PBPrimate BiologyPBPrimate Biol.2363-4715Copernicus PublicationsGöttingen, Germany10.5194/pb-3-9-2016The common marmoset (Callithrix jacchus): a relevant preclinical model of human (auto)immune-mediated inflammatory disease of the brain't HartBert A.DunhamJordonJagessarS. AnwarKapYolanda S.Department Immunobiology, Biomedical Primate Research Centre, Rijswijk, the NetherlandsBert A. t'Hart (hart@bprc.nl)12February2016319225November20157January201611January2016This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://pb.copernicus.org/articles/3/9/2016/pb-3-9-2016.htmlThe full text article is available as a PDF file from https://pb.copernicus.org/articles/3/9/2016/pb-3-9-2016.pdf
The increasing prevalence of chronic autoimmune-mediated inflammatory disorders (AIMIDs)
in aging human populations creates a high unmet need for safe and
effective medications. However, thus far the translation of pathogenic
concepts developed in animal models into effective treatments for the
patient has been notoriously difficult. The main reason is that currently
used mouse-based animal models for the pipeline selection of promising new
treatments were insufficiently predictive for clinical success. Regarding
the high immunological similarity between human and non-human primates (NHPs),
AIMID models in NHPs can help to bridge the translational gap between
rodent and man. Here we will review the preclinical relevance of the
experimental autoimmune encephalomyelitis (EAE) model in common marmosets
(Callithrix jacchus), a small-bodied neotropical primate. EAE is a generic AIMID model
projected on the human autoimmune neuro-inflammatory disease multiple
sclerosis (MS).
Introduction
Aging Western societies are facing an increasing prevalence of chronic
(auto)immune-mediated inflammatory diseases (AIMIDs) for which an effective
treatment is either lacking or associated with detrimental side effects.
This challenging situation creates a need for translationally relevant
animal models in which the (immuno)pathogenesis of AIMID can be investigated
and in which safe and effective treatments can be developed. At the same
time there has been an impressive development in the field of biotechnology,
which now enables the transformation of biological molecules into highly
specific therapeutic bullets, monoclonal antibodies (mAbs) for example, with
which pathogenic cells and molecules can be eliminated. However, although
several biological drugs are now successfully used in the clinic, there is a
long list of promising candidates which failed to reproduce positive
effects observed in animal models when they were tested in patients.
Furthermore, several clinically approved therapeutics are plagued with
detrimental side effects.
One of the recognized causes for this frustrating situation is the poor
predictive validity for safety and efficacy of the animal models used in
preclinical research. By far the largest proportion of preclinical research
in AIMID is based on a limited number of inbred and specific pathogen-free (SPF)-bred rodent strains.
While rodent models are very useful in terms of reliability and
reproducibility of the models, relatively low costs, the plethora of
available reagents for research, and the availability of genetically modified
substrains, the shortcomings of laboratory rodents as preclinical AIMID
models are a clear handicap. Due to the lack of genetic diversity and
exposure to environmental pathogens, the immune system of inbred SPF mice is
immature and poorly comparable to the robust pathogen-educated human immune
system (Davis, 2008).
We postulate here that the translational gap between disease models in mice
and the corresponding human disease can be bridged by using non-human
primate (NHP) disease models. The focus of this publication will be on
experimental autoimmune encephalomyelitis (EAE), which models autoimmune
pathogenic mechanisms of the human neuro-inflammatory disease multiple
sclerosis (MS) and has been well established in common marmosets
(Callithrix jacchus) (Vierboom et al., 2010; 't Hart et al., 2004a).
Pros and cons of the marmoset as a model of human autoimmune disease
There are some challenging limitations to usage of the marmoset for
immunological research:
The small body size (±400 g at adult age) precludes the sampling
of large blood volumes. As a rule of thumb, the maximum volume that can be
collected per month without harming the well-being of the animals equals
1 % of the bodyweight.
As the marmoset is not an established model for immunological research,
reagents are scarce. In the past 15 years we have compiled a useful set of
reagents and methods for immunological research in the marmoset (Jagessar et al., 2013a).
The relatively long time (1.5–2 years) to reach sexual maturity compared to
rodent species – together with the special housing, handling, and dietary
needs of the marmose – increase costs of research substantially.
Facility requirements limit the ability of many research institutes to
perform research using the marmoset. Additionally, animal handlers require
specialized training.
Regulatory restrictions due to the NHP status of the marmoset, and welfare
considerations, are a serious limiting factor in research performed. These
limitations can be overcome by careful study design and daily veterinary care.
There are also several clear advantages of this model:
Despite the small body size (Jagessar et al., 2013a) it is possible to obtain meaningful
longitudinal information about the monkey's immune status by using
micromethods detailed elsewhere (Jagessar et al., 2013).
The marmoset is a friendly animal that adapts well to experimental
handlings, without the need of sedation, and responds well to training
procedures designed for testing cognitive function.
Marmosets usually give birth to two or three non-identical siblings. During
the in utero development the placental blood stream of twin siblings is fused,
which implies that they have a chimeric bone marrow (Haig, 1999; Picus et al., 1985). The ensuing
stable allo-tolerance creates the possibility to adoptively transfer immune
cells between fraternal siblings. Moreover, as the immune systems of such
twins have been educated in the same thymic environment, they are highly
similar. This chimeric bone marrow status is highly advantageous for
placebo-controlled studies designed to test potential therapeutics as the
fraternal sibling can be used as an optimal control. Such a paired approach
reduces the number of monkeys needed for research.
Marmosets in captivity can reach a high age, above 20 years. It is unknown,
however, whether the immune system ages in the same way as the human immune
system. Importantly, marmosets are naturally infected with similar viruses
to humans, including those related to viruses implicated in the aging of
the human immune system, namely the β-herpesvirus CMV (cytomegalovirus) and the
γ-herpesvirus EBV (Epstein–Barr Virus) (Nigida et al., 1979; Cho et al., 2001).
Just like humans, marmosets held in captive colonies under conventional
conditions have a robust pathogen-educated immune system, which comprises
specificities engaged in the control of chronic latent herpesvirus
infection. We will discuss in this publication that the marmoset versions of
CMV and EBV have a central role in the pathogenesis of MS-like disease in marmosets.
The need to formulate the immunizing antigen with strong immune stimulators,
such as the complete Freund's adjuvant (CFA), for reproducible induction of
AIMID in laboratory animals raises concerns. CFA is notorious for the
adverse effects, which cause serious discomfort to the animals
(Billiau and Matthys, 2001). Moreover, CFA causes skewing of the T cell response to
the immunizing (auto)antigen into a pro-inflammatory profile that is
dominated by Th1 and Th17 cells. The thus far-reaching disappointing effects in MS
therapies targeting these subsets indicate that CFA-based EAE models may be
less representative for MS. We recently discovered that full-blown clinical
EAE in marmosets can be induced with myelin antigen formulated with the much
less detrimental incomplete Freund's adjuvant (IFA) (Jagessar et al., 2010, 2015). IFA lacks
the mycobacteria that cause the adverse effects.
Multiple sclerosis (MS)
MS is an inflammatory/demyelinating disease of the human central nervous
system (CNS). In the vast majority of patients (±85 %) the disease
follows initially a relapsing–remitting course characterized by alternating
episodes of disease activity and recovery. In about 50% of these
relapsing–remitting MS (RRMS) cases and after a variable period of time the
disease worsens progressively and remissions are less frequently observed,
i.e., secondary progressive disease (SPMS). In 15 % of the patients the
disease is progressive from the onset, i.e., primary progressive disease (PPMS)
(Dendrou et al., 2015). The cause of MS is unknown, but, once
established, disease evolution is driven by the combined activity of
autoreactive T and B cells specific for components of the myelin sheaths
that wrap around axons in the CNS white matter. Genome-wide association
studies and the beneficial effect of immunomodulatory therapies support a
central pathogenic role of the immune system. However, immune-modulatory or
immune-suppressive therapies mainly have an effect in the
relapsing-remitting phase of MS; it is therefore believed that the immune
system exerts its main pathogenic functions during this phase and is less
important for the progressive phase.
It is generally believed that autoimmunity in MS is caused by the
interaction of genetic and environmental risk factors. Low vitamin D, EBV
infection, and smoking are all widely considered environmental risk factors
associated with MS (Ascherio and Munger, 2007a, b). Genome-wide association studies have shown
that more than 100 genes contribute to the genetic susceptibility (Sawcer et al., 2011).
The strongest genetic influence is exerted by the major histocompatibility
complex (MHC) class II region. The individual contribution of the other
genes, which all encode an immunological function, is more modest. MHC II
molecules are expressed on antigen-presenting cells (APCs) and present
antigens to CD4 +T cells. Although this association is suggestive for a
core pathogenic role of CD4 +T cells in MS, therapies targeting this cell
type lack convincing efficacy in RRMS (see below).
Despite decades of intensive research a viral or bacterial trigger, which
withstands Koch's postulates, has not been found. There is strong
evidence for a pathogenic role of EBV infection, but the discrepancy between
the high prevalence of EBV infection in the human population (> 90 %)
and the low incidence of MS (±0.1 %) is unexplained
(Ascherio et al., 2012). The risk for MS increases 2–3-fold with
development of infectious mononucleosis compared to EBV-seropositive people,
and an increased IgG1 titer against EBV proteins EBNA-1 is predictive of
disease exacerbation (Ascherio and Munger, 2015). MS development
is virtually not seen in EBV-seronegative individuals (Pakppor et al., 2013), providing
further evidence of a link between EBV and MS. Critical to this discussion,
and as previously mentioned, is the fact that marmosets are naturally
infected with an EBV homolog (CalHV3). We will discuss in following
paragraphs data obtained in the marmoset EAE model, which may provide a
mechanistic explanation of the enigmatic association between MS and EBV infection.
The uncertainty about an external cause of MS has led others
(Stys et al., 2012) and us ('t Hart er al., 2009) to propose that
the cause may be inside the CNS. Our “response-to-injury” concept of MS has
been inspired by the old “primary lesion hypothesis” proposed by Wilkin (1989).
According to this hypothesis, “autoimmunity is a genetically predisposed hyper-response
to self antigens released by tissue injury (= the primary lesion) due to an antecedent pathogenic event”. We will
explain in the following sections that unique aspects of the marmoset model
provide the opportunity for testing hypotheses on the enigmatic trigger of
the primary lesion and the ensuing autoimmune reactions.
Modeling the cause of the primary lesion
It is generally assumed that the pathogenic event that elicits MS predates
the presentation of neurological deficits for many years. This obviously
complicates the identification of that elusive event. However, in a recent
publication remarkable observations were reported that may provide useful
insights ('t Hart, 2015): (1) while MS has always been rare in Japan, the annual
incidence curve has dramatically increased since World War II. This
increment has been attributed to the introduction of the Western diet
(Yamamura and Miyake, 2013). (2) MS seems to affect only humans. Even our closest living
relatives, chimpanzees and bonobos, are not affected by the disease despite
the high genetic similarity and the fact that they are naturally affected
with similar pathogens to humans. This suggests that MS is due to a
pathogenic condition that is only present in humans.
Analysis of the earliest pathological changes in the myelin of MS patients
(called pre-active lesions; van der Valk and Amor, 2009) revealed aggregates of
microglia cells around abnormal axons, called microglia nodules
(Singh et al., 2013). At the electron microscopic level demyelination
seems to start with dissociation of the inner myelin lamellae from the axon
(Rodriguez and Scheithauer, 1994). This has led to the concept that the primary MS
lesion may start as instability of axon–myelin units
(Stys et al., 2012). One of the consequences of the dissociation of
myelin from axons is that the trophic support of axons with essential
monocarboxylate nutrients (lactate, pyruvate) is disturbed. This inevitably
results in the degeneration of axon and neuron (Saab et al., 2013; Simons et al., 2014).
Experiments in mice have shown that the interaction of myelin-associated
glycoprotein (MAG) with gangliosides on the axon surface is essential for
the stability of axon–myelin units (Schnaar, 2010; Schnaar et al., 2014). MAG is a sialic-acid-binding
lectin (Siglec-4) with high specificity for N-acetylneuraminic acid (Neu5Ac)
(Collins et al., 1997). Neu5Ac is the substrate for another
prevalent sialic acid, N-glycolylneuraminic acid (Neu5Gc). Interestingly,
expression of the Neu5Ac into Neu5Gc-converting enzyme,
CMP-N-acetylneuraminic acid hydroxylase (CMAH), is suppressed in the CNS,
suggesting that expression of Neu5Gc in the brain and spinal cord is
detrimental for homeostasis (Davies and Varki, 2013).
Varki et al. reported a genetic deficiency of the enzyme CMAH in humans that
is not present in hominoid apes (Varki, 2001). Due to this deficiency humans are
unable to synthesize Neu5GC anywhere in their whole body, while chimpanzees
produce it in all body cells except the CNS. Neu5GC is, however, present in
abundance in the red meat of lifestock (cow, pig, goat), which is consumed
in large quantities via the Western diet, but it is absent in fish, poultry
and vegetables (Arnon et al., 1996; Wang, 2009). Intriguingly, the steep increase of MS
prevalence in Japan after World War II has been attributed to the
replacement of the Neu5GC-poor traditional Japanese food by the Neu5GC-rich
Western diet (Yamamura and Miyake, 2013). Experiments in CMAH-deficient mice have shown that
dietary Neu5Gc is incorporated in the gut flora and in the glycocalyx of
various body tissues (Taylor et al., 2010). The dietary modified gut flora induces the
production of heterophilic antibodies, which can bind Neu5Gc incorporated in
tissue glycocalyx and in this way interfere with cellular interactions and
communication (Taylor et al., 2010; Samraj et al., 2014).
The questions whether dietary Neu5Gc is incorporated also in the
gangliosides of the CNS and whether peripheral anti-Neu5Gc antibodies gain
access to the CNS and bind thus-modified gangliosides have not been
resolved. Interestingly, the common marmoset has the same genetic CMAH
deficiency as humans (Springer et al., 2014) and therefore provides an exquisite model to
examine this unexplored ethiogenic mechanism in further detail.
Modeling response to myelin injury
Studies in rats where CNS white-matter injury was inflicted via a cryolesion
have shown that myelin debris can be found within CNS-draining lymphoid
organs (cervical and lumbar lymph nodes and the spleen) (Philips et al., 1997). Similar
observations have been reported for MS (Fabriek et al., 2005) and for EAE-affected mice
(van Zwam et al., 2009a) and marmosets (de Vos et al., 2002). Moreover, surgical removal of cervical and
lumbar lymph nodes, which drain the spinal cord, impairs chronic relapsing
EAE development in Biozzi ABH mice (van Zwam et al., 2009b). These
finding support the concept that (under certain conditions) myelin released
from injured CNS can be a trigger of encephalitogenic immune reactions.
We have analyzed the response of the pathogen-educated immune system of
marmosets to CNS myelin from an MS patient in great detail. Marmosets
immunized with MS myelin formulated with CFA developed a chronic
neurological disease that resembles MS in clinical and neuropathological
presentation ('t Hart et al., 1998). Just like in MS, we observed
with T2-weighted MRI formation of focal hyperintense regions, which at
histological examination represented focal areas of primary demyelination,
characterized by variable degrees of inflammation, axonal pathology,
astrogliosis, and even remyelination (Fig. 1). In later studies others and we
observed that MS-like lesions were also present in the cortical grey matter,
a phenomenon usually not observed in rodent EAE models (Pomeroy et al., 2008; Kap et al., 2011b). Indeed,
the lack of cortical grey-matter pathology observed in the MS patient is a
major pitfall of rodent EAE models. Currently research is being undertaken
for more in-depth pathological characterization of the marmoset EAE model.
Preliminary results indicate that, in addition to grey-matter pathology, many
of the desired pathological features of MS, not reflected in rodent EAE, are
replicated in the marmoset EAE models (Dunham et al., 2016).
Brain lesions visualized with MRI
The anatomy of the marmoset brain, including the grey / white-matter ratio,
resembles the human brain. This is especially important for studies on
diseases associated with white- and grey-matter pathology. Magnetic resonance
imaging is the imaging technique of choice for the visualization of MS
pathology in the brain and spinal cord and is an accepted primary outcome
measure in clinical trials of new therapies (Barkhof et al., 2012). The most commonly
applied T2-weighted MRI technique, which we also used in our first studies,
is highly sensitive to altered tissue distribution of water, vasogenic
edema for example (Fig. 1a). Figure 1b shows usage of the same technique for
longitudinal imaging of brain lesion development. In later years more
specific imaging techniques were developed for scanners with higher field
strength, such as the 4.7T lab animal scanner at the Imaging Center in
Utrecht, the Netherlands. Figure 1c shows techniques used for a quantitative
description of the in vivo detectable brain pathology developing in the marmoset
EAE model ('t Hart et al., 2004b). Quantitative MRI techniques are ideal
for determining the effect of a new therapeutic mAb on ongoing disease
('t Hart et al., 2006).
Modeling the T cell response to injury
The high response of the pathogen-educated immune system of marmosets to CNS
myelin and the multi-factorial immune reactions that underlie the
neurological problems have been examined in great detail
('t Hart et al., 2015). Myelin is a complex tissue composed of (glyco)lipids,
lipoproteins, proteolipids, and (glyco)proteins. We could show by
immunization of Biozzi ABH mice (Smith et al., 2005) and marmosets
(Jagessar et al., 2008) with myelin from mice lacking myelin
oligodendrocyte glycoprotein (MOG) that this quantitatively minor
constitutent (< 1 % of the protein fraction) is essential for the
evolution of chronic EAE.
Magnetic resonance imaging of brain lesions in marmoset experimental
autoimmune encephalomyelitis (EAE). (a) Marmosets were immunized with human myelin in CFA. The depicted case was
sacrificed at the height of the disease with serious clinical deficits. The
brain was carefully removed, briefly fixed with 4 % buffered formalin and
MRI-scanned (T2-weighted, 4.7 T) to detect the spatial distribution of
lesions. Lesions are indicated with capitals (a, b). Stainings of individual
lesions with Klüver–Barrera for myelin are depicted in (c)–(f). (b) The brain
of a single marmoset in which EAE was induced with rhMOG/CFA was
longitudinally scanned (T2-weighted, 4.7 T) to illustrate the dissemination
of lesion formation in time and space. In the insert the time after
immunization (psd: post-sensitization day) is given. (c) Different imaging
modalities were developed for in vivo characterization of lesions: T2-weighted
images for high contrast anatomy, T1 and T2 relaxation time (RT) images to
quantify changes in the signal intensities associated brain injury and
inflammation, magnetic transfer ratio (MTR) images which provide essentially
the same information and finally images created by subtracting T1-RT images
generated before and 10 min after infusion of the paramagnetic contrast
agent gadolinium-DTPA. This modality visualizes lesions with a leaky blood–brain
barrier (BBB) caused by inflammation. The message of this figure is
that a lesion first visible as a small region of BBB leakage appears 10 days
later as a large hyperintense region in the T2 and MTR images.
MOG is only present in the CNS, where it is expressed as a homodimer on the
surface of oligodendrocytes and myelin sheaths. The likely natural function
of MOG is maintenance of an anti-inflammatory brain milieu. This role
depends on the interaction of the fucosylated glycan residue, which is
linked to the asparagine residue at position 31 of the extracellular domain,
with the C-type lectin receptor (CLR) DC-SIGN. DC-SIGN is surface-expressed
on resident APCs (microglia) and APCs within the CNS draining cervical and
lumbar lymph nodes (Garcia-Vallejo et al., 2014). Thus, as a glycosylated protein MOG is
tolerogenic, while as a non-glycosylated protein MOG is strongly
immunogenic. The latter is illustrated by the recently reported observation
that immunization of marmosets with recombinant human MOG (produced in
E. coli) formulated with IFA induced full-blown clinical EAE (Jagessar et al., 2015).
We used a panel of synthetic 23-amino-acid-long peptides overlapping by 10
derived from the extracellular domain of human MOG (residues 1–125) to probe
the T cell response profile in a genetically heterogeneous population of
rhMOG-sensitized marmosets (Brok et al., 2000; Kap et al., 2008). The same panel had been used for
probing the T cell repertoire of MS patients. The reactivity patterns that
we found in EAE-affected marmosets were remarkably similar to the reactivity
patterns found by others in MS patients (Kerlero de Rosbo and Ben-Nun, 1998). We observed
in monkeys immunized with rhMOG/CFA T cell reactivity against two
immuno-dominant T cell epitopes in rhMOG, which are juxtaposed in a
highly conserved part of the extracellular domain, residues 24–36 and 40–48.
In marmosets immunized with rhMOG/IFA only T cell reactivity against two
overlapping peptides that define the first epitope could be detected, namely 14–36
and 24–46 (Jagessar et al., 2015). Neither T cells nor antibodies reacting against the
MOG34–56 peptide could be detected. However, marmosets immunized with
MOG34–56 peptide in IFA developed full-blown clinical EAE, characterized by
MS-like pathology in the white and the grey matter (Jagessar et al., 2010). This observation
demonstrates that MOG40–48 specific T cells are present in the normal
repertoire. Apparently these highly pathogenic T cells were not activated
when the monkeys were immunized with the full-length rhMOG protein in IFA.
Where could these effector memory T cell specificities originate from?
In the classical EAE model induced with rhMOG in CFA, CD8 + ve T cells
against the MOG40–48 epitope were found to emerge late in the disease, in
association with the expression of clinical signs (Kap et al., 2008). This finding
indicates pathogenic dominance of this epitope. We further characterized
these late-appearing T cells in marmosets immunized with peptide MOG34–56
formulated with IFA, in which clinical signs and widespread demyelination of
white and grey matter developed without the induction of myelin-binding
autoantibodies (Jagessar et al., 2010). It was shown that the T cells are specific for the
MOG40–48 epitope, which is presented via MHC class I/Caja-E molecules; that
the responding T cells have an NK–CTL (natural killer–cytotoxic T lymphocyte) phenotype (CD3 + CD8 + CD56 +); and
that they display cytotoxic activity against EBV-infected B cells pulsed
with MOG34–56 (Jagessar et al., 2012a). This type of T cells has been found in humans in the
repertoire of effector memory T cells that keep chronic latent infection
with CMV under control (Pietra et al., 2003). Interestingly, we also
found that the MOG40–48 epitope shares high sequence similarity with a
peptide encoded in the UL86 ORF of human CMV and that T cells raised against
the CMV peptide cross-react with MOG34–56 (Brok et al., 2007). As marmosets are naturally
infected with a human CMV-related β-herpesvirus, it is tempting to
speculate that the effector memory T cells that cause MS-like disease in
marmosets immunized with MOG34–56/IFA originate from the anti-CMV repertoire.
In the EAE model induced with rhMOG in CFA, the sequence 24–36 was defined
as the specific epitope of MHC class II/Caja-DRB*W1201 restricted T helper
(Th) 1 cells (Brok et al., 2000). The epitope contains at the Asn31 residue the N-linked
glycosylation site where the fucosylated glycan epitope of DC-SIGN is
attached (Johns and Bernard, 1999). Regarding the putative role of MOG–DC-SIGN
interaction in the control of tolerance and autoimmunity (Garcia-Vallejo et al., 2014; Garcia-Vallejo and van Kooyk, 2009), it is
well possible that the anti-MOG24–36 Th cells detected in the rhMOG/IFA
model are effector memory regulatory T (Treg) cells present in the normal
repertoire. In the presence of danger signals from mycobacteria in CFA these
might have been skewed towards a pro-inflammatory Th1 function. Indeed, in
marmosets immunized with the long MOG20–50 peptide, which contains both
epitopes, formulated with IFA EAE development was suppressed. In these
monkeys, T cell and antibody reactivity against the MOG24–36 epitope was
clearly detectable, while reactivity against the MOG40–48 epitope was
suppressed (Jagessar et al., 2015). By contrast, in monkeys immunized with a mixture of the
MOG14–36 and MOG34–56 peptides in IFA, clinical EAE developed and T cell
reactivity against both peptides was detectable. This experiment hints at
the possibility that Treg cells re-activated by the MOG24–36 epitope may
control the activation of CTL against MOG40–48 via linked suppression (Fig. 2).
Putative interaction of the immunodominant MOG T cell epitopes.
To test the possible interaction of the two MOG T cell epitopes 24–36
and 40–48, we immunized marmoset twins twice (psd 0 and 28) with the long peptide
MOG20–50 in which both epitopes were physically linked or with an equimolar
mixture of the two short peptides MOG14–36 and MOG34–56 in which the two
epitopes were on separate molecules (non-linked). All monkeys were
challenged twice (psd 56 and 84) with an encephalitogenic dose of MOG34–56.
The adjuvant was in all cases IFA. All twin siblings (3/3) immunized with
the non-linked epitopes developed clinically evident EAE, while two of
three siblings immunized with the linked epitopes were resistant to EAE. The
message of this figure is that T regulator cells (Tr1) specific for MOG24–36
and T effector CTL may need to interact during their reactivation by the same APC.
EAE development in marmosets involves two pathways
The findings discussed thus far led us to postulate that the core pathogenic
process in myelin-immunized marmosets involves two pathways, which develop
sequentially but can also be activated separately ('t Hart et al., 2011) (Fig. 3).
The EAE initiation pathway: the observation that marmosets immunized with
MOG-deficient myelin develop acute EAE warrants the conclusion that EAE can
be initiated independent of autoimmunity against MOG. Indeed, clinical EAE
with small-sized lesions of a mainly inflammatory nature that resembled
those found in the MBP-induced Lewis rat model could be induced by
immunization with MBP or PLP in CFA (Massacesi et al., 1995). In these
models Th1 cells are activated, but demyelination is not induced unless
anti-MOG antibodies are present (Genain et al., 1995; McFarland et al., 1999). Similarly, EAE initiation in
the rhMOG/CFA model initially involves Th1 cell activation and induction of
anti-MOG antibodies (Brok et al., 2000).
B cells contribute to the initiation pathway by the production of
autoantibodies that bind and opsonize CNS myelin. The immune complexes
elicit myelin injury via complemental and/or macrophage-mediated cytotoxicity
reactions (ADCC, CDC) (Noseworthy et al., 2000). The observation that sera
from marmosets immunized with MOG-deficient myelin do not contain
myelin-binding IgG specificities shows that MOG is an important primary
target of the antibody opsonization (Jagessar et al., 2008). Antibodies
capable of opsonizing myelin bind conformational epitopes (Jagessar et al., 2015; Menge et al., 2007). It
has been shown that adoptive transfer of anti-MOG antibodies into
MBP/CFA-sensitized marmosets induces widespread demyelination of
inflammatory lesions (Genain et al., 1995).
The EAE perpetuation pathway: for the progression of EAE, autoimmunity
against MOG is essential. NK–CTLs against the MOG epitope 40–48 seem to play
an important role in late-stage disease. These T cells can be induced by
immunization of marmosets with MOG peptide 34–56 in IFA (Jagessar et al., 2012a), a condition
where myelin-binding antibodies are not formed (Jagessar et al., 2015). The pathogenic role
of these cytotoxic cells in MS is not exactly clear. It is of considerable
interest, however, that a similar type of T cells, albeit specific for another
myelin antigen (MBP), has been found in MS lesions, where they were found
engaged in the killing of HLA-E+ ve oligodendrocytes (Zaguia et al., 2013).
In the EAE perpetuation pathway B cells have en essential pathogenic role,
but not via the production of autoantibodies. Instead B cells have an
essential role in the activation of the autoaggressive CTLs that cause direct
demyelination ('t Hart et al., 2013).
Validation of the marmoset EAE model with clinically relevant immunotherapies
The evolutionary distance between marmosets and humans has been estimated at
±55 million years. This relative proximity in comparison with mice is
reflected by the relatively high cross-reaction of mAbs raised against CD
markers of human leukocytes with leukocytes of marmosets (Jagessar et al., 2013a; Brok et al., 2001). This
makes the marmoset a potentially useful model for the safety and efficacy
evaluation of biological therapeutics, mAbs for example, which for
specificity reasons cannot be tested in lower species. The term “potentially
useful” is used here on purpose as cross-reaction of a test compound alone
is not a sufficient criterion for a translationally relevant preclinical
model of MS. The model will only be relevant for MS when the
immuno-pathogenic mechanisms resemble those in the human disease. One way to
find this out is to test the activity of treatments, which have proven
effective or not effective in mouse EAE or RRMS, also in the marmoset model.
Figure 4 gives a graphical representation of this strategy.
Treatments targeting T cells
Rodent EAE models show a central pathogenic role of CD4 +T cells. This
concept prompted a small-sized clinical trial in which the anti-human CD4
mAb MT-412 was tested in RRMS patients (van Oosten et al., 1997). The treatment induced
profound depletion of CD4 +T cells but nevertheless exerted no clear
effect on lesion activity detected with MRI, suggesting that CD4 +T cells
may have a different pathogenic role in RRMS than in the EAE model.
The two pathogenic pathways in the rhMOG/CFA-induced marmoset experimental
autoimmune encephalomyelitis (EAE) model. In all marmosets immunized with rhMOG/CFA, initiation of EAE involves the
activation of CD4 +T helper 1 cells specific for the epitope 24–36.
Activation of the T cells is restricted by the monomorphic allele
Caja-DRB*W1201, which all marmosets have in common. After a variable period
of time CD4 + CD8 + CD56 + cytotoxic T cells specific for the epitope 40–48
are activated; activation is restricted by the invariant non-classical MHC
class Ib allele Caja-E, which all marmosets have in common.
Validation of the marmoset experimental autoimmune encephalomyelitis (EAE)
model. Therapies showing promising clinical effects in the EAE model, of mouse and
marmosets alike, are tested in MS patients. When this forward translation
proves successful, it confirms the validity of the targeted pathogenic
process EAE model for MS. In the case of forward translation showing that
promising clinical effects observed in MS cannot be reproduced in MS, the
treatment should be re-evaluated in the EAE model to examine why the
targeted process seems irrelevant for MS.
The differentiation of CD4 +T cells into functionally polarized
pro-inflammatory subtypes (Th1 and Th17) is steered by two related
cytokines, IL-12 and IL-23. Both cytokines are heterodimers, composed of a
shared p40 subunit and a specific subunit, respectively p35 and p19.
Ustekinumab is a fully human mAb directed against the shared p40 subunit of
human IL-12/IL-23. The mAb binds the cytokines from marmosets but not from
lower species, precluding efficacy testing in rodent EAE models.
Unexpectedly, the mAb lacked clinical efficacy in RRMS (Segal et al., 2008), but it has
found a new clinical target in psoriasis and possibly inflammatory bowel
disease. In a first experiment marmosets immunized with human myelin in CFA
were treated with the mAb from 1 day before the immunization, in contrast to
RRMS, resulting in complete protection against
EAE (Brok et al., 2002). This encouraging finding prompted us to
test the mAb also in a clinically more relevant study design, namely
starting during the chronic phase of the disease ('t Hart et al., 2005). In brief,
marmosets immunized with rhMOG/CFA were subjected to T2-weighted brain MRI
to visualize white-matter lesions, as depicted in Fig. 1b. Once lesions of
sufficient size for quantification of inflammatory activity were detected,
treatment with the antibody or placebo was started. We observed that –
although the increase of T2 signal intensity, as a measure of inflammation,
and of the lesion enlargement, as a measure of demyelination, were
completely suppressed – the onset of clinical signs was only temporarily
delayed, similar to RRMS. It is tempting to speculate that the delayed
exacerbation of clinical signs, while MRI-detectable lesion inflammation
appears suppressed, is due to demyelination induced by the autoaggressive
CTLs, which drive the EAE perpetuation pathway (see above). Importantly,
these studies highlight the profound need for EAE study design to be as
clinically relevant as possible and further provide evidence that the
marmoset EAE model is a valid pre-clinical model with the correct study design.
The signature and immuno-active cytokine of Th1 cells secreted after
stimulation with IL-12 is interferon (IFN)γ. Clinical studies in
RRMS showed that administration of IFNγ exacerbated disease activity
(Panitch et al., 1987) while treatment with anti-IFNγ antibody had
only minor clinical effects. In the mouse EAE model both positive and
negative effects of IFNγ neutralization have been reported, often
depending on the timing of the treatment (Sanvito et al., 2010). We have tested
whether early (psd 0–25) or late (psd 56–81) treatment with IFNγ
would alter the EAE course in marmosets immunized with huMOG34–56 peptide in
IFA. We observed that neither of the treatment regimens had a positive
effect on the disease course, but the disease exacerbation observed in RRMS
was not seen. Remarkably,Th1-associated humoral and cellular autoimmune
parameters were affected (Jagessar et al., 2012b).
The signature cytokine of Th17 cells is IL-17A, but the pathogenic role of
the cytokine in EAE is unclear. In the rhMOG/CFA marmoset EAE model, IL-17A
is prominently expressed by the late-acting CTLs, which induce cortical
grey-matter demyelination and irreversible neurological deficit (Jagessar et al., 2010). We
tested a IgG4κ mAb raised against human IL-17A in the rhMOG/CFA
marmoset EAE model (Kap et al., 2011a). Treatment with the mAb was started 1 day
before the immunization and continued to the end of the experiment. The
antibody caused only a moderate delay of the disease onset.
IL-7 is a hemopoietic growth factor that is produced by stromal cells in
bone marrow and thymus and has a crucial role in lymphopoiesis. In the
periphery IL-7 is produced by a variety of cells, including APCs (dendritic
cells). IL-7 signals through CD127 for maintenance of T and B cell
homeostasis. Blockade of IL-7 binding to CD127 ameliorates disease in mouse
EAE models and is currently under evaluation in RRMS patients (personal
communication). Importantly, EBV-infected B cells, which are the requisite
APCs for the late-acting autoaggressive CTLs in the rhMOG/CFA marmoset EAE
model, produce high levels of IL-7. These findings prompted us to test a new
chimeric IgG1κ mAb against human CD127 in marmosets immunized with
MOG34–56/IFA (Dunham et al., 2016). Unexpectedly, we observed
a dichotomous clinical effect of the treatment. In twins with an early
disease onset, we observed a markedly prolonged asymptomatic period in the
mAb-treated sibling, whereas in twins with a late disease onset no evident
clinical effect was found. It is unclear how IL-7R blockade will perform in
the clinic, although it has been suggested that IL-7R may be an attractive
target for a subset of MS patients. Interestingly, we found in monkeys
responding to the treatment that the increase of circulating CD20 + CD40 +B cells
induced by the immunization was suppressed. This subpopulation was
also prominently depleted by the anti-CD20 mAb but spared in the monkeys
treated with anti-BlyS/APRIL (see below). This led us to postulate that
among the CD20 + veCD40 + ve Bcells there are the virus-infected APCs of the
autoaggressive CTLs.
Collectively, these observations corroborate the concept that the pathogenic
role of pro-inflammatory CD4 + Th1/Th17 cells is confined to EAE
initiation, whereas disease progression is driven by a different pathogenic
process. Nevertheless, several tested treatments failed to reproduce
promising effects observed in mouse EAE when they were tested in the
marmoset EAE model and validate the marmoset EAE model as a pre-clinical
model for MS. One explanation for these paradoxical effects may be that the
immune process most closely linked with the induction of neurological
problems is the activation of autoaggressive effector memory CTLs. These
cells may already have been committed to their pathogenic function and may
be refractory to immunomodulatory treatment. For this reason, we became
interested in the B cells that are responsible for the activation of the CTLs.
Treatments targeting B cells
CD20 is a broadly expressed surface molecule in the B cell lineage. Only
pro-B cells and terminally differentiated plasma cells, which are the main
producers of antibodies, do not express CD20. The collective results of
clinical trials with three mAbs against human CD20, one chimeric (rituximab)
and two fully human (ofatumumab and ocrelizumab), show a remarkably brisk
clinical effect, which seems to persist for months (Barun and Bar-Or, 2012).
However, as plasma levels of antibodies capable to mediate demyelination
were not affected, the exact working mechanism is not understood.
HuMab7D8 is a clonal variant of ofatumumab that is also fully human
(IgG1κ) and cross-reacts well with marmoset B cells. The mAb was
tested in the rhMOG/CFA marmoset EAE model starting well after the single
immunization (psd 21)
(Kap et al., 2010). The treatment induced a similar brisk
and persistent depletion of B cells to that observed in RRMS patients. Moreover,
robust and persistent suppression of clinical signs was observed. Detailed
examination of the effect on EAE pathology showed that B cells were also
eliminated from the brain, while the formation of MS-like lesions in the
white and grey matter of brain and spinal cord was markedly reduced (Kap et al., 2011b).
A clear difference with the RRMS clinical trial was that in the EAE model
plasma levels of autoantibodies binding MOG protein, potentially capable of
mediating demyelination, were suppressed. This prompted us to perform an
additional experiment in the marmoset EAE model induced with MOG34–56/IFA in
which MOG protein-binding antibodies are not formed. Also in this model we
observed a robust and persistent beneficial effect of anti-CD20 mAb, both on
the clinical signs and on the underlying pathology (Jagessar et al., 2012c). The inevitable
conclusion of this experiment is that B cells are directly involved in the
activation and/or function of the autoaggressive NK–CTLs that drive EAE
development in this model.
The survival and differentiation of B cells depend on the cytokines
BAFF/BlyS and APRIL (Jagessar et al., 2012). The two cytokines are physiologically active as
BAFF–BAFF or APRIL–APRIL homodimers or as BAFF–APRIL heterodimer. There are
three receptors for these cytokines expressed on B cells: BAFF-R, BCMA, and
TACI. BAFF-R is only bound by BAFF, while both homodimers bind to BCMA and
TACI, but only TACI binds the heterodimer. Regarding the impressive clinical
effect in RRMS of B cell depletion via anti-CD20 mAb, it was a reasonable
expectation that B cell depletion via the capture of BlyS/BAFF and/or APRIL
might also have a beneficial clinical effect. However, this was not observed
as a clinical trial in RRMS with atacicept; a recombinant fusion protein of
human TACI and IgG unexpectedly increased MRI-detectable lesion activity
despite the depletion of peripheral B cells (Kappos et al., 2014).
We used a somewhat different approach in the marmoset EAE model to replicate
these findings, namely using a mAb against human BAFF (belimumab) and a mAb
against human APRIL (Jagessar et al., 2012d). We observed that both mAbs caused only a
moderate, albeit statistically significant, delay of the disease onset in
marmosets immunized with rhMOG/CFA.
The reason for the unexpected paradoxical clinical effects of B cell
depletion via anti-CD20 or inactivation of BlyS or APRIL is not known but
seems not restricted to MS. Also in rheumatoid arthritis the anti-CD20 mAb
rituximab exerted a remarkable clinical effect, whereas atacicept and the
anti-BlyS mAb belimumab were only marginally effective (Dorner et al, 2010; Richez et al., 2014). A
possible explanation was found in the marmoset EAE model. We observed that
in monkeys treated with anti-CD20 mAb the copy number of CalHV3 DNA in lymph
nodes and spleen was strongly reduced while the copy number was increased in
monkeys treated with anti-BlyS or anti-APRIL mAb (Jagessar et al., 2013b). Our interpretation
of this finding is that B cells immortalized by the EBV-related γ-herpesvirus
CalHV3 may not need BAFF or APRIL for their survival and thus
ignore inactivation of the cytokines, while they express CD20 and can thus
still be depleted with anti-CD20 mAb.
Further research in the model also provided a possible explanation for the
brisk clinical effect of anti-CD20 mAb. We observed that the space left
inside lymph nodes by the depletion of CD20+ ve B cells is replenished
by activated T cells expressing the lymph node homing receptor CCR7. This
was not observed in monkeys treated with anti-BlyS or anti-APRIL mAb
(Kap et al., 2014). This finding may suggest that the anti-CD20
treatment may interfere with the licensing of activated autoreactive T cells
to egress the lymph nodes.
A marker that is constitutively expressed on B cells and induced after
activation on myeloid APCs is CD40 (Laman et al.,
1996; van Kooten and Banchereau, 1997). As
mentioned earlier, the CD20 + APC of the core pathogenic autoaggressive
CTLs prominently express CD40. The ligation of CD40 on B cells with its
counter structure on T cells (CD154) elicits bi-directional signaling that
is important for B cell activation and differentiation; antibody isotype
switch also depends on CD40–CD154 interaction. In EBV-infected B cells the
major oncoprotein LMP1 mimics constitutively activated CD40, but recent data
suggest that activation signals can still be relayed through CD40 (Ma et al., 2015).
In agreement, we observed that incubation of EBV-infected marmoset B cells
with a stimulatory anti-CD40 mAb enhances IgG production (own unpublished observation).
The mAb 5D12 is a non-stimulatory mouse IgG2b against human CD40 that binds
marmoset CD40 and blocks its interaction with human CD40
ligand/CD154 (Laman et al., 2002). Marmosets immunized with human
myelin/CFA were treated early (psd 14–42) or later (psd 25–53) with the
mAb. The treatment suppressed the expression of EAE symptoms during presence
of the mAb but did not abrogate EAE development after the treatment was
stopped, which might be due to the development of a neutralizing antibody
response (Laman et al., 2002). For this reason, a second experiment was
performed testing a novel mouse–human chimeric version of 5D12 in the
rhMOG/CFA model. Also in the mAb-treated monkeys clinical signs did not
develop during the 40-day observation period, while all placebo-treated
monkeys displayed clinical EAE (Boon et al., 2001). Importantly, the treatment effect
was reflected by alteration of the antibody reactivity profile with a panel
of rhMOG peptides.
Concluding remarks
The marmoset EAE model tells a different story about the putative
immunopathogenesis of human multiple sclerosis than the corresponding models
in inbred/SPF mice. The most important lesson is that the pathogen-educated
T cell repertoire of marmosets contains effector memory cytotoxic
specificities that can readily be activated by an antigen without the need of
danger co-signaling. These CTLs mediate a
pathogenic pathway that has not (yet) been found in mouse EAE models but
that seem to be relevant for ongoing disease in MS patients. The finding
that the EBV-infected B cells are involved in the activation of these highly
pathogenic cells may provide a mechanistic explanation for the elusive
association of this γ1 herpesvirus with susceptibility to MS
(Lunemann and Munz, 2009; Pakpoor et al., 2012). Another important finding has been that in the marmoset EAE model
MS-like cortical grey-matter pathology develops. Essentially all lesion
stages present in the grey matter of MS patients can be found in marmosets
(Pomeroy et al., 2005). Moreover, at a late disease stage also grey-matter
atrophy develops (Pomeroy et al., 2008). This implies that the
marmoset provides a highly desired experimental model for the study of
grey-matter pathology development in progressive MS (Mahad et al., 2015).
Acknowledgements
The authors would like to thank Henk van Westbroek for the artwork.
Edited by: E. Fuchs
Reviewed by: two anonymous referees
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