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J. Exp. Med. Vol. 206 No. 6 1303-1316
Autoimmune diseases can affect most organs of
the body including liver, heart, the endocrine
system, the musculoskeletal apparatus, and the
central nervous system (CNS). They commonly
start off at a young age and then last throughout
life, often resulting in severe disability. The fac-
tors that trigger the onset, modulate the course,
and determine the clinical character of autoim-
mune diseases have remained obscure, a deficit
of knowledge which sets limits to the design of
specific and efficient therapies.
Yet there is increasing evidence that organ-
specific autoimmune diseases, such as rheuma-
toid arthritis, type 1 diabetes mellitus, and multiple
sclerosis (MS), are the result of a pathogenic inter-
action of autoimmune T and B cells. There is
substantial information on the role of T cells in
organ-specific autoimmunity. Some act as ef-
fector cells attacking self-tissues, either directly
or via recruiting accessory cells like macrophages.
Other T cells regulate the time course of the
response and still others provide help to auto-
antibody-producing B cells. The contribution of
autoimmune B cells to the inflammatory patho-
genesis seems to be complex as well. Beyond
producing humoral autoantibodies, B cells serve
as APCs activating pathogenic T cells, and,
through their capacity of releasing cytokines,
B cells are involved in shaping local microenvi-
ronments favorable to evolving cellular auto-
Florian C. Kurschus:
Abbreviations used: cDNA,
complementary DNA; CNS,
central nervous system; EAE,
experimental autoimmune en-
cephalomyelitis; MOG, myelin
MS, multiple sclerosis; NTL,
nontransgenic littermate; OSE,
opticospinal EAE; OSMS, opti-
cospinal MS; PI, propidium
iodide; rMOG, recombinant rat
MOG; RR, relapsing-remitting.
A. Holz’s present address is Department of Cellular and Mo-
lecular Biology, Technical University of Braunschweig,
D-38106 Braunschweig, Germany.
B. Pllinger’s present address is Novartis Pharma AG,
CH-4056 Basel, Switzerland.
F.C. Kurschus’s present address is I. Medizinische Klinik
und Poliklinik, Johannes Gutenberg Universität, D-55131
Spontaneous relapsing-remitting EAE
in the SJL/J mouse: MOG-reactive
transgenic T cells recruit endogenous
MOG-specific B cells
Bernadette Pöllinger,1 Gurumoorthy Krishnamoorthy,1 Kerstin Berer,1
Hans Lassmann,3 Michael R. Bösl,2 Robert Dunn,4 Helena S. Domingues,1
Andreas Holz,1 Florian C. Kurschus,1 and Hartmut Wekerle1
1Department of Neuroimmunology and 2Transgenic Service, Max Planck Institute of Neurobiology, D-82152 Martinsried, Germany
3Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria
4Department of Immunology, Biogen Idec, San Diego, CA 92122
We describe new T cell receptor (TCR) transgenic mice (relapsing-remitting [RR] mice)
carrying a TCR specific for myelin oligodendrocyte glycoprotein (MOG) peptide 92–106 in
the context of I-As. Backcrossed to the SJL/J background, most RR mice spontaneously
develop RR experimental autoimmune encephalomyelitis (EAE) with episodes often altering
between different central nervous system tissues like the cerebellum, optic nerve, and
spinal cord. Development of spontaneous EAE depends on the presence of an intact B cell
compartment and on the expression of MOG autoantigen. There is no spontaneous EAE
development in B cell–depleted mice or in transgenic mice lacking MOG. Transgenic T cells
seem to expand MOG autoreactive B cells from the endogenous repertoire. The expanded
autoreactive B cells produce autoantibodies binding to a conformational epitope on the
native MOG protein while ignoring the T cell target peptide. The secreted autoantibodies
are pathogenic, enhancing demyelinating EAE episodes. RR mice constitute the first spon-
taneous animal model for the most common form of multiple sclerosis (MS), RR MS.
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The Journal of Experimental Medicine
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
JEM VOL. 206, June 8, 2009
Deciphering the interactions between T and B cells in the
spontaneous development of organ-specific autoimmune re-
sponses requires suitable animal models. Naturally occurring
models are available for type 1 diabetes mellitus and systemic
lupus erythematosus but not for autoimmunity in the CNS
(1). Recently, we and others described a double-transgenic
mouse model, which simulates opticospinal MS (OSMS) re-
markably well, a variant of which is also known as Devic’s
disease (2, 3). These mice, termed opticospinal experimental
autoimmune encephalomyelitis (EAE [OSE]) mice, express
myelin oligodendrocyte glycoprotein (MOG)–specific recep-
tors on T and B cells and spontaneously develop demyelinat-
ing inflammatory disease at frequencies >50%. Like in human
OSMS (4), the lesions in affected mice are restricted to optic
nerve and spinal cord, and, in most cases, the disease takes a
chronic progressive course without remissions and marked re-
lapses. It should, however, be noted that the type of MS that
most prevalently affects Caucasian populations differs funda-
mentally from OSMS (5). Typically, MS starts out with a re-
lapsing-remitting (RR) course, where disease episodes may
completely resolve only to be followed by a subsequent re-
lapse. In this disease variant, the pathogenic lesions, demyelin-
ating plaques, may be located throughout the CNS, thus
causing the notoriously varied neurological defect patterns.
In this paper, we describe a new transgenic mouse model
that spontaneously develops RR-EAE and, thus, recapitulates
the “Western” variant of MS. Furthermore, and most impor-
tantly, we found that in these mice transgenic autoimmune
T cells expand autoimmune B cells from the endogenous
immune repertoire and guide them to produce antibodies
against conformational epitopes of the MOG protein, which,
together with complement, may initiate the destruction of
MOG-expressing target cells.
New MOG-specific TCR transgenic SJL/J mice
We generated transgenic mice expressing a TCR specific for
the rat/mouse MOG peptide 92–106 in the context of I-As.
This TCR, which uses V8.3 and V4 genes, was derived
from a MOG-specific encephalitogenic Th1-CD4+ T cell clone
isolated from a WT SJL/J mouse immunized against recom-
binant rat MOG (rMOG; Fig. S1). We selected three founder
lines differing in markedly distinct proportions of transgenic
V8.3+/V4+ CD4+ T cells in central and peripheral immune
repertoires (Fig. 1 A and Fig. S2). In low frequency TCR1586
mice, 18% of single-positive CD4+CD8 thymocytes ex-
pressed the transgenic TCR. The proportion was 75% in
medium frequency TCR1639 mice and 99% in high frequency
TCR1640 mice (Fig. 1 A). In all three transgenic mouse lines,
transgene expression levels in the peripheral immune system
were proportional to the ones in the central thymic reper-
toires (Fig. S2).
The density of the transgenic TCR on the surface of ma-
ture CD4+ T cells in the spleen varied markedly (Fig. S2).
Especially in high frequency TCR1640 spleens, T cells could
be distinguished based on the low and high densities of sur-
face TCR (dim and bright TCR). The dim TCR population
expressed higher levels of CD25 and CD69 and lower levels
of CD62L (unpublished data).
Stimulation of nonimmunized transgenic spleen cells with
rMOG or MOG92-106 peptide in vitro led to dose-dependent pro-
liferative responses (Fig. 1 B). Concomitant cytokine responses
largely followed the proliferation pattern. Although the secretion
of proinflammatory IFN-, IL-17, and antiinflammatory IL-10
was similar in TCR1640 and TCR1639 lines, IL-2 secretion was
higher in TCR1639 T cells. The Th2-related cytokines IL-4 and
IL-5 were absent in nearly all samples tested (Fig. 1 C).
Spontaneous EAE in single- and double-transgenic SJL/J
anti-MOG mice: RR course with varied clinical syndromes
While continuously backcrossing the line TCR1640 into the
SJL/J background, we noted spontaneous EAE-like disease de-
veloping at a high frequency (Fig. 2 A and Table S1). In the
eighth backcross generation, >80% of all females developed
EAE within 160 d. During the same period, the EAE rate in
males was >60%. EAE was rare in transgenic SJL/J mice with
low frequency TCR1586 and surprisingly absent in medium fre-
quency TCR1639 mice and in Mog-deficient TCR1640 (TCR1640
× Mog/) mice (Table S1).
We and others previously described a double-transgenic
mouse strain, OSE mice (2, 3), which expressed a MOG-
specific TCR transgene, 2D2 (specific for MOG peptide
35–55 in context of I-Ab), along with the gene coding for the
rearranged IgH variable chain of the classical anti-MOG
monoclonal antibody, 8.18-C5 (IgHMOG mice) (6). We have
now created similar double-transgenic mice by mating
TCR1640 SJL/J mice to IgHMOG on SJL/J background. Dou-
ble-transgenic TCR1640 × IgHMOG mice developed sponta-
neous EAE very similar to TCR1640 single transgenics, with
some apparently minor differences. Almost all double-trans-
genic females came down with disease by 120 d of age,
whereas males of the same age showed an EAE rate of <40%.
This gender gap was highly significant but narrowed during
the subsequent weeks (Fig. 2 A).
Figure 1. Characterization of a new MOG-specific TCR transgenic SJL/J mouse. (A) Thymocytes from 8–10-wk-old TCR transgenic mice or NTLs
were stained with antibodies to CD4, CD8, TCR-V4, and TCR-V8.3 and cells were analyzed by flow cytometry. Representative figures of three to seven
analyzed mice are shown. Transgenic V8.3 and V4 are shown on cells gated as indicated, either CD4+/CD8 or CD4/ CD8+. (B) Proliferative response to
recombinant MOG protein (rMOG) or MOG peptide 92–106 measured as 3H-thymidine incorporation. Splenocytes from TCR transgenic mice (8–10-wk-old
healthy) were cultured with indicated concentrations of rMOG (top) and MOG92-106 (bottom). (C) Cytokine responses to rMOG. Indicated cytokines were
measured in supernatants harvested 48 h after rMOG activation by ELISA. Pooled data from three independent experiments are shown. Error bars indicate
SEM. B and C: TCR1640, n = 3; TCR1639, n = 3; TCR1586, n = 3; NTL, n = 2.
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
The CNS disease spontaneously developing in TCR trans-
genic SJL/J mice differed dramatically from spontaneous OSE
seen in OSE mice (2, 3). In TCR transgenic SJL/J mice, single-
transgenic TCR1640 mice, and double-transgenic TCR1640 ×
IgHMOG mice, disease was extremely variable both in course and
We also crossed IgHMOG mice to the medium and low fre-
quency lines TCR1639 and TCR1586, respectively. Only 1 out
of 17 medium frequency TCR1639 × IgHMOG mice displayed
EAE and none of the low frequency TCR1586 × IgHMOG showed
any clinical symptoms (Table S1).
Figure 2. Spontaneous RR-EAE in TCR transgenic SJL/J mice. (A) Male and female single-transgenic TCR1640 compared with double-transgenic
TCR1640 × IgHMOG mice. Shown is the spontaneous incidence of first signs of ataxia or classical EAE-like symptoms in TCR1640 (left) and double-transgenic
TCR1640 × IgHMOG (right) mice. TCR1640 females (f), n = 12; males (m), n = 27; TCR1640 × IgHMOG females, n = 20; males, n = 26; TCR1640 × Mog/ mice,
n = 6. Disease kinetic of genders in TCR1640 mice was not statistically significant (P = 0.304) but differed significantly between sexes of TCR1640 × IgHMOG
mice (P = 0.0004). (B) Histological analysis of cerebellum and spinal cord from a sick TCR1640 mouse (RR mouse) with ataxia and classical paralysis. Cer-
ebellum (I–IV) and spinal cord (V–VIII) showed severe infiltration, demyelination, and axonal damage as visualized by immunohistochemistry using anti-
CD3 (I and V) and anti–Mac3 antibodies (II and VI), Luxol fast blue staining (III and VII), and Bielschowsky silver impregnation (IV and VIII). I, II, V, and VI
were counterstained by hematoxylin and eosin (H&E). Magnification: ×17 (cerebellum) and ×30 (spinal cord). Bars, 1 mm. Data are representative of at
least two independent experiments consisting of more than three mice per group.
Table I. Spontaneous EAE in TCR transgenic SJL/J mice: course of disease
Mice (gender) RR (n) Progressive (n)
With full remission With partial remission
TCR1640 × IgHMOG (f)
TCR1640 × IgHMOG (m)
rMOG imm. SJL/J (f)
f, female; m, male; imm., immunized.
JEM VOL. 206, June 8, 2009
clinical nature. Typically, in females EAE started with an RR
course. Often, the first attacks resolved completely but were
followed by further bouts. Intriguingly, individual EAE bouts
often differed radically in their neurological defects. In a sub-
stantial number of cases, the initial bout was dominated by
ataxia rather than by classical EAE defects. Affected mice
were unable to walk along a straight line, deviating and fall-
ing to the side, but did not show limb weakness or paralysis
(Video 1). In many cases, the mice recovered completely un-
til a first relapse, which commonly showed a completely dif-
ferent clinical picture, for example, hind limb paralysis as seen
in typical EAE. Again, this second disease episode subsided,
giving way to partial remission. Later disease episodes mostly
showed classical paralytic EAE (Table I and Fig. S3).
Although there was no clear distinction between single- and
double-transgenic SJL/J mice, EAE course differed between
females and males. As detailed in Table I, RR disease was more
common in females. A minority of female mice developed pro-
gressive EAE from the beginning, but more than half of the
males developed primary progressive EAE.
CNS changes representing different clinical EAE episodes
The clinical deficiencies developing in affected mice were
reflected by the location and character of underlying CNS
lesions. Ataxic mice displayed large inflammatory and demy-
elinated lesions in cerebellum and brain stem (Fig. 2 B). In
contrast, in mice suffering from conventional EAE, lesions
were distributed throughout the spinal cord, brain stem, and
optic nerve (Table S2).
Irrespective of their location within the CNS, the inflam-
matory lesions were characterized by numerous CD3+ T cells
in the middle of a plethora of Mac3-positive activated macro-
phage-like cells (Fig. 2 B). The inflammatory infiltrates were
embedded in large areas of demyelination and axon destruc-
tion. In line with their comparable clinical score, the histolog-
ical patterns were similar between TCR1640 single-transgenic
and TCR1640 × IgHMOG double-transgenic mice (Table S2).
Cytofluorimetric analyses confirmed that, within the CNS,
the major infiltrating inflammatory lymphocytes were CD4+
T cells and B cells together with a minor population of mac-
rophages and CD8+ T cells (Fig. 3, A–C), with only minor
contributions from CD8+ T cells. Notably, most transgenic
CD4+ T cells infiltrating the CNS were activated. Many cells
expressed the activation marker CD25 and were CD62L nega-
tive (Fig. 3 D). Additionally, infiltrating CD4+ T cells ex-
pressed the activation markers CD44, VLA4, and ICOS
(unpublished data). Furthermore, a high proportion (>65%)
of CNS-infiltrating transgenic T cells partly down-modulated
Figure 3. Inflammatory cell infiltrates in RR-EAE lesions.
(A–C) Cellular infiltrate into the CNS of sick TCR1640 mice (score 3) is com-
posed of macrophages, T cells, and B cells. CNS mononuclear cells were
isolated from a sick TCR1640 mouse and stained against CD11b and CD45.1
(A), together with CD8 and CD4 (B) or together with CD19 and B220
(C). Cells in B and C were analyzed among gated CD45.1+CD11b cells (red
region as indicated). (D–F) Activation and Th1/Th17 cytokine expression of
infiltrating CD4+ T cells. (D) CNS infiltrate cells express CD25, but not
CD62L, and partially down-modulate their TCR (V8.3 and V4). Activa-
tion status and TCR expression was compared between splenocytes (left)
and CNS-isolated cells (right). (E) CD4+CD3+ T cells infiltrating the CNS of
sick TCR1640 mice predominantly expressed the pathogenic TCR composed
of V8.3 and V4 chains in a low expression level (TCRlow) compared with
the spleen. Numbers indicate the percentage of stained cells in the re-
spective quadrant or region. (F) Intracellular cytokine staining after stimu-
lation with PMA/ionomycin in brefeldin A. Th1 (IFN-+/IL-17) and Th17
(IFN-/IL-17+) cells are enriched in CNS infiltrates of sick TCR1640 (score
3.5) mice compared with splenocytes. D–F were analyzed among gated
CD45.1+CD4+ cells. Flow cytometry data are representative of three to five
sick TCR1640 mice analyzed in three to five independent experiments.
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
CD4 in diseased mice. Interestingly, we found no major differ-
ences between immunized WT SJL/J mice and TCR1640 mice,
which suffered from classical paralytic EAE. In line with their
clinical picture, ataxic mice showed higher expression levels of
the analyzed genes in brain (which also includes cerebellum and
brain stem) than did paralytic relapsed mice. Conversely, expres-
sion in spinal cord was higher in paralytic relapsed mice than in
ataxic mice. Intriguingly, most analyzed factors were elevated
even in healthy TCR1640 mice (score 0) as compared with
healthy nontransgenic littermate (NTL) mice, likely indicating
subclinical inflammation. Finally, during the remission phase,
expression of all analyzed genes dropped down to similar values
as in score 0–TCR1640 mice (Fig. S4).
Expansion of MOG-specific B cells in TCR1640
single-transgenic SJL/J mice
Single-transgenic TCR1640 mice showed evidence of a strong
MOG-specific B cell response. Spontaneous EAE lesions of sin-
gle-transgenic TCR1640 animals displayed prominent deposits of
Ig and some activated complement (Fig. 4). Furthermore, the
inflamed CNS tissue contained, besides CD4+ and CD8+ lym-
phocytes, substantial numbers of B cells (5–40% of infiltrated
lymphocytes) expressing CD19 or B220 as revealed by immuno-
cytochemistry and flow cytometry (Fig. 3 and Fig. 4 E).
their transgenic V4 and V8.3 determinants, which is in
contrast to peripheral splenic populations, where the modu-
lated subpopulations comprised less than half of the entire
transgenic CD4+ T cell subset (Fig. 3 E).
In agreement with previous EAE studies (7–10), we found
CNS-infiltrating Th1 (IFN-+/IL-17) and Th17 (IFN-/
IL-17+) cells. Compared with spleen, Th17 cells were enriched
in CNS threefold and Th1 cells more than twofold (Fig. 3 F).
A substantial part of the activated CD4+CD25+ infiltrate T cells
express transcription factor Foxp3, a marker of regulatory
T cells (not depicted) (11).
Next, we measured the cytokine milieu in brain and spinal
cord of TCR1640 mice in different disease stages by real-time
PCR in comparison to rMOG immunized SJL/J mice (Fig. S4).
Most cytokines followed an infiltration pattern proportional to
CD4 expression. As expected from our infiltrate analysis (Fig. 3),
we found expression of both IFN- and IL-17 in diseased mice.
In accordance with this, TNF- and IFN- inducible protein
10 (IP-10/CXCL10) were strongly up-regulated during disease.
Of the Th2 cytokines analyzed (IL-4, IL-5, and IL-13), only
IL-5 was at measurable levels. This cytokine may support B cell
proliferation, differentiation, and antibody secretion (12). The
levels of the regulatory cytokine IL-10 and of the T reg cell
transcription factor FoxP3 paralleled the observed expression of
Figure 4. Ig deposition and B cell infiltrates in spinal cord of sick TCR1640 × IgHMOG and TCR1640 mice. (A–D) Histological analysis of spinal cord
using anti-Ig antibodies shows deposition of Ig in sick TCR1640 × IgHMOG (A and B) and TCR1640 (C and D) mice (B and D show magnifications of marked
areas in A and C, respectively). (E) Anti-B220 staining revealed some B cells among cellular infiltrates in sick TCR1640 mice. (F) Deposition of complement
C9neo within CNS lesions of TCR1640 mice. Mononuclear cells were stained with H&E. Magnifications: A and C, ×50; B, ×425; D and E, ×200; F, ×450. Bars:
(A and C) 1 mm; (B and D–F) 100 µm. Data are representative of at least two independent experiments consisting of more than three mice per group.
JEM VOL. 206, June 8, 2009
Double-transgenic TCR1640 × IgHMOG animals showed an
anti-MOG antibody response dominated by the transgene-
specific allotype Igha. In addition, however, a minor part of the
MOG-specific antibodies expressed the endogenous nontrans-
genic Ighb allotype (Fig. 5 B). Both antibody species were
switched from IgM to IgG1 (Fig. 5, A and B).
Single-transgenic TCR1640 mice also produced anti-MOG
autoantibodies (of endogenous Ighb-allotype) reaching titers sim-
ilar to those from WT mice immunized with rMOG (Fig. 6 A).
The anti-MOG Igs were mainly of IgG1 and IgG2a/b isotypes,
with very little IgM. The spontaneously appearing anti-MOG
transgenic high frequency TCR1640 mice formed autoantibodies
irrespective of their EAE status. This contrasted with low fre-
quency single-transgenic TCR1586, where anti-MOG autoanti-
bodies were noted solely in the few animals with spontaneous
EAE but not in resistant mice (Fig. 6 B). Also, mice of the EAE-
resistant medium frequency line TCR1639 did not produce spon-
taneous MOG-specific antibodies. Development of spontaneous
MOG antibodies was dependent on the presence of the autoan-
tigen. Sera of MOG-deficient TCR1640 mice (bred on the Mog-
deficient [Mog/] background [reference 13]) did not contain
any MOG-specific antibodies (Fig. 6 B). The B cell response in
TCR1640 mice was specific to MOG and did not extend to other
autoantigens (Fig. 6 C). In single-transgenic TCR1640 mice, au-
toantibodies became demonstrable from 5 wk of life and, in our
series of measurements, persisted up to 9–10 wk, but very young
mice, up to 4 wk of age, had no anti-MOG Igs (IgG1 and
IgG2a/b isotypes) (Fig. 6 D).
Contribution of endogenous B cells and anti-MOG
autoantibodies to RR spontaneous EAE
There is evidence that only autoantibodies directed against
conformational epitopes, not linear epitopes, are involved in
the pathogenesis of EAE (14) and that SJL/J mice, but not
C57BL/6 mice, are able to produce such antibodies upon im-
munization with rMOG in CFA (15). The anti-MOG auto-
antibodies formed spontaneously in TCR1640 mice also bound
to correctly folded MOG expressed on the surface of trans-
duced EL4 cells and, thus, behaved like the classical anticon-
formational MOG mAb 8.18-C5 and its H chain transgene in
TCR1640 × IgHMOG mice (16) (Fig. 7 A). Binding of autoan-
tibodies from TCR1640 and sick TCR1586 mice to EL4-MOG
cells (dilutions of 1:20 and 1:200) followed by rabbit comple-
ment (Fig. 7 B) resulted in the specific lysis of the target cells.
Sera from healthy TCR1586 and TCR1639 or Mog-deficient
TCR1640 did not support lysis (unpublished data).
We confirmed the pathogenic potential of antibodies, in
vivo. We immunized WT SJL/J mice with a low dose of PLP
139–151 and with incipient clinical EAE symptoms (between
scores 0.5 and 1), we transferred serum either from TCR1640
mice (high titers of anti-MOG antibodies documented by
ELISA) or NTLs, and compared the effects with the standard
Figure 5. MOG-specific antibodies in double-transgenic TCR1640 ×
IgHMOG and single TCR transgenic mice. (A) MOG binding allotype a
autoantibodies were detected by ELISA in sera of indicated groups (each
five to six mice). (B) Spontaneous anti-MOG IgG1 allotype b antibodies in
TCR1640 and TCR1640 × IgHMOG but not in NTL (n = 5 for each group). Sera
at the indicated dilutions were incubated with plates precoated with
rMOG. Bound anti-MOG Ig was detected by allotype-specific antibodies as
indicated. Mean absorbance at OD 405 nm is shown. Error bars indicate
SEM. Data are representative of two to three independent experiments.
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
TCR1640 mice were treated with anti-CD20 or isotype con-
trol (mouse IgG2a) every 2 wk starting on day 3. This proto-
col removed the B cells from peripheral blood, as well as
spleen and lymph nodes, but left significant numbers of B cells
in the bone marrow (Fig. S5, A and B). B cell depletion went
along with either complete or partial loss of anti-MOG IgG1
anti-MOG monoclonal antibody 8.18C-5. In mice that had re-
ceived TCR1640 serum, we noted a statistically significant increase
in EAE severity similar to that of 8.18C-5 mAb (Fig. 8 A).
To examine the importance of B cells in the develop-
ment of spontaneous EAE, we depleted B cells in TCR1640
mice using a monoclonal mouse anti–mouse CD20 antibody.
Figure 6. MOG-specific antibodies in single TCR transgenic mice. (A) Endogenous MOG binding antibodies of allotype b were measured by ELISA in
sera of NTLs, healthy and sick TCR1640, and rMOG-immunized WT SJL/J (n = 6 for each group). (B) Anti-MOG Igs of IgG1b isotype are formed in TCR1640 and sick
TCR1586 but not in TCR1639 or TCR1586 mice. Control NTLs, as well as TCR1640 mice deficient for MOG (Mog/), are devoid of MOG binding autoantibodies (each
three to five mice per group). (C) Autoantibodies in sera from TCR1640 mice are specific for MOG. Anti-MOG Ig antibodies, but not anti-MBP or anti–DNA-
specific antibodies, were detected by ELISA in sera from TCR1640 mice (1/50 diluted) using IgG1b-specific antibodies (n = 5). (D) Appearance of anti-MOG IgG1b
and IgG2a/bb in TCR1640 mice by 5 wk of age. MOG binding antibodies were measured in 1/100 diluted sera from mice of different ages, each with three to five
mice per group. Sera at the indicated dilutions were incubated with plates precoated with rMOG. Bound anti-MOG Ig was detected by allotype-specific anti-
bodies as indicated. Mean absorbance at OD 405 nm is shown. Error bars indicate SEM. Data are representative of two independent experiments.
JEM VOL. 206, June 8, 2009
The course and clinical expression of autoimmune dis-
eases are determined by diverse factors, both genetic and en-
vironmental. Indeed, SJL/J mice are predisposed to develop
fluctuating disease. Depending on the protocol applied, active
immunization against encephalitogenic proteins may trigger
RR disease (18). Clearly, this response pattern is facilitated by
distinct regulatory genes (19), which may also influence spon-
taneous EAE developing in RR mice. So far, our breeding
results suggest that B10.S mice, which have a distinct genetic
background but share the MHC and the TCR transgene with
SJL/J RR mice, very rarely develop spontaneous EAE, and in
the few cases observed, the disease took a chronic rather than
RR course (unpublished data).
Most variants of induced EAE present a stereotypical clini-
cal syndrome with motor deficiencies starting at tail and hind
limbs and progressing cranially with ongoing disease. Histolog-
ically, this clinical picture is reflected by a caudocranial gradient
of inflammatory lesions (20). In a few models, however, addi-
tional parts of the CNS are affected. For example, in C57BL/6
mice, MOG-induced EAE has a predilection to affect the optic
nerve (2, 3), and cerebellar disease was noted in further anti-
gen/mouse combinations (21, 22). Our RR mouse models are
the first to show immune attacks against different CNS parts in
subsequent inflammatory relapses. They thus provide a unique
paradigm to study the mechanisms underlying the targeting of
MS lesions within the CNS during ongoing disease.
serum autoantibodies (Fig. S5 C). Within an observation pe-
riod of up to 30 wk, only one out of nine anti-CD20–treated
mice presented mild and transient EAE symptoms, whereas in
mice receiving control antibody, spontaneous EAE was noted
in >85% of the cases (Fig. 8 B and Table S3).
The RR mouse, as described in this paper, is the first transgenic
mouse model that recapitulates the major features of RR-MS,
the most prevalent type of human inflammatory demyelination.
The disease starts spontaneously and, in contrast to OSE mice
described previously (2, 3), in most cases takes an RR course,
with clinical signs differing between individual bouts. Impor-
tantly, development of spontaneous disease goes along with the
expansion of myelin autoimmune B cells from the endogenous
RR mice are transgenic SJL/J mice expressing at high frequency
a TCR specific for MOG peptide 92–106 in the context of I-As.
RR mice differ substantially from previously created transgenic
SJL/J mice expressing TCRs recognizing a dominant peptide of
PLP (17). These animals, kept under specific pathogen-free con-
ditions, developed spontaneous disease but, in contrast to RR
mice, the disease was mostly progressive without remissions and
Figure 7. MOG-specific autoantibodies bind MOG-expressing cells and activate complement. (A) Binding of anti-MOG Ig from indicated mice to
correctly conformed MOG on the cell surface of transduced EL4 cells (EL4-MOG) shown by flow cytometry. EL4 cells (top) and MOG-expressing EL4-MOG
cells (bottom) were incubated with 1/200 diluted sera obtained from the indicated mice or 0.5 µg/ml 8.18-C5 mAb. Bound antibodies were detected by
FACS using biotinylated anti–IgG1-specific antibody (allotype unspecific) and streptavidin-PE (SA-PE). A representative plot of three independent experi-
ments is shown. (B) Complement activating capability of MOG binding antibodies in sera from transgenic mice. EL4 and EL4-MOG cells were incubated
with sera (1/20 and 1/200 diluted) obtained from the indicated mice, each with three to five per group. Complement activating capability was measured
by cell lysis using propidium iodide (PI) staining after incubation of sera-bound EL4 cells with rabbit complement. Background values were subtracted,
and shown are mean values with the SEM of one experiment representative of three similar experiments.
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
than MOG p92-106, has not been patent in spontaneous dis-
ease of our RR mice.
Expansion of MOG-specific B cells
from endogenous repertoire
As described recently, in the C57BL/6 OSE model, MOG-
reactive TCR transgenic T cells require the presence of MOG-
specific B cells to kindle spontaneous EAE at high frequency.
In this model, the MOG-specific B cells were transgenic with
their germline IgJ region replaced by the rearranged H chain of
a MOG-specific mAb (2, 3).
Based on these previous observations, we initially created
double-transgenic mice by crossing our new MOG TCR
Several investigators reported that in RR-EAE actively
induced by immunization with encephalitogenic peptides,
relapses went along with the T cell reactivity against new epi-
topes (23), a phenomenon termed “determinant spreading”
by Lehmann et al. (24). Recent work by McMahon et al. (25)
suggests that in actively induced chronic EAE, initial bouts of
CNS inflammation result in the induction of professional
APCs within or near the affected CNS target tissue. By taking
up myelin antigens and presenting crucial epitope compo-
nents to newly arrived naive autoreactive T cells, the reper-
toire of target autoantigens spreads (25). It should, however,
be mentioned that determinant spreading, i.e., recruitment of
potentially encephalitogenic T cells specific for epitopes other
Figure 8. B cells and anti-MOG antibodies are essential for spontaneous EAE. (A) Spontaneously developed MOG-specific autoantibodies from
TCR1640 mice have pathogenic potential. EAE was induced in WT SJL/J mice by low-dose PLP 139–151 immunization. Serum from TCR1640 mice, NTL mice,
or 8.18c5 mAb were transferred after mice showed first clinical symptoms. Serum from TCR1640 mice significantly increased disease severity in recipient
mice compared with serum from NTL mice. Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were pooled from two independent ex-
periments, each six to seven per group. (B) B cell depletion protects TCR1640 mice from spontaneous EAE. B cells were depleted from TCR1640 mice by twice
weekly injections of anti-CD20 antibodies from day 3 after birth, and control mice received mouse IgG2a antibodies. The development of spontaneous
EAE was monitored regularly. Although 85% of isotype control antibody-treated mice developed spontaneous EAE, treatment with CD20 antibody pro-
tected TCR1640 mice from disease development. Shown are the disease course (left) and the spontaneous EAE incidence (right) of TCR1640 mice treated with
these antibodies. Data were pooled from two independent experiments, each with six to seven per group.
JEM VOL. 206, June 8, 2009
virus-specific B cell was noted in the presence of high num-
bers of virus-specific transgenic CD4+ T cells and a viral
pseudo-autoantigen expressed throughout the organism in
cell types including DCs and macrophages (30).
Several mechanisms could be involved in MOG-specific
B cell expansion. MOG-specific T cells could intrude into the
naive CNS and create immunogenic conditions that allow the
activation and expansion of naive B cells in the local milieu.
This scenario has been forwarded recently as an explanation of
epitope spreading in actively induced EAE (25). Alternatively,
immunogenic MOG and/or myelin debris could be trans-
ported to local lymph nodes, either as cell-free material or by
phagocytes, to be presented there to autoreactive B cells (31).
Although MOG is predominantly expressed in the CNS, its
messenger RNA was also found in some non-CNS organs,
such as thymus, spleen, and liver (13, 32, 33), and low protein
levels were found in the peripheral nervous system (34). Pre-
sentation of very low amounts of peripherally available MOG
remains, at least in theory, an alternative mechanism of B cell
expansion in RR mice.
Finally, animal models with spontaneously developing EAE
show promise for the discovery of new drugs and the validation
of existing medications. Thus, the opticospinal type of EAE de-
scribed previously may represent essential features of human
OSMS/Devic’s disease (2), a disease variant which is rarely found
in high prevalence populations. The spontaneous RR-EAE,
with its conspicuous variation of neurological deficiencies, will
provide a model for the most frequent type of MS, as it is seen
typically in early phases of the disease.
MateRials and Methods
Mice. SJL/J mice were purchased from Charles River Laboratories. Mog KO
mice (13) were backcrossed into SJL/J for seven generations, crossed with
TCR1640 transgenic SJL/J mice, and then intercrossed to generate TCR1640
transgenic Mog KO mice. All mouse strains were bred in the animal facilities
of the Max Planck Institute of Neurobiology (Martinsried, Germany).
Generation of TCR transgenic mice. TCR donor clone (C3) was
selected from a panel of MOG-specific T cell clones (series SL48) derived
from a SJL/J mouse immunized with rMOG in CFA. The encephalitogenic
clone C3 uses V8.3 along with V4 and recognizes MOG92-106 bound
to I-As. Total RNA of clone C3 was isolated by TRI reagent extraction
(Sigma-Aldrich) and, after DNase I treatment, was converted into comple-
mentary DNA (cDNA) using hexanucleotide primers and Superscript II re-
verse transcription (Invitrogen). The rearranged cDNA of TCR-8.3 and
TCR-4 chains were amplified with the following primer pairs: TCR-8.3
sense with SalI restriction site, 5-AGGTGTCGACCTTCCATGAACAT-
GCGTCCTGACACC-3; TCR- C terminus with BamHI restriction
site, 5-ATAGGATCCTCAACTGGACCACAGCCTCAGC-3; TCR-4
sense with SalI restriction site, 5-AGGTGTCGACTGACACTGCTATGG-
GCTCCATTTTCCTC-3; and TCR- C2 terminus with BamHI restriction
site, 5-ATAGGATCCGGGTGAAGAACGGCTCAGGATGC-3 (all syn-
thesized at Metabion). The rearranged TCR-8.3 and TCR-4 chains from
C3 cDNA were verified by sequencing. To eliminate a XhoI restriction
site (CTC GAG) in TCR-4 at aa 116, GAG was changed into GAA, both
coding for glutamate, using a site-directed mutagenesis kit (Agilent Tech-
nologies) with the following primer pair: V4 XhoI–Mut sense, 5-CCAGA-
CTGACTGTTCTCGAAGATCTGAGAAATGTG-3; and V4 XhoI–Mut,
amino acid sequence of the CDR3 regions is as follows: TC8.3, LYY
transgenic SJL/J strain with MOG-specific B cell knock-in
SJL/J mice. These mice presented with spontaneous RR-EAE
at high frequency. Less expected spontaneous EAE was noted
also in TCR single-transgenic SJL/J mice with a high propor-
tion of MOG-reactive CD4 T cells but without transgenic B
cells. It turned out that in these TCR single-transgenic mice,
MOG-specific B cells were expanded from the endogenous
B cell compartment. Substantial numbers of B cells were ob-
served to invade the inflammatory lesions, and there were
heavy local deposits of Ig along with some activated comple-
ment. Most importantly, TCR single transgenics produced
high titers of anti-MOG autoantibodies of IgG1 and IgG2 iso-
types, which bound to correctly conformed membrane bound
autoantigen, destroyed MOG-expressing target cells, and, thus,
have demyelinating potential (16).
MOG-reactive B cells are essential for the spontaneous
development of RR-EAE. Depletion of B cells by treatment
with anti-CD20 mAb profoundly suppressed RR-EAE when
started at young age. Treatment of adult mice produced much
more inconsistent results, a phenomenon which was recently
noted in other investigations in actively induced EAE (26).
B cells contribute to immune and autoimmune responses by
secreting humoral antibodies, by acting as APCs, and by releas-
ing cytokines. RR mice, both single and double transgenic, pro-
duce MOG-specific autoantibodies that share their dominant
isotype, IgG1, with the original demyelinating anti-MOG mAb
8.18-C5 and that, in vivo, aggravate EAE when transferred i.v.
(27). Importantly, we found large Ig deposits in the demyelinat-
ing lesions of single- and double-transgenic RR mice. At least
part of these antibodies may have been produced by local infil-
trating B cells. In fact, B cells isolated from CNS infiltrates were
found to secrete anti-MOG antibodies at high levels.
Beyond autoantibody secretion, MOG-specific B cells pro-
foundly influence autoimmune responses by directly interacting
with autoimmune T cells. In OSE mice, as well as in TCR1640
× IgHMOG mice (unpublished data), MOG-specific B cells con-
centrate highly diluted MOG and present the antigen to MOG-
reactive T cells. Hence, antigen-presenting B cells may contribute
to the initiation and progression of spontaneous EAE. But, in
addition, B cells may act into the opposite direction; they were
shown to limit EAE by releasing IL-10 (28) and by influencing
function of regulatory T cells (29).
The spontaneous recruitment and expansion of autoim-
mune B cells from the endogenous repertoire by organ-specific
autoimmune T cells in the absence of exogenous autoantigen
represents a mechanism of autoimmunity that has not yet been
described. The MOG-specific B cell expansion process criti-
cally requires the presence of the endogenous target autoanti-
gen, as Mog-deficient TCR transgenic SJL/J mice never
produced anti-MOG autoantibodies, nor did they develop
The role of endogenous MOG is intriguing, considering
that this target autoantigen is predominantly confined to the
CNS tissues, which are largely inaccessible to circulating T
and B cells. This hidden localization distinguishes the RR
mouse paradigm from transgenic mice, where activation of
SPONTANEOUS RELAPSING-REMITTING EAE | Pöllinger et al.
fluorochrome-labeled antibodies were purchased from BD or eBioscience:
CD3 (145-2C11), CD4 (RM4-5), CD8 (53–6.7), V8.3 (B21.14), V4
(KT4), CD11b (M1/70), CD45.1 (A20), CD19 (1D3), B220 (RA3-6B2),
CD62L (MEL-14), CD25 (PC61), IFN- (XMG1.2), IL-17 (TC11-
18H10), and IgG1 (A85-1).
Histological analysis. Brain and spinal cord tissue from mice fixed by perfu-
sion with 4% phosphate buffered paraformaldehyde were embedded in paraf-
fin. Paraffin sections were stained with H&E, with Luxol fast blue for myelin,
and by Bielschowsky silver impregnation for visualization of neurons and ax-
ons. Adjacent serial sections were stained by immunocytochemistry, using pri-
mary antibodies directed against CD3, B220, Mac-3, mouse Ig, and rat/mouse
complement C9 neoantigen. Immunocytochemistry was performed with a bi-
otin avidin technique.
Quantitative real-time TaqMan PCR analysis. Real time quantitative
PCR analysis was performed as previously described (2). Quantification of the
expression of mouse immune and housekeeping genes was performed by
Taqman PCR using the following primer/probe combinations: GAPDH
sense, 5-TCACCACCATGGAGAAGGC-3; GAPDH antisense, 5-GCT-
AAGCAGTTGGTGGTGCA-3; GAPDH probe, 5-ATGCCCCCATG-
TTTGTGATGGGTGT-3; mIL-5 sense, 5-CCGCTCACCGAGCTCT-
GTT-3; mIL-5 antisense, 5-AGATTTCTCCAATGCATAGCTGG-3;
IL-5 probe, 5-CAGGAAGCCTCATCGTCTCATTGCTTGT-3; mIL-
10 sense, 5-CAGAGAAGCATGGCCCAGAA-3; mIL-10 antisense, 5-
TGCTCCACTGCCTTGCTCTT-3; mIL-10 probe, 5-TGAGGCGCT-
GTCATCGATTTCTCCC-3; mIL-17 sense, 5-AACTCCCTTGGCG-
CAAAAGT-3; mIL-17 antisense, 5-GGCACTGAGCTTCCCAGATC-
3; mIL-17 probe, 5-CCACGTCACCCTGGACTCTCCACC-3; mCD4
sense, 5-CGTTTCCTCTCATCATCAATAAACTTA-3; mCD4 antisense,
5-GGCTGGTACCCGGACTGAAG-3; mCD4 probe, 5-CACTTTG-
AACACCCACAACTCCACCTCCT-3; TCR-V8.3 sense, 5-CCAC-
GCCACTCTCCATAAGAG-3; TCR-V8.3 antisense, 5-CAGTAG-
TACAGGCCAGAGTCTGACA-3; TCR-V8.3 probe, 5-CCTGAGC-
CAAAATACAGCGTTT-3; TCR-V sense, 5-TGATGACTCGGCC-
ACATACTTC-3; TCR-V4 antisense, 5-AGCAGCTCCTTCCATCT-
GCAGAAGTCC-3; TCR-V4 probe, 5-TGCCAGCAGCCAAGAACG-
GACAGAT-3; mFoxP3 sense, 5-AGGAGAAGCTGGGAGCTATGC-3;
mFoxP3 antisense, 5-TGGCTACGATGCAGCAAGAG-3; and mFoxP3
probe, 5-AAGGCTCCATCTGTGGCCTCAATGGA-3. IFN-, TNF-,
IL-4, IL-13, and IP-10 Taqman probes were synthesized as described in
Giulietti et al. (37).
Cell surface serum binding and complement assay. For EL4-MOG cells,
the mouse Mog cDNA was cloned into the retroviral vector pLXSN (Clontech
Laboratories, Inc.) and transformed into a GP+E-86 packaging cell line. Virus-
containing supernatant was used to stably transduce the mouse EL4 lymphoma
cell line. EL4 and MOG-transduced EL4 cells (EL4-MOG; 2 × 105/well) were
incubated with sera at the indicated dilutions for 30–45 min at 4°C, washed in-
tensively, and thereafter either stained with biotinylated anti–mouse IgG1 (BD)
and streptavidin–PE diluted at 1/150 and PI at 1 µg/ml or incubated with
LOW-TOX-M rabbit complement (Cedarlane Laboratories) at a 1/10 dilution
for 90 min at 37°C and thereafter analyzed for lysis by staining with PI. For
analysis of binding only live (PI) cells are gated for the histograms.
Depletion of B cells. For B cell depletion, 20 µg of mouse anti–mouse CD20
(18B12; IgG2a; Biogen Idec) (38) or IgG2a control antibody (Sigma-Aldrich)
was injected s.c. into 3-d-old TCR1640 mice. 2 wk later, pups were injected with
80 µg and, at 4 wk old, with 250 µg of respective antibodies. Injections were re-
peated thereafter at 2-wk intervals with 250 µg of respective antibodies.
Serum transfer. Sera were obtained through retroorbital bleeding from TCR1640
or NTL. WT SJL/J mice were immunized with 100 µg PLP139-151 in 5 mg/ml
CFA. 200 ng of Pertussis toxin was injected on days 0 and 2. When mice showed
clinical EAE signs (score 1.0), they received i.p. injections of 250 µl TCR1640
serum or NTL serum or 8.18c5 mAb three times at 48-h intervals.
CALSGGNNAPR FG (V8.3J4); and TCR-4, CASS QERT DSAE TLY
(V4D1J2.3C2). The complete rearranged TCR chains were cloned into
the pHSE3 vector (35), leading to expression under the control of the transgenic
MHC class I H2-Kb promoter. XhoI-linearized TCR-containing plasmids were
coinjected into the pronuclei of fertilized FVB oocytes. Transgenic founders
were identified by PCR using a specific primer for C3 V-J or V-J in
combination with a pHSE3-specific primer (TCR- chain: mTCR-V8.3-
CDR3 sense, 5-CTCCATAAGAGCAGCAGCTCC-3, and hu -globin
antisense, 5-CGTCTGTTTCCCATTCTAAACTGTACC-3; TCR- chain:
PH-2Kb sense, 5-CTGGATATAAAGTCCACGCAGCC-3, and TCR-
V4-CDR3 antisense, 5-CAATCTCTGCTTTTGATGGCTCAAAC-3).
Transgenic mice were backcrossed for at least eight generations into the
EAE induction and scoring. Mice were injected s.c. at the back with 200 µl
of recombinant MOG (200 µg), which was emulsified in Freund’s adjuvant and
supplemented with 3 mg/ml Mycobacterium tuberculosis (strain H37Ra). 200 ng
of pertussis toxin was injected i.p. on days 0 and 2 after immunization. Clinical
scoring of classical paralytic EAE was performed as follows: score 0, healthy; 1,
flaccid tail; 1.5, flaccid tail and impaired righting reflex; 2, impaired righting re-
flex and hind limb weakness; 2.5, one hind leg paralyzed; 3, both hind legs par-
alyzed with residual mobility in both legs; 3.5, both hind legs completely
paralyzed; 4, both hind legs completely paralyzed and beginning front limb par-
alysis; and 5, moribund or death of the animal after preceding clinical disease.
Ataxic scoring was as follows: score 0, healthy; 1, mouse partly tilted, feet fall
into cage fence; 2, tilted and tumbles; 3, mouse heavily tilted and moves in cir-
cles; 4, inability to walk, is only rolling. and 5, moribund. All animal procedures
used were in accordance with guidelines of the committee on animals of the
Max Planck Institute of Neurobiology and with the license of the Regierung
von Oberbayern (Munich, Germany).
Antigens. Recombinant histidine-tagged rat MOG protein (aa 1–125) was pu-
rified from bacterial inclusion bodies (36). MOG peptide 90–110 (SDEGGYTCF-
FRDHSYQEEAA), MOG 1–26 (GQFRVIGPGYPIRALVGDEAELPCRI),
and MOG 92–106 (DEGGYTCFFRDHSYQ) were synthesized at the core fa-
cility of the Max Planck Institute of Biochemistry (Martinsried, Germany) or
BioTrend (Köln, Germany). All antigens used in the study were of >95% purity,
analyzed by silver staining of PAGE (proteins) or HPLC analysis (peptides).
In vitro proliferation assay. Proliferation assays were performed as previ-
ously described (2). Each sample was run in triplicates, the proliferative re-
sponse of which is represented by the mean ± SEM. All assays were replicated
at least three times.
ELISA. Cytokine measurements and determination of serum titers of MOG-
specific antibodies was performed as previously described (2). Antibodies were
obtained from BD, except the anti–IL-17 which was obtained from R&D
Systems. Each assay was repeated at least three times.
Flow cytometric analysis. Single cell suspensions were prepared from spleen,
lymph node, thymus, or CNS tissue by mechanical disruption via forcing trough
40-µm cell strainers (BD). Erythrocytes of spleen cell suspensions were lysed by
hypotonic treatment. CNS infiltrate cells were purified by Percoll gradient cen-
trifugation. Cells were resuspended in Percoll (GE Healthcare) at a density of
1.035 g/ml and centrifuged over Percoll of a 1.08-g/ml density for 30 min at
20°C. The interphase was recovered and subjected to FACS analysis. For detec-
tion of cell surface markers by flow cytometric analysis, cells were stained in
FACS medium (PBS containing 1% BSA and 0.1% NaN3) with different fluo-
rochrome-labeled MAbs. For cytofluorometry, we used the FACSCalibur sys-
tem with CellQuest software (BD), and for analysis we used WinMDI 2.9
software (Joe Trotter, The Scripps Institute, La Jolla, CA) was used. For intracel-
lular cytokine staining, cells were first surface stained and then fixed and permea-
bilized in 4% PFA/0.1% saponin in Hepes-buffered HBSS, stained intracellularly,
and washed. Before staining, cells were activated with 50 ng/ml PMA/500 ng/
ml ionomycin in the presence of 5 µg/ml brefeldin A for 4 h. The following
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Statistics. Differential spontaneous EAE incidence of female to males was
analyzed by logrank test (by an in-built survival curve analysis function from
Prism 4 [Graph Pad Software, Inc.]), and the effect of serum transfer on EAE
development was analyzed by two-way ANOVA. P-values < 0.05 were
considered to be significant.
Online supplemental material. Fig. S1 shows the nature and encephalito-
genicity of TCR donor clone C3. Fig. S2 shows the TCR expression in
TCR1640 transgenic mice splenocytes. Fig. S3 illustrates the spontaneous dis-
ease course of individual transgenic TCR1640 animals. Fig. S4 shows the CNS
messenger RNA expression analysis of spontaneous EAE- and MOG-immu-
nized mice. Fig. S5 shows the efficiency of B cell depletion in TCR1640 mice.
Video S1 shows one diseased double-transgenic mouse in different EAE stages.
Table S1 shows a summary of spontaneous EAE in TCR transgenic SJL/J
mice. Table S2 shows a summary of histological analysis of representative indi-
vidual mice with spontaneous EAE. Table S3 summarizes spontaneous EAE
incidence and mortality in TCR1640 mice treated with anti-CD20 and control
isotype antibodies. Online supplemental material is available at http://www
We are grateful to Irene Arnold-Ammer, Lydia Penner, Iris Jarsch, Michaela Krug,
Marianne Leiszer, and Ulrike Köck for excellent technical support.
This project was supported by the Deutsche Forschungsgemeinschaft (SFB 571,
Project B6) and the Max Planck society. S.H. Domingues is supported by a PhD
fellowship (Portuguese FCT program SFRH/BD/15885/2005).
The authors have no conflicting financial interests.
submitted: 6 February 2009
accepted: 3 May 2009
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