The employment of the animal model experimental autoimmune encephalomyelitis (EAE) to further unravel the multifaceted etiology of multiple sclerosis (MS) has received both acclamation and criticism. The complexity of MS is owing to its association with a repertoire of genetic variants (1) in combination with the confounding influence of exogenous environmental factors (reviewed in Refs. 2, 3). Attempting to develop a model of MS in its entirety is therefore ambitious, and it is perhaps such attempts that have led to erroneous and, at times, misleading results that have raised uncertainties over their value. In stark contrast, using the EAE model to test a defined hypothesis has proven that it can provide a resourceful platform with which to understand specific disease pathways and processes implicated not only in MS, but in autoimmune diseases as a whole (4). This is exemplified by the wealth of knowledge that this model has imparted during the last few decades and the therapies that have been developed as a result (5).

In the early 1990s the murine model of EAE gained momentum by harnessing the development of transgenic technologies in which the murine genome could be manipulated to magnify a subtle effect into one that could be more easily investigated. In 1993 and then a year later, two independent groups—Goverman et al. (6) and Lafaille et al. (7), respectively—published their findings in Cell in which they generated a transgenic mouse model for which all CD4+ T cells expressed a TCR specific for the dominant myelin basic protein (MBP) epitope, MBP1–11, in mice bearing the H-2u haplotype (6). In generating a transgenic model for which all CD4+ T cells responded uniformly to one Ag, specific hypotheses could be tested pertaining to both tolerance mechanisms and the effects posed by the surrounding environment.

Classical EAE induction requires s.c. administration of a myelin-specific peptide emulsified in CFA, in addition to i.v. or i.p. injection of Bordatella pertussis toxin. Depending on the strain, disease will develop within 1–2 wk and initiates with a limp tail, progressing to eventual hind limb paralysis (8). Using the TCR-transgenic mouse model, Goverman et al. were able to determine which components of this immunizing regimen were essential for disease induction and, moreover, how this information could be used to further stratify variation observed in disease severity across the strain. All transgenic mice were highly susceptible to disease induction, and, furthermore, disease was exacerbated compared with the nontransgenic controls, thus providing compelling evidence that central or peripheral tolerance mechanisms were not effective against the MBP1–11 epitope.

Sequentially omitting components from the immunizing protocol revealed that the key catalyst in the susceptibility of the transgenic line was their exposure to pertussis toxin, conferring a comparable disease incidence to that observed when administered in combination with MBP and CFA. Importantly, this demonstrated that although a high level of precursor cell frequency was required, the ability to detect the autoantigen was not the sole determinant in disease onset. In fact, these data implied that what were necessary were the danger signals and access of the immune cells to the CNS under inflammatory conditions to break through the disease induction threshold. In corroboration with these observations, several TCR-transgenic mice within the colony, which was housed in a nonsterile facility, developed spontaneous disease, whereas those housed under specific pathogen-free conditions remained disease free. Furthermore, whereas transgenic mice developed EAE when immunized with MBP and CFA without pertussis toxin, albeit to a lower severity than that observed with the addition of pertussis, no mice developed disease when following this immunizing protocol under specific pathogen-free conditions. Goverman et al. hypothesized that an asymptomatic infection may have been influencing this outcome, despite being unable to uncover a specific culprit.

Both clinical experience and scientific data, including those described herein, have implicated the role of not only opportunistic infections (9) but also that of commensal gut bacteria as potential immune triggers in predisposed individuals (10, 11). A breakdown in the homeostasis between the gut microbiome and the immune system has been associated with several autoimmune disorders, including inflammatory bowel disease (12), type 1 diabetes (13), and MS (reviewed in Ref. 14). Moreover, studies have also indicated that the immune system is actually dependent on the presence of intestinal bacteria to fully mature (1517). Although Goverman et al. did not explore an association with commensal bacteria in their study, a follow-up study demonstrated that variation in the gut microbiome across the housing facilities was a viable candidate for the correlation observed with disease incidence (18). More recently, supporting evidence for this has demonstrated that mice that are housed in germ-free conditions, and therefore have no gut flora, are protected against both spontaneous (19) and inducible (20) EAE. However, the extent of protection upon active immunization was variable between research groups, with one study showing partial disease protection (19), whereas that in another was nearly complete (20). Such discordance may be explained by genetic differences between the strains that were used for each study, and so highlights the importance of considering genetic background when correlating any environmental stimuli with disease risk.

Although disease burden in the TCR-transgenic mice was exacerbated compared with nontransgenic controls, owing to the overwhelming abundance of MBP-specific autoreactive T cells that had escaped negative selection, Goverman et al. highlighted that this may also be a result of the absence of regulatory CD8+ T cells owing to their selection disadvantage during thymic development. In support of these findings, several other murine EAE models have identified that CD8+ T cells can have both regulatory (21, 22) and pathogenic roles (23) in MS. Initial studies using CD8+ knockout mice showed that CD8+ T cells could be regulatory, as exacerbated EAE disease was observed in such mice compared with CD8+ T cell–sufficient mice (21). Furthermore, the presence of CD8+ T cells was required for protection against a second round of immunization (22). More recently, a population of regulatory CD8+ T cells has been identified that is able to suppress both Ab- (24) and CD4+ T cell–mediated responses in EAE (25). These cells are restricted by the MHC class Ib molecule, Qa-1 (HLA-E in humans), which upon interaction with either the TCR or CD94/NKG2A complex on CD8+ T cells will lead to their activation or attenuation, respectively (reviewed in Ref. 26).

A year following Goverman et al.’s seminal work, Lafaille et al. published the role of MBP-specific CD4+ T cells in Rag1-deficient mice (7), in which endogenous CD4+ and CD8+ T cells do not develop (27). By stripping back this model to its main component, this study provided an opportunity to investigate the pathogenic power of autoreactive CD4+ T cells in the absence of contributing regulatory or pathogenic factors. In doing so, Lafaille et al. discovered that autoreactive CD4+ T cells were able to invoke spontaneous disease independently in 100% of TCR-transgenic Rag1-deficient mice, in comparison with only 14% of their Rag1-sufficient littermates. Histological analysis revealed that disease occurred as a result of CNS-infiltrating CD4+ T cells and an abundance of MHC class II–expressing cells (macrophages/microglia). Interestingly, phenotyping the CD4+ T cells showed that they were only active at the site of injury and remained inactive in the periphery. Despite the CNS being an immune privileged site, this observation gave credence to the notion that a low level of immune surveillance of the CNS occurs routinely, and in the absence of regulation, autoreactive cells that see their cognate Ag have the capacity to mount an attack. These important findings perpetuated the idea that understanding immune regulation was as fundamentally important to resolving disease burden as being able to identify the cells that posed the risk. Successive studies using this model were able to further contribute to this understanding, and, in particular, provided a platform with which to characterize the role of regulatory CD4+ T cells (reviewed in Ref. 28).

An intriguing, and likely unpredicted, observation made by both groups throughout their studies was that the development of spontaneous disease was not uniform across the mouse line; some mice developed EAE at an early stage of adulthood, whereas others developed disease several months later. It is not possible to exclude any exogenous influence on these findings. However, owing to the fact that mice within the same cage showed variability in time to disease onset, this is an unlikely explanation. Genetic variation within the same strain, either by subtle differences inherited from the original founder lines or via epigenetic modifications, may provide a possible explanation. Although outside the scope of either of these studies, using these models to pinpoint the genes or epistatic interactions that control such variability could provide a rich resource of information (29).

It is clear from both of these papers that making a transgenic mouse model often bestows more outcomes than simply testing the hypothesis, and importantly, because of this, in vivo models provide a key to understanding events that previous biological experience could not foresee. In the case of Goverman et al. and Lafaille et al., their models provided a pivotal platform from which to dissect underlying disease mechanisms in EAE that relate not only to interactions within the immune system but also to the relationship of these mechanisms to the environment.

Abbreviations used in this article:

EAE

experimental autoimmune encephalomyelitis

MBP

myelin basic protein

MS

multiple sclerosis.

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The authors have no financial conflicts of interest.