Experimental autoimmune encephalomyelitis in the neotropical primate common marmoset (Callithrix jacchus) is a relevant autoimmune animal model of multiple sclerosis. T cells specific for peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) have a central pathogenic role in this model. The aim of this study was to assess the requirement for innate immune stimulation for activation of this core pathogenic autoimmune mechanism. Marmoset monkeys were sensitized against synthetic MOG34-56 peptide alone or in combination with the nonencephalitogenic peptide MOG74-96 formulated in incomplete Freund adjuvant, which lacks microbial components. Experimental autoimmune encephalomyelitis development was recorded by monitoring neurological signs, brain magnetic resonance imaging, and longitudinal profiling of cellular and humoral immune parameters. All monkeys developed autoimmune inflammatory/demyelinating central nervous system disease characterized by massive brain and spinal cord demyelinating white matter lesions with activated macrophages and CD3+ T cells. Immune profiling ex vivo demonstrated the activation of mainly CD3+CD4+/8+CD56+ T cells against MOG34-56. Upon ex vivo stimulation, these T cells produced more interleukin 17A compared with TH1 cytokines (e.g. interferon-gamma) and displayed peptide-specific cytolytic activity. These results indicate that the full spectrum of marmoset experimental autoimmune encephalomyelitis can be induced by sensitization against a single MOG peptide in incomplete Freund adjuvant lacking microbial compounds for innate immune activation and by eliciting antigen-specific T-cell cytolytic activity.

Despite lack of classical lymphatic vessels in the central nervous system (CNS), cells and antigens do reach the CNS-draining lymph nodes. These lymph nodes are specialized to mediate mucosal immune tolerance, but can also generate T- and B-cell immunity. Their role in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) therefore remains elusive. We hypothesized that drainage of CNS antigens to the CNS-draining lymph nodes is vital for the recurrent episodes of CNS inflammation. To test this, we surgically removed the superficial cervical lymph nodes, deep cervical lymph nodes, and the lumbar lymph nodes prior to disease induction in three mouse EAE models, representing acute, chronic, and chronic-relapsing EAE. Excision of the CNS-draining lymph nodes in chronic-relapsing EAE reduced and delayed the relapse burden and EAE pathology within the spinal cord, which suggests initiation of CNS antigen-specific immune responses within the CNS-draining lymph nodes. Indeed, superficial cervical lymph nodes from EAE-affected mice demonstrated proliferation against the immunizing peptide, and the deep cervical lymph nodes, lumbar lymph nodes, and spleen demonstrated additional proliferation against other myelin antigen epitopes. This indicates that intermolecular epitope spreading occurs and that CNS antigen-specific immune responses are differentially generated within the different CNS-draining lymphoid organs. Proliferation of splenocytes from lymphadenectomized and sham-operated mice against the immunizing peptide was similar. These data suggest a role for CNS-draining lymph nodes in the induction of detrimental immune responses in EAE relapses, and conclusively demonstrate that the tolerance-inducing capability of cervical lymph nodes is not involved in EAE.

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).

Axonal damage is the major cause of irreversible neurologic disability in patients with multiple sclerosis. Although axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we show that Biozzi ABH mice immunized with NF-L protein develop neurologic disease characterized by spastic paresis and paralysis concomitant with axonal degeneration and inflammation primarily in the dorsal column of the spinal cord. The inflammatory central nervous system lesions were dominated by F4/80+ macrophages/microglia and relatively low numbers of CD4+ and CD8+ T-cells. In splenocyte cultures, proliferation to NF-L was observed in CD4+ T-cells accompanied by the production of the proinflammatory cytokine interferon-gamma. Elevated levels of circulating antibodies recognizing recombinant mouse NF-L were present in the serum, and immunoglobulin deposits were observed within axons in spinal cord lesions of mice exhibiting clinical disease. These data provide evidence that autoimmunity to NF-L protein induces axonal degeneration and clinical neurologic disease in mice, indicating that autoimmunity to axonal antigens, as described in multiple sclerosis, may be pathogenic rather than acting merely as a surrogate marker for axonal degeneration.

Induction of experimental autoimmune encephalomyelitis (EAE) has been documented in common marmosets using peptide 34-56 from human myelin/oligodendrocyte glycoprotein ) in incomplete Freund's adjuvant (IFA). Here, we report that this EAE model is associated with widespread demyelination of grey and white matter. We performed an in-depth analysis of the specificity, MHC restriction and functions of the activated T cells in the model, which likely cause EAE in an autoantibody-independent manner. T-cell lines isolated from blood and lymphoid organs of animals immunized with MOG 34-56 displayed high production of IL-17A and specific lysis of MOG 34-56 -pulsed EBV B-lymphoblastoid cells as typical hallmarks. Cytotoxicity was directed at the epitope MOG [40][41][42][43][44][45][46][47][48] presented by the non-classical MHC class Ib allele Caja-E, which is orthologue to HLA-E and is expressed in non-inflamed brain. In vivo activated T cells identified by flow cytometry in cultures with MOG 34-56, CD561 T cells also expressed CD27, but CD16, CD45RO, CD28 and CCR7 were absent. These results show that, in the MOG34-56/IFA marmoset EAE model, a Caja-E-restricted population of autoreactive cytotoxic T cells plays a key role in the process of demyelination in the grey and white matter.Key words: Common marmoset . Cytotoxicity . HLA-E . Oligodendrocytes . Natural killer-cytotoxic T lymphocyte (NK-CTL)Supporting Information available online IntroductionMultiple sclerosis (MS) is a chronic inflammatory disease of the human central nervous system (CNS) of unknown aetiology. The pathological hallmark of MS is the lesion. Lesions are regions of usually focal demyelination of variable size localized in the grey and/or white matter of the brain and spinal cord, formed by a combined cellular and humoral autoimmune attack. CNS-targeting autoimmune reactions are thought to be induced as a response to infection (response-to-infection paradigm) [1], although the pathogen(s) that elicit this pathogenic process in MS has not been identified. We have proposed a responseto-damage paradigm for MS, based on the work in a unique nonhuman primate model of MS, experimental autoimmune encephalomyelitis (EAE) in common marmosets (Callithrix jacchus), [2]. The new concept postulates that autoimmunity in MS patients is caused by a genetically predisposed hyper-response to myelin antigens released from damaged white matter due to an unknown antecedent event. We showed that the most important anti-myelin reactivity for the induction of neurological deficit is mediated by antigen-experienced T-cells specific for peptides 34-56 of myelin/oligodendrocyte glycoprotein ) [3].Subsequently it was demonstrated that these cells could be activated in vivo by immunization of marmosets with MOG in incomplete Freund's adjuvant (IFA) [4]. A peptide in IFA emulsion is a more common formulation for the in vivo activation of antigen-experienced T-cells than for autoreactive T-cells as bacterial ligands of innate antigen receptors are usually required. It is no...

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EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E–restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20+ spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40–48 core epitope. Finally, LCV infection also induced expression of LC3-II+ cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40–48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40–48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs.

The robust and rapid clinical effect of depleting anti-CD20 monoclonal antibodies (mAb) in multiple sclerosis (MS) demonstrates a critical pathogenic contribution of B cells. The clinical effect of anti-CD20 mAb has been replicated in a relevant preclinical MS model, experimental autoimmune encephalomyelitis (EAE) in marmoset monkeys (Callithrix jacchus). By contrast, treatment with mAbs against two essential cytokines in B cell activation growth and survival, i.e. BlyS/BAFF and APRIL, was only partially effective. All three mAbs induced depletion of CD20+ B cells from the circulation, albeit with different kinetics and based on distinct mechanisms of action. In the current study we analyzed whether the different clinical effect of anti-CD20 mAb or the anti-BLyS and anti-APRIL mAbs is due to different depletion of B cells infected with the EBV of marmosets, CalHV3. Employing a novel PCR-based assay, half of the colony of group-housed marmosets was tested positive for CalHV3 DNA in secondary lymphoid organs. The same prevalence was observed in placebo-treated monkeys. In marmosets treated with anti-CD20 mAb the load of CalHV3 DNA in lymphoid organs was substantially reduced, while this was not observed in the monkeys treated with anti-BLyS or anti-APRIL mAbs. To examine the pathogenic role of virus-transformed B cells, we infused EBV-transformed B lymphoblastic cell (BLC) lines presenting the immunodominant MOG34-56 peptide. We observed in the recipients of MOG34-56 pulsed BLC, but not in their fraternal siblings infused with non-pulsed BLC, activation of anti-MOG34-56 T cells and meningeal inflammation. Collectively, the data show that among CD20+ B cells, the herpesvirus-transformed subset has a particularly important pathogenic role in the marmoset EAE model.