Download Peptides Based on MHC-TCR Binding Motifs Ordered Autoimmune

Immunomodulation of Experimental
Autoimmune Encephalomyelitis with Ordered
Peptides Based on MHC-TCR Binding Motifs
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of June 14, 2017.
Pedro J. Ruiz, Jason J. DeVoss, Louis-Vu T. Nguyen, Paulo
P. Fontoura, David L. Hirschberg, Dennis J. Mitchell, K.
Christopher Garcia and Lawrence Steinman
J Immunol 2001; 167:2688-2693; ;
doi: 10.4049/jimmunol.167.5.2688
http://www.jimmunol.org/content/167/5/2688
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Copyright © 2001 by The American Association of
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References
Immunomodulation of Experimental Autoimmune
Encephalomyelitis with Ordered Peptides Based on MHC-TCR
Binding Motifs1
Pedro J. Ruiz,2,3* Jason J. DeVoss,2* Louis-Vu T. Nguyen,* Paulo P. Fontoura,*
David L. Hirschberg,* Dennis J. Mitchell,* K. Christopher Garcia,† and Lawrence Steinman*
T
he T cell recognition of myelin peptides embedded in
MHC class II mediates the autoimmune attack in the human disease multiple sclerosis (MS)4 (1). In MS patients,
immune responses are detected against various components of the
myelin sheath. Many of these myelin components can also induce
experimental autoimmune encephalomyelitis (EAE), an animal
model of MS (1). Amino acids 85–99 of myelin basic protein
(MBP) contain the immunodominant epitope for T cells and autoantibodies in MS brain lesions (3– 6). The core motif from MBP
peptide (MBPp)85–99, HFFK, is the predominant epitope recognized by T cells and autoantibodies in the brain of MS patients
who are HLA-DRB1*1501 DQB1*0602 (HLA-DR2) (6). In the
Lewis rat, MBPp85–99 induces EAE in a monophasic, self-limiting
form. Acute paralysis lasts for a few days, followed by near total
clinical recovery. In the Lewis rat model of EAE, the encephalogenicity of MBPp85–99 is restricted by the RT1.Dl MHC class II
allele (DR like) (7).
Previously, we compared the structural requirements for autoantibody recognition to those of T cell clones reactive to
MBPp85–99 in humans. Anti-MBP Abs were affinity-purified from
CNS lesions of 12 postmortem cases studied. The MBPp87–99 was
immunodominant in all cases and inhibited Ab binding to MBP by
Departments of *Neurology and Neurological Sciences and †Structural Biology, Stanford University School of Medicine, Stanford, CA 94305
Received for publication April 9, 2001. Accepted for publication June 19, 2001.
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 in part by National Research Service Award AI07290-15
to P.J.R. from the National Institutes of Health and a National Science Foundation
Graduate Research Fellowship to J.J.D. from the National Science Foundation.
2
P.J.R. and J.J.D. contributed equally to this report.
3
Address correspondence and reprint requests to Dr. Pedro J. Ruiz, Department of
Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305. E-mail address: [email protected]
4
Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MBPp, MBP peptide; APL,
altered peptide ligand; GA, glatiramer acetate; gpSCH, guinea pig spinal cord
homogenate.
Copyright © 2001 by The American Association of Immunologists
⬎95% (6). Ab-binding residues were located in a 10-aa segment at
position 86 –95 (VVHFFKNIVT) that contained the MHC-TCR
contact residues for T cells recognizing MBP in the context of
DRB1*1501 and DQB1*0602. In the epitope center, the same residues (VHFFK) were important for T cell binding and MHC recognition (6).
The crystal structure of HLA-DR2 with MBPp85–99 confirmed
that Phe90 is a major anchor for the hydrophobic P4 pocket of the
MHC molecule, whereas Lys91 is the major TCR contact residue
(2). Substitution at Lys91 creates an altered peptide ligand (APL)
that can reverse paralysis and reduce proinflammatory cytokine
production in the Lewis rat (8). One such APL, MBPp87–99 (Lys91Ala), neither binds anti-MBP Abs nor triggers MBP-specific T
cells. The characteristics of this molecule have guided APL clinical trials for therapy in MS patients (9). The search for optimal
APLs can provide new therapeutic tools in the treatment of autoimmune diseases.
We have designed peptides containing repetitive amino acid
sequences that, according to computer modeling, bind the pockets of MS-related MHC molecules and potentially interfere with
the activation of pathogenic T cells. These peptides incorporate
the four amino acids, glutamate, tyrosine, lysine, and alanine,
used to create the random copolymer, glatiramer acetate (GA).
GA, known as Copaxone, is currently approved for the treatment of MS. The molar ratio of Ala:Lys:Glu:Tyr in GA is 4.5:
3.6:1.5:1, respectively; the copolymer has an average length of
40 –100 aa. The ratio of these four amino acids was chosen to
reflect their relative composition in MBP. Directly labeled GA
efficiently binds to different murine and human MHC class II
molecules (10, 11).
We report here that one possible sequence from these four
amino acids, EYYKEYYKEYYK, is effective in preventing and
treating EAE in the Lewis rat, an animal model of MS. It may thus
be possible to simplify and define the active components of a random copolymer like GA. We have designed an APL based on
computer modeling. This strategy, designing molecules based on
their predicted capacity to bind MHC class II, may prove effective
for the design of other molecules useful for intervention in a variety of immune-related diseases.
0022-1767/01/$02.00
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T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major
T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84
and 103 of myelin basic protein (1). The crystal structure of HLA-DR2 complexed with myelin basic protein84 –102 confirmed that
Lys91 is the major TCR contact site, whereas Phe90 is a major anchor to MHC and binds the hydrophobic P4 pocket (2). We have
tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation.
One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model
of MS. The Journal of Immunology, 2001, 167: 2688 –2693.
The Journal of Immunology
Materials and Methods
Animals
Female Lewis rats (6 – 8 wk old) were purchased from Harlan Sprague
Dawley (Indianapolis, IN)
Peptides
For immunization and disease reversal, peptides were synthesized on a
peptide synthesizer (model 9050; MilliGen, Burlington, MA) by standard
9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC.
Structure was confirmed by amino acid analysis and mass spectroscopy.
Peptides used for the experiments were: ENPVVHFFKNIVTPR
(MBPp85–99), ENPVVHFFANIVTPR (MBPp85–99 K⬎A), EYYKEYY
KEYYK (EYYK), KYYKYYKYYKYY (KYY), and AYEKAYEKAYEK
(AYEK).
Peptide-MHC binding assay
Computer modeling
The structural modeling of the peptide-MHC complexes was performed
using Insight II (Accelerys, San Diego, CA). The crystal structure of HLADR2 complexed with a peptide from human MBP (2) was used as a template for all studies. Substitutions in the native MBP sequence were done
using the residue replace function of InsightII. The backbone and side
chain torsion angles of the original residues were retained. Steric clashes
and chemical incompatibilities were assessed manually. A RasMol-readable script was then exported from InsightII and imported into RasMol for
rapid imaging.
Alignment of human and rat MHC class II
Homology analysis was done using ClustalW with a Blosum matrix (12).
The protein sequences are identified in GenBank as: 60497 for RT1.Dl and
1BX2B for HLA-DR2. The ClustalW output was formatted in BoxShade
for presentation.
EAE induction
II molecule. The ordered peptides EYYK and KYY were incubated with APCs and allowed to bind the MHC cleft. After 1 h of
incubation, biotinylated MBPp85–99 was added and incubated for
4 h more. The APCs were analyzed by flow cytometry to determine the amount of biotinylated MBPp85–99 bound to MHC class
II. As shown in Fig. 1, the ordered peptide EYYK is capable of
blocking MBPp85–99 from the Ag-binding cleft of MHC class II.
The IC50 value of EYYK calculated from a linear regression curve
was 83.0 ␮M.
Molecular modeling of binding interactions
Because EYYK was capable of displacing MBPp85–99, we created
a hypothetical model of the bound structure of EYYK in the HLADR2 peptide-binding groove. The previously published crystal
structure of HLA-DR2 complexed with MBPp85–99 was used as a
template. The sequence of MBP residues His90, Phe91, and Phe92
were changed to Glu90, Tyr91, and Tyr92; Lys93 remained the same
(Fig. 2). Although energy minimization or interatomic contacts
were not calculated, obvious steric clashes and chemical incompatibilities were avoided.
In our model, residues 90, 91, and 93 project out of the cleft and
are implicated in TCR recognition. The glutamate that replaces
His90 carries a negative charge and may block TCR signaling by
electrostatic repulsion. The addition of a polar hydroxyl group onto
a hydrophobic TCR contact residue (Phe913 Tyr) could also be
deleterious to TCR engagement. In our model, the Phe923 Tyr
change results in the submersion of the aromatic ring into the cleft
(Fig. 3). In the native peptide, Phe92 is important in anchoring the
peptide to the P4 hydrophobic pocket of the MHC class II molecule (2). However, the P4 pocket is not entirely hydrophobic, and
several residues could form hydrophilic interactions with the polar
tyrosine hydroxyl of EYYK. Hydrophilic residues that could potentially form hydrogen bonds with tyrosine in the EYYK peptide
are shown in Fig. 4. These residues may stabilize the binding of
EYYK in the MHC cleft, helping it block native MBPp85–99.
The residues derived from the crystal structure are based on a
human HLA-DR molecule (2). Because we conducted our experiments in Lewis rats and the residues are not identical, we tested
the homology of the two sequences. The rat (RT1-Dl) and human
MHC class II (DRB1*1501) molecules were aligned and analyzed
to determine whether peptide-MHC interactions are conserved.
The result of this alignment is shown in Fig. 5. In humans, the
Guinea pig spinal cord homogenate (gpSCH) or synthetic MBPp85–99 was
dissolved in 1 M PBS to a final concentration of 5 mg/ml or 2 mg/ml,
respectively, and emulsified with an equal volume of IFA supplemented
with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). Rats were injected s.c. with 0.1 ml of the peptide emulsion.
Experimental animals were assessed for signs of clinical disease as follows: 0, no clinical disease; 1, tail weakness or paralysis; 2, hind limb
weakness; 3, hind limb paralysis; 4, forelimb weakness or paralysis; and 5,
moribund or dead animal.
Peptide treatment
For disease prevention, peptides were emulsified in IFA to a final concentration of 1 mg/ml. A total of 0.1 ml was injected intradermally, once on
day ⫺20 and again on day ⫺10. Animals were challenged for EAE with
gpSCH at day 0. For treatment of ongoing disease, EAE was induced with
a 0.1-mg emulsion of MBPp85–99 in CFA. On the first day of acute clinical
disease, rats were injected i.p. with a dose of 0.5 mg peptide (EYYK or
KYY) in 1 ml 1 M PBS.
Results
The ordered peptide EYYKEYYKEYYK inhibits MBPp85–99
binding to rat MHC class II molecules
A competition assay was performed to test whether the ordered
peptide EYYK inhibits the binding of MPB85–99 to the MHC class
FIGURE 1. MHC-binding competition of EYYK (f) and KYY (⽧).
The ability of both peptides to inhibit binding of biotin-labeled MBPp85–99
at concentrations ranging from 0 to 80 ␮M was measured and presented as
percentage of inhibition of mean relative binding. The data represent four
separate experiments (n ⫽ 4). EYYK binds Lewis rat MHC with an IC50
value of 83.0 ␮M. Avidity effects due to the repeat structure of EYYK
likely lead to competitive binding.
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Peptide binding to class II molecules was measured as described before (8)
with some modifications. Briefly, APCs were purified from spleen cells by
negative selection using magnetic beads (Dynal, Oslo, Norway) conjugated
with Abs specific for T cells (CD52), macrophages (HIS36), and NK cells
(NKR-P1A) (BD PharMingen, San Diego, CA). After selection, cells were
plated at a concentration of 0.5 ⫻ 106 cells/well in flat-bottom 96-well
microtiter plates (Corning Costar, Corning, NY). EYYK and KYY peptides
were added to the wells at different concentrations (ranging from 0.01 to
0.24 mM) in a volume of 0.05 ml. After 1 h of incubation at 37°C, 0.01 mM
biotinylated MBPp85–99 was added. After 4 h more of incubation, cells
were harvested, and binding of PE-avidin to the cell surface was analyzed
by FACS (BD Biosciences, San Jose, CA). To determine the IC50 value of
EYYK, a linear regression curve was calculated using data points from
0.01 to 0.80 mM. Additional data points were not included because the
reaction saturated at concentrations above 80 ␮M.
2689
2690
MHC-TCR BINDING MOTIF-BASED PEPTIDES TO TREAT EAE
genic epitope can be controlled by the previous exposure of the
immune system to an APL derived from one of those epitopes (14,
15). To measure the degree of immunomodulation by the ordered
peptide EYYK on EAE induced by gpSCH known to contain the
dominant epitope MBPp72– 88, the experimental animals were injected on days 0 and 10 intradermally with a 0.1-ml emulsion of
0.1 mg ordered peptide in IFA. Ten days after the last injection,
rats were immunized with an emulsion of 0.5 mg gpSCH in CFA.
As seen in Fig. 6, injection of EYYK in IFA has a protective effect
when EAE is induced with an emulsion of gpSCH and CFA, as
compared with animals immunized with KYY peptide or IFA
alone ( p ⫽ 0.052).
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FIGURE 2. Stick models of the major contact residues of (A)
MBPp85–99 and (B) EYYK with HLA-DR2. The MBPp85–99 contains a
positively charged amino acid at the 90th position (histidine), whereas
EYYK consists of a negative charge (glutamic acid). Both phenylalanines
have been modified to tyrosines. The lysine residue remains unchanged.
residues lining the P4 pocket are Arg13, Phe26, Asp28, Gln70,
Ala71, and Tyr78. In rats, these residues are Gly, Leu, Ala, Gln,
Leu, and Ala, respectively. The presence of the large aromatic
residues in the human molecule may decrease the size of the P4
pocket. However, the HLA-DR2 P4 pocket can accommodate a
phenylalanine residue, so size is most likely not a factor. Based on
our modeling, the hydrophilic residues in the predominantly hydrophobic P4 binding pocket may anchor EYYK to the MHC.
Hydrophilic residues that could stabilize EYYK in the MHC cleft
are found in both molecules. The crystal structure of HLA-DR2
with MBPp85–99 showed that Gln70 is positioned over the P4
pocket (2). Gln70 is conserved in RT1.Dl, making it capable of
hydrophilic interactions with EYYK.
The EYYK ordered peptide inhibits the development of EAE
We tested the potential of the predicted peptide sequences to prevent or to revert the development of EAE in Lewis rats. Induction
of EAE in Lewis rats can be achieved by immunization with SCH,
whole MBP, or MBPps. Responses to MBPp68 – 88 are restricted by
the RT1.B MHC class II allele, and responses to MBPp85–99 are
restricted by the RT1.D allele (13). It has been demonstrated that
autoimmune responses to Ags containing more than one patho-
FIGURE 3. A molecular model of the binding region of (A) MBPp85–99
in the cleft of HLA-DR2 and (B) the modified EYYK peptide with HLADR2. The peptides are shown as stick models; the MHC class II molecule
is a rendered surface. The charged residues, His90 in MBPp85–99 and Glu in
EYYK, stick out of the cleft and are implicated in involvement with the
TCR. The second tyrosine of EYYK is embedded in the cleft, potentially
interacting with hydrophilic residues of HLA-DR2 and RT1.Dl. Gln70 of
the MHC ␤-chain is positioned over the embedded tyrosine and may contribute to binding via hydrophilic interactions.
The Journal of Immunology
We then tested whether this inhibition was specific for a blockade of anti-MBPp85–99 responses. In MBPp85–99-immunized
Lewis rats, we delivered a single dose of EYYK (a solution of 0.5
mg peptide in 1 ml 1 M PBS i.p.) on the day when the first signs
of clinical disease became apparent. As seen in Fig. 7, administration of EYYK suppresses the development of clinical disease in
experimental animals when compared with KYY- or PBS-treated
animals.
Discussion
In this report, we used MHC class II binding motifs of MBP to
design optimized analogs containing repetitive peptide sequences
that prevent and suppress EAE in Lewis rats.
One currently approved drug for MS therapy is a random synthetic amino acid copolymer, GA (poly Tyr, Glu, Ala, and Lys)
(16). Glatiramer suppresses EAE in rodents, slows the progression
of disability, and reduces the relapse rate in MS. The mechanism
of action is likely MHC blocking and TCR antagonism of
MBPp82–100-specific T cells (16). Binding-motif analyses of glatiramer after elution from HLA-DR molecules showed tyrosine at
the first anchor position followed by alanine in the subsequent
FIGURE 6. Ordered peptide EYYKEYYKEYYK prevents the induction of EAE in Lewis rats. Animals were injected with a 0.1-mg emulsion
of EYYKEYYKEYYK (f), KYYKYYKYYKYY (⽧), or PBS (F) in IFA
at day ⫺20 and again at day ⫺10. At day 0, the animals were challenged
with gpSCH in CFA. Results are expressed as mean disease score of a
group of six rats.
pockets (17). Although glatiramer is a random sequence of amino
acids and each batch has considerable variability, Fridkis-Hareli et
al. (17) have suggested that substitutions of valine or phenylalanine for tyrosine may improve glatiramer’s efficacy for binding to
the P1 pocket of HLA molecules.
APLs, in contrast to glatiramer, have a defined sequence and
change autoimmune responses in part by altering cytokine production in autoreactive T cells (18 –20). Previously, an APL of
MBPp85–99 was designed based on TCR-MHC contact residues
(21). Administration of MBPp87–99 (Lys913 Ala), an APL in
which the primary TCR contact residue is changed, reversed EAE
induced paralysis in the Lewis rat and reduced production of the
proinflammatory cytokine TNF-␣ (8). In a recent report, the minimal structural requirements for a peptide to tolerize animals with
ongoing EAE were determined (22). Using a panel of truncated
variants of the MBPp87–99, a 7-aa peptide, FKNIVPT, was found
to induce remission of EAE in the Lewis rat model. These results
indicate that only one MHC anchor and one TCR binding site are
essential for initiation of Ag-specific T cell tolerance (22). However, these peptides closely resemble the original self peptide and
have the potential to induce cross-reactive adverse side effects
when used for therapeutic purposes.
The peptides we designed contain repetitive sequences of three
(KYY) or four (EYYK and AYEK) amino acids and resemble the
FIGURE 5. Sequence alignment of HLA-DR2 (DRB1*1501) ␤-chain and rat RT1.Dl ␤-chain. Identical residues are shaded black; similar residues are
shaded gray. Residues indicated by an asterisk comprise the P4 binding pocket of HLA-DR2. The human P4 pocket consists of amino acids with larger
R groups, but is capable of accommodating a phenylalanine inside of it.
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FIGURE 4. Modified crystal structure of EYYK in the binding cleft of
HLA-DR2. All residues are shown as stick models. The binding residues of
EYYK are yellow, and those of HLA-DR2 are blue. Residues colored red
represent hydrophilic residues of HLA-DR2 in close proximity with the
tyrosine of EYYK. These residues could stabilize the polar head group of
tyrosine through hydrophilic interactions and/or hydrogen bonding. Gln70
is conserved in both rat RT1.Dl and human HLA-DR2.
2691
2692
MBPp85–99 core motif. The ordered peptide EYYKEYYKEYYK
is effective in preventing and reverting the development of EAE in
the Lewis rat. Although prevention of disease may be related to
immune deviation, the abrogation of disease progression by the
ordered peptide is correlated in vitro with competitive binding of
the peptide to the rat class II MHC. Computer modeling reveals
that the substitution of His90 by Glu may be an important alteration
from the original HFFK TCR-MHC motif. The presence of the
glutamate residue may affect the stability of TCR binding, whereas
the two tyrosines and the lysine may resemble the original FFK
structure.
Previously, MBPp86 –100 was oligomerized into a 16-mer. This
oligomerized peptide treated ongoing disease and prevented disease when administered before EAE induction. Interestingly, the
peptide also induced EAE in traditionally “resistant” strains (23).
In this study, we have minimized the number of amino acids required to treat and prevent disease. In addition, we have created a
peptide that is not homologous to MBP and does not induce EAE
or cause B or T cell cross-reactivity to MBP (data not shown).
EYYK and AYEK are tetrad repeats, similar to the native HFFK
structure. AYEK, a similar tetrad structure, resulted in partial abrogation of EAE (data not shown). Unlike AYEK or EYYK, KYY
is a triplet repeat and contains fewer HFFK-analogous binding
regions (Fig. 8). KYY did not bind MHC or prevent disease. This
observation suggests that there is a minimum of four residues per
repeat required for efficient APL binding.
The oligomerized MBPp86 –100 improved the response of low
avidity T cell clones (23). The repetitive structure may effectively
raise the local concentration of peptide. If the tyrosine residues of
EYYK are structurally predisposed to bind in pockets P3 and P4 of
the rat MHC class II, then EYYK contains three potential binding
regions (the first, second, and third repeat of EYYK). When EYYK
binds and then releases from the MHC cleft, two other EYYK
binding regions are immediately adjacent and can bind the MHC
molecule (Fig. 8). This would give the peptide a fast on/off rate and
a high avidity for binding.
Further studies should be conducted to determine the importance of the first amino acid of the repeat in abrogating EAE. In the
native HFFK sequence, histidine is first and carries a positive
charge (His90). KYY, which starts with another positively charged
amino acid, resulted in a slight increase in EAE scores. Electro-
FIGURE 8. The EYYK peptide can bind in multiple positions. The local concentration of EYYK is increased because the peptide has three potential binding sites. When one block of EYYK releases, two others are
immediately adjacent to bind MHC. This may raise the avidity of the interaction and result in stronger, sustained binding. In contrast, the KYY
peptide has only two binding sites.
static interactions between His90 and TCR residues may trigger a
nontolerogenic signal. Altering the charge of this residue can affect
the stimulatory capacity; when converted to a nonpolar alanine
(AYEK), partial abrogation of EAE is observed. Furthermore,
when changed to the negatively charged amino acid glutamate
(EYYK), there is a significant reduction in EAE scores.
The P4 pocket is a major anchor for the peptide to the MHC
cleft. Although the residues that make up the cleft differ between
human and rat MHC, the crystal structure of human HLA-DR2
suggests that tyrosine can act as an anchor residue in the P4 pocket
(2). The polar end group of tyrosine potentially interacts with
Gln71, a residue positioned over the P4 pocket in human and rat
MHC. The EYYK peptide was designed with the structure of rat
RT1.Dl in mind, where the P4 pocket is composed of mainly nonpolar residues that could interact with the aromatic portion of tyrosine. In humans, the P4 pocket consists of multiple polar and
charged residues. Despite structural studies suggesting that tyrosine will fit in the human P4 pocket, peptides designed for human use could be engineered with a polar residue. MBP analogs
that contain histidine, arginine, lysine, glutamine, or asparagine
instead of Phe92 bind the P4 pocket (2, 4).
The possibility of using crystal structures of HLA molecules to
design TCR-MHC antagonists should improve the pace of discovery for new therapies in autoimmune diseases. As the interactions
between MHC, peptide, and TCR are defined, disease- and genotype-specific APLs will become possible.
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FIGURE 7. Ordered peptide EYYKEYYKEYYK prevents the development of EAE in Lewis rats. Animals were injected with a 0.1-mg emulsion
of MBPp85–99 in CFA for EAE induction. When the clinical manifestations
of disease became apparent (day 10), a single i.p. dose of peptide
EYYKEYYKEYYK (f), KYYKYYKYYKYY (⽧), or PBS (F) was administered. Results are expressed as mean disease score of groups of six
animals. The data shown are representative of three experiments.
MHC-TCR BINDING MOTIF-BASED PEPTIDES TO TREAT EAE
The Journal of Immunology
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