Aging Western societies are facing an increasing prevalence of chronic
autoimmune-mediated inflammatory disorders (AIMIDs) for which treatments that are safe and effective are scarce. One of the
main reasons for this situation is the lack of animal models, which accurately replicate
clinical and pathological aspects of the human diseases. One important AIMID is the
neuroinflammatory disease multiple sclerosis (MS), for which the mouse experimental
autoimmune encephalomyelitis (EAE) model has been frequently used in preclinical
research. Despite some successes, there is a long list of experimental treatments that
have failed to reproduce promising effects observed in murine EAE models when they were
tested in the clinic. This frustrating situation indicates a wide validity gap between
mouse EAE and MS. This monography describes the development of an EAE model in nonhuman
primates, which may help to bridge the gap.
Foreword
The marmoset experimental autoimmune encephalomyelitis (EAE) model was first
documented in 1995 by the Florentine neurologist Dr. Luca Massacesi, who pioneered the
model in the laboratory of Prof. Steve Hauser at UCSF (University of California, San
Francisco, USA). During my research at the Biomedical Primate Research Centre (Rijswijk,
the Netherlands) I had already some experience with an EAE model in rhesus monkeys (see
review; Brok et al., 2001; 't Hart et al., 2005a), but was rather unhappy with the an
acute clinical course of the model and the destructive neuropathology, which more closely
resembled acute postinfectious demyelinating disease, such as acute disseminated
encephalomyelitis (ADEM) than multiple sclerosis (MS; 't Hart et al., 2005a). The
description of the new model in marmosets looked much better than our rhesus monkey EAE
model and this became clearer once we started collecting our own data.
An important success factor for the model has been our choice to focus on
translational research into the pathogenesis as well as the treatment of MS.
Our research thus stood on two legs, an exploratory leg where we unraveled
(immuno)pathogenic mechanisms and an applied leg where the efficacy and
safety of new therapies were tested (see Fig. 1). The underlying thought
was that via this strategy we could use information from the applied leg to
validate new pathogenic concepts developed in the exploratory leg.
Translational research: an iterative process. The main goal of our
exploratory preclinical research has been to find new targets in the pathogenic process
for safer and more effective therapies. The translation of a new scientific discovery
into a safe and effective innovative treatment for patients is indicated as forward
translation. In the applied arm of our research, new therapies are tested. Results from
such tests can be used to validate scientific concepts. When the process of forward
translation fails, the reasons for failure should be investigated and this information
should be fed back (i.e., reverse translation) to the animal model in order to make the
necessary corrections in the scientific concept and/or the animal model itself.
At the start of our MS research in the marmoset EAE model, our thinking was strongly
influenced by concepts developed in well-established EAE models in immunologically
naïve rodents (i.e., specific pathogen-free, SPF, mice and rats). Indeed, we assumed that just like in the rodent
models, autoreactive T cells in marmosets are naïve and require strong stimulation
with danger signals for escaping regulatory mechanisms that keep them inactive
(Matzinger, 1994). It took us many years to realize that this rather ignorant view was
fundamentally wrong and that the immune systems of conventionally reared marmosets and
SPF-bred mice or rats are for a large part incomparable. The same is true for the human
immune system, as we can learn from the seminal work of Mark Davis and colleagues at
Stanford University (CA, USA) (Davis, 2008; Brodin and Davis, 2017). There is now also
mounting evidence that environmental factors have a profound influence on the
immunocompetence of animal disease models. As an example, cohousing of SPF-bred
laboratory mice with mice from pet shops or the wild creates a more human-like immune
system (Beura et al., 2016). As stated by the authors of this hallmark publication, it is
indeed ironic that an immunologically inexperienced 10–12-week-old mouse has become de
rigueur for studies on the complex human immune system in health and disease (Beura et
al., 2016).
We made the exciting discovery that EAE in marmosets is not driven by a single pathway
but by two fundamentally different autoimmune pathways, one of which is more or less
identical to the one in mouse EAE, while the other is completely different. The
observation that the T cells that drive disease progression in the marmoset EAE model
might be recruited from a repertoire of antigen-experienced T cells, which are absent in
inexperienced SPF-bred rodents, underlies the concept that autoimmunity in MS might not
be elicited by an infection (response-to-infection paradigm), but by primary injury
inside the central nervous system (CNS; response-to-injury paradigm) ('t Hart et al.,
2009).
Clinical and pathological aspects of marmoset EAE induced with MS myelin/CFA.(a) The graphs
show the protracted clinical course, which is variable among individual animals. Case EH
has relapsing–remitting disease that could be followed for almost 1 year. The other four
cases transit to progressive disease, which can worsen quickly (EK, EL) or more slowly
(EI, EJ). (b) Case EI was subjected to in vivo magnetic resonance imaging
(MRI) just before sacrifice. The middle and bottom rows show two horizontal brain slices from
a T2-weighted image (middle) and a postcontrast (triple dose Gadolinium-DTPA; bottom)
with the position of corresponding lesions indicated. After the scans were made, the
monkey was humanely killed, the brain was removed and fixated in toto. Then a new
postmortem T2-weighted scan was made. This allowed us to determine the exact position of
all lesions that were detected in vivo. (c) The top row shows two coronal
sections of the same MRI scan with lesions indicated. The middle and bottom rows show a
magnification of individual lesions, which allowed us to conclude that brain lesions in
this model are presented in different stages. (d) The top image shows an MRP14
staining of macrophages from lesions I and J, illustrating their inflammatory active
nature. Notice that lesion I is one of the two gadolinium contrast-enhancing lesions. The
three images below show the histological aspect of lesion J, which is characterized by
primary demyelination (LFB staining), sparing of axons (Bielschowsky silver impregnation)
and inflammation (MRP14). The macrophage staining shows the heterogeneity of this lesion,
which is suggestive of confluent lesions of different age.
Our first experiments addressed basic questions on the diagnosis of disease symptoms and
the visualization of CNS pathology in living animals. We designed a panel of MRI
sequences that could be used for visualization and quantification of brain pathology in
sedated EAE marmosets. A picture of the equipment can be found in the book The Laboratory Primate ('t Hart et al., 2005d). That work brought us a nice
multidisciplinary publication, in which MRI, neuropathology and immunology were
integrated ('t Hart et al., 1998). Figure 2 shows a compilation of figures from this
publication and illustrates the strategy that we followed for obtaining histological
information on MRI-detectable abnormalities in the EAE-affected marmoset brain.
Particularly stimulating was the support from MS neuropathologists, who appreciated the
remarkable neuropathological similarity of the model with MS. In retrospect, it is
regrettable that we restricted our neuropathology examination in the first paper to the
white matter. If we would have performed immunostaining for proteolipid protein (PLP), we might have been the first to report on the dramatic
demyelination of cortical grey matter, a now very important pathological feature of MS
that was then unknown (see Fig. 3).
Characteristic brain pathology of marmoset EAE induced with human MS myelin/CFA. One brain hemisphere was immunostained for PLP to visualize demyelination
and for MRP14 (macrophages/microglia) to visualize inflammation. For comparison, a brain
hemisphere of a healthy marmoset was stained for PLP. Clearly visible is the different
pathological aspect of white and cortical grey matter (cGM).
The questions for subsequent experiments unfolded more or less logically:
Which immunopathogenic mechanisms are operational in the periphery and
within the lesions?
What is the fine specificity of the T cells and antibodies that mediate the
autoimmune attack on the CNS?
Can the EAE induction protocol be refined by eliminating the role of
nonspecifically acting factors, such as components of the adjuvant?
Can methods be developed for longitudinal monitoring of the
(immuno)pathogenic process?
Can the MRI protocol be refined to get more information on lesion
development in the intact animal?
The only research groups performing immunological research on marmosets were Prof. Claude
Genain's group at UCSF (who stopped in 2007) and our group at the Biomedical Primate
Research Centre (BPRC). Hence, we needed to work hard on the filling of our research toolbox with
reagents (monoclonal antibodies, cytokine assays, PCR probes and primers) that could be
used for immune profiling of our marmoset EAE model. We also had to develop assays
suitable for the availability of only 3 mL venous blood per month. An overview of the
currently used tools has been published (Jagessar et al., 2013b). The stream of
publications in the peer-reviewed literature summarized in this monography shows that we
have achieved most of our goals.
The question of whether there is a future for the marmoset EAE model has to be asked in
these days, where biomedical research using live animals, primates in particular, is
heavily debated in Europe and beyond. Scientists using nonhuman primates for the study of
serious human diseases need to deal with increasing political and societal pressure as
well as shortage of funding, both creating huge challenges. Our only arguments in the
fight are the results of our research, which cannot be obtained in models in lower
species, nor in patients, nor in cell or organ cultures.
Our research in the marmoset EAE model has provided highly relevant information for
better understanding of MS as a basis for more effective therapies. In the past 5 years
we have even entered the terra incognita of progressive MS, for which there is no valid
alternative animal model and no effective treatment. I sincerely hope that this
monography will enhance the appreciation of the translational relevance of nonhuman
primate disease models among our diverse stakeholders. In addition, it might provide
useful information in the dialogue with sectors of the human population criticizing
nonhuman primate usage in biomedical research.
Preface
Due to their close phylogenetic relationship with humans, nonhuman primates (NHPs)
provide translationally relevant models of a variety of diseases that threaten the aging
human population (Mansfield, 2003; Tardif et al., 2011; 't Hart et al., 2012). The high
degree of biological similarity of NHPs and humans is reflected in the immune system,
which according to most experts is a central driver of the autoimmune neurological
disease multiple sclerosis (MS). The translational relevance of NHP disease models should
always be weighed against the high costs and justified ethical concerns, i.e., whether
the same information can be obtained in a lower-ranked model or without animals. On the
other side of the scale is the interest of the MS patients, who are desperately waiting
for an effective treatment for their disease. I posit here that MS models in NHPs are
indispensable in preclinical MS research as they can provide novel insights into
mechanisms operating in the initiation and especially the progression of the disease that
cannot be obtained in any other MS animal model, nor in the patient. Obviously, NHPs will
never replace corresponding experimental models in adolescent (8–12 weeks old)
inbred/SPF laboratory mice, but they definitely add complementary translational relevance
to these models.
This monography reviews insights that we have gained during 25 years
(1995–2020) of research in a model of experimental autoimmune
encephalomyelitis (EAE) in the common marmoset (Callithrix jacchus). In this publication only
the highlights can be discussed. For the underlying hard data, the reader is
referred to the original publications given in the reference list.
Introduction
Aging Western societies are facing a steadily increasing prevalence of
autoimmune-mediated inflammatory disorders (AIMIDs), for which no effective treatments
exist, such as MS and type I diabetes. Despite significantly increased investment in the
development of innovative therapies, the rate of drug candidates that reproduce promising
effects observed in animal models when they are tested in the clinic, remains
disappointingly low. The reasons for the low efficiency rate in therapy development for
AIMID are unforeseen toxicity and a lack of efficacy, indicating that the predictive
value of animal models for the clinical success of a candidate treatment is insufficient
(Kola and Landis, 2004). However, funding for research aiming at understanding the
reasons why treatments developed via forward translation (from the laboratory to the
clinic) fail is limited. This is an unfortunate situation, as much can be learned from
reverse translation research (from the clinic back to the laboratory) and the new
insights can be used to improve currently used animal models ('t Hart et al., 2014).
The subject of this monography is the EAE model, an experimentally induced autoimmune
disease in (genetically) susceptible laboratory animals that is projected on MS. EAE is
not only the most frequently used animal model in preclinical therapy research for MS but
has also often been used by immunologists as a generic model for testing basic concepts
of immune tolerance and autoimmunity (Hohlfeld and Wekerle, 2004; Gold et al., 2006).
However, mounting evidence indicates a wide immunological gap between frequently used EAE
models in adolescent (8–12 weeks of age) mice or rats, which are bred and housed under
very clean (SPF) conditions, and the MS patient. The important influence of the
environment is elegantly illustrated by cohousing of SPF-bred mice with dirty mice, e.g.,
those purchased from the pet shop, which gives the SPF mice a more human-like immune
system (Beura et al., 2016). The decisive influence of gut microbiota on EAE
susceptibility is also illustrated in studies by Berer et al., who showed that EAE-prone
transgenic mice, which do not develop evident disease under germ-free conditions, become
spontaneously sick after administration of normal commensal gut microbiota (Berer et al.,
2011). These and other studies underline the strong influence that gut microbiota can
have on the competence of the murine immune system.
The fact that nonhuman primates are bred and raised under conventional conditions implies
that, similar to the situation in humans, their immune system has been trained from early
life onwards by genetic diversity and the exposure to environmental pathogens, gut
microbiota as well as to chronic latent infection with herpes- and polyomavirus. Of note
is that the gut microbiota of marmosets is highly enriched with bifidobacteria, which
most likely is an adaptation to their specific dietary habits (e.g., gum) (Kap et al.,
2018b); it most closely resembles the microbiome of human neonates (own unpublished
observation). The fact that marmosets are naturally infected with β- and γ-herpesviruses is also particularly important; these viruses closely resemble those
infecting humans (Nigida et al., 1979; Rivailler et al., 2002). It has been well
established that endogenous and exogenous microbiota have a profound influence on the
human immune system, as can be seen in aged people for example (Vallejo et al., 2004; 't
Hart et al., 2013; Goronzy and Weyand, 2013; Vanheusden et al., 2015).
In summary, the fact that in nonhuman primates, autoimmune reactions develop within a
pathogen-trained immune context marks an important difference with the situation in
immunologically naïve inbred/SPF mice.
MS in a nutshell
MS is a chronic progressive neurological disease that specifically affects the central
nervous system (CNS), which comprises the brain and spinal cord. Clinically, MS is
characterized by increasing defects of sensory, motoric and/or cognitive functions. For
quantification of the disease severity, symptoms are ranked on an expanded disability
scoring scale (EDSS) (Fig. 4a). The EDSS is commonly used in clinical trials for
quantifying the effect of a new treatment on the disease (Hohlfeld and Wekerle, 2004;
Compston and Coles, 2008).
Clinical aspects of multiple sclerosis.(a) For quantification of the degree of disability, for example in the clinical
assessment of new treatments, the expanded disability status scale (EDSS) has been
developed (Kurtzke, 1983). (b) A graphical representation of the most common MS
phenotype. In the beginning MS is asymptomatic, but with advanced imaging techniques
(contrast-enhanced MRI for example) focal abnormalities due to inflammation can be
observed inside the brain white matter. This is followed by a period of variable length
between patients where the inflammation increases in severity causing discrete episodes
of disability (relapse) alternating with complete recovery (remission). In about 50 %
of the patients with relapsing–remitting MS the disease becomes progressive, where
remissions disappear and neurological functions decrease progressively. Based on data
from the marmoset EAE model, we posit that the degeneration of oligodendrocyte/myelin
complexes can be differentiated into three types: (1) normal age-appropriate progressive
degeneration; (2) progressive degeneration amplified by a newly discovered T cell attack
on oligodendrocytes; (3) reversible degeneration induced by a classical autoimmune attack
of pro-inflammatory T cells and autoantibodies on myelin sheaths.
Conceptually, the disease course, as seen in the majority of MS patients (±85 %), can be divided into three phases that vary in length between individual
patients (Fig. 4b). In the early, pre-symptomatic phase abnormalities can be detected in
the brain white matter with the help of sophisticated imaging techniques, such as
magnetic resonance imaging (MRI), but these pathological features are usually not
expressed clinically. The subsequent phase, which can last for 5 to 20 years, is
characterized by alternating episodes of neurological defects (relapse) and recovery
(remission). This relapsing–remitting (RR) phase is followed by a secondary progressive
(SP) phase, where symptoms worsen progressively and remissions disappear gradually. Note,
there is no sharp separation between the RR and SP phases, and transition from RRMS to
SPMS is usually assessed retrospectively (Lublin et al., 2014). In a minority of the
patients (±15 %) the disease is progressive from the onset; this disease type is
indicated as primary progressive MS (PPMS).
Based on evidence obtained in nonhuman primate EAE models, which will be discussed in
more detail later, we posit that full development of disease activity in MS is caused by
at least two distinct (and possibly sequential) pathological processes. We identified a
classical combination of humoral and cellular autoimmune reactions, which induce episodic
loss of neurological functions (relapses). In addition, we identified a novel cytopathic
mechanism that seems to amplify the normal age-associated degeneration of
myelin/oligodendrocyte complexes (Fig. 4b).
The exact nature of the events occurring at the transition from pre-symptomatic to
relapsing–remitting MS (transition 1) and from relapsing–remitting to secondary
progressive MS (transition 2) is debated. The simplest, albeit not necessarily correct,
explanation may be as proposed in Fig. 4b, namely that transition 1 occurs when the
mounting inflammatory pathology caused by the autoimmune attack exceeds a clinical
threshold, whereas transition 2 occurs when the pathogenic influence of the immune system
declines and progressive loss of function by irreversible degeneration of neuro-axonal
and oligodendrocyte–myelin complexes becomes dominant. It is also proposed that in MS
the age-related degeneration of oligodendrocyte–myelin complexes is enhanced by the pathological process. As was already mentioned and will be
further discussed later, data from the marmoset EAE model revealed that this amplified
degeneration may be mediated by a newly discovered pathogenic mechanism resembling
neurodegenerative diseases (Alzheimer, Parkinson) (Bartzokis, 2004).
The pathological hallmark of MS and the most likely cause of the accumulating
neurological deficits is the lesion, a usually well-defined area in the CNS white matter
(WM), where the insulating myelin layers around axons are destroyed, but myelin-forming
oligodendrocytes are spared. Depending on their age, activity and localization, lesions
contain variable degrees of inflammation, repair (remyelination), astrogliosis (scar
formation) and neuro-axonal pathology (Frohman et al., 2006). The presence of immune
cells and molecules (antibodies, complement) in inflammatory active lesions, which are
usually characterized by enhanced permeability of the blood–brain barrier (BBB),
indicates involvement of the immune system in lesion formation. While MS was initially
considered to be a typical autoimmune disease of the WM, it is now clear that the grey
matter (GM) is affected as well, maybe even to a greater extent than WM (Geurts and
Barkhof, 2008). In progressive MS the extent of cortical GM (cGM) demyelination, which
can be focal in leukocortical and intracortical lesions or widespread in subpial lesions,
often exceeds the load of WM lesions. Lesions in the cGM are often paucicellular, except
in the very beginning of the disease (Lucchinetti et al., 2011), with only a rim of
activated microglia cells. It is therefore suspected that demyelination of white and grey
matter is caused by different pathogenic mechanisms. This view is supported by data from
the marmoset EAE model, as will be discussed later in this monography.
In debates on the start of MS the terms cause and trigger are often used as synonyms, but
I believe that these terms indicate clearly different entities. I like to use the First
World War (WW1) as a metaphor to explain the difference. The cause of WW1 was a complex
set of factors, including mounting economic problems in European countries and the
increasing militarization of Imperial Germany. The trigger of WW1, however, was a single
factor, namely the assassination of Archduke Franz Ferdinand of Austria and his wife
Sophie by the Bosnian-Serb Gavrilo Princip. Whether the assassination would have sparked
such a tragedy if the economic and social situation in Europe were more stable remains an
open question.
Mutatis mutandis, the same may hold true for MS. While an undisputed trigger of MS has
not been identified thus far, the disease risk profile indicates that there is a variety
of causal factors that influence MS susceptibility; these can be genetic or environmental
(Ascherio et al., 2012). Genomewide association studies (GWASs) reveal that the vast
majority of genetic risk factors have a function in the immune system, although the
individual contribution of each gene to the pathogenic process is often not clear.
Environmental risk factors are diverse and include exposure to infectious (with
Epstein–Barr virus) as well as noninfectious factors (smoking, sunshine exposure)
(Ascherio and Munger, 2007; Ascherio et al., 2012). In the course of our research the
question of whether MS might be triggered by an endogenous factor or process emerged.
Our work in the marmoset EAE model has been mainly focused on investigating
the immunobiology of these MS risk factors, as potential causes of MS. The
underlying thought was that via the analysis of MS risk factors insight
could be gained into critical steps in the disease process.
MS and EAE: different or overlapping pathologies?
In the literature, two essentially complementary etiological concepts for MS are
presented (Fig. 5) (Stys et al., 2012). According to an outside-in paradigm, autoimmunity
in MS is triggered in genetically predisposed individuals by infection with an exogenous
pathogen; various mechanisms have been proposed, such as molecular mimicry and bystander
activation (Fujinami et al., 2006). An inside-out paradigm proposes that MS starts as a
degenerative process inside CNS myelin, indicated here as a primary lesion. Conceptually,
the immune system reacts against antigens released from this primary lesion. It must be
emphasized here that both concepts are based on dynamic interactions of genetic and
environmental factors. However, while in the outside-in paradigm pathogens are the direct
trigger of the autoimmune process, in the inside-out concept gene–pathogen interactions
are a cause of MS, but not a trigger. Instead, they set the immune system in a
hyper-responsive state to injury, which is triggered by another event ('t Hart et al.,
2009). We will discuss later that data obtained in the marmoset EAE model are strongly
supportive for the inside-out paradigm.
Two opposing paradigms explaining the cause of autoimmunity in MS. The
prevailing concept is the outside-in paradigm, namely that infection of individuals who
are genetically predisposed to MS with an as yet unidentified microorganism activates
autoreactive T and B cells present in the normal immune repertoire. The autoimmune attack
on the CNS induces cytodegeneration. Less commonly accepted is the inside-out paradigm,
which states that a pathogenic event inside the CNS elicits the release of myelin
antigens that activate autoreactive T and B cells present in the normal immune
repertoire. The principle difference between both paradigms is that in the outside-in
paradigm infection is the direct trigger of autoimmunity, whereas in the inside-out
paradigm infections create a higher responsive state of the immune system.
The inside-out paradigm harmonizes nicely with a much older “primary lesion” theory of
Terence Wilkin (Wilkin, 1990). This theory essentially implies that
autoimmunity is not itself an entity, but a physiological response to
sustained excess antigen turnover in diseased tissues (the primary lesion).
those who develop clinical disease are viewed as high responders to
critical antigens.
The first proposition implicates a mechanism that amplifies the normal age-associated
degeneration of myelin-oligodendrocyte and neuro-axonal complexes (as postulated in
Fig. 4). As will be discussed later, such a mechanism has been found in the marmoset EAE
model. The second proposition implies a hyper-immune response of MS patients against
myelin components compared to healthy individuals. This has been shown by several authors
(Kerlero de Rosbo et al., 1993; Bielekova et al., 2004). As will be discussed later, we
found a dominant antigenic role of the quantitatively minor constituent myelin
oligodendrocyte glycoprotein (MOG) in the marmoset EAE model.
Epstein–Barr virus
(EBV).
It is heavily debated whether studies in an EAE model can provide relevant insights into
the mechanisms that trigger and/or perpetuate MS (Sriram and Steiner, 2005; Steinman and
Zamvil, 2005; 't Hart et al., 2011; Ransohoff, 2006). The essential difference between MS
and EAE is that MS develops spontaneously in (genetically) predisposed individuals, while
EAE is experimentally induced in otherwise healthy animals by means of rather artificial
procedures (Laman et al., 2017). Historical data indicate, however, that there may be no
sharp separation between the two diseases. Humans were found to be susceptible to EAE. In
fact, the EAE model stems from the observation by Pasteur that humans vaccinated with
inactivated rabies virus grown on rabbit brains, developed brain pathology reminiscent of
an acute demyelinating disease in humans, called acute disseminated encephalomyelitis
(ADEM) (Pasteur, 1885). Subsequent work by Rivers et al. (1933), Rivers and
Schwenkter (1935) and Kabat et al. (1947) in rhesus macaques clarified that this
neurological disease was not caused by the virus, but induced by components from the
rabbit brain that contaminated the vaccine. Collectively, the published data imply that
sensitization of humans and nonhuman primates (i.c., rhesus monkeys) against antigens
present in the rabbit brain elicits similar ADEM-like pathology and disease. On the other
hand, there is a report from the Oregon National Primate Research Center describing cases of spontaneous MS-like disease
in a colony of Japanese snow monkeys (Macaca fuscata). The disease could be
attributed to infection with a new γ2-herpesvirus that is most closely related to
a rhesus macaque rhadinovirus (Axthelm et al., 2011). Interestingly, also in the mouse a
rhadinovirus, murine gammaherpesvirus-86, has been implicated as a trigger of MS-like
disease (Marquez and Horwitz, 2015). It remains an intriguing question why MS-like
disease in animals is associated with a γ2-herpesvirus, while the human disease
is associated with a γ1-herpesvirus, the lymphocryptovirus (LCV) EBV (Box 1).
To our knowledge there is no clear evidence that γ2-herpesviruses,
Kaposi sarcoma herpesvirus (KSHV) for example, have a pathogenic role in
MS. Although both γ1- and γ2-herpesviruses infect neuronal cells as well
as B cells in vitro, such as in Hodgkin's and primary effusion lymphoma, respectively
(Jha et al., 2015), it is unclear at this stage whether γ2-herpesviruses activate
cellular pathways relevant to T-cell-mediated autoimmune disease, such as those described
recently for EBV-infected B cells (peptide citrullination and activation of the autophagy
pathway) ('t Hart et al., 2016). The infected snow monkeys display MS-like pathology in
the white matter of the cerebellum as well as oligoclonal bands in their cerebrospinal
fluid (CSF) (Blair et al., 2016). However, the paper does not explicitly mention that
demyelination was detected in cortical grey matter (cGM), which might mean that this
pathological hallmark of MS is absent. Our own data in the marmoset EAE model (reviewed
in the following paragraphs) indicate that cGM and WM pathology may be distinct entities
induced via different pathogenic mechanisms (see below) and that the γ1-herpesvirus is implicated in the pathway leading to GM pathology ('t Hart et al.,
2017a).
Nonhuman primate models of EAE
Rivers and Schwenkter (1935) reported that macaques
given repeated (60 or more) intramuscular injections of alcohol-ether extracts from
normal rabbit brain developed similar ADEM-like pathology as observed in earlier studies
(Rivers and Schwenkter, 1935). Subsequently, Kabat et al. (1947) observed that ADEM-like disease could also be induced with only a few
injections (∼3) of heterologous (rabbit)
or even autologous (rhesus monkey) brain extracts, provided that the extracts were mixed
with paraffin oil in which heat-killed dried tubercle bacteria were suspended (Kabat et
al., 1947). This powerful immune-potentiating paraffin oil/mycobacteria mixture is
nowadays commercially available as complete Freund's adjuvant (CFA) and is the most
frequently used, albeit disputed, adjuvant in EAE studies (Laman et al., 2017).
The good thing of CFA is that it elicits both cellular and humoral autoimmune reactions
against coformulated antigens. The bad thing is that the adjuvant skews the
differentiation of antigen-activated CD4+ T cells towards a pro-inflammatory profile
(Billiau and Matthys, 2001), which often does not necessarily reflect the unbiased
adaptive immune response of the inoculated animal. Moreover, the injection of CFA into
the skin provokes ulcerative lesions at the injection sites, which causes serious
discomfort to the animals. Recently we reported that robust clinical EAE could be induced
in three nonhuman primate species – rhesus monkey, cynomolgus monkey and common marmoset
– with the myelin antigen myelin oligodendrocyte glycoprotein (MOG) formulated with only
the oil without mycobacteria (incomplete Freund's adjuvant; IFA) (Haanstra et al.,
2013b). This discovery not only implies a major reduction of discomfort for the animals
(refinement), but also enables interrogation of the animal's immune repertoire for the
presence of pathogenic cells without the need of aspecific co-stimulation.
The EAE research at the Netherland's primate center (in 1994 renamed to BPRC) was
initiated by Margreet Jonker, who set up the model in rhesus monkeys (van Lambalgen and
Jonker, 1987b, a; Jonker et al., 1991). Observations by Rose et al. (1994) could be replicated, namely that
intracutaneous injection with myelin or with the quantitatively major
myelin basic protein (MBP) formulated with CFA elicited an acute
neurological disease with serious inflammatory/necrotic lesions. Disease development in
this model was associated with increased numbers of neutrophilic granulocytes in the
blood as well as in the lesions. Interestingly, the same formulations elicited EAE with
only mild inflammatory lesions in marmosets (Brok et al., 2000). The beneficial effect of
anti-CD4 monoclonal antibody in the rhesus monkey EAE model indicates that there must be
an underlying adaptive autoimmune process (van Lambalgen and Jonker, 1987b, a), which may
be aggravated by innate immune reactions against components of the adjuvant that cause
the serious damage. Recent work by Dunham et al. (2017b) shows that the combination of intra-CNS oxyradical production by infiltrated
neutrophils and redistribution of iron generates toxic oxygen (O2-,
H2O2) and nitrogen (NO) species that can seriously aggravate tissue
destruction in the lesions (Dunham et al., 2017b). Note, the different severity of tissue
destruction is reflected at the antigen/CFA inoculation sites in the skin; in rhesus
monkeys serious granulomas are formed, while these are much less severe in marmosets. The
beneficial effect of the neutrophil oxidative burst antagonist apocynin on the skin
granuloma formation in rats demonstrates that neutrophils have a prominent role in tissue
destruction ('t Hart et al., 1992).
Although the rhesus monkey EAE model has been useful for the efficacy
testing of therapeutic agents, which due to insufficient cross-reactivity
could not be tested in marmosets (Haanstra et al., 2013a,
2015), we believe that the EAE model in marmosets is more relevant for MS.
EAE in marmosets: a superior primate MS modelWhat is a marmoset?
Marmosets are small-bodied nonprotected primates, weighing 350–400 g at adult age,
which have their natural habitat in the Amazon forest. Figure 6a shows the features and
size of these animals. As a detailed description of the biology of marmosets and their
usage in preclinical research is beyond the scope of this monography, we refer the reader
to reviews published elsewhere (Haig, 1999; Mansfield, 2003; Ludlage and Mansfield, 2003;
Tardif et al., 2003, 2011; 't Hart et al., 2012). To name a few important aspects,
marmosets breed well in captivity, where they can give birth to two nonidentical twins
per year. Due to the sharing of the placental bloodstream, the immune systems of
fraternal siblings are educated in the same thymic compartment and are therefore very
comparable. This creates an attractive experimental setting for two-leg studies comparing
the effect of an experimental variable, a new therapy for example, with a control
preparation.
What is a marmoset? Depicted is an artist's impression of two
marmosets (Callithrix jacchus) showing their small body size (oil on canvas
painting by Sir Edwin Landseer, 1803–1873, the Royal Collection, United Kingdom). The
lower part of the figure depicts the phylogenetic tree of primates with the evolutionary
distance to humans.
Dual role of MOG, as a tolerogen or immunogen.
The BPRC houses a large self-sustaining colony of marmosets, comprising between 100 and
150 animals. The monkeys are kept in family groups that are housed in spacious cages with
indoor and outdoor enclosures. This means that our marmosets are exposed from birth to
environmental microbes as well as to intestinal microbiota and chronic latent infections
(EBV; cytomegalovirus, CMV) carried by family members. Evidence shows that these
microorganisms have an important effect on the immune status; in other words marmosets
have a pathogen-educated immune system. Mounting evidence indicates that this daily
contact with bacteria and viruses from in- and outside the body generates immune
functions that are complementary to the classical ones found in the inbred/SPF laboratory
mice used in most immunological research.
Some immunological characteristics of the marmoset relevant to preclinical studies in the EAE model
MHC. Genomewide association studies in MS and results obtained in rodent
EAE models show that the highly polymorphic major histocompatibility complex (MHC) class
II region exerts the strongest genetic influence on disease susceptibility (Sawcer et
al., 2011, 2014). The main biological function of MHC class II molecules is to present
antigens to CD4+ T cells,
which dominate the immunopathology in mouse EAE models, but seem to have a less prominent
role in MS (Lassmann and Ransohoff, 2004). Alleles constituting the HLA-DR2 haplotype,
HLA-DRB1*1501/-DRB5*0101/-DQB1*0602, have the strongest risk
association with MS in the Caucasian populations of northern Europe. Disease associations
with the MHC class I region have been much less intensively investigated, which is
remarkable as CD8+ T cells, which receive their antigens presented by MHC class I, are
the dominant T-cell subtype in MS lesions.
The MHC class II region of marmosets comprises the equivalents of HLA-DR and -DQ, which
are respectively indicated with the acronyms Caja-DR and -DQ (from Callithrix jacchus); Caja-DP genes are absent (Antunes et al., 1998; Doxiadis et al., 2006). Three
Caja-DR loci have been identified: two produce functional transcripts, namely the
monomorphic Caja-DRB*W1201 and the polymorphic Caja-DRB*W16 loci, while the Caja-DRB1*03
locus contains only pseudogenes. Two hybrid Caja-DRB transcripts were found, which are
composed of exon 1 and 3 of Caja-DRB*W16 alleles with exon 2 of Caja-DRB1*03 alleles.
Furthermore, oligomorphic Caja-DQA1 (2 alleles), -DQB1 (3 alleles) and -DQB2 (2 alleles)
genes were found. The invariant Caja-DRB*W1201 product is particularly relevant for the
EAE model as it restricts the activation of CD4+ T cells specific for rhMOG epitope
24–36, which have a key function in the initiation of the disease (Brok et al., 2000).
The MHC class I region of marmosets has been less well characterized (van der Wiel et
al., 2013). Sequences comparable to the classical HLA-A, -B and -C genes have not been
found. Evidence was found for functional transcripts of the oligomorphic Caja-E locus (2
alleles). Caja-E has an important role in the marmoset EAE model as it restricts the
activation of autoaggressive cytotoxic T cells (CTL) specific for rhMOG epitope 40–48 (Jagessar et al., 2012d). The classical
antigen presentation function to CD8+ T cells seems to be executed by products from the
polymorphic Caja-G locus (van der Wiel et al., 2013).
TCR. The repertoire of T cell antigen receptors (TCRs) has been unraveled (in
part) (Uccelli et al., 1997). Overall a high similarity of marmoset and
human TCRBV-D-J-C sequences was observed, illustrating the close
phylogenetic relationship between the species.
BCR. The repertoire of B cell antigen receptors (BCR) has been partly
unraveled as well (von Budingen et al., 2001). Also at the level of the immunoglobulin
heavy variable (IGHV) gene repertoire a high similarity was found between marmosets and
humans.
Other relevant immune molecules. The initiation and perpetuation of
(auto)immune reactions involves interaction of various cell types via multiple cell-bound
or secreted molecules. Some insight into the degree of relatedness between those
molecules from humans and marmosets can be gained by testing whether monoclonal
antibodies (mAbs) raised against human surface-expressed CD molecules cross-react with
marmoset cells. It is obviously a major effort to do this for all immune molecules, but
for several the data have been published and the list is periodically updated (Jagessar
et al., 2013b).
In conclusion, the relatively close evolutionary distance of marmosets and humans, which
has been estimated at about 30 million years (Fig. 6b), is reflected by a high degree of
immunological similarity. This makes the marmoset an exquisitely attractive animal model
of human AIMIDs.
EAE induction in marmosets
The first report on successful EAE induction in marmosets was published in 1995
(Massacesi et al., 1995). The authors used a rather harsh immunization protocol,
involving human brain homogenate formulated with CFA containing
3 mg mL-1Mycobacterium tuberculosis. In addition, they gave
intravenous injections of 1010 inactivated Bordetella pertussis particles
at the day of immunization and 2 d later. This resulted in a relapsing–remitting (RR)
disease course with inflammatory–demyelinating pathology. The authors also showed that
EAE could be transferred within (bone marrow chimeric) twins from an EAE-affected monkey
to its fraternal sibling using MBP-specific T cell lines. In later studies by this group,
which were led by the neurologist Dr. Claude Genain, several important findings were
made, such as the detailed unraveling of the repertoire of immune reactions against MOG,
the crucial role of anti-MOG antibodies for demyelination, the presence of anti-MOG
antibodies inside lesions and the unexpected late exacerbation of neurological disease
occurring after tolerization against MOG (reviewed in Genain and Hauser, 2001).
The harsh experimental procedures used by Genain and Hauser (2001) are a clear reflection of the prevailing SPF rodent-based concepts
on EAE and MS in the 1990s, without taking into account that the immune systems from SPF
rodents and non-SPF primates might differ fundamentally. Our group at the BPRC chose
another approach, namely a stepwise refinement of the marmoset EAE model to the minimal
components needed for the induction of MS-like pathology and disease. These efforts are
summarized in the following.
Inspired by the seminal publication of Massacesi et al. (1995), we used the same EAE
induction protocol in marmosets from our own purpose-bred colony ('t Hart et al., 1998).
The only modification was that we used myelin isolated from an MS brain, which we
purchased from the Netherlands Brain Bank (Amsterdam, the Netherlands). Similar to the original study, we
obtained relapsing–remitting (RR) EAE with severe inflammatory pathology. However, we
noticed that clinically evident EAE, albeit with a more protracted course and less
destructive pathology, could be obtained when we used a commercially available CFA
preparation (Difco) containing only 1 mg mL-1 mycobacteria and left out the
Bordetella particle injections. The huge advantage of this modified protocol for the
animals was that the inoculum provoked considerably less skin ulceration.
The observation by McFarland et al. (1999), that demyelination in marmosets immunized with a chimeric
MBP/PLP protein in the adjuvant Titermax was always associated with spreading of the
immune response to MOG, underscores the pathogenic relevance of this quantitatively minor
CNS myelin component. Data summarized in Box 2 indicate that MOG is a remarkable molecule
with a Dr. Jekyll/Mr. Hyde-type of role in the brain, which depends on its glycosylation
state. We tested whether immunity to MOG is indeed essential for EAE development. Our
approach was to immunize fraternal twin siblings with myelin from B6 mice, which were
either wild-type or MOG-deficient mice. MOG-deficient mice had been developed by
Delarasse et al. (2003). We observed that in one twin with acute onset EAE the absence or presence of MOG
in the myelin inoculum made no difference for the EAE presentation, while in the other
twin, development of chronic EAE was impaired
when MOG was absent. At the pathological level, demyelination was reduced albeit not
absent (surprisingly) in the sibling immunized with MOG-deficient myelin (Jagessar et
al., 2008)!
Clinical and pathological aspects of the marmoset EAE model induced with rhMOG/CFA. (a) Depicted is the EAE course in a representative selection of
30 unrelated marmosets receiving a single immunization with
rhMOG/CFA on day 0. All monkeys developed clinically evident EAE, but the time of onset
varied from 2 to 16 weeks. (b) Serial imaging of a case with late EAE onset,
showing early onset of brain lesion formation. The white arrow points to the first
detectable lesion. Clearly visible is that formation of new lesions in the depicted brain
slice (0.5 mm) is disseminated in time and space. Around the time that clinical signs
were diagnosed, lesion colonization of cortical grey matter is detectable (inserted
magnifications). (c) PLP staining of a brain hemisphere shows the dramatic
demyelination in white and grey matter (a). Different grey matter lesion types
identified in the MS brain can be distinguished (b). Lesions are paucicellular
with regard to T (CD3) and B (CD20) cells, but contain abundant MRP14+ myeloid cells,
representing microglia and macrophages.
The previous findings show that autoimmunity against MOG is indispensable for the
development of chronic EAE in marmosets. To further investigate the anticipated complex
patterns of cellular and humoral autoimmunity against MOG in the genetically outbred
marmoset EAE model, we immunized marmosets with a nonglycosylated recombinant protein
expressed in Escherichia coli bacteria
that represents the extracellular domain (residues 1–125). Possible epitopes located in
the transmembrane and intracellular parts of the molecule, which were identified for mice
and rats, were excluded from our studies ('t Hart et al., 2011). The protein was
formulated with CFA and the emulsion was injected once at day 0. The clinical course
pattern in a representative cohort of 30 monkeys immunized with
rhMOG/CFA is depicted in Fig. 7a, showing that the disease incidence is 100 %; while
in this collection of EAE cases the time of EAE onset varied from 2 to 16 weeks. Our
original idea that this variation reflects the genetic heterogeneity of the monkeys
proved to be incorrect as was shown by serial magnetic resonance brain imaging (MRI). In
the depicted example (Fig. 7b), T2 lesion development was detectable already within a few
weeks after immunization, while clinical signs were diagnosed many weeks later. It was
noticed in this example that very late in the disease and around the time that clinical
signs could be diagnosed, lesions started to “colonize” the grey matter of the cerebral
cortex. We drew the tentative conclusion from these observations that the formation of
CNS lesions and the induction of neurological deficits might be based on distinct
immunopathogenic mechanisms (for details see also Box 3). Immunostaining of a cerebral
hemisphere from an rhMOG/CFA-immunized monkey for PLP confirmed the presence of
demyelination in WM and cGM (Fig. 7c).
Why are the MOG24-36 and MOG40-48 immunodominant?
The immunological analysis of the rhMOG/CFA model revealed two distinct pathogenic
mechanisms (Fig. 8). We found that the 100 % EAE incidence maps to a common
autoimmune mechanism, namely the Caja-DRB*W1201-restricted activation of Th1 cells
specific for epitope MOG24-36 (Brok et al., 2000). By contrast, we found that the
variable EAE course was related to the degree of diversity of the T-cell response against
MOG peptides (Kap et al., 2008). Counterintuitively, monkeys that progressed relatively
fast to clinically definitive EAE were characterized by a broader T-cell reactivity with
rhMOG peptides than slow progressor cases. This is opposite to mouse models, where EAE
progression is associated with progressive diversification of autoreactive T-cell
reactivity, a phenomenon known as “epitope spreading” (Vanderlugt et al., 1998). Our
interpretation were that while fishing in the pool of MOG breakdown products, certain MHC
alleles present in the repertoire of fast progressor monkey bind peptides that can
activate T cells with the capacity to accelerate EAE development. Later studies showed a
role of IL-7 in the pathogenic function of these accelerator T cells (Dunham et al.,
2016). Candidate epitopes emerging from T-cell reactivity scans were MOG4-26, MOG34-56
and MOG64-86/74-96 (Brok et al., 2000; Kap et al., 2008). The encephalitogenic activity
of MOG4-26 was not (yet) tested. For the other two, we observed potent encephalitogenic
activity for MOG34-56, while MOG74-86/74-96 was inactive (Kap et al., 2008). Subsequent
studies revealed that the core pathogenic epitope is MOG40-48 and that this epitope can
be presented to (antigen experienced) cytotoxic T cells (CTLs) via Caja-E molecules
(Jagessar et al., 2012d). Besides the MHC-E restriction of epitope recognition, these
CTLs express the natural killer (NK) cell marker CD56 and the
MOG40-48 epitope shares almost sequence similarity with a peptide derived from the major
capsid protein of cytomegalovirus (Brok et al., 2007) we tentatively identified these
CTLs as being potentially related to or similar to CMV-induced effector memory T cells
(Pietra et al., 2003).
Two autoimmune pathways. EAE development in the rhMOG/CFA model
involves two distinct pathogenic mechanisms. The 100 % EAE incidence (see Fig. 7a)
maps to the MHC class II/Caja-DRB*W1201-restricted activation of Th1 cells specific for
MOG epitope 24–36. The signature cytokine of this pathway, which essentially replicates
mouse EAE models, is IFNγ. Early blockade of IL-12/IL-23 with a mAb against
IL-12p40 abrogates the activation of this pathway. The observation that the EAE
initiation pathway is activated in rhMOG/IFA model indicates that the responsive Th1
cells may be antigen-experienced cells. The variable EAE onset maps to another pathogenic
mechanism, namely the MHC class I/Caja-E-restricted activation of CTL specific for MOG
epitope 40–48, which shares almost complete sequence similarity with an epitope from the
major capsid protein of CMV. The signature cytokine of this pathway is IL-17A. The
characteristics and specificity of the CTLs suggest that they originate from the effector
memory T cells that keep CMV under control. We hypothesize that the variable activation
of the latter pathway is induced by antigens released from white matter lesions induced
by the former pathway.
In summary, we showed two independent pathogenic mechanisms in the rhMOG/CFA marmoset EAE
model: a mouse EAE-like pathway operating early in the disease and a novel pathway
starting late in the disease and mediating EAE progression.
Based on findings discussed above we immunized marmosets with MOG34-56 or MOG74-96, each
formulated with CFA. Of these two, MOG34-56
emerged as the superior encephalitogenic peptide (Kap et al., 2008). Intriguingly, we
observed 100 % EAE incidence in marmosets sensitized against MOG34-56 together with
widespread demyelination in the white and grey matter of brain and spinal cord (Fig. 9),
despite the absence of detectable levels of myelin-binding antibodies in the serum. In
addition, we observed that CFA could even be replaced by IFA, which is only the mineral
oil without mycobacteria (Jagessar et al., 2010). A comparable mechanism has not been
found in any other EAE model, indicating that this might represent a novel pathogenic
mechanism that may be confined to the pathogen-educated primate immune system. Indeed,
characterization of T-cell responses in this model revealed that they phenotypically
resemble IL-17A+ve natural killer cytotoxic T lymphocyte (CD8+CD16-CD56+;
NK-CTL) and that the core epitope (residues
40–48; henceforth indicated as MOG40-48) is presented via nonclassical Caja-E molecules.
Furthermore, we observed cytotoxic activity of the NK-CTL towards B cells infected with
EBV (strain 95-8) (Jagessar
et al., 2012d). Intriguingly, NK-CTLs have been found in MS lesions in the vicinity of
HLA-E expressing oligodendrocytes, suggesting cytotoxic interaction (Zaguia et al.,
2013). The observation that the MOG40-48 sequence shares sequence homology with a peptide
from the major capsid protein of cytomegalovirus (CMV) sheds light on the possible origin
of the encephalitogenic NK-CTL, namely the repertoire of T cells that suppress
exacerbation of CMV (Pietra et al., 2003; Mazzarino et al., 2005).
Clinical and pathological aspects of an atypical EAE model induced with MOG34-56/IFA. Immunization with MOG34-56/IFA at a 28 d interval (arrows) induces
100 % clinical EAE with a variable time of onset. Notice that the only information
relayed to the marmoset's immune system is the sequence of 23 letters (CTL core epitope
in red). Immunostaining for PLP shows lesions in the white and grey
matter (A). Rectangle 1 indicates a demyelinated region in the cingulate cortex, which
is characterized by complete demyelination (B), presence at the lesion edge of
macrophages containing a PLP+ inclusion particle (C), activated MRP14+
microglia (E) and depletion of oligodendrocytes (F). Immune cells (CD3)
were only detected in meninges. Rectangle 2 (A) indicates a leukocortical and an
intracortical lesion (G), which are also depleted from
oligodendrocytes (H) and contain abundant MRP14+
microglia. Mf is macrophage, TPPP is tubulin polymerization-promoting protein, ODC is oligodendrocyte.
A serendipitous finding revealed which of the two pathways described in Box 3 can be
associated with GM pathology. We observed in an atypical marmoset EAE model induced with
recombinant human (rh) MOG formulated with incomplete Freund's adjuvant (IFA) the
activation of T cells against MOG24-36 together with antimyelin antibodies. However, in
these animals T- and B-cell reactivity with the MOG34-56 peptide was lacking (Jagessar et
al., 2015). Histological analysis of the brain showed profound demyelination of white
matter (Jagessar et al., 2015), while cortical grey matter demyelination was minimal
(own unpublished results). This suggests a
causal relation between the absence of autoimmunity against MOG34-56 and the absence of
grey matter pathology. Indeed, sensitization of marmosets against the MOG34-56 peptide in
IFA induced fulminant cortical grey matter demyelination, with a crucial role of
EBV-infected B cells ('t Hart et al., 2017a, see below for further discussion).
The consideration that MS is obviously not elicited by injection of an
antigen/adjuvant formulation but develops spontaneously raises at least two
important questions:
What is the nature of the primary lesion that starts the release of myelin
antigens?
What is the nature of the antigen-presenting cells (APCs) that capture and
process these antigens, with a special focus on MOG, and present them to encephalitogenic
T cell (response-to-injury paradigm)?
Considerations on the primary lesion
The review of immune responses against CNS myelin injury will be briefly interrupted here
for a brief summary of our current insights into the primary lesion. The
response-to-damage/inside-out paradigm is based on the assumption that some pathological
event inside the CNS precedes the activation of autoreactive T and B cells that may be
more responsive to the damage due to prior antigenic experience. In the EAE model the
antigenic experience comes from active or passive immunization; in MS this might rather
be infection with a pathogen sharing molecular mimicry with a CNS antigen.
Already in 1990, Terence Wilkin postulated in his primary lesion theory that
“autoimmunity is a genetically predisposed immune hyper-reaction to the excess of
autoantigen released from a primary lesion” (Wilkin, 1990). Shedding of fragments from
aging myelin sheaths seems to be a normal physiological process (Safaiyan et al., 2016).
Our own data show that this may not inevitably lead to autoimmunity, as binding to
myeloid APCs is mediated by normally glycosylated MOG. It was observed that MOG isolated
from a nonpathological brain binds to the C-type lectin receptor DC-SIGN (Garcia-Vallejo
et al., 2014), which is expressed within the human CNS on microglia and within primate
cervical lymph nodes (CLNs) on the phagocytes that have captured the myelin
(Garcia-Vallejo et al., 2014). DC-SIGN is best known for its role in antigen capture and
presentation by myeloid dendritic cells (mDCs). Ligand activation of DC-SIGN relays
inhibitory signals to the dendritic cells (DCs), which keeps them
in an immature, tolerogenic state (Geijtenbeek et al., 2004) and prevents the assembly of
inflammasomes, which are needed for the production of bioactive IL-1β
(Garcia-Vallejo et al., 2014). Note, studies in Alzheimer mice identified inflammasome
activation in microglia as a crucial step towards neurodegeneration (Heneka et al.,
2013).
The inflammasome response of cells is regulated by autophagy; more specifically,
inflammasomes can be removed from cells via autophagy (Takahama et al., 2018). Mice
defective in autophagy genes display intracellular accumulation of protein aggregates in
the cytosol of neurons and hepatocytes. This raises the question of whether MS patients
may be defective in autophagy. Although this subject has not been intensively studied, a
publication by Igci et al. (2016) reported altered expression levels for several genes
encoding autophagy-related proteins in MS versus healthy controls.
These (and other) findings led to the concept that myelin binding to phagocytes in
draining lymph nodes is mediated via MOG, which via its N-linked glycan attached to Asn31
bind the C-type lectin receptor DC-SIGN. The response of the APC to myelin debris
therefore depends on whether MOG exposed on the myelin particles is normally or
abnormally glycosylated (see also 't Hart and van Kooyk, 2004). This concept may narrow
the problem of autoimmunity initiation in MS to the question of what causes the
disturbance of normal MOG glycosylation. Intriguingly, we observed that myelin produced
under inflammatory conditions (exposure of oligodendrocytes to TNF-α) has a
different glycan make-up and fails to counteract DC activation via
Toll-like receptors (TLRs) (see Box 2).
Why do humans develop MS and chimpanzees not? A hypothesis.
The first histological evidence of neuro-inflammation in MS is the presence of microglia
aggregates in normal-appearing white matter in the absence of other inflammatory cells or
demyelination. These aggregates are indicated as preactive lesions (van der Valk and
Amor, 2009) a.k.a. microglia nodules (Singh et al., 2013) a.k.a. newly forming lesions
(Barnett and Prineas, 2004), but likely represent the same pathological entity (Sato et
al., 2015). Interestingly, similar aggregates of microglia were found in the EAE models
in marmosets (Maggi et al., 2014) and in rhesus monkeys (Burm et al., 2016). In the
marmoset EAE model the non-demyelinating inflammatory microglia nodules correlated with
small-sized prelesional MRI-detectable blood–brain-barrier leakage and were
“contaminated” with some immune cells (T cells, macrophages) (Maggi et al., 2014). It
is currently unclear whether they are completely comparable to the pure microglia nodules
in MS. The observation by Singh et al. (2013), that microglia nodules are formed around a
degenerating axon, draws the interest towards the axo-myelinic synapse, a new functional
concept for the axon–myelin unit (Micu et al., 2017). A graphic representation of the unit is
depicted in Box 4. As discussed elsewhere, an intact unit is essential for adequate
trophic support to high nutrient demanding axons ('t Hart, 2016b). We posit that these
findings underlie the inside-out concept that the primary cause of MS may be instability
of the axon–myelin unit (Stys et al., 2012). One can only speculate why this happens. It
could have an internal cause, such as a genetic or metabolic defect and subtle molecular
changes (Caprariello et al., 2018), or an external cause, which could be associated with
a Western lifestyle ('t Hart, 2016b) (see Box 4), an infection or a combination of all
features. It is pertinent to emphasize, however, that our choice for the inside-out
paradigm as a working concept does not negate the possibility that MS is triggered by an
external factor.
Antigen-presenting cells mediating the response-to-injury paradigmAntigen capture by phagocytes in CNS draining cervical and lumbar lymph nodes
Conceptually, the cells that capture, process and present antigens to the immune system
have a decisive influence on the way the immune system responds to injury. The original
thought that the CNS lacks lymphatic vessels via which CSF and interstitial fluids can be
drained to the lymphoid system needed to be adjusted after the discovery of such
structures in the meninges that surround the brain and spinal cord (Louveau et al., 2015;
Aspelund et al., 2015). Note, similar structures were found in the marmoset brain
(Absinta et al., 2017). Via these lymph vessels, fluids draining from the brain and
spinal cord respectively pass through the cervical and lumbar lymph nodes (CLNs and
LLNs), where antigens might be captured by local APCs. Via in situ immunochemistry we
could demonstrate the localization of myelin antigens within DC-SIGN+ phagocytic cells
of EAE-affected mice and marmosets (de Vos et al., 2002). Moreover, we observed that
surgical removal of CLNs and LLNs impairs the chronic relapsing EAE course in
MOG-sensitized Biozzi/ABH mice (van Zwam et al., 2009). These findings suggest that the
induction of T-cell reactivity against injury may take place in the CLNs and LLNs.
Indeed, we have frequently detected ex vivo T-cell reactivity (proliferation) against
MOG peptides in mononuclear cell suspensions from CLNs and LLNs of EAE marmosets.
B cells as requisite antigen-presenting cells (APCs)
For many years, translational therapy research in MS was mainly focused on the modulation
or suppression of autoreactive CD4+ T cell functions (Ransohoff et al., 2015). However,
in the past 10 years the field has changed rather dramatically, sparked by the discovery
that depletion of B cells via administration of the anti-CD20 mAb rituximab had an
unexpected positive clinical effect in RRMS (Hauser et al., 2008). This remarkable
finding has since then been confirmed with two fully human anti-CD20 mAbs, ofatumumab and
ocrelizumab (Barun and Bar-Or, 2012). The latter mAb even exerted a positive clinical
effect in patients with primary progressive MS (PPMS) (Montalban et al., 2017).
Collectively, these findings indicate that B cells have a much more prominent pathogenic
role in MS than just the production of autoantibodies. We investigated via which
mechanism(s) B cells exert their core pathogenic role in the marmoset EAE model and
whether this role is executed by all mature B cells or only by a certain subset.
An alternative method for B cell depletion is by capturing cytokines that B cells need
for survival and differentiation, i.e., BlyS/BAFF or APRIL (Dillon et al., 2006). To
achieve this, the chimeric protein atacicept has been developed by joining TACI, the
joint receptor for BlyS and APRIL on B cells, with an Fc fragment of human immunoglobulin G. Unexpectedly, atacicept showed no
relevant clinical effect in RRMS and even worsened lesion activity, necessitating the
termination of two clinical trials (Kappos et al., 2014).
These paradoxical clinical findings in MS sparked our interest in comparing the effect of
mAbs against CD20, BlyS and APRIL in the marmoset EAE model; we used the models induced
with rhMOG/CFA or with MOG34-56/IFA. As rituximab failed to bind marmoset B cells, we
selected a clonal variant of ofatumumab (HuMab7D8), which cross-reacted with marmoset B
cells. We observed that late-stage treatment (from psd 21, post-sensitization day) with the anti-CD20 mAb abrogated EAE development in marmosets immunized with
rhMOG/CFA (Kap et al., 2010), whereas the anti-BlyS and anti-APRIL mAbs only moderately
delayed EAE onset (Jagessar et al., 2012e). As depletion of B cells from the circulation
was observed in both types of treatment, we focused our analysis on the secondary
lymphoid organs (SLOs), i.e., lymph nodes and spleen. Intriguingly, we observed that in
the monkeys treated with anti-CD20 mAb, but not in those treated with anti-BlyS or
anti-APRIL mAbs, T cells within SLOs remained strongly positive for CCR7, indicating that
their release into the circulation might have been impaired (Kap et al., 2014).
Furthermore, we observed that in the monkeys treated with anti-CD20 mAb, but not in those
treated with anti-BlyS or anti-APRIL mAb, the copy numbers of CalHV3 DNA, being a
lymphocryptovirus of marmosets, was strongly reduced, indicating the depletion of
CalHV3-infected B cells (Jagessar et al., 2013a).
Collectively, these data underlie the following concepts:
that B cells act as requisite antigen-presenting cells for the T cells that
initiate and perpetuate EAE in marmosets,
that this role may be primarily executed by the CalHV3-infected B cell
fraction.
This concept was further tested in the MOG34-56/IFA EAE model in which lesion development
in WM and cGM occurs independent of anti-MOG antibodies (Jagessar et al., 2010). As
expected, we also observed that the development of clinical signs and CNS pathology in
this model was substantially reduced by the treatment with anti-CD20
mAb, hinting at a
direct role of B cells in the activation of pathogenic T cells (Jagessar et al., 2012b).
Antigen processing and presentation
We then asked why LCV/CalHV3 infection of B cells is important for their role as APCs in
the marmoset EAE model, in particular for presentation of the vulnerable MOG34-56 peptide
to pathogenic T cells. Inspired by a seminal publication from Manoury et al. (2002), who
first presented the concept of destructive processing of autoantigen (MBP) in thymic APCs
as an explanation for why certain autoreactive T cells escape thymic selection, we
proposed that this might also be the case for MOG ('t Hart et al., 2016). Our hypothesis
was supported by recent studies in mice, indicating that autoreactive T cells specific
for the MOG40-48 epitope escape negative thymic selection because the epitope is
destroyed by a thymus-specific serine protease (TSSP), which is expressed in thymic APCs.
TSSP cleaves at SP/PP residues within the MOG40-48 epitope (see Box 3). We proposed that
peripheral activation of these escaped autoreactive T cells is prohibited by B cells
expressing the lysosomal serine protease cathepsin G (catG). CatG cleaves the epitope at
the Arg41 and Arg46 residues ('t Hart et al., 2016). We also proposed that this
peripheral back-up tolerance mechanism might be impaired when the B cells are infected by
EBV/CalHV3. This concept was tested in nonhuman primate B lymphoblastoid cell (BLC)
lines, which were obtained by ex vivo infection of blood mononuclear cells with EBV
isolate 95-8 (for marmoset) or herpesvirus papio
(for rhesus monkey).
We observed that infection of B cells with the EBV-related lymphocryptoviruses (LCVs)
induced a variety of cell biological changes that potentiate their pathogenic role in the
EAE model. These include the following:
Malignant transformation. Transformed B cells are constitutively in an
activated state and are relatively resistant to apoptosis. Moreover, they have the
capacity to migrate across the BBB into the CNS (Pender, 2003). We indeed observed that
infusion of marmosets with autologous BLC, which were prepulsed in vitro with MOG34-56,
evoked mononuclear cell infiltrates in meninges and small-sized perivascular inflammatory
lesions (Haanstra et al., 2013c; Jagessar et al., 2013a).
Immune evasion. One of the immune escape mechanisms employed by LCVs is to
avoid T-cell recognition of viral antigens by cytotoxic T cells through mitigation of
antigen presentation via MHC class Ia (MHC-A, -B, -C) and MHC class II (MHC-DR) molecules
(Ressing et al., 2008). The absence of MHC class I molecules evokes an attack by NK cells
on the B cells, which can be repelled by the upregulation of MHC-E molecules. To achieve
this, MHC-E must be occupied by epitopes expressing a so-called QdM motif, which directs
the interaction of MHC-E towards inhibitory CD94-NKG2A heterodimeric NK receptors
(Vales-Gomez et al., 1999). Binding of peptides lacking a QdM binding motif directs MHC-E
binding to activating CD94/NKG2C heterodimeric NK receptors (Miller et al., 2003).
Interestingly, the MOG40-48 epitope was found to bind with high affinity to HLA-E and
Caja-E molecules expressed by transfectant K562 cells (Jagessar et al., 2012d).
Antigen cross-presentation. LCV-infected B cells have the capacity to
cross-present peptide antigens via MHC class Ib/Caja-E molecules to CD8+
cytotoxic T cells (Jagessar et al., 2012d).
Citrullination. The upregulation of peptidylarginine deïminase (PAD)
enzymes in LCV-infected B cells enables the conversion of arginine residues
into citrullines (Ireland and Unanue, 2011, 2012).
Autophagy. The autophagy pathway seems to be constitutively activated in
LCV-infected B cells. Evidence from EBV-infected B cells suggests that constitutive
activation is induced by the viral antigen LMP2A, which drives B cell function and
survival (Caldwell et al., 1998). LMP2A replaces the B-cell receptor as inducer of a
signaling cascade also involving Bruton's tyrosine kinase (Btk) (Merchant and Longnecker,
2001).
Each of the above-listed capacities of LCV-infected B cells can contribute to the still
elusive association of MS with EBV infection, especially around adolescent age. However,
evidence was found that the combination of antigen citrullination and autophagy
activation underlies the presentation of the potent encephalitogenic MOG34-56 peptide by
EBV-infected B cells (Ireland and Unanue, 2011). Our specific interest for antigen
processing and presentation in B cells was sparked by some remarkable observations.
First, we observed that immunization with MOG34-56/CFA elicited severe clinical EAE in
rhesus monkeys (Brok et al., 2007), while in monkeys immunized with MOG34-56/IFA neither
clinical signs nor T- or B-cell responses against the peptide could be measured (own
unpublished observation). However, when the peptide was administered via ex vivo-pulsed
LCV-infected B lymphoblastoid cell lines, T-cell reactivity with the peptide could be
measured ex vivo. Second, we observed that T- or B-cell reactivity with the MOG34-56
peptide was conspicuously absent in marmosets immunized with rhMOG/IFA (Jagessar et al.,
2015), while T- and B-cell immunity is clearly detectable in marmosets immunized with
MOG34-56 peptide in IFA (Jagessar et al., 2010). Moreover, we observed that infusion with
LCV-infected B lymphoblastoid cells pulsed ex vivo with the peptide elicited T- and
B-cell reactivity in marmosets (Jagessar et al., 2013a).
Destructive and productive processing of MOG34-56.
Collectively, these findings led to us to hypothesize that the MOG40-48 epitope might be
destroyed during normal processing of rhMOG in APCs and that LCV infection of B cells
might cause the conversion of destructive processing of the MOG40-48 epitope into
productive processing of the epitope from the immunizing MOG34-56 peptide and
presentation via MHC-E molecules (see Box 5 for details). This concept was tested in
EBV-infected and CpG-stimulated human B cells (Morandi et al., 2017a). An important
discovery was that LCV infection of B cells induces activation of the autophagy pathway
(see Box 6). In brief, we observed that the replacement of only the Arg46 residue in
the MOG40-48 epitope by citrulline had a profound effect on the degradation of the
peptide by catG in intact EBV-infected B cells, while the
Arg41→ citrulline substitution
exerted only a minor effect. In particular, we observed that activation of the autophagy
pathway with the mammalian target of rapamycine (mTOR) inhibitor reduced degradation of the peptide, while
inhibition of autophagy with 3-methyladenine (3-MA) increased degradation. Finally, we could detect
localization of rhMOG within autophagosomes, which were visualized by staining cells for
the docking autophagosome molecule microtubule-associated protein light chain 3 (LC3)
(Morandi et al., 2017b).
Autophagy.
Our interpretation of these data was that citrullination of Arg46 may protect a
putative F-LIR (LC3-interacting region) motif within the MOG40-48 epitope (xSxFSRVx)
against cleavage by catG (see Birgisdottir et al., 2013). Conceptually, the association
of the MOG34-56 peptide with autophagosomes may explain the protection of the MOG40-48
epitope against fast degradation in lysosomes, which leads to enhanced immunogenicity
(Delamarre et al., 2006) and facilitates cross-presentation (Munz, 2009). Moreover,
evidence suggests that HLA-E localizes in autophagosomes, where uploading with the
peptide could take place (Camilli et al., 2016). Collectively, these data provide a
plausible novel explanation for the EBV-MS association and for the pathogenic role of the
EBV-infected B cell.
In discussions about the causal association of EBV infection with MS the counter argument
is often raised that the EBV infection prevalence in the human population (90 %) is
much higher than the prevalence of MS (±0.1 %). Data from the marmoset EAE model
warrant the hypothesis that the MS risk may not map to events triggered by the infection,
but to capacities that B cells with a meaningful specificity for MS acquire by the
infection with EBV. In this context it is important to notice that in EBV carriers, the
frequency of B cells that contain the virus was estimated at between 1 and 50 per
106, i.e., < 0.005 % (Khan et al., 1996). So the question of whether
myelin-reactive B cells are more often infected with EBV in MS than in healthy controls
or patients with other diseases arises. We are not aware of any published direct
evidence. However, it has been reported that autoantibody responses against native MOG
were found in about 20 % of adolescents with
infectious mononucleosis, i.e., symptomatic EBV infection, while this antibody
specificity was not found in healthy control cases (Kakalacheva et al., 2016).
MOG-specific IgG responses declined after clinical resolution of infectious mononucleosis (IM), indicating the antibodies were either produced by EBV-infected B
cells themselves or a bystander product of noninfected B cells induced by factors from
EBV-infected B cells. Note, MOG-specific IgG antibodies are found in 40 % of
adolescents with an autoimmune CNS demyelinating disease (ADEM, MS, NMOSD), but not in
healthy control subjects. These data hint at the possibility that MOG-specific B cells
are more frequently infected with EBV in MS than in healthy age-matched adolescent
controls.
T and B cell epitopes plotted on the 3-D structure of MOG monomer.
(a) The conformational antibody epitope is formed by the three loops that
connect the B–C, C'–C” and F–G β sheets. Notice that the B–C connecting loop
(residues 27–36) overlaps with the CD4 T cell epitope and contains the Asn31 residue to
which in the native molecule the N-linked glycan is attached. (b) The positions
of the three dominant T cell epitopes and a linear antibody epitope are indicated. Notice
that these epitopes all overlap with β-sheet connecting loops. (c) An
excision of the critical MOG34-56 peptide, which appears to be composed of two large
antiparallel β sheets and a small one. Notice that the three critical Arg residues
in the MOG34-56 peptide where the peptide can be cleaved by cathepsin G (positions 41, 46
and 52) are located at contact points of loop and β sheet. (d) Depicts a
space-filling model of monomeric MOG (pdb accession number 1PKO) in molecular
surface representation, colored according to B factor (blue low rms/rigid; red high
rms/flexible. The surface-exposed MOG40-48 epitope (YRSPFSRVV) is indicated in
white/purple. The P43 and F44 residues, which stick out of the plane towards the reader,
are not resolved in the structure, probably due to the high flexibility of this part of
the sequence resulting in a diffuse diffraction pattern (Breithaupt et al., 2003), the
V48 residue is buried in the interior of the protein, and therefore not visible. The
putative LIR motif (xSxF43SRV47), which is part of the 40–48 epitope is shown
in purple. The surface exposure of this motif enables interaction with the LC3 docking
molecule of autophagosomes. (e) In a ribbon representation of monomeric MOG the
three Arg residues are highlighted. It is clear from this figure that the Arg46 and
Arg52 residues stick out while the Arg41 residue is somewhat buried.
Transfer of antigen from myeloid APCs to B cells?
The observations discussed thus far imply that myelin antigens (MOG for example) are
captured by myeloid APCs in CLNs or LLNs, while the B cell depletion studies reveal a key
function of LCV-infected B cells in the EAE model. This somewhat paradoxical situation
can be understood when MOG is in some way transferred from the myeloid APCs to the
LCV-infected B cells for further processing. However, in the absence of direct evidence,
this explanation can only be speculative. The fact that pathogenic anti-MOG antibodies
bind to a conformational epitope formed by three loops at the apical tip of the molecule
(Fig. 10a), implies that the B cells should receive via their BCR conformationally intact
MOG from the DCs. A publication by MacPherson (MacPherson et al., 1999) seems to be
important in this context. The author reports that part of the protein antigens that are
taken up by DCs is not processed but transferred to B cells in the intact form (Fig. 11).
In the rhMOG/IFA model only CD4+ T cell activation against the MOG24-36 epitope could
be detected, while the MOG34-56 peptide seemed to be destroyed, as neither T cell nor
antibody reactivity against this peptide could be detected (Jagessar et al., 2015). The
model in Fig. 11 may explain how the two EAE pathways can be activated independent from
each other. When MOG is processed through endolysosomal catG within the DC-like
phagocytes, a sufficient part of the MOG24-36 epitope remains intact for occupation of
the Caja-DRB*1201 epitope binding cleft, while the MOG40-48 epitope is destroyed. Part of
the MOG molecules is transferred relatively intact (possibly because the Arg residues are
citrullinated) to the B cells, which via the
infection with LCV have acquired the capacity to cross-present the protease-sensitive
MOG40-48 epitope via the autophagy pathway (Box 6) and Caja-E to CD8+ T cells (reviewed
in 't Hart et al., 2016). It is tempting to speculate that Arg residues in critical
epitopes of MOG released from lesions are citrullinated before they are captured by the
APC, thus ensuring that epitopes resist destructive processing. Molecular modeling shows
that the Arg46 and Arg52 residues are surface-exposed on MOG, while the
Arg41 residue is buried (Fig. 10d). The accessible Arg46 and Arg52
residues can be citrullinated enzymatically via PAD or iNOS.
Concept for T cell activation within the CLNs. (a) MOG draining via interstitial fluid (ISF) or cerebrospinal fluid (CSF) to the CLNs is captured by DC-like phagocytic cells (de Vos et al.,
2002). (b, c) T cells against the two dominant epitopes (24–36 and 40–48) are
probably not deleted during thymic negative selection as both epitopes are vulnerable to
destruction by TSSP, which cleaves at SP/PP residues present in both epitopes (Serre et
al., 2017). Processing of MOG in myeloid APCs and B cells is led by catG, which cleaves
at arginine (R) residues (Jagessar et al., 2016). This mechanism prevents peripheral
activation of autoaggressive T cells that have escaped thymic selection. (b) The
peptide sequence left after cleavage of the MOG40-48 epitope at Arg41 and Arg46
(the 4-mer 42–45) is too short for filling the peptide-binding cleft of the MHC class I
molecule. In the BLC, however, the MOG40-48 epitope is protected against destructive
processing. (c) Cleavage of the MOG24-36 epitope at Arg25 may have little
effect as a peptide of sufficient length (the 9-mer 26–34) is left intact for filling
the peptide-binding cleft of the MHC class II molecule.
Regarding PAD, it was discussed that an
increasing proportion of the myelin antigens in the MS brain is citrullinated through the
upregulation of PAD2 by inflammatory factors and an increase in [Ca2+] as
essential cofactor (Carrillo-Vico et al., 2010; Bradford et al., 2014). Regarding iNOS,
it was shown by Dunham et al. (2017a) that enzymes involved in oxyradical
(NOX2) and nitric oxide (iNOS) production are
upregulated in the inflammatory active lesions in marmoset EAE brain. During NO*
synthesis, arginine is converted into citrulline, but it needs to be tested whether this
can also occur when Arg is embedded in a peptide or protein (Rath et al., 2014).
In the mouse EAE model, the Arg residues are contact sites for the T cell antigen
receptor. Hence, their post-translational modification (PTM) potentially reduces the
amount of available antigen for the induction of relapses by pro-inflammatory T cells.
This notion underlies the new concept that as a consequence of PTM the autoreactive T-
and B-cell-driven inflammatory demyelination will gradually fade out and be replaced by a
pathogenic process inducing progressive disease.
A non-immunological role of B cells in (progressive) MSB cells in progressive MS
Data discussed thus far posit a central pathogenic role of LCV-infected B cells in the
autoimmune pathway(s) leading to cortical grey matter (cGM) pathology (reviewed in 't
Hart et al., 2017a). Their originally proposed role is the activation of effector memory
CTL specific for MOG40-48, which can kill oligodendrocytes ('t Hart et al., 2017a).
However, in contrast to observations in MS lesions (Zaguia et al., 2013), co-localization
of T cells with MHC-E expressing oligodendrocytes could not yet be detected in the
marmoset model. In fact, T cells were rare, if present at all, in subpial cGM lesions,
but they were frequently found in the adjacent meninges ('t Hart et al., 2017a).
We were inspired by the concept that the interaction of T and B cells in meningeal
infiltrates might produce (a) cytopathic factor(s) capable of directly inducing cGM
demyelination (Howell et al., 2011; Choi et al., 2012; Gardner et al., 2013). In the rat
model this activity was found to be mediated by pro-inflammatory cytokines, IFNγ
and TNFα in particular (Gardner et al., 2013). Intriguingly, data from Lisak et
al. (2012) suggest yet another mechanism, namely that
oligodendrocyte death may be induced by B-cell-derived large molecular factors (Lisak et
al., 2012), probably even extracellular vesicles (poster at American Academy of Neurology
2017 meeting; see https://www.youtube.com/watch?v=-3k1ubpq0Zo, last access:
17 April 2019). This theory is in remarkable
agreement with observations reported 40 years ago on the detection of a high molecular
transmissible factor in the MS brain, termed MS-associated agent (MAA), that is absent in
the healthy brain and that was found to induce depression of neutrophils in mice
(Koldovsky et al., 1975; Editorial, 1976).
These findings prompted the following experiments:
to test whether infusion of autologous
LCV-infected B cells prepulsed in vitro with MOG34-56 peptide induces EAE pathology in
rhesus monkeys and marmosets
to examine whether the interaction of LCV-infected B cells with or
without MOG34-56 peptide with T cells from EAE marmosets in vitro elicits production of
cytotoxic factors that may be implicated in cGM demyelination (Gardner et al., 2013).
Concerning study 1, we observed the formation of T and B cells containing meningeal
infiltrates (in both species), together with small-sized perivascular inflammatory
lesions (Jagessar et al., 2013a; Haanstra et al., 2013c).
Concerning study 2, we observed peptide-dependent production of IL-17A,
while IFNγ and TNFα were produced at a negligible level
(Dunham et al., 2017c), which essentially
reproduces earlier findings in the EAE model induced with MOG34-56/IFA
(Jagessar et al., 2012d). The combination of these observations indicates
that MOG34-56 loaded LCV-infected B cells are indeed capable of eliciting
neuro-inflammation, but that cytokines implicated in cGM demyelination in
the rat model seem not to be produced in sufficient quantity for eliciting
oligodendrocyte death in primate EAE.
Antigenicity and cytotoxicity of citrullination MOG34-56. EBV-BLC
cocultures with lymph node cells from EAE marmosets. (a) Marmoset EBV-induced
BLCs were lethally irradiated and incubated for 1 h with titrating concentrations of
unmodified MOG34-56 (peptide 10), or
peptide citrullinated at positions 41+46 (peptide 11) or positions 46+52 (peptide 12). Subsequently, lymph node or
spleen cells from marmosets immunized with MOG34–56 were added. T-cell responses to the peptides were assayed by proliferation
and are expressed as stimulation index per culture condition. The experiment was
conducted with N=6 (marmosets) and with three biological replicates. Data are presented
as mean ± standard error of the mean (SEM). (b) To test which
cell type is targeted by the peptides, EBV BLCs (from two marmosets, M1 and M2,
respectively) were incubated with Celltrace dye before incubation with peptide (white
circles) and mixture with the spleen/lymph node cells (red triangles). Lymphocytes that
are not subjected to coculturing were used as controls (black circles). Cultured cells
were harvested and stained for Annexin V as a marker of late apoptotic/dead cells. Final
analysis was done utilizing flow cytometry.
There is another argument against the induction of oligodendrocyte depletion by
T-cell-derived cytokines. The necessity to citrullinate the Arg residues within the
MOG40-48 epitope for protection of the immunizing MOG34-56 peptide against destructive
processing may affect T-cell recognition of the epitope. As was already mentioned, it has
been shown in B6 mice that the Arg residues at positions 41 and 46 contact the antigen
receptor of I-Ab restricted encephalitic CD4+ T cells and that T cell lines raised
against the noncitrullinated epitope cannot be stimulated by the citrullinated epitope
vice versa (Carrillo-Vico et al., 2010). We tested whether the same residues are needed
for T-cell recognition in the marmoset model. Indeed, we found that while the unmodified
peptides induced dose-dependent proliferation of T cells from marmosets immunized with
MOG34-56/IFA, peptides with the Arg residues at positions 41+46 or 46+52 replaced by
citrulline seem to exert cytotoxic activity (Fig. 12). It is therefore posited that the
incrementing citrullination of myelin inevitably leads to reduced autoimmune activation
and an increasing role of a novel B-cell-mediated cytopathic mechanism, which is
discussed in the next paragraph.
A novel EBV B-cell-dependent cytopathic mechanism
The observations discussed thus far seem to dispute the concept that the depletion of
oligodendrocytes in cGM lesions occurs by physical interaction with cytotoxic T cells or
by T-cell-derived cytotoxic factors. Hence, we tested an alternative option, namely
whether the MOG34-56 peptide itself or a metabolite thereof might be the elusive
cytopathic factor.
Figure 10a depicts the 3-D configuration of monomeric human MOG as elucidated by
Breithaupt et al. (2003). The structure is rich in β sheets, which are connected
by α-helical loops. Indicated is the conformational epitope to which the
monoclonal antibody 8.18C5 binds, with which demyelination has been induced in marmosets
with inflammatory EAE (Genain et al., 1995). In Fig. 10b the position of the linear T
cell and antibody epitopes are indicated. Note that the two dominant T cell epitopes
(MOG24-36 and MOG40-48) overlap with β-sheet connecting loops. The
encephalitogenic sequence 34–56 comprises three β sheets (C, C', C”), which are
connected by α-helical loops (41–46 and 52–56) (Fig. 10c). Notice also that the
Arg41, Arg46 or Arg52 residues are positioned where the loops and sheets
are connected. Hypothetically, cleavage by catG at these positions separates the β
sheets.
We observed the following: (1) replacement of these residues by citrulline protects the
MOG34-56 peptide against cleavage by catG and (2) that citrullination promotes
spontaneous aggregation of the peptide in the following order:
MOG34-56 < MOG34-56c41,46 ≪ MOG34-56c46,52. Moreover, the oligomeric
aggregates show amyloid-like seeding behavior (Araman et al., 2019).
These findings may shed an entirely new light on the role of LCV-infected B cells in cGM
demyelination. It has been well-established that EBV-infected B cells secrete
antigen-containing vesicles for spreading of an immune response (Raposo et al., 1996).
The notion that the MOG34-56 peptide associates with autophagosomes via LC3 raises the
question of whether LCV-infected B cells might secrete LC3+ vesicles. Figure 13 shows a
picture suggesting that this may indeed be the case.
LC3 staining of EBV-infected human B lymphoblastoid cells (BLCs).
Cytospin preparations of stably growing cells were prepared, fixed and stained with
anti-LC3 + GAM-FITC conjugate. Arrows point to small LC3+ bodies, which seem to be
shed from the BLCs.
The next question was whether the vesicles contain MOG34-56 peptide. This was tested
using synthetic peptides containing a bio-orthogonal group for attachment of a
fluorochrome to peptide inside (fixed and permeabilized) cells. The peptides were
unmodified (CA-1) or contained citrulline at the positions 41 and 46 (CA-2) or 46 and 52
(CA-3). Titrating doses of the peptides were fed to human EBV-infected BLCs for 48 h in
serum-free medium.
Figure 14 shows stainings of cytospin preparations from BLCs incubated with 6.2
or 25 µM peptide in serum-free medium. Several noticeable
observations can be made:
Column (a) shows that BLC incubated without peptide express only low staining
of LC3.
Column (b) shows the same incubation conditions of the peptides, but without
BLC. In the absence of BLC no precipitate of CA-1 is detectable, while
aggregates of peptides CA-2 and CA-3 were spun down on the glass slides.
Column (c) shows BLC incubated with two doses of CA-1, -2 and -3, specifically 6.2
and 25 µM.
Incubates of CA-1 with BLC contained globular structures staining positively for the
peptide (arrows) are detectable between high-LC3 expressing cells. These structures do
not contain a detectable nucleus (DAPI staining), while preliminary data show that
peptide is enclosed within a membrane that binds anti-MHC class II (not shown). This may
imply that the spherical structures are vesicles that have budded off from the B cells.
The reason why the vesicles were spun down at relatively low centrifugal speed (3 min at
28 g) may be that they are attached to the much heavier nucleated BLC. At the higher
peptide concentration LC3 expression of BLC was reduced and globular structures
associated with diffuse aggregates of CA-1 were detectable.
Also in the incubations with peptide CA-2 (citrullination at positions 41
and 46), albeit at lower concentration than CA-1, globular structures
associated with diffuse aggregates of CA-1 were detectable with only scarce
LC3+ BLC.
In the incubations of CA-3 (citrullination at positions 46 and 52) with the B cells
mainly large-sized aggregates were found surrounded by LC3-expressing BLCs.
Monitoring the uptake and aggregation of bio-orthogonal, site-specific citrullinated MOG peptides via confocal microscopy. Human EBV-infected BLCs were
incubated for 48 h with either none (peptide conc. 0 µM), 6.2 or
25 µM of unmodified MOG34-56
(peptide 10), or peptide citrullinated at positions 41+46 (peptide 11) or positions
46+52 (peptide 12) (highlighted in yellow). Cells were fixed with 4 %
paraformaldehyde and processed for immunofluorescence with
the following primary antibodies; the nucleus was stained with DAPI (blue) and LC-3 was
used as an autophagosome marker (green). The bio-orthogonal peptides were stained using
CuAAC chemistry with azide Alexa-647 (Thermo Fisher, the Netherlands).
These data strongly suggest that the CA-1 peptide has successfully survived passage
through the B cells, which according to previous insights (Jagessar et al., 2016; Morandi
et al., 2017a) implies that position 46 has been citrullinated, possibly via secreted
PAD. In the cultures containing the globular structures, reduction of LC3 staining was
observed, which points at a possible toxic effect.
In summary, we make the following four points:
We observed that immunization of marmosets with MOG34-56/IFA activates a
pathway that leads to demyelination of white and cortical grey matter of the brain. The
heart of this nonclassical pathogenic mechanism is formed by CD8+CD56+ effector
memory cytotoxic T cells, which are activated by LCV-infected B cells presenting the
epitope MOG40-48 via Caja-E molecules (Jagessar et al., 2010, 2012d). The similarity with
data from the McGill group motivated us to posit that the CTL induce demyelination by
killing oligodendrocytes (Zaguia et al., 2013). This concept was challenged by the
finding that the MOG40-48 epitope is highly sensitive to destructive processing by
cathepsin G, a leading endolysosomal protease in autoantigen processing in B cells
(Burster et al., 2004).
We observed a special relevance of the citrullination of the Arg46 residue,
which is located within a surface-exposed putative F-LIR motif that enables association
of the immunizing MOG34-56 peptide with autophagosomes, as shown in EBV-infected human B
lymphoblastoid cells fed with the peptide. In this way the peptide is protected against
fast endolysosomal degradation, which enhances immunogenicity (Delamarre et al., 2006).
The observations that treatment of the cells with rapamycine, a stimulator of autophagy,
enhances protection of the peptide against degradation and treatment with 3MA, an
inhibitor of autophagy, enhances degradation of the peptide support this concept
(Jagessar et al., 2016; Morandi et al., 2017a).
The replacement of arginine residues by citrullines impaired T-cell stimulation
(proliferation). This finding argues against demyelination induction via cytotoxic T
cells.
The paradoxical situation that citrullination of the Arg46 residue is
essential for epitope survival in B cells but impairs TCR recognition necessitated the
formulation of a new concept. Figure 15 shows staining of human EBV BLCs with an anti-LC3
mAb. Besides the expected punctate staining pattern, representing autophagosomes,
LC3+ vesicles were also seen, which seem to have been shed from the cells. It is
possible that these are extracellular vesicles (EVs) that BLCs produce for spreading
ingested antigen with the aim to enhance the immune response (Raposo et al., 1996).
Conceptually, citrullinated MOG34-56 can gain access to these LC3+ EVs via the
association with the autophagy pathway, where they may form aggregates (to be
determined).
Another line of research involved the serendipitous discovery that
citrullinated MOG34-56 spontaneously aggregates to multimeric structures, which display
seeding behavior comparable to amyloid-β. Note, the peptide contains three Arg
residues, which are at positions 41, 46 and 52. As discussed above, the Arg46
residue mediates interaction of the peptide with LC3. Substitution of the Arg41 or
Arg52 residue induces self-aggregation, although MOG34-56c46,52 is more
aggregation-prone than MOG34-56c41,46.
Effect of the dietary modification (2014) on incidence of EAE and CalHV3 infection in the BPRC marmoset colony. Since the discovery in 2012 that full-blown
clinical EAE could be induced with the rhMOG/IFA model, 19 marmosets have been tested.
(a) Time to clinical score 2 (disease-free survival) or the clinical endpoint
(overall survival) of monkeys tested before (n=13 marmosets, water-based supplement is
WBS) and after (n=6 marmosets) the introduction of the yogurt-based supplement (YBS).
The graphs show a significant effect of the dietary modification on the incidence and
course of EAE (log-rank test). (b) Periodic blood samples were collected from
the colony at the dates on the x axis and tested for the presence of CalHV3 DNA using
qPCR. The percentages of monkeys tested positive and negative for the viral DNA are given
on the y axis. The figure shows that after the dietary change the percentage of monkeys
in which CalHV3 could be detected was reduced from ±70 % to ±30 %.
Based on these collective data I posit that EBV-infected B cells may function as a Trojan
horse in the progressive phase of MS, bringing MOG peptide oligomeric aggregates into the
CNS and releasing them locally as packages within cytotoxic vesicles.
Studies in the effector arm of the marmoset EAE model
It was already recognized in the beginning of the model development that the
human-like architecture of the marmoset brain and the MS-like histology of
the EAE lesions might be relevant for studies on the monitoring of brain
pathology development and treatment with magnetic resonance imaging (MRI)
('t Hart et al., 1998, 2004, 2006).
Our group at BPRC has developed a variety of sequences for visualizing and quantifying
MRI sequences that were subsequently used for testing the radiological and clinical
effects of therapeutic antibodies against human IL-12p40 and CD40 ('t Hart et al., 2005b,
c). In addition, we used magnetic resonance (MR) spectroscopy of 24 h urine samples collected at 7 d
intervals during the disease course. The demonstration that urine samples collected
before, shortly after and late after EAE onset have different “metabolite fingerprints”
indicates the presence of urinary biomarkers for the different pathological processes ('t
Hart et al., 2003). However, the primary focus of our work has been on the afferent arm
of the disease, being the initiation and perpetuation of the autoimmune mechanisms that
cause the pathology. We mainly used the information from the therapy trials to assess the
relevance of an immunological mechanism for MS and to adjust the model where needed ('t
Hart et al., 2014, 2017b).
The effector arm of the disease has been studied in great detail by others. In studies by
Absinta et al. (2016) and Maggi et
al. (2017) a myelin-induced marmoset EAE model was examined
with high-contrast MR images recorded at 7 T to monitor the initiation and development of lesions in the white
matter. An extensive discussion of results obtained by these authors is beyond the scope
of this monography. Hence, they will be briefly summarized. For more detail the
interested reader is referred to publications from this group (see reviews by Absinta et
al., 2016; Maggi et al., 2017, and references to primary publications therein).
As discussed above, it is highly difficult to assess whether inflammation precedes or
follows lesion onset in the MS brain. In brief, analysis of the temporal development of
brain white matter lesions in a myelin-induced marmoset EAE model show that lesions start
as a small-sized focal enhancement of blood–brain barrier permeability developing into
an acute inflammatory lesion (< 1 week old), which is cuffed around a central
parenchymal vein and is composed of Iba-1+ microglia, CD3+ T cells, MRP14+
macrophages and (few) CD20+ B cells (Maggi et al., 2014). In lesions of older age (1 to
5 weeks), indicated as subacute, permeability of the blood–brain barrier was
substantially reduced and demyelination was clearly noticed. The lesions typically
contained Iba-1+ microglia, MRP14+ macrophages, CD3+ T lymphocytes and GFAP+
activated astrocytes. Substantial damage to axons was found in these lesions as well. The
clear association of demyelination with macrophage infiltration indicates a prominent
pathogenic role of innate immune mechanisms at this stage. Around such lesions a rim of
activated astrocytes can sometimes be found (Laman et al., 1998). Similar to our own
studies ('t Hart et al., 1998), the study by Maggi et al. (2014) revealed a substantial
number of lesions without strong inflammatory activity or signs of remyelination and with
moderate axonal damage, which they named late subacute lesions. Interestingly, on T2W MR
images these latter lesions usually have a smaller size than the acute and subacute
lesions, which may be due to the resorption of tissue edema and/or repair ('t Hart and
Massacesi, 2009; Maggi et al., 2017).
The main message from these studies is that perivenular inflammation, i.e., lymphocyte
cuffs and microglia activation, precedes the formation of lesions. Lesions develop by
centrifugal expansion from non-demyelinated nodules of lymphoid and myeloid cells around
a leaky blood vessel via which they have infiltrated the CNS parenchyma. Although these
observations clearly support a primary inflammatory ontogeny of MS lesions, they do not
answer the question of where autoimmunity is induced.
Targeted dietary intervention in the marmoset EAE model: was Metchnikoff
right?
Research discussed thus far has led to the conclusion that the heart of the marmoset EAE
model is formed by pathogenic functions that B cells acquire via LCV infection:
protection of the vulnerable MOG40-48 epitope against fast degradation,
cross-presentation of the epitope via Caja-E to effector memory CTLs and secretion of
post-translationally modified MOG34-56 as toxic aggregates, either packaged or not
packaged in vesicles. This concept may provide a plausible mechanistic explanation for
the association of EBV infection with MS susceptibility.
The clear core pathogenic role of LCV-infected B cells raises the question of whether
other environmental factors that influence the disease expression in the model may exert
their effect via LCV-infected B cells. A serendipitous finding showed that this may be
the case for (a) component(s) of a new dietary supplement that was introduced into the
marmoset colony at BPRC in January 2014. Recent literature is flooded with publications
about the role of a gut–immune–brain axis in the initiation and course of EAE (Joscelyn
and Kasper, 2014; Shahi et al., 2017; Wekerle, 2017). At this point the marmoset EAE
model has also yielded some intriguing new insights, which are summarized in the
following paragraphs.
A direct consequence of the high level of refinement of the marmoset EAE model has been
the loss of robustness of the model, which is probably due to higher sensitivity for the
variable influence of genetic and environmental factors. This has led to increased
variation in the response to the immunizing antigen or to an experimental treatment
(e.g., anti-CD127 mAb), including nonresponders, and the potential loss of statistical
power ('t Hart, 2016a). In the course of 4 years (2014–2018) we were facing an
unexplained sudden decrement in the disease incidence of the atypical EAE models induced
with MOG34-56/IFA (from 90 % to 65 %) or rhMOG/IFA (from 100 % to 60 %)
(Fig. 15a). The reduced EAE susceptibility coincided with a sharp decrease in the number
of marmosets in the BPRC colony testing positive for CalHV3 (Fig. 15b). It has taken us
considerable time to find out that the sudden drop of EAE susceptibility coincided with
the modification of a dietary supplement, which was introduced in January 2014. The
modification was rather modest, namely from a water-based to a yogurt-based formulation,
which also contained the double dose of B vitamins. Nevertheless, the effects on the
model were rather dramatic (Kap et al., 2018a).
These enigmatic observations prompted a controlled study in marmoset twins,
which had been fed the new dietary supplement from weaning onwards. For the
experiment, one sibling of each twin was reverted to the old water-based
supplement (WBS), starting 8 weeks prior to EAE induction, while the other
sibling remained on the new yogurt-based supplement (YBS). In brief, the EAE
incidence in the old WBS and new YBS groups differed, albeit not
significantly (8/8 in the old diet group, 6/8 in the new diet
group) (Kap et al., 2018a). In addition, we
observed a significant reduction of spinal cord demyelination, markedly
altered expression of genes involved in apoptosis and myelination in the
brain, a marked reduction of CalHV3 expression and alteration of cellular
immune parameters, which in previous studies have been implicated in the EAE
pathogenesis. As expected, the dietary modification was reflected by a
marked alteration of gut microbiota composition. However, diversification of
gut microbiota between the WBS and YBS groups became first detectable only 3
weeks after rhMOG/IFA inoculation.
It is unclear at this moment which components of the YBS were responsible for the effects
on the model. One candidate is the yogurt. The health benefits of sour milk have been
known since biblical times, but a deeper mechanistic understanding comes from the work of
the 1908 Nobel Prize winner for physiology or medicine, Ilya Metchnikoff. He proposed
that health can be enhanced and aging postponed through manipulation of the gut
microbiota with host-friendly bacteria present in fermented milk (Mackowiak, 2013). This
is exactly what we found in the twin study (Kap et al., 2018a). We observed a positive
effect of the YBS on the proportion of bifidobacteria in the marmoset stool. According to
another theory, yogurt is a dietary source of tryptophan, which can be metabolized by the
marmoset gut microbiota, the bifidobacteria in particular. Tryptophan can be converted by
probiotic gut microbiota into high-affinity ligands of the aryl-hydrocarbon receptor
(AHR), such as kynurenine (O'Mahony et al., 2015). Ligand-activated AHR counteracts the increased expression of
genes relevant for B-cell growth and function, such as early B-cell factor 1 (EBF1), in
EBV-infected B cells (Li et al., 2017). Upregulation of EBF1 induced by EBNA1 is
associated with EBNA1's role in maintaining the viability of infected B cells and keeping
the EBV genome intact during latent infection (Tempera et al., 2015). This suggests that
the yogurt intake in combination with higher levels of bifidobacteria in the gut may lead
to the production of factors mediating AHR activation, which might have caused the
reduced survival of CalHV3-infected B cells.
The translational relevance of these findings for the MS patient may be that
the pathogen-educated immune system of adult outbred marmosets is
susceptible to dietary modification in such a way that immune responses
against MOG released from a primary lesion in the CNS are reduced. Further
work should reveal which components in the YBS diet are responsible for this
effect.
Preclinical efficacy assessment of candidate treatments
One of the strongest arguments for developing nonhuman primate EAE models of MS is that
they provide relevant systems for translational research into the pathogenesis and
treatment of the disease. Relevant features in this line of research are that many
biologicals developed for immunosuppressive or immunomodulatory therapy in MS cross-react
between marmosets and humans and that marmosets and humans share a high level of
immunological similarity. Indeed, reports from others (Maggi et al., 2017) and us ('t
Hart et al., 2015) documented the remarkable similarities in the afferent and effector
arms between marmoset EAE and MS.
The diagram in Fig. 1 shows that the integration of exploratory research of pathogenic
mechanisms and applied research of new therapies creates a powerful research strategy.
The integration of forward and reverse translation creates an iterative scientific
process that potentially provides deep insight into the critical steps in the pathogenic
process and delivers better therapies. We have used this iterative strategy in the
marmoset EAE model. More specifically, we tested therapies that worked or did not work in
the clinic for eliminating pathogenic mechanisms in the EAE model that are irrelevant for
the human disease. The ultimate aim of this strategy was to create the most relevant
preclinical model for MS ('t Hart, 2015, 2017b).
Figure 16 gives a graphical presentation of relevant factors in the pathogenic
process inside the CNS, each of which could be a target for therapeutic
intervention. Several of these targets have been validated in the marmoset
EAE model:
It is believed that the immunopathogenic process within the CNS starts with
the infiltration of peripherally activated CD4+ T cells with an inflammatory phenotype
(a). One type of therapy targets the attachment of the T cells to endothelial cells of
the blood–brain barrier, which is mediated by adhesion molecules. An example of a drug
approved for clinical use is Natalizumab, a monoclonal antibody directed against α4β1 integrin (VLA-4), which mediated autoreactive T cell infiltration into the
CNS in mouse EAE studies (Steinman, 2005) (see insert).
T cells that have achieved transmigration of the blood–brain barrier need to
engage in cognate interaction with local antigen-presenting cells (APCs) for unfolding
their complete pathogenic activity. This interaction comprises three signals: signal 1,
the formation of a trimolecular complex composed of a peptide presented by MHC molecules
and the antigen receptor of T cells; signal 2, the interaction of co-stimulatory
molecules expressed by T cells and APCs, such as CD28 with CD80/86 or CD40 ligand with
CD40; and signal 3, cytokines that steer functional polarization of the activated T
cells, such as IL12, IL-23 and IL-7. A frequently tested approach in the broad spectrum
of immune-based inflammatory disorders is the functional ablation of inflammatory T cells
by blocking signal 2.
The infiltration of CD4+ T cells is followed by a second wave of
infiltrating immune cells, in particular B cells (b) and macrophages. Macrophages are a
dominant cell type in inflammatory active lesions. We
actually found that the MRI markers of inflammation in lesions correlate reasonably well
with macrophage infiltration in marmoset EAE (Blezer et al., 2007). Mounting evidence
indicates that B cells have a multifaceted pathogenic role within the CNS, although there
is only solid evidence for production of autoantibodies capable of binding myelin (Genain
et al., 1999).
Mounting evidence indicates that MS patients have an inherently deficient
repair capacity for myelin damage by recruitment of oligodendrocyte precursor cells (c)
into lesions, where they are supposed to differentiate into mature oligodendrocytes which
are capable of producing new myelin sheaths.
The demyelination process focusses on the oligodendrocyte/myelin–axon units via two
mechanisms, namely cytotoxic damage to myelin sheaths via complement (CDC) or macrophages
(ADCC) and T-cell-mediated cytotoxicity towards oligodendrocytes.
Below follows an overview of the types of therapy that were tested in the
marmoset EAE model.
Schematic overview of immune processes in the EAE model.(a) Autoreactive CD4+ T cells that have been activated in peripheral lymphoid
organs by the injection of antigen-adjuvant emulsion infiltrate the CNS via passage
through the blood–brain barrier. This passage is mediated by interaction adhesion
molecules, such as the α4β1-integrin VLA-4 with ICAM-1 on blood–brain
barrier endothelial cells (see insert). By local cognate interactions with local
antigen-presenting cells, including dendritic cells, macrophages and microglia, the T
cells elicit a cascade of pathophysiological reactions leading to inflammation and
demyelination. (b) A second infiltrating lymphocyte type is the B cell, which
secrete antibodies that, via binding to myelin sheaths and oligodendrocytes, elicit
macrophage-mediated and complement-dependent cytotoxicity (ADCC and CDC, respectively).
However, this classical role of B cells requires adjustment, because B cells have a much
more elaborate pathogenic role. (c) The target of the autoimmune process is the
axon–myelin unit, which comprises axons, the enwrapping myelin sheaths and the
myelin-forming oligodendrocytes. In the healthy CNS, damaged oligodendrocytes can be
replaced by infiltrating oligodendrocyte precursor cells (OPCs), but this repair capacity
seems impaired in MS.
Category 1: therapies targeting T cells. Rodent EAE models show a central
pathogenic role of two types of pro-inflammatory CD4+ T helper (Th) cells, which can be
distinguished on the basis of their cytokine signature: namely IFNγ+ Th1 cells
and IL-17A+ Th17 cells (Gran et al., 2004; Damsker et al., 2010). The differentiation
of these functional subtypes from precursor Th0 cells is directed by cytokines produced
by the APCs, i.e., IL-12 for Th1 and IL-23 for Th17 (Hunter, 2005). There is ample
evidence in the literature that functional or physical elimination of Th1 and/or Th17
cells mitigates the incidence and/or course of EAE in SPF-rodent models.
IL-12/IL-23. The CD4+ Th cell-centered concept of MS stimulated the
development of a fully human mAb (ustekinumab) against the shared p40 subunit of IL-12
dimer (p35/p40) and IL-23 dimer (p19/p40). However, contrary to expectation, the antibody
failed to show relevant activity in RRMS (Segal et al., 2008). Control tests showed that
the mAb cross-reacts with marmoset IL-12p40. We have assessed the clinical efficacy of
the mAb in two different treatment modes, namely starting treatment just before the
immunization (prophylactic) or once brain lesions of sufficient size could be detected
and quantified with MRI (therapeutic). In brief, in the prophylactic experiment (n=2×5) EAE was induced with myelin/CFA. All monkeys treated with a placebo
preparation (phosphate buffered saline; PBS) developed clinically evident EAE with
complete EAE pathology. In the prophylactic experiment EAE was induced with MS
myelin/CFA. In all five monkeys treated with the ustekinumab mAb we observed complete
suppression of clinical and pathological aspects of EAE (Brok et al., 2002). In the
therapeutic experiment (n=2×5) the time of appearance of brain lesions,
recorded with MRI, differed between individual animals. All monkeys developed clinical
EAE as well as severe pathology. It was observed that MRI parameters for lesion activity
(post Gado-T1, T2, lesion surface enlargement) measured after infusion of the mAb did not
further change, indicating that pathogenic processes inside the CNS were stopped for the
remaining time of the experiment. Nevertheless, the onset time of clinical signs was only
delayed for a few weeks ('t Hart et al., 2005c).
In conclusion, the two experiments show that the mAb had a strong beneficial effect when
treatment was started early in the disease, while the treatment was less effective once
the disease was ongoing. Moreover, we observed a dissociation between the strong effect
of the mAb on white matter pathology and the modest, albeit statistically significant,
effect on the clinical signs. An explanation for this clinical–pathological paradox may
be the existence of two immunopathogenic pathways, an initiation pathway driven by
IFNγ+ CD4+ T cells and a progression pathway driven by IL-17+ CD8+ T cells
('t Hart et al., 2011).
IFNγ. In a second experiment, we tested the effect of human IFNγ
in the MOG34-56/IFA model (Jagessar et al., 2012a). In murine EAE, promising effects of
this treatment have been obtained (Sanvito et al., 2010). The cytokine was administered
from day 0–25 (prophylactic) or day 65–81 (therapeutic). We observed that the early
treatment caused a moderate, albeit statistically not significant, delay of disease onset
(from day 98±36 in the control group to day 118±40 in the cytokine-treated
group). The late treatment had a more modest effect on EAE onset (from day 98±36 in
the control group to day 105±50 in the cytokine-treated group). Moreover, we
observed a clear effect of the early treatment on humoral and cellular autoimmune
parameters.
In conclusion, these observations argue against an important pathogenic role
of Th1 cells in the marmoset EAE model.
IL-17A. In a third experiment, we tested the effect of a humanized IgG4κ
mAb against human IL-17A in the rhMOG/CFA model. Biocore tests showed that the reactivity
with marmoset IL-17A was about 2-fold lower than with human IL-17A, whereas in a bioassay
the activity was 4-fold lower (Kap et al., 2011b). The mAb was tested in two doses in the
marmoset EAE model, 3 or 30 mg kg-1, and the effect was compared with PBS as
placebo. The data showed a trend towards delayed EAE onset in the low-dose group (mean
day 69.3; 48–113) compared with the control group (mean day 57.1; 39–91) and the
high-dose group (mean day 60; 51–69). Moreover, the EAE progression time from EAE score
2 (ataxia) to score 3 (paraplegia) was faster in the control group (mean 10.8 d; 3–20)
than in the low (mean 12.9 d; range 5–28) and high (mean 13.7; 6–31) mAb dose groups.
These differences were statistically not significant. Also, the pathological examination
showed no significant effect of the mAb.
In conclusion, we found that treatment with anti-IL-17A antibody induced a
moderate delay of clinical EAE in marmosets, but EAE development was not
completely abrogated. This finding hints at a pathogenic role for IL-17A in
the marmoset EAE model and maybe in MS, but IL-17A may not be the only key
pathogenic cytokine.
IL-7 receptor/CD127. IL-7 is a cytokine produced by stroma cells that has a
broad activity range. In the bone marrow, IL-7 stimulates the differentiation of
hemopoietic stem cells into lymphoid progenitor cells. Moreover, the cytokine stimulates
proliferation and differentiation of T cells, B cells and NK cells. We have tested a
chimeric IgG mAb raised against marmoset IL-7 in the MOG34-56/IFA model (Dunham et al.,
2016). Treatment of seven twins with mAb or PBS intravenously administered was started at
21 d after the first immunization. Although the mAb blocked CD127 function in all
monkeys, as assessed by IL-7-induced STAT5 phosphorylation, we observed a beneficial
effect on the disease course only in twins with fast-progressing disease. This remarkable
dichotomous effect indicates that even in the highly refined MOG34-56/IFA model disease
heterogeneity can be observed.
In conclusion, The IL-7 pathway seems a relevant target of therapy for a subset of MS
patients.
Overall conclusion of this part. These experiments seem to confirm observations
in MS clinical trials, namely that therapies targeting only CD4+ T cells are only
moderately effective.
Therapies targeting B cells. The remarkable results of the anti-CD20 mAb
rituximab in RRMS (Hauser et al., 2008) created a paradigm shift in MS. While B cells
were for a long time viewed only as producers of myelin opsonizing antibodies, which
induce demyelination via complement- (CDC) or macrophage-dependent (ADCC) cytotoxicity
reactions, they have now gained a central position in the MS pathogenic process (von
Budingen et al., 2015). These findings prompted us to test the second generation
anti-CD20 mAb HuMab7D8, which is a clonal variant of the human mAb ofatumumab with
confirmed cross-reactivity with marmoset B cells in the EAE model. We made a number of
noticeable observations.
CD20. We tested the efficacy of HuMab7D8 in the rhMOG/CFA model, starting weekly
treatment at 21 d postimmunization. The antibody induced almost complete and persistent
depletion of CD20+ B cells from blood and lymphoid organs as well as from the CNS (Kap
et al., 2010). The treatment led to almost complete suppression of clinical signs of EAE
as well of CNS pathology in WM and cGM (Kap et al., 2010, 2011a). Analysis of autoimmune
reactions revealed suppression of antibody production as well as T-cell responses against
the immunizing rhMOG protein (Kap et al., 2010). Intriguingly, the emptied B cell areas
in secondary lymphoid organs of mAb-treated monkeys were replenished by T cells
expressing CD127 (IL-7 receptor) and CCR7 (Kap et al., 2014). As discussed above,
treatment with anti-CD127 mAb induced substantially delayed EAE onset in fast disease
progressor monkeys (Dunham et al., 2016), which was a hallmark of the EAE progression
pathway (Kap et al., 2008). These findings combined indicate that treatment with
anti-CD20 mAb prohibits the release of activated autoaggressive T cells from the
secondary lymphoid organs.
One of the presumed roles of B cells in MS is antigen presentation to
autoaggressive T cells (von Budingen et al.,
2015). This was tested in the MOG34-56/IFA model, in which pathology and
disease is induced by the action of autoaggressive T cells, without the
support of myelin-opsonizing autoantibodies. Also in this model, we observed
profound suppression of EAE symptoms and pathology, as well as suppression
of humoral and cellular autoimmune parameters (Jagessar et al., 2012b).
BlyS and APRIL. An alternative method for B cell depletion is the capture of
cytokines that B cells need for survival and differentiation, i.e., BlyS/BAFF or APRIL
(Dillon et al., 2006). This prompted us to test two mAbs in the rhMOG/CFA model, namely
anti-human BlyS mAb (belimumab) and an
anti-human APRIL mAb. Treatment was again started at 21 d after the immunization. Both
antibodies caused only a moderate, albeit statistically significant, delay of the EAE
onset (Jagessar et al., 2012c). Note, examination of the secondary lymphoid organs did
not reveal retention of activated autoaggressive T cells (Kap et al., 2014).
CD40. The treatments with anti-BlyS/APRIL and anti-CD127 mAbs hinted at a
special role of B cells expressing high levels of CD40 in the EAE model (see above). This
conclusion is supported by data from earlier studies in which we tested murine, chimeric
and humanized variants of the human CD40 blocking mAb 5D12 in the EAE model. In brief, we
observed in a marmoset EAE model induced with human myelin/CFA that treatment with a
mouse anti-human CD40 mAb (5D12) delays the onset of clinical signs (Laman et al., 2002).
In the same study, we observed that the mAb gains access to inflammatory active lesions.
To assess whether treatment efficacy might be limited by the formation of antidrug
antibodies we gave a single intravenous injection of the murine mAb and tested serum
antibody levels at several time points thereafter. Indeed, free test substance levels
were substantially lower in mAb-treatment cases than in a naïve monkey.
Based on these encouraging findings, a chimeric mouse–human IgG4 mAb was developed
(ch5D12), which was tested in the EAE model induced with rhMOG/CFA (Boon et al., 2002).
Also in this experiment, we observed profound suppression of EAE clinical signs and
partial suppression of brain and spinal cord pathology. As was expected, the induction of
anti-rhMOG IgG antibodies was suppressed as well.
Overall conclusion. These immunotherapy findings support a core pathogenic role
of CD20+CD40+ B cells in the marmoset EAE model.
Cell-based regenerative therapies. One of the great challenges in MS research is
to develop treatments with which CNS damage can be repaired once the (immuno)pathogenic
process has been stopped. Much is expected from treatment with stem cells (SCs), which
are now available in different flavors, such as hemopoietic SCs, neural stem/precursor
cells (NPCs), mesenchymal SCs or induced pluripotent stem cells (iPSs) (Martino et al.,
2010; Di Ruscio et al., 2015). We have tested two types of stem cells in the marmoset EAE
model, namely human NPC, grown as homogenous cell line, and iPS cells derived from
reprogrammed human skin fibroblasts, differentiated into oligodendrocyte precursor cells
(OPCs).
NPC therapy. This form of therapy is based on the administration of
self-renewing, multipotent cells isolated from the CNS. For our study, we used human
eGFP-transduced NPC, grown as a homogenous cell line, to enable the detection of injected
cells in postmortem brain tissue (Pluchino et al., 2009). The cells were administered to
rhMOG/CFA-immunized monkeys either intra-CSF via injection into the cisterna magna or
into the blood stream via the tail vein. We injected a low dose of cyclosporine A (CsA;
10 mg kg-1) to prevent immediate rejection of the xenogeneic (human) NPCs.
Interestingly, the administration of CsA transformed the natural, rapidly progressing
disease course into a chronic relapsing course. Significant clinical amelioration was
observed both with the intravenous and the intra-CSF treatments, over an observation
period of 90 d. Even after this long time interval, eGFP+ve cells could still be detected in the marmoset CNS. However, we did not observe
signs of newly formed neurons or glia cells.
OPC therapy. The scientific impact of the Nobel Prize-winning new technology
for reprogramming of committed somatic cells into pluripotent stem cells from which other
cell lineages can be generated has been immense (Takahashi and Yamanaka, 2006). We have
used this exciting technology to generate OPCs from reprogrammed human skin fibroblasts
(Thiruvalluvan et al., 2016). The potency of these (GFP-labeled) OPCs to remyelinate
axons that were denuded by autoimmune demyelination was tested in the MOG34-56/IFA
marmoset EAE model, The OPC graft was injected into the brain at some distance from an
MRI-detectable (T2) lesion. Like in the previous experiment, low dose CsA
(10 mg kg-1) was injected to prevent xenorejection of the human OPC. Our study
showed that the injected OPC migrated from the injection site to the lesion, where they
differentiated into mature oligodendrocytes, which made contact with a denuded axon and
started forming a new myelin sheath (Thiruvalluvan et al., 2016).
Conclusion. the marmoset EAE model has proven its value for the efficacy
assessment of innovative treatments. Relevant features for this line of research are
(1) the immunological similarity between marmosets and humans and (2) the neuroanatomical
similarity of marmoset and humans.
Ethical considerations on nonhuman primates as a model of human
disease3 or 4 R's?
The leading principles in preclinical research with living animals have been formulated
by Russell and Burch as the “3 R's”: replacement, reduction and
refinement (Russell and Burch, 1959). Strict adherence to these principles is
now common practice, especially in research with sentient animals, such as NHPs. However,
it is rather remarkable that an obviously important fourth R is lacking, namely the
(clinical) relevance of an animal model. In the following, these four R's will be briefly
discussed as well as why difficulties encountered when harmonizing them have made the
generation of a clinically relevant EAE model an acrobatic balancing act ('t Hart,
2016a).
The replacement principle encourages scientists to use methods in their research that
avoid or replace the use of living animals. Although in vitro models with cells or
tissues and in silico models of human pathology are increasingly used in preclinical
research, it is generally felt that the complexity of a pathological process in relation
with other physiological body systems is only displayed in living animals. In that case
the lowest animal species from which relevant information can be obtained should be
selected. This consideration underlies the increasing use of evolutionary distant model
systems, such as zebrafish (Brachydanio rerio), Drosophila flies and
Caenorhabditis elegans worms. However,
although important insights into novel disease mechanisms can be obtained from these
models, it is felt that model systems more closely related to the patient are needed for
the integration of all information into a coherent pathogenic concept that can be
translated into new therapies.
The reduction principle encourages scientists to use research methods that provide either
comparable levels of information from fewer animals or more information from the same
number of animals. We achieved the latter goal by the application of nuclear magnetic
resonance (NMR) imaging techniques ('t Hart et al., 2004), but compliance with the former
condition appeared to be more problematic. Reviewers of projects and publications often
limit the interpretation of the reduction principle only to the minimal number of animals
that should be used for a “usable” result. Power calculations are applied to determine
the minimal size of experimental and control groups, so that the effect of an
experimental variable can be statistically tested. Critical variables in power
calculations are the disease incidence, the anticipated treatment effect and the expected
variation in read-out parameters, such as the time to clinically evident disease or the
disease severity. The outbred nature of NHPs and their conventional housing conditions
imply a variable influence of genetic background and environmental factors, ironically
being dominant autoimmune disease risk factors in the human population. The inevitable
consequence of variation is that experimental groups may contain clinically low
responders or nonresponders, which reduces disease incidence and thus requires larger
group size for statistical power. In rodent disease models this dilemma is bypassed by
using well-established genetically homogeneous (inbred) SPF-bred strains and the usage of
potent adjuvants to obtain high disease incidence and a synchronous disease course.
However, a recent study raised questions on the relevance of immunologically immature SPF
mice as models of complex human diseases (Beura et al., 2016). Moreover, the usage of
strong bacterial adjuvants clearly conflicts with the refinement and relevance principles
(see below).
The refinement principle encourages scientists to employ research methods that minimize
the discomfort from experimental procedures to the animals. A major concern with respect
to the EAE model is that for reproducible disease induction strong adjuvants need to be
used to pepper the immunogenic potency of self-antigens by disrupting the regulatory
mechanisms that keep autoaggressive T and B cells under control (Matzinger, 1994). The
most frequently used is complete Freund's adjuvant (CFA), which is an emulsion of
heat-killed mycobacteria (M. tuberculosis or butyricum) in mineral oil.
The mineral oil, also known as incomplete Freund's adjuvant (IFA), forms a depot for the
finely dispersed antigen solution in aqueous buffer; the mycobacteria provide danger
signals needed for “awakening” of the tolerized T and B cells. However, CFA is
notorious for its seriously detrimental side effects, in particular the induction of
severe ulcerative skin lesions at the injection sites, which are clearly caused by the
mycobacteria. Usage of CFA in nonhuman primates is therefore discouraged. As discussed
above, in the marmoset EAE model IFA can be used, which implies a major reduction of
discomfort to immunized animals compared to animals immunized with CFA.
The forgotten fourth R of relevance: the usage of CFA also introduces a mechanistic bias
in the EAE model as immune responses against antigens formulated with CFA are skewed
towards a pro-inflammatory profile that is dominated by CD4+ T cells (Billiau and
Matthys, 2001). The poor translation record of experimental therapies targeting CD4+ T
cells from EAE to MS indicates that this lymphocyte subset may be less relevant for the
human disease than in the animal model (Hohlfeld et al., 2015). However, this does not
preclude a pathogenic role of CD4+ T cells early in the disease process, i.e., before
the diagnosis has been made.
Compliance of the marmoset EAE model with the fourth R has been achieved via an iterative
strategy depicted in Fig. 13. In brief, research in the exploratory arm aimed at maximum
refinement of the original EAE model, which was induced by immunization with myelin
isolated from the brain of an MS patient formulated with the bacterial adjuvant CFA ('t
Hart et al., 1998). The stepwise refinement of this complex model towards the minimally
needed components yielded a highly refined model that displays essential pathological
aspects of MS ('t Hart et al., 2015) (Fig. 7). In the applied arm, the consecutive steps
of the refinement process were validated with clinically relevant therapeutic mAbs. The
in depth characterization of this atypical EAE model is still ongoing, but
preliminary data show that the MHC-E-restricted CTL that mediate the development of
chronic EAE in marmosets (Jagessar et al., 2012d) can also be found in MS lesions (Zaguia
et al., 2013).
The replacement of CFA for IFA in the translationally relevant atypical EAE
models had various important consequences:
The discomfort to the animals in experiment was reduced,
The immunogenicity of administered biological therapeutics, which limited
their activity window, was reduced,
The dogma that danger signals are absolutely needed for autoimmunity
induction may need to be adjusted,
A new pathogenic mechanism was discovered.
Concerning the third issue a caveat may be needed as the injection of antigen/IFA
emulsion will certainly cause damage and induce the release of damage-associated
molecular patterns, which can relay danger signals to APCs through DAMP receptors (Kono
and Rock, 2008). The released DAMPs cause visible skin irritation. However, these signals appear too
weak for the elicitation of EAE in genetically susceptible, but immunologically immature,
SPF mice (Jagessar et al., 2010).
A conflict among the R's
Compliance of the primate EAE model with the refinement and relevance principles
introduced an unforeseen conflict with the reduction principle. It was observed that the
replacement of CFA with IFA produced less robust EAE models, which were more prone to
variation in the response against immunization as well as in the response to treatment. A
likely explanation is that these refined disease models are more sensitive to the
variable influences of genetic and environmental factors. An experiment in which we
encountered the consequences was reported recently (Dunham et al., 2016). In brief, we
tested the efficacy of a mAb raised against the human IL-7 receptor CD127 in a powered
two-leg study in marmoset twins (n=7) immunized with MOG peptide 34-56/IFA (Dunham et
al., 2016). One sibling of each twin received the therapeutic mAb and the other a placebo
preparation. We observed that one twin pair did not develop clinical EAE within the
150 d observation period, while in the six twin pairs that did develop EAE no
statistically significant effect was observed at the group level. However, at the
individual twin level we observed that with a fast disease evolution in three twins the
treatment had a clear clinical effect, while no effect of the mAb was observed in three
twins with a slowly evolving disease. The lack of statistical significance at the group
level made publication of the data highly problematic.
How should we interpret this experiment? Should further development of the mAb be stopped
because a significant effect of the treatment could not be proven, even when the number
of animals per group is doubled or tripled? Or might the mAb be clinically relevant for a
subset of the patients, namely those with fast disease progression? This is not a
theoretical issue as patients variation in the response to treatment (with
interferon-β) has been also observed in MS (Axtell et al., 2010).
The prisoner's dilemma here is that when there are no markers for disease progression
rate that can be used for preselection of high responder animals, the only way to achieve
statistical significance is increasing the group size. This obviously creates a conflict
with the reduction principle.
There seems to be no easy solution for the apparent conflict between (statistical)
significance and (clinical) relevance for the highly refined EAE models. One solution
could be that reviewers of grants and publications on proof of concept studies choose not
to apply the normal sample size dogmas developed for homogeneous models, such as those in
inbred/SPF mice, to studies in more complex and more heterogeneous disease models in
nonhuman primates (Bacchetti et al., 2011, 2012). The central argument is that the value
of a study does not increase proportionally with the number of animals added to a test
group, while the burden from discomfort on study participants does increment
proportionally (Bacchetti et al., 2005).
Concluding remarks and open questions for further research
A final relevant question is whether insights that we gained through studies
of the marmoset EAE model may help explain the disease course depicted in
Fig. 4b. I posit the concept that the course of EAE in marmosets is driven by
post-translational modifications of the immunodominant myelin component MOG.
From tolerance to autoimmunity. We showed that MOG in the healthy brain
maintains homeostasis via the interaction of its glycan epitope attached at the
asparagine (Asn/N) 31 residue with the C-type lectin receptor DC-SIGN, which is expressed
on microglia and dendritic cells in the draining (cervical and lumbar) lymph nodes.
Modification of this glycan, which may either have a genetic or a pathological cause
(inflammation for example) uncovers the pathogenic role of MOG (Garcia-Vallejo et al.,
2014). Studies in the marmoset EAE model show that nonglycosylated rhMOG protein or MOG
peptide are strongly immunogenic and encephalitogenic. It is therefore proposed that
autoimmunity against MOG is induced when the protein loses its tolerogenic glycan
epitope.
From autoimmunity to relapsing disease. We identified two epitopes, MOG24-36
(CD4+ T cells) and MOG40-48 (CD8+ T cells). A recent study in mice may help
understand why these two epitopes are immunodominant. It was shown that epitopes
containing an SP or PP motif are destroyed in the thymus by the serine protease TSSP
(Serre et al., 2017). MOG contains three SP motifs: residues 27 and 28, which are located
in the CD4 epitope; residues 42 and 43, which are located in the CD8 epitope and residues
123 and 124, which have not been located in a known T cell epitope in marmosets, although
it is a T cell epitope in B6 mice (MOG119-128) (Shetty et al., 2014). Thus, the two
marmoset T cell epitopes contain an SP motif, which may implicate their destruction in
thymic epithelial cells, as a consequence of which T cells against these specificities
escape negative selection from the immune repertoire.
A second relevant post-translational modification of myelin induced under inflammatory
conditions is the replacement of arginine residues by citrulline, a process indicated as
citrullination. Enzymatic citrullination is mediated by peptidyl-arginine deïminase
(PAD) enzymes, which exist in five isoforms (PAD1, 2, 3, 4/5 and 6); PAD2 is the most
prevalent in the CNS (Vossenaar et al., 2003). Full activity of PADs depends on local
[Ca2+]. The replacement of positively charged arginine residues in
peptides/proteins by neutrally charged citrulline can affect the conformation and
function of proteins. It has been shown that the proportion of citrullinated MBP is
substantially higher in the MS brain than in healthy tissue (45 % vs. 18 %),
while in Marburg's acute MS as much as 90 % of MBP is citrullinated (Moscarello et
al., 1994; Wood et al., 2008). In the early phase of MS, and at a young age of the
patient, only a minor fraction of MOG is citrullinated. It can thus be envisaged that in
the early phase of MS the immune system sees only a low citrullination grade of myelin
antigens.
Data obtained in the rhMOG/IFA model show that the immune response against the
noncitrullinated protein is restricted to pro-inflammatory CD4+ T cells against
MOG24-36 and antibodies against the conformational epitope located at the apical end of
monomeric MOG. The combined activity of these factors mediates the initial autoimmune
attack on the myelin sheaths in the white matter. As oligodendrocytes are spared,
remyelination can occur. T-cell reactivity against MOG34-56 is not induced, which is
probably due to the destructive processing of the peptide by the endolysosomal serine
protease cathepsin G. Conceptually, once the autoimmune inflammation is strong enough to
exceed a clinical threshold, neurological deficits are detectable (relapse), which are
antagonized by counter-regulatory mechanisms that suppress T cell inflammation, such as
Treg cells, corticosteroids and anti-inflammatory cytokines (TGFβ, IL-10). We
posit that the recurrent activation and suppression of this autoimmune–inflammatory
pathology underlies the relapsing–remitting course. An important finding has been the
discovery that engagement of T cells against the MOG34-56 peptide strongly accelerates
EAE development (Kap et al., 2008). Intriguingly, blockade of the IL-7 receptor with an
anti-human CD127 MoAb abrogated accelerated EAE development (Dunham et al., 2016). This
resembles the situation in MS where IL-7-responsive T cells specific for MOG34-56 were
identified as one of six specificities distinguishing MS patients from healthy controls
(Bielekova et al., 2004).
In silico 3-D modeling of human MOG (Fig. 10) shows that the sequence 34–56 is exposed
at the surface of the molecule. The thee Arg residues are highlighted, showing that the
Arg46 and Arg52 residues are freely accessible, while the Arg41 residue is
somewhat buried. Citrullination of the Arg46 residue, which is relatively more surface
exposed than Arg41 is sufficient for the association of the peptide with
autophagosomes in APCs. The ensuing protection of the peptide against fast degradation
warrants availability of the epitope for cross-presentation via Caja-E to CD8+CD56+
CTL. This CTL activation requires the involvement of LCV-infected B cells, which possess
mechanisms (autophagy, citrullination) to protect the MOG40-48 epitope against fast
degradation by cathepsin G and cross-presentation. In this phase of the disease both
CD4+ and CD8+ T-cell responses can be measured. Via the CTL a novel pathogenic
pathway is activated, which mediates an attack on the cortical grey matter.
From relapsing to progressive disease. Literature data suggest that with the
progression of time (and disease severity) the proportion of myelin protein that is
citrullinated increases (Moscarello et al., 1994). By the citrullination, T-cell
recognition of the antigen is disturbed, while the tendency to spontaneously form
amyloid-type aggregates increases. Citrullination of the Arg52 residue appears to be
particularly important for aggregation as this modification stabilizes the β
sheets within the encephalitogenic MOG34-56 peptide. This feature activates a third
pathogenic mechanism in which LCV-infected B cells have a central, albeit
non-immunological, role. The available data, albeit still scarce, suggest that
EBV-infected B cells have a role in aggregate formation and in the packaging of
aggregates in vesicles. The aggregates seem to induce apoptosis in cocultures of
EBV-infected B cells and MOG-reactive T cells. However, it still remains to be proven
whether aggregates secreted by the EBV-infected B cells are toxic for CNS cells.
Nevertheless, we believe that the data provide a potentially relevant concept for
progressive MS.
Open questions. The marmoset EAE model is now regarded by many as the
translationally most relevant animal model of MS. Important aspects of the model are the
clear pathogenic role of B cells infected with the lymphocryptovirus CalHV3. However, it
is still an open question of whether the mechanisms uncovered in the model are relevant
for the human disease.
The EAE model in marmosets is pathologically characterized by lesions in the white and
grey matter of the brain and the spinal cord. The model also revealed that lesions in
white and grey matter are induced via distinct pathogenic mechanisms. Also, the question
of whether the mechanisms that we have explored are translatable to MS is still open.
An intriguing new finding is that toxic amyloid-like aggregates are formed in the
interaction between citrullinated MOG34-56 peptide with EBV-infected B cells. It is still
an open question as to whether these aggregates are secreted by the B cells, in which
form this occurs and whether the aggregates are toxic towards neurons and glia cells.
Data availability
The author of this monography has retired and has no access to primary data. Primary data on the pathological characterization of the model can be found in reference 't Hart et al. (1998). Primary immunology data of the model can be found in references Brok et al. (2000), Kap et al. (2008) and Jagessar et al. (2010, 2012d). Papers presenting primary data on the pathogenic role of EBV-infected B cells are Jagessar et al. (2013a), (2016) and Morandi (2017b) and Araman et al. (2019).
Competing interests
The author is a member of the editorial board of Primate Biology. No other competing interests are reported.
Acknowledgements
The author would like to thank Francesca van Hassel for the artwork.
Review statement
This paper was edited by Eberhard Fuchs and reviewed by Luca Massacesi and Che Serguera.
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