Hafler Et Al-2005-Immunological Reviews

David A.
Hafler
Jacqueline M. Slavik
David E. Anderson
Kevin C. OConnor
Philip De Jager
Clare Baecher-Allan
Multiple sclerosis
Authors addresses
David A. Hafler1,2, Jacqueline M. Slavik1, David E.
Anderson1, Kevin C. OConnor1, Philip De Jager1,2, Clare
Baecher-Allan1
1
Laboratory of Molecular Immunology, Center
for Neurologic Diseases, Brigham and
Womens Hospital and Harvard Medical
School, Boston, MA, USA.
2
The Broad Institute, Massachusetts Institute of
Technology and Harvard University,
Cambridge, MA, USA.
Summary: Multiple sclerosis (MS) is a complex genetic disease associated

with inflammation in the central nervous system (CNS) white matter and
is thought to be mediated by autoimmune processes. Clonal expansion of
B cells, their antibody products, and T cells, hallmarks of inflammation in
the CNS, are found in MS. The association of the disease with major
histocompatibility complex genes, the inflammatory white matter
infiltrates, similarities with animal models, and the observation that MS
can be treated with immunomodulatory and immunosuppressive
therapies support the hypothesis that autoimmunity plays a major role
in the disease pathology.
This review discusses the immunopathology of MS with particular focus
given to regulatory T cells and the role of B cells and antibodies, immunomodulatory therapeutics, and finally new directions in MS research,
particularly new methods to define the molecular pathology of human
disease with high-throughput examination of germline DNA haplotypes,
RNA expression, and protein structures that will allow the generation of a
new series of hypotheses that can be tested to develop better understandings and therapies for this disease.
Correspondence to:
Dr David A. Hafler, MD
Center for Neurologic Diseases
Harvard Medical School
77 Avenue Louis Pasteur
Boston, MA 02115
USA
Tel: 617 525 5330
Fax: 617 525 5333
E-mail: dhafler@rics.bwh.harvard.edu
Overview
Immunological Reviews 2005

Vol. 204: 208231
Printed in Singapore. All rights reserved
Copyright Blackwell Munksgaard 2005
Immunological Reviews
0105-2896
208
Multiple sclerosis (MS) was first described in 1868 noting the

accumulation of inflammatory cells in a perivascular distribution within the brain and spinal cord white matter of patients
with intermittent episodes of neurologic dysfunction (1, 2).
This led to the term sclerose en plaques disseminees or MS. The more
recent observation in 1948 by Elvin Kabat (3) of increases in
oligoclonal immunoglobulin in the cerebro spinal (CSF) of
patients with MS provided further evidence of an inflammatory nature to the disease (3). In the past half-century, several
large population-based MS twin studies demonstrated a strong
genetic basis to this clinical-pathologic entity (4). Lastly, the
demonstration of an autoimmune, at times demyelinating,
disease in mammals with immunization of central nervous
system (CNS) myelin [experimental autoimmune encephalomyelitis (EAE)], first made by Thomas Rivers (5) at the Rockefeller Institute in 1933 with the repeated injection of rabbit
brain and spinal cord into primates, has led to the generally
accepted hypothesis that MS is secondary to an autoimmune
Hafler et al Multiple sclerosis
response to self-antigens in a genetically susceptible host. It

should be pointed out that although the inflammation found
in the CNS of patients with MS is thought to represent an
autoimmune response, this belief is based on negative results
with the inability to consistently isolate a microbial agent from
the tissue of diseased patients. Nevertheless, primary viral
infections in the CNS may induce an autoimmune response
(6), and the recurring lesson from the EAE model is that the
minimal requirement for inducing inflammatory, autoimmune CNS-demyelinating disease is the activation of myelinreactive T cells in the peripheral immune system (7, 8).
epitope spreading to a number of antigens implicated in MS,

including a-crystalline and proteolipid protein (PLP) (Altman D,
Kuchroo V, Hafler D, manuscript submitted). As we have
observed high expression of B7.1 costimulatory molecules in
the CNS white matter of patients with MS (11) and most
patients exhibit T-cell reactivity to a number of myelin
antigens (12), it is likely that by the time a patient develops
clinical MS there has been epitope spreading, with reactivity
to multiple myelin epitopes. However, the presence of clonally expanded T cells in the CSF and brain tissue of patients
with the disease raises the issue that there may be clonal
reactivity to just a few myelin antigens. Single cell cloning
of T cells from the inflamed CNS tissue screened against
combinatorial and protein libraries may allow new insights
into pathophysiology of MS.
Data from a number of laboratories combined with experimental data in the EAE model where myelin antigen is injected
with adjuvant into mammals indicate that there are autoreactive T cells recognizing myelin antigens in the circulation
of mammals. It appears that the activation of these T cells is the
critical event in inducing autoimmune disease (Fig. 1). We and
others first demonstrated over a decade ago that T-cell clones
isolated from the blood of patients with MS frequently exhibit
exquisite specificity for the immunodominant p8599 epitope
of MBP (1214). However, while the TCR appears to be
highly specific in recognizing this peptide, altering the peptide
ligand can change the TCR conformation to yield a higher
degree of T-cell cross-reactivity (15). Using combinatorial
chemistry, even a greater degree of cross-reactivity could be
demonstrated, and a number of viral epitopes were identified
Immunopathophysiology of MS
A critical lesson from the EAE model is that of epitope spreading, first observed by Eli Sercarz (9). With the injection of a
single myelin protein epitope into mice with subsequent
development of EAE, it was observed that T cells became
activated against other epitopes of the same protein; this was
followed by T-cell activation to other myelin proteins that
become capable of adoptively transferring the disease to
nave mice. The epitope spreading requires costimulation
with B7/CD28, suggesting that with tissue damage in the
CNS, an adjuvant is created in the CNS with the expression
of high amounts of B7.1 costimulatory molecules associated
with antigen release (10). Moreover, we have recently
observed that a transgenic mouse expressing DR2
(DRB1*1501) and a T-cell receptor (TCR) (Ob1A12) cloned
from the blood of a patient with MS-recognizing myelin basic
protein (MBP) 8599 spontaneously developed EAE with
Antigen-induced, adoptive
regulatory T cells
autoagreactive
Th3
Autoimmune response
APC
IL-10
IL-2
IFN-
TNF-
IL-10
TGF-
IL-12
B7-1
autoagreactive
Tr1
CD28
CD28
autoagreactive
ThP
autoagreactive
Th1
B7-1
autoagreactive
TH1
CD154 CD40
IL-12
CD4+
CD25+
Local antigenpresenting cells
NK T
cells
Innate, non-induced regulatory T cells
autoantigens
Fig. 1. Induced and innate regulatory T cells.

In a genetically susceptible host, common
microbes activate the antigen-presenting cell
(APC) through Toll receptors and contain
protein sequences cross-reactive with selfantigens. The underlying immunoregulatory
defects of regulatory T cells allow the further
propagation of autoreactive T cells. Activated
autoreactive T cells migrate into the tissue and
recognize antigens presented by local antigenpresenting cells, secrete Th1 cytokines and
initiate the inflammatory cascade. Regulation of
autoimmune responses: naturally occurring
mechanisms may exist to regulate autoimmune
responses including the induction of autoreactive
Th3 (TGF-b) and Tr1 (IL-10) cytokine secreting
T cells that migrate to the inflamed tissue and
downregulate inflammatory Th1 autoreactive
T cells, whereas the naturally occurring
CD4CD25 and NK T cells may regulate initial
T-cell activation without prior induction.
Immunological Reviews 204/2005
209
that could trigger autoreactive T-cell clones in a manner that

would not be predicted by simple algorithms (16). Indeed,
one MBP-reactive T-cell clone recognized an epitope of an
entirely different self-protein, the myelin oligodendrocyte
glycoprotein. Hence, a significant degree of functional
degeneracy exists in the recognition of self-antigens by T
cells. This finding is consistent with the hypothesis that MS is
triggered by autoreactive T cells activated by microbial
antigens cross-reactive with myelin (17). The high frequency
of activated, myelin-reactive T cells in the circulation and CSF
of patients with MS is further consistent with this hypothesis.
CNS-specific regulation of inflammation

A variety of peripheral mechanisms regulate CNS autoimmunity, whether it be editing of the autoreactive T-cell
repertoire, limiting activation of autoreactive T cells, or suppressing autoreactive T-cell activity with a variety of regulatory
T-cell populations. Nevertheless, the unique anatomy and cell
types of the CNS together provide additional mechanisms that
limit CNS inflammation and autoimmunity. Recent advances
in neurobiology and immunology, aided by technical advances
in gene expression analysis and in vivo imaging, have begun to
provide greater insight into the molecular events occurring
within and around MS plaques, and this knowledge should
soon help elucidate the relative roles resident cells in the CNS
play in regulating inflammation.
Cellular events occurring within and around MS plaques
Gross examination of MS brain tissue reveals multiple sharply

demarcated plaques in the CNS white matter with a predilection to the optic nerves and white matter tracts of the periventricular regions, brain stem, and spinal cord. As was
recognized early on and so elegantly investigated in more
recent studies, substantial axonal injuries with axonal transections are abundant throughout active MS lesions (18).
The inflammatory cell profile of active lesions is characterized by perivascular infiltration of oligoclonal T cells (19)
consisting of CD4/CD8 a/b (20, 21) and g/d (22) T cells
and monocytes with occasional B cells and infrequent plasma
cells (23). Lymphocytes may be found in normal appearing
white matter beyond the margin of active demyelination (24).
Macrophages are most prominent in the center of the plaques
and are seen to contain myelin debris, while oligodendrocyte
counts are reduced. In chronic active lesions, the inflammatory
cell infiltrate is less prominent and may be largely restricted to
the rim of the plaque, suggesting the presence of ongoing
210
inflammatory activity along the lesion edge. Recently, four

pathologic categories of the disease were described related to
potentially different pathophysiologic disease mechanisms,
though this has yet to be demonstrated at a molecular level (25).
It was of interest that the pattern of pathology tended to be
the same in multiple lesions from any single individual with
MS.
Molecular events occurring within and around MS plaques

Our own early studies of inflammatory gene expressions
revealed high levels of interleukin-12 (IL-12) mRNA in the
early plaque tissue of patients with MS (11). Many subsequent
studies have individually identified one or a few cell types or
molecules associated with MS plaques. More recently, gene
expression analysis has been used to identify molecules associated with plaques in a more comprehensive and unbiased
fashion (2635). Generally, the studies have failed to demonstrate consistent patterns of gene regulation, with a few notable
exceptions. Genes associated directly or indirectly with cellular
and humoral immunity [e.g. CD4, human leukocyte antigen
(HLA) class II, immunoglobulin (Ig) heavy chain, and IL-6R]
are upregulated in lesions compared to normal brain tissue.
The failure to identify larger numbers of genes that are
similarly regulated across multiple studies is likely due in
part to challenges involved with comparing gene expression
analyses among studies that have used slightly different
methodologies (35). Of perhaps greater importance, many
of these studies used materials from only one or a few patients,
or from patients with different types of MS, and did not
account for the great heterogeneity among different types of
MS plaques.
Histology and/or immunohistochemistry can be used to
define several different types of lesions, characterized by
focal destruction of myelin, axonal loss, and different degrees
of inflammatory infiltrate (36). Inflammatory infiltrates are
found throughout early active lesions, whereas inflammatory
activity is localized to the edge of chronic active lesions.
The centers of chronic lesions are less cellular and marked by
gliosis, relative to the marginal zone of lesions. Chronic
plaques with only sparse evidence of inflammation are deemed
chronic inactive lesions.
The more recent gene expression studies have analyzed
distinct regions of plaques (margin versus center) and/or
with different degrees of activity (active versus chronic)
(3135). This more precise analysis has the potential to
provide greater insight regarding molecules/pathways
associated with pathogenesis (e.g. genes upregulated in the
margins of chronic active lesions compared to the margins of

inactive lesions) or with tissue repair and/or immunoregulation (e.g. genes upregulated in the center of chronic
inactive lesions compared to the center regions of active
lesions). For example, transcripts for CD4 and CD19 were
upregulated in the margins of chronic active versus inactivate
lesions but were not differentially expressed when comparing
the center regions of these lesions (32). In contrast, IL-10 was
upregulated in the center region of chronic inactive lesions
compared to the center of active lesions, but it was not
differentially expressed when comparing marginal zones of
these lesions. Thus, potentially important differences in gene
expression, such as the role of T-helper (Th) cells and B cells in
the pathogenesis of MS or the role of IL-10 in regulation of the
response, may be obscured if care is not given to the tissue and
cell populations used in the analyses. Indeed, data gleaned
from future studies of gene expression profiles associated
with MS plaques will be of greatest value if they can distinguish between molecules associated with pathogenesis and
those associated with regulation of the disease. As laser capture
microdissection and single-cell gene expression methodologies improve, it should soon be possible to assess gene
regulation of defined cell types (for example, infiltrating
T cells or resident astrocytes or microglia) that are present
within MS lesions.
Numerous studies employing recent advances in magnetic
resonance imaging techniques have clearly demonstrated that
there are abnormalities in normal appearing white matter
(NAWM) and gray matter that surround MS lesions (37),
and recent gene expression analyses of MS lesions and
NAWM confirm these observations (35, 38). Seventy percent
of differentially expressed genes in lesions or NAWM relative
to brain tissue from patients not affected by primary neurological disease were modulated in the same manner (35). In
MS lesions, the greatest proportion of differentially regulated
genes (37%) were those involved with neuronal homeostasis,
compared to only 21% of genes associated with humoral or
cellular immunity. In contrast, immune-related genes
comprised the greatest proportion of genes (40%) regulated
in NAWM. Downregulation of genes associated with antiinflammatory function [e.g. transforming growth factor b3
(TGF-b3) and Cre-bp1] was apparent in both lesions and
NAWM, suggesting widespread dysregulation of the CNS
inflammatory response. Consistent with other gene expression
studies of MS lesions, the upregulation of genes associated
with humoral immunity distinguished active demyelinating
lesions from NAWM, implicating antibodies in lesion formation and MS pathogenesis.
Regulation of CNS inflammation by resident cells of the CNS
For well over a decade, there has been considerable debate

about the relative influences microglia and astrocytes have on
CNS inflammation associated with MS. Many studies have
characterized the relative abilities of astrocytes and microglia
to process and present antigen or to influence Th-cell activation, differentiation, or apoptosis (39, 40). Increasingly, focus
is shifting to the roles of glial cells in regulation of CNS
inflammation. Glial cells may respond in predetermined manners to inflammatory cues, with the glial responses having
indirect but profound consequences on neurons and infiltrating lymphocytes. For example, lipopolysaccharide (LPS),
a molecular component of Gram-negative bacteria, has been
shown to induce neuronal and axonal loss both in vitro and in vivo
due to activation of microglia through Toll-like receptor 4 (41).
In another study, peripheral administration of LPS to female
rats resulted in a 240% increase in the density of activated
microglia in the dentate gyrus of the hippocampus, which
correlated with a 35% decrease in hippocampal neurogenesis
(42). Co-culture of neural progenitor cells with LPS-stimulated but not resting microglia inhibited neurogenesis in vitro
by approximately 50%, an effect that was mediated by
microglial secretion of IL-6. The effect of peripheral LPS
administration on neurogenesis was completely blocked by
systemic administration of the non-steroidal anti-inflammatory drug indomethacin. Similar results have been obtained
in another study, which documented an inverse correlation
between the number of activated microglia and new neurons in the hippocampus after CNS delivery of LPS (43).
Again, suppression of microglia activation, this time with
systemic administration of minocycline, reduced the number
of activated microglia and increased the number of new
neurons in the hippocampus. With respect to MS lesion
formation, IL-6 is upregulated in active lesions relative to
inactive lesions (29, 32) and could contribute to axonal
destruction. Indeed, IL-6-deficient mice are completely
resistant to EAE (44).
While both microglia and astrocytes become activated in
response to CNS inflammation, they may respond in different
ways to inflammatory stimuli (45). For example, microglia
but not astrocytes are responsible for production of the
Th1-promoting cytokine IL-12 (39). Aloisi et al. (46) have
demonstrated that astrocytes inhibit IL-12 secretion by in vitroactivated microglia, while others have demonstrated using
primary astrocyte/microglia co-cultures that astrocyte-derived
IL-10 inhibits LPS-induced secretion of the proinflammatory
molecules nitric oxide (NO), IL-6, tumor necrosis factor-a
(TNF-a), and IL-1b (47). Orian and colleagues (48) have
211
recently demonstrated that depending on the mouse strain used

(NOD/Lt or C57Bl/6), injection with encephalitogenic myelin
oligodendrocyte glycoprotein (MOG) peptide 3555 induces
disease that resembles relapsing/remitting or primary progressive
forms of MS. Subsequently, they have found that in the relapsing/
remitting form of disease induced in NOD/Lt mice, there is
evidence of both astrocyte and microglial activation prior to the
first clinical sign of disease (49). In contrast, microglia but not
astrocytes appear activated in the primary progressive form of
disease induced in C57Bl/6 mice. While preliminary, the data
suggest that differences in microglial and astrocytic responses to
inflammation could influence the pathogenesis of MS.
Astrocytes may also prove to indirectly suppress CNS inflammation by induction of regulatory populations of T cells.
A recent report demonstrated that primary rat astrocytes (or
astrocytoma cell lines) cultured for 24 h with primary T cells
inhibited T-cell secretion of interferon (IFN)-g in a cell
contact-dependent manner (50). T cells cultured with astrocytes in turn acquired a regulatory phenotype in that they
could inhibit Con A-induced T-cell proliferation in vitro and
ameliorate the severity of EAE induced with rat spinal cord
homogenate in vivo. Using ex vivo human malignant glioma
tumor specimens, we have recently demonstrated that astrocytederived tumor cells are notable for their secretion of the
immunosuppressive cytokines IL-10 and TGF-b and that
CD4CD25 effector cells that infiltrate the tumors are notable
for their secretion of IL-10 in the near absence of IFN-g
(D. Anderson, unpublished observations). Due to their
propensity to secrete IL-10, it is tempting to speculate that
astrocytes may naturally promote generation of IL-10-secreting
type 1 T-regulatory cells that could help suppress CNS
inflammation (51). It remains to be seen whether nontransformed reactive astrocytes can similarly induce regulatory
populations of lymphocytes and in which CNS inflammatory
diseases this may arise.
Regulatory T cells in MS
Clonal deletion of self-reactive T cells in the thymus and induction
of T-cell anergy alone do not explain the maintenance of immunologic self-tolerance, as potentially pathogenic autoreactive T
cells are present in the periphery of healthy individuals (12,
52). Thus, other regulatory mechanisms exist to prevent autoreactive T cells from causing immune disorders. Active suppression by regulatory T cells plays a key role in the control of
self-antigen-reactive T cells and the induction of peripheral tolerance in vivo (53, 54). Seminal experiments performed by Sakaguchi
et al. (55) have shown that depletion of CD4CD25 suppressor
212
cells results in the onset of systemic autoimmune diseases in

mice. Furthermore, cotransfer of these cells with CD4CD25
cells prevents the development of experimentally induced autoimmune diseases such as colitis, gastritis, insulin-dependent
autoimmune diabetes, and thyroiditis (56). We and others
have recently described a population of CD4CD25hi regulatory
T cells in human peripheral blood and thymus (57, 58). Human
CD4CD25hi T cells, similar to the mouse CD4CD25
suppressor cells, are anergic to in vitro antigenic stimulation and
strongly suppress the proliferation of responder T cells upon coculture (59). CD4CD25 T cells are among the best-characterized
immune regulatory subsets shown to prevent activation and
effector function of activated responder T cells (60).
While autoreactive T cells are present in healthy individuals
and patients with autoimmune disorders, autoreactive T cells
found in patients with autoimmune disease are more easily
activated as compared to those from normal subjects (60).
This finding led us to hypothesize that either deficient
generation or reduced effector function of CD4CD25hi regulatory T cells plays a role in the autoimmune state of patients
with MS. Thus, we recently compared the frequency and
function of CD4CD25hi T-regulatory cells derived from a
group of untreated patients who have relapsing/remitting
MS with those from age-matched healthy control subjects (61).
CD4CD25hi T cells are present with the same frequency in

healthy donors and patients with MS
We stained whole mononuclear cells from freshly drawn human

blood with different combination of anti-CD4-CyChrome,
anti-CD25-phosphatidylethanolamine, and a cocktail of fluorescein isothiocyanate (FITC) labeled anti-CD14, anti-CD32,
and anti-CD116. The cells were gated on lymphocytes via
their forward- and side-scatter features, and all FITC-labeled
cells were negatively selected during sorting. Human
peripheral blood contains a heterogeneous population of
CD4CD25 T cells that express either moderate levels of
CD25 consisting of non-regulatory T cells or high levels
of CD25 that exhibit regulatory function (57). As there are
no other known cell-surface makers able to identify regulatory
T cells ex vivo, we used CD25 expression to discriminate
regulatory T cells in humans. We analyzed the mean fluorescent
intensity of the CD25 population in both patients with MS and
control subjects and found no differences between the two groups.
Approximately 10% of CD4 T cells express the a-chain of IL-2
receptor CD25, but only 12% are CD25hi. No differences in the
frequency of CD4CD25hi T cells were found between patients and
healthy controls.
CD4CD25hi regulatory T cells display impaired function in

patients with MS
We also examined the production of cytokines in all the

cultures and the ability to inhibit their secretion by CD4CD25hi
T cells co-cultured with CD4CD25 responder cells. The secretion of the Th1 cytokine IFN-g, resulting from the activation of
destructive autoreactive T cells, was suppressed by CD4CD25hi
T-cell co-cultures from healthy controls but not in co-cultures
derived from patients with MS. IL-10 was variably secreted,
predominantly by the CD4CD25 T cells. The secretion of
IL-10 was often reduced upon co-culture with CD4CD25hi
T cells, excluding a potential role of this cytokine in mediating
this regulatory suppressor function. In this ex vivo model of
suppression, blocking IL-10 or TGF-b does not result in loss
of suppressor function by CD4CD25 regulatory T cells.
It was important to examine whether the loss of regulatory
function was due to a decrease in CD4CD25hi T-cell function
or an increase in the resistance of activated CD4CD25
responder T cells to inhibition. Thus, we performed mixing
experiments in which patient and control regulatory
CD4CD25hi T cells were co-cultured with the autologous
and the converse target cells isolated from either healthy
subjects or patients with MS. Regulatory T cells from patients
with MS could not suppress the proliferative response of target
responder T cells from either patients or healthy controls
(suppression 23%). In the reciprocal experiments, regulatory
CD4CD25hi T cells from healthy controls suppressed the
proliferative response of target CD4CD25 T cells derived
from both controls and patients with MS (suppression 78%).
These data indicate that the primary regulatory defect is in the
function of CD4CD25hi T cells isolated from the circulation
of patients with MS.
As it was critical to examine the regulatory T-cell function, we

isolated highly pure CD4CD25hi regulatory and CD4CD25
responder cell populations by high speed flow cytometric sorting. CD4CD25 responder cells from both patients with MS
and healthy individuals responded similarly in a dose-dependent
fashion to varying concentration of plate-bound anti-CD3
monoclonal antibody (mAb). CD4CD25hi T cells isolated
from both groups were anergic to stimulation at all doses of
plate-bound anti-CD3, indicating that CD4CD25hi T cells isolated from patients with MS do exhibit this regulatory property.
To quantitate their regulatory function, CD4CD25hi T cells
were co-cultured with autologous responder cells (2.5 103/
well) at different ratios (responder/suppressor ratio 1 : 1, 1 : 1/
2, 1 : 1/4, and 1 : 1/8). As previously reported in healthy
individuals, CD4CD25hi T cells consistently suppressed
proliferation at a 1 : 1 ratio (Fig 1). Increasing the ratio of
responder/suppressor T cells resulted in less suppression. In
striking contrast, the regulatory CD4CD25hi T cells isolated
from the circulation of patients with MS, while normal in
frequency as compared to healthy controls, poorly inhibited
responder CD4CD25 T-cell proliferation. As we have
previously shown that increased strength of signal inhibits
regulation (62), we stimulated co-cultures of regulatory
and responder T cells from patients with MS and healthy
controls with a maximal concentration (2.5 mg/ml) of platebound anti-CD3 mAb. As predicted, the CD4CD25hi regulatory T cells no longer suppressed the proliferation of responder
T cells (Fig. 2).
Percentage of suppression
of proliferation
80
(0.1 g/ml)
60
(2.5 g/ml)
(0.5 g/ml)
P = 0.002
Healthy controls
Patients with MS
P = 0.04
P = 0.003
P = 0.0008
P = 0.01
40
P = 0.01
20
0
1:0
1 : 1/4
1 : 1/2
1:1
1:0
CD4+
hi
1 : 1/4
CD25
Fig. 2. Summary of CD4 CD25 T-cell regulatory function that is

altered in patients with multiple sclerosis (MS). The mean percent
inhibition of the proliferative response by CD4CD25hi cells derived
from 15 MS patients (open circles) and 21 healthy controls (filled
squares) was calculated. CD4CD25 and CD4CD25hi populations were
stimulated with plate-bound anti-CD3 mAb alone or co-cultured at
varying ratios. The proliferative response was inhibited upon addition of
1 : 1/2
/CD4+
1:1
CD25hi
1:0
1 : 1/4
1 : 1/2
1:1
ratio
CD4 CD25 cells to the CD4CD25 responder cells at 1 : 1 ratio in

normal controls. Regulatory T cells from patients with MS exhibited
significantly less suppressive activity. Decreasing the number of
CD4CD25hi T cells (responder/suppressor ratios 1 : 1/2 and 1 : 1/4)
resulted in less suppression in all the conditions examined. Reprinted
with permission from the Journal of Experimental Medicine,
2004;199:971979.
hi
213
CD62L expression on CD4CD25hi regulatory T cells
Although there were no differences in the frequency of

CD4CD25hi T cells or in their proliferative or cytokine secretion in response to different stimuli between healthy subjects
and patients with MS, it was important to determine whether
an increase in the frequency of activated CD4 T cells in the
circulation was diluting the regulatory CD4CD25hi T cells.
Therefore, we used CD62L expression to further purify
regulatory from the activated T cells, because l-selectin
expression is downregulated upon activation. We isolated
CD4CD25hiCD62L and total CD4CD25hi regulatory T cells
from healthy subjects and patients with MS. Whereas in
the healthy controls both populations were able to suppress
the proliferative response to anti-CD3 stimulation, the
CD4CD25hi regulatory cells isolated from patients with MS,
although further depleted of the potentially activated CD62L
T cells, were still unable to inhibit the proliferation of the
CD4CD25 responder population. These data strongly
confirm a defect in the highly purified regulatory subset in
patients with MS.
Discussion of CD4CD25 T cells in patients with MS
An important aspect of these investigations was the measurement of regulatory T-cell function as opposed to simple
phenotypic measurement of CD4CD25 T-cell frequency.
Unlike 6-week-old mice raised in clean facilities, whose total
CD4CD25 T-cell population manifests regulatory properties, humans are exposed to a myriad of infections and show a
significant population of CD4CD25medium/low T cells that do
not exhibit regulatory function (57). Thus, it was critical to
examine the functional state of the regulatory T cells expressing high levels of CD25. We have previously demonstrated
that the strength of signal delivered through the TCR of target
T cells is one factor determining whether regulatory
CD4CD25hi T cells can suppress the responder T-cell proliferation (62). Thus, to properly examine the function of
regulatory CD4CD25hi T cells, we used a number of different
strengths of stimulatory signals in these experiments.
We observed that the strong signal provided by maximal
concentration of plate-bound anti-CD3 mAb similarly
abrogated suppression in both patient and control co-cultures.
In contrast, lower concentrations of plate-bound anti-CD3
delivered a signal that resulted in the appearance of a significant defect in the suppressive function of this subset of
regulatory cells derived from patients with MS.
The use of different stimulatory conditions allowed us to
reveal alterations in the regulatory function of CD4CD25hi
214
T cells while still demonstrating that they are CD25 regulatory T cells as opposed to activated responder cells expressing
CD25. Stimulation of cultures with soluble anti-CD3 and antiCD28, which has previously shown to be the most permissive
for enabling co-culture suppression, gave equivalent levels of
suppression in patients and control subjects when co-cultured
at 1 : 1 ratios. In contrast, the stimulation provided by platebound anti-CD3 at 0.1 and 0.5 mg/ml resulted in a threefold
decrease of suppression by CD4CD25hi cells derived from
patients with MS as compared to normal controls. Previous
experiments showed that delivering a low strength TCR signal
to responder T cells, such as that provided by self-antigens as
compared to microbial antigens, resulted in a greater sensitivity to suppression. Thus, the present findings may help explain
defects in suppression of autoreactive T cells in autoimmune
patients as compared to T cells stimulated by microbial antigens during infections. An important control to note in all
these experiments is the anergy or lack of thymidine incorporation resulting from stimulation of CD4CD25hi T cells
cultured alone. This anergy indicates that CD4CD25hi
T cells isolated from patients with MS are not CD25 activated
T cells; such cells would not exhibit regulatory activity but
rather enhance proliferation. It was critical to determine
whether the decrease in T-cell regulatory function observed
in patients with MS was due to a defect in the CD4CD25hi
T-cell subset or whether the responder CD4CD25 T cells
were refractory to suppression. By performing co-mixing
experiments, we could clearly demonstrate that the defect
lies in the CD4CD25hi T-cell function, as opposed to
enhanced responder T-cell resistance in patients with MS.
Although in vitro measurement of biologic function in
patients with autoimmune diseases will always be correlative,
these in vitro experiments, based on in vitro and in vivo
experimentation in mouse models of autoimmune disease,
provide the first definitive evidence for a defect in regulatory
T-cell function in a human autoimmune disease. Ultimately,
monitoring the effects of immunomodulatory drugs on this
regulatory T-cell subset will help define their pathogenic role
in MS and other human autoimmune diseases.
Autoantibodies in patients with MS

While accumulating studies of MS and EAE have led to the
characterization of MS as a cell-mediated immune disorder and
have directed the focus to the role of T cells in disease pathogenesis, an abnormal humoral immune response has also been well
described in MS patients. A renewed interest in the possible
contribution of B cells to MS immunopathology has been
sparked with more recent findings in MS pathological studies. In

the past, the role of B cells in MS pathology was viewed by some
as an epi-phenomenon of a dysregulated immune system. More
recently, convincing data is mounting that has shaped a picture
in which the abnormal humoral responses described in MS are
directly involved in disease pathogenesis.
The humoral response in the MS CNS
Intensive investigation into the role of the humoral response in

the pathogenesis of MS followed Kabats report (3) in the
1940s that the CSF of patients with the disease contained
elevated levels of Igs. This seminal observation has been
consistently reproduced, such that the detection of elevated
levels of CSF Ig is still used to support the diagnosis of MS in
some cases. The elevated Ig levels are mostly due to increased
synthesis of IgG (63), with lesser elevations also observed for
the IgM (64), IgD (65), and IgA (66) isotypes.
B cells and plasma cells have been identified in MS lesions
(23, 67), along with antibodies (68, 69) and Ig transcripts
(31). MS is heterogeneous not only in its clinical course but
also in the histological appearance of the lesions. As discussed
above, recent work by Lassmann and colleagues (70) demonstrated that MS lesions can be grouped into four distinct patterns, based primarily on the degree of demyelination and
oligodendrocyte loss. The most common patterns (I II) are
characterized by perivenular accumulation of T cells, macrophages, plasma cells, and perivenular demyelination. Pattern II
differs from pattern I by the prominent deposition of Igs
(primarily IgG) and the terminal complement complex
C9neo on the myelin sheath, which is indicative of
antibody/complement-mediated destruction of the myelin
sheath. In lesions classified as patterns III and IV, oligodendrocyte
loss is pronounced while demyelination is less prominent.
Molecular studies of B cells and plasma cells within CNS tissue
and CSF uncover characteristics strongly indicating that these cells
have undergone T-cell-mediated antigen-driven clonal expansion
(reviewed in 71, 72) (Table 1). This expansion includes intrathecal
Ig synthesis (73) and the presence of IgG oligoclonal bands in the
CSF (74, 75). Studies designed to characterize the variable regions
(VH) of Igs expressed in MS plaques (7679) and CSF (8082)
revealed that the variable regions are biased in family representation, extensively mutated, and oligoclonal.
CSF Ig reactivity
Substantial effort has been invested to elucidate the antigenic
specificity of MS CSF Ig. Much of the earlier research in this
field focused on exogenous antigens and on the identification
of antibodies within the elevated CSF Ig directed against viruses

and bacteria. This investigation was fueled by early reports that
measles antibodies could be detected in MS sera (83).
Since then, oligoclonal IgG antibodies that react with a range
of viruses, including measles, mumps, herpes simplex virus
type-1 (HSV-1), varicella-zoster virus, cytomegalovirus, and
rotavirus, have been reported in some MS patients (84).
Antibodies reactive towards EpsteinBarr virus antigens are
present in greater than 80% of CSF samples from MS patients
(85). Antibodies directed against b-hemolytic Streptococcus,
Hemophilus influenzae type B, Escherichia coli, and Enterococcus have
also been reported (86). However, in all these studies, the viral
and bacterial antibodies constitute only a minor fraction of the
elevated MS CSF Ig (87). In contrast, in subacute sclerosing
panencephalitis (SSPE), much of the intrathecal Ig is directed
against the measles virus (88), and in HSV-1 encephalitis, the
elevated CSF Ig is largely accounted for by antibodies directed
against the herpes virus (89). Furthermore, the anti-microbial
antibodies identified in MS CSF have a lower antigen affinity
than their counterparts in SSPE and HSV encephalomyelitis
(90). These observations imply that the production of CSF Ig
in SSPE and HSV-1 is antigen driven and directed against the
causative infectious agent. In MS, however, the driving
mechanism behind the Ig elevation has not been successfully
assigned to a single exogenous antigen.
The possibility that CSF Ig in MS patients is generated as a
response to myelin self-antigens has also been considered.
Antibodies specific for MBP were in fact first identified some
25 years ago in the CSF (91, 92) as well as in CNS tissue (93)
of patients with MS. In another study, autoantibodies to MBP
were detected in the CSF of >90% of MS patients with active
disease, while they were undetectable in 98% of non-MS CSF
(94). These results contrast others; several studies report their
absence (95, 96). Adding further uncertainty, screening of
antigen libraries has revealed that MBP epitopes were not
among those identified as having reactivity with CSF-derived
Ig (79, 97).
Antibodies directed to PLP are also found in MS CSF, though,
curiously, it seems that anti-MBP and anti-PLP antibodies are not
present simultaneously in a given patient (98, 99). Whether this
observation reflects the gradual spread of the antigenic focus in MS,
(epitope spreading) remains to be established. Autoantibodies reactive with myelin-associated glycoprotein (100), the
enzyme transaldoase (TAL) (101) and with oligodendrocytespecific protein (OSP) (102) have also been detected in the CSF
of some MS patients. In one study, high-affinity autoantibodies
directed against TAL were detectable in CSF of 15/20 patients
with MS, while absent in 145 normal individuals and patients
215
Table 1. Summary of reports providing evidence for a role of B cells in the immunopathology of multiple sclerosis (MS). This summary is
mostly limited to studies focusing on the central nervous system (CNS) rather than the periphery where the role of B cells and antibodies
is more controversial
Evidence supporting a role for B cells in the immunopathology of MS
References
Intrathecal immunoglobulin synthesis

The presence of IgG oligoclonal bands (used as a diagnostic test)
B cells and plasma cells in active and late MS lesions
(73)
(74, 75)
(23, 67, 208) OConnor KC,
Bregoli L, and Bailey GP
(unpublished observations)
(68)
Antibodies are found in active multiple sclerosis CNS lesions

B cells in the cerebral spinal fluid (CSF) and brain lesions of multiple sclerosis patients suggest that there is
a T-cell mediated, antigen-driven clonal expansion of B cells. Induction of immune effector mechanisms by these
antibodies is evidenced by:
(1) capping of surface IgG on macrophages involved in myelin breakdown;
(2) codeposition of IgG and complement, particularly the activated terminal lytic complex at plaque borders;
(3) the presence in CSF of membrane attack complex-enriched membrane vesicles, indicating a role for
complement-mediated injury in MS.
The H-chain V regions (VH) of IgG expressed in MS plaques and CSF revealed a limited repertoire with features
of a targeted B-cell response: VH sequences from MS plaques and CSF are oligoclonal, extensively mutated, and
derived in part from clonally expanded B-cell populations, indicative of antigenic stimulation
Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) were found bound to disintegrating myelin
segments in MS lesions, providing evidence that myelin autoantibodies may, in part, mediate CNS tissue damage
IgG isolated from CNS tissue binds to myelin basic protein (MBP) in solid-phase assays, and an MBP (8397)
peptide was capable of significantly inhibiting this binding
The specificity of two recombinant antibodies derived from the CNS lesions of patients with MS was
determined to be directed toward double-stranded DNA
CSF-derived IgG reacted with antigens unrelated to any known protein (using phage-displayed random peptides).
IgG from different MS patients bound different peptides, authors to speculate that the CSF antibody
repertoire is individual-specific
Plasma cells present in the CSF of patients with optic neuritis express Ig with features of antigen-driven stimulation.
ON often precedes MS. This may be early MS or a CIS
Ig isolated from MS CNS lesions recognize folded MOG first study demonstrating binding to MOG in native
conformation including glycosylation by Ig isolated from lesions
Serum IgM antibodies to MOG may serve as markers for conversion of CIS to MS
MS lesion pathology is categorized into four subtypes. The most common, type II, is characterized (in part) by
Ig deposits, complement and B cells
Ig transcripts are abundant in MS lesions
CIS, clinically isolated syndrome.
with other autoimmune and neurological diseases (101). TAL is

expressed in oligodendrocytes and is of interest because it shares
amino acid sequence homologies with core proteins of human
retroviruses. The putative B-cell epitope of OSP shares sequence
homology with several viral peptides. These reports suggest a
role for molecular mimicry in the development of MS CSF Igs. In
all of these studies, however, only a minor component of the
total Ig measured in MS patient CSF samples could be explained
by antibodies directed against the myelin self-antigens under
question.
Serum autoantibodies in MS
The detection of autoantibodies directed against myelin antigens in the serum of patients with MS has been elusive.
This difficulty may in part relate to a tendency to form
immune complexes which hampers their detection (103).
Serum anti-MBP antibodies have been reported (104, 105),
though they were not detected in all studies (95, 106, 107).
Investigators have reasoned that negative results reported from
216
1. (209)
2. (69, 70, 210)
3. (211)
(7682)
(212)
(93)
(79)
(97)
(213)
(143)
(130)
(70)
(31)
searches for autoantibodies may be due to the deposition of

the relevant antibodies in the CNS that may act as a sink.
Certainly, low affinity and low concentration may be hampering detection. Nearly all of the investigations concerning MBP
autoantibodies in MS have used assays in which the antigen
is immobilized on either a plastic plate [enzyme-linked
immunosorbent assay (ELISA)/solid phase radioimmunoassay
(RIA)] or a membrane (Western/immunoblot). Immobilization of protein antigens can result in the loss of epitopes due to
protein denaturation; immobilization may also expose new
epitopes not otherwise present in the native conformation
(108110). In addition to high affinity antibodies, ELISAs
are likely to measure antibodies of low affinity due to the
excess antigen fixed to the plate and the multimeric presentation of the antigen to the Ig (110).
We characterized autoantibodies to MBP by comparing a
solution-phase assay to a solid-phase assay. The solutionphase assay consistently measured autoantibodies in serum
from human subjects with Semple rabies vaccine (SRV)induced demyelinating disease or from MBP-immunized
rodents. Autoantibodies capable of solution-phase binding to

recombinant or human MBP were not detected in the serum or
CSF of patients with MS. Using an ELISA technique, sera- but
not CSF-autoantibodies to MBP were detected in a small number of patients with MS. Additional solution-phase measurements revealed that anti-MBP antibodies from individuals with
SRV-induced demyelinating disease or from an immunized
rodent shared the same binding affinity profile expected for
polyclonal antibodies with a range of affinities from low to
high. In contrast, antibodies to MBP in the serum of MS
patients detected by ELISA did not bind soluble MBP in the
same assay (Fig. 3). These results indicate that the humoral
response in patients with MS does not include moderate- or
high-affinity autoantibodies to the immunodominant antigen
MBP in the serum or CSF.
There are sporadic reports detecting serum autoantibodies
against recombinant TAL (101), and B cells secreting autoanti-
500
450
400
350
cpm
300
250
200
150
100
50
0
n=
NHD
(23)
MS
(53)
OND
(17)
APL
(7)
SRV
(6)
Fig. 3. The level of serum autoantibodies to myelin basic protein

(MBP) from multiple sclerosis (MS) patients and controls, measured
by a solution phase radioimmunoassay (RIA). Each data point
represents the mean of two separate experiments. NHD, normal
healthy donors; MS, multiple sclerosis; OND, other neurological diseases;
APL, patients who received an altered peptide ligand of MBP; SRV, sera
from individuals with Semple rabies vaccine-induced demyelinating
disease. Anti-MBP serum from an immunized rabbit showed in a Dcpm
value of 8000 within the same assay; serum from a non-immunized rabbit
serum produced a Dcpm value of 0.00. Reprinted with permission by the
Journal of Neuroimmunology, OConner KC et al. 2003;12:140148.
bodies directed against PLP have been identified in the peripheral

blood of MS patients (111). Autoantibodies to other putative MS
antigens, such as myelin oligodendrocytic basic protein (MOBP),
CNPase, ab-crystalline, and S-100b, have not been analyzed in
sera (112). Autoantibodies directed against non-myelin antigens
have also been detected in the sera of some patients with MS.
Anti-nuclear antibodies (113), anti-cardiolipin antibodies (114),
and antibodies against b-2-glycoprotein I (115) are detected in
MS sera more frequently than in normal individuals. Recently,
elevated autoantibody levels towards a large panel of organ- and
non-organ-specific antigens were measured in MS patients relative to controls (116). The pathogenic relevance of these observations is not established, and a form of non-specific systemic
immune dysregulation is suspected.
MOG as a B-cell antigen in MS

Among the candidate antigens, MS, MOG arguably remains the
most important, because it is the only one that can induce
demyelinating EAE through both B- and T-cell driven mechanisms. MOG is accessible to autoantibodies in intact myelin as it
is located on the surface of the myelin sheath and the plasma
membrane of oligodendrocytes. MOG is a type I membrane
protein that represents a minor fraction of the total protein in
the myelin membrane, and it is expressed exclusively in the
CNS. The extracellular domain adopts a classical Ig fold and
contains a single site for N-linked glycosylation (117).
B cells and antibodies are not absolutely required for the
development of EAE, as spontaneous lesions develop in MBP
TCR transgenic recombination activating gene (RAG)-1/
mice, which lack B cells (118), and the disease thus can be
induced in B-cell deficient mice (119). However, autoantibodies to myelin antigens may nevertheless be highly relevant
in CNS demyelinating diseases in the more complex setting of
an intact immune system. The potential of MOG autoantibodies to induce severe demyelination and oligodendrocyte
loss was convincingly demonstrated by passive transfer of antiMOG mAb (818C5) into mice or rats with mild clinical signs
of EAE (120, 121), and demyelination was shown to be dependent on the coordinate action of myelin-specific T cells and
autoantibodies. Antibodies to MOG also appear to play an
important role in the chronic, relapsing/remitting disease
process in the marmoset EAE model induced by immunization
with the extracellular domain of MOG (122, 123), and large
demyelinated lesions are observed in this model that resemble
MS plaques.
Of course, a question central to the role of B cells in MS
pathology concerns the specificity of the autoantibodies, and
217
due to its role in EAE, a significant amount of effort in this

regard has centered on MOG. The presence of high-affinity,
specific autoantibodies, almost invariably seen in other organspecific autoimmune diseases, has been difficult to demonstrate as only low-affinity, poorly characterized antibodies
binding to MOG have been reported. In some studies, antibodies directed against MOG are not detectable in the serum of
patients with MS (124, 125). Conversely, in other studies,
they were detected; however, they were also found in nonMS controls, including normal subjects and/or patients with
other neurological diseases (104, 126129). Berger and colleagues (130) have reported a series of studies in which they
examined serum and CSF samples using Western blot analysis
of recombinant MOG extracellular domain. Antibodies to
MOG were detected in 38% of MS patients, 53% of patients
with other inflammatory CNS diseases and 3% of patients
with non-inflammatory CNS diseases. The major limitations
of this method are that large amounts of recombinant protein
were loaded per lane, and that no control proteins were
included on these blots to assess the level of background
binding, an issue that is relevant because colorimetric detection is subjective. A recent study by Berger et al. (130) reported
that the development of clinically definite MS can be predicted
based on serum IgM antibodies to MOG in patients with a
clinically isolated syndrome. A recent study from another
group failed to confirm these findings (124). Patient populations are often cited as cause for such discrepancies, but the
basis for incongruity in the results is likely to lie in the antigen
preparation and/or the assay conditions.
All of these reports utilized either MOG peptides or denatured MOG protein. The antigen thus lacked its native secondary structure or was deliberately denatured by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for
Western blot analysis. The importance of protein conformation for MOG autoantibody binding was demonstrated by
Haase et al. (131), who used the extracellular domain of
MOG isolated from inclusion bodies expressed in E. coli to
affinity-purify antibodies from serum samples. The recombinant protein used for affinity purification had not been
refolded to create the disulfide bond that is critical for the
structure of an Ig domain, and only one of 17 samples bound
to native MOG on the surface of transfected cells, even though
antibodies from all MS patients and control subjects bound
synthetic MOG peptides.
Post-translational modifications, such as glycosylation, can
affect the three-dimensional structure and the antigenic properties of proteins including MOG (132134). Human MOG
antibodies identified with denatured protein or synthetic pep-
218
tides may not bind to the native protein and may thus not be
capable of inducing demyelination. The importance of native
structure is highlighted by numerous EAE studies demonstrating that pathogenic antibodies bind to conformationdependent epitopes (135137) and that such antibodies are
essential factors in CNS disease dissemination (138). In other
human autoimmune diseases, it is well established that autoantibodies recognize conformational epitopes of self-antigens,
and relevant examples are GAD65, transglutaminase and the
acetylcholine receptor (139141). The crystal structure of
MOG with the Fab fragment of the demyelinating 818C5
antibody demonstrated that the antibody binds to a conformational epitope created by three loops on the membrane-distal
side of MOG (142). The glycosylation site of MOG, Asn-31, is
located in a loop at the top of the membrane-distal side of
MOG and the contribution of this post-translational modification, therefore, needs to be considered in an evaluation of
MOG autoantibodies in MS.
Considering these essential criteria for pathogenic antibodies, our laboratory tested the hypothesis that autoantibodies recognizing MOG are produced in the CNS of patients
with MS. We isolated IgG from the CNS parenchyma of MS
cases and examined autoantibody binding with preparations of
folded MOG protein in both solid-phase and solution-phase
assays. The results demonstrate that antibodies to MOG are
primarily found in the CNS parenchyma of patients with MS,
rather than CSF or blood (143). Antibodies synthesized at this
site may diffuse into the CSF, but considerable dilution is likely
to take place given the large volumes of CSF that are produced
daily. Therefore, detection in this fluid or sera is difficult.
Novel therapeutics in MS
Therapy overview
The majority of patients present with relapsing/remitting

disease, often with a single clinical event. Unlike in other autoimmune diseases such as diabetes where the majority of the
pancreatic islet cells are destroyed with clinical presentation,
patients with MS are now identified relatively early in the
disease, allowing manipulation of the immune system in disease prevention. In relapsing/remitting MS, patients have clinical exacerbations followed by partial or complete recovery of
function. Whereas with progressive MS, patients have a gradual accumulation of disability with or without superimposed
relapses. Progressive disease can further be divided into primary progressive, where progression is present from onset, or
secondary progressive, where the patients start with a relapsing/remitting disease before progressing.
Genetic factors associated with the development of MS are

discussed later in this review; it has been postulated that one
environmental factor contributing to the development of MS is
an immune response to a foreign antigen that is cross-reactive
to self-antigen(s) (MBP or MOG). As discussed at length
above, it is thought that T-cell recognition of peptides derived
from myelin proteins in the context of major histocompatibility complex (MHC) presented by microglia, the local antigenpresenting cells, is involved in the diseases pathogenesis.
There are only six FDA approved treatments for relapsing/
remitting MS: two IFN-b1a agents (Avonex1 and Rebif1), one
IFN-b1b (Betaseron1), glatiramer acetate (Copaxone1), antiVLA-4 monoclonal antibody (Tysabri1) and mitoxanthrone
(Novantrone1) for advanced cases. For patients with secondary
progressive disease, immunosuppressive treatments such as cyclophosphamide and mitoxanthron are prescribed. These treatments
are only modestly efficacious and can be associated with significant toxicity, often causing patients to delay therapy for significant
lengths of time. A potential exception is the anti-VLA-4 monoclonal antibody treatment, that may be a significant advance in the
therapy of patients with MS. Thus, there is a need for developing
therapies with low toxicities that can be administered early during
the disease course with the potential for arresting the disease.
With our ever more complete yet continually evolving understanding of the origins and mechanisms of MS, the development
of such novel therapies is a common goal in the field.
Earlier in this review, we discussed in detail how the cells of the
immune system are involved in damaging the CNS, resulting in
the varied and debilitating symptoms of MS. An ideal therapy
seeks to prevent the initiation of such attacks, to halt such attacks if
in progress, and optimally, to reverse the damage done. Because
the disease is the result of a dysregulated immune system, rational
therapy would be aimed at correcting the underlying dysfunction.
Immunotherapy refers to therapy seeking to alter the immune
response to prevent or treat the disease in question. Thus, unlike
treatment of other diseases, such as some types of cancer, in the
case of MS almost any promising therapy will be termed an
immunotherapy, due to the autoimmune nature of the disease.
We can envision several broadly defined categories of immunotherapies with potential for the successful treatment of MS.
We next summarize the treatment options for MS and discuss
the rationale for each, where applicable summarizing data that has
accumulated regarding use of a particular therapy.
Cytokines and costimulatory signals: manipulating the milieu

It is well known that the cytokine milieu affects the outcome
of T-cell stimulation. EAE, an animal model of MS, is mediated
by Th1 type T cells secreting the cytokines IL-2, IFN-g, TNF-a,

and lymphotoxin. Disease recovery is associated with Th2 type
T cells secreting the cytokines IL-4, IL-10, and TGF-b1 (144,
145). The type of effector T cell that is generated is determined by the cytokine and costimulatory microenvironment
at the time of primary stimulation (146). IL-12 and B7-1
costimulation is important for differentiation into Th1 cells,
whereas IL-4 and B7-2 induce a Th2 response (60, 147149).
These types of treatments aim to influence the milieu in
which the antigen-specific T cells responsible for CNS damage
are stimulated. This manipulation could be accomplished in
several ways, including preventing the interaction of specific
inflammatory cytokines with their receptors or by targeting
the cytokines themselves with antibodies, such that they are
degraded and ineffective. Those cytokines involved in exacerbating disease and promoting inflammation would be neutralized. Interfering with TNF-a has been used to treat
rheumatoid arthritis (RA) with great success, and anti-IFN-g
may also work in RA (150, 151). However, attempts to
systemically block TNF have been ineffective in treating MS;
such treatment worsened disease (152). In addition to its
proinflammatory characteristics, TNF also appears to be
capable of immunosuppressive activity. Because the latter
qualities of TNF do not require the p55 TNF receptor while
the former appear to, specific blockade of the p55 TNFR may
be a more effective treatment option (153).
b-IFNs have similarly had an impact on the treatment of
relapsing/remitting MS, although whether they can prevent
the transition to secondary progressive MS is not yet known.
The mechanism of action of b-IFN is also not as yet worked
out, and it likely involves alterations of a number of different
pathways including induction of IL-10 and inhibition of T-cell
traffic by blocking metalloproteinases (154). Clinical trials that
block the common IL-12 and IL-23p40 chain are about to
begin, as are efforts to block costimulatory signals provided by
B7CD28 interactions using cytotoxic T-lymphocyte antigen
(CTLA)-4-Ig fusion protein.
Antibodies to the IL-2 receptor are also strong therapeutic
candidates in this category. A clinical trial with anti-IL2R
showed promising results (155). Another theoretical approach
to manipulating the cytokine milieu would be to introduce a
cytokine that reduces inflammation, thereby inhibiting or
redirecting the immune response.
Peptides bound to MHC as therapeutic options
It was recognized almost a decade ago that the strength of

signal delivered through the TCR determines which cytokines
219
are secreted by the T cell (156). The cell apparently measures

affinity in part by timing the engagement between the TCR
and the peptide/MHC complex. With longer engagement, a
qualitatively different TCR complex has time to form, and the
extent of z-chain phosphorylation increases correspondingly
(157). Altered peptide ligands (APLs), which bind with low
affinity to the TCR, weaken this signal. The ability of APLs to
change the cytokine program of a T cell from a Th1 to a Th2
response was exploited first by Kuchroo and coworkers (158)
as a therapy for autoimmune disease. Using the EAE model of
MS, these authors showed that APLs can activate IL4 secretion
by both encephalitogenic T cells and nave T-cell clones that
cross-react with self-antigens.
Injection of APLs is of clear therapeutic value in treating
different models of EAE (159, 160), and autoreactive human
T-cell clones can also be induced to secrete the anti-inflammatory cytokines IL-4 and TGF-b after TCR engagement by APLs
(161, 162). However, it was noted that while APLs can induce
Th2 cytokine secretion of MBP-reactive T cells isolated from
the peripheral blood of patients with MS, they can also induce
a heteroclitic response in some patients, activating these MBPreactive T cells against the patients own tissues (163). These
data provide a strong rationale for the therapeutic use of APLs
in patients with autoimmune disease. However, they also raise
the issue that in some instances, highly degenerate TCRs can
recognize APLs as self-antigens.
A recently published phase II clinical trial testing an altered
MBP p8599 peptide confirms both of these conclusions. At
the higher peptide dosage tested, two of seven MS patients
developed remarkably high frequencies of MBP-reactive T cells,
and these responses were likely associated with significant
increases in magnetic resonance imaging (MRI)-detectable
lesions (164). In contrast, patients treated with lower doses
of the APL showed no such disease flare-ups and indeed may
have exhibited some degree of immune deviation towards
increases in IL-4 secretion of MBP-reactive T cells (164,
165). Thus, APLs represent a classic double-edged sword. In
our outbred population, given the high degree of degeneracy
in the immune system, it is unclear whether it is possible to
find APLs of self peptides that pose no risk of cross-reactivity
with self.
An alternative approach to the use of a single APL is the
administration of peptide mixtures that contain many different
antigen specificities. The use of random copolymers that contain amino acids commonly used as MHC anchors and TCR
contact residues have been proposed as possible universal
APLs. Glatiramer acetate (Copaxone1) is a random sequence
polypeptide consisting of four amino acids [alanine (A), lysine
220
(166), glutamate (E), and tyrosine (Y) at a molar ratio of

A:K:E:Y of 4.5 : 3.6 : 1.5 : 1] with an average length of
40100 amino acids (167). Directly labeled GA binds
efficiently to different murine H-2 I-A molecules, as well as
to their human counterparts, the MHC class II DR molecules,
but it does not bind MHC class II DQ or MHC class I molecules
in vitro (168). In phase III clinical trials, GA, subcutaneously
administered to patients with relapsing/remitting MS,
decreases the rate of exacerbations and prevents the appearance
of new lesions detectable by MRI (169, 170). This study
represents perhaps the first successful use of an agent that
ameliorates autoimmune disease by altering signals through
the TCR.
A universal antigen containing multiple epitopes would be
expected to induce proliferation in vitro in nave T cells from
the circulation, due to its expected high degree of cross-reactivity with other peptide antigens. Indeed, GA induces strong
MHC class II DR-restricted proliferative responses in T cells
isolated from MS patients or from healthy controls (168). We
and others found that in most patients, daily injection with GA
causes a striking loss of responsiveness to this polymer antigen, accompanied by greater secretion of IL-5 and IL-13 by
CD4 T cells, indicating a shift toward a Th2 response (171
174) (Fig. 4). In addition, the surviving GA-reactive T cells
exhibit a high degree of degeneracy, as measured by their
ability to cross-react with a large variety of peptides represented in a combinatorial library (172).
In vivo administration of GA induces highly cross-reactive
CD4 T cells that are immune-deviated to secrete Th2 cytokines. We have proposed that GA-induced migration of highly
cross-reactive Th2 (and perhaps Th3) cells to sites of inflammation allows their highly degenerate TCRs to contact selfantigens, which they recognize as weak agonists, much like
APLs. These T cells then apparently secrete suppressive, Th2/
Th3 cytokines, thus restricting local inflammation. Knowledge
of the strong genetic association for MHC in patients with MS
has indirectly led to a number of therapeutic trials and new
insights into the disease.
Prevent trafficking
Because local responses of T cells are central to the symptoms
of the disease, preventing the T cells from gaining access to the
potential site of inflammation (CNS) would be an effective
therapy. It was shown over 20 years ago that the very late
activation (VLA) antigens were expressed on T cells from
patients with MS (175) and almost a decade ago that the
VLA-4 a4b7 integrin, was critical for T-cell traffic into the
Proliferation
pre
Exp. 3
120
90
60
30
0
120
90
60
30
0
n.d.
1 10 100 0 1 10 100 0 1 10 100

2 stimulation : glatiramer acetate [g/ml]
0
B
pre
1 month
3 months
10000
7500
5000
2500
0
10000
7500
5000
2500
0
pg/ml
cpm 103
Exp. 2
10000
7500
5000
2500
0
0
1 10 100 0 1 10 100 0 1 10 100

IFN-
4000
3000
2000
1000
0
Exp. 1
pg /ml
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Exp. 3
3 month
120
90
60
30
0
Exp. 1
IL-13
C
1 month
4000
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0
[Glatiramer acetate] :1 stimulation

no antigen
1 g/ml
4000
3000
2000
1000
0
n.d.
10 g/ml
100 g/ml
1 10 100 0 1 10 100 0 1 10 100

Fig. 4. GA-specific secretion of cytokines is polarized toward a Th2

response after daily injections of GA. The GA-specific secretion of the
cytokines interleukin (IL)-5 and interferon (IFN)-g was measured in
T-cell lines by two methods: ELISPOT (A) and ELISA (B) assays. Each
symbol represents the difference of spots counted or Dpg/ml measure in
split-well assays between the GA (20 mg/ml) condition and the
no-antigen control. The limits of detection were 1 spot and 10 pg/ml,

respectively. Numbers represent the percentage of T-cell lines in each
quadrant with a minimum difference in spots of twice the SD of
the negative controls for IL-5 and IFN-g, respectively. Reprinted
with permission by the Journal of Clinical Investigation,
2000;105(7):967976.
CNS of mice with EAE (176). The results were a highly

successful phase two trials and phase three trials of anti-VLA-4
in patients with recurring remittent MS (176), which is now
in phase three investigations. This excellent example shows
how the EAE model, if used to ask the correct question, might
be highly useful in developing therapies for MS.
MS (61) (from previous section). These cells are thought to

have a role in immune homeostasis, that is, in tempering the
immune response and preventing postimmune response
inflammation, etc. Therefore, a functional lack of CD4CD25hi
T cells in MS patients is likely to contribute to the pathophysiology of the disease. Thus, it follows that reversing this lack of
functional regulatory cells would be a viable and productive
treatment avenue. Unfortunately, while a great deal is known
about the requirements and characteristics of these cells in mice,
much less is known about them in humans. Once we have a
more complete understanding of the regulatory T-cell subsets in
Regulatory T cells
We have shown that the regulatory function of the
CD4CD25hi T-cell subpopulation is defective in patients with
221
humans, this area will be an attractive one for intervention or

immunotherapy with a goal of increasing either the number or
the functionality of these T cells in MS patients.
center is currently involved in a Phase I/II study to determine

the efficacy of sirulimus (Rapammune1) in treating relapsing/remitting MS.
Immunosuppressants
The immunosuppressant rapamycin has been shown to
improve autoimmune diseases in animal models such as EAE,
the non-obese diabetic mouse, and adjuvant-induced arthritis
(178, 179). More recent studies demonstrated successful isletcell transplantation in patients with type I diabetes treated with
low dose rapamycin in combination with humanized antiIL2R mAb and FK506, where autoimmune destruction of
islet cells was prevented (180). Previously, we found that
depending on the strength of the signal delivered to the T cell
via both the TCR and the costimulatory molecule CD28, CD8
T cells are capable of rapamycin-resistant proliferation (181)
(Fig. 5). The selective immunosuppressive effect of rapamycin
in human CD8 T-cell populations could be predictive of a
selective effect allowing cytotoxic responses during microbial
infections, where there are strong signals associated with high
affinity TCRs and costimulatory second signals. In contrast, the
weaker autoimmune and perhaps allogeneic responses can be
selectively inhibited by the actions of rapamycin (182). Our
Combination therapies
One largely unexplored therapeutic frontier for the treatment
of MS is that of combination therapies. Transplant patients
routinely receive complex cocktails of drugs to prolong graft
survival, and even patients with other autoimmune diseases
such as RA are often treated with a regimen of several drugs at
once. This area is a promising yet untapped one in MS therapy,
as many of the treatments discussed herein work by different,
potentially complementary, mechanisms. This fact might
allow the use of them in combinations in smaller doses,
which would minimize side-effects and toxicity while maximizing protection from disease.
MS remains a clinically defined syndrome within which

several subtypes have been defined on a clinical basis. There
are, for example, primary progressive and relapsing/remitting
anti-CD28 (5 g/ml)
anti-CD28 (1 g/ml)
A
50 000
50 000
40 000
[3H]-Thymidine
incorporation
[3H]-Thymidine
incorporation
New directions in MS research: understanding the

genetic basis of MS
30 000
20 000
10 000
40 000
control
30 000
Rapa 1 nM
20 000
Rapa 10 nM
10 000
Rapa 100 nM
0
0
0.1
10
50
anti-CD28 (1 g/ml)
50 000
40 000
40 000
[3H]-Thymidine
incorporation
[3H]-Thymidine
incorporation
10
50
anti-CD28 (5 g/ml)
50 000
30 000
20 000
10 000
control
30 000
Rapa 1 nM
20 000
Rapa 10 nM
10 000
Rapa 100 nM
0
0
0.1
10
50
anti-CD3 (g/ml)
Fig. 5. Simultaneously crosslinking CD3 and CD28 on human CD8+

peripheral blood T cells results in rapamycin-resistant proliferation.
Plates were coated with anti-CD3 (A) or with anti-CD3 plus anti-CD28
(B) at indicated concentrations. CD8 PBT cells were pretreated with
222
anti-CD3 (g/ml)
anti-CD3 (g/ml)
B
0.1
0.1
10
50
anti-CD3 (g/ml)
rapamycin or vehicle control (ethanol) then plated with the indicated

concentration of rapamycin plus soluble anti-CD28 mAb (A).
Proliferation was assessed at 72 h by the incorporation of
[3H]-thymidine.
forms as well as benign and malignant courses of the disease.

These clinical observations suggest that MS is a syndrome
regrouping several related diseases, each of which may
respond differently to a particular therapy. Studying the genetics of MS is one approach that will yield biomarkers with
which to guide prognostication and therapeutic selection.
However, understanding what genetic variants contribute to
MS susceptibility will also have a much more profound impact
in opening new avenues of research into the pathophysiology
of the disease.
Is MS a genetic disease? Epidemiological studies suggest that
there are both environmental (183, 184) and genetic (4, 185)
risk factors that contribute to MS susceptibility. Because there
is no clear pattern of inheritance, we consider MS to be a
genetically complex disease in which susceptible individuals
encounter environmental triggers that initiate a chronic process of demyelination and degeneration. The clearest evidence
for genetic risk factors comes from the fact that siblings and
fraternal twins of patients have a 2040 times higher risk for
the disease and that identical twins a 150300 times greater
risk than unrelated individuals in the population (4, 184189).
It is likely that many genes or loci have variants that increase
risk of MS, but, so far, only the HLA locus on chromosome
6p21 has been definitively implicated as a contributor to
genetic risk in MS (186, 187).
Comprehensive studies of genetic susceptibility to MS have
been attempted but with limited success. Over the past
10 years, 10 linkage scans involving a total of 1524 affected
subjects have failed to identify non-HLA genetic risk factors for
MS (189195). A recent meta-analysis of these studies did not
offer further insights (GAMES, 2003). The only risk haplotype
that has been definitively identified is the HLA DRB1*1501DQB1*0602 haplotype on chromosome 6, which explains
between 14%-50% of the genetic risk (186, 188194).
New methods are therefore required to identify the remaining
genetic risk factors for MS. One promising method is an
association study design, which is thought to have more
statistical power than a traditional linkage study (189) and
can be applied in different ways to both genome-wide and
focused genetic analyses.
There are two major approaches to gene discovery efforts in
human complex traits such as a diagnosis of MS; one can
pursue either a narrow hypothesis such as asking whether a
particular gene is involved in a disease or an unbiased
approach that asks which part of the human genome is relevant to the disease process. Each approach has its advantages,
and we view them as complementary strategies. A genomewide scan has an intrinsic appeal given its lack of assumptions
as to which gene or chromosomal segment is important.

However, a statistically well-powered study only now is
becoming a reality, thanks to our evolving knowledge of the
human genome as well as technological advances in genotyping and subsequent decreases in genotyping costs. Below, we
discuss the three different genome-wide scans that we are
pursuing; they include a high-resolution linkage study, an
admixture study, and a haplotype-based study. Linkage studies
have yet to yield their full complement of data to the effort of
understanding genetic susceptibility to MS and will pave the
way for the definitive haplotypebased association study. The
admixture scan is another intermediate study that will also
help to identify chromosomal regions that contain susceptibility loci and can be fully characterized using the haplotypebased approach. Overall, these genome-wide scans are longterm large-scale projects, which leave room for focused but
locally comprehensive gene discovery efforts that will also be
discussed below.
The structure of human genetic variation

Several recent scientific advances have dramatically changed
our ability to examine genetic variation as it relates to human
disease. The first key advance is the completion of a draft
sequence of the human genome (190). This advance provides
a direct way to connect a chromosomal region with its DNA
sequence and gene content. The second key advance is the
successful effort to define DNA sequence variation in the
human genome. Specifically, a genome-wide single nucleotide
polymorphism (SNP) map has grown from an initial version
with 4000 SNPs in 1999 (191) to a map with more than 1.4
million SNPs by early 2001 (192) and several million more
SNPs today. The third key advance is shown in recent studies
that define the long-range extent of correlations among SNPs
in the human genome and identify the existence of haplotype
blocks in the genome (193196).
These studies (193) of the inheritance patterns of genetic
variants within small segments of the genome revealed the
relative simplicity of the underlying patterns of human variation. The original study focused on a 500-kb region within the
cytokine gene cluster on 5q31 and demonstrated that there
were chromosomal segments within which neighboring
sequence variations were observed in only a limited number
of combinations in human populations. Each unique combination of alleles is called a haplotype, and so these chromosomal segments, in which typically 46 common (frequency
>0.05) haplotypes were observed, were called haplotype
blocks. Between these blocks, there are short intervals where
223
recombination is apparently most active in creating assortments of these haplotype blocks. Thus, rather than each individual chromosome carrying a unique combination of SNP
alleles, each chromosome is a simple mosaic of long common
patterns of SNP alleles. Put another way, the quantum element
of genetic variation on a population-wide scale is not the
individual base pair or SNP, but rather 5100 kb blocks of
sequence unbroken by recombination in modern human evolution. The clustering is suggestive of local hotspots of recombination. Similar observations made for the class II region of
the HLA locus on 6p21.3 that have been shown to arise directly
from strong inhomogeneity of recombination rate (195).
Block-like haplotype structure is now confirmed in 60
different genomic regions. A subsequent large-scale study by
Gabriel and colleagues (196) confirmed the existence of
haplotype block structure in all chromosomes using publicly
available SNP databases. Their study shows that haplotype
blocks are a general feature of the genomic landscape, with
much more than 80% of the entire genome falling into very
tightly linked blocks. These blocks are consistently large in size
across the genomein fact, half of all randomly ascertained
20-kb genomic segments show no evidence of historical
recombination and consequently display very low haplotype
diversity in a population of European descent. Collectively,
these findings conclusively show that all common haplotypes
within a block can be identified with a small subset of variants
within that block, known as haplotype tagging SNPs (htSNPs)
(197). Gabriel and colleagues also showed in association tests
that these common haplotype patterns serve as an excellent
surrogate for all common genetic variation within a block
regardless of whether all variants have been discovered
(196). These discoveries have culminated in the creation of a
new, comprehensive paradigm for studying genetic variation
across a region and discovering its association to phenotype
(198): the haplotypebased association study.
These discoveries prompted the creation of the International
Haplotype Map (Hap Map) Consortium (http://www.
hapmap.org/) that has begun to assemble a map of all
haplotype blocks and will eventually create a road map to
guide investigators in selecting htSNPs to interrogate all common genetic variation in any given chromosomal segment.
This guide is a tremendous advance for the field, but it should
not be viewed as a panacea. Some regions of the genome will
probably not be adequately captured by haplotype blocks. In
addition, the current definitions of haplotype blocks depend
on the frequency of recombination events and are only useful
in assessing common (frequency >0.05) variation in the
human genome. Rarer genetic variants will not be adequately
224
captured by the current map. Despite these important limitations, the Hap Map promises to be a very powerful tool in our
assessment of a large fraction of human genetic variation.
Quantitating genetic risk in autoimmune disease

The identification of loci affecting susceptibility to other
genetically complex diseases such as type 1 and type 2 diabetes
mellitus (DM), inflammatory bowel disease (IBD), and autoimmune hypothyroidism (AIH), has given us much more
realistic estimates of the amount of disease risk that a single
allele can carry. Indeed, the genotype risk ratio (GRR), which
is the increase in risk attributable to the disease allele, in these
well-defined complex disease risk alleles, such as those in
PPARg (type 2 DM), IBD5 (IBD), and CTLA-4 (type 1 DM
and AIH), are on the order of 1.251.5, a range that is much
smaller than originally anticipated (199203).
Power calculations can be performed to estimate the sample
sizes necessary to identify loci with these GRRs. To discover a
risk allele or risk haplotype with a GRR of 1.5, 3295 affected
subject/biological parents trios are needed to achieve 95%
power in searching for a risk allele with a frequency of 0.05 in
the subject population, given the correction for the multiple
hypothesis testing associated with a genome-wide scan
(http://www.broad.mit.edu/personal/mjdaly/assoc.html).
Thus, it is not surprising that studies until now have not been
able to find a definitive association between a non-HLA locus
and MS susceptibility. All studies have been underpowered,
which was not done consciously. The study of complex traits
has evolved dramatically over the past 10 years, and study
designs once thought to be powered adequately, based on
earlier assumptions, have now been revealed to be inadequate
(189).
These power calculations also underline another important
fact: the required study size is now so large that no one group
of investigators can carry out the study by themselves. A
relative mass of financial resources as well as varied clinical,
technical, and statistical expertise must be assembled and driven by a team of talented investigators to ensure the successful
completion of a well-designed and well-powered study of
genetic susceptibility in MS.
The emergence of MS genetic consortia

In response to the need for multicenter collaboration, consortia have emerged for the purpose of discovering genes
involved in genetically complex traits such as disease susceptibility. Many of these consortia exist for a variety of different
diseases and some of them are functional. The field of MS

genetics has two such consortia.
The first consortium to emerge in the field of MS genetics
was the Genetic Analysis of MS in Europeans (GAMES) collaboration. This remarkable effort brought together 16 different
research groups from across Europe and Australia to perform
the analysis of 6000 markers in 3376 subjects with MS, 3409
control subjects, and 948 trio families with one child affected
with MS. The genotyping has been performed, but the analysis
of the data is ongoing (201). This collaboration is the largest
association study in MS so far, and while the number of
markers is now deemed to be insufficient for a genome-wide
association screen, this intermediate scan should nonetheless
offer new insights as to which regions may contain loci
relevant to MS susceptibility.
In parallel with the GAMES effort, several groups, including
our own, came together to establish the International Multiple
Sclerosis Genetics Consortium (IMSGC). Its stated purpose is to
work toward the completion of a genome-wide haplotypebased association screen for loci influencing susceptibility to
MS. Concrete progress has been made in establishing a shared
resource of genetic material as well as a centralized database for
data storage and dissemination. The member groups are now
pursuing the first phase of the project, which is focused on
resolving the issue of which genetic variant(s) within the HLA
DRB1*1501-DQB1*0602 haplotype is responsible for the association of this haplotype to MS susceptibility. The IMSGC is,
therefore, functional and holds great promise as the vehicle that
will discover risk alleles as part of the first genome-wide scan in
MS to assess the vast majority of common genetic variation.
Linkage study
A haplotype-based scan of the human genome is quickly
becoming a reality; however, it cannot be done today. As a
result, the IMSGC has undertaken a new linkage study to
identify chromosomal regions that show suggestive evidence
for being involved in the disease. Several such regions have
been described in the past, but linkage scores have remained
low for any given locus, even in the meta-analysis (187). One
could argue, therefore, that another linkage study is unlikely to
yield much additional information. However, there is mounting evidence that prior linkage studies have been severely
limited in their statistical power, not only because of the
limited number of markers involved in these studies but also
because of high error rates in genotyping. Simulations have
shown that even a 1% error ratea rate often seen with wellbehaved microsatellite markerscan reduce LOD scores by as
much as 50% (202). Because many microsatellite markers

have error rates greater than 1% and the existing linkage
studies have predominantly used this type of genetic marker,
missing data have been a significant issue that has a particularly large impact on linkage studies. Indeed, the meta-analysis
of linkage screens in MS revealed that the mean information
extraction was only 44% across the genome and that the point
of greatest information extraction was only 68% (187, 203).
There is, therefore, room to improve the analysis of existing
linkage sample collections.
Recently, comparisons of genotyping platforms have
revealed that the newer SNP-based methods are much more
robust in terms of genotyping error. These results can be
extrapolated to show that SNP-based genotyping should
improve information extraction by 50% (203, 204). These
encouraging preliminary results have prompted the IMSGC to
launch a new SNP-based genome-wide linkage screen in MS.
However, the goal of the study is not to identify specific genes
involved in MS. Rather, we take the more measured perspective that this effort will help to identify regions within which
the susceptibility genes may lie. The results of the linkage
study will, therefore, be critical in stratifying the genome
into areas that have higher-or-lower prior probabilities of
containing MS susceptibility loci. This study will allow us to
focus on the higher probability areas early in the course of the
haplotype-based scan.
Admixture study
An improved linkage study is not the only way to stratify
the genome into high- and low-yield regions in terms of
MS susceptibility; a method of association mapping called
admixture mapping has emerged as a powerful technique
with which to identify disease susceptibility loci. This technique can be used when 1) two human populations have
different prevalence rates of the same disease and 2) a third
population exists that is the result of admixture between
the two parent populations. In the case of MS, AfricanAmerican individuals have a prevalence rate that is 40%
that of European-Americans, and Africans are thought to
have a prevalence rate of approximately 1% that of
European-Americans (183). Thus, a number of MS susceptibility loci may exist in the European genome and may be
absent in the African genome. One can, therefore, look at a
population of African-American individuals with MS and
search for regions that have an increased proportion of
European ancestry because they may contain a risk allele of
European origin.
225
The idea of performing an admixture analysis in AfricanAmericans was first published 16 years ago (205), but it is
only with recent technological advances and the availability of
vast numbers of SNPs that an admixture study became a reality
(206, 207). MS is the first disease to be studied using admixture mapping. A genome-wide scan using this technique is
under way and is already showing promising results. This scan
will define chromosomal segments of European ancestry
associated with risk of MS; however, projections suggest that
these admixture-defined loci will be large (510 Mb) (207).
Thus, a haplotype-based approach is a necessary adjunct to an
admixture scan to perform the detailed dissection of the
implicated chromosomal region.
Haplotype-based study
An association study based on the use of haplotype-tagging
SNPs is predicted to be very powerful in assessing all common
human genetic variation that falls within haplotype blocks.
Estimates vary, but it is thought that approximately 300 000
htSNPs will allow us to extract approximately 80% of the
genome-wide information using 1500 MS trios. The resulting
quantity of genotyping and extent of data analysis is not
practical today, but the relevant technologies are evolving
quickly. Such a haplotype-based study will become a reality
within 5 years and will yield a thorough evaluation of the role
of a majority of common genetic variation in determining
susceptibility to MS.
Targeted haplotype-based scans of chromosomal segments
are possible today. Given an average haplotype block size of
approximately 20 kb and 10 htSNPs per block, one can estimate that 2500 htSNPs would be sufficient to cover a 10-Mb
chromosomal segment and query all common genetic variation within that segment. This is the predicted size of an
admixture scan-defined segment, and even a 20-Mb segment
identified by linkage analysis can be scanned using this
technique with todays genotyping technology. Thus, the
high-resolution linkage scan and admixture scans play a
critical role in enabling a haplotype-based approach in
restricted areas of the genome that have a higher probability
of containing susceptibility loci. Our first target for haplotypebased analysis is the HLA locus, the only locus that has been
clearly implicated in MS.
The limitations of a haplotype-based approach have already
been mentioned. First, a sizable fraction of the genome may
not fall into haplotype blocks. Genetic variants in these regions
will therefore not be assayed. Second, genetic variants which
226
are present at a frequency <0.05 in human populations will be

captured poorly by the haplotype-based analysis. Both of these
problems will be solved by the next level of genetic characterization: whole genome sequencing. However, this level of
genome-wide genetic analysis will not be practical for the
foreseeable future.
Candidate gene study
The limitations of the haplotype-based approach and its

need for a consortium-level group to be implemented
leave an opening for candidate gene studies. Indeed, at this
level, a haplotype-based analysis can be done rapidly and
relatively cheaply. Comprehensive assessments not of single
genes but of pathways implicated in the disease are now a
reality and will yield valuable insights, especially if resources
do not allow for the more comprehensive genome-wide
approach. The key will be to power the study effectively;
the use of a few hundred samples will yield studies of only
marginal interest, whose negative results cannot be interpreted and positive results are difficult to replicate at the
same level. Candidate gene studies have a role to play when
substantial collections of 5001000 affected subjects are
used as a screening tool and a replication set of similar or
preferably greater size is available. This type of study, by
looking much more deeply into the genetic variation of a
small chromosomal segment, will also prove to be particularly valuable in assessing the role of genes or loci that are
not captured by the haplotype block structure and of rare
variants that are discovered by sequencing affected
subjects within the restricted chromosomal segment. At
this time, many suggestive associations to MS have been
made using candidate gene studies, but, given the sample
sizes typically used, it is not yet clear whether any of them
are real.
Summary
The field of complex trait genetics has learned much from its
rare successes and many failures in the 1990s. The resulting
advances in the theory of statistical genetics is now being
coupled to the unparalleled resources of the human genome
sequence and the emerging Hap Map to usher in a new period of
exciting advances in human genetics. As high-throughput genotyping continues to improve and becomes affordable over the
next 5 years, we will see the definitive identification of several
non-HLA loci that are associated with susceptibility to MS.
References
1. Charcot J. Histologic de la sclerose en
plaque. Gaz Hop 1868;41:554566.
2. Charcot J. Lectures on the diseases of the
nervous system. London: New Sydenham
Society, 1877.
3. Kabat EA, Glusman M, Knaub V. Quantitative
estimation of the albumin and gamma
globulin in normal and pathologic
cerebrospinal fluid by immunochemical
methods. Am J Med 1948;4:653662.
4. Mackay RP, Myrianthopoulos NC. Multiple
sclerosis in twins and their relatives. Arch
Neurol 1966;15:449462.
5. Rivers TM, Sprunt DH, Berry GP.
Observations on attempts to produce acute
disseminated encephalomyelitis in monkeys.
J Exp Med 1933;58:3953.
6. Hafler DA. The distinction blurs between an
autoimmune versus microbial hypothesis in
multiple sclerosis. J Clin Invest
1999;104:527529.
7. Ben-Nun A, Wekerle H, Cohen IR. The rapid
isolation of clonable antigen-specific T
lymphocyte lines capable of mediating
autoimmune encephalomyelitis. Eur J
Immunol 1981;11:195199.
8. Goverman J, et al. Transgenic mice that
express a myelin basic protein-specific T cell
receptor develop spontaneous
autoimmunity. Cell 1993;72:551560.
9. Lehmann PV, et al. Spreading of T-cell
autoimmunity to cryptic determinants of an
autoantigen. Nature 1992;358:155157.
10. Miller SD, et al. Blockade of CD28/B71
interaction prevents epitope spreading and
clinical relapses of murine EAE. Immunity
1995;3:739745.
11. Windhagen A, et al. Expression of
costimulatory molecules B71 (CD80),
B72 (CD86), and interleukin 12 cytokine
in multiple sclerosis lesions. J Exp Med
1995;182:19851996.
12. Ota K, et al. T-cell recognition of an
immunodominant myelin basic protein
epitope in multiple sclerosis. Nature
1990;346:183187.
13. Pette M, et al. Myelin basic protein-specific
T lymphocyte lines from MS patients and
healthy individuals. Neurology
1990;40:17701776.
14. Martin R, et al. Fine specificity and HLA
restriction of myelin basic protein-specific
cytotoxic T cell lines from multiple sclerosis
patients and healthy individuals. J Immunol
1990;145:540548.
15. Ausubel LJ, et al. Complementary mutations
in an antigenic peptide allow for
crossreactivity of autoreactive T-cell clones.
Proc Natl Acad Sci USA
1996;93:1531715322.
16. Hemmer B, et al. Identification of high

potency microbial and self ligands for a
human autoreactive class II-restricted T cell
clone. J Exp Med 1997;185:16511659.
17. Wucherpfennig KW, Strominger JL.
Molecular mimicry in T cell-mediated
autoimmunity: viral peptides activate human
T cell clones specific for myelin basic
protein. Cell 1995;80:695705.
18. Trapp BD, et al. Axonal transection in the
lesions of multiple. N Engl J Med
1998;338:278285.
19. Wucherpfennig KW, et al. T cell receptor V
alpha-V beta repertoire and cytokine gene
expression in active multiple sclerosis
lesions. J Exp Med 1992;175:9931002.
20. Traugott U, Reinherz EL, Raine CS. Multiple
sclerosis. distribution of T cell subsets within
active chronic lesions. Science
1983;219:308310.
21. Hauser SL, et al. Immunohistochemical
analysis of the cellular infiltrate in multiple
sclerosis lesions. Ann Neurol
1986;19:578587.
22. Wucherpfennig KW, et al. Gamma delta
T-cell receptor repertoire in acute multiple
sclerosis lesions. Proc Natl Acad Sci USA
1992;89:45884592.
23. Prineas JW, Wright RG. Macrophages,
lymphocytes, and plasma cells in the
perivascular compartment in chronic
multiple sclerosis. Lab Invest
1978;38:409421.
24. Prineas J. Pathology of the early lesion in
multiple sclerosis. Hum Pathol
1975;6:531554.
25. Lucchinetti CF, et al. Distinct patterns of
multiple sclerosis pathology indicates
heterogeneity on pathogenesis. Brain Pathol
1996;6:259274.
26. Becker KG, et al. Analysis of a sequenced
cDNA library from multiple sclerosis lesions.
J Neuroimmunol 1997;77:2738.
27. Whitney LW, et al. Microarray analysis of
gene expression in multiple sclerosis and
EAE identifies 5-lipoxygenase as a
component of inflammatory lesions.
28. Whitney LW, et al. Analysis of gene
expression in multiple sclerosis lesions using
cDNA microarrays. Ann Neurol
1999;46:425428.
29. Baranzini SE, et al. Transcriptional analysis of
multiple sclerosis brain lesions reveals a
complex pattern of cytokine expression.
J Immunol 2000;165:65766582.
30. Chabas D, et al. The influence of the
proinflammatory cytokine, osteopontin, on
autoimmune demyelinating disease. Science
2001;294:17311735.
31. Lock C, et al. Gene-microarray analysis of

multiple sclerosis lesions yields new targets
validated in autoimmune encephalomyelitis.
Nat Med 2002;8:500508.
32. Mycko MP, et al. Microarray gene expression
profiling of chronic active and inactive
lesions in multiple sclerosis. Clin Neurol
Neurosurg 2004;106:223229.
33. Mycko MP, et al. cDNA microarray analysis
in multiple sclerosis lesions: detection of
genes associated with disease activity. Brain
2003;126:10481057.
34. Tajouri L, et al. Quantitative and qualitative
changes in gene expression patterns
characterize the activity of plaques in
multiple sclerosis. Brain Res Mol Brain Res
2003;119:170183.
35. Lindberg RL, et al. Multiple sclerosis as a
generalized CNS disease-comparative
microarray analysis of normal appearing
white matter and lesions in secondary
progressive MS. J Neuroimmunol
2004;152:154167.
36. Lassmann H, et al. Immunopathology of
multiple sclerosis: report on an international
meeting held at the Institute of Neurology of
the University of Vienna. J Neuroimmunol
1998;86:213217.
37. Miller DH, Thompson AJ, Filippi M.
Magnetic resonance studies of abnormalities
in the normal appearing white matter and
grey matter in multiple sclerosis. J Neurol
2003;250:14071419.
38. Graumann U, et al. Molecular changes in
normal appearing white matter in multiple
sclerosis are characteristic of neuroprotective
mechanisms against hypoxic insult. Brain
Pathol 2003;13:554573.
39. Becher B, et al. Soluble tumor necrosis factor
receptor inhibits interleukin 12 production
by stimulated human adult microglial cells
in vitro. J Clin Invest 1996;98:15391543.
40. Dong Y, Benveniste EN. Immune function of
astrocytes. Glia 2001;36:180190.
41. Lehnardt S, et al. Activation of innate
immunity in the CNS triggers
neurodegeneration through a Toll-like
receptor 4-dependent pathway. Proc Natl
Acad Sci USA 2003;100:85148519.
42. Monje ML, Toda H, Palmer TD.
Inflammatory blockade restores adult
hippocampal neurogenesis. Science
2003;302:17601765.
43. Ekdahl CT, et al. Inflammation is detrimental
for neurogenesis in adult brain. Proc Natl
Acad Sci USA 2003;100:1363213637.
44. Eugster HP, et al. IL-6-deficient mice resist
myelin oligodendrocyte glycoproteininduced autoimmune encephalomyelitis. Eur
J Immunol 1998;28:21782187.
227
45. Aloisi F, Ria F, Adorini L. Regulation of

T-cell responses by CNS antigen-presenting
cells: different roles for microglia and
astrocytes. Immunol Today
2000;21:141147.
46. Aloisi F, et al. IL-12 production by central
nervous system microglia is inhibited by
astrocytes. J Immunol
1997;159:16041612.
47. Ledeboer A, et al. Interleukin-10, interleukin-4,
and transforming growth factor-beta
differentially regulate lipopolysaccharideinduced production of pro-inflammatory
cytokines and nitric oxide in co-cultures
of rat astroglial and microglial cells. Glia
2000;30:134142.
48. Onuki M, et al. Axonal degeneration is an
early pathological feature in autoimmunemediated demyelination in mice. Microsc
Res Tech 2001;52:731739.
49. Ayers MM, et al. Early glial responses in
murine models of multiple sclerosis.
Neurochem Int 2004;45:409419.
50. Trajkovic V, et al. Astrocyte-induced
regulatory T cells mitigate CNS
autoimmunity. Glia 2004;47:168179.
51. Roncarolo MG, et al. Type 1 T regulatory
cells. Immunol Rev 2001;182:6879.
52. Pullen AM, Marrack P, Kappler JW. Evidence
that Mls-2 antigens which delete V beta 3
T cells are controlled by multiple genes.
J Immunol 1989;142:30333037.
53. Sakaguchi S. Regulatory T cells: key
controllers of immunologic self-tolerance.
Cell 2000;101:455458.
54. Shevach EM, et al. Control of T-cell
activation by CD4 CD25 suppressor
T cells. Immunol Rev 2001;182:5867.
55. Sakaguchi S, et al. Organ-specific
autoimmune diseases induced in mice by
elimination of T cell subset. I. Evidence for
the active participation of T cells in natural
self-tolerance; deficit of a T cell subset as a
possible cause of autoimmune disease. J Exp
Med 1985;161:7287.
56. Read S, Malmstrom V, Powrie F. Cytotoxic
T lymphocyte-associated antigen 4 plays an
essential role in the function of CD25()
CD4() regulatory cells that control
intestinal inflammation. J Exp Med
2000;192:295302.
57. Baecher-Allan C, et al. CD4CD25high
regulatory cells in human peripheral blood.
J Immunol 2001;167:12451253.
58. Dieckmann D, et al. Ex vivo isolation and
characterization of CD4() CD25() T cells
with regulatory properties from human
blood. J Exp Med 2001;193:13031310.
59. Kalsi JK, et al. Functional and modelling
studies binding human monoclonal anti-dna
antibodies DNA. Mol lmmunol 1996;
33:471483.
228
60. Reijonen H, et al. Detection of

GAD65-specific T-cells by major
histocompatibility complex class II tetramers
in type 1 diabetic patients and at-risk
subjects. Diabetes 2002;51:13751382.
61. Viglieta V, et al. Loss of functional
suppression by CD4 CD25 regulatory
T cells in patients with multiple sclerosis.
J Exp Med 2004;199:971979.
62. Baecher-Allan C, Viglietta V, Hafler DA.
Inhibition of human CD4() CD25(high)
regulatory T cell function. J Immunol
2002;169:62106217.
63. Tourtellotte WW. The cerebrospinal fluid in
multiple sclerosis. In: Vinken PJ, et al., eds.
Handbook of Clinical Neurology.
Amsterdam, New York: Elsevier Science,
1985; 79130.
64. Sharief MK, Thompson EJ. Intrathecal
immunoglobulin M synthesis in multiple
sclerosis. Relationship with clinical and
cerebrospinal fluid parameters. Brain
1991;114:181195.
65. Sharief MK, Hentges R. Importance of
intrathecal synthesis of IgD in multiple
sclerosis. A combined clinical,
immunologic, and magnetic resonance
imaging study. Arch Neurol
1991;48:10761079.
66. Walsh MJ, et al. Immunoglobulin G, A, and
M clonal restriction in multiple sclerosis
cerebrospinal fluid and serum analysis by
two-dimensional electrophoresis. Clin
Immunol Immunopathol 1985;35:313327.
67. Esiri MM. Immunoglobulin-containing cells
in multiple-sclerosis plaques. Lancet
1977;2:478.
68. Mehta PD, et al. Bound antibody in multiple
sclerosis brains. J Neurol Sci 1981;49:9198.
69. Storch MK, et al. Multiple sclerosis: in situ
evidence for antibody- and complementmediated demyelination. Ann Neurol
1998;43:465471.
70. Lucchinetti C, et al. Heterogeneity of
multiple sclerosis lesions: implications for
the pathogenesis of demyelination. Ann
Neurol 2000;47:707717.
71. OConnor KC, Bar-Or A, Hafler DA. The
neuroimmunology of multiple sclerosis:
possible roles of T and B lymphocytes in
immunopathogenesis. J Clin Immunol
2001;21:8192.
72. Cross AH, Trotter JL, Lyons J. B cells and
antibodies in CNS demyelinating disease.
73. Kabat EA, et al. A study of the crystalline
albumin, gamma globulin and the total
protein in the cerebrospinal fluid of one
hundred cases of multiple sclerosis and other
diseases. Am J Med Sci 1950;219:5564.
74. Lowenthal A, Van Sande M, Karcher D. The

differential diagnosis of neurological
diseases by fractionating electrophoretically
the CSF G-globulins. J Neurochem
1960;235:229233.
75. Johnson KP, et al. Agarose electrophoresis of
cerebrospinal fluid in multiple sclerosis.
A simplified method for demonstrating
cerebrospinal fluid oligoclonal
immunoglobulin bands. Neurology
1977;27:273277.
76. Owens GP, et al. Restricted use of VH4
germline segments in an acute multiple
sclerosis brain. Ann Neurol
1998;43:236243.
77. Baranzini SE, et al. B cell repertoire diversity
and clonal expansion in multiple sclerosis
brain lesions. J Immunol
1999;163:51335144.
78. Smith-Jensen T, et al. Comparison of
immunoglobulin G heavy-chain sequences
in MS and SSPE brains reveals an antigendriven response. Neurology
2000;54:12271232.
79. Williamson RA, et al. Anti-DNA antibodies
are a major component of the intrathecal B
cell response in multiple sclerosis. Proc Natl
Acad Sci USA 2001;98:17931798.
80. Owens GP, et al. Single-cell repertoire
analysis demonstrates that clonal expansion
is a prominent feature of the B cell response
in multiple sclerosis cerebrospinal fluid.
J Immunol 2003;171:27252733.
81. Qin Y, et al. Clonal expansion and somatic
hypermutation of V (H) genes of B cells
from cerebrospinal fluid in multiple
sclerosis. J Clin Invest
1998;102:10451050.
82. Colombo M, et al. Accumulation of clonally
related B lymphocytes in the cerebrospinal
fluid of multiple sclerosis patients.
J Immunol 2000;164:27822789.
83. Adams JM, Imagawa DT. Measles antibodies
in multiple sclerosis. Proc Soc Exp Biol Med
1962;111:562566.
84. Sandberg-Wollheim M, et al. The intrathecal
immune response in the early stage of
multiple sclerosis. J Neurol Sci
1987;81:4553.
85. Bray PF, et al. Antibodies against EpsteinBarr nuclear antigen (EBNA) in multiple
sclerosis CSF, and two pentapeptide
sequence identities between EBNA and
myelin basic protein. Neurology
1992;42:17981804.
86. Vartdal F, Vandvik B, Norrby E. Viral and
bacterial antibody responses in multiple
sclerosis. Ann Neurol 1980;8:248255.
87. Vartdal F, Vandvik B. Multiple sclerosis.
Electrofocused bands of oligoclonal CSF
IgG do not carry antibody activity against
measles, varicella-zoster or rotaviruses.
J Neurol Sci 1982;54:99107.
88. Vandvik B, et al. Oligoclonal measles virusspecific IgG antibodies isolated from
cerebrospinal fluids, brain extracts, and sera
from patients with subacute sclerosing
panencephalitis and multiple sclerosis. Scand
J Immunol 1976;5:979992.
89. Vandvik B, Vartdal F, Norrby E. Herpes
simplex virus encephalitis. intrathecal
synthesis of oligoclonal virus-specific IgG,
IgA and IgM antibodies. J Neurol
1982;228:2538.
90. Luxton RW, et al. Affinity of antigen-specific
IgG distinguishes multiple sclerosis from
encephalitis. J Neurol Sci 1995;132:1119.
91. Panitch HS, et al. Antibodies to myelin basic
protein in cerebrospinal fluid of patients
with multiple scierosis. In: Bova H, ed.
Progress in multiple scierosis. Berlin:
Springer Verlag, 1980;62.
92. Warren KG, Catz I. A correlation between
cerebrospinal fluid myelin basic protein and
anti-myelin basic protein in multiple
sclerosis patients. Ann Neurol
1987;21:183189.
93. Warren KG, Catz I. Autoantibodies to myelin
basic protein within multiple sclerosis
central nervous system tissue. J Neurol Sci
1993;115:169176.
94. Warren KG, Catz I. An extensive search for
autoantibodies to myelin basic protein in
cerebrospinal fluid of non-multiple-sclerosis
patients: implications for the pathogenesis of
multiple sclerosis. Eur Neurol
1999;42:95104.
95. Brokstad KA, et al. Autoantibodies to myelin
basic protein are not present in the serum
and CSF of MS patients. Acta Neurol Scand
1994;89:407411.
96. Chou CH, Tourtellotte WW, Kibler RF.
Failure to detect antibodies to myelin basic
protein or peptic fragments of myelin basic
protein in CSF of patients with MS.
Neurology 1983;33:2428.
97. Cortese I, et al. Identification of peptides
specific for cerebrospinal fluid antibodies in
multiple sclerosis by using phage libraries.
1996;93:1106311067.
98. Warren KG, Catz I. Relative frequency of
autoantibodies to myelin basic protein and
proteolipid protein in optic neuritis and
multiple sclerosis cerebrospinal fluid.
J Neurol Sci 1994;121:6673.
99. Warren KG, et al. Anti-myelin basic protein
and anti-proteolipid protein specific forms
of multiple sclerosis. Ann Neurol
1994;35:280289.
100. Moller JR, et al. Antibodies to myelinassociated glycoprotein (MAG) in the
cerebrospinal fluid of multiple sclerosis
patients. J Neuroimmunol 1989;22:5561.
101. Banki K, et al. Oligodendrocyte-specific

expression and autoantigenicity of
transaldolase in multiple sclerosis. J Exp Med
1994;180:16491663.
102. Bronstein JM, et al. A humoral response to
oligodendrocyte-specific protein in MS.
a potential molecular mimic. Neurology
1999;53:154161.
103. Dasgupta MK, et al. Myelin basic protein:
a component of circulating immune
complexes in multiple sclerosis. Can J Neurol
Sci 1983;10:239243.
104. Reindl M, et al. Antibodies against the
myelin oligodendrocyte glycoprotein and
the myelin basic protein in multiple sclerosis
and other neurological diseases: a
comparative study. Brain
1999;122:20472056.
105. Terryberry JW, Thor G, Peter JB.
Autoantibodies in neurodegenerative
diseases: antigen-specific frequencies and
intrathecal analysis. Neurobiol Aging
1998;19:205216.
106. Olsson T, et al. Antimyelin basic protein and
antimyelin antibody-producing cells in
multiple sclerosis. Ann Neurol
1990;27:132136.
107. Colombo E, et al. Comparative analysis of
antibody and cell-mediated autoimmunity
to transaldolase and myelin basic protein in
patients with multiple sclerosis. J Clin Invest
1997;99:12381250.
108. Butler JE. The behavior of antigens and
antibodies immobilized on a solid phase. In:
Van Regenmortel MHV, ed. Structure of
Antigens. Boca Raton: CRC Press, 1992:
209260.
109. Butler JE, et al. The immunochemistry of
sandwich ELISAs VI. Greater than 90% of
monoclonal and 75% of polyclonal antifluorescyl capture antibodies (CAbs) are
denatured by passive adsorption. Mol
Immunol 1993;30:11651175.
110. Sodoyez-Goffaux F, et al. Advantages and
pitfalls of radioimmune and enzyme linked
immunosorbent assays of insulin antibodies.
Diabetologia 1988;31:694702.
111. Sun JB, et al. Autoreactive T and B cells
responding to myelin proteolipid protein in
multiple sclerosis and controls. Eur J
Immunol 1991;21:14611468.
112. Schmidt S. Candidate autoantigens in
multiple sclerosis. Mult Scler
1999;5:147160.
113. Barned S, Goodman AD, Mattson DH.
Frequency of anti-nuclear antibodies in
multiple sclerosis. Neurology
1995;45:384385.
114. Colaco CB, Scadding GK, Lockhart S. Anticardiolipin antibodies in neurological
disorders: cross-reaction with anti-single
stranded DNA activity. Clin Exp Immunol
1987;68:313319.
115. Roussel V, et al. Prevalence and clinical

significance of anti-phospholipid antibodies
in multiple sclerosis: a study of 89 patients.
J Autoimmun 2000;14:259265.
116. Spadaro M, et al. Autoimmunity in multiple
sclerosis: study of a wide spectrum of
autoantibodies. Mult Scler 1999;5:121125.
117. von Budingen HC, et al. Immune responses
against the myelin/oligodendrocyte
glycoprotein in experimental autoimmune
demyelination. J Clin Immunol
2001;21:155170.
118. Lafaille JJ, et al. High incidence of
spontaneous autoimmune encephalomyelitis
in immunodeficient anti-myelin basic
protein T cell receptor transgenic mice. Cell
1994;78:399408.
119. Wolf SD, et al. Experimental autoimmune
encephalomyelitis induction in genetically
B cell-deficient mice. J Exp Med
1996;184:22712278.
120. Schluesener HJ, et al. A monoclonal
antibody against a myelin oligodendrocyte
glycoprotein induces relapses and
demyelination in central nervous system
autoimmune disease. J Immunol
1987;139:40164021.
121. Piddlesden SJ, et al. The demyelinating
potential of antibodies to myelin
oligodendrocyte glycoprotein is related to
their ability to fix complement. Am J Pathol
1993;143:555564.
122. Raine CS, et al. Demyelination in primate
autoimmune encephalomyelitis and acute
multiple sclerosis lesions: a case for antigenspecific antibody mediation. Ann Neurol
1999;46:144160.
123. Genain CP, et al. Antibody facilitation of
multiple sclerosis-like lesions in a
nonhuman primate. J Clin Invest
1995;96:29662974.
124. Lampasona V, et al. Similar low frequency of
anti-MOG IgG and IgM in MS patients and
healthy subjects. Neurology
2004;62:20922094.
125. Xiao BG, Linington C, Link H. Antibodies to
myelin-oligodendrocyte glycoprotein in
cerebrospinal fluid from patients with
multiple sclerosis and controls.
126. Karni A, et al. Elevated levels of antibody to
myelin oligodendrocyte glycoprotein is not
specific for patients with multiple sclerosis.
Arch Neurol 1999;56:311315.
127. Lindert RB, et al. Multiple sclerosis: B- and
T-cell responses to the extracellular domain
of the myelin oligodendrocyte glycoprotein.
Brain 1999;122:20892100.
229
128. Schmidt S, et al. Serum autoantibody

responses to myelin oligodendrocyte
glycoprotein and myelin basic protein in
X-linked adrenoleukodystrophy and
multiple sclerosis. J Neuroimmunol
2001;119:8894.
129. Guggenmos J, et al. Antibody crossreactivity between myelin oligodendrocyte
glycoprotein and the milk protein
butyrophilin in multiple sclerosis. J Immunol
2004;172:661668.
130. Berger T, et al. Antimyelin antibodies as a
predictor of clinically definite multiple
sclerosis after a first demyelinating event. N
Engl J Med 2003;349:139145.
131. Haase CG, et al. The fine specificity of the
myelin oligodendrocyte glycoprotein
autoantibody response in patients with
multiple sclerosis and normal healthy
controls. J Neuroimmunol
2001;114:220225.
132. Huang X, et al. Glycosylation affects both the
three-dimensional structure and antibody
binding properties of the HIV-1IIIB GP120
peptide RP135. Biochemistry
1997;36:1084610856.
133. Mazzucco S, et al. A synthetic glycopeptide
of human myelin oligodendrocyte
glycoprotein to detect antibody responses in
multiple sclerosis and other neurological
diseases. Bioorg Med Chem Lett
1999;9:167172.
134. Carotenuto A, et al. Conformational analysis
of a glycosylated human myelin
oligodendrocyte glycoprotein peptide
epitope able to detect antibody response in
multiple sclerosis. J Med Chem
2001;44:23782381.
135. von Budingen HC, et al. Molecular
characterization of antibody specificities
against myelin/oligodendrocyte
glycoprotein in autoimmune demyelination.
2002;99:82078212.
136. Bourquin C, et al. Myelin oligodendrocyte
glycoprotein-DNA vaccination induces
antibody-mediated autoaggression in
experimental autoimmune
encephalomyelitis. Eur J Immunol
2000;30:36633671.
137. Brehm U, et al. Epitope specificity of
demyelinating monoclonal autoantibodies
directed against the human myelin
oligodendrocyte glycoprotein (MOG).
138. von Budingen HC, et al. Epitope recognition
in the myelin/oligodendrocyte glycoprotein
differentially influences disease phenotype
and antibody effector functions in
autoimmune demyelination. Eur J Immunol
2004;34:20722083.
230
139. Sblattero D, et al. The analysis of the fine

specificity of celiac disease antibodies using
tissue transglutaminase fragments. Eur J
Biochem 2002;269:51755181.
140. Richter W, Shi Y, Baekkeskov S. Autoreactive
epitopes defined by diabetes-associated
human monoclonal antibodies are localized
in the middle and C-terminal domains of the
smaller form of glutamate decarboxylase.
1993;90:28322836.
141. Psaridi-Linardaki L, Mamalaki A, Tzartos SJ.
Future therapeutic strategies in autoimmune
myasthenia gravis. Ann N Y Acad Sci
2003;998:539548.
142. Breithaupt C, et al. Structural insights into
the antigenicity of myelin oligodendrocyte
glycoprotein. Proc Natl Acad Sci USA
2003;100:94469451.
143. OConnor KC, et al. Isolation of
autoantibodies recognizing MOG from CNS
tissue of patients with MS [Submitted,
2004].
144. Khoury SJ, Hancock WW, Weiner HL. Oral
tolerance to myelin basic protein and natural
recovery from experimental autoimmune
encephalomyelitis are associated with
downregulation of inflammatory cytokines
and differential upregulation of
transforming growth factor beta, interleukin
4, and prostaglandin E expression in the
brain. J Exp Med 1992;176:13551364.
145. Powrie F, Menon S, Coffman RL.
Interleukin-4 and interleukin-10 synergize
to inhibit cell-mediated immunity in vivo.
Eur J Immunol 1993;23:30433049.
146. Seder RA, et al. Interleukin 12 acts directly
on CD4 T cells to enhance priming for
interferon gamma production and
diminishes interleukin 4 inhibition of such
priming. Proc Natl Acad Sci USA
1993;90:1018810192.
147. Mocci S, Coffman RL. Induction of a Th2
population from a polarized Leishmaniaspecific Th1 population by in vitro culture
with IL-4. J Immunol
1995;154:37793787.
148. Kuchroo VK, et al. B71 and B72
costimulatory molecules activate
differentially the Th1/Th2 developmental
pathways: application to autoimmune
disease therapy. Cell 1995;80:707718.
149. Nicholson LB, Kuchroo VK. Manipulation of
the Th1/Th2 balance in autoimmune
disease. Curr Opin Immunol
1996;8:837842.
150. Elliott MJ, et al. Randomised double-blind
comparison of chimeric monoclonal
antibody to tumour necrosis factor alpha
(cA2) versus placebo in rheumatoid
arthritis. Lancet 1994;344:11051110.
151. Moreland LW, et al. Treatment of

rheumatoid arthritis with a recombinant
human tumor necrosis factor receptor (p75)
-Fc fusion protein. N Engl J Med
1997;337:141147.
152. van Oosten BW, et al. Increased MRI activity
and immune activation in two multiple
sclerosis patients treated with the
monoclonal anti-tumor necrosis factor
antibody cA2. Neurology
1996;47:15311534.
153. Kassiotis G, Kollias G. Uncoupling the
proinflammatory from the
immunosuppressive properties of tumor
necrosis factor (TNF) at the p55 TNF
receptor level: implications for pathogenesis
and therapy of autoimmune demyelination.
J Exp Med 2001;193:427434.
154. Stuve O, et al. Interferon beta-1b decreases
the migration of T lymphocytes in vitro:
effects on matrix metalloproteinase-9. Ann
Neurol 1996;40:853863.
155. Bielekova B, et al. Humanized anti-CD25
(daclizumab) inhibits disease activity in
multiple sclerosis patients failing to respond
to interferon beta. Proc Natl Acad Sci USA
2004;101:87058708.
156. Evavold BD, Allen PM. Separation of IL-4
production from Th cell proliferation by an
altered T cell receptor ligand. Science
1991;252:13081310.
157. Sloan-Lancaster J, et al. Partial T cell
signaling: altered phospho-zeta and lack of
zap70 recruitment in APL-induced T cell
anergy. Cell 1994;79:913922.
158. Nicholson LB, et al. A T cell receptor
antagonist peptide induces T cells that
mediate bystander suppression and prevent
autoimmune encephalomyelitis induced
with multiple myelin antigens. Proc Natl
Acad Sci USA 1997;94:92799284.
159. Nicholson LB, et al. An altered peptide
ligand mediates immune deviation and
prevents autoimmune encephalomyelitis.
Immunity 1995;3:397405.
160. Brocke S, et al. Treatment of experimental
encephalomyelitis with a peptide analogue
of myelin basic protein. Nature
1996;379:343346.
161. Windhagen A, et al. Modulation of cytokine
patterns of human autoreactive T cell clones
by a single amino acid substitution of their
peptide ligand. Immunity 1995;2:373380.
162. Ausubel LJ, Krieger JL, Hafler DA. Changes
in cytokine secretion induced by altered
peptide ligands of myelin basic protein
peptide 8599. J Immunol
1997;159:25022512.
163. Ausubel LJ, Bieganowska KD, Hafler DA.
Cross-reactivity of T-cell clones specific for
altered peptide ligands of myelin basic
protein. Cell Immunol 1999;193:99107.
164. Bielekova B, et al. Encephalitogenic potential

of the myelin basic protein peptide (amino
acids 8399) in multiple sclerosis: results of
a phase II clinical trial with an altered
peptide ligand. Nat Med
2000;6:11671175.
165. Kappos L, et al. Induction of a nonencephalitogenic type 2 T helper-cell
autoimmune response in multiple sclerosis
after administration of an altered peptide
ligand in a placebo-controlled, randomized
phase II trial. The Altered Peptide Ligand
Relapsing MS Study Group. Nat Med
2000;6:11761182.
166. Murate T, et al. Cell type-specific localization
of sphingosine kinase 1a in human tissues.
J Histochem Cytochem 2001;49:845855.
167. Bornstein MB, et al. A pilot trial of Cop 1 in
exacerbating-remitting multiple sclerosis.
N Engl J Med 1987;41:533539.
168. Fridkis-Hareli M, Strominger JL.
Promiscuous binding of synthetic
copolymer 1 to purified HLA-DR molecules.
J Immunol 1998;160:43864397.
169. Johnson KP, et al. Extended use of glatiramer
acetate (Copaxone) is well tolerated and
maintains its clinical effect on multiple
sclerosis relapse rate and degree of disability.
Copolymer 1 Multiple Sclerosis Study
Group. Neurology 1998;50:701708.
170. Filippi M, et al. Glatiramer acetate reduces
the proportion of new MS lesions evolving
into black holes. Neurology
2001;57:731733.
171. Duda PW, et al. Human and murine CD4
T cell reactivity to a complex antigen:
recognition of the synthetic random
polypeptide glatiramer acetate. J Immunol
2000;165:73007307.
172. Duda PW, et al. Glatiramer acetate
(Copaxone) induces degenerate,
Th2-polarized immune responses in patients
with multiple sclerosis. J Clin Invest
2000;105:967976.
173. Qin Y, et al. Characterization of T cell lines
derived from glatiramer-acetate-treated
multiple sclerosis patients. J Neuroimmunol
2000;108:201206.
174. Dabbert D, et al. Glatiramer acetate
(copolymer-1) -specific, human T cell lines:
cytokine profile and suppression of T cell
lines reactive against myelin basic protein.
Neurosci Lett 2000;289:205208.
175. Hafter DA, et al. Investigation of in vivo
activated T cells in multiple sclerosis and
inflammatory central nervous system
diseases. Clin Immunol Immunopathol
1985;37:163171.
176. Yednock TA, et al. Prevention of
experimental autoimmune
encephalomyelitis by antibodies against
alpha 4 beta 1 integrin. Nature
1992;356:6366.
177. Miller DH, et al. A controlled trial of

natalizumab for relapsing multiple sclerosis.
N Engl J Med 2003;348:1523.
178. Martel RR, Klicius J, Galet S. Inhibition of
the immune response by rapamycin, a new
antifungal antibiotic. Can J Physiol
Pharmacol 1977;55:4851.
179. Kahan BD, Chang JY, Sehgal SN. Preclinical
evaluation of a new potent
immunosuppressive agent, rapamycin.
Transplantation 1991;52:185191.
180. Shapiro AM, et al. Islet transplantation in
seven patients with type 1 diabetes mellitus
using a glucocorticoid-free
immunosuppressive regimen. N Engl J Med
2000;343:230238.
181. Slavik JM, et al. Uncoupling p70(s6) kinase
activation and proliferation: rapamycinresistant proliferation of human CD8()
T lymphocytes. J Immunol
2001;166:32013209.
182. Slavik JM, et al. Rapamycin-resistant
proliferation of CD8 T cells correlates with
p27kip1 downregulation and bcl-xL
induction, and is prevented by an inhibitor
of phosphoinositide 3-kinase activity. J Biol
Chem 2004;279:910919.
183. Dean G, et al. Multiple sclerosis among
immigrants in greater London. Br Med J
1976;1:861864.
184. Kurtzke JF, Gudmundsson KR, Bergmann S.
MS in Iceland: 1. Evidence of a post-war
epidemic. Neurology 1982;32:143150.
185. Sadovnick A, Baird P, Ward R. Multiple
sclerosis: updated risks for relatives. Am J
Med Genet 1988;39:533541.
186. Jersild C, et al. Histocompatibility
determinants in multiple sclerosis, with
special reference to clinical course. Lancet
1973;2:12211225.
187. GAMES: Transatlantic multiple sclerosis
genetics cooperative. A meta-analysis of
whole genome linkage screens in multiple
sclerosis. J Neuroimmunol
2003;143:3946.
188. Allen M, et al. Association of susceptibility to
multiple sclerosis in Sweden with HLA class
II DRB1 and DQB1 alleles. Hum Immunol
1994;39:4148.
189. Risch N, Merikangas K. The future of genetic
studies of complex human diseases. Science
1996;273:15161517.
190. Consortium G.I.H.G.S. Initial Sequencing
and analysis of the human genome. Nature
2001;409:860921.
191. Wang DG, et al. Large-scale identification,
mapping, and genotyping of singlenucleotide polymorphisms in the human
genome. Science 1998;280:10771082.
192. Sachidanandam R, et al. A map of human
genome sequence variation containing 1.42
million single nucleotide polymorphisms.
Nature 2001;409:928933.
193. Rioux JD, et al. Genetic variation in the 5q31

cytokine gene cluster confers susceptibility
to Crohn disease. Nat Genet
2001;29:223228.
194. Daly MJ, et al. High-resolution haplotype
structure in the human genome. Nat Genet
2001;29:229232.
195. Jeffreys AJ, Kauppi L, Neumann R. Intensely
punctate meiotic recombination in the class
II region of the major histocompatibility
complex. Nat Genet 2001;29:217222.
196. Gabriel SB, et al. The structure of haplotype
blocks in the human genome. Science
2002;296:22252229.
197. Johnson GC, et al. Haplotype tagging for the
identification of common disease genes. Nat
Genet 2001;29:233237.
198. Goldstein DB. Islands of linkage
disequilibrium. Nat Genet
2001;29:109111.
199. Altshuler D, et al. The common PPARgamma
Pro12Ala polymorphism is associated with
decreased risk of type 2 diabetes. Nat Genet
2000;26:7680.
200. Giallourakis C, et al. IBD5 is a general risk
factor for inflammatory bowel disease:
replication of association with Crohn disease
and identification of a novel association with
ulcerative colitis. Am J Hum Genet
2003;73:205211.
201. Sawcer S, Compston A. The genetic analysis
of multiple sclerosis in Europeans: concepts
and design. J Neuroimmunol
2003;143:1316.
202. Abecasis GR, Cherny SS, Cardon LR. The
impact of genotyping error on family-based
analysis of quantitative traits. Eur J Hum
Genet 2001;9:130134.
203. Sawcer SJ, et al. Enhancing linkage analysis
of complex disorders: An evaluation of highdensity genotyping. Hum Mol Genet
2004;13:19431949.
204. Middleton FA, et al. Genomewide linkage
analysis of bipolar disorder by use of a highdensity single-nucleotide-polymorphism
(SNP) genotyping assay: a comparison with
microsatellite marker assays and finding of
significant linkage to chromosome 6q22.
Am J Hum Genet 2004;74:886897.
205. Chakraborty R, Weiss WK. Admixture as a
tool for finding linked genes and detecting
that difference from allelic association
between loci. Proc Natl Acad Sci USA
1988;85:91199123.
206. Patterson N, et al. Methods for high-density
admixture mapping of disease genes. Am J
Hum Genet 2004;74:9791000.
207. Smith MW, et al. A high-density admixture
map for disease gene discovery in African
Americans. Am J Hum Genet
2004;74:10011013.
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David A.

Summary: Multiple sclerosis (MS) is a complex genetic disease associated

Immunological Reviews 2005

Copyright Blackwell Munksgaard 2005

Multiple sclerosis (MS) was first described in 1868 noting the

Hafler et al Multiple sclerosis

response to self-antigens in a genetically susceptible host. It

epitope spreading to a number of antigens implicated in MS,

Local antigenpresenting cells

Innate, non-induced regulatory T cells

Fig. 1. Induced and innate regulatory T cells.

Immunological Reviews 204/2005

Hafler et al Multiple sclerosis

that could trigger autoreactive T-cell clones in a manner that

CNS-specific regulation of inflammation

Cellular events occurring within and around MS plaques

Gross examination of MS brain tissue reveals multiple sharply

Immunological Reviews 204/2005

inflammatory activity along the lesion edge. Recently, four

Molecular events occurring within and around MS plaques

Hafler et al Multiple sclerosis

margins of chronic active lesions compared to the margins of

Regulation of CNS inflammation by resident cells of the CNS

For well over a decade, there has been considerable debate

Hafler et al Multiple sclerosis

recently demonstrated that depending on the mouse strain used

Immunological Reviews 204/2005

cells results in the onset of systemic autoimmune diseases in

CD4CD25hi T cells are present with the same frequency in

We stained whole mononuclear cells from freshly drawn human

Hafler et al Multiple sclerosis

CD4CD25hi regulatory T cells display impaired function in

We also examined the production of cytokines in all the

As it was critical to examine the regulatory T-cell function, we

Fig. 2. Summary of CD4 CD25 T-cell regulatory function that is

CD4 CD25 cells to the CD4CD25 responder cells at 1 : 1 ratio in

Immunological Reviews 204/2005

Hafler et al Multiple sclerosis

CD62L expression on CD4CD25hi regulatory T cells

Although there were no differences in the frequency of

Discussion of CD4CD25 T cells in patients with MS

Immunological Reviews 204/2005

Autoantibodies in patients with MS

Hafler et al Multiple sclerosis

sparked with more recent findings in MS pathological studies. In

The humoral response in the MS CNS

Intensive investigation into the role of the humoral response in

of antibodies within the elevated CSF Ig directed against viruses

Hafler et al Multiple sclerosis

Intrathecal immunoglobulin synthesis

Antibodies are found in active multiple sclerosis CNS lesions

with other autoimmune and neurological diseases (101). TAL is

Immunological Reviews 204/2005

searches for autoantibodies may be due to the deposition of

Hafler et al Multiple sclerosis

rodents. Autoantibodies capable of solution-phase binding to

Fig. 3. The level of serum autoantibodies to myelin basic protein

bodies directed against PLP have been identified in the peripheral

MOG as a B-cell antigen in MS

Hafler et al Multiple sclerosis

due to its role in EAE, a significant amount of effort in this

Immunological Reviews 204/2005

The majority of patients present with relapsing/remitting

Hafler et al Multiple sclerosis