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Keywords:

  • Animal models;
  • Autoimmunity;
  • EAE;
  • MS

Abstract

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

EAE comes in many shapes and colors. Individual variants of EAE present a baffling complexity of different aspects and traits, clinical, immunological, and structural. But, embedded in this seemingly chaotic image, the educated eye will discern patterns that retrace fundamental features of immune response, in particular, autoimmunity and self-tolerance. EAE and its variants thus can be likened to an autostereogram, i.e. they are an immunologist's magic eye.

This brief review intends to serve as a guide through the diversity of EAE models. First, it sums up the basics of animal models for EAE and the use of these models in biomedical research. Second, there is a brief summary of discoveries that have been made using EAE models and that have fundamentally shaped our present day's concepts of the immune pathogenesis of MS, and, beyond this, of immune reactivity, self-tolerance, and autoimmunity, in general.

Variant models of EAE

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

Modes of induction

EAE has been actively induced in susceptible experimental animals via several routes. Initially, active immunization with CNS homogenates or myelin components suspended in strong adjuvant was used. Later, especially with the advent of T-cellline technology, EAE was passively transferred by in vitro-activated autoimmune T-cell lines. More recently, spontaneous EAE models have been developed with transgenic mice in which a large proportion of T cells are myelin-specific (Tables 1 and 2).

Actively induced EAE (aEAE) is the easiest inducible model. It yields fast and robust results in studies screening the effects of drugs on autoimmune inflammation or the function of particular genes, for example, in transgenic/knock-out mice. These models are the “gold standard” for studying the encephalitogenic potential of myelin and neuronal autoantigens. Another important application of aEAE is in studies with nonhuman primates, whose immune systems resemble that of humans much more closely than that of rodents. Interpretation of aEAE data, however, is compromised by the mode of induction, which requires the inoculation of large deposits of adjuvant that may globally distort immune reactivity in the treated individual.

Table 1. Commonly used active and passive EAE models and their applications
Species/strainKnown encephalitogenic autoantigen/epitopesCommon studies and applications
 aEAEpEAE 
Mice
C57BL/6MOG 35–555; OSP, OSP 179–207 42; rNogo-66 9NF-M 18–30 10; MOG 35–55 5Commonly used strain for transgenic mice construction and many transgenic and knock-out mice available; Th1/Th17 CD4+ T-cell-mediated pathogenesis; pre-clinical validation of therapeutic compounds; CD8+ T-cell-mediated CNS damage
SJL/JMOG, MOG 92–106 43; PLP 139–151 44; PLP 178–191 45; PLP 104–117 46; OSP, OSP 57–72 47; MOBP 37–60 48; MOBP 15–36 49; MBP 50; MBP 89–10151; Nogo-66 9MOG 27; PLP 139–151 52; PLP 178–191 53; OSP 54; MOBP 15–36 49; MBP 89–101 55Study of relapse mechanisms; genetic control of autoimmune disease; epitope spreading; antibody-mediated demyelination; gender influence on autoimmunity; pre-clinical validation of therapeutic compounds
Biozzi ABHMOG, MOG 1–22, MOG 43–57, MOG 134–148 43; PLP 56–70 56; MBP 21–35 51; MAG 97–112 47; αβ-crystallin 1–16 57; NF-L 11; GFAP 58Study of relapse mechanisms; antibody-mediated demyelination; pre-clinical validation of therapeutic compounds
B10.PL and PL/JMBP Ac1–11 59, 60; MOG 35–55 61MBP Ac1–11 59, 60; MOG 35–55 61;Study of Treg cells; molecular mimicry and environmental triggers; T-cell self-tolerance; pre-clinical validation of therapeutic compounds
C3H/HeJPLP 215–232, PLP 53MBP 79–87 62CD8+ T-cell-mediated CNS damage; T-cell self-tolerance
Rat
LewisMBP, MBP 29–84, MBP 61–82, MBP 80–105, MBP 170–186 2, 3; β-Synuclein 93–111 8MBP 2, 3; β-Synuclein 93–111 8Study of migratory behavior of autoimmune T cells; genetic control of autoimmunity; pre-clinical validation of therapeutic compounds
DA, BNMOG 63MOG 74–90, MOG 93–107 64Antibody-mediated demyelination; molecular mimicry; genetic control of autoimmunity
Primates
Rhesus monkey (Macaca mulatta)MBP, MOG 34–56 65Environmental triggers of CNS autoimmunity; axonal pathology; antibody-mediated demyelination; pre-clinical validation of therapeutic compounds
Common marmoset (Callithrix jacchus)MOG, MOG 14–36 66 
Table 2. Spontaneous EAE mouse models
StrainEpitopeModelSpontaneous autoimmunity and clinical characteristicsApplications
C57BL/6MOG 35–55CD4+ TCR tg 67Paralytic EAE and optic neuritis (4 and 30%)Study of autoimmune mechanisms developing without exogenous manipulation (spontaneous T-cell activation, B-cell responses, innate immune mechanisms, pre-clinical validation of therapeutic compounds)
C57BL/6MOG 35–55 and MOGCD4+ TCR tg × BCR tg 29Paralytic EAE and optic neuritis (50%) 
B10.PLMBP Ac1–9CD4+ TCR tg 68Paralytic EAE (100% in RAG-deficient background) 
B10.PLMBP Ac1–11CD4+ TCR tg 22Paralytic EAE (14–44%) 
SJL/JPLP 139–151CD4+ TCR tg 69Paralytic EAE (45–83%) 
SJL/JMOG 92–106CD4+ TCR tg 27Paralytic and ataxic EAE, relapsing –remitting (60–90%) 
C57BL/6Neo-self antigen OVAODC-OVA tg × OT-I (CD8+) TCR tg 70Paralytic EAE and locomotor defects (90% in normal background and 100% in RAG-deficient background) 
Humanized Tg (HLA-DR2)hMBP 84–102Human CD4+ TCR tg (with HLA DR2) 71Classical EAE (4% in normal background and 100% in RAG-deficient background)To test human immune mechanisms in an in vivo context (genetic control, effector mechanisms)
Humanized Tg (HLA A3)hPLP 45–53Human CD8+ TCR tg (with HLA A3) 41Motor deficits (4%) 
Humanized Tg (HLA-DR2)hMBP 85–99Human CD4+ TCR tg (with or without HLA-DR2) 40Progressive EAE (86%) without HLA-DR2 and relapsing EAE with HLA-DR2 (54%) in RAG-deficient background 

Passively transferred EAE (pEAE) have also been useful in drug screening and functional gene characterization. New applications include the use of genetically modified T-cell lines expressing fluorescent protein markers for real-time imaging. pEAE studies are, however, hampered by potential artifacts produced by in vitro activation and the bulk transfer of large numbers of maximally activated effector populations.

The problems inherent in adjuvant inoculation and bulk transfers of in vitro-activated T cells are circumvented in TCR transgenic mice that develop spontaneous EAE without experimental manipulation 1. Within these models, “humanized” mice carrying TCR from human T-cell clones are of great use in directly investigating the effects of human TCR on disease course.

Antigens/animal combinations

There are rodent strains reputed to be particularly susceptible to aEAE, and others appear resistant, but in each animal strain, EAE responsiveness critically depends on the nature of the autoantigen applied. Some CNS proteins are highly encephalitogenic in one animal strain, but not so in another. Lewis rats, for example, are highly responsive to autoimmunization against myelin basic protein (MBP) 2, 3, but hardly respond to myelin oligodendrocyte glycoprotein (MOG) 4, while reactivity of C57BL/6 mice is the exact opposite 5, 6.

Initial attempts to establish definitive target autoantigen in MS were largely focused on major myelin proteins such as MBP and proteolipid protein (PLP) as these are considered to be the logical targets. Over the years, however, other myelin and nonmyelin proteins were also studied. The ever-growing list of potential autoantigens in MS and EAE now includes myelin antigens (MBP, PLP, MOG, MOBP, MAG, OSP, Nogo-A, CNPase), glial antigens (GFAP, S100ß, αβ-crystallin), and neuronal antigens (Neurofilament-L, Neurofilament-M, β-Synuclein, Contactin-2, Neurofascin) 7–14.

Until a few years ago, Lewis rats were the most popular animals used for EAE studies, because of their 100% responsiveness to EAE induction by active immunization with MBP containing CFA, and the stability of anti-MBP-specific T-cell lines isolated from primed donors. More recently, C57BL/6 mice have become the animals of choice, especially for studies involving transgenic mice. Lewis rats and C57BL/6 mice develop either self-limited monophasic or chronic EAE. In addition to these models, EAE with interchanging relapses and remissions, reminiscent of early human MS, can be induced in SJL/J or Biozzi ABH mice. Finally, primate models of EAE have been used. As closer relatives to humans, primate models share clinical and pathological similarities to human MS 15. The main fields of application of these EAE variants are summarized in Tables 1 and 2.

Insights from EAE studies

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

Early discoveries: Freund's adjuvant and myelin autoantigens

Rivers et al. 16 repeatedly injected rabbit brain homogenate into rhesus monkey and observed in two out of eight treated animals the development of paralytic disease, a condition later termed EAE. Similar treatment did not cause EAE in smaller experimental animals. EAE was, however, reliably inducible in Guinea pigs or rodents by subcutaneous injection of brain matter suspended in a water/oil emulsion with CFA 17. Experiments using such protocols identified the molecular structures in brain white matter, such as PLP 18 and MBP 19, as the targets of the inflammatory response. Thus, early EAE experiments laid the base to our understanding of autoimmune reactions against individual tissue-specific molecules, and, practically, they produced the most widely used experimental immune adjuvant.

Autoimmune T-cell clones in the normal T-cell repertoire

EAE induced by MBP immunization in the Lewis rat was the first paradigm of experimental autoimmunity to allow the isolation of pure lines of autoimmune effector T cells 20. The finding that CD4+ T cells exclusively specific for MBP were able to transfer classical EAE to naïve syngeneic hosts established that T cells were the driving force in at least this model of organ-specific autoimmune disease. Moreover, as T-cell receptor sequences remain stable (without somatic mutation) throughout immune responses, one could conclude that the T-cell line progenitors preexisted in the original donor animal. This was definitively established when encephalitogenic MBP-specific T-cell lines were isolated from naïve, nonimmunized rats 21. The T-cell-receptor genes of MBP-specific mouse T cells were cloned and inserted into the germline of transgenic mice 22. While the majority of all CD4+ T cells in these transgenics expressed the autoreactive T-cell receptor, only a minority of the animals spontaneously developed EAE. These experiments were the first to directly demonstrate potentially autoaggressive T-cell clones in the immune system of healthy experimental animals. Later studies confirmed the presence of such cells in primates and humans. This necessitated a modification of the clonal selection theory, which ascribed immunological self-tolerance exclusively to the physical elimination of self-reactive immune cell clones, thus prohibiting the existence of additional regulatory mechanisms such as Treg (vide infra).

Treg cells and effector T-cell lineages

As mentioned, transgenic mice with MBP-specific T cells rarely develop spontaneous EAE when bred on an immunocompetent background. In the absence of endogenous immune cells (e.g. RAG knock-out mice), however, these animals develop lethal EAE, which can be rescued by transfers of WT CD4+ T cells 23. The T cells responsible for protection against EAE were ultimately identified as Treg, cells that express the transcription factor FoxP3 as a lineage-specific genetic marker 24. Paradoxically, CNS-specific Th17 and Treg both require TGF-β as key inducing factor. The key cytokine turns out to be IL-6: it drives naïve CD4 T cells toward Th17 differentiation, while in its absence Treg differentiation prevails 25.

These basics of Treg biology and Th17 cells have been recognized in EAE, but, although Treg-like cells have been described in human disease, including MS, their relevance and their biology remain still largely uncharted.

B-cell effector functions (autoantibody, cytokine, APC)

The classic early work established that CD4+ T cells are the critical effectors of CNS autoimmune disease, but at the same time, soon it became clear that autoimmune B cells and their antibody products have essential roles too. Injection of monoclonal antibodies against a myelin surface glycoprotein, MOG, aggravated actively induced or T-cell-transfer EAE by enhancing demyelination 26. The role of autoimmune B cell became more visible with protocols that allow for depletion of B cells, and, especially, with the advent of transgenic mice, expressing immunoglobulin H chain-binding MOG autoantigen. Numerous studies have revealed that B cells have diverse functions in the pathogenesis of CNS autoimmune disease. A complementary transgenic model, the SJL/J mice with transgenic MOG-specific T-cell receptors, showed that autoimmune T cells select and expand myelin-specific B lymphocytes from the endogenous immune repertoire 27.

Apart from secreting demyelinating autoantibodies, B cells preferentially present myelin autoantigen by concentrating local autoantigen with their surface immunoglobulin receptors 28, 29. B cells also contribute to create particular cytokine milieus 30, 31. With these potentials, B cells seem to contribute to MS lesions (pattern 2), with Ig decorated myelin debris and complement activation, and B cell infiltrates, even follicle-like structures in meningeal areas.

Migration pathways

Much of our knowledge on the fate of autoimmune T cells in vivo comes from EAE studies. Earliest work with T-cell lines showed a lag period between transfer of encephalitogenic T cells and onset of clinical disease. More recently, genetically labeled, fluorescent T cells were used for detailed studies of effector cells in situ. The work revealed strict migratory pathways, with activated effector cells first traveling to the lungs, then to lymphoid organs, until they reach their CNS destination via the blood–brain barrier. T cells changed their gene expression profiles in each of their surrounding milieus 32. Imaging techniques also illustrated through which mechanisms autoimmune T cells interact with the endothelial blood–brain barrier 33–35, and how the T cells navigate through the CNS tissue 36, 37. These studies go beyond EAE, establishing basic principles of autoimmune T-cell behavior such as local recognition of autoantigens by the invading T cells and potentially revealing new therapeutic targets.

Genetic control

EAE studies have been valid predictors of genes, which later were confirmed to control susceptibility in human MS, too. One recent example is the gene for the alpha chain of the IL-7 receptor. Large genome-wide screenings identified IL-7Rα as a risk factor gene for conferring susceptibility to chronic EAE in mice 38, and were later shown to be also relevant in humans 39. Another example documenting the value of genetic EAE research comes from humanized transgenic mice carrying human myelin-specific TCR along with human HLA products. A recent report detailed the epistatic interactions between products of an MS-favoring HLA-DR haplotype 40 and the modulatory effects of HLA-A products 41.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

EAE is an experimental paradigm most popular among cellular immunologists. A simple search in Medline documents more than 6500 articles carrying EAE or its full term in the title. EAE's attractiveness is owed to several assets. First of all, EAE is technically simple and reliable, involving important cellular immune mechanisms such as regulation of autoimmunity. Second, with the development of a stable EAE model in the C57BL/6 mouse, EAE became the paradigm of choice to examine gene effects on autoimmunity and T-cell responsiveness in transgenic/knock-out mice. Third, it is useful for the retrieval of organ-specific targets and represents essential aspects of MS, an important human disease, and to evaluate novel therapies although they cannot guarantee a biological safety in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereiche (SFB) 571 and the Max Planck Society.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Variant models of EAE
  4. Insights from EAE studies
  5. Concluding remarks
  6. Acknowledgements
  7. References
  8. Supporting Information

See accompanying article: http://dx.doi.org/10.1002/eji.200939545

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