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Coadministration of Plasmid DNA Constructs
Encoding an Encephalitogenic Determinant
and IL-10 Elicits Regulatory T Cell-Mediated
Protective Immunity in the Central Nervous
System
Sagie Schif-Zuck, Gizi Wildbaum and Nathan Karin
J Immunol 2006; 177:8241-8247; ;
doi: 10.4049/jimmunol.177.11.8241
http://www.jimmunol.org/content/177/11/8241
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Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
The Journal of Immunology
Coadministration of Plasmid DNA Constructs Encoding an
Encephalitogenic Determinant and IL-10 Elicits Regulatory
T Cell-Mediated Protective Immunity in the Central
Nervous System1
Sagie Schif-Zuck, Gizi Wildbaum, and Nathan Karin2
E
xperimental autoimmune encephalomyelitis (EAE)3 is a T
cell-mediated autoimmune disease of the CNS that serves
as an animal model for human multiple sclerosis (MS),
given that in both diseases autoimmune cells home to the CNS to
attack myelin components (1, 2). EAE is transferable by CD4⫹ T
cells selected in response to CNS Ags (1). Depending on their
cytokine profile, the CD4⫹ T cells fall into different subsets including: Th1 cells that produce large amounts of IFN-␥ and TNF-␣
and low levels of IL-4; Th2 cells that mostly produce IL-4, IL-5,
and IL-13 and, to a much lesser extent, IFN-␥ and TNF-␣ (3– 6);
Th3 cells that produce high levels of TGF-␤ and to a much lesser
extent other cytokines (7, 8); regulatory T cells (Tr1) that produce
high levels of IL-10 (9); CD4⫹CD25⫹-Trl (10 –13); and the very
newly defined Th17 cells, selected in response to IL-23 and producing high levels of the inflammatory cytokine IL-17 (14 –20)
and are likely to contribute to the pathogenic manifestation of inflammatory autoimmune diseases, including EAE (14, 17). The
pivotal role of Th1 cells in the initiation and progression of the
inflammatory process in EAE and other T cell-mediated autoimmune diseases has been well documented. Not only can these cells
Department of Immunology, Bruce Rappaport Faculty of Medicine and Rappaport
Family Institute for Research in the Medical Sciences, Haifa, Israel
Received for publication March 23, 2006. Accepted for publication September
14, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Israel Science Academy, the Ministry
of Health Chief Scientist, and the Rappaport Institute.
2
Address correspondence and reprint requests to Dr. Nathan Karin, Rappaport Family
Institute for Research in the Medical Sciences, P.O. Box 9697, Haifa 31096, Israel.
E-mail address: [email protected]
3
Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; Tr1, regulatory T cell; MBP, myelin basic protein; EAN,
experimental autoimmune neuritis; HEV, high endothelial venule.
Copyright © 2006 by The American Association of Immunologists, Inc.
adoptively transfer EAE, but also blockade of their migratory
properties rapidly suppresses an established disease (21, 22). Th17
cells are likely to be highly potent Ag-specific inflammatory cells
as well (15–20). Th3 cells and Tr1 cells produce suppressor/regulatory cytokines (TGF-␤, IL-10) in response to their target Ag (7,
9). Their ability to suppress different autoimmune diseases has
been well documented in adoptive transfer experiments (7, 9, 23).
CD4⫹CD25⫹ are natural suppressor T cells (12, 13). Their absence enhances the development of T cell-mediated autoimmunity,
whereas their adoptive transfer suppresses these diseases (24, 25).
We have previously shown that during inflammatory autoimmune diseases the immune system mounts a beneficial autoantibody response against selected number of inflammatory mediators
that direct the pathogenesis of each disease and that these responses could be rapidly amplified by targeted DNA plasmids encoding each relevant mediator (26 –34). One of these studies has
also been extended to humans (28). In this particular study, we
have also shown that this response is directed exclusively to inflammatory, but not regulatory mediators such as IL-4, IL-10, or
TGF-␤ and that under these conditions administration of plasmid
DNA encoding a regulatory mediator would not lead to production
of autoantibodies to this mediator (28). Tentatively, under these
conditions, the function of this mediator encoded by plasmid DNA
would not be neutralized and could even be functional. Independently, the group of Lawrence Steinman, at Stanford University
(Stanford, CA), developed a novel therapeutic strategy in which
the DNA plasmid encoding the self-autoimmune determinant is
coadministered with another plasmid encoding IL-4 (35). Under
these conditions, the IL-4 produced at the site of immunization is
not neutralized and therefore could effectively shift the polarization of Ag T cells activated in response to the target autoimmune
Ag into IL-4-producing T cells to suppress the disease (35). In
clinical trials, Steinman, Garren and colleagues have accomplished
0022-1767/06/$02.00
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We have previously shown that Ag-specific IL-10-producing regulatory T cells (Tr1) participate in the regulation of experimental
autoimmune encephalomyelitis and that their specificity undergoes determinant spread in a reciprocal manner to effector T cell
specificity. The current study shows that coadministration of plasmid DNA vaccines encoding IL-10 together with a plasmid
encoding a myelin basic protein (MBP) encephalitogenic determinant during an ongoing disease rapidly amplifies this Tr1mediated response, in a disease-specific manner. Thus, coadministration of both plasmids, but not the plasmid DNA encoding MBP
alone, rapidly suppresses an ongoing disease. Tolerance included elevation in Ag-specific T cells producing IL-10 and an increase
in apoptosis of cells around high endothelial venules in the CNS after successful therapy. Tolerance could be transferred by
MBP-specific primary T cells isolated from protected donors and reversed by neutralizing Abs to IL-10 but not to IL-4. Due to
the nature of determinant spread in this model, we could bring about evidence implying that rapid and effective induction of
Tr1-induced active tolerance is dependent on redirecting the Tr1 response to the epitope to which the effector function dominates
the response at a given time. The consequences of these findings to multiple sclerosis, and possibly other inflammatory autoimmune
diseases are discussed. The Journal of Immunology, 2006, 177: 8241– 8247.
8242
a similar feat in early phase 1/2 studies in patients with relapsing
remitting MS (36).
We have recently shown that shifting the Ag-specific Th1/Th2
balance into Th2 suppresses EAE by an indirect mechanism because Ag-specific Th2 cells are incapable of transferring the beneficial effect resulting from driving the T cell balance into Th2
(37). In another study, we have shown that Ag-specific IL-10producing Tr1 cells participate in the regulation of EAE and that
their specificity undergoes determinant spread in a reciprocal manner to effector T cell specificity (23). The current study explores
for the first time the possibility of using combined DNA vaccine
strategy to selectively amplify the regulatory function of these Agspecific Tr1 cells during ongoing EAE.
Materials and Methods
Animals
Female Lewis rats ⬃6 wk old were purchased from Harlan Biotech Israel
and maintained under special pathogen-free conditions in our animal
facility.
Myelin basic protein (MBP) peptide 68 – 86 (YGSLPQKSQRSQDENPV)
and P257– 81 (FKNTEISKLGQEFEETTADNRKTK) were synthesized on a
MilliGen 9050 peptide synthesizer by standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC. Sequence was confirmed by amino acid analysis and mass spectroscopy. Only peptides that
were ⬎95% pure were used in our study.
Active induction of EAE or experimental autoimmune
neuritis (EAN)
MBP68 – 86 or P257– 81 at a concentration of 1 mg/ml were dissolved in PBS
and emulsified with an equal volume of IFA supplemented with 4 mg/ml
heat-killed Mycobacterium tuberculosis H37Ra in oil (Difco Laboratories).
Rats were immunized s.c. in the hind footpads with 0.1 ml of the emulsion
and monitored daily for clinical signs by an observer blind to the treatment
protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail;
2, hind limb paralysis; 3, front and hind limb paralysis; 4, total body paralysis. EAN was scored as follows: 0, clinically normal; 1, flaccid tail; 2,
hind limb paralysis.
Selection of MBP-specific T cell lines
MBP68 – 86-specific T cell lines were selected according to the method developed by Ben-Nun et al. (1), with our minor modifications (38).
Generation of IL-10- and MBP-encoding DNA plasmids
The cDNA encoding rat MBP and rat IL-10 were obtained from CNS
samples of inflamed EAE brain and subjected to RT-PCR amplification of
the IL-10 and the MBP encoding genes using the oligonucleotide primers
IL-10 (forward 5⬘-ATGCTTGGCTCAGCAC-3⬘, reverse 5⬘-TCAATTTTT
CATTTTGAGTGT-3⬘) and MBP (forward 5⬘-ATGGCATCACAGAAGA
GA-3⬘, reverse 5⬘-TCAGCGTCTTGCCATGGGAG-3⬘) according the protocol described elsewhere (38), and cloned into a different pcDNA3
constructs as described (34). The oligonucleotide encoding rat MBP68 – 86
and rat MBP87–99 were synthesized according to the sequences MBP68 – 86,
5⬘-CTACGGCTCCCTGCCCCAGAAGTCGCAGAGGACCCAAGATGA
AAACCCAGT-3⬘, and MBP87–99 5⬘-GTCCACTTCTTCAAGAACATT
GTTGACACCTCGTACACCC-3⬘ and cloned into pcDNA3 constructs.
Cytokine determination in primary cultures
Spleen cells or lymph node cells from EAE donors were stimulated in vitro
(107 cells/ml) in 24-well plates (Nalge Nunc International) with 100 ␮M
MBP68 – 86. After 72 h of stimulation, supernatants were assayed for the
protein level of various cytokines using the commercially available ELISA
kits as specified: for IL-10 (Diaclone Research); for IL-4, rat IL-4 Eli-pair
(Diaclone Research).
Western blot analysis
Draining lymph nodes were mechanically homogenized in 1 ml of the
following buffer: 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.4), 0.001 M EDTA,
1 ␮g/ml aprotinin, and 1.6 ␮M Pefabloc SC (Boehringer Mannheim). Protein concentration was determined using a BCA protein assay (Pierce
Chemical), and proteins were subjected to Western blot analysis in which
membranes were hybridized with anti-phospho-STAT3 Ab or STAT3 Ab
(New England Biolabs) diluted 1/1000 in TBST, flowing HRP-conjugated
secondary Ab (Jackson ImmunoResearch Laboratories). The membranes
were then processed as in the ECL protocol (Amersham Life Sciences).
Isolation of CNS mononuclear cells
Cells were isolated according to protocol described previously by KatzLevy et al. (39).
FACS analysis
FACS analysis was conducted according to the basic protocol we used
previously (33). Intracellular staining of IL-4, IL-10, and TGF-␤ were done
using a commercially available kit: LEUCOPERM, BUF9 (Serotec). PElabeled mouse anti-rat IL-10 mAb, FITC-labeled mouse anti-rat TGF-␤
mAb, and FITC-labeled mouse anti-rat IL-4 mAb (BD Pharmingen, BD
Biosciences) were used for direct staining. Brefeldin A was added to cells
for the last 6 h as described previously by Openshaw et al. (40). Cells were
analyzed using a FACSCalibur (BD Biosciences). Data were collected for
10,000 events and analyzed using a CellQuest software program (BD
Biosciences).
Histopathology
Frozen sections of the same area of the lumbar region of the spinal cord
were stained with H&E. Each section was evaluated without knowledge of
the treatment status of the animal. The following scale was used: 0, no
perivascular lesions; 1, 1–5 perivascular lesions per section with minimal
parenchymal infiltration; 2, 5–10 perivascular lesions per section with parenchymal infiltration; and 3, ⬎10 perivascular lesions per section with
extensive parenchymal infiltration.
Immunohistochemistry
Immunohistochemical detection of active caspase 3 was conducted using a
commercially available kit (APO active 3TM; Cell Technology) according
to the manufacturer’s protocol.
Statistical analysis
Significance of differences was examined using Student’s t test. A value of
p ⬍ 0.05 was considered significant. The Mann-Whitney sum of ranks test
was used to evaluate significance of differences in mean of maximal clinical score. A value of p ⬍ 0.05 was considered significant.
Results
Coadministration of plasmid DNA vaccines encoding MBP68 – 86
and IL-10 suppresses EAE in a disease-specific manner
Groups of six Lewis rats were subjected to active induction of
EAE induced by MBP68 – 86/CFA. At the onset of disease, these
rats were treated with a plasmid DNA encoding MBP68 – 86, a plasmid DNA encoding IL-10, or both plasmids coadministered. Control rats were treated with PBS or with an empty vector. Of these
groups only rats given coadministration of the IL-10-encoding
DNA plasmid together with the MBP-encoding plasmids went into
fast remission (Fig. 1A; mean maximal score of 0.3 ⫾ 0.166 compared with 4 ⫾ 0.66 in control group; p ⬍ 0.001). Within the other
groups, only those treated with plasmid DNA encoding IL-10 displayed a lower mean maximal score (2.6 ⫾ 0.5, compared with
4 ⫾ 0.66 in the control group; p ⬍ 0.05), which was significantly
higher than the one recorded in the group subjected to coadministration of plasmids (0.3 ⫾ 0.166 compared with 2.6 ⫾ 0.5; p ⬍
0.001). On day 14 (peak of disease in the control group; Fig. 1A),
representative rats were scarified (three per group). Lumbar spinal
cord samples were subjected to histological evaluation (Fig. 1B).
The mean histological score of control EAE rats (Fig. 1Bc) was
2.1 ⫾ 0.66 and did not differ from that of rats injected with an
empty vector (2 ⫾ 0.5, Fig. 1Bb). Both were significantly higher
( p ⬍ 0.001) than the histological score observed in rats treated
with MBP and IL-10-encoding plasmids (Fig. 1Bd; 0.66 ⫾ 0.166;
p ⬍ 0.001), which closely resembled sections from naive healthy
rats (Fig. 1Ba). Thus, histological evaluation could verify the beneficial effect that was clinically observed in the group subjected to
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Peptide Ags
COADMINISTRATION OF DNA PLASMIDS
The Journal of Immunology
8243
coadministration of both plasmids (Fig. 1A). We have subjected
the lumbar spinal cord sections of each group to active caspase 3,
a whole mark of apoptosis, immunohistochemistry. Our results
(Fig. 1C) clearly show that: 1) during EAE apoptosis of cells
around high endothelial venules (HEV) is apparent; 2) the combined therapy with plasmids DNA encoding IL-10 and MBP significantly increases the number of caspase 3⫹ cells around HEV as
compared with control EAE, EAE rats treated with IL-10-encoding
plasmid alone, or MBP-encoding plasmid alone. Fig. 1C shows a
representative section from each group. Statistical analysis of 18
sections per group, 3 fields per section, showed significant differences between mice subjected to combined therapy with DNA
plasmids encoding IL-10 and MBP (Fig. 1Ce) to control EAE (Fig.
1Cb), EAE rats treated with IL-10-encoding plasmid alone (Fig.
1Cc) or MBP-encoding plasmid alone (Fig. 1Cd; 20.6 ⫾ 2.4 compared with 5.2 ⫾ 0.6 and 7.4 ⫾ 0.8 and 5.66, respectively; p ⬍
0.01). These data suggest that rapid apoptosis of invading leuko-
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FIGURE 1. Lewis rats were subjected to
active induction of EAE. On day 10, these
rats were separated into five groups of equally
sick animals (six rats per group) and repeatedly administered (two times, days 10 and 12;
300 ␮g per rat per injection) with either plasmid DNA (pcDNA) encoding MBP68 – 86 (E),
plasmid DNA encoding IL-10 (f), or coadministered with both plasmids (Œ). Control
rats were treated with PBS (䡺) or with an
empty vector (F). Rats were monitored daily
for clinical signs by an observer blind to the
experimental protocol. A, Results of one of
three independent experiments with similar
data are shown as mean maximal score ⫾ SE.
B, Histological analysis of lumbar spinal cord
section of naive healthy rats (a), rats treated
with an empty vector (b), PBS (c), or the
combined vaccine comprised of MBP- and
IL-10-encoding plasmids (d). C, Immunohistological staining of active caspase 3 of naive
healthy rats (a), rats treated with an empty
vector (b), rats treated with plasmid DNA encoding IL-10 alone (c), rats treated with plasmid DNA encoding MBP alone (d), or the
combined vaccine comprised of MBP and
IL-10 encoding plasmids (e). D, Primary cultures of spleen cells, isolated at this time,
were subjected to in vitro stimulation with the
MBP68 – 86 target Ag, and levels of IL-4 and
IL-10 were determined by ELISA. E, Additional groups (six rats each) were subjected to
the induction of active EAE with MBP
(MBP87–99; a) or to EAN (b). On days 10 and
12, both groups were subjected to the codelivery of plasmid DNA vaccines encoding
MBP68 – 86 ⫹ IL-10 (E). Control rats were
treated with PBS (䡺) or empty vector (f). In
a reciprocal experiment (c), rats (six per
group) were subjected to EAE induced by
MBP68 – 86 and at the onset of disease to
combined DNA vaccines including: plasmid DNA encoding MBP68 – 86 ⫹ plasmid
DNA encoding IL-10 (F), plasmid DNA
encoding MBP87–99 ⫹ plasmid DNA encoding IL-10 (f), plasmid DNA encoding
whole MBP ⫹ plasmid DNA encoding
IL-10 (F), or PBS (䡺). Results of one of
three independent experiments with similar
data are shown as mean maximal score ⫾ SE.
cytes within the CNS contributes to the rapid recovery from the
disease. Spleen cells from each group were subjected to in vitro
stimulation with the MBP68 – 86 target Ag. Levels of IL-4 and
IL-10 were then recorded (Fig. 1D). We show here that primary
cultures from EAE rats treated with the combined constructs (encoding MBP and IL-10) displayed an elevated production of IL-10
in response in comparison with each of the other groups (Fig. 1D;
2.33 ⫾ 0.5 ␮g/ml compared with 1.2 ⫾ 0.2 ␮g/ml in the control
group; p ⬍ 0.001). No significant difference could be observed in
IL-4 production between these groups.
In a complementary experiment (Fig. 1E), rats were subjected to
either active EAE induced by the secondary determinant of MBP
(peptide 87–99) or to EAN, a paralytic disease of the peripheral
nervous system (41). Both groups were subjected to the codelivery of
plasmid DNA vaccines encoding MBP68 – 86 together with a plasmid
DNA encoding IL-10. Fig. 1E clearly shows that this targeted DNA
therapy provides protective immunity in a disease-specific manner.
8244
COADMINISTRATION OF DNA PLASMIDS
The therapeutic effect acquired by coadministration of plasmid
DNA vaccines encoding MBP68 – 86 and IL-10 is IL-10
dependent
In a subsequent experiment, conducted under the same conditions
as described in the legend to Fig. 1A, groups of six Lewis rats each
were subjected to active induction of EAE induced by MBP68 – 86/
CFA. At the onset of disease, these rats were injected with plasmid
DNA encoding MBP68 – 86, plasmid DNA encoding IL-10, or both
plasmids coadministered. On day 15 (peak of disease in control
groups), the anterior muscle of the tibia was subjected to RT-PCR
of IL-10 transcription (Fig. 2A) and cervical lymph node cells for
Western blot analysis for the expression of STAT-3 and pSTAT-3
(Fig. 2B), which has been well implicated with IL-10 production
(44, 45). Our results clearly show a significant elevation in
pSTAT-3 expression as well as IL-10 transcription in rats subjected to the administration of plasmid DNA encoding IL-10, either alone (Fig. 2B, lane b) or together with the MBP-encoding
plasmid (Fig. 2B, lane d), but not in control (Fig. 2B, lane a), or
following the administration of plasmid DNA encoding MBP
alone (Fig. 2B, lane c). Primary cultures from the cervical lymph
node cells of each group were cultured with the target MBP68 – 86
epitope followed by intracellular flow cytometry analysis (gated on
CD4⫹ T cells) of intracellular cytokine production. Fig. 2C shows
analysis of one of three independent experiments with similar results. It shows that: 1) during EAE, the number of IL-10highIL4low-producing cells in the control EAE group elevates (Fig. 2C;
1.2% compared with 0.7%); 2) administration of either plasmid
DNA encoding IL-10 alone, or MBP68 – 86 alone (but not an empty
vector; data not shown) significantly increases the relative number
of these cells (3.7 and 3.4% compared with 1.2%), albeit not
enough to suppress an ongoing disease. One possibility is that the
MBP-encoding plasmid further promotes the proliferation of
MBP-specific Tr1 cells, among other MBP-specific T cells. As for
the IL-10-encoding plasmid, IL-10 transcribed by this plasmid
may provide an enrichment milieu for Tr1 cells (9), and therefore
their relative number increases; 3) only within the group subjected
to the coadministration of both plasmids does the number of
CD4⫹IL-10highIL-4low-producing cells substantially increase (Fig.
2C; 16.9% compared with 3.7%, 3.4% in groups treated with sin-
FIGURE 2. Lewis rats were subjected to active induction of EAE. On
day 10, these rats were separated into four groups of equally sick animals
(six rats per group) and repeatedly treated with PBS (Aa and Ba), plasmid
DNA encoding IL-10 (Ab and Bb), plasmid DNA encoding MBP68 – 86 (Ac
and Bc), or coadministration of plasmid DNA encoding MBP68 – 86 and
plasmid DNA encoding IL-10 (Ad and Bd). Fifteen days after disease induction, rats were sacrificed. The anterior muscle of the tibia was subjected
to PCR for IL-10 transcription (A). Cervical lymph nodes were harvested
and either subjected to Western blot analysis for the expression of Stat-3
and pStat-3 (B) or cultured with MBP68 – 86 epitope and then subjected to
intracellular flow cytometry analysis (gated on CD4⫹ T cells) for IL-4 and
IL-10 production (C) and for TGF-␤ and IL-10 production (D) as follows:
a, naive rats; b, EAE rats; c, EAE rats treated with plasmid DNA encoding
MBP; d, EAE rats treated with plasmid DNA encoding IL-10; e, EAE rats
treated with plasmid DNA encoding MBP ⫹ plasmid DNA encoding IL10. These cells were then tested for their ability to transfer disease resistance (E). Three groups of EAE rats (six rats per group) were subjected to
the administration of 3 ⫻ 107 MBP-activated spleen cells isolated from
PBS (䡺), control EAE rats (E), or those coadministered with plasmid
DNA encoding MBP68 – 86 and plasmid DNA encoding IL-10 (f). An observer blind to the experimental protocol monitored clinical manifestation
of disease. Results of one of three independent experiments with similar
data are shown as mean maximal score ⫾ SE. F, On day 15, cervical lymph
node cells from representative rats from each group were tested for their
ability to proliferate in response to the target Ag (MBP68 – 86). Results are
shown as mean stimulation index ⫾ SE.
gle plasmid DNA vaccines). We could not observe increased intracellular expression of TGF-␤ in either CD4⫹IL-10high or
CD4⫹IL-10low-producing T cells (Fig. 2D). A direct ELISA further substantiated this observation (data not shown). Cytokine levels in supernatants of cultured primary cervical lymph node T cells
proliferating in response to their target Ag were 10.5 ⫾ 0.5, 12.7 ⫾
0.7, 11.6 ⫾ 0.4, and 11.9 ⫾ 0.6 pg/ml in control EAE rats, rats
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That is, rats subjected to disease induction with one determinant of
MBP acquire resistance to tolerance induced by a plasmid DNA
encoded by the other determinant (Fig. 1Ea; mean maximal score
of 0.66 ⫾ 0.166 compared with 2.5 ⫾ 0.5 in the control group; p ⬍
0.01), but not to a disease resulting from an autoimmune response
against an irrelevant target Ag (i.e., P2 in Fig. 1Eb). Very similar
results were obtained using plasmid DNA encoding the whole
MBP together with plasmid DNA encoding IL-10 (data not
shown). In a reciprocal experiment (Fig. 1Ec) rats were subjected to EAE induced by MBP68 – 86 and at the onset of disease
to combined DNA vaccines including: plasmid DNA encoding
MBP68 – 86 ⫹ plasmid DNA encoding IL-10; plasmid DNA encoding MBP87–99 ⫹ plasmid DNA encoding IL-10; plasmid DNA
encoding whole MBP ⫹ plasmid DNA encoding IL-10; or PBS.
We have previously shown that in this model of disease at the
onset of disease the immune response spreads from the secondary
epitope (MBP87–99) to the major one (MBP68 – 86), but not vice
versa (23, 42, 43). Fig. 1Ec shows that at this time combined DNA
plasmids encoding the major encephalitogenic determinant and IL10, but not the secondary determinant, and IL-10 rapidly suppress
the disease. This strongly suggests that rapid and effective induction of Tr1-induced active tolerance is dependent on redirecting
the Tr1 response to the epitope to which the effector function dominates the response.
The Journal of Immunology
Discussion
The cascade of events leading to the development of MS is not
fully understood. Yet, it is believed that the disease involves an
autoimmune destructive process (46), and as such nonspecific and
specific approaches for interfering in the multistep events initiating
and promoting the inflammatory process of the disease may lead to
disease regression. Fourteen years ago, we have demonstrated that
the ␣4␤1 integrin VLA-4 is a key adhesion molecule that directs
inflammatory leukocyte trafficking to the CNS and that Ab blockade of this molecule may reverse EAE (21). This particular study
has been successfully extended in clinical trials (47) and became a
Food and Drug Administration-approved drug for the disease (47).
Nevertheless, it has recently been found that such a successful
nonselective inhibition of T cell and monocyte migration to the
FIGURE 3. Lewis rats were subjected to active induction of EAE. At the
onset of disease, these rats were separated into five groups of equally sick
animals (six rats per group) that were or were not (䡺) treated by coadministration of plasmid DNA encoding MBP68 – 86 and plasmid DNA encoding IL10. One day after the onset of disease, these rats were or were not (F) subjected to repeated administration (every other day) of 100 ␮g/ml rabbit anti-rat
IL-10 (f), rabbit anti-rat IL-4 polyclonal Abs (E), or polyclonal Abs from
preimmunized rabbit (Œ). An observer blind to the experimental protocol monitored clinical manifestation of disease. Results of one of three experiments
with similar data are shown as mean score ⫾ SE.
CNS may apparently lead to fatal infections (48). This experience
further demonstrates the importance of developing novel Ag-specific means of therapy. The need to develop Ag-specific therapy is
compelling and motivated us to explore DNA vaccination-based
therapy that would select highly specific Trl capable of suppressing the disease. Ag-specific Tr1 cells were discovered by Groux et
al. (9) 9 years ago. We have later shown that these IL-10-producing Tr1 cells participate in the natural regulation of EAE and that
their function could be amplified in a beneficial manner by soluble
peptide therapy (23). The promising use of DNA vaccines as future
means of intervention in various diseases motivated us to investigate whether codelivery of plasmid DNA encoding IL-10 together
with a plasmid encoding the self-autoimmune Ag would effectively select IL-10-producing Trl capable of suppressing the disease. The data presented in the current manuscript show that indeed these combined vaccines select Ag-specific Tr1 cells capable
of suppressing EAE, and their beneficial function is reversible by
anti IL-10 Abs. Three questions may arise from the data presented
in Fig. 1. Why does the combined vaccine affect the manifestation
of disease so rapidly? Why is it selective against disease induced
by the other encephalitogenic determinant, but not against an irrelevant autoimmune disease (EAN; Fig. 1E)? And why do the
combined vaccines encoding one target epitope and IL-10 suppress
disease induced by the other epitope and not vice versa? We have
previously shown that during EAE the immune system mounts a
Tr1-mediated response, not only against the determinant with
which disease was induced, but also against the determinants to
which the pathogenic response spreads (23), and that this regulatory response restrains the severity, albeit not totally suppress its
manifestation (23). The data presented here clearly show, once
again, an apparent increase in IL10highIL-4low-producing T cells in
cervical lymph nodes during EAE (Fig. 2C) that is profoundly
increased following coadministration of plasmid DNA encoding
MBP ⫹ plasmid DNA encoding IL-10. Tolerance was associated
with marked cell apoptosis around HEV (Fig. 1C). These data
suggest that rapid apoptosis of invading leukocytes within the CNS
either contributes, or is the outcome, of the rapid recovery from the
disease. A positive association between successful therapy of
EAE, including Trl-based therapy, had previously been observed
by others (49, 50). We have previously shown that leukocytes
apoptosis at the autoimmune site is accelerated before and during
spontaneous remission from the disease, and that neutralization of
Fas ligand inhibits this process and delays recovery (33). It has
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treated with plasmid DNA encoding MBP68 – 86 alone, plasmid
DNA encoding IL-10 alone, or their combination, respectively.
We have previously shown that Tr1 cells selectively accumulate
in the cervical lymph nodes that drain the CNS and to a much
lesser extent in spleen and other lymph nodes (23). Nevertheless,
the total number of these cells in cervical lymph nodes is not
sufficient for adoptive transfer experiments. Before extending our
study to adoptive transfer experiments, we have recorded the relative number of these cells following combined DNA plasmid
therapy in cervical lymph nodes, in comparison with spleen and
inguinal lymph nodes. These organs were removed 2 days after the
second administration of the combined DNA plasmid therapy. The
relative number of IL-10highCD4⫹ T cells in cervical lymph nodes
was ⬃16%, and that in spleen and inguinal lymph nodes was
⬃6%. The relative number of these cells in control EAE rats was
⬃1–1.5% in each organ (data not shown). Thus, accumulation of
regulatory cells is selective. However, due to their limited total
number in cervical lymph nodes, continuing adoptive transfer experiments were conducted using primary spleen cells (see below).
We have then tested the ability of these primary T cells, activated in vitro with MBP68 – 86, to affect the kinetics of ongoing
EAE, in an adoptive transfer experiment (Fig. 2E). Just after the
onset of disease, three groups of equally sick EAE rats (six rats per
group) were subjected to the administration of 3 ⫻ 107 MBPactivated spleen cells isolated from either control EAE rats or
those treated by coadministration of plasmid DNA encoding
MBP68 – 86 and plasmid DNA encoding IL-10. Only the adoptive
transfer of MBP-specific primary T cells from protected donors,
subjected to coadministration of plasmid DNA encoding MBP68 – 86
and plasmid DNA encoding IL-10 rapidly suppressed ongoing EAE
(Fig. 2E; mean maximal score of 0.66 ⫾ 0.16 compared with 2.33 ⫾
0.3; p ⬍ 0.01). On day 15, cervical lymph node cells from representative rats from each group were tested for their ability to proliferate in response to the target Ag (MBP68 – 86). Fig. 2F shows a
significant decrease in the proliferative response of T cells from
protected donors, as compared with both control groups (mean
stimulation index ⫾ SE, 2.5 ⫾ 0.4 compared with 5.8 ⫾ 0.8 and
7.2 ⫾ 1.2, respectively; p ⬍ 0.01).
Finally, to learn whether therapy is indeed IL-10 dependent, five
groups of Lewis rats (six rats each) were subjected to the induction
of active EAE and at the onset of disease were, or were not, treated
by coadministration of plasmid DNA encoding MBP68 – 86 and
plasmid DNA encoding IL-10. These rats were then subjected to
repeated administration of 100 ␮g/ml commercially available
(PeproTech) rabbit anti-rat IL-10, rabbit anti-rat IL-4 polyclonal
Abs, or polyclonal Abs from preimmunized rabbit (PeproTech).
Our results (Fig. 3) show that only the administration of neutralizing Abs to IL-10 could reverse the tolerance induced by the
above combined plasmid DNA therapy.
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Disclosures
The authors have no financial conflict of interest.
References
1. Ben-Nun, A., H. Wekerle, and I. R. Cohen. 1981. The rapid isolation of clonable
antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11: 195–199.
2. Raine, C. S. 1991. Multiple sclerosis: a pivotal role for the T cell in lesion
development. Neuropathol. Appl. Neurobiol. 17: 265–274.
3. O’Garra, A., L. Steinman, and K. Gijbels. 1997. CD4⫹ T-cell subsets in autoimmunity. Curr. Opin. Immunol. 9: 872– 883.
4. O’Garra, A., and K. Murphy. 1994. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 6: 458 – 466.
5. Mosmann, T. R., and R. L. Coffman. 1989. Th1 and Th2 cells: different patterns
of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 9: 145–173.
6. Cash, E., A. Minty, P. Ferrara, D. Caput, D. Fradelizi, and O. Rott. 1994. Macrophage-inactivating IL-13 suppresses experimental autoimmune encephalomyelitis in rats. J. Immunol. 153: 4258 – 4267.
7. Chen, Y., V. K. Kuchroo, J. Inobe, D. Hafler, and H. L. Weiner. 1994. Regulatory
T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265: 1237–1240.
8. Fukaura, H., S. C. Kent, M. J. Pietrusewicz, S. J. Khoury, H. L. Weiner, and
D. A. Hafler. 1996. Induction of circulating myelin basic protein and proteolipid
protein-specific transforming growth factor-␤1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98: 70 –77.
9. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, and
M. G. Roncarolo. 1997. A CD4⫹ T-cell subset inhibits antigen-specific T-cell
responses and prevents colitis. Nature 389: 737–742.
10. Schwartz, R. H. 2005. Natural regulatory T cells and self-tolerance. Nat. Immunol. 6: 327–330.
11. Sakaguchi, S., and N. Sakaguchi. 2005. Regulatory T cells in immunologic selftolerance and autoimmune disease. Int. Rev. Immunol. 24: 211–226.
12. Shevach, E. M. 2002. CD4⫹CD25⫹ suppressor T cells: more questions than
answers. Nat. Rev. Immunol. 2: 389 – 400.
13. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh,
Y. Kuniyasu, T. Nomura, M. Toda, and T. Takahashi. 2001. Immunologic tolerance maintained by CD25⫹CD4⫹ regulatory T cells: their common role in
controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182: 18 –32.
14. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang,
L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells
regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6:
1133–1141.
15. McGeachy, M. J., and S. M. Anderton. 2005. Cytokines in the induction and
resolution of experimental autoimmune encephalomyelitis. Cytokine 32: 81– 84.
16. Lubberts, E., M. I. Koenders, and W. B. van den Berg. 2005. The role of T cell
interleukin-17 in conducting destructive arthritis: lessons from animal models.
Arthritis Res. Ther. 7: 29 –37.
17. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham,
J. D. Sedgwick, T. McClanahan, R. A. Kastelein, and D. J. Cua. 2005. IL-23
drives a pathogenic T cell population that induces autoimmune inflammation.
J. Exp. Med. 201: 233–240.
18. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy,
K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4⫹ effector
T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat.
Immunol. 6: 1123–1132.
19. Bettelli, E., and V. K. Kuchroo. 2005. IL-12- and IL-23-induced T helper cell
subsets: birds of the same feather flock together. J. Exp. Med. 201: 169 –171.
20. Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, and A. L. Gurney. 2003.
Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the
production of interleukin-17. J. Biol. Chem. 278: 1910 –1914.
21. Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, and
N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by
antibodies against ␣4␤1 integrin. Nature 356: 63– 66.
22. Kent, S. J., S. J. Karlik, C. Cannon, D. K. Hines, T. A. Yednock, L. C. Fritz, and
H. C. Horner. 1995. A monoclonal antibody to ␣4 integrin suppresses and reverses
active experimental allergic encephalomyelitis. J. Neuroimmunol. 58: 1–10.
23. Wildbaum, G., N. Netzer, and N. Karin. 2002. Tr1 cell-dependent active tolerance blunts the pathogenic effects of determinant spreading. J. Clin. Invest. 110:
701–710.
24. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, and
J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of
the CD4⫹CD25⫹ immunoregulatory T cells that control autoimmune diabetes.
Immunity 12: 431– 440.
25. Kohm, A. P., P. A. Carpentier, H. A. Anger, and S. D. Miller. 2002. Cutting edge:
CD4⫹CD25⫹ regulatory T cells suppress antigen-specific autoreactive immune
responses and central nervous system inflammation during active experimental
autoimmune encephalomyelitis. J. Immunol. 169: 4712– 4716.
26. Goldberg, R., Y. Zohar, G. Wildbaum, Y. Geron, G. Maor, and N. Karin. 2004.
Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27. J. Immunol. 173: 6465– 6471.
27. Goldberg, R., G. Wildbaum, Y. Zohar, G. Maor, and N. Karin. 2004. Suppression
of ongoing adjuvant-induced arthritis by neutralizing the function of the p28
subunit of IL-27. J. Immunol. 173: 1171–1178.
28. Wildbaum, G., M. Nahir, and N. Karin. 2003. Beneficial autoimmunity to proinflammatory mediators restrains the consequences of self-destructive immunity.
Immunity 19: 679 – 688.
29. Wildbaum, G., N. Netzer, and N. Karin. 2002. Plasmid DNA encoding IFN-␥inducible protein 10 redirects antigen-specific T cell polarization and suppresses
experimental autoimmune encephalomyelitis. J. Immunol. 168: 5885–5892.
30. Salomon, I., N. Netzer, G. Wildbaum, S. Schif-Zuck, G. Maor, and N. Karin.
2002. Targeting the function of IFN-␥-inducible protein 10 suppresses ongoing
adjuvant arthritis. J. Immunol. 169: 2685–2693.
31. Youssef, S., G. Maor, G. Wildbaum, N. Grabie, A. Gour-Lavie, and N. Karin.
2000. C-C chemokine-encoding DNA vaccines enhance breakdown of tolerance
to their gene products and treat ongoing adjuvant arthritis. J. Clin. Invest. 106:
361–371.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
also been suggested that during recovery from EAE CD4⫹ T cells
do not leave the CNS but rather undergo apoptosis there (51, 52).
Following tolerance induction of apoptosis is likely to be amplified. This may explain, in part, the mechanistic basis of increased
apoptosis in treated mice (Fig. 1C).
Combined plasmid DNA vaccines encoding MBP68 – 86 ⫹ IL-10
could rapidly suppress disease induced by the major epitope
(MBP68 – 86) and the other, noncross-reactive epitope (MBP87–99),
but not EAN (Fig. 1E). One possible explanation is that both the
MBP87–99-specific effector T cells, initially activated after active
induction of disease, and the MBP68 – 86-specific Trl, induced after
protective therapy, home to the CNS and cointact, in the microenvironment there, in part via IL-10 functioning as a suppressor
factor (bystander suppression). This may well explain why the
combined DNA vaccine protects from EAE but not EAN. The
other option is that the combined vaccine acts, during determinant
spread, on nonpolarized MBP68 – 86-specific T cells, undergoing
Ag-specific T cell activation, and redirects their polarization into
Ag-specific regulatory, rather than effector T cells. On this subject,
we have previously shown in numerous studies that the MBP68 – 86,
as a major epitope, is extremely dominant and that the MBP-specific response spreads very fast from the secondary to the major
epitope, but not the opposite, and that the onset of disease is
dependent on the response to the primary, but not necessarily
the secondary epitope (23, 42, 43). In two consecutive experiments, we tried to subject rats with MBP68 – 86-induced disease to
MBP87–99 and IL-10-encoding DNA plasmids and failed to induce
tolerance (data not shown). Nevertheless, in these rats we could
not record any significant response in cervical lymph nodes to
MBP87–99 (data not shown). Therefore, this model is probably not
the best model for distinguishing direct from bystander effects of
active tolerance. A possible implication for human MS could be
that a tolerance induction during determinant spread should focus
on the determinant to which the immune response spreads, at a
given time point.
STAT3 was elevated in rats subjected to plasmid DNA encoding
IL-10 alone, as well as to those subjected to the combined plasmids. Yet only the administration of the combined plasmid was
highly protective. It is likely that only the combination would provide the immune system to favor the polarization of Ag-specific T
cells into regulatory cells, which may also explain the specificity of
therapy (Fig. 1E).
Finally, we do not exclude the possibility that other types of Tr,
particularly (CD25⫹ T cells, are also being activated and contribute to the resistant state achieved by plasmid DNA therapy. Nevertheless, we think that the Lewis rats system is well set up for
studying the biological functions of Tr1 cells (23), and to a much
lesser extend the biological functions of CD25⫹ cells. For this
purpose, we intend to extend our studies, in the near future, to
myelin oligodendrocyte glycoprotein-induced disease in C57BL/6
mice.
COADMINISTRATION OF DNA PLASMIDS
The Journal of Immunology
42. Grabie, N., I. Wohl, S. Youssef, G. Wildbaum, and N. Karin. 1999. Expansion of
neonatal tolerance to self in adult life. I. The role of a bacterial adjuvant in
tolerance spread. Int. Immunol. 11: 899 –906.
43. Grabie, N., and N. Karin. 1999. Expansion of neonatal tolerance to self in adult
life. II. Tolerance preferentially spreads in an intramolecular manner. Int. Immunol. 11: 907–913.
44. Donnelly, R. P., H. Dickensheets, and D. S. Finbloom. 1999. The interleukin-10
signal transduction pathway and regulation of gene expression in mononuclear
phagocytes. J. Interferon Cytokine Res. 19: 563–573.
45. Wehinger, J., F. Gouilleux, B. Groner, J. Finke, R. Mertelsmann, and R. M. WeberNordt. 1996. IL-10 induces DNA binding activity of three STAT proteins (Stat1,
Stat3, and Stat5) and their distinct combinatorial assembly in the promoters of
selected genes. FEBS Lett. 394: 365–370.
46. Pedotti, R., D. Mitchell, J. Wedemeyer, M. Karpuj, D. Chabas, E. M. Hattab,
M. Tsai, S. J. Galli, and L. Steinman. 2001. An unexpected version of horror
autotoxicus: anaphylactic shock to a self-peptide. Nat. Immunol. 2: 216 –222.
47. Miller, D. H., O. A. Khan, W. A. Sheremata, L. D. Blumhardt, G. P. Rice,
M. A. Libonati, A. J. Willmer-Hulme, C. M. Dalton, K. A. Miszkiel, and P. W.
O’Connor. 2003. A controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 348: 15–23.
48. Steinman, L. 2005. Blocking adhesion molecules as therapy for multiple sclerosis: natalizumab. Nat. Rev. Drug Discov. 4: 510 –518.
49. Kohm, A. P., J. S. Williams, A. L. Bickford, J. S. McMahon, L. Chatenoud,
J. F. Bach, J. A. Bluestone, and S. D. Miller. 2005. Treatment with nonmitogenic
anti-CD3 monoclonal antibody induces CD4⫹ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis. J. Immunol. 174: 4525– 4534.
50. Madakamutil, L. T., I. Maricic, E. Sercarz, and V. Kumar. 2003. Regulatory T
cells control autoimmunity in vivo by inducing apoptotic depletion of activated
pathogenic lymphocytes. J. Immunol. 170: 2985–2992.
51. Schmied, M., H. Breitschopf, R. Gold, H. Zischler, G. Rothe, H. Wekerle, and
H. Lassmann. 1993. Apoptosis of T lymphocytes in experimental autoimmune
encephalomyelitis: evidence for programmed cell death as a mechanism to control inflammation in the brain. Am. J. Pathol. 143: 446 – 451.
52. Flugel, A., T. Berkowicz, T. Ritter, M. Labeur, D. E. Jenne, Z. Li, J. W. Ellwart,
M. Willem, H. Lassmann, and H. Wekerle. 2001. Migratory activity and functional changes of green fluorescent effector cells before and during experimental
autoimmune encephalomyelitis. Immunity 14: 547–560.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
32. Wildbaum, G., S. Youssef, and N. Karin. 2000. A targeted DNA vaccine augments the natural immune response to self TNF-␣ and suppresses ongoing adjuvant arthritis. J. Immunol. 165: 5860 –5866.
33. Wildbaum, G., J. Westermann, G. Maor, and N. Karin. 2000. A targeted DNA
vaccine encoding Fas ligand defines its dual role in the regulation of experimental
autoimmune encephalomyelitis. J. Clin. Invest. 106: 671– 679.
34. Youssef, S., G. Wildbaum, G. Maor, N. Lanir, A. Gour-Lavie, N. Grabie, and
N. Karin. 1998. Long-lasting protective immunity to experimental autoimmune
encephalomyelitis following vaccination with naked DNA encoding C-C chemokines. J. Immunol. 161: 3870 –3879.
35. Garren, H., P. J. Ruiz, T. A. Watkins, P. Fontoura, L. T. Nguyen, E. R. Estline,
D. L. Hirschberg, and L. Steinman. 2001. Combination of gene delivery and
DNA vaccination to protect from and reverse Th1 autoimmune disease via deviation to the Th2 pathway. Immunity 15: 15–22.
36. Garren, H., H. Antel, A. Bar-Or, C. A. Bodner, D. Campagnolo, J. Gianettoni,
F. Jalili, N. Kachuck, Y. Lapierre, M. Niino, et al. 2006. Phase I/II trial of a MBP
encoding DNA plasmid (BHT-3009) alone or combined with atorvastatin for
treatment of multiple sclerosis. In European Neurology Society, 2006 meeting.
Springer Medizin Verlag, Lausanne, pp. II/27–II/28.
37. Schif-Zuck, S., J. Westermann, N. Netzer, Y. Zohar, M. Meiron, G. Wildbaum,
and N. Karin. 2005. Targeted overexpression of IL-18 binding protein at the
central nervous system overrides flexibility in functional polarization of antigenspecific Th2 cells. J. Immunol. 174: 4307– 4315.
38. Karin, N., F. Szafer, D. Mitchell, D. P. Gold, and L. Steinman. 1993. Selective
and nonselective stages in homing of T lymphocytes to the central nervous
system during experimental allergic encephalomyelitis. J. Immunol. 150:
4116 – 4124.
39. Katz-Levy, Y., K. L. Neville, J. Padilla, S. Rahbe, W. S. Begolka, A. M. Girvin,
J. K. Olson, C. L. Vanderlugt, and S. D. Miller. 2000. Temporal development of
autoreactive Th1 responses and endogenous presentation of self myelin epitopes
by central nervous system-resident APCs in Theiler’s virus-infected mice. J. Immunol. 165: 5304 –5314.
40. Openshaw, P., E. E. Murphy, N. A. Hosken, V. Maino, K. Davis, K. Murphy, and
A. O’Garra. 1995. Heterogeneity of intracellular cytokine synthesis at the singlecell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182:
1357–1367.
41. Rostami, A., and S. K. Gregorian. 1991. Peptide 53–78 of myelin P2 protein is
a T cell epitope for the induction of experimental autoimmune neuritis. Cell.
Immunol. 132: 433– 441.
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