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Suppression of Experimental Autoimmune Encephalomyelitis
Using Peptide Mimics of CD28
1
Mythily Srinivasan,* Ingrid E. Gienapp,
§
Scott S. Stuckman,
§
Connie J. Rogers,
§
Scott D. Jewell,
†
Pravin T. P. Kaumaya,*
‡
and Caroline C. Whitacre
2§
The B7:CD28/CTLA-4 costimulatory pathway plays a critical role in regulating the immune response and thus provides an ideal
target for therapeutic manipulation of autoimmune disease. Previous studies have shown that blockade of CD28 signaling by mAbs
can both prevent and exacerbate experimental autoimmune encephalomyelitis (EAE). In this study, we have designed two CD28
peptide mimics that selectively block B7:CD28 interactions. By surface plasmon resonance, both the end group-blocked CD28
peptide (EL-CD28) and its retro-inverso isomer (RI-CD28) compete effectively with the extracellular domain of CD28 for binding
to B7-1. Both the CD28 peptide mimics inhibited expansion of encephalitogenic T cells in vitro. A single administration of
EL-CD28 or RI-CD28 peptide significantly reduced disease severity in EAE. Importantly, we show that either CD28 peptide mimic
administered during acute disease dramatically improved clinical signs of EAE, suppressing ongoing disease. The ratio of CD80:
CD86 expression was significantly lower on CD4
ⴙ
and F4/80
ⴙ
spleen cells in CD28 peptide-treated mice. Peripheral deletion of
Ag-specific CD4
ⴙ
T cells occurs following in vivo blockade of CD28 with synthetic CD28 peptides. The Journal of Immunology,
2002, 169: 2180–2188.
Multiple sclerosis (MS)
3
is a demyelinating disease of
the CNS characterized by myelin damage accompa-
nied by inflammation and axonal severing. Experi-
mental autoimmune encephalomyelitis (EAE) shares many of the
clinical and histopathological features of MS and thus serves as a
useful animal model. Considerable evidence suggests a central role
for T cell-mediated immune responses in the pathogenesis of MS
and EAE (1).
Activation of T cells requires a primary signal delivered via the
TCR-CD3 complex interacting with a MHC-peptide complex on an
APC and a second costimulatory signal provided primarily by CD28
interacting with the B7 molecules on the APC (2). Signaling via
CD28 leads to transcriptional activation of several cytokine genes,
principally IL-2, and up-regulation of anti-apoptotic molecules such
as Bcl-x
L
. High levels of IL-2 are required during the expansion
phases of both primary and secondary T cell responses. Bcl-x
L
reg-
ulates proliferation and survival of naive T cells (3). CTLA-4, ex-
pressed on activated T cells, also binds the B7 ligands on the APC,
transmitting negative signals to terminate the immune response (4).
B7/CD28:CTLA-4 interactions play a critical role in the patho-
genesis and/or regulation of EAE and MS (5–7). Mice genetically
deficient in B7-1, B7-2, or CD28 are highly resistant to EAE (8).
Therapeutic intervention in the B7:CD28/CTLA-4 pathway has led
to varied results. Although administration of CTLA-4 Ig or anti-
B7-1 mAb prevented the induction of EAE, CTLA-4 Ig was not
effective in the treatment of established disease. Anti-B7-2 or anti-
CTLA-4 mAb treatment exacerbated the clinical course of EAE
(9–13). Interference with the delivery of negative signals via
CTLA-4 may account for the differences in clinical outcome in
these studies. Recently, Perrin et al. (14) have shown that treatment
with Fab of CD28 mAb that specifically blocks B7:CD28 interac-
tions ameliorates the clinical course of EAE.
Knowledge of the molecular topology of interacting surfaces
can be used to develop antagonists of protein-protein interactions.
For example, a peptide analog derived from the complementarity-
determining region (CDR)-3-like region of CD4 inhibits T cell
responses (15, 16). The premise is that the side-chain functional
groups of the key residues of the binding epitope can be transferred
to a much smaller fragment without loss of binding efficiency (17).
A surface binding pocket consisting of CC⬘or CDR-3 loops has
been shown to mediate the binding of CD4 to MHC class II and of
CD2 to LFA-3 (18). In the present study, we evaluated the ther-
apeutic efficacy of a peptide antagonist for interfering with B7:
CD28 interactions. Based on the differences in the kinetics of in-
teraction of CD28 and CTLA-4 with B7 ligands, we hypothesized
that a peptide derived from the ligand binding region of CD28 will
selectively block B7:CD28 interactions without affecting the
higher affinity B7:CTLA-4 interactions.
CD28 is a member of the Ig supergene family with a single
IgV-like domain (19, 20). The residues implicated in ligand bind-
ing include the conserved hydrophobic motif “MYPPPY” and the
adjacent charged residues, localized in the solvent-exposed CDR3-
like loop region of CD28 (19–21). Similar proline-rich sequences
in accessible regions of other globular proteins have been impli-
cated in protein-protein interactions (22). Recently, we identified
two biologically active CD28 peptide mimics derived from the
*Department of Microbiology, College of Biological Sciences, and Departments of
†
Pathology,
‡
Obstetrics and Gynecology, and
§
Molecular Virology, Immunology, and
Medical Genetics, College of Medicine and Public Health, Ohio State University,
Columbus, OH 43210
Received for publication August 1, 2001. Accepted for publication June 10, 2002.
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 Institutes of Health Grants R01
AI40302 (to P.T.P.K.) and R01 AI43376 (to C.C.W.) and Multiple Sclerosis Society
Grants RG3091 and RG3272 (to C.C.W.).
2
Address correspondence and reprint requests to Dr. Caroline C. Whitacre, Depart-
ment of Molecular Virology, Immunology, and Medical Genetics, Ohio State Uni-
versity, 2078 Graves Hall, 333 West Tenth Avenue, Columbus, OH 43210-1239.
E-mail address: whitacre.3@osu.edu
3
Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental auto-
immune encephalomyelitis; CDR, complementarity-determining region; MBP, mye-
lin basic protein; LNC, lymph node cell; PLP, proteolipid protein.
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00
CD28 CDR-3-like region. An end group-blocked CD28 peptide
(EL-CD28) and its retro-inverso isomer (RI-CD28) were synthe-
sized and characterized for their binding properties and biological
activity (23). The synthetic CD28 peptides exhibited similar ki-
netics of interaction as CD28 for binding the CD80 ligand. More-
over, the CD28 peptides were observed to interfere with the acti-
vation of encephalitogenic T cells in vitro (23).
In the present study, we investigated the potential of the peptide
mimics to suppress EAE in vivo. Our results indicate that treat-
ment with the CD28 peptides prevented EAE induction and ame-
liorated established disease. Suppression of EAE was greater with
the retro-inverso CD28 mimic than the L peptide analog of the
parent sequence. The observed protection was accompanied by a
decrease in the CD80:86 ratio on T cells and macrophages as well
as apoptosis of Ag-specific CD4
⫹
T cells.
Materials and Methods
Peptide design and synthesis
All L- and D- peptides corresponding to the CD28 CDR3-like region were
assembled by solid-phase peptide synthesis using F-moc/dicyclohexylcar-
bodiimide/hydroxybenzotriazole methodology on a fully automated pep-
tide synthesizer as described (23). The free L-CD28 peptide was assembled
on 4-methylbenzhydrylamine resin. The end-group blocked L-CD28 (EL-
CD28) peptide, the retro-inverso CD28 (RI-CD28) peptide and the control
peptides were assembled as peptide amides on Rink amide resin (Advanced
ChemTech, Louisville, KY). The free NH
2
group of the terminal amino
acid residue was acetylated with acetylimidazole and confirmed by a neg-
ative Ninhydrin test. The retro-inverso peptide was assembled in reverse
order with respect to the parent peptide using F-moc-D-amino acid deriv-
atives. The control peptides included an all Dpeptide, consisting of Damino
acids in the forward sequence, and a reverse L-peptide, consisting of L
amino acids in the reverse sequence as that of the parent peptide (Table I).
The peptides were purified by semipreparative reverse-phase HPLC (Vy-
dac, Hesperia, CA) and the identity of the purified peptide was confirmed
by matrix-assisted laser desorption/ionization time of flight mass
spectrometry.
Binding experiments
Competitive kinetic analyses between CD28-Ig or CTLA-4 Ig and the syn-
thetic CD28 peptides for binding the B7-1 ligand were conducted using the
BIAcore biosensor (Pharmacia Biosensor, Uppsala, Sweden) that employs
surface plasmon resonance for directly measuring intermolecular interac-
tions. CD80-Ig (282–368 resonance units (RU)) was immobilized indi-
rectly via anti-mouse Fc (1210–1390 RU) to the sensor chip as described
(24). Initially, the affinity constant of the interaction between CD28-Ig (the
extracellular domain of CD28 fused to mouse IgG2a, a gift from Dr. Y.
Liu, Ohio State University, Columbus, OH) or CTLA-4 Ig and CD80-Ig
was determined by direct kinetic analysis. Competition was assessed by
injecting a series of solutions containing a constant concentration of
CD28-Ig (3.2
M) or CTLA-4 Ig (0.8
M) and increasing concentrations
of EL-CD28 or RI-CD28 peptides. The CD80-Ig surface was regenerated
between injections by washing with 5 mM NaOH. As control, the solutions
were also injected onto an empty flow cell with no ligand immobilized.
Data was analyzed using BIAevaluation software 2.1 and BIAsimula-
tion software version 2.1 (Pharmacia Biosensor). Before kinetic analysis, a
zero baseline level was obtained by subtracting the background responses
from injection of the analytes through a control flow cell with no ligand
immobilized. In competitive kinetic experiments, when two analytes
(CD28 peptides and CD28-Ig) were injected at the same time and react
with the same site on the immobilized ligand (CD80-Ig), the observed
response is the sum of the contributions from both analytes. The binding
data was analyzed as previously described (24).
Antigens
Myelin basic protein (MBP) was extracted from guinea pig spinal cords
(Harlan Sprague Dawley, Indianapolis, IN) as previously described (25).
For EAE induction, MBP was further purified on a Sephadex G-50 column
eluted with 0.01N HCl. The purified protein was dialyzed against water and
lyophilized.
Induction of EAE and CD28 peptide treatment
Female B10.PL mice, 6–8 wk old, obtained from The Jackson Laboratory
(Bar Harbor, ME) were injected s.c. over four sites on the flank with 200
g of MBP in CFA containing 200
g killed Mycobacterium tuberculosis,
Jamaica strain. Pertussis toxin (List Biological Laboratories, Campbell,
CA), 150 ng in 0.2 ml of PBS, was given i.p. at the time of immunization
and 48 h later. Animals were observed daily for clinical signs and scored
as follows: 0, no clinical signs; 1, limp tail or waddling gait with tail
tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial
paralysis of one limb; 3, partial hind-limb paralysis; 3.5, full paralysis of
one limb with partial paralysis of the second limb; 4, full paralysis of two
limbs; 4.5 moribund; and 5, death. For treatment of EAE, mice were in-
jected i.v. with 500
g CD28 peptide (reconstituted in sterile PBS) either
on the day of MBP immunization or 14 days later.
Proliferation analysis
Vehicle-, synthetic CD28 peptide-, and control peptide-treated mice were
sacrificed either 10 or 26 days after immunization and in vitro proliferation
of lymphocytes was assessed as described (26). Briefly, single cell suspen-
sions prepared from spleens, mesenteric lymph nodes, and peripheral
lymph nodes (inguinal, axillary, brachial, cervical, popliteal) were cultured
in RPMI 1640 medium containing 10% FCS, 25 mM HEPES, 2 mM L-
glutamine, 50 U/ml penicillin, 50
g/ml streptomycin, and 5 ⫻10
⫺5
M
2-ME in round-bottom 96-well plates (4 ⫻10
5
cells/well) and stimulated
with MBP (40
g/ml) or medium for 72 h, including a final 18-h pulse with
[
3
H]thymidine. Cultures were harvested onto glass-fiber mats using a Ska-
tron harvester (Skatron, Sterling, VA) and counted by liquid scintillation
using a Wallac betaplate (Wallac, Rockville, MD). The results are ex-
pressed as ⌬cpm (mean cpm of cultures with Ag ⫺mean cpm of cultures
with medium alone) ⫾SE.
ELISPOT analysis for cytokine-producing cells
ELISPOT analysis was performed as described (26). Briefly, 96-well uni-
filter plates (Polyfiltronics, Rockland, MD) were coated with anti-mouse
IL-2 (clone JES6-1A12) or anti-mouse IFN-
␥
(clone R46A2) (BD PharM-
ingen, San Diego, CA) at 4
g/ml overnight at 4°C. After blocking with
1% BSA in DMEM for2hatroom temperature, isolated lymph node cells
(LNC) (5 ⫻10
5
cells/well) were resuspended in HL-1 medium (BioWhit-
taker, Walkersville, MD) and added to the plates in triplicate in the pres-
ence or absence of 40
g/ml MBP. Following incubation for 24 h at 37°C,
the plates were washed with PBS containing Tween 20 (1:2000) and cy-
tokine-specific secondary Abs, biotinylated anti mouse-IL-2 (clone JES6-
5H4) or biotinylated anti-mouse IFN-
␥
(clone XMG1.2) (BD PharMin-
gen), 2
g/ml, were added. After overnight incubation at 4°C, the plates
were incubated with goat anti-biotin Ab conjugated to alkaline phosphatase
(Vector Laboratories, Burlingame, CA) for2hatroom temperature. The
spots were visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/
nitroblue tetrazolium phosphatase substrate (Kirkegaard & Perry Labora-
tories, Gaithersburg, MD). Image analysis of ELISPOT plates was per-
formed using the KS ELISPOT system (Zeiss, Oberkochen, Germany).
Data are expressed as the frequency of MBP-responsive cytokine produc-
ing cells per million ⫾SEM.
Detection of apoptosis
The terminal deoxynucleotidyl transferase assay was performed as de-
scribed (27). LNC (10
6
) from peptide- and control-treated mice were
stained with PE-labeled anti-mouse CD4 (L3T4, clone RM 4-4; BD
PharMingen) for 30 min at 4°C. After washing, the cells were fixed in 4%
paraformaldehyde for 30 min at room temperature, followed by permeabi-
lization with 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min
at 4°C. The cells were then incubated with FITC-labeled TdT reaction
mixture for 60 min at 37°C using an in situ cell death detection kit (Boehr-
inger Mannheim, Mannheim, Germany) according to manufacturer’s in-
structions. As a positive control, cells were incubated in DNase (1
g/ml)
for 10 min at room temperature before incubation with TdT. A negative
Table I. Amino acid sequences of synthetic CD28 and control peptides
Abbreviation Sequence
a
L-CD28 NH
2
KIEFMYPPPYLDNERSNGTICOOH
EL-CD28 CH
3
CO-L[KIEFMYPPPYLDNERSNGTI]-CONH
2
RI-CD28 CH
3
CO-D[ITGNSRENDLYPPPYMFEIK]-CONH
2
D-CD28 CH
3
CO-D[KIEFMYPPPYLDNERSNGTI]-CONH
2
RL-CD28 CH
3
CO-L[ITGNSRENDLYPPPYMFEIK]-CONH
2
a
Italicized Land Drefer to Land Damino acid residues, respectively.
2181The Journal of Immunology
control consisted of cells incubated without TdT. Cells were then washed,
resuspended in PBS, and analyzed by flow cytometry.
mAb Staining and flow cytometric analysis
Single cell suspensions of LNC and spleen cells (0.5 ⫻10
6
) were incu-
bated with 1
g of PE-conjugated anti-CD80 (clone 16-10A1), anti-CD86
(clone GL-1), anti-CD28 (clone 37.51), FITC-conjugated anti-CD11c
(clone HL3), anti-CD19 (clone 1D3), or anti-CD4 (clone RM4-5) for 30
min at 4°C. For macrophages, a three-step staining procedure was used.
LNC and splenocytes were incubated with 1
g of anti-F4/80 (clone C1:
A3-1) (R&D Systems, Minneapolis, MN) followed by washing and incu-
bation in FITC-conjugated anti-rat IgG2b (clone A95-1) and PE-conju-
gated anti-CD80 or anti-CD86. All staining reactions were accompanied by
appropriately matched isotype control reactions. All mAbs (unless speci-
fied) were purchased from BD PharMingen. Labeled cells were washed,
resuspended in 1% paraformaldehyde, and analyzed by flow cytometry on
an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA).
Statistical analysis
For the mean clinical score, proliferation, and TUNEL assays, a one-way
ANOVA with Tukey’s posthoc was performed to determine the differences
between the groups. Results were considered statistically significant at
p⬍0.05.
Results
Peptide design and synthesis
Mutagenesis and molecular modeling of the extracellular domain
of CD28 were used to design a 20 residue CD28 peptide mimic
encompassing the “MYPPPY”motif and the delineated flanking
sequence (19–21). The charges at the end groups of the synthetic
CD28 peptide were blocked (EL-CD28) to resemble the termini of
the ligand binding epitope of the parent CD28 molecule (Table I).
In addition, this modification stabilizes the secondary structure,
which may be important for its functional interaction. A retro-
inverso isomer of the CD28 peptide mimic (RI-CD28) was also
synthesized. The use of Damino acids results in inverted chirality
and the reversed order of amide bonds (-NHCO- instead of
-CONH-) creates an analog that regenerates both the planarity of
peptide bonds and spatial orientation of side chains closely related
to that of the original peptide (28).
CD28 peptides compete with CD28 extracellular domain to bind
CD80-Ig
Kinetic analysis of the sensograms for the binding of CD28-Ig or
CTLA-4 Ig to CD80-Ig yielded a K
d
of 3.17 and 0.8
M, respec-
tively (data not shown). Competitive binding assays were per-
formed by injecting a mixture of CD28-Ig (3.2
M) or CTLA-Ig
(0.8
M) at a constant concentration together with increasing con-
centrations of CD28 peptides as analytes over immobilized CD80-
Ig. Representative sensograms of the competition between
CD28-Ig or CTLA-4Ig and EL-CD28 are shown in Fig. 1. The top
curve represents the interaction of CD28-Ig (Fig. 1A) or CTLA-4Ig
(Fig. 1B) alone with the CD80-Ig. In Fig. 1A, the response level
gradually decreases as the concentration of peptide mimic in-
creases, indicating that the EL-CD28 peptide competes with
CD28-Ig for binding CD80-Ig. No appreciable decrease in re-
sponse units was observed when EL-CD28 peptide at 51.6–804
M was competed with CTLA-4-Ig for binding the CD80-Ig (Fig.
1B). A similar response was observed with RI-CD28 peptide (23).
However, a decrease of 168 RU was observed when EL-CD28 at
1237
M or 3 mg/ml was competed with CTLA-4Ig (Fig. 1B). The
RI-CD28 peptide also exhibited a decrease of 189 RU at a 1237
M concentration (23), suggesting that at very high concentra-
tions, both the EL-CD28 and the RI-CD28 peptides may compete
weakly with the CTLA-4 fusion protein for binding the B7-1
ligand.
Costimulatory blockade in vivo by CD28 peptides suppresses
clinical EAE in B10.PL mice
To evaluate the biological activity of synthetic CD28 peptide mim-
ics during Ag priming, B10.PL mice were immunized with MBP
and received i.v. a single injection of EL-CD28, RI-CD28, control
CD28 peptides, or PBS on the day of immunization. The vehicle-
treated and control (L-CD28, RL-CD28, and D-CD28) peptide-
treated mice exhibited maximum disease incidence of 100, 100,
91.5, and 91.5% and mean cumulative scores of 50.2, 47.6, 44.9,
and 52.3, respectively (Table II). In contrast, significant suppres-
sion of EAE was observed in mice treated with EL-CD28 and
RI-CD28 peptides with a mean cumulative score of 29.2 and 19.4,
respectively (Fig. 2, Aand B). We also observed a trend of de-
creased mean maximal score for mice treated with EL-CD28 and
RI-CD28 peptides compared with controls (Table II). The RI-
CD28 peptide produced greater protection that lasted for the du-
ration of the observation period (Fig. 2).
Synthetic CD28 peptide treatment ameliorates established EAE
We assessed the ability of CD28 peptide mimics to suppress on-
going established clinical disease. Mice immunized with MBP in
CFA and showing clinical signs of disease by 14 days postimmu-
nization were randomly distributed into six groups, such that the
mean clinical score for each group was approximately the same.
Groups of mice were injected i.v. with PBS or 500
gofEL-
CD28, RI-CD28, or control CD28 peptides. The mean clinical
score of vehicle-treated mice continued to increase, reaching a
maximum of 3.4 on day 20. Similarly the disease continued to
FIGURE 1. Competition between EL-CD28 and CD28Ig or CTLA-4 Ig
for binding CD80-Ig. Overlay of sensograms obtained from injection of a
mixture of (A) CD28-Ig (3.2
M) or (B) CTLA-4 Ig (0.8
M) and EL-
CD28 peptide at varying concentrations (10.3–1237
M) at 5
l/min over
aflow cell with bound CD80-Ig. In both sensograms, the top curve rep-
resents the binding of the respective fusion protein alone in the absence of
the competing peptide. The response of (A) CD28-Ig binding decreases
with increasing concentrations of EL-CD28 (10.3–412
M). The response
of (B) CTLA-4 Ig binding decreases appreciably (168 RU) only at the
highest concentration (1237
M) of EL-CD28 peptide.
2182 SUPPRESSION OF EAE WITH CD28 PEPTIDES
progress in mice treated with control CD28 peptides, viz, L-CD28,
RL-CD28 and D-CD28, reaching a mean maximal clinical score of
3.8 (day 18), 3.4 (day 16), and 3.7 (day 16), respectively (Table
III). In contrast, mice treated with EL-CD28 or RI-CD28 peptides
showed clinical improvement from day 16 throughout the obser-
vation period (26 days postimmunization) (Fig. 3, Aand B). These
results demonstrate that blockade of CD28 costimulation can at-
tenuate the progression of ongoing disease in EAE.
CD28 peptides suppress Ag-specific proliferation and IL-2
production by MBP-primed T cells
To determine the effect of costimulatory blockade in vivo by CD28
peptides, we assessed the proliferation of MBP-primed T cells
upon restimulation in vitro. Splenocytes from mice with EAE
treated with PBS, EL-CD28, RI-CD28, or control CD28 peptide
analogs on the day of immunization were collected 10 days
postimmunization and stimulated in vitro with MBP. The prolif-
erative response to MBP, but not an irrelevant Ag, tetanus toxoid,
was significantly decreased in splenocytes from mice treated with
EL-CD28 or RI-CD28 peptide as compared with PBS-treated mice
(Fig. 4, Aand B). We observed a relatively high degree of variance
in the MBP-specific proliferative response of spleen cells from
D-CD28 peptide and L-CD28 peptide-treated mice, although this
variance was not observed in the response from RL-CD28-treated
mice (Fig. 4, Aand C). Splenocytes from mice treated with RI-
CD28 14 days after MBP immunization also exhibited reduced
proliferation in response to MBP as compared with controls (Fig.
4C). Separate groups of animals were sacrificed 26 days after im-
munization and MBP-specific proliferative responses remained
suppressed (data not shown).
We evaluated the effect of synthetic CD28 peptides on T cell
cytokine secretion. Mice were treated with CD28 peptides on the
day of immunization and ELISPOT was used to assess in vitro
cytokine production by LNC upon restimulation with MBP. The
frequency of IL-2-secreting LNC decreased significantly with EL-
CD28 and RI-CD28 treatment as compared with PBS and control
peptide treatment (Fig. 4D). The frequency of IFN-
␥
secreting
LNC and splenocytes at this time was equivalent in all cultures
(data not shown). Perrin et al. (12) have also reported similar find-
ings of decreased IL-2 and unaltered IFN-
␥
production after co-
stimulatory blockade of MBP-specific T cells.
In vivo blockade of CD28 costimulation induces apoptosis in
Ag-specific T cells
Our previous studies demonstrated that MBP NAc1-11 specific
V

8.2
⫹
T cells showed decreased proliferation when stimulated in
vitro with MBP peptide in the presence of CD28 peptides (23).
One possibility to explain this in vitro observation and the sup-
pression of EAE observed in this study is that peptide treatment
induces apoptosis of disease-relevant T cells. We measured the
DNA strand breaks in CD4
⫹
lymphocytes from peptide-treated
mice using enzymatic labeling of nicked DNA. A significantly
higher percentage of CD4
⫹
T cells were apoptotic in mice treated
on the day of immunization with RI-CD28 peptide (16%) as com-
pared with PBS-treated mice (8.7%) or control peptide-treated
mice (10.2%) (Fig. 5A). Similarly, an increase in the percent of
apoptotic cells was observed in mice treated with RI-CD28 peptide
(8.3%) during acute disease as compared with vehicle (6%) and
control peptide-treated mice (2.6%), although the difference did
not reach statistical significance (Fig. 5B). Fig. 5, Cand D, are
representative histograms showing increased apoptosis of LNC
from mice treated with RI-CD28 peptide on days 0 and 14, re-
spectively. This suggests that the CD28 peptide mimic engages the
FIGURE 2. CD28 peptide treatment inhibits EAE. B10.PL mice were
immunized for EAE as described in Materials and Methods.A, The data
are presented as the mean clinical score per group over time. B, The se-
verity of EAE is depicted as the mean score per day, which is the cumu-
lative score for each animal divided by the number of days that animal was
observed. The mean of these values was calculated for each group. EL-
CD28- and RI-CD28 peptide-treated mice had significantly reduced scores
(⫹,p⬍0.05; ⴱ,p⬍0.01 by ANOVA) as compared with control mice. No
significant differences were observed between PBS- and control CD28 pep-
tide-treated mice. Data represents pooled values from two experiments.
Table II. EAE clinical signs in CD28 peptide-treated mice treated on the day of MBP immunization
Group No. of
Animals Mean Cumulative
Score Mean Score Per
Day
a
Mean Maximal
Score
b
PBS 11 50.23 ⫾4.71 1.93 ⫾0.18 4.05 ⫾0.17
L-CD28 6 47.6 ⫾2.02 1.83 ⫾0.09 4.2 ⫾0.2
EL-CD28 10 29.2 ⫾7.27
c
1.12 ⫾0.28
c
2.55 ⫾0.47
RI-CD28 9 19.39 ⫾6.57
d
0.74 ⫾0.25
d
2.05 ⫾0.55
RL-CD28 6 44.9 ⫾3.62 1.73 ⫾0.14 3.8 ⫾0.2
D-CD28 6 52.25 ⫾9.5 2.01 ⫾0.37 3.5 ⫾0.67
a
Average of the cumulative clinical score of each animal divided by the total number of days.
b
Average of the highest score of all mice in each group.
c
p⬍0.05 by ANOVA.
d
p⬍0.01 by ANOVA.
2183The Journal of Immunology
B7-ligands on the APC and effectively blocks the costimulatory
signal required for sustained activation and long-term survival of
CD4
⫹
T cells.
Treatment with CD28 peptides down-regulates the CD80:CD86
ratio in CD4
⫹
T cells and F4/80
⫹
macrophages
Previous studies on the in vivo expression of B7 during the course
of EAE have shown that CD80 is up-regulated on spleen cells
during clinical disease (29). To investigate whether the observed
protection following treatment with CD28 peptides is a result of
reduced costimulation, we analyzed the expression of CD80,
CD86, and CD28 on T cells and APCs in the spleen.
Consistent with previous reports, lymphoid cells from naive
B10.PL mice express higher levels of CD86 than CD80 (30–32).
Immunization with MBP results in the up-regulation of CD80 on
both CD4
⫹
LNC and spleen cells. However, CD86 expression is
down-regulated on CD4
⫹
spleen cells from mice with EAE as
FIGURE 3. CD28 peptide mimics attenuate established EAE. B10. PL
mice were immunized for EAE as in Fig. 2. A, Mice that received EL-
CD28 or RI-CD28 peptide 14 days postimmunization showed significant
clinical improvement. B, The clinical severity was significantly reduced in
EL-CD28- and RI-CD28- (⫹,p⬍0.05 by ANOVA) treated mice as com-
pared with PBS-treated mice. There were no significant differences be-
tween control peptide and PBS-treated mice. Data represents pooled values
from two experiments.
FIGURE 4. Proliferative responses and IL-2-secreting cells are reduced
in mice treated in vivo with CD28 peptides. A, Splenocytes collected 10
days postimmunization from mice treated with RI-CD28 and EL-CD28
peptide on the day of immunization showed significant inhibition of pro-
liferation in response to MBP. ⴙ,p⬍0.05 as compared with PBS-, RL-
CD28-, and L-CD28 peptide-treated mice; ⴱ,p⬍0.01 compared with
PBS-treated and all control peptide-treated mice by ANOVA. B, Spleno-
cytes from peptide-treated mice showed no differences in response to tet-
anus toxoid. C, Splenocytes collected 16 days postimmunization from mice
treated with RI-CD28 peptide during acute disease showed significant in-
hibition of proliferation in response to MBP compared with cells from
PBS- or control peptide-treated mice. D, The frequency of IL-2-producing
cells (as measured by ELISPOT) in response to MBP stimulation is de-
creased in mice treated on the day of immunization with EL-CD28 and
RI-CD28 peptide as compared with PBS-treated and L-CD28- and RL-
CD28 peptide-treated mice. (⫹,p⬍0.05; ⴱ,p⬍0.01 compared with
PBS-, L-CD28-, and RL-CD28 peptide-treated mice; @, p⬍0.01 as com-
pared with PBS-treated mice by ANOVA).
Table III. EAE clinical signs in CD28 peptide-treated mice treated during acute disease
a
Group No. of
Animals Mean Cumulative
Score Mean Score Per
Day
b
Mean Maximal
Score
c
PBS 12 37.56 ⫾3.1 3.13 ⫾1.04 4 ⫾0.25
L-CD28 6 17.06 ⫾2.67 2.84 ⫾1.5 4.17 ⫾0.17
EL-CD28 12 19.24 ⫾2.82
d
1.92 ⫾0.98
d
2.92 ⫾0.34
RI-CD28 11 19.38 ⫾2.47
d
1.76 ⫾0.87
d
3.23 ⫾0.35
RL-CD28 5 12.82 ⫾3.97 2.56 ⫾0.91 3.7 ⫾0.3
D-CD28 5 13 ⫾3.96 2.6 ⫾0.92 3.9 ⫾0.4
a
Fourteen days postimmunization; disease scores included beginning from time of treatment initiation.
b
Average of the cumulative clinical score of each animal divided by the total number of days.
c
Average of the highest score of all mice in each group.
d
p⬍0.05 by ANOVA.
2184 SUPPRESSION OF EAE WITH CD28 PEPTIDES
compared with naive animals. The CD80 expression is only mildly
elevated in CD4
⫹
spleen cells of mice treated with RI-CD28 pep-
tide with CD86 expression at levels similar to that of unimmunized
animals (Fig. 6A). The difference in the expression of CD80 was
less marked between EL-CD28 peptide and control peptide-treated
mice. Thus, the CD80/CD86 ratio was significantly reduced in
CD4
⫹
spleen cells of RI-CD28 peptide-treated mice relative to
control or PBS-treated mice (Fig. 6, C–F). A lowered CD80:CD86
ratio was observed in EL-CD28 and RI-CD28 peptide-treated mice
even at 26 days postimmunization (data not shown). In general,
CD28 expression was lower in CD4
⫹
LNC and splenocytes from
mice treated with EL-CD28 or RI-CD28 peptide as compared with
PBS-treated mice.
F4/80
⫹
spleen cells from naive mice expressed higher levels of
CD86 than CD80, as previously reported (29). Following immu-
nization with MBP, the expression of CD80 was up-regulated on
F4/80
⫹
macrophages in all groups of mice. However, the F4/80
⫹
macrophages in the EL-CD28-treated mice had higher CD86 ex-
pression than in the control groups. Thus, the CD80/CD86 ratio
was significantly reduced in F4/80
⫹
macrophages in the EL-CD28
peptide-treated mice relative to the control peptide or PBS-treated
mice (Fig. 6B). We performed similar analyses for CD11c
⫹
den-
dritic cells and CD19
⫹
B lymphocytes. No differences in CD80:
CD86 ratios were observed in either cell type (data not shown).
Interestingly, we observed a decrease in the number of CD11c
⫹
,
but not CD19
⫹
, spleen cells in the EL-CD28 peptide-treated mice
relative to the PBS-treated controls (data not shown).
Discussion
CD28 and CTLA-4 on the T cell surface bind the same ligands
CD80 and CD86 on APC. However, CTLA4 has a faster on-rate of
binding and higher avidity than CD28 for both ligands (33, 34).
We took advantage of the differential binding kinetics in the design
of peptides to selectively block B7:CD28 interactions. The syn-
thetic peptides likely produce a steric hindrance preventing the cell
surface CD28 from binding the B7 ligands. This likely reduces the
degree of CD28 aggregation or “receptor capping”required for
signal transduction and T cell activation (35). The cell surface
CTLA-4 can still potentially down-regulate the immune response
by overriding the competition from the synthetic CD28 peptide
due to its higher affinity for the same ligands.
The highly conserved nature of the hydrophobic motif in CD28
and its localization in the solvent exposed CDR3-like region
strongly suggests a functional significance (21). Mutagenesis of
the “MYPPPYLDN”resulted in loss of binding of CD28 with B7
ligands without affecting the cell surface expression of these moi-
eties (20). Rather than providing a structurally defined complex,
the proline-rich regions in cytoplasmic proteins are thought to play
a role in bringing proteins together such that subsequent interac-
tions are probable. Typically, these polyproline sequences adopt a
polyproline type II helical conformation, an extended structure
with three residues per turn (22). Structural characterization by
circular dichroism showed that the synthetic CD28 peptide mimics
adopt a typical spectrum of a polyproline type II helix (23).
In addition to structural integrity, the biological activity of the
peptide mimics depends on the duration of stability. Marini et al.
(16) reported that a synthetic CD4 peptide, effective in inhibiting
the clinical signs of EAE in the SJL mouse, had a half-life of 45
min (16). Both the EL-CD28 and RI-CD28 peptides, when ex-
posed to mouse serum, were stable in vitro for over 4 wk as de-
termined by HPLC (data not shown).
We have previously shown that the CD28 peptide mimics bind
B7-1 with low affinity and fast kinetics (23). Significantly, the
synthetic CD28 peptide mimics effectively compete with CD28-Ig
to bind B7-1 ligands, indicating that the selected peptide sequence
represents a ligand binding epitope of CD28. Competitive kinetic
studies showed that the CD28 peptide mimics exhibit a much
lower affinity than the CTLA-4 Ig for the B7-1 ligand. Collec-
tively, these results suggest that the CD28 peptides carry a greater
potential to selectively block B7:CD28 interactions while main-
taining the higher affinity B7:CTLA-4 interactions largely intact.
Treatment with synthetic CD28 mimic at the time of immuni-
zation protects B10.PL mice from EAE. The retro-inverso isomer
(RI-CD28) inhibited EAE development more effectively, with the
treated mice exhibiting lower disease incidence and significantly
decreased clinical severity than the end group-blocked parent pep-
tide. This difference in activity may be attributed to a decreased
susceptibility of a peptide composed of D-amino acids to proteases
in vivo. Furthermore, the lack of protection following treatment
with L-CD28 peptide can be attributed to rapid proteolytic cleav-
age in vivo of peptide acids by amino and carboxyl-peptidases.
Importantly, a single administration of synthetic CD28 peptide
mimic ameliorated ongoing disease in B10.PL mice. The clinical
signs of EAE were dramatically improved within 24–48hofad-
ministration of RI-CD28 or EL-CD28 peptide. All mice treated
with the CD28 peptide mimics showed a decrease in clinical score
throughout the period of observation, but there was not complete
clinical recovery.
If CD80 and CD86 are able to direct T cell differentiation and
activation, the outcome of an immune response will depend upon
the level of expression of the two molecules on the APC. Our
observation of increased expression of B7-1 relative to B7-2 on
splenocytes in mice immunized for EAE is consistent with the
studies of Karandikar et al. (29) who reported similar findings in
FIGURE 5. CD28 peptide treatment in vivo induces apoptosis in T
cells. Apoptosis among CD4
⫹
cells was assayed by TUNEL as described
in Materials and Methods.A, A higher percentage of CD4
⫹
LNC was
apoptotic in mice treated with RI-CD28 peptide on the day of immuniza-
tion (⫹,p⬍0.05 by one-way ANOVA) vs PBS-treated mice (n⫽3). B,
A greater percent apoptosis was also observed in CD4
⫹
LNC collected 16
days postimmunization from mice treated with RI-CD28 peptide on day 14
(n⫽3). Cand D, Representative histograms showing an increase in
TUNEL-positive cells in mice treated with RI-CD28 peptide on the day of
immunization (day 0) (C) or after disease development (day 14) (D).
2185The Journal of Immunology
proteolipid protein (PLP) peptide-induced EAE. However, in con-
trast to their studies, we observed a similar trend of increased B7-1
expression in peripheral LNC as well. This difference may be at-
tributed to different genetic backgrounds of the mice or the method
of EAE induction (PLP vs MBP). CD4
⫹
splenocytes and LNC of
mice treated with the RI-CD28 peptide exhibited significantly
lower B7-1 expression relative to B7-2 during both the initial and
late effector phases of EAE. These data, together with a significant
decrease in the frequency of IL-2-secreting cells and cell surface
expression of CD25 (data not shown), suggest that there are fewer
activated encephalitogenic T cells in the periphery of mice treated
with RI-CD28 peptide. Collectively, our findings can be viewed as
FIGURE 6. CD28 peptide treatment alters the
expression of CD80 and CD86 on CD4
⫹
T cells
and F4/80
⫹
macrophages following induction of
EAE. A significant decrease in the CD80:CD86 ra-
tio is observed in (A) CD4
⫹
splenocytes from mice
treated with RI-CD28 peptide and (B) macrophages
from mice treated with EL-CD28 peptide on the
day of immunization relative to PBS-treated con-
trols (ⴱ,p⬍0.05). Representative histograms
showing CD80 (Cand E) and CD86 (Dand F)
expression on CD4
⫹
splenocytes collected 10 days
postimmunization from PBS (Cand D)orRI-
CD28 peptide-treated mice (Eand F) on the day of
immunization. The figures indicate the mean chan-
nel fluorescence intensity of the histograms. ⌬MFI
indicates detectable surface expression above back-
ground (light gray line).
2186 SUPPRESSION OF EAE WITH CD28 PEPTIDES
supportive of the hypothesis proposed by Cross et al. (30) that T
cells expressing B7-1 preferentially migrate to the CNS during
acute EAE.
Infiltration of T cells alone into the CNS is not sufficient for
induction of clinical EAE (36). It has been suggested that the in-
filtrating T cells recruit peripheral macrophages, which eventually
cause tissue damage. The number of infiltrating macrophages cor-
relates well with disease and the depletion of peripheral macro-
phages prevents EAE (36–38). Although CD86 is constitutively
expressed on a variety of APCs, the expression of both CD80 and
CD86 is elevated upon activation (2). Increased expression of
CD80 on APCs is correlated with disease progression in EAE (30,
31). Consistently, we observed elevated levels of CD80 on mac-
rophages in all groups of mice. However, CD86 expression was
not down-regulated on F4/80
⫹
macrophages in the EL-CD28 pep-
tide-treated mice, resulting in a significantly decreased CD80:
CD86 ratio in these mice. In this context, it is pertinent to note that
in mice, CTLA-4 has been shown to preferentially bind CD86
(39). Furthermore, CTLA-4 ligation on activated CD4
⫹
T cells has
been shown to induce apoptosis (40). These observations, together
with the data presented in this study, suggest that the CD80:CD86
ratio on CD4
⫹
T cells and macrophages, rather than the expression
of each molecule alone, is a dynamic factor that plays a significant
role in determining the pathogenic cell response following Ag
stimulation.
Previously, Kearney et al. (41) have shown that in vivo treat-
ment with a combination of anti-B7-1 and anti-B7-2 mAbs
blocked clonal expansion with subsequent loss of these cells pre-
sumably due to programmed cell death. Our observation of in-
creased apoptosis of CD4
⫹
T cells from the lymph nodes of mice
treated with RI-CD28 peptide suggests clonal deletion as the
mechanism of protection against EAE. This is supported by the
absence of up-regulation of CD25 and CD28 on CD4
⫹
T cells
from the peripheral lymphoid organs of CD28 peptide-treated mice
as compared with the activated phenotype of CD4
⫹
T cells from
vehicle-treated mice.
The therapeutic potential of CD28 blockade in autoimmune dis-
eases is exemplified by the fact that within a decade of demon-
stration of its potential immunosuppressive effects, CTLA4-Ig has
entered phase I clinical trials for the treatment of psoriasis (42).
Perrin et al. (14) have shown that specific blockade of CD28 sig-
naling induced less severe disease in PLP-induced EAE in PL ⫻
SJL F
1
mice. Whereas systemic administration of CTLA-4 Ig had
minimal effect, local CNS delivery of CTL4-Ig using a nonrepli-
cative adenoviral vector has been shown to ameliorate ongoing
EAE (12, 43). Taken together, our data and those of others suggest
that short-term CD28 blockade may provide a means to ameliorate
an established autoimmune disease. The advantage of small pep-
tide-based therapeutics over mAbs lies in their lack of immuno-
genicity with the potential for use over long periods. In addition,
the peptides have a substantially lower m.w. than the Abs or fusion
proteins and perhaps have greater accessibility to the tissues of
the CNS.
In conclusion, we have integrated the results of mutagenesis
experiments, binding kinetics, molecular modeling, and structural
characterization of T cell costimulatory molecules together with
peptidomimetics in the design of two biologically active peptide
mimics that selectively block CD28 costimulation in vivo. The
potential therapeutic application of these peptides can be extended
to most T cell-mediated autoimmune disorders and graft-vs-host
disease where the aim is to down-regulate T cell responses and
accelerate recovery.
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2188 SUPPRESSION OF EAE WITH CD28 PEPTIDES
