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Antibodies to native myelin oligodendrocyte glycoprotein in children with inflammatory demyelinating central nervous system disease.

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Antibodies to Native Myelin
Oligodendrocyte Glycoprotein in Children
with Inflammatory Demyelinating Central
Nervous System Disease
Fabienne Brilot, PhD,1 Russell C. Dale, PhD,1 Rebecca C. Selter,2 Verena Grummel,2
Sudhakar Reddy Kalluri, MSc,2 Muhammad Aslam, M Phil,2 Verena Busch, MD,2,3 Dun Zhou, PhD,2
Sabine Cepok, PhD,2 and Bernhard Hemmer, MD2
Objective: Myelin oligodendrocyte glycoprotein (MOG) is a candidate target antigen in demyelinating diseases of the central
nervous system (CNS). Although MOG is encephalitogenic in different animal models, the relevance of this antigen in human
autoimmune diseases of the CNS is still controversial.
Methods: We investigated the occurrence and biological activity of antibodies to native MOG (nMOG) in 47 children during
a first episode of CNS demyelination (acute disseminated encephalomyelitis [ADEM], n ⫽ 19 and clinical isolated syndrome
[CIS], n ⫽ 28) by a cell-based bioassay.
Results: High serum immunoglobulin G (IgG) titers to nMOG were detected in 40% of children with CIS/ADEM but 0% of
the control children affected by other neurological diseases, healthy children, or adults with inflammatory demyelinating diseases,
respectively. By contrast, IgM antibodies to nMOG occurred in only 3 children affected by ADEM. Children with high
anti-nMOG IgG titer were significantly younger than those with low IgG titer. Anti-nMOG IgG titers did not differ between
the ADEM and CIS group, and did not predict conversion from CIS to MS during a mean 2-year follow-up. However,
intrathecal IgG anti-MOG antibody synthesis was only seen in CIS children. IgG antibodies to nMOG not only bound to the
extracellular domain of nMOG, but also induced natural killer cell-mediated killing of nMOG-expressing cells in vitro.
Interpretation: Overall, these findings suggest nMOG as a major target of the humoral immune response in a subgroup of
children affected by inflammatory demyelinating diseases of the CNS. Children may provide valuable insight into the earliest
immune mechanisms of CNS demyelination.
Ann Neurol 2009;66:833– 842
Demyelinating diseases of the central nervous system
(CNS) are an important cause of neurological disability
in children and young adults. A first CNS demyelinating event in adults is most likely to represent multiple
sclerosis (MS). A first demyelinating event in children
when accompanied by encephalopathy is termed acute
disseminated encephalomyelitis (ADEM) and has a
lower risk of progression to MS.1 The other first CNS
demyelinating event in children is a clinically isolated
syndrome (CIS), which has a higher risk of progression
to MS.2–5 A recent study testing new international criteria of pediatric demyelination has found that ADEM
has a significantly lower progression to MS than CIS
after 2-year follow-up.5 Although the presence of encephalopathy makes the development of MS less likely,
it has been recognized that very young children can
have encephalopathy during MS presentation.6 Although the cause of demyelinating disease is largely unknown, several lines of evidence suggest an underlying
autoimmune process.7,8 Myelin antigens, such as myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP), have been proposed as targets of
the immune response in MS and ADEM.9 –11 MOG is
expressed on the surface of oligodendrocytes and
readily accessible to autoantibodies. Immunization with
MOG induces experimental autoimmune encephalo-
From the 1Neuroimmunology Group, Institute for Neuroscience
and Muscle Research, the Kids Research Institute at the Children’s
Hospital at Westmead, University of Sydney, Sydney, Australia;
2
Department of Neurology, Klinikum rechts der Isar, Technical
University Munich, Munich, Germany; and 3Children Hospital,
Technical University Munich, Munich, Germany.
Potential conflict of interest: Nothing to report.
Address correspondence to Dr Hemmer, Department of Neurology,
Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany.
Received Jun 29, 2009, and in revised form Oct 25. Accepted for
publication Oct 30, 2009. Published online in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21916
© 2009 American Neurological Association
833
myelitis in several animal species.11 In the rat and marmoset model, an antibody response to MOG is observed, which contributes to CNS demyelination.12,13
These antibodies target a conformational epitope of
MOG, which is located on the extracellular domain of
the protein.14 In humans, immunoglobulin (Ig) G and
IgM antibodies to denatured MOG protein have been
detected in sera of adult MS patients and controls, although serum levels are rather low and do not seem to
correlate with disease progression in patients with
CIS.15–17 More recently, cell-based assays have been
developed, with MOG being properly expressed on the
membrane, which enable quantification of antibodies
to native human MOG (nMOG).18 –20 Although IgG
antibodies to nMOG are elevated in MS patient sera
compared with controls, only a small number of adult
MS patients show high antibody titer.18
In this study, we used cell-based bioassays to determine IgG and IgM antibodies to native MOG, PLP,
and aquaporin-4 (AQP4), 3 putative autoantigens in
demyelinating CNS diseases. We focused on a welldefined group of children with a first inflammatory demyelinating event who were prospectively followed for
a mean of 2 years to monitor ongoing disease activity
and conversion to MS. Antibody titers were compared
to control groups. We observed a unique antibody response in a subgroup of children with a first demyelinating event.
(LN18-PLP) and AQP4 (LN18-AQP4) were established using the same strategy.21
Subjects and Methods
Patients and Controls
The following bioassay was used to quantify antibody reactivity of sera and cerebrospinal fluid (CSF) to native MOG,
PLP, and AQP4. Serum was added to 30,000 LN18-MOG,
LN18-AQP4, LN18-PLP, or LN18-CTR cells in RPMI1640, yielding a final serum dilution of 1:100. Cells were
incubated on ice on an orbital shaker for 20 minutes and
washed twice with washing buffer (1% fetal calf serum [FCS]
in phosphate-buffered saline [PBS]). Cells were then stained
with Alexa Fluor 488-labeled goat anti-human IgG or IgM
secondary antibody (Invitrogen) for 20 minutes on ice,
washed again twice, and then resuspended in washing buffer.
Analysis of cell surface staining was determined by flow cytometry using CyAn ADP (Beckman Coulter, Fullerton, CA)
and Summit software (Beckman Coulter). Levels of antibody
titers are expressed by ⌬-median fluorescence intensity
(MFI). ⌬MFI was determined by the subtraction of MFI
obtained with LN18-CTR cells from the MFI obtained with
LN18-MOG, LN18-AQP4, or LN18-PLP cells. To detect
anti-nMOG antibodies in CSF, IgG concentration of sera
and corresponding CSF were measured by nephelometry
(BN ProSpec, Siemens, Erlangen, Germany). Both sera and
CSF were adjusted to a final IgG concentration of 5mg/l,
and the reactivity to nMOG was determined by flow cytometry as described above.
All pediatric patients and controls were recruited in the UK.
Adult MS patients were recruited at the neurology departments in Düsseldorf and Munich.
Ethics approval for this pediatric study was granted by
both the Children’s Hospital at Westmead, Australia, and
the Institute of Child Health, Great Ormond Street, NHS
Trust, UK ethics committee. Ethics approval for the adult
study was obtained by the local ethics committees in Düsseldorf and Munich.
Cloning and Expression of Human CNS Proteins
The bioassay was established as described previously.18
Briefly, full-length human MOG cDNA, synthesized out of
a human brain total RNA (BD Biosciences, San Jose, CA)
was used to transduce a human glioblastoma cell line LN18.
The primers 5⬘-ATTGAGATCTGAGATGGCAAG-3⬘ and
5⬘-GAGATCTCAGAAGGGATTTCG-3⬘ were used to add
BglII restriction sites at 5⬘ and 3⬘ ends of the MOG cDNA.
The polymerase chain reaction product was then cloned into
the plasmid pLenti6/V5 (Invitrogen, Carlsbad, CA), and a
293FT cell line was transfected by Lipofectamine (Invitrogen) using the pLenti6/V5-MOG. Virus-containing supernatant was used to transduce the LN18 glioblastoma cell line
(LN18-MOG). A control cell line (LN18-CTR) was obtained by transducing the LN18 cell line with an empty vector pLenti6/V5. Additional cell lines expressing human PLP
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Assessment of Surface Expression of Human CNS
Native Proteins
Surface expression of MOG and PLP on transduced LN18
cells was determined after staining with a monoclonal antiMOG antibody (mAb 8-18C5), or anti-PLP antibody (mAb
O10) in combination with an Alexa Fluor 488-conjugated
goat antimouse IgG antibody (Invitrogen) as secondary antibody. AQP4 expression was confirmed by intracellular staining using polyclonal rabbit anti-AQP4 antibody (1mg/ml,
Sigma, St. Louis, MO) and Alexa-488-labeled anti-rabbit
(Molecular Probes, Eugene, OR; Invitrogen) (data not
shown, Reddy et al, submitted). Cell surface staining was
also analyzed by flow cytometry. Immunocytostaining was
performed on 3% paraformaldehyde-fixed cells using described protocols.18
Sensitivity and reproducibility of the assay were evaluated
by titration experiments using the mAb 8-18C5, mAb O10,
or rabbit anti-AQP4 –specific polyclonal antibody, respectively. For titration experiments, transduced LN18 or LN18CTR cells were incubated with diluted mAbs (10 dilutions
from 0.001 to 1mg/ml) or anti-AQP4 –specific polyclonal
antibody (10 dilutions from 0.02 to 10␮g/ml), followed by
staining with secondary antibodies as described above. Experiments were done in duplicates. Nonspecific binding and
polyreactivity of anti-MOG–positive sera to cell surface proteins were excluded by using LN18-CTR.
Cell-Based Assay for Quantification of Antibody
Reactivity in Sera and Cerebrospinal Fluid
In Vitro Antibody Dependent Cellular
Cytotoxicity Assay
IgG was purified from sera using Protein G-Sepharose (GE
Healthcare, Piscataway, NJ) according to the manufacturer’s
protocol. The titer of anti-nMOG antibodies in purified IgG
was determined by the cell-based assay as described above.
Peripheral blood mononuclear cells of healthy donors were
separated by density gradient centrifugation (Biocoll Separating Solution, Biochrom, Berlin, Germany). Natural killer
(NK) cells were stained with bead-conjugated anti-CD56 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD56⫹ cells
were then separated by positive selection using an AutoMACS (Miltenyi Biotec). Purity of ⬎95% NK cells was
obtained.
Six micrograms of purified IgG diluted in RPMI-1640
was added to 30,000 LN18-CTR or LN18-MOG cells and
incubated on ice for 25 minutes under agitation. Cells were
then washed twice with fluorescent-activated cell sorter analysis (FACS) buffer (1% FCS in PBS) and once with RPMI1640, and were added to 60,000 CD56⫹ NK cells resuspended in RPMI-1640 in a final volume of 150␮l. After
incubation at 37°C in a humidified CO2 incubator for 12
hours, the supernatant was collected, and remaining cells
were detached by trypsin-ethylenediamine tetraacetic acid.
Cell number and viability were then analyzed using FACSbased cell counting (CyAn ADP, Beckman Coulter).
Table. Clinical Diagnoses of Patients with First
Episode of CNS Demyelination (n ⴝ 47), Mean
Follow-up Duration, and Progression to MS
First CNS
Demyelination
Episode
Diagnosis
ADEM,
n ⴝ 19
CIS,
n ⴝ 28
Sex; mean age,
yr (range)
13 boys;
5.2 (1.8–10.5)
12 boys;
9.2 (2.5–15.5)
0/19
8/28a
Mean duration
of follow-up,
yr (range)
1.96 (0.5–4.6)
2.02 (1.0–4.0)
Progression to
MSb
1/18
12/28
Intrathecal
OCB at
onset
a
Six of 8 CIS patients with intrathecal OCB progressed to MS
in follow-up.
b
Diagnosis based on clinical relapse.
CNS ⫽ central nervous system; MS ⫽ multiple sclerosis;
ADEM ⫽ acute disseminated encephalomyelitis; CIS ⫽
clinically isolated syndrome; OCB ⫽ oligoclonal
immunoglobulin G bands.
Statistical Analysis
Due to the fact the data were not normally distributed, the
Mann-Whitney U test and Kruskal-Wallis test were used to
compare antibody titer between patients and controls. For
correlation analysis between anti-nMOG titer and cytotoxicity, the Spearman rank correlation was applied.
Results
Patients with First CNS Demyelinating Episode
Forty-seven children (25 boys; mean age, 7.63 years;
range, 1.8 –15.5 years) were recruited with a first episode of acute onset CNS demyelination between 2002
and 2008 (Table). Patients were retrospectively defined
using 2007 International consensus definitions for a
first episode of CNS demyelination in children.22
Pediatric ADEM (n ⫽ 19) was defined as a first
clinical event of inflammatory demyelination with
acute or subacute onset that affected multifocal areas of
the CNS. The event was polysymptomatic and included encephalopathy (defined as behavioral change
or change in consciousness). Magnetic resonance imaging (MRI) showed focal or multifocal lesions predominantly involving the white matter.
Pediatric CIS (n ⫽ 28) was defined as a first event
of inflammatory demyelination that was either focal
(optic neuritis, n ⫽ 7; hemispheric syndrome, n ⫽ 6;
brainstem syndrome, n ⫽ 2; cerebellar syndrome, n ⫽
2; transverse myelitis, n ⫽ 1) or multifocal (n ⫽ 10),
but did not include encephalopathy. The pediatric CIS
classification was based on clinical, rather than radiological features.22
Investigation and Management of First CNS
Demyelinating Episode
All serum samples from children with pediatric ADEM
or CIS (n ⫽ 47) were taken within the first 3 weeks of
neurological onset (mean, 7.8 days; median, 7 days);
15 presented fulminantly and were sampled within 3
days, 29 presented subacutely and were sampled within
7 days, and 7 presented indolently and were sampled
within 3 weeks. All serum and CSF samples were taken
at the same time, and before treatment. Patients were
routinely tested for CSF/serum oligoclonal bands (see
Table). MRI demonstrated typical inflammatory demyelinating lesions disseminated throughout the CNS,
but predominantly involving white matter: Forty-five
of 47 had supratentorial white matter lesions (1 had
isolated radiological optic neuritis; 1 had isolated radiological transverse myelitis). The majority of patients
(43/47) received steroid treatment (intravenous
methyl-prednisolone 30mg/kg for 3 days, then oral
prednisolone over 4 weeks in the case of incomplete
recovery). The 4 pediatric patients who did not receive
steroid therapy had CIS, and were spontaneously improving at the time of assessment. Three children received 2g/kg body weight of intravenous immunoglobulin due to disease deterioration despite steroid
therapy.
Follow-up and Multiple Sclerosis
Patients were followed-up for a mean duration of 2.0
years (range, 0.5– 4.6 years). The patient classification
of the first demyelinating episode was pediatric ADEM
Brilot et al: MOG Antibodies in Children
835
or CIS. A child was reclassified as having MS based on
the presence of clinical relapse, rather than radiological
evidence of new lesions.22,23
Pediatric MS diagnosis required multiple episodes of
CNS demyelination separated in time and space, as for
adults.22 A relapse involved a new site and occurred
⬎4 weeks after the first event. All patients had repeat
magnetic resonance neuroimaging that demonstrated
new lesions. During that time, 13 patients relapsed and
fulfilled a diagnosis of pediatric MS. Pediatric CIS had
a higher risk of relapse and progression to MS.4 Only 1
patient with pediatric ADEM had multiple nonADEM relapses and was diagnosed with MS (see Table). The MS-defining relapse occurred a mean of 10
months and median of 7.5 months after the first event.
Ten of 13 pediatric MS patients had their MS-defining
relapse within the first year (range, 1– 42 months).
Eight of 13 pediatric MS patients are taking betainterferon disease-modifying therapy.
Controls
The pediatric control group included children with
other noninflammatory neurological diseases (ONDs)
(n ⫽ 28; 16 boys; mean age, 6.0 years; range, 0.3–14
years), such as developmental delay (n ⫽ 8), CNS malformations (n ⫽ 5), neurodegeneration (n ⫽ 8), and
epilepsy syndromes (n ⫽ 7). Serum from communityacquired healthy children (HC) (n ⫽ 30; 15 boys;
mean age, 11.0 years; range, 9 –13 years) and serum
from type 1 diabetes (n ⫽ 15; mean age, 10.9 years;
range, 2–16 years) were also used in this study. The
pediatric control groups were not age-matched due to
the difficulty in obtaining blood samples in healthy
young children. However, the pediatric OND group
included children with a broad age range, including
very young children. The mean, median, and age range
of the pediatric OND group were not significantly different from our patient group. The adult MS patient
group consisted of 54 patients. One patient had CIS,
33 were relapsing-remitting, 1 was primary, and 19
were secondary progressive MS (mean age, 40 years).
High Antibody Titer to Native MOG
in Children with a First Episode of CNS
Inflammatory Demyelination
We studied the presence of autoantibodies to native
PLP, AQP4, and MOG in children affected by a first
inflammatory demyelinating event compatible with
childhood ADEM or CIS. We used a sensitive bioassay
for antibody detection based on a glial tumor cell line
transduced with full-length MOG (LN18-MOG)-,
PLP (LN18-PLP)-, and AQP4 (LN18-AQP4)containing lentiviruses. This assay allows us to determine the binding of antibodies to the extracellular domain of proteins and preserves conformational
epitopes, which are essential for antibody binding in
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Fig 1. Cell-based assay to determine antibodies to native
central nervous system (CNS) proteins. CNS proteins were
expressed in the human glioblastoma cell line LN18. (A)
Immunohistochemistry of the LN18-CTR, LN18-MOG, and
LN18-PLP cell lines. Expression of native proteins was confirmed after staining of LN18-MOG, LN18-PLP, and
LN18-CTR with monoclonal antibody (mAb) 8-18C5
against myelin oligodendrocyte glycoprotein (MOG) (staining
shown for LN18-CTR and LN18-MOG) and mAb O10
against proteolipid protein (PLP) (staining shown for LN18PLP) followed by Alexa Fluor 488-conjugated secondary antimouse immunoglobulin G antibodies. Representative images
(original magnification, ⫻600) and flow cytometry histograms are shown. Surface expression was confirmed by flow
cytometry as shown for mAb anti-MOG 8-18C5 with the
LN18-MOG (red line), LN18-PLP (green line), and the
LN18-CTR (blue line) cell lines. (B) Titration of mAb
demonstrates the correlation between concentration of the
antibody and median fluorescence intensity. Flow cytometry
on different transfected cells was used to quantify the concentration of the mAbs against MOG (left) and PLP (right) at
a wide range of antibody concentrations. Means and standard deviations of 3 measurements for each concentration
are shown.
vivo. Protein expression was confirmed by immunohistochemistry (shown for MOG and PLP, Fig 1A and
B). An empty virus was used as control (LN18-CTR),
and was negative for MOG, PLP, and AQP4 stainings
(see Fig 1A and B, and data not shown). The intensity
of the fluorescence expressed in MFI was correlated to
the concentration of antibody used to stain the cells,
suggesting that MFI is directly dependent on antibody
titer (see Fig 1C).
When we applied sera from all children groups at a
dilution of 1:100, we observed a significant IgG antibody reactivity to nMOG (Fig 2A and C), but could
not detect any significant binding to native AQP4
(nAQP4) and native PLP (nPLP) (see Fig 2C), except
in 1 pediatric CIS patient who was weakly positive for
anti-nAQP4 antibodies. Furthermore, IgM antibodies
to MOG, but not the other autoantigens, were also
observed in only 3 of 47 children (see Figs 2B). These
findings suggest that a specific immune response to
nMOG but not to the other antigens occurs in children.
Next, we compared the serum IgG antibody reactivity to nMOG between children with a first inflammatory demyelinating event (DEM) and controls, either
children with ONDs or HC (see Fig 3). Antibody titers were higher in DEM children (median ⌬MFI,
24.5; range, 1.0 –1896.3) than pediatric HC (median
⌬MFI, 2.2; range, 0 –10.7), pediatric OND (median
⌬MFI, 3.4; range, 0.3–107.1), and a group of adult
patients with MS (median ⌬MFI, 2.1; range,
2.1–21.5) (see Fig 3). Based on the titer observed in
pediatric controls, we determined a cutoff for nMOG
IgG seropositivity at ⌬MFI ⫽ 38.1 (median ⫹ 95%
Š
Fig 2. Serum antibody titer to human native myelin oligodendrocyte glycoprotein (nMOG), native proteolipid protein
(nPLP), and native aquaporin-4 (nAQP4) in children. Antibody reactivity to human native central nervous system proteins was determined in sera from all patients and controls by
incubating LN18-MOG, LN18-PLP, LN18-AQP4, and
LN18-CTR cells with serum at a dilution of 1:100. Secondary staining was performed with Alexa Fluor 488-conjugated
antihuman immunoglobulin (Ig) G antibody. Representative
flow cytometry histograms for negative (upper histogram) and
positive (lower histogram) staining for (A) IgG and (B) IgM
specific for nMOG are shown (LN18-MOG, red line; LN18PLP, green line; and LN18-CTR, blue line). ⌬MFI is the
difference between median fluorescence intensity obtained with
the cell line expressing the target protein and the MFI obtained with LN18-CTR cells. (C) IgG antibody titer to
nPLP, nAQP4, and nMOG in all childhood patients (children with a first inflammatory demyelinating event) and controls (healthy children and children with other noninflammatory neurological diseases). Antibody titers were compared
among groups by the Kruskal-Wallis test. Plain bars display
median titer from the whole group, and dotted bars display
median titer of positive samples.
Brilot et al: MOG Antibodies in Children
837
percentile of pediatric HC and ONDs), and defined a
“high” titer as ⌬MFI ⫽ 109.8 (median ⫹ 99% percentile). We detected IgG antibodies to nMOG with
⌬MFI ⬎ 38.1 in 46.8% of DEM children (22 of 47)
and 6.9% (2 of 29) of OND children. No pediatric
HC or adult MS patient was found to be positive for
IgG antibody according to this cutoff. Additionally, we
also determined nMOG IgG titers in sera from a cohort of 15 type 1 diabetes children. All type 1 diabetes
sera were negative (median ⌬MFI, 2.34; range,
0 –16.52, data not shown), suggesting that IgG to
nMOG antibodies were not evidence of general immune dysregulation, but rather a specific finding of
CNS demyelination. High titers were only found in
40.4% of DEM children (18 of 47 patients), but were
not detected in the 3 childhood and adult control
groups. These findings in adult patients were in line
with our previous published findings on adult MS and
unpublished findings on anti-nMOG titer in adult CIS
patients from the Benefit trial.18,24 Titers with MFI
⬎100 were found in ⬍3%, and titers ⬎500 in ⬍1%
of adult CIS and MS patients. Anti-nMOG IgG antibodies in some DEM children were still detected at a
serum dilution as low as 1:10,000 (data not shown).
We also investigated IgM serum antibodies to
nMOG (see Fig 3B). Positive IgM antibody reactivity
to nMOG was observed in only 3 DEM children (not
significant) and no controls. All 3 children also had
high IgG serum antibody titer to nMOG.
Fig 3. Serum antibody titer to native human myelin oligodendrocyte glycoprotein (nMOG) in children with a first inflammatory demyelinating event (DEM). (A) Immunoglobulin (Ig)
G and (B) IgM antibody titers to nMOG were determined in
sera of healthy children (HC), children with other noninflammatory neurological diseases (ONDs), children with a first
inflammatory demyelinating event (DEM), and adults with
multiple sclerosis (MS). Antibody titers were compared among
groups by the Kruskal-Wallis. Plain bars display median titer
from the whole group, and dotted bars display median titer of
positive samples. p Values comparing DEM children with
other groups are given. MFI ⫽ median fluorescence intensity;
ns ⫽ not significant; nd ⫽ not determined.
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IgG Antibodies to nMOG Are Found in Children
Both with CIS and with ADEM, Whereas IgM
Antibodies Are Only Present in ADEM
Based on current consensus definitions, we stratified
DEM children into pediatric CIS or ADEM. Children
were followed up for a mean of 2 years after the initial
demyelinating event to determine whether they developed MS or had no further disease activity (see Table).
Similar IgG antibody titers to nMOG were observed in
children with ADEM and CIS (Fig 4A). The frequency
of children with high titer did not differ between
groups. Furthermore, the presence and titer of antinMOG IgG antibodies did not predict progression to
MS, because similar titers were found in the pediatric
DEM group that progressed to MS and those that did
not progress to MS (see Fig 4B). Next, we compared
the age of the patients with high anti-nMOG IgG
(⌬MFI ⬎ 109.8) versus low or negative IgG antibody
titer to nMOG. Both pediatric ADEM and CIS patients with high titer were significantly younger than
children without nMOG serum antibodies or low titer
(see Fig 4C). In children with anti-nMOG antibodies
(cutoff, ⌬MFI ⫽ 38.1), serum titer and age correlated
negatively (r ⫽ ⫺0.46, p ⫽ 0.023, data not shown).
Interestingly, IgM antibodies to nMOG were only
found in 3 of 19 children affected by ADEM but none
of those affected by CIS (see Fig 3B and data not
shown).
diatric ADEM and 5 pediatric CIS), in whom enough
CSF was available for analysis. Anti-nMOG IgG antibodies were detected in all CSF tested. To determine
whether IgG anti-nMOG antibodies are produced intrathecally, we adjusted serum and CSF of each patient
to the same IgG concentration. Paired samples were
then analyzed for nMOG titer. In all children affected
by ADEM, the antibody titer was lower in CSF than
serum. In 3 of 5 CIS children, an intrathecal IgG antibody production specific for nMOG was observed (titer was higher in CSF than serum), 1 of whom has
progressed to MS (Fig 5).
IgG Antibodies to Native MOG Are Produced in the
Periphery and the CNS
To understand whether the antibodies specific for
nMOG are produced in the periphery alone, or also
locally in the CNS, we determined the antibody titers
in the CSF of 8 children from the same cohort (3 pe-
Antibodies to nMOG Are Cytotoxic
in DEM Children
The binding of antibodies to nMOG at the surface of
LN18-MOG cells suggests that they might fix complement or induce activation of antibody-dependent cellular cytotoxicity (ADCC) in MOG-expressing cells;
this has been proposed as a mechanism by which antibodies can have detrimental effect.25 To determine
the biological activity of these antibodies on LN18MOG and LN18-CTR cells, we performed ADCC assays with purified IgG from sera of children with high
or low anti-nMOG antibodies. Survival of LN18-CTR
was similar after incubation with IgG from sera with
high anti-nMOG antibodies and negative anti-nMOG
antibodies (Fig 6a). On the contrary, IgG from patients with high nMOG antibodies induced NKmediated killing of LN18-MOG cells compared with
IgG from patients with negative anti-nMOG antibodies, suggesting that the binding of IgG to nMOG promotes a cytotoxic effect (see Fig 6a). We also observed
a strong correlation between the IgG nMOG titer and
the extent of ADCC (see Fig 6b).
Discussion
Several lines of evidence suggest that antibodies and B
cells play an important role in autoimmune diseases of
Š
Fig 4. Relation between disease course, age of onset, and antihuman native myelin oligodendrocyte glycoprotein (nMOG)
antibodies. (A) Comparison of immunoglobulin G anti-MOG
antibody titer in children with acute disseminated encephalomyelitis (ADEM) and clinically isolated syndrome (CIS), and
(B) children who did and who did not progress to multiple
sclerosis (MS). Plain bars display median titer from the whole
group, and dotted bars display median titer of positive samples. p Values are given. (C) Children with a first inflammatory demyelinating event were stratified into 2 groups, with
either high anti-MOG titer in serum (⌬-median fluorescence
intensity [MFI] ⬎ 108.9) or negative/low titer, and were
compared according to age of disease onset. The p value is
displayed on the right side of the graph. Antibody titers were
compared between groups using the Mann-Whitney U test.
ns ⫽ not significant.
Brilot et al: MOG Antibodies in Children
839
against native MOG in children with a first episode of
demyelination was performed by O’Connor et al.19
Using a different method, O’Connor found that a significant subgroup of ADEM patients have very high
antibodies against native MOG, higher than adult MS
patients. Importantly, the O’Connor cohort used pa-
Fig 5. Intrathecal synthesis of anti-native human myelin oligodendrocyte glycoprotein (nMOG) antibodies in acute disseminated encephalomyelitis (ADEM) and clinically isolated syndrome (CIS). The extent of specific intrathecal antibody
production was determined in individual pediatric ADEM
(n ⫽ 3) and CIS patients (n ⫽ 5, including 1 CIS patient
who progressed to multiple sclerosis). Cerebrospinal fluid (CSF)
and serum immunoglobulin G (IgG) were normalized to
5mg/l, and the ratio of anti-nMOG reactivity was determined. We defined intrathecal IgG production as a ratio
above 1.5 (dotted line).
the CNS. An increasing number of autoantibodies specific for rare inflammatory diseases of the CNS have
been identified such as antibodies to AQP4 in neuromyelitis optica,26 GABA, and amphiphysin in stiff person syndrome,27,28 voltage-gated potassium channels or
NMDA-receptor in limbic and paraneoplastic encephalitis.29 The discovery of these antibodies has had an
important impact on our understanding of the underlying pathomechanisms, but has also enriched our diagnostic repertoire.30,31
In MS, a number of myelin antigens have been proposed as targets of the autoimmune response. Among
them, MOG has been the most persuasive candidate.
In particular, its expression on the surface of the myelin sheath and its ability to induce encephalitis in many
animal models emphasizes a possible role of MOG in
CNS autoimmunity.11 However, the results from
many studies have not provided a conclusive picture on
the possible role of MOG in autoimmune diseases of
the CNS.
One of the reasons for the controversial results
might have been the different technical approaches to
measure antibodies against MOG. Although many
studies have shown that biologically relevant antibodies
target conformational epitopes of MOG, most studies
have measured antibody responses to denatured MOG
protein.15–17 Only recently have assays been established
that enabled us to measure antibody responses to native MOG reflecting the correct properties of the protein in vivo.18 –20
The only other report investigating antibodies
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Annals of Neurology
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December 2009
Fig 6. Biological activity of anti–native human myelin oligodendrocyte glycoprotein (nMOG) antibodies. Antibodydependent cellular cytotoxicity was performed with purified
CD56⫹ natural killer cells and purified immunoglobulin G
(IgG) from acute disseminated encephalomyelitis and clinically
isolated syndrome serum. (A) Survival rate of LN18-CTR and
LN18-MOG cells after overnight incubation with patient
IgG. Patients with high IgG anti-nMOG titer (Ab⫹) and
patients with negative anti-nMOG IgG antibodies in serum
(Ab⫺) were used. Symbols represent cell survival obtained in
the presence of individual sera. (B) Comparison between antinMOG antibody titer and specific cytotoxic activity of serum
in vitro is shown. Specific cell survival for each serum was
determined as follows: % cell survival of LN18-CTR ⫺ %
cell survival of LN18-MOG; Spearman rank correlation test
was used. r and p values are displayed. DEM ⫽ children
with a first inflammatory demyelinating event.
tients from multiple countries, did not apply 2007 International Consensus criteria, and did not follow up
patients. This is relevant because the 2007 International Consensus is the first published guidelines establishing strict clinical criteria to help in the diagnosis of
CIS and ADEM. In our study, we investigated systematically the antibody response in a 2007 International
Consensus-defined and prospectively followed group of
children affected by a first CNS demyelinating event.
We also present the first evidence that these antibodies
against nMOG in children can participate in a pathogenic mechanism via antibody-dependent cellularmediated cytotoxicity.
In contrast to adults affected by MS, we observed a
very strong IgG response to nMOG in a subgroup of
these children. In adults, subclinical disease activity
may precede the first disease episode by years. By contrast, in children it is conceivable that the onset of clinical symptoms and signs occurs much more rapidly after initiation of CNS demyelination,6 supporting acute
inflammation and antibody production. Therefore,
children may be a better model of early immunological
changes in CNS demyelination. The inverse correlation
between antibody titer and age also suggests that profound antibody responses to nMOG are more common in early childhood and do not seem to persist
during later stages of CNS autoimmunity. The antibody response against MOG was primarily of the IgG
isotype. MOG IgM was observed rarely, and only in
ADEM children, possibly related to the hyperacute
postinfectious nature of ADEM, compared with CIS
and MS. Such high levels of anti-nMOG antibodies
were not seen in age-matched controls, in children affected by other neurological diseases, or in type 1 diabetes children.
The cohort only had a short mean follow-up, and
prolonged follow-up is important not only for diagnosis purposes, but also to define the association between
antibodies and first episode of demyelination further.
However, it is worth noting that the majority of children (10/13) had their MS-defining relapse within the
first year. This is in line with previous data reporting
that most children ⬎10 years old experience their second attack within 12 months of the first attack.6
nMOG IgG was also present in the CSF of a limited
number of patients tested, although intrathecal production was observed in 3 CIS patients, 1 of whom progressed to MS. However, high intrathecal synthesis of
MOG IgG may be an important precondition for relapse of CNS demyelination, as in MS. This is in line
with reports in adult MS patients suggesting that antiMOG antibodies are enriched in CNS lesions compared with the periphery.32,33 Further CSF investigation will be needed to clarify this point.
Interestingly, the response in patients with pediatric
demyelination was specifically against nMOG, because
no significant antibodies were observed to other candidate membrane autoantigens, such as PLP or AQP4.
Future studies could include other autoimmune
control groups, such as type 1 diabetes systemic lupus
erythematosus.
The detection of antibodies per se does not mean
they are important in causing disease, as they can be
present as an epiphenomenon to cell damage. However, we demonstrated that anti-nMOG antibodies
were associated with NK cell-mediated cytotoxicity. In
the human brain, cells such as microglia and macrophages have been shown to be recruited at MS lesions,34 and to have the potential to mediate ADCC
and to participate in antibody-induced myelin damage.35 Other mechanisms of antibody pathogenicity,
such as complement-mediated cytotoxicity, were not
examined in this report, and represent an alternate
pathogenic mechanism.18 These findings suggest that a
peripheral immune response to nMOG occurs in a
subgroup of children affected by a first demyelinating
event, which might be pathologically relevant in the
demyelination process.
The initial events involved in the generation of antibodies to nMOG are not understood. Exposure to
viral antigens can lead to autoimmunity via molecular
mimicry.36 –38 Further studies are warranted to clarify
how antibodies to MOG are generated and whether
MOG is the key target of the first inflammatory demyelinating event in children.
In summary, we identified a unique and highly specific antibody response to nMOG in a subgroup of
children affected by the first demyelinating event. The
identification of this early autoimmune response significantly adds to our understanding of the autoantigens
involved in early CNS autoimmunity.
This study was supported by University of Sydney postdoctoral fellowships (R.C.D., F.B.); the Brain Foundation, Australia (R.C.D.,
F.B.); the Deutsche Forschungsgemeinschaft (grant He2386/7-1;
B.H. and S.C.); the German Ministry of Education, Science, Research, and Technology (krankheitsbezogenes Kompetenznetz MS;
B.H., S.C., R.S., V.K., V.G., S.R.K.); the Gemeinnützige Hertie
Stiftung (D.Z.); the Düsseldorf chapter of the Multiple Sclerosis
Society and the DAAD (M.A.).
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