EAE: imperfect but useful models of multiple sclerosis

Opinion

EAE: imperfect but useful models of multiple sclerosis Bert A. ’t Hart1,2,3,4, Bruno Gran5 and Robert Weissert6 1

Department of Immunobiology, Biomedical Primate Research Center, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands Department of Medical Physiology, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Immunology, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 4 MS Center ErasMS, Rotterdam, The Netherlands 5 Division of Clinical Neurology, University of Nottingham, Nottingham, United Kingdom 6 Department of Neurology, University of Geneva, Geneva, Switzerland 2

The high failure rate of immunotherapies in multiple sclerosis (MS) clinical trials demonstrates problems in translating new treatment concepts from animal models to the patient. One main reason for this ‘immunotherapy gap’ is the usage of immunologically immature, microbiologically clean and genetically homogeneous rodent strains. Another reason is the artificial nature of the experimental autoimmune encephalomyelitis model, which favors CD4+ T cell driven autoimmune mechanisms, whereas CD8+ T cells are prevalent in MS lesions. In this paper, we discuss preclinical models in humanized rodents and non-human primates that are genetically closer to MS. We also discuss models that best reproduce specific aspects of MS pathology and how these can potentially improve preclinical selection of promising therapies from the discovery pipeline. EAE: a relevant preclinical model of multiple sclerosis Animal models have a dual role in preclinical research, including that of multiple sclerosis (MS), an (auto)immune driven neurological disease specifically affecting the central nervous system (CNS). Inbred and specific pathogen free (SPF) laboratory strains of mice and rats provide a frequently used animal model, experimental autoimmune encephalomyelitis (EAE). EAE is widely regarded as a useful general MS model for preclinical therapy development. EAE has helped clarify immunopathogenic mechanisms for the development of immunotherapies; indeed, several treatments were successfully translated from EAE to MS, including glatiramer acetate, a random polymer of four amino acids of myelin basic protein (MBP) that induces regulatory T cells; the antineoplastic drug mitoxantrone, which targets type II topoisomerase and disrupts DNA synthesis; FTY720 (fingolimod), a sphingosine analog which sequesters lymphocytes within lymphoid organs; and natalizumab, a monoclonal antibody (mAb) against a4b1 integrin that inhibits accumulation of lympho-/monocytes in the CNS [1]. However, these successes are matched with failures. Examples include anti-CD40L mAb, the clinical development of which was halted after thromboembolic complications [2] in spite of encouraging results in relapsing–remitting MS (RRMS); tumor necrosis factor-a Corresponding author: ’t Hart, B.A. ([email protected]).

neutralizing agents, which induced MS-like pathology [3,4], and the anti-interleukin(IL)-12/23p40 mAb ustekinumab, which lacked efficacy in RRMS [5]. Moreover, MS patients in the secondary progressive phase and even more with MS patients in the primary progressive experience little or no benefit from currently available treatments. This ‘immunotherapy gap’ between EAE and MS justifies the question as to whether immunopathogenic processes observed in classical EAE models can be extrapolated to chronic MS. In this paper, we first discuss immune mechanisms driving acute inflammation, which is the pathological hallmark of RRMS. Then we discuss alternative immune mechanisms involved in chronic disease, which is characterized by widespread demyelination of hemispheric and spinal cord white matter (WM), as well as cortical, spinal and deep gray matter (GM). Of special interest are T cells that mediate antibody-independent demyelination in non-human primate EAE, but not in rodent models. EAE models incorporating these alternative immune mechanisms will be an essential tool for developing therapies targeting chronic MS. Similarities and discrepancies between EAE and MS Commonly used EAE models are based on inbred mouse and rat strains (see Boxes 1 and 2, respectively). One or both species serve to prove efficacy of new drug candidates, to test for potential side effects, to analyze pharmacokinetic/pharmacodynamic characteristics and to predict the human dose. This procedure has worked reasonably well for the safety and efficacy testing of new chemical entities or small molecules, such as FTY720. However, there are substantial limitations with regard to biological agents such as mAb, owing to the specificity of mAbs for their human target structure. Moreover, the artificial methods used to induce EAE in rodents creates a potential problem when these are used for development of therapies based on modulating pathogenic mechanisms as these can differ between MS and EAE. Immunopathogenic mechanisms inducing CNS inflammation in classical mouse and rat EAE models comprise two proinflammatory T cell lineages, interferon (IFN)-g-producing T helper (Th)1 and IL-17A-producing Th17 cells. It is unresolved whether one cytokine dominates over the other or whether both cytokines are equally

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Opinion Box 1. Overview of current mouse EAE models used in MS research

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Box 2. Overview of current rat models used in MS research

Active EAE is elicited by combined activation of adaptive and innate immune reactions. The antigen required for autoimmunity induction varies depending on genetics of the strain and the adjuvant used. MHC and non-MHC genes exert promoting or protective effects in EAE [14]. The most frequently used mouse models for studying disease pathogenesis and screening of drug candidates are RR EAE induced with PLP(139–151) in SJL mice and chronic EAE induced with MOG(35–55) in C57BL/6 mice. This latter model is often considered chronic but the disease course can vary depending on details of immunization procedures. After initial paralysis disease severity worsens to reach a peak within a few days, which is typically followed by incomplete recovery. Passive EAE is established by transfer of myelin-specific T cells from animals with actively induced disease to a suitable naive recipient, usually being an individual from the same strain. The variety of passive EAE models in mice are typically based on transfer of Th1 cells against myelin peptides, such as PLP(139–151), MBP(84–102) or MOG(35–55), inducing mainly inflammation [43– 45]. Demyelination can be induced by injection of IgG antibody, usually against MOG [46]. Active and passive EAE models using genetically modified C57BL/ 6 mice overexpressing proinflammatory mediators such as cytokines at defined locations are used for unraveling pathogenic mechanisms [47]. An intriguing group of new disease models in C57BL/6 mice overexpress receptors of MOG(35–55) specific T cells as transgene (TCRMOG35–55). These spontaneously develop clinical EAE characterized by MS-like pathology in brain and spinal cord although at low incidence [48]. Intriguingly double-transgenic mice, which in addition to the TCRMOG35–55 overexpress the heavy chain of anti-MOG IgG, develop opticospinal EAE at high frequency [49]. These models allowed validation of earlier observations regarding the separate and synergistic contributions of T cells and B cells in the pathogenesis of chronic EAE in non-genetically manipulated mice. There are also attempts to use humanized mice when the immune system of the mouse is replaced with a human system [50]. However, hurdles remain because the newly introduced human immune system molecules need to operate in the context of the mouse proteome.

Although the rat has become a less frequently used EAE model, various active EAE models have been developed. Some display remarkable clinical and pathological similarity with MS, the rrMOG/ CFA-induced EAE models in DA and Brown Norway (BN) rats in particular [32,51]. DA rats develop mainly focal spinal cord and cerebellar lesions, as well as optic neuritis, whereas BN rats develop a clinical picture reminiscent of neuromyelitis optica. In LEW.1AR1 rats, cortical lesions can also be modeled. For the routine screening of drug candidates, the most frequently used rat model is the monophasic EAE in LEW rats. However, rrMOG/CFA-induced models in DA and BN rats are increasingly used for preclinical evaluation of new MS therapies. DA rats offer a relatively unique EAE model, namely full-blown clinical and pathological EAE induced by immunization with rrMOG in IFA, a formulation that lacks ligands of innate receptors (Box 3). This is an ethical improvement, because mycobacteria causing serious skin lesions are omitted, but also challenges the concept that innate immune stimulation of APCs is indispensable for in vivo activation of autoreactive Th1 cells in naı¨ve rats. Usage of this rrMOG/IFA-induced EAE model removes one bias, namely artificial stimulation of innate immune functions with strong bacterial antigens. A classical passive EAE model in LEW rats uses transfer of MBP specific CD4+ T cells. MBP-specific T cells infiltrate the CNS via defined routes involving cognate interactions with APCs within peripheral lymphoid organs and CNS [52]. Transfer EAE models are mainly characterized by CNS inflammation, but express little or no demyelination, unless autoantibodies are cotransferred [53]. Examples of therapy trials include widespread demyelination and axonal loss in optic nerve of BN rats immunized with rrMOG/CFA. After fluorogold labeling of retinal ganglion cell and subsequent EAE induction, positive effects on retinal ganglion cells survival were observed. The model has been used to design neuroprotective strategies for translation into MS, with erythropoietin, for example [54,55]. FTY720 has been used in a prophylactic and therapeutic setting for treatment of EAE induced in DA rats with whole spinal cord and rrMOG. The results demonstrated strong efficacy of this therapeutic approach and the rat was well suited to assess functional readouts of disease as assessed by electrophysiological assessments [56].

important in the disease [6,7]. The activation of both subsets requires engagement of innate immune activation inducing two related heterodimeric cytokines: IL12 (p35/ p40) for induction of Th1 and IL-23 (p19/p35) together with other cytokines [(IL-1, IL-6, transforming growth factor b (TGF-b)] for induction of Th17 [8]. It is unclear to what extent pathogenic mechanisms operating within EAE lesions, being driven by CD4+ and MHC class II-restricted T cells are representative of MS lesions, in which MHC class I-restricted CD8+ T cells are more numerous [9]. Discrepancies between an EAE-affected mouse or rat and an MS patient exist at various levels: (i) immunological differences in innate and adaptive immune functions reflect the large evolutionary distance between rodents and man [10]; (ii) rodent EAE models comprise a limited number of genetically homogeneous (inbred) mouse or rat strains, which are bred under SPF conditions; (iii) the immune system of such SPF strains is not exposed to the environmental factors/pathogens that shape the human immune system [11]; and (iv) MS develops spontaneously without an obvious external trigger(although the existence of such a trigger has not been ruled out). By contrast, the induction requires an artificial procedure, the injection of autoantigen formulated in potent bacterial adjuvants (Box 3).

Revising the EAE model Reducing the gap between EAE and MS could be achieved by creating new and refined EAE models in humanized mice or perhaps by switching to species more closely related to humans, such as the common marmoset (Callithrix jacchus). This is a small-sized Neotropical primate with high genetic similarity to humans. Its mature immune system, shaped by life-long exposure to environmental and latent infections, resembles the human immune system. The MS-like disease phenotype of marmoset EAE is particularly useful to investigate treatment approaches in RR and chronic forms of MS. Rodent and marmoset EAE models induced with myelin antigen in complete Freund’s adjuvant (CFA; Box 3) reproduce various features of MS. In these models, lesions are formed by Th1 and/or Th17 cells, inducing CNS inflammation, together with autoantibodies inducing demyelination by opsonization of myelin, triggering complementand/or macrophage-mediated cytotoxicity. However, discrepancies with chronic MS are clear, including differences in the composition of T cell infiltrates in lesions (i.e. CD4 in EAE and CD8 in MS) and the lack of efficacy of IL-12/23p40 neutralizing antibody in humans [5]. Remarkably, marmosets immunized with myelin oligodendrocyte glycoprotein 34–56 [MOG(34–56)] in incomplete

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Box 3. Adjuvant usage in the EAE model Adjuvants were classically viewed as ‘the immunologist’s dirty little secret’ [57], used to amplify antigen-specific immune reactions without understanding the mode of action. Some of the mysteries have been resolved after the discovery that APCs are equipped with pathogen recognition receptors, which relay activation signals for autoreactive cells (reviewed in [58]). In EAE models, adjuvants are used to activate innate immune mechanisms that support the induction of autoimmune diseases. Commonly used are CFA, an emulsion of mineral oil (IFA) supplemented with inactivated mycobacteria and inactivated particles or toxin of Bordetella pertussis (BP and BPT). The role of adjuvant in the EAE model is probably multifaceted, exceeding only the induction of co-stimulatory signals by peripheral APCs. Co-stimulatory signals from APCs delivered by membrane-expressed molecules (CD40, CD80/86) and soluble factors (e.g. IL-12, IL-23) are needed for disrupting the regulatory mechanism that keep autoreactive T cells in an inactive state. Adjuvants are also involved in the induction of antibody production by autoreactive B cells and in the activation of APC within the target organ [59–61]. BP particles or BPT are often injected in addition to CFA for boosting the action of proinflammatory T cells [62], to enhance antigen presentation [63] and to help T cells transmigrate the blood–brain barrier [64,65]. In addition, BPT can prevent the induction of anergy in autoreactive T cells [66] and also break established tolerance [67]. BPT has also been shown to upregulate P-selectin by TLR4-dependent mechanisms [68].

Freund’s adjuvant (IFA; Box 3) developed similar clinically evident EAE [12] as induced with MOG(34–56) in CFA [13]. The model is characterized by an almost 100% incidence of RR EAE, followed by secondary progressive disease, inflammatory/demyelination of WM and widespread demyelination of cortical GM with limited inflammation. Several immunological hallmarks distinguish the MOG(34–56)/IFA model from the corresponding MOG (34–56)/CFA model [12]. Phenotyping of in vivo activated T cells revealed that in vivo activated cells were CD3 + CD56 + CD16– and CD4 single or CD4/8 double positive. Specific T cell lines grown from lymphoid organs collected during active MOG(34–56)/IFA-induced EAE produced high levels of IL-17A. By contrast, IFN-g, which is the signature cytokine of Th1-driven models induced with CFA, was undetectable. These cell lines displayed specific granzyme/perforin-mediated cytotoxicity towards MOG(34–56) pulsed Epstein Barr virus-infected B lymphocytes. MOG protein binding antibodies, which are potent effectors of demyelination [14], were undetectable. Thus, marmoset monkeys can develop a similar type of MS-like disease and pathology along two nonoverlapping pathways (Figure 1). One pathway seems dominant in EAE models

[()TD$FIG] (a)

Pathway 1

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ROS TNF

+ CD8 CTL

Oligodendrocyte

Oligodendrocyte TRENDS in Molecular Medicine

Figure 1. Two nonoverlapping immune pathways leading to CNS demyelination. (a) Pathway 1 includes the mechanisms of inflammation mediated by autoreactive Th1 cells and demyelination mediated by autoantibodies. This pathway is observed in classical EAE model in rodents and marmosets, which are induced with recombinant MOG in CFA. (b) Pathway 2 is observed in marmosets immunized with MOG(34–56) in IFA. In this model Th17 cells induce inflammation by the activation of microglia cells and demyelination by MOG-specific cytotoxic T cells. We propose that pathway 1 models pathogenic mechanisms operating in WM lesion formation in the early phase of MS, whereas pathway 2 models pathogenic mechanisms operating in cortical GM lesion formation in chronic MS. Abbreviations used: APC = antigen presenting cell; CTL = cytotoxic T lymphocyte; Mf = macrophage; ROS = reactive oxygen species; Th17 = T helper 17 cell; TLR = Toll-like receptor.

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Opinion induced with recombinant human MOG (rhMOG) or MOG(34–56) in CFA, reproducing pathogenic mechanisms observed in classical mouse and rat EAE models (i.e. TLR, Th1 cells and autoantibodies). The second pathway, typical for EAE induced with IFA, is driven by CD4 + CD56+ and/or CD4 + CD8 + CD56+ cells. Notably, CD4 + CD56 + cytolytic T cells capable of inducing demyelination have been described in MS [15]. Translating EAE pathways to MS Both postulated pathogenic routes seem relevant for MS. Pathway 1 can primarily model the initial RRMS phenotype associated with inflammatory pathology and blood– brain barrier leakage in WM. Pathway 2 seems more relevant for late progressive MS and could be activated by the release of CNS antigens from injured CNS WM owing to Th1-induced injury [16]. The existence of two separate pathways could help explain why IFN-b treatment is particularly effective in the early Th1-driven RR phase (pathway 1), but less effective when patients convert to the Th17/NK-CTL-driven progressive phase of the disease (pathway 2). Importantly, Th1 cells are more sensitive to inhibition by IFN-b than Th17 cells, and there is evidence that IFN-b can even exacerbate Th17-dependent neuroinflammation [17]. This dichotomy is supported by preclinical therapy trials in the marmoset EAE model. Based on a substantial body of literature showing that mAbs against the shared p40 subunit of IL-23 and IL23 (IL-12p40) suppress rodent EAE models, a fully human IgG1k mAb was developed. Intravenous treatment, started before EAE induction with myelin/CFA, completely protected against EAE [18]. When marmosets immunized with rhMOG/CFA were treated with this mAb after brain WM lesions had been detected with magnetic resonance imaging (MRI), complete suppression of MRI-detectable lesion enlargement and inflammation was observed, but only delayed onset of clinical signs [19]. Similar to several mouse EAE models, Th17 cells have a central pathogenic role, thus the role of this cytokine was tested in marmoset EAE. Intravenous treatment starting one day before immunization with rhMOG/ CFA did not protect against EAE and induced only a moderately delayed onset of neurological signs without a detectable effect on lesion pathology [20]. These data suggest that neutralization of IL-12p40 is most effective early in EAE, whereas neutralization of IL-17A could be more effective in late-stage disease. IgG autoantibodies are considered essential for demyelination in rodent and marmoset EAE models. Hence, mAbs targeting two constitutively expressed surface markers of human B cells, namely CD20 and CD40, were investigated as a potential treatment. CD40 is a co-stimulatory molecule of antigen presenting cells (APCs) with pleiotropic functions in T cell activation and antibody induction, constitutively expressed on B cells and activation-induced on myeloid APCs (i.e. dendritic cells and monocytes/macrophages). The CD40 mAb suppressed clinical EAE in rhMOG/CFA-immunized marmosets associated with altered B cell responses [21]. Intravenous treatment of CD20 mAb in marmosets starting 3 weeks after immunization with rhMOG/CFA caused profound 122

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and long-lasting depletion of CD20+ B cells from the circulation, lymphoid organs and CNS [22]. The treatment induced complete suppression of clinical EAE and lesion formation in the CNS WM and had marked modulatory effects on humoral and cellular immune parameters. The studies in the marmoset EAE model indicate a central pathogenic role of B cells in EAE, which is exerted via canonical functions, such as autoantibody production, as well as noncanonical mechanisms, such as the activation of autoreactive T cells. These experiments in the marmoset confirm pathogenic concepts gained from rodent EAE models, but raise two important questions with regard to translation to MS. First, why is the anti-IL-12p40 antibody so much more effective than the anti-IL-17A mAb in the rhMOG/CFA EAE model and why is the same antibody ineffective in MS? Second, why is the B cell depleting antibody so effective in the rhMOG/CFA EAE model, even in the presence of activated T cells capable of inducing widespread demyelination independent of autoantibodies? The answer to both questions might be in the artificial procedures used for EAE induction with strong bacterial adjuvants present in CFA. T cells producing IL-17 and having specific cytotoxic activity that seem to mediate demyelination in the MOG(34–56)/IFA model in marmosets were not found in mouse EAE models [12]. However, there are some similarities between marmoset and mouse EAE models. For example, IL-17A-producing CD4+ (Th17) and CD8+ (Tc17) cells are induced by the same cytokines (i.e. TGFb, IL-6 and IL-23), but with an additional requirement of IL21 for Tc17 induction in the mouse [23]. Whether IL17Aproducing CD4+ and CD8+ T cells have a central pathogenic role in all mouse EAE models is debated. Tc17 cells might not have an EAE promoting but rather a suppressive role in EAE [24], whereas the pathogenic CD8+ T cells that induced EAE in MOG(35–55) sensitized C57BL/ 6 mice resemble classical CTL and fail to produce IL-17A [25]. In MS the presence of (MBP-specific) CD4+CD56+ T cells with in vitro cytolytic activity towards oligodendrocytes has been documented [15,26]. Both reports postulate a nonMHC restricted and thus peptide-independent cytolytic mechanism, which is compatible with the absence of MHC class II expression by oligodendrocytes in noninflamed CNS. More recently, MOG(34–56) specific CD8+ T cells were identified in MS, but the capacity of these cells to induce demyelination was not investigated [27]. Implications for therapy development for MS The immunopathogenesis of MS is probably compartmentalized, with relevant components of the immune response operating primarily in the periphery beside lesion-related components. It is unlikely that this pathogenic complexity can be reproduced in a single disease model. Rather, different EAE models should be developed which highlight specific aspects of MS immunobiology and neuropathology. Cases where translation of immunomodulatory agents from EAE to MS was successful (e.g. glatiramer acetate, natalizumab or FTY720) concerned treatments mainly effective in RRMS. Regarding mechanism of action, RRMS seems driven by infiltration of peripherally activated

Opinion immune cells into the CNS. The effectiveness of IFN-b in this phase suggests that infiltrating T cells are mainly of the Th1 phenotype [17]. The lack of efficacy of the IL-12p40 neutralizing mAb ustekinumab in RRMS is more difficult to understand because the current EAE models clearly demonstrate that IL-12 and IL-23 are key factors in the induction of Th1 and Th17 cells. As an explanation, we postulate here that autoreactive Th1 cells activated in the initiation of MS could be derived from a pre-existing and committed population of antigen-experienced Th1 memory cells. Non-human primate EAE models show that such preexisting populations exist in a mature immune repertoire and can be generated in response to persistent herpesvirus infection [28]. It is tempting to speculate that the different specificities of (antigen-experienced) T cells specific for MBP, proteolipid protein (PLP) and MOG epitopes that were identified in the T cell antigen receptor (TCR) repertoire of MS patients [29–31] were shaped and tuned by infection However, direct evidence is lacking. The notion that autoreactive T cells could originate from a virus-induced repertoire of antigen-experienced T cells marks an important difference with actively induced rodent EAE models. In the latter models, naı¨ve autoreactive T cells need to be primed and mobilized from an immune repertoire with limited antigen experience. Activation of mouse autoreactive T cells requires the help of strong bacterial adjuvants such as CFA and BP or BPT (Box 3). Such models are useful for development of therapies aiming at the modulation of Th1 effector mechanisms, but the experience with ustekinumab illustrates that they can yield ‘false-positive’ results. With progression of MS, the efficacy of the current Th1cell directed therapies declines, but (CD8+) T cells are still prominent in WM lesions. It has therefore been argued that in late-stage progressive MS, cortical GM lesions and progressive neurodegeneration could be pathogenetically more important and can develop without an obvious role of autoreactive T cells. Innate immune mechanisms are thought to be operative in these phases, with increasing involvement of microglia cells and astrocytes. Studies in unique sets of congenic LEW based rat strains show a clear association between the presence of cortical GM demyelination and certain MHC class II alleles, suggesting an adaptive immune cause [32]. In addition, demyelination of cortical GM is consistently found in marmosets with EAE [33,34]. Immunological characterization of T cells mediating EAE in the MOG(34–56)/IFA marmoset model shows clear functional differences with Th1 cells, namely a combination of high IL-17A production and cytolytic activity. The marmoset EAE model shows the first MRI-detectable signs of CNS inflammation around the lateral ventricles in proximity to the choroid plexus. This mimics what is seen in MS patients, where Th17 cells can infiltrate a noninflamed brain and gain access to the cerebrospinal fluid (CSF) via CCR6–CCL20 interactions in the choroid plexus [35]. A recent report suggests that Th17 cells can also induce GM injury by activating microglia [36]. In addition, Th17 cells can facilitate CNS infiltration by cytolytic T cells via the same route, which then could attack the GM from the CSF. Thus, the marmoset model reflects the dynamic pathogenesis of MS

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and shows that different pathogenic phases of MS can be driven by distinct T cell specificities and functionalities. Concluding remarks The marmoset model more closely mimics the antigenexperienced nature of the human immune system than available rodent systems. The high costs and ethical constraints, however, make non-human primates an improbable model for the routine screening of large amounts of compounds from development pipelines. A highly promising development enabling the generation of better disease models is by humanization of mouse strains. Although in their infancy, these models have already been of great value for specific aspects of MS pathology and therapy development. Mice carrying functional human genes, cells and tissues have been used as models of inflammatory demyelination, with particular focus on the functional role of antigen presentation and T cell activation in autoreactivity to myelin. For this purpose, single or multiple transgenes have been expressed in the mouse, such as HLA-DR1, DR2 (DRB1*1501, DRB5*0101), DR4 (DRB1*0401), DR3, DQ (QB1*0602), CD4 a humanized myelin basic protein-HLADR2-zeta chimeric receptor, a MBP(85–99)-specific T cell receptor, a MBP(111–129)-specific T cell clone, and CD52 [37]. For example, triple transgenic mice expressing HLADRB5*0101 (DR2a) and DRB1*1501 (DR2b), the TCR of a T cell clone derived from an MS patient and specific for MBP(84–102) bound to HLA-DRB1*1501, and the human CD4 coreceptor were used to study the relevance of such molecules to CNS inflammation in vivo in the absence of endogenous conventional mouse MHC class II genes. Upon immunization with the immunodominant MBP(84–102) peptide (identical in mouse and human MBP), almost all transgenic mice developed neurologic disease, with a variety of clinical manifestations that was reminiscent of CNS lesions seen in human MS [38]. In a more recent study, it was shown in mice transgenic for the human HLA-DR15 haplotype (DRB1*1501, DRB5*0101 and DQB1*0602) that it is DQB1*0602 rather than DRB1*1501 which confers susceptibility to EAE induced by two peptides of a different myelin protein, myelin-associated oligodendrocytic basic protein, MOBP(15–36) and MOBP(55–7), by inducing encephalitogenic Th1/Th17 cells instead of nonpathogenic Th2 cells [39]. In addition, a hCD52 transgenic mouse (CD52 is not expressed in murine immune cells) is proving instrumental in characterizing the involvement of neutrophils and NK cells in the activity of the potent diseasemodifying treatment, alemtuzumab. In spite of difficulties related to the introduction of human immune system molecules into the mouse proteome context, such humanized models are expected to be of increasing value to clarify specific aspects of MS pathology and therapeutics. Preclinical models should preferably account for the antigen-experienced state of the human immune system. This could be achieved by using mouse or rat models based on prior induction of antigen-experienced autoreactive T cells, such as SJL mice infected with Theiler’s murine encephalitis virus followed by immunization with PLP(139–151) or of Biozzi ABH mice infected with Semliki Forest virus followed by immunization with MOG(8–21). 123

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Box 4. Outstanding questions  Are relapsing–remitting and progressive MS driven by T cell mediated immune mechanisms differing in specificity and quality?  Does CNS injury induce the recruitment of T cells from a virusinduced memory repertoire? How and where are these cells activated? What is their specific role in cortical gray matter lesion induction? Can this mechanism be targeted pharmacologically?  Is disease progression in chronic MS driven by these recruited T cells?  Are these T cells capable of inducing antibody-independent demyelination?  What is the functional interplay between CD4+ and CD8+ autoreactive T cells in the induction and evolution of lesions?  How relevant to human pathology are the known protective aspects of T cell mediated CNS inflammation?

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As mentioned, the Dark Agouti (DA) rat EAE model could be useful because the induction of full-blown EAE with spinal cord homogenate/IFA or rrMOG/IFA suggests a preexisting antigen-experienced state of the pathogenic MBPspecific Th1 cells [40,41] resembling observations in marmosets. These models can be particularly useful for analyzing whether novel therapeutic strategies, such as Treg cells or altered peptide ligands, are also capable of modulating the activity of antigen-experienced effector T cells. As a final preclinical step, the EAE model in marmosets can help to assess whether effective lead compounds in rodent EAE models are also effective in higher species. It is common practice in the transplantation field that new therapies are validated in non-human primates before they are tested in patients [42]. We believe that translation of this principle to therapy development for MS should be considered (Box 4).

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