesoid-X-receptor in myeloid cells controls CNS autoimmunity in an IL-10-dependent fashion.

Innate immune responses by myeloid cells decisively contribute to perpetuation of central nervous system (CNS) autoimmunity and their pharmacologic modulation represents a promising strategy to prevent disease progression in Multiple Sclerosis (MS). Based on our observation that peripheral immune cells from relapsing-remitting and primary progressive MS patients exhibited strongly decreased levels of the bile acid receptor FXR (farnesoid-X-receptor, NR1H4), we evaluated its potential relevance as therapeutic target for control of established CNS autoimmunity. Pharmacological FXR activation promoted generation of anti-inflammatory macrophages characterized by arginase-1, increased IL-10 production, and suppression of T cell responses. In mice, FXR activation ameliorated CNS autoimmunity in an IL-10-dependent fashion and even suppressed advanced clinical disease upon therapeutic administration. In analogy to rodents, pharmacological FXR activation in human monocytes from healthy controls and MS patients induced an anti-inflammatory phenotype with suppressive properties including control of effector T cell proliferation. We therefore, propose an important role of FXR in control of T cell-mediated autoimmunity by promoting anti-inflammatory macrophage responses.

Myelinating Cultures: An In Vitro Tool to Identify Demyelinating and Axopathic Autoantibodies.

Myelinating cultures derived from embryonic spinal cord provide an invaluable tool to detect demyelinating and axopathic autoantibodies in clinical samples. A single preparation will provide a minimum of 200 individual cultures allowing 60 or more samples to be assayed in triplicate.

MOG-induced experimental autoimmune encephalomyelitis in the rat species triggers anti-neurofascin antibody response that is genetically regulated.

BACKGROUND: Ιn multiple sclerosis (MS), axonal damage leads to permanent neurological disabilities and the spreading of the autoimmune response to axonal antigens is implicated in disease progression. Experimental autoimmune encephalomyelitis (EAE) provides an animal model that mimics MS. Using different EAE models, we investigated the pathophysiological basis of epitope spreading to neurofascin, a protein localized at the node of Ranvier and its regulation by non-MHC genes. METHODS: We used two different EAE models in DA rat; one which is induced with myelin oligodendrocyte glycoprotein (MOG) which leads to disease characterized by profound demyelination, and the second which is induced with myelin basic protein (MBP) peptide 63-88 which results in severe central nervous system (CNS) inflammation but little or no demyelination. We determined anti-neurofascin antibody levels during the course of disease. Furthermore, the anti-neurofascin IgG response was correlated with clinical parameters in 333 (DAxPVG.1AV1) x DA rats on which we performed linkage analysis to determine if epitope spreading to neurofascin was affected by non-MHC genes. RESULTS: Spreading of the antibody response to neurofascin occurred in demyelinating MOG-induced EAE but not in EAE induced with MBP peptide 63-88. Anti-neurofascin IgG levels correlated with disease severity in (DAxPVG.1AV1) x DA rats, and a genomic region on chromosome 3 was found to influence this response. CONCLUSIONS: Inter-molecular epitope spreading to neurofascin correlates with disease severity in MOG-EAE is dependent on extensive demyelination and is influenced by non-MHC genes. The findings presented here may shed light on factors involved in the severity of MS and its genetics.

Fibroblast growth factor signalling in multiple sclerosis: inhibition of myelination and induction of pro-inflammatory environment by FGF9.

Remyelination failure plays an important role in the pathophysiology of multiple sclerosis, but the underlying cellular and molecular mechanisms remain poorly understood. We now report actively demyelinating lesions in patients with multiple sclerosis are associated with increased glial expression of fibroblast growth factor 9 (FGF9), which we demonstrate inhibits myelination and remyelination in vitro. This inhibitory activity is associated with the appearance of multi-branched 'pre-myelinating' MBP+ / PLP+ oligodendrocytes that interact with axons but fail to assemble myelin sheaths; an oligodendrocyte phenotype described previously in chronically demyelinated multiple sclerosis lesions. This inhibitory activity is not due to a direct effect of FGF9 on cells of the oligodendrocyte lineage but is mediated by factors secreted by astrocytes. Transcriptional profiling and functional validation studies demonstrate that these include effects dependent on increased expression of tissue inhibitor of metalloproteinase-sensitive proteases, enzymes more commonly associated with extracellular matrix remodelling. Further, we found that FGF9 induces expression of Ccl2 and Ccl7, two pro-inflammatory chemokines that contribute to recruitment of microglia and macrophages into multiple sclerosis lesions. These data indicate glial expression of FGF9 can initiate a complex astrocyte-dependent response that contributes to two distinct pathogenic pathways involved in the development of multiple sclerosis lesions. Namely, induction of a pro-inflammatory environment and failure of remyelination; a combination of effects predicted to exacerbate axonal injury and loss in patients.

Maternal neurofascin-specific autoantibodies bind to structures of the fetal nervous system during pregnancy, but have no long term effect on development in the rat.

UNLABELLED: Neurofascin was recently reported as a target for axopathic autoantibodies in patients with multiple sclerosis (MS), a response that will exacerbate axonal pathology and disease severity in an animal model of multiple sclerosis. As transplacental transfer of maternal autoantibodies can permanently damage the developing nervous system we investigated whether intrauterine exposure to this neurofascin-specific response had any detrimental effect on white matter tract development. To address this question we intravenously injected pregnant rats with either a pathogenic anti-neurofascin monoclonal antibody or an appropriate isotype control on days 15 and 18 of pregnancy, respectively, to mimic the physiological concentration of maternal antibodies in the circulation of the fetus towards the end of pregnancy. Pups were monitored daily with respect to litter size, birth weight, growth and motor development. Histological studies were performed on E20 embryos and pups sacrificed on days 2, 10, 21, 32 and 45 days post partum. RESULTS: Immunohistochemistry for light and confocal microscopy confirmed passively transferred anti-neurofascin antibody had crossed the placenta to bind to distinct structures in the developing cortex and cerebellum. However, this did not result in any significant differences in litter size, birth weight, or general physical development between litters from control mothers or those treated with the neurofascin-specific antibody. Histological analysis also failed to identify any neuronal or white matter tract abnormalities induced by the neurofascin-specific antibody. CONCLUSIONS: We show that transplacental transfer of circulating anti-neurofascin antibodies can occur and targets specific structures in the CNS of the developing fetus. However, this did not result in any pre- or post-natal abnormalities in the offspring of the treated mothers. These results assure that even if anti-neurofascin responses are detected in pregnant women with multiple sclerosis these are unlikely to have a negative effect on their children.

Neurofascin 186 specific autoantibodies induce axonal injury and exacerbate disease severity in experimental autoimmune encephalomyelitis.

Axonal injury is considered the major cause of chronic disability in multiple sclerosis (MS) patients, however the mechanisms behind remain still unclear. Recently, it was demonstrated that autoantibodies against Neurofascin, a cell adhesion molecule within the adult nervous system, can contribute to the development of axonal pathology in some patients. We compared the ability of the two different isoforms of Neurofascin, Nfasc155 and Nfasc186, to induce a pathogenic antibody response in the Dark Agouti (DA) rat. Animals were immunized with recombinant proteins prior to induction of experimental autoimmune encephalomyelitis (EAE) by adoptive transfer of activated MOG-specific T cells. Only Nfasc186 induced an axopathic autoantibody response in vivo, despite extensive cross reactivity between the two isoforms as shown by ELISA and flow cytometry. In this case, using transfected cell lines failed to differentiate between pathogenic and non-pathogenic responses. These findings have important implications with respect to the usage of cell based assays as an approach to detect pathologically relevant autoantibodies in clinical samples.

Functional identification of pathogenic autoantibody responses in patients with multiple sclerosis.

Pathological and clinical studies implicate antibody-dependent mechanisms in the immunopathogenesis of multiple sclerosis. We tested this hypothesis directly by investigating the ability of patient-derived immunoglobulins to mediate demyelination and axonal injury in vitro. Using a myelinating culture system, we developed a sensitive and reproducible bioassay to detect and quantify these effects and applied this to investigate the pathogenic potential of immunoglobulin G preparations obtained from patients with multiple sclerosis (n = 37), other neurological diseases (n = 10) and healthy control donors (n = 13). This identified complement-dependent demyelinating immunoglobulin G responses in approximately 30% of patients with multiple sclerosis, which in two cases was accompanied by significant complement-dependent antibody mediated axonal loss. No pathogenic immunoglobulin G responses were detected in patients with other neurological disease or healthy controls, indicating that the presence of these demyelinating/axopathic autoantibodies is specific for a subset of patients with multiple sclerosis. Immunofluorescence microscopy revealed immunoglobulin G preparations with demyelinating activity contained antibodies that specifically decorated the surface of myelinating oligodendrocytes and their contiguous myelin sheaths. No other binding was observed indicating that the response is restricted to autoantigens expressed by terminally differentiated myelinating oligodendrocytes. In conclusion, our study identifies axopathic and/or demyelinating autoantibody responses in a subset of patients with multiple sclerosis. This observation underlines the mechanistic heterogeneity of multiple sclerosis and provides a rational explanation why some patients benefit from antibody depleting treatments.

Cerebellar cortical demyelination in the murine cuprizone model.

In multiple sclerosis, demyelination occurs beside the white-matter structures and in the cerebral and cerebellar cortex. We have previously shown that, in the cuprizone model, demyelination is present not only in the corpus callosum but also in the cerebral cortex. Here, we have performed a detailed analysis of the dynamics of de- and remyelination in the cerebellar cortex and white matter at nine timepoints in two cerebellar regions. To induce demyelination, C57BL/6 mice were fed with 0.2% cuprizone for 12 weeks followed by a recovery of 8 weeks. Both cortex and white-matter structures were significantly demyelinated after 12 weeks of cuprizone feeding. Remyelination occurred after withdrawal of cuprizone but was less prominent in the more caudal cerebellar region. Microglia infiltration was prominent in all analyzed cerebellar areas, preceding demyelination by approximately 2-4 weeks, and was delayed in the more caudal cerebellar region. Astrogliosis was also seen but did not reach the extent observed in the cerebrum. In summary, cuprizone feeding provides an excellent model for the investigation of de- and remyelination processes in the cerebellar cortex and white matter. Furthermore, demyelination, microglia and astrocyte changes were different in the cerebellum as compared with the cerebrum, indicating region-dependent pathomechanisms.

Recombinant TCR ligand reverses clinical signs and CNS damage of EAE induced by recombinant human MOG.

Increasing evidence suggests that in addition to T cell-dependent effector mechanisms, autoantibodies are also involved in the pathogenesis of MS, including demyelinating antibodies specific for myelin oligodendrocyte glycoprotein (MOG). Our previous studies have demonstrated that recombinant T cell receptor ligands (RTLs) are very effective for treating T cell-mediated experimental autoimmune encephalomyelitis (EAE). In order to expand the scope of RTL therapy in MS patients, it was of interest to study RTL treatment of EAE involving a demyelinating antibody component. Therefore, we evaluated the therapeutic effects of RTL551, specific for T cells reactive to mouse (m)MOG-35-55 peptide, on EAE induced with recombinant human (rh)MOG in C57BL/6 mice. We report that RTL551 therapy can reverse disease progression and reduce demyelination and axonal damage induced by rhMOG without suppressing the anti-MOG antibody response. This result suggests that T cell-mediated inflammation and associated blood-brain barrier dysfunction are the central contributors to EAE pathogenesis and that successful regulation of these key players restricts potential damage by demyelinating antibodies. The results of our study lend support for the use of RTL therapy for treatment of MS subjects whose disease includes inflammatory T cells as well as those with an additional antibody component.