Recombinant Human Myelin Oligodendrocyte Glycoprotein Research Articles

CD40 Drives Central Nervous System Autoimmune Disease by Inducing Complementary Effector Programs via B Cells and Dendritic Cells.

Costimulatory CD40 plays an essential role in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, how CD40 drives autoimmune disease pathogenesis is not well defined. Here, we used a conditional knockout approach to determine how CD40 orchestrates a CNS autoimmune disease induced by recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We found that deletion of CD40 in either dendritic cells (DCs) or B cells profoundly reduced EAE disease pathogenesis. Mechanistically, CD40 expression on DCs was required for priming pathogenic Th cells in peripheral draining lymph nodes and promoting their appearance in the CNS. By contrast, B cell CD40 was essential for class-switched MOG-specific Ab production, which played a crucial role in disease pathogenesis. In fact, passive transfer of MOG-immune serum or IgG into mice lacking CD40 on B cells but not DCs reconstituted autoimmune disease, which was associated with inundation of the spinal cord parenchyma by Ig and complement. These data demonstrate that CD40 supports distinct effector programs in B cells and DCs that converge to drive a CNS autoimmune disease and identify targets for intervention.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.The authors describe quantitatively and qualitatively different forms of experimental autoimmune encephalitis (EAE) in cynomolgus macaques. They found that bacterial contaminants within recombinant human myelin oligodendrocyte glycoprotein seemed to aggravate disease evolution. They provide anatomopathological features of fulminant and progressive forms of EAE, allowing them to distinguish specific factors influencing the evolution of this model of autoimmune demyelinating disease.

Efficient Distribution of a Novel Zirconium-89 Labeled Anti-cd20 Antibody Following Subcutaneous and Intravenous Administration in Control and Experimental Autoimmune Encephalomyelitis-Variant Mice.

Objective: To investigate the imaging and biodistribution of a novel zirconium-89 (89Zr)-labeled mouse anti-cd20 monoclonal antibody (mAb) in control and experimental autoimmune encephalomyelitis (EAE) mice following subcutaneous (s. c.) and intravenous (i.v.) administration.Background: Anti-cd20-mediated B-cell depletion using mAbs is a promising therapy for multiple sclerosis. Recombinant human myelin oligodendrocyte glycoprotein (rhMOG)-induced EAE involves B-cell-mediated inflammation and demyelination in mice.Design/Methods: C57BL/6J mice (n = 39) were EAE-induced using rhMOG. On Day 14 post EAE induction, 89Zr-labeled-anti-cd20 mAb was injected in control and EAE mice in the right lower flank (s.c.) or tail vein (i.v.). Positron emission tomography/computed tomography (PET/CT) imaging and gamma counting (ex vivo) were performed on Days 1, 3, and 7 to quantify tracer accumulation in the major organs, lymphatics, and central nervous system (CNS). A preliminary study was conducted in healthy mice to elucidate full and early kinetics of the tracer that were subsequently applied in the EAE and control mice study.Results: 89Zr-labeled anti-cd20 mAb was effectively absorbed from s.c. and i.v. injection sites and distributed to all major organs in the EAE and control mice. There was a good correlation between in vivo PET/CT data and ex vivo quantification of biodistribution of the tracer. From gamma counting studies, initial tracer uptake within the lymphatic system was found to be higher in the draining lymph nodes (inguinal or subiliac and sciatic) following s.c. vs. i.v. administration; within the CNS a significantly higher tracer uptake was observed at 24 h in the cerebellum, cerebrum, and thoracic spinal cord (p < 0.05 for all) following s.c. vs. i.v. administration.Conclusions: The preclinical data suggest that initial tracer uptake was significantly higher in the draining lymph nodes (subiliac and sciatic) and parts of CNS (the cerebellum and cerebrum) when administered s.c. compared with i.v in EAE mice.

Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques.

BackgroundAutoimmune demyelinating diseases (ADD) are a major cause of neurological disability due to autoreactive cellular and humoral immune responses against brain antigens. A cure for chronic ADD could be obtained by appropriate immunomodulation.MethodsWe implemented a preclinical scheme to foster immune tolerance to myelin oligodendrocyte glycoprotein (MOG), in a cynomolgus-macaque model of experimental autoimmune encephalomyelitis (EAE), in which administration of recombinant human MOG (rhMOG) elicits brain inflammation mediated by MOG-autoreactive CD4+ lymphocytes and anti-MOG IgG. For immunotherapy, we used a recombinant antibody (Ab) directed against the dendritic cell-asialoglycoprotein receptor (DC-ASGPR) fused either to MOG or a control antigen PSA (prostate-specific antigen).FindingsrhMOG and the anti-DC-ASGPR-MOG were respectively detected in CD1a+ DCs or CD163+ cells in the skin of macaques. Intradermal administration of anti-DC-ASGPR-MOG, but not control anti-DC-ASGPR-PSA, was protective against EAE. The treatment prevented the CD4+ T cell activation and proinflammatory cytokine production observed in controls. Moreover, the administration of anti-DC-ASGPR-MOG induced MOG-specific CD4+CD25+FOXP3+CD39+ regulatory lymphocytes and favoured an upsurge in systemic TGFβ and IL-8 upon rhMOG re-administration in vivo.InterpretationWe show that the delivery of an anti-DC-ASGPR-MOG allows antigen-specific adaptive immune modulation to prevent the breach of immune tolerance to MOG. Our findings pave the way for therapeutic vaccines for long-lasting remission to grave encephalomyelitis with identified autoantigens, such as ADD associated with anti-MOG autoantibodies.FundWork supported by the French ANR (ANR-11-INBS-0008 and ANR-10-EQPX-02-01), NIH (NIH 1 R01 AI 105066), the Baylor Scott and White Healthcare System funding and Roche Research Collaborative grants.

11C]PIB PET imaging can detect white and grey matter demyelination in a non-human primate model of progressive multiple sclerosis

BackgroundMultiple sclerosis (MS) is a demyelinating and inflammatory disease of the central nervous system. Its diagnosis is clinical, often confirmed by magnetic resonance imaging. This image modality, however, is not ideal for discrimination of demyelination in grey and white matter regions from inflammatory lesions. Positron Emission Tomography (PET), using specific radiopharmaceuticals, can be a tool to differentiate between these processes. The radiopharmaceutical [11C]PIB is widely used for detection of β-amyloid plaques, but has also been suggested for the analysis of myelin content due to its consistent uptake in white matter. The aim of this study was to evaluate [11C]PIB PET imaging as a tool for detecting demyelinated regions in white and grey matter of non-human primate model of progressive MS. MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in marmosets by injection of recombinant human myelin oligodendrocyte glycoprotein (rhMOG) emulsified in either Incomplete Freund's Adjuvant (IFA) or Complete Freund's Adjuvant (CFA). [11C]PIB PET images were acquired prior to immunization (baseline) and after symptoms were present (end of experiment). Brain tissue was isolated for histochemical analysis. ResultsAll rhMOG/IFA-treated and rhMOG/CFA-treated animals showed clinical signs of EAE. The rhMOG/CFA group presented a significant [11C]PIB uptake reduction only in the left motor cortex (9%, P = 0.011). For the rhMOG/IFA group, significant decrease in [11C]PIB uptake was observed in the whole brain (15%, P = 0.015), in the right hemisphere of body of corpus callosum (34%, P = 0.02), splenium of corpus callosum (38%, P = 0.004), hippocampus (19%, P = 0.036), optic tract (13%, P = 0.025), thalamus (14%, P = 0.041), Globus pallidus (23%, P = 0.017), head of caudate nucleus (25%, P = 0.045), tail of caudate nucleus (29%, P = 0.003), putamen (28%, P = 0.047) and left hemisphere of body of corpus callosum (14%, P = 0.037) and head of caudate nucleus (23%, P = 0.023). [11C]PIB uptake significantly correlated with luxol fast blue histology (myelin marker), both in the rhMOG/IFA (r2= 0.32, P < 0.0001) and the rhMOG/CFA group (r2= 0.46, P < 0.0001). Conclusion[11C]PIB PET imaging is an efficient tool for detecting demyelination in grey and white matter, in a non-human primate model of progressive MS.

Targeted Diet Modification Reduces Multiple Sclerosis-like Disease in Adult Marmoset Monkeys from an Outbred Colony.

Experimental autoimmune encephalomyelitis (EAE) in common marmosets is a translationally relevant model of the chronic neurologic disease multiple sclerosis. Following the introduction of a new dietary supplement in our purpose-bred marmoset colony, the percentage of marmosets in which clinically evident EAE could be induced by sensitization against recombinant human myelin oligodendrocyte glycoprotein in IFA decreased from 100 to 65%. The reduced EAE susceptibility after the dietary change coincided with reduced Callitrichine herpesvirus 3 expression in the colony, an EBV-related γ1-herpesvirus associated with EAE. We then investigated, in a controlled study in marmoset twins, which disease-relevant parameters were affected by the dietary change. The selected twins had been raised on the new diet for at least 12 mo prior to the study. In twin siblings reverted to the original diet 8 wk prior to EAE induction, 100% disease prevalence (eight out of eight) was restored, whereas in siblings remaining on the new diet the EAE prevalence was 75% (six out of eight). Spinal cord demyelination, a classical hallmark of the disease, was significantly lower in new-diet monkeys than in monkeys reverted to the original diet. In new-diet monkeys, the proinflammatory T cell response to recombinant human myelin oligodendrocyte glycoprotein was significantly reduced, and RNA-sequencing revealed reduced apoptosis and enhanced myelination in the brain. Systematic typing of the marmoset gut microbiota using 16S rRNA sequencing demonstrated a unique, Bifidobacteria-dominated composition, which changed after disease induction. In conclusion, targeted dietary intervention exerts positive effects on EAE-related parameters in multiple compartments of the marmoset's gut-immune-CNS axis.

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda-/- and Ung-/- Mice.

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda-/- mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in Aicda-/- mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice (Ung-/-) that exhibit a defect primarily in CSR. We observed that Ung-/- mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG1 class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.

Experimental Autoimmune Encephalomyelitis (EAE) Model of Cynomolgus Macaques Induced by Recombinant Human MOG1-125 (rhMOG1-125) Protein and MOG34-56 Peptide.

Experimental autoimmune encephalomyelitis (EAE) induced by self-myelin antigen is a widely used in multiple sclerosis (MS) model for preclinical studies of new therapeutics and potential pathogenesis. By comparison with rodent EAE models, EAE models in primates are more similar to MS. Some groups have developed EAE models in primates by using common marmoset (Callithrix jacchus). However, this model has some limitations. EAE in cynomolgus monkey (Macaque fasciculrais) could overcome these limitations. The main objective of this study was to establish an ideal EAE cynomolgus monkey model resembling human MS by immunizing human myelin oligodendrocyte glycoprotein (MOG) protein. In this study, six female cynomolgus monkeys were divided into two separate experiment groups and one monkey as the control. EAE was induced in cynomolgus monkey by immunizing with the recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG1-125) and a synthetic peptide, representing peptide 34-56 of human myelin oligodendrocyte glycoprotein (MOG34-56) in complete Freund's adjuvant (CFA) by subcutaneous multipoint immunization in the armpit and inguinal region and in combination with intravenous injection of pertussis toxin, and subsequent booster immunizations with the same dose of antigen in incomplete Freund's adjuvant (IFA) until the animals developed clinically significant EAE (score≥2). The body weight, clinical scores, magnetic resonance imaging (MRI), histopathology, antibody, cytokine profiling and antigen-specific lymphocyte proliferation were evaluated. The results showed that despite the different time intervals between onset and significant neurological deficits, all the cynomolgus monkeys immunized with rhMOG1-125 or MOG34-56 developed clinically significant acute fulminant or mild forms of EAE, with a success rate of 100%. The clinical courses were obvious heterogeneity which closely resembles MS. MOG34-56 immunization was much milder compared with rhMOG1-125 immunized individuals, at least in cynomolgus monkeys. Inflammation and demyelinated lesions were present in the brains and spinal cords. Immune profiling revealed high IgG levels associated with early onset of EAE but not the course of the disease. Significant antigen-specific T lymphocyte responses against immunodominant epitopes of MOG were also detected. Our results indicated rhMOG1-125 and MOG34-56 could induce successfully EAE models resembling MS in cynomolgus monkeys. The synergistic action of anti-myelin T cells and Abs during the pathogenesis of EAE could establish a more ideal EAE model.

Immune profile of an atypical EAE model in marmoset monkeys immunized with recombinant human myelin oligodendrocyte glycoprotein in incomplete Freund's adjuvant.

BackgroundExperimental autoimmune encephalomyelitis (EAE) in the common marmoset monkey (Callithrix jacchus) is a relevant preclinical model for translational research into immunopathogenic mechanisms operating in multiple sclerosis (MS). Prior studies showed a core pathogenic role of T and B cells specific for myelin oligodendrocyte glycoprotein (MOG). However, in those studies, the quality of the response against MOG epitopes was strongly biased by bacterial antigens in the complete Freund’s adjuvant (CFA), in which the immunizing recombinant human (rh) MOG protein had been formulated. In response to the need of a more refined EAE model, we have tested whether disease could also be induced with rhMOG in incomplete Freund’s adjuvant (IFA).MethodMarmosets were immunized with rhMOG emulsified in IFA in the dorsal skin. Monkeys that did not develop neurological deficit were given booster immunizations at 28-day interval with the same antigen preparation. In a second experiment, three marmoset twin pairs were sensitized against MOG peptides in IFA to study a possibility for suppressive activity towards pathogenic T cells directed against the encephalitogenic epitope MOG40-48.ResultsDespite the absence of strong danger signals in the rhMOG/IFA inoculum, all monkeys developed clinically evident EAE symptoms. Moreover, in all monkeys, demyelinated lesions were present in the white matter and in two cases also in the cortical grey matter. Immune profiling at height of the disease showed a dominant T cell response against the overlapping peptides 14–36 and 24–46, but reactivity against the pathogenically most relevant peptide 34–56 was conspicuously absent. In the second experiment, there was an indication for a possible suppressive mechanism.ConclusionsImmunization of marmoset monkeys with rhMOG in IFA elicits clinical EAE in all animals. Moreover, rhMOG contains pathogenic and regulatory epitopes, but the pathogenic hierarchy of rhMOG epitopes is strongly influenced by the adjuvant in which the protein is formulated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0378-5) contains supplementary material, which is available to authorized users.

Selective Blockade of CD28-Mediated T Cell Costimulation Protects Rhesus Monkeys against Acute Fatal Experimental Autoimmune Encephalomyelitis

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the β-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies

Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B-MHC II(-/-)), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)-specific B cell receptor (BCR; IgH(MOG-mem)) but cannot secrete antibodies. B-MHC II(-/-) mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell- and B cell-dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B-MHC II(-/-) mice. In the absence of antibodies, IgH(MOG-mem) mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle-like structures were observed in IgH(MOG-mem) mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell-targeted therapies.

The Encephalitogenic, Human Myelin Oligodendrocyte Glycoprotein–Induced Antibody Repertoire Is Directed toward Multiple Epitopes in C57BL/6-Immunized Mice

Although Abs specific for myelin oligodendrocyte glycoprotein (MOG) have been detected in patients with multiple sclerosis (MS), their contribution to pathogenesis remains poorly understood. Immunization of C57BL/6 mice with recombinant human MOG (hMOG) results in experimental autoimmune encephalomyelitis involving MOG-specific, demyelinating Abs. This model is therefore informative for understanding anti-MOG humoral responses in MS. In the current study, we have characterized the hMOG-specific Ab repertoire in immunized C57BL/6 mice using both in vitro and in vivo approaches. We demonstrate that hMOG-specific mAbs are not focused on one specific region of MOG, but instead target multiple epitopes. Encephalitogenicity of the mAbs, assessed by the ability of the mAbs to exacerbate experimental autoimmune encephalomyelitis in mice, correlates with the activity of the mAbs in binding to CNS tissue sections, but not with other in vitro assays. The targeting of different MOG epitopes by encephalitogenic Abs has implications for disease pathogenesis, because it could result in MOG cross linking on oligodendrocytes and/or immune complex formation. These studies reveal several novel features concerning pathogenic, humoral responses that may have relevance to human MS.

Induction of Experimental Autoimmune Encephalomyelitis With Recombinant Human Myelin Oligodendrocyte Glycoprotein in Incomplete Freund’s Adjuvant in Three Non-human Primate Species

The experimental autoimmune encephalitis (EAE) model is used for preclinical research into the pathogenesis of multiple sclerosis (MS), mostly in inbred, specific pathogen free (SPF)-raised laboratory mice. However, the naive state of the laboratory mouse immune system is considered a major hurdle in the translation of principles from the EAE model to the MS patient. Non-human primates (NHP) have an immune system harboring T- and B-cell memory against environmental antigens, similar as in humans. We sought to further refine existing NHP EAE models, which may help to bridge the gab between mouse EAE models and MS. We report here on new EAE models in three NHP species: rhesus monkeys (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis) and common marmosets (Callithrix jacchus). EAE was induced with recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1–125) (rhMOG) formulated in incomplete Freund’s adjuvant (IFA). IFA lacks the bacterial antigens that are present in complete Freund’s adjuvant (CFA), which are notorious for the induction of discomforting side effects. Clinically evident EAE could be induced in two out of five rhesus monkeys, six out of six cynomolgus monkeys and six out of six common marmosets. In each of these species, the presence of an early, high anti-rhMOG IgM response is correlated with EAE with an earlier onset and more severe disease course. Animals without an early high IgM response either did not develop disease (rhesus monkeys) or developed only mild signs of neurological deficit (marmoset and cynomolgus monkeys).

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

BackgroundClinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.MethodsActivation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).ConclusionsAID−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.

Antagonizing the α4β1 Integrin, but Not α4β7, Inhibits Leukocytic Infiltration of the Central Nervous System in Rhesus Monkey Experimental Autoimmune Encephalomyelitis

The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the α(4) integrins expressed by T lymphocytes. The α(4)β(1) integrin mediates migration of memory T lymphocytes into the CNS, whereas the α(4)β(7) integrin mediates migration preferentially into gastrointestinal tissue. This paradigm was established primarily from investigations in rodents; thus, the objective of this investigation was to determine if blocking the α(4)β(7) integrin exclusively would affect migration of T lymphocytes into the CNS of primates. The effects of the dual α(4)β(1) and α(4)β(7) antagonist natalizumab were compared with those of the α(4)β(7) antagonist vedolizumab on experimental autoimmune encephalomyelitis in the rhesus monkey. Animals received an initial i.v. bolus of placebo, natalizumab (30 mg/kg), or vedolizumab (30 mg/kg) before intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein and then Ab once weekly thereafter. Natalizumab prevented CNS inflammation and demyelination significantly (p < 0.05), compared with time-matched placebo control animals, whereas vedolizumab did not inhibit these effects, despite saturating the α(4)β(7) integrin in each animal for the duration of the investigation. These results demonstrate that blocking α(4)β(7) exclusively does not inhibit immune surveillance of the CNS in primates.

Autoantigen Conformation Influences Both B- and T-cell Responses and Encephalitogenicity

It has become increasingly clear that only antibodies recognizing conformation-dependent epitopes of myelin oligodendrocyte glycoprotein (MOG) have a demyelinating potential in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Nevertheless, for the induction of EAE, most studies to date have used MOG peptides or bacterially expressed MOG, neither of which contain the tertiary structure of the native antigen. Non-refolded recombinant human MOG does not induce EAE in DA rats. Therefore, we refolded this protein in order to assess the influence of MOG conformation on its pathogenicity in DA rats. DA rats immunized with refolded human MOG developed severe acute EAE. As expected, rats immunized with the refolded protein had a higher amount of conformational MOG antibodies present in serum. But in addition, a striking effect of MOG refolding on the generation of T-cell responses was found. Indeed, T-cell responses against the encephalitogenic MOG 91-108 epitope were greatly enhanced after refolding. Therefore, we conclude that refolding of MOG increases its pathogenicity both by generating conformation-dependent MOG antibodies and by enhancing its processing or/and presentation on MHC molecules. These data are important in regard to investigations of the pathogenic potential of many (auto)antigens.

B-Cell Depletion Attenuates White and Gray Matter Pathology in Marmoset Experimental Autoimmune Encephalomyelitis

This study investigated the effect of CD20-positive B-cell depletion on central nervous system (CNS) white and gray matter pathology in experimental autoimmune encephalomyelitis in common marmosets, a relevant preclinical model of multiple sclerosis. Experimental autoimmune encephalomyelitis was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein in complete Freund adjuvant. At 21 days after immunization, B-cell depletion was achieved by weekly intravenous injections of HuMab 7D8, a human-anti-human CD20 antibody that cross-reacts with marmoset CD20. In vivo magnetic resonance imaging showed widespread brain white matter demyelination in control marmosets that was absent in CD20 antibody-treated marmosets. High-contrast postmortem magnetic resonance imaging showed white matter lesions in 4of the 7 antibody-treated marmosets, but these were significantly smaller than those in controls. The same technique revealed gray matter lesions in 5 control marmosets, but none in antibody-treated marmosets. Histologic analysis confirmed that inflammation, demyelination, and axonal damage were substantially reduced in brain, spinal cord, and optic nerves of CD20 antibody-treated marmosets. In conclusion, CD20-postive B-cell depletion by HuMab 7D8 profoundly reduced the development of both white and gray matter lesions in the marmoset CNS. These data underline the central role of B cells in CNS inflammatory-demyelinating disease.

Late B Cell Depletion with a Human Anti-Human CD20 IgG1κ Monoclonal Antibody Halts the Development of Experimental Autoimmune Encephalomyelitis in Marmosets

Depletion of CD20(+) B cells has been related to reduced clinical activity in relapsing-remitting multiple sclerosis. The underlying mechanism is not understood, because serum IgG levels were unaltered by the treatment. We report the effect of late B cell depletion on cellular and humoral immune mechanisms in a preclinical multiple sclerosis model (i.e., experimental autoimmune encephalomyelitis [EAE] in the common marmoset). We used a novel human anti-human CD20 IgG1κ mAb (HuMab 7D8) that cross-reacts with marmoset CD20. EAE was induced in 14 marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein (MOG) in CFA. After 21 d, B cells were depleted in seven monkeys by HuMab 7D8, and seven control monkeys received PBS. The Ab induced profound and long-lasting B cell depletion from PBMCs and lymphoid organs throughout the observation period of 106 d. Whereas all of the control monkeys developed clinically evident EAE, overt neurologic deficits were reduced substantially in three HuMab 7D8-treated monkeys, and four HuMab 7D8-treated monkeys remained completely asymptomatic. The effect of HuMab 7D8 was confirmed on magnetic resonance images, detecting only small lesions in HuMab 7D8-treated monkeys. The infusion of HuMab 7D8 arrested the progressive increase of anti-MOG IgG Abs. Although CD3(+) T cell numbers in lymphoid organs were increased, their proliferation and cytokine production were impaired significantly. Most notable were the substantially reduced mRNA levels of IL-7 and proinflammatory cytokines (IL-6, IL-17A, IFN-γ, and TNF-α). In conclusion, B cell depletion prevents the development of clinical and pathological signs of EAE, which is associated with impaired activation of MOG-reactive T cells in lymphoid organs.

Effects of Early IL-17A Neutralization on Disease Induction in a Primate Model of Experimental Autoimmune Encephalomyelitis

We report on the effect of antibody-mediated neutralization of interleukin (IL)-17A in a non-human primate experimental autoimmune encephalomyelitis (EAE) model induced with recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We tested a human-anti-human IL-17A-antibody in two doses (3 and 30 mg/kg) against placebo (PBS). The treatment was started 1 day before EAE induction and continued throughout the experiment. Although all monkeys developed clinically evident EAE, the onset of neurological signs was delayed in some monkeys from both treatment groups. Total CNS lesion volumes, demyelination, or inflammation did not differ between the different groups. Immune profiling revealed an altered distribution of IL-17A producing cells in the lymphoid organs of antibody-treated monkeys. Comparable numbers of IL-17A producing cells were observed in the brain. RhMOG-induced T cell proliferation in the lymph nodes was slightly reduced after anti-IL-17A antibody treatment. To summarize, we found that anti-IL-17A antibody as a single treatment from disease induction effects a trend towards delayed neurological disease progression in the marmoset EAE model, although the effect did not reach statistical significance. This suggests a role of IL-17A in late stage disease in the marmoset EAE model, but IL-17A may not be the dominant pathogenic cytokine.

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.