The Journal of Experimental Medicine
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 206 No. 3 507-514
BRIEF DEFINITIVE REPORT
Guillain-Barr é syndrome (GBS) and chronic
infl ammatory demyelinating polyneuropathy
(CIDP) are autoimmune diseases characterized by
infl ammatory demyelinating lesions of the periph-
eral nervous system (PNS) that cause devastating
neurological defi cits and paralysis. Multifocal de-
myelination associated with perivascular mono-
nuclear cell infi ltrates in the PNS is the pathologi-
cal hallmark of GBS and CIDP, and autoreactive
T and B cell responses are believed to be essential
in both diseases ( 1 ). Defi ning self-antigens tar-
geted by the autoimmune response can lead to a
better understanding of the immunopathology of
the disease and can potentially lead to antigen-
specifi c tolerogenic therapies ( 2 ); however, prog-
ress has been limited by the paucity of animal
models. We previously described the fi rst sponta-
neous model of autoimmune disease of the PNS,
called spontaneous autoimmune peripheral poly-
neuropathy, in autoimmune-prone nonobese
diabetic (NOD) mice defi cient for the co-stimu-
latory molecule B7-2 (NOD-B7-2KO mice) or
mimicked many pathophysiological characteris-
tics of GBS and CIDP ( 3 ). NOD-B7-2KO mice
exhibit a progressive and generalized limb paraly-
sis associated with severe demyelination and axo-
nal damage caused by an autoimmune attack of
the PNS ( 3 ). Infl ammatory CD4 + T cells are es-
sential for the development of autoimmune
peripheral neuropathy, and NOD-B7-2KO mice
defi cient for IFN- ? were protected from disease
( 3, 4 ). In this report, we examined the antigen
specifi city of autoreactive T cells infi ltrating the
nerves of neuropathic mice and found that
myelin protein 0 (P0) is a dominant self-antigen
recognized by T cells and autoantibodies. We
generated a TCR transgenic (TCR-Tg) mouse
specifi c for a P0 epitope and demonstrated that
these T cells were suffi cient to induce a fulminant
form of peripheral neuropathy.
Jeffrey A. Bluestone:
H. Bour-Jordan and J.A. Bluestone contributed equally to
A novel myelin P0 – specifi c T cell receptor
transgenic mouse develops a fulminant
autoimmune peripheral neuropathy
C é dric Louvet , Beniwende G. Kabre , Dan W. Davini , Nicolas Martinier ,
Maureen A. Su , Jason J. DeVoss , Wendy L. Rosenthal , Mark S. Anderson ,
H é l è ne Bour-Jordan , and Jeff rey A. Bluestone
Diabetes Center and the Department of Medicine, University of California, San Francisco, San Francisco, CA 94143
Autoimmune-prone nonobese diabetic mice defi cient for B7-2 spontaneously develop an
autoimmune peripheral neuropathy mediated by infl ammatory CD4 + T cells that is reminis-
cent of Guillain-Barr é syndrome and chronic infl ammatory demyelinating polyneuropathy.
To determine the etiology of this disease, CD4 + T cell hybridomas were generated from
infl amed tissue – derived CD4 + T cells. A majority of T cell hybridomas were specifi c for
myelin protein 0 (P0), which was the principal target of autoantibody responses targeting
nerve proteins. To determine whether P0-specifi c T cell responses were suffi cient to medi-
ate disease, we generated a novel myelin P0 – specifi c T cell receptor transgenic (POT)
mouse. POT T cells were not tolerized or deleted during thymic development and prolifer-
ated in response to P0 in vitro. Importantly, when bred onto a recombination activating
gene knockout background, POT mice developed a fulminant form of peripheral neuropathy
that affected all mice by weaning age and led to their premature death by 3 – 5 wk of age.
This abrupt disease was associated with the production of interferon ? by P0-specifi c
T cells and a lack of CD4 + Foxp3 + regulatory T cells. Collectively, our data suggest that
myelin P0 is a major autoantigen in autoimmune peripheral neuropathy.
© 2009 Louvet et al. This article is distributed under the terms of an Attribu-
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NEUROPATHY IN NOVEL P0-SPECIFIC TCR-TG MICE | Louvet et al.
regulation of CD69 ( Fig. 1 D ). 1 out of these 11 hybridoma
responded to an antigen shared between the PNS and central
nervous system (CNS; Table S1, available at http://www.jem
.org/cgi/content/full/jem.20082113/DC1), supporting the
previous fi nding that neuropathy in NOD-B7-2KO mice
preferentially targeted the PNS without aff ecting CNS tissues
( 3 ). As expected, up-regulation of CD69 on T cell hybridomas
in response to nerve lysate was inhibited in the presence of
anti – MHC class II blocking mAbs (Fig. S1), confi rming that
the response of T cell hybridomas to nerve antigens was de-
pendent on recognition of a peptide – MHC class II complex.
Antigen specifi city of nerve-derived CD4 + T cell hybridomas
Neural proteins targeted by autoantibodies in NOD-B7-2KO
mice were determined to identify potential targets of autore-
active T cells ( 7, 8 ). Protein extracts prepared from the sciatic
nerve were immunoblotted with sera from neuropathic mice
( 4 ). The majority of sera (27 out of 50; 54%) from individual
neuropathic T reg cell – depleted NOD-B7-2KO mice re-
acted strongly against an antigen migrating at ? 25 – 30 kD
( Fig. 2 A ). Immunoprecipitation was performed on columns
containing protein G agarose coupled to sera from neuro-
pathic animals, followed by peptide mass fi ngerprinting, and
unequivocally identifi ed the 28-kD protein P0, a major com-
ponent of the myelin sheath expressed exclusively in the PNS
but not in the CNS. 6 out of 11 hybridomas that were acti-
vated by peripheral nerve lysate up-regulated CD69 in the
presence of P0 peptides, suggesting that P0 is a major autoan-
tigen targeted by T cells in neuropathic mice ( Fig. 2 B ). Of
these six P0-specifi c hybridomas, four recognized P0 peptides
in the putatively extracellular portion of the protein (peptide
pool #1), whereas the other two hybridomas were specifi c
for P0 peptides located in the cytoplasmic portion of the pro-
tein (peptide pool #4; Fig. 2 B ). Three out of the four extra-
cellular P0-specifi c hybridomas were responsive to the same
P0 peptide (amino acids 1 – 25), suggesting the presence of an
immunodominant epitope in this portion of the P0 protein
( Fig. 2 C and not depicted). We should emphasize that the
four hybridomas specifi c for P0 1 – 25 were generated from
distinct T cell clones, because the TCR V ? and V ? combi-
nation was unique to each hybridoma (Table S1).
Generation of a myelin P0 – specifi c TCR-Tg (POT) mouse
Next, we generated a POT mouse by cloning the rearranged
TCR ? (V ? 9) and ? (V ? 11) chains from hybridoma 2E7,
which was strongly activated in response to peripheral nerve ly-
sate and P0 immunodominant peptide 1 – 25 in vitro ( Fig. 2 C ).
The POT mice showed no diff erence in the total cell num-
bers of the spleen cells or thymocytes as compared with non-
Tg littermates (unpublished data). The percentages of CD4
and CD8 single-positive cells were decreased in the thymus
in POT mice as compared with control littermates, and the
CD4/CD8 ratio was slightly increased (4.9 ± 0.8% compared
with 3.6 ± 0.2%, respectively; P < 0.05; Fig. 3 A , left). Simi-
lar data were observed in the spleen ( Fig. 3 A , right). Analysis
of TCR expression confi rmed the expression of the Tg TCR
RESULTS AND DISCUSSION
Generation of peripheral nerve – specifi c T cell hybridomas
We previously demonstrated that CD4 + T cells from neu-
ropathic NOD-B7-2KO mice could transfer disease, sug-
gesting that the site of infi ltration and tissue destruction
would be enriched in pathogenic autoreactive T cells spe-
cifi c for nerve antigens. CD4 + T cells were isolated from the
infi ltrated nerves of neuropathic NOD-B7-2KO mice ( Fig. 1 )
and were expanded with anti-CD3 and anti-CD28 mAbs,
and IL-2 by 20 – 100-fold after 2 wk in culture, as previously
described ( Fig. 1 B ) ( 5 ).
Transfer of 2 – 5 × 10 6 expanded CD4 + T cells into immuno-
defi cient recipients led to neuropathy within 5 wk ( Fig. 1 C
and not depicted). Expanded CD4 + T cells from nerves were
fused with TCR ? BW1100 cells ( 6 ) to generate antigen-
specifi c T cell hybridomas. Stable CD4 + TCR + T cell hybrid-
omas, responsive to anti-CD3 stimulation, were tested further
for their ability to be activated by protein lysates prepared from
sciatic nerves. A small subset of CD4 + T cell hybridomas (11
out of 56) derived from the infi ltrated nerves of neuropathic
mice responded to sciatic nerve antigens as measured by up-
Figure 1. Generation of peripheral nerve – specifi c T cell hybrid-
oma. (A and B) CD4 + T cells were sorted from the nerves of neuropathic
NOD-B7-2KO mice (A) and expanded in vitro (B). Data are representative
of 15 sorts and expansions. (C) 2 – 5 × 10 6 expanded cells were transferred
into NOD-SCID or NOD-RAGKO immunodefi cient mice, and recipients
were followed for the development of neuropathy ( n = 5 mice from two
independent experiments). (D) CD4 + T cell hybridomas were stimulated
with APCs alone (open histogram) or in the presence of the indicated
stimulus (shaded histograms), and CD69 up-regulation was assessed.
Clone 2E7 is shown. Data are representative of four experiments.
JEM VOL. 206, March 16, 2009
BRIEF DEFINITIVE REPORT
confi rmed that TCR-Tg T cells were positively selected in the
thymus, although the cell numbers were greatly reduced com-
pared with RAG +/ ? or POT-RAG +/ ? mice (Fig. S2, available
suggesting a more eff ective negative selection induced by P0-
expressing thymic stromal cells in this setting ( 10 ). Although
the total spleen cell number was reduced in POT-RAGKO
compared with POT mice, the majority of mature T cells
from POT-RAGKO mice were CD4 + V ? 11 + T cells, with
few CD8 + V ? 11 + T cells (a mean ratio of 10.6 ± 1.2% versus
3.7 ± 0.3% in POT-RAG +/ ? mice; Fig. 3 B and Fig. S2).
Thus, P0-specifi c POT TCR-Tg T cells successfully sur-
vived thymic selection to generate a population of mature
autoreactive T cells in the periphery even on a RAGKO
background. Finally, we examined the presence of T reg cells
and the activation status of TCR-Tg T cells in POT mice.
There was no diff erence in the expression of CD69, CD62L,
and CD44 on CD4 + T cells isolated from POT mice com-
pared with non-Tg littermates (unpublished data), showing
that POT T cells were not activated in the periphery. Fur-
thermore, a similar percentage of CD4 + T cells expressed the
and showed very effi cient allelic exclusion in POT mice
( Fig. 3 A , right). Indeed, > 90% of CD4 + T cells were V ? 11 + in
the spleen of POT mice versus < 10% in non-Tg littermates
(90.6 ± 5.4% in POT mice versus 6.5 ± 0.5% in controls).
Conversely, POT splenic CD4 + T cells contained < 2% V ? 8 +
T cells, whereas V ? 8 + T cells represented > 20% of CD4 + T
cells in control littermates (1.2 ± 1.3% in POT mice versus
22.8 ± 1.2% in controls). These results suggested that T cells
expressing the POT TCR were positively selected to gener-
ate a normal population of mature T cells in the periphery,
similar to other TCR-Tg mice specifi c for autoantigens such
as islet-specifi c BDC2.5 ( 9 ). Analysis of POT mice, crossed
onto a RAGKO background to eliminate the potential infl u-
ence of endogenous TCR ? chains on the thymic selection,
Figure 2. Myelin P0 is targeted by the autoimmune response in
NOD-B7-2KO mice. (A) Immunoblotting of sciatic nerve extracts with
serum from individual anti – B7-1 – treated NOD-B7-2KO neuropathic mice
(lanes 1 – 9), nonneuropathic NOD mice (lanes 10 and 11), or BALB/c con-
trols (lanes 12 and 13). Data are representative of fi ve to nine indepen-
dent experiments for each serum. The predominant 25 – 30-kD reactivity is
indicated with an arrow. (B) Nerve-specifi c CD4 + T cell hybridomas were
examined for reactivity to P0 as in Fig. 1 D , using pools of overlapping
peptides spanning the P0 protein. Data are representative of two experi-
ments. Arrows indicate reactive clones to a specifi c pool of peptide.
(C) Activation of the 2E7 hybridoma by individual P0 peptides from pools
#1 and #4. Data are representative of two experiments.
Figure 3. Phenotypic analysis of POT TCR-Tg mice. (A) Phenotyping
of CD4 + and CD8 + T cells in the thymus (left) and spleen (right) of NOD-POT
TCR-Tg mice (top) and transgene-negative littermates (bottom). (B) Pheno-
typing of CD4 + T cells in the spleens of NOD-RAGKO-POT TCR-Tg mice.
(C) Foxp3 expression in the spleens of the indicated 12-wk-old mice (histo-
grams are gated on CD4 + T cells). Numbers indicate percentages of Foxp3 +
cells in the CD4 population. (A – C). (D) CD25 and Foxp3 expression among
CD4 + cells in the spleens of the indicated 19-d-old mice. Percentages ± SD
are indicated. Data are representative of two or more experiments.
NEUROPATHY IN NOVEL P0-SPECIFIC TCR-TG MICE | Louvet et al.
the development of diabetes versus neuropathy directly cor-
relates with the ability of islet versus nerve antigen-specifi c
autoreactive T cells to proliferate in relevant lymph nodes
( Fig. 4 B ) ( 11 ). Finally, we detected high levels of IFN- ? and
IL-17 in the culture supernatant of POT T cells upon in vitro
stimulation in the absence of any skewing culture conditions
( Fig. 4 C ). Surprisingly, intracellular cytokine staining re-
vealed that 40 – 50% of T cells isolated from POT-RAGKO
mice produced IFN- ? but no IL-17 ex vivo, whereas only
2.5% of POT T cells on a RAG-suffi cient background pro-
duced IFN- ? and < 1% produced IL-17, similar to what was
observed in NOD littermates ( Fig. 4 D ). This result suggested
that cytokine production observed in a RAG-suffi cient back-
ground was infl uenced by endogenous TCR ? chains and/or
regulatory cell populations that could limit the activation and
diff erentiation of autoreactive POT T cells.
Pathogenicity of P0-specifi c TCR-Tg T cells in vivo
Although mononuclear infi ltration could be detected in the
nerves of some POT mice as early as 3 wk of age ( Fig. 5 A ),
many POT mice did not show any sign of infl ammation in
sciatic nerves or clinically detectable peripheral neuropathy
by 15 – 17 wk of age ( Fig. 5 A and not depicted). However,
adoptive transfer of in vitro – activated or CD25-depleted
POT T cells led to peripheral nerve infi ltration and induced
T reg cell marker Foxp3 in POT mice compared with con-
trols ( Fig. 3 C ). However, CD4 + Foxp3 + T reg cells could
not be detected in the spleen and thymus of POT-RAGKO
mice ( Fig. 3 D ; and Figs. S3 and S4), thus confi rming that the
thymic selection of T reg cells in POT mice involved TCRs
using endogenous ? chains.
Proliferation and cytokine production of P0-specifi c
TCR-Tg T cells
POT CD4 + T cells proliferated strongly in response to pro-
tein extracts prepared from sciatic nerves but not spinal cord
or brain, confi rming their strict PNS specifi city ( Fig. 4 A ).
Furthermore, POT T cells displayed a similarly strong pro-
liferation when cultured with P0 protein (including the P0
peptide 1 – 25) but not with another myelin protein (P2), con-
fi rming the P0 specifi city and demonstrating that the T cells
are fully functional and not anergic in the periphery. We
examined the capacity of activation of PNS-specifi c auto-
reactive T cells to proliferate in NOD (resistant) versus NOD-B7-
2KO (susceptible) mice. POT T cells proliferated extensively
in several lymph nodes in NOD-B7-2KO but not NOD
recipients, including brachial, axillary, and inguinal lymph
nodes ( Fig. 4 B ; and Fig. S5, available at http://www.jem
.org/cgi/content/full/jem.20082113/DC1), illustrating a
clear dichotomy between NOD and NOD-B7-2KO mice, as
Figure 4. Proliferation and cytokine production of P0-specifi c NOD-POT T cells. (A) Proliferation of CFSE-labeled NOD-POT T cells in the presence
of APCs alone (gray histogram) or APCs and the indicated stimulus (black histograms). Histograms are gated on the CD4 + population. (B) Proliferation of
CFSE-labeled NOD-POT spleen cells in NOD or NOD-B7-2KO recipients (22 wk of age) after 4 d (inguinal lymph nodes are shown). Histograms are gated
on the Thy1.1 + CD4 + population. (C) Production of IFN- ? (left) and IL-17 (right) in the culture supernatant of NOD-POT cells activated by the indicated
stimulus. (D) Production of IFN- ? and IL-17 after stimulation of spleen cells from the indicated mice with PMA and ionomycin. Dot plots are gated on
CD4 + T cells. Numbers indicate percentages of cells in the quadrants. Data are representative of two or more experiments.
JEM VOL. 206, March 16, 2009
BRIEF DEFINITIVE REPORT
and D ). The spontaneous peripheral neuropathy induced
by POT T cells led to motor dysfunction indicated by clasp-
ing of the limbs and diffi culty walking ( Fig. 5 C ; and Videos
1 – 3, available at http://www.jem.org/cgi/content/full/jem
.20082113/DC1), similar to what we originally observed in
NOD-B7-2KO mice ( 3 ). Development of neuropathy was
accompanied by a heavy infi ltrate in the sciatic nerves of
NOD-RAGKO-POT mice ( Fig. 5, A and E ), as well as my-
elin destruction (Fig. S6). The infi ltration in the sciatic nerves
of NOD-RAGKO-POT mice was strikingly higher than in
their RAG-suffi cient counterparts at 3 – 4 wk of age (P = 0.01
using a Mann-Whitney U test; Fig. 5 A ), correlating with
neuropathy in immunodeficient recipients (unpublished
data). These results suggested that the lack of spontaneous
neuropathy in POT mice may have been a consequence of
increased numbers of antigen-specifi c Tg + Foxp3 + T reg cells
as compared with Tg-negative NOD mice. Thus, we exam-
ined T reg cell – defi cient POT-RAGKO mice for tissue in-
fi ltration and clinical disease ( Fig. 3 D ; and Figs. S3 and S4).
POT-RAGKO mice displayed a dramatic phenotype charac-
terized by early weight loss and premature death by 3 – 5 wk
of age ( Fig. 5, B and C ; and not depicted), and spontaneously
developed a fulminant form of neuropathy that started as
early as 18 d and aff ected all mice by 3 – 4 wk of age ( Fig. 5, C
Figure 5. Spontaneous development of a fulminant form of neuropathy in NOD-RAGKO-POT mice. (A) Quantifi cation of infi ltration in the
nerves of indicated mice at 3 – 4 wk of age. Horizontal bars indicate means. *, P < 0.05. (B) Weight in the indicated mice as a function of age. Results rep-
resent cumulated data from two different experiments. Data are means ± SEM. (C) Clasping phenotype in a representative NOD-RAGKO-POT mouse com-
pared with the normal plantar reaction in a NOD-RAGKO littermate, and weight defi cit in NOD-RAGKO-POT mice at 3 – 4 wk of age. (D) Neuropathy in the
indicated mice as a function of age. (E) Intense infi ltrate in the sciatic nerve of NOD-RAGKO-POT mice. Bars, 250 μ m. (F) Adoptive transfer of neuropathy
by spleen cells from NOD-RAGKO-POT mice into NOD-SCID recipients ( n = 4). Data are representative of two independent transfers. Videos 1 – 3 are avail-
able at http://www.jem.org/cgi/content/full/jem.20082113/DC1.
NEUROPATHY IN NOVEL P0-SPECIFIC TCR-TG MICE | Louvet et al.
ing a specifi c P0 peptide. Although the peptide specifi city (P0
180 – 199) was diff erent from the POT TCR epitope (P0 1 – 25),
the epitope overlapped with two of our peripheral nerve –
specifi c hybridoma clones ( Fig. 2 B ; and Table S2, available at
In their report, the adoptive transfer of a P0-specifi c line only
led to very mild disease (grade 1). The diff erence with our re-
sults may be because of the specifi city diff erences or their use
of a T cell line.
Our results suggest that neuropathy induced by P0-spe-
cifi c POT T cells involves mainly IFN- ? – producing Th1
cells in agreement with a central role of IFN- ? in the devel-
opment of peripheral neuropathy in NOD-B7-2KO mice
( 4 ). Furthermore, elevated serum levels of IFN- ? have been
reported in GBS patients, and the percentage of IFN- ? +
CD4 + T cells was markedly increased in the cerebrospinal
fl uid of CIDP patients ( 23, 24 ). Th17 cells have been sug-
gested to play a major role in multiple sclerosis and its animal
model experimental autoimmune encephalomyelitis ( 25 ).
Although increased levels of IL-17 have been described in
the spinal fl uid of CIDP patients ( 24 ), it is still unclear whether
Th17 cells are important for autoimmune peripheral neurop-
athies. We found that low levels of IL-17 were produced by
POT T cells on a RAG-suffi cient background in vitro. It was
recently reported that Th1 and Th17 cells may have diff erent
abilities to infi ltrate the CNS and cause experimental autoim-
mune encephalomyelitis depending on the level of local tis-
sue infl ammation ( 26 ). Thus, our report does not preclude a
role for IL-17 in spontaneous autoimmune peripheral poly-
neuropathy and other PNS autoimmune diseases but suggests
that IFN- ? is the dominant cytokine associated with periph-
eral neuropathy induced by P0-specifi c POT T cells.
Another important insight revealed by our report is the
control of P0-specifi c T cells and, hence, the development of
neuropathy by CD4 + Foxp3 + T reg cells. Indeed, the develop-
ment of the fulminant form of neuropathy in POT-RAGKO
mice was associated with a complete absence of CD4 + Foxp3 +
T reg cells, suggesting that antigen-specifi c T reg cells are key
to controlling the disease. Because the percentage of T reg cells
has been shown to be lower in the peripheral blood of GBS
and CIDP patients ( 27 – 29 ), our fi ndings suggest that immuno-
therapy using P0-specifi c T reg cells may be an important fu-
ture therapeutic option for these patients, as similar treatments
are now being developed for several autoimmune diseases ( 2 ).
MATERIALS AND METHODS
Mice. NOD and NOD-SCID mice were purchased from Taconic. NOD-
B7-2KO ( 3 ), NOD-TCR ? KO, and NOD-RAG2KO mice were bred in
our facility. Neuropathy was assessed weekly ( 3 ). All mice were housed in a
specifi c pathogen-free University of California, San Francisco (UCSF) facil-
ity. All animal experiments were approved by the Institutional Animal Care
and Use Committee of UCSF.
Peripheral nerve – specifi c hybridomas. Cells from sciatic nerves of
neuropathic NOD-B7-2KO mice were prepared as previously described
( 3 ), stained with CD4-FITC and CD8-allophycocyanin. CD4 + CD8 ? cells
were sorted on a cytometer (MoFlo; Dako) and expanded with anti-CD3 –
and anti-CD28 – coated beads supplemented with 2,000 IU/ml rhIL-2, as
clinical disease. Indeed, adoptive transfer of 1.3 × 10 6 spleno-
cytes from a 25-d-old neuropathic NOD-RAGKO-POT
mouse induced severe neuropathy in all NOD-SCID recipi-
ents in < 2 wk ( Fig. 5 F ). Finally, the peripheral neuropathy
induced by P0-specifi c POT TCR-Tg T cells was extremely
severe and led to death of the animals, usually within 24 h
after clinical disease onset, either spontaneously in NOD-
RAGKO-POT mice or after adoptive transfer of T cells
from NOD-RAGKO-POT or NOD-POT mice (unpub-
Collectively, our data demonstrate that myelin P0 is a
major autoantigen targeted by autoantibodies and autoreac-
tive T cells in a spontaneous mouse model of peripheral
neuropathy. Importantly, the development of an abrupt form
of disease in NOD-RAGKO-POT mice reveals that P0-
specifi c CD4 + T cells were suffi cient to induce catastrophic
damage in the PNS and suggests that P0-specifi c T cells
could play a central role in inducing tissue destruction in au-
toimmune neuropathy. P0 is a major component of the PNS
that represents > 50% of the peripheral myelin protein con-
tent. Experimental autoimmune neuritis can be induced in
rats and mice by immunization with P0 protein or peptides
in adjuvant or by adoptive transfer of P0-specifi c T cell lines
( 1 ). However, these models involve active immunization
and powerful adjuvants, which does not mirror the sponta-
neous disease in individuals genetically prone to autoimmu-
nity. Reports of immune responses to P0 in GBS and CIDP
patients have generated confl icting results that could be ex-
plained by diff erent techniques and patient populations.
Nevertheless, of all neural proteins tested, P0 has been the
most consistent reactivity detected in the serum of patients
with active GBS or CIDP ( 12 – 14 ). Khalili-Shirazi et al. ini-
tially reported proliferative responses to P0 protein or peptides
in 9 out of 19 GBS patients ( 15 ). More recently, P0-specifi c
T cell responses measured by proliferation or ELISPOT
were not statistically diff erent in GBS and CIDP patients as
compared with nonautoimmune neuropathies ( 16 ), whereas
another report described P0-specifi c IL-10 production in
GBS patients ( 17 ), raising the possibility that immune re-
sponses to P0 may be regulatory rather than pathogenic.
These fi ndings were reminiscent of the glutamic acid decar-
boxylase 65-kD isoform (GAD65) autoantigen in type 1 di-
abetes. Similar to P0, autoantibodies to GAD were readily
detected in type 1 diabetes patients and in the NOD mouse
( 18, 19 ), and GAD-specifi c T cells could adoptively transfer
diabetes in the NOD mouse model ( 20 ). However, GAD-
specifi c TCR-Tg or retrogenic mice revealed that GAD-spe-
cifi c T cells did not induce diabetes but rather exerted a
protective eff ect ( 21 ). In contrast, our report of an aggressive
form of peripheral neuropathy spontaneously occurring in
P0-specifi c POT TCR-Tg mice strengthens the potential
role of P0 as a central autoantigen involved in disease devel-
opment and tissue damage in autoimmune neuro pathy.
While this paper was being reviewed, Kim et al. published
a report ( 22 ) showing that autoimmune neuropathy in NOD-
B7-2KO mice could be prevented by tolerance induction us-
JEM VOL. 206, March 16, 2009
BRIEF DEFINITIVE REPORT
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previously described ( 5 ). The expanded CD4 + T cells were fused to the
TCR-negative thymoma partner BW1100 (provided by E. Palmer, Univer-
sity Hospital Basel, Basel, Switzerland) using standard protocols. TCR-
expressing hybridoma clones were tested for antigen specifi city by incubating
50,000 cells with 0.2 × 10 6 APCs (NOD-TCR ? KO spleen cells, irradiated
NOD spleen cells, or NOD BM-derived DCs) with the stimulus indicated
in the fi gures for 16 – 24 h, followed by staining with CD69-PE. Overlapping
peptides spanning the sequence of human P0 (which is 94% homologous to
mouse P0 at the amino-acid level) were provided by N. Gregson (King ’ s
College London, London, England, UK; Table S2).
Generation of POT TCR-Tg mice. The full-length coding sequences
for the TCR ? and ? chains were cloned by PCR from 2E7 hybridoma
cDNA using the indicated primers (Table S3, available at http://www.jem
.org/cgi/content/full/jem.20082113/DC1), and were subcloned into a CD2
and CD4 expression vector, respectively ( 30 ), allowing expression of the
transgenes in both CD4 and CD8 T cells. Tg mice were generated by micro-
injection of CD2-TCR ? and CD4-TCR ? constructs into NOD embryos.
Vectors were provided by N. Killeen (UCSF, San Francisco, CA).
Statistical analysis. All statistical analyses were performed using an un-
paired two-tailed Mann-Whitney U test with Prism software (GraphPad
Software, Inc.). P ≤ 0.05 was considered signifi cant.
Online supplemental material. Table S1 shows the characteristics of nerve-
specifi c hybridomas. Tables S2 and S3 show the sequences of peptides and
primers used in this report. Fig. S1 shows the inhibition of hybridoma activa-
tion by anti – MHC class II mAbs. Figs. S2 – S4 show CD4, CD8, V ? , CD25,
and Foxp3 FACS analysis of thymus and spleen cells from NOD-RAGKO-
POT mice. Fig. S5 shows the enhanced proliferation of NOD-POT cells in
NOD-B7-2KO mice compared with NOD recipients. Fig. S6 shows sciatic
nerve demyelination in NOD-RAGKO-POT mice. Videos 1 and 2 show a
grid test to assess neuropathy in a NOD-RAGKO-POT mouse and a NOD-
RAGKO littermate, respectively. Video 3 shows the clasping phenotype and
weight defi cit in a NOD-RAGKO-POT mouse compared with the normal
plantar reaction in a NOD-RAGKO littermate. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20082113/DC1.
The authors wish to thank H. Lu and N. Killeen for the vectors and microinjections,
C. Austin for histology, S. Jiang for cell sorting, D. Vignali and A. Burton for
discussions, N. Gregson for providing P0 peptides, and A. Weishaupt and R. Gold for
providing P0 and P2 recombinant proteins.
This work was supported by National Institutes of Health grant R01 AI50834, a
UCSF Research Evaluation and Allocation Committee grant, and a Neuropathy
Association scientifi c research grant.
The authors have no confl icting fi nancial interests.
Submitted: 23 September 2008
Accepted: 23 January 2009
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