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ImmuLogic Pharmaceutical Corporation, Waltham, MA 02154
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most widely used animal model of MS is experimental autoimmune encephalomyelitis (EAE), in which disease is induced by immunization with myelin proteins in adjuvant. EAE has been induced with MBP, PLP, and MOG (10, 11, 12, 13, 14), and the syndromes that develop bear varying degrees of resemblance to human MS. These models have been used to test the feasibility of treatment of MS with soluble peptides containing major T cell epitopes of the inducing myelin Ag (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). MBP-, PLP-, and MOG-induced EAE all have been prevented or treated postdisease induction by administration of MBP, PLP, or MOG peptides, respectively. These experiments suggest that it may be possible to develop a therapy for human MS based on T cell epitope-containing myelin peptides.
In several different experimental systems, regulatory cells specific for one Ag have been observed to down-regulate responsiveness to a second Ag in a bystander fashion (20, 25, 26, 27, 28). In all of these experiments, pretreatment with the first or tolerizing Ag induced a suppressor or Th2 response, and this population was then able to negatively influence the outcome of the subsequent priming event to the second Ag. This down-regulation of priming appears to depend on recognition of the tolerizing Ag and the priming Ag occurring in the same microenvironment, or on the surface of the same APC, a phenomenon known as linked recognition. In the EAE model, the tolerizing Ag has been either an exogenous, non-self protein (26) or a myelin protein distinct from that used to induce EAE (20, 25). In either case, subsequent disease induction with the priming myelin Ag was blocked. However, in the human autoimmune disease situation, the opportunity to intervene occurs in the postpriming phase of the disease. It is therefore important to determine whether the tolerance that can be induced to single Ags after disease induction would mediate bystander effects and down-regulate other preprimed encephalitogenic responses via a linked recognition type of event.
EAE induced with a homogenate of spinal cord has been used as a model of an antigenically complex disease in which peptide therapy can be assessed. Indeed, it has been shown that a peptide derived from a single myelin protein will treat EAE in this model (17, 24, and D. Smilek, unpublished observation). However, it could not be demonstrated that more than one myelin component in the homogenate had encephalitogenic activity in the mouse strains used in these experiments. Recently, adoptive transfer models of EAE have been used in an attempt to address the question as well, and the conclusions have been contradictory (21, 29).
We describe in this work a novel EAE model in which two different myelin proteins, MBP and MOG, administered in combination, induce a more severe form of EAE than that generated by either protein alone. In this model, lymphocytes specific for each of the two proteins appear to act together to produce the disease state. Furthermore, we show that this disease can be reduced significantly postinduction by parenteral administration of myelin epitope-containing peptides, either from one or both of the disease-inducing proteins. The results suggest that it may be possible to down-regulate a complex autoimmune disease by parenteral administration of T cell epitope peptides, even if the T cell response to only one of the initiating Ags in that disease has been identified and targeted for therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were initiated with 8- to 12-wk-old female (PL/J x SJL)F1 mice purchased from The Jackson Laboratory (Bar Harbor, ME), and housed in microisolator cages in the animal facility at ImmuLogic Pharmaceutical (Waltham, MA).
Peptides and proteins
MBP Ac1–11[4Y] (ASQYRPSQRHG), and the peptides corresponding to human MOG amino acids 1–20, 11–30, 21–40, 31–50, 41–60, 51–70, 61–80, 71–90, 81–100, 91–110, 101–120, and 111–130 were prepared by standard 9-fluorenylmethoxycarbonyl chemistry. The peptide corresponding to human MOG amino acids 41–60 was also synthesized using tert-butyloxycarbonyl chemistry, which resulted in higher peptide yields. Peptides were purified by reverse-phase HPLC (>80%), and amino acid composition of each peptide was confirmed by amino acid analysis.
A truncated form of human recombinant MOG (thr-MOG), spanning the first 120 amino acids lacking the transmembrane regions, and containing a His 6 segment, was purified from supernatant of insect Hi-5 cells (infected with high-titered stock of plaque-purified baculovirus) by passage over a nickel-nitrilotriacetate agarose column (Qiagen, Chatsworth, CA), as previously described (23). Purity was assessed by SDS-PAGE, and protein content was determined by micro bicinchoninic acid protein assay (Pierce, Rockford, IL). The thr-MOG protein was dialyzed against 20 mM NH4HCO3, pH 8.5, then lyophilized.
MBP was prepared from guinea pig (gpMBP) spinal cords (Harlan Bioproducts for Science, Indianapolis, IN) by a modification of a previously described method (30). Briefly, MBP was extracted from isolated myelin membranes using chloroform and methanol, precipitated with potassium citrate, acid extracted, and lyophilized. SDS-PAGE analysis of the protein showed the major band at the expected m.w. of 18.5 kDa. Protein content was determined by micro bicinchoninic acid protein assay. Proteins were stored at -20°C until resuspension in Dulbecco’s PBS at 4 to 8 mg/ml, then used immediately or stored frozen at -80°C.
Induction of EAE and treatment with peptide
EAE was induced by s.c. immunization at the base of the tail with 75 to 200 µg gpMBP, or 100 to 400 µg thr-MOG, or the combination of both 75 µg gpMBP and 100 µg thr-MOG, emulsified in CFA (Life Technologies, Grand Island, NY). Emulsions were supplemented with 400 µg Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) per mouse. Three hundred nanograms of pertussis toxin (List Biologic Laboratories, Campbell, CA) was administered i.v. at the time of immunization, and again 48 h later.
Therapeutic peptides or PBS were administered at the times indicated in the figure legends. Lyophilized peptides were reconstituted in PBS (Life Technologies, Grand Island, NY) to the appropriate concentration and were administered i.v. in a volume of 0.2 ml. A total of 250 nmol equals 332 µg of MBP Ac1–11[4Y] or 589 µg of MOG 41–60.
Mice were scored daily for clinical signs of disease based on the following scale: 0, normal; 1, limp tail; 2, partial hind limb paralysis or ataxic gait; 3, complete hind limb paralysis; 4, partial or complete forelimb paralysis; and 5, moribund or dead. Food was made accessible to immobile animals, and moribund animals were sacrificed on the third day with a score of 5. A score of 5 was included in the calculation of daily mean clinical score only during periods of morbidity and on day of death, and was thereafter excluded from these calculations. In some experiments, mice were weighed daily, at the time their clinical scores were assessed.
Several parameters of disease were examined to evaluate the severity of
EAE and the success of peptide therapy: mean clinical score (MCS), the
mean of clinical scores for all mice within a group on a given day;
mean day of onset, the mean day that affected mice within a group first
developed clinical signs of disease; percentage of mortality, the
number of mice within a group that died or were sacrificed as a result
of severe EAE, expressed as a percentage of the starting number of mice
in that group; percentage of incidence, the number of mice within a
group that developed a clinical score of 1 or greater, expressed as a
percentage of the starting number of mice in that group; mean maximum
severity, the mean of the maximum daily score that each mouse in a
group developed over the course of the experiment; and disease index,
the sum of the daily mean clinical scores for a group over a given
number of days, divided by that number of days. The percentage of
observations with a score of 0 or percentage of observations with a
score 
3 was calculated as the total number of such observations over
a given period of time, divided by the total number of live animal
observations recorded in that time period.
T cell proliferation assays
Draining lymph nodes (LN; inguinal, popliteal, and periaortic) were removed from mice 10 days after s.c. immunization with myelin proteins, as described above, in CFA supplemented with 400 µg M. tuberculosis H37Ra. Tissue was forced through fine wire mesh to form single cell suspensions into RPMI 1640 supplemented with 10% heat-inactivated FCS, penicillin-streptomycin, L-glutamine, and 5 x 10-5 M 2-ME. The LN cell suspensions were cultured in 96-well flat-bottom microtiter plates at 4 x 105 cells/well with the peptide or protein Ags listed in the figure legends. Controls included cells stimulated with media, Con A, and purified protein derivative of mycobacteria. Proliferation assay microwells were pulsed on day 4 with 1 µCi of [3H]TdR for 12 h. Cells were harvested onto glass fiber filters, and [3H]TdR incorporation was assessed by scintillation counting. The stimulation index was calculated by dividing the number of counts incorporated in response to Ag by the number of counts incorporated in the absence of any Ag.
Statistical analysis
Significant differences in daily MCS and disease index were
determined by ANOVA (p 
0.001) or repeated
measures ANOVA (p 0.001), respectively, with
post hoc comparisons by the Scheffé’s F test
(p 0.05). Differences in mean maximum
severity were determined using the Mann-Whitney U test.
Differences in mean day of onset were determined using Student’s
t test. Differences in all percentages were determined using
the 
2 test, or Fisher’s exact test when cell sizes
lower than 5 were observed. Because all non-ANOVA comparisons involved
repeated pairwise comparisons within a data set, a lower p
value (p < 0.01) was required for
significance. Data analysis was performed using Abacus Concepts,
Statview software (Abacus Concepts, Berkeley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EAE was induced in (PL/J x SJL)F1 mice with
varying doses of thr-MOG, gpMBP, or a combination of the two to create
an antigenically complex EAE model. As shown in Figure 1
A, the disease induced by the
combination of 50 µg thr-MOG plus 50 µg gpMBP was more severe than
that induced by 200 µg of either Ag alone. When mice were immunized
with suboptimal doses of either thr-MOG or gpMBP alone, the
contribution of the other to the combination disease was made quite
apparent (Fig. 1B). Table I illustrates the higher incidence,
severity, and mortality that resulted from immunization with 100 µg
thr-MOG plus 75 µg gpMBP compared to immunization with either of
those Ags alone, indicating that reactivity to both components
contributed to the underlying pathology of the combination disease
process.
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also confirms, in additional experiments, that the severity of
the disease induced by the combination of MOG plus MBP exceeded that
induced by the same total dose of either immunizing Ag alone. The
higher incidence associated with gpMBP EAE and the earlier onset
associated with thr-MOG EAE appear to combine to produce a higher
disease index in the thr-MOG plus gpMBP EAE than that found in disease
induced with 200 µg of either protein alone. This disease index is
not simply a reflection of the significantly higher mortality that
accompanied the combination disease, as clinical scores of mice that
die are dropped from the calculation of disease index after the day of
death.
The disease induced by the combination of Ags displayed a
relapsing/progressive character. Mice that survived the unusually high
mortality associated with the acute phase of this disease went on to
demonstrate remissions and relapses of variable duration, as assessed
by both disease score and animal weight. Clinical scores and weights
from three representative animals are illustrated in Figure 2, A–C. At disease onset,
increases in symptom scores were always accompanied by substantial
decreases in weight. Although lost weight was recovered within 2 wk,
indicating a remission period, disease scores showed varied improvement
from animal to animal in the same time frame. Eventual progression to
persistently high disease scores was observed in 19 of 28 surviving
mice that were followed for 45 days postimmunization. Continued
fluctuations in weight revealed additional disease remissions and
relapses in these mice that were not evident from examination of
clinical scores alone.
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T cell responsiveness in mice immunized with a combination of MOG and MBP
The results above suggest that both myelin proteins induce an
immune response in MOG plus MBP EAE. Before the specificity of this
response could be assessed, MOG T cell epitopes had to be defined in
the (PL/J x SJL)F1 strain. Peptide-specific
proliferation of LN cells from thr-MOG-primed mice was examined using a
panel of MOG peptides 20 amino acids long, spanning the thr-MOG
sequence and overlapping each other by 10 amino acids. Figure 3A illustrates that peptides
from at least three separate regions of the MOG protein induced T cell
proliferation. Those regions were located within amino acids 1–20,
31–60, and 81–110. The same regions were identified when IL-2
production was used as an assay of T cell responsiveness (data not
shown).
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A) was unexpected. Examination of the amino
acid sequence of this peptide reveals three substitutions in the human
sequence at residues 9, 10, and 20 compared with native mouse MOG (Gly,
Tyr in mouse to Arg, His in human at 9 and 10; Ala in mouse to Val in
human at 20) (33, 34). Figure 3B illustrates that the 1–20
peptide-specific response in mice immunized with thr-MOG did not
cross-react on the murine MOG 1–20 sequence. Therefore, the dominant
response to human MOG 1–20 was not an autoimmune response in the
mouse, but rather a response to the non-self human MOG
substitutions.
Mice immunized with the combination of thr-MOG plus gpMBP mounted
proliferative responses to both proteins (Fig. 4A). These data confirm
that both proteins induce T cell reactivity in this model system. The
dominant epitopes in these responses matched those found in responses
to either immunizing Ag used alone (Fig. 4, B and
C). Reactivity to thr-MOG was dominated by the high
response to the human sequence MOG 1–20 peptide (again with no
cross-reactivity on murine 1–20; data not shown), with small, but
detectable (stimulation index greater than 2) responses to peptides in
the 31–60 region. The response to gpMBP was directed primarily to the
acetylated amino terminus of that molecule (35, 36), with no detectable
reactivity to the minor determinant reported in the 31–47 region (16)
or the H-2s-restricted determinant in the 89–101 sequence
(14, 37). Therefore, immunization with the two Ags together induces T
cells of comparable specificity with those induced by immunization with
each Ag alone.
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Treatment of EAE induced with MOG in (PL/J x SJL)F1
It was of interest to evaluate the ability of peptides derived
from both thr-MOG and gpMBP to treat the symptoms associated with the
combination disease. Although the MOG peptide 91–110 has been shown to
be effective in treating MOG-induced EAE in SJL mice (23), treatment of
MOG-induced EAE has not been studied previously in (PL/J x
SJL)F1 mice. The peptides identified as the major
epitopes of thr-MOG in the studies described above were used to treat
disease induced with thr-MOG in (PL/J x SJL)F1 mice.
Figure 5A shows that MOG
41–60 effectively treated thr-MOG-induced EAE, while the other major
epitope peptides did not. The success of treatment with the 41–60
peptide was somewhat variable, with Figure 5B illustrating a
less optimal therapeutic effect. Five experiments produced treatment
effects similar to those seen in Figure 5, while two additional
experiments demonstrated no discernible benefit from MOG 41–60 using
the same protocol. The success of therapy in this disease appeared to
be more dependent on the severity of disease in the control group (the
most severe disease predicting a poor treatment outcome), rather than
the number or schedule of the peptide injections. The MBP peptide
Ac1–11[4Y], although effective at preventing gpMBP-induced EAE in
this strain (19, 22), did not have a measurable effect on
thr-MOG-induced disease in any experiment (Fig. 5B).
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Intravenous administration of the MBP peptide Ac1–11[4Y] and
the MOG peptide 41–60 together was effective in treating MOG plus MBP
EAE. Figure 6A illustrates the
reduction in daily MCS obtained upon administration of 125 nmol of each
of these peptides in combination, starting 9 days after the
disease-inducing immunization with both myelin proteins in adjuvant.
Data from three experiments, including the experiment illustrated in
Figure 6, were compiled for the purpose of statistical analysis. The
daily clinical scores of mice in the group treated with the MBP/MOG
peptide combination were significantly different from those of the
PBS-treated controls on 18 of the 25 observation days following the
initiation of peptide treatments. Table II illustrates that reductions in
clinical incidence, disease index, and mean maximum score in this
peptide-treated group were all statistically significant when compared
with the PBS-treated control group. Additional parameters to assess
disease burden included the calculation of percentage of observations
with high clinical scores (3 or greater) or with clinical scores of 0
(no symptoms). These reveal a dramatic and significant reduction in the
number of days with severe disease, with a corresponding increase in
the number of disease-free days, in MBP/MOG peptide-treated
mice.
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A). In the
statistical analysis, mice treated with the 125 nmol dose of MBP
Ac1–11[4Y] alone had daily clinical scores significantly different
from PBS-treated controls on only 7 of the 25 observation days
following the initiation of peptide treatments. This dose of
MBP Ac1–11[4Y] alone did not produce statistically significant
reductions in clinical incidence, disease index, or mean maximum score
compared with PBS-treated controls (Table II). These results suggest
that both the MBP Ac1–11[4Y] and MOG 41–60 peptides contributed to
the treatment effect observed when 125 nmol of each peptide was used in
combination to treat MOG plus MBP EAE.
To adequately compare the efficacy of treatment with a single
peptide with treatment with a peptide combination, however, it is
important to consider the total peptide dose, as we have observed that
peptide treatment of MBP-induced EAE is dose dependent (22, and data
not shown). Figure 6B illustrates the effects of
administration of either the MOG 41–60 or the MBP Ac1–11[4Y]
peptide alone when the dose of peptide was increased to 250 nmol (same
total nmol dose as the MBP Ac1–11[4Y] + MOG 41–60 peptide at 125
nmol each). Daily MCS obtained with the 250 nmol dose of the MBP
peptide alone were similar to those obtained using the combination of
125 nmol each of MBP and MOG peptides, with no statistically
significant differences between these two groups on any of the 25
observation days following the initiation of peptide treatment. In
Table II, it can be seen that all measures of disease were reduced in
mice treated with 250 nmol of MBP Ac1–11[4Y] compared with 125 nmol
of the same peptide, indicating a dose-dependent therapeutic effect.
Although treatment with 250 nmol of MOG 41–60 alone significantly
reduced the number of days with severe disease, with a corresponding
increase in disease-free days, significant reductions in clinical
incidence, overall disease index, and mean maximum score were not
apparent (Table II).
All of the same disease outcome measures that were reduced by the MBP/MOG combination peptide treatment were also significantly impacted by administration of 250 nmol of MBP Ac1–11[4Y] alone. Although mortality was reduced by both of these treatment protocols, the reductions were not statistically significant in either case. In addition, the day of disease onset in mice that did develop symptoms was not delayed significantly by either protocol (data not shown). Thus, the MBP peptide alone was capable of reducing the incidence and severity of EAE induced with a combination of two myelin proteins in a dose-dependent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An interesting feature of the disease induced by the combination of thr-MOG plus gpMBP was that severity by any measure was greater than would be produced by the same total dose of either Ag alone. A synergy of effects is inferred. It previously has been shown that Ab to MOG can induce demyelination in vivo when administered to animals that have been immunized with MBP, or that have received T cells specific for MBP or MOG (38, 39, 40). In the new EAE model described in this work, it is possible that the primary contribution of the MOG immunization is the induction of anti-MOG Abs that act in combination with T cells specific for MBP to cause the severe, and often fatal, disease that was observed. Alternatively, the pathogenic potential of two nonoverlapping T cell populations may be greater than that of a larger single population.
The primary goal of this study was to determine the capacity of
immunodominant MOG and MBP peptides, administered together or
individually, to treat EAE induced with the MOG plus MBP Ag
combination. This required some initial studies in the MOG EAE system,
as MOG peptides have not been assessed previously for treatment of
MOG-induced EAE in (PL/J x SJL)F1 mice, although
the MOG 91–110 peptide has been shown to reduce disease in SJL mice
(23). The MOG T cell epitopes identified using a T cell proliferation
assay and thr-MOG-immunized F1 mice corresponded to
epitopes previously identified in the parent strains (12, 23, 31). The
dominance of the MOG 1–20 epitope was surprising, as it has been
reported previously to be an epitope in SJL (23, 31), and the
H-2u of the PL/J parent was expected to dictate the
dominant epitopes in the F1 (32). Further analysis of this
response in thr-MOG-primed mice revealed no cross-reaction to the mouse
MOG 1–20 sequence, which differs from human at residues 9, 10, and 20.
The response to human MOG 1–20 therefore should have no pathologic
consequences, consistent with the observation that human MOG 1–20 did
not treat thr-MOG-induced EAE (Fig. 5) and, like the corresponding
mouse MOG sequence (12), was not encephalitogenic (B. Devaux and D.
Smilek, unpublished observation).
Of the remaining antigenic MOG peptides identified in the
F1 (Fig. 3), MOG 41–60, but not 31–50, was used
successfully to treat MOG-induced EAE in this strain. An
encephalitogenic H-2u-restricted determinant has been
reported previously by Kerlero de Rosbo et al. (31) in the 35–55
region of MOG, although it was proposed that the T cell epitope resided
in the amino-terminal half of that peptide. It is not clear why the
human sequence MOG 31–50 peptide used in the current study did not
confer protection from disease; a difference in the human to mouse MOG
sequences in that region (Pro to Ser at residue 42) may again be
significant. Finally, although MOG 91–110 reduced MOG EAE in SJL mice,
this peptide was not effective in treating MOG-induced disease in
(PL/J x SJL)F1 mice. This H-2s-restricted
epitope is a minor one in the F1 strain, confirming earlier
observations in the MBP disease model that major epitopes are
relatively better therapeutics than minor epitopes (16).
With this knowledge, it was possible to determine the capacity of MOG and MBP peptides, administered together or individually, to treat EAE induced with the MOG plus MBP Ag combination. Based on the data discussed above, the MOG 41–60 peptide was chosen for these studies. With respect to MBP peptides, both Ac1–11 and Ac1–11[4Y] have been shown to be effective in treating MBP-induced EAE (19, 22). MBP Ac1–11 is a dominant T cell epitope in the PL/J and (PL/J x SJL)F1 mouse strains (35, 36). The substituted MBP peptide Ac1–11[4Y], in which tyrosine replaces lysine at residue 4, is a strong agonist peptide, demonstrating improved antigenicity compared with Ac1–11 (41, 42), most likely because the tyrosine at residue 4 enhances binding of the peptide to the I-Au molecule (42, 43). Of these two peptides, MBP Ac1–11[4Y] was selected for these experiments because it is also more potent as a therapeutic (19, 22), and was expected to be more effective in treating the severe MOG plus MBP EAE.
A combination of the two therapeutic peptides, MBP Ac1–11[4Y] and MOG 41–60, successfully treated MOG plus MBP EAE, with statistically significant reductions in daily and maximum clinical scores, clinical incidence, and disease index. Both peptides appeared to contribute to the optimal therapeutic effect, because MBP Ac1–11[4Y] alone, administered at the same 125 nmol dose used in the combination, was not as effective as the combination of the two peptides. This result provides further evidence that T lymphocytes responsive to both MBP and MOG are involved in the propagation of EAE induced with the combination of these two myelin proteins. When the dose of MBP Ac1–11[4Y] was increased to 250 nmol, therapeutic benefit was increased, and approached that observed with the combination of MOG and MBP peptides at 125 nmol each. Even this higher dose of MBP Ac1–11[4Y] was not effective in treating EAE induced with MOG alone, indicating that the treatment effect of Ac1–11[4Y] in MOG plus MBP disease was not due to cross-reactivity between these two epitopes at the T cell level.
The most significant benefit for any of the peptide treatment groups with the combination disease was a reduction in disease severity, assessed by a number of different measurements. Although clinical incidence was not reduced to zero in the combination peptide or the high dose MBP peptide treatment groups, the degree of physical impairment exhibited by affected animals was dramatically lower than that observed in the PBS control group. Reductions in disease-associated mortality, although obvious, were not statistically significant in any peptide treatment group. The mean day of death was not delayed by peptide treatment (range of mean = 14.7 to 17.2), generally occurring 3 to 4 days after disease onset. Despite the persistence of deaths in peptide treatment groups, measures of clinical severity showed the same statistically significant improvements over PBS-treated controls whether these measures were calculated with a score of 5 recorded on the day of death only, or retained through the length of the experiment.
The results of these studies suggest that a sufficiently high dose of a peptide derived from one myelin protein can effectively treat established EAE that results from an immune response to multiple myelin components, when the peptide is derived from one of the immunizing myelin components. The failure to completely reduce the combination disease is consistent with the results of Brocke et al. (21) in the treatment of disease induced by adoptive transfer of two different T cell clones. The mechanism of the therapeutic effect we have generated is not clear. It is significant, in this respect, that the MBP peptide was not capable of alleviating EAE induced only with MOG. This stands in contrast to the findings of Nicholson et al. (20) and Al-Sabbagh et al. (25), in which preimmunization or feeding with one myelin Ag led to protection from EAE induced with a second unrelated myelin Ag, due to the generation of regulatory Th2 cells capable of influencing the outcome of the disease-inducing immunization by virtue of colocalization to the CNS. Either Th2 cells are not induced utilizing the treatment protocol used in the current studies, or such cells do not have an impact on encephalitogenic responses postpriming.
It is possible that the treatment protocol used in the current studies produces a state of anergy in the targeted cells (16, 44, 45). This does not rule out a role for suppression through linked recognition, as anergized cells have been shown to serve as cytokine sinks for concurrent responses (46). The observation that the MOG plus MBP disease itself was significantly more severe than that induced by the individual components makes it difficult to distinguish the lower level of disease in successfully treated animals, from EAE induced with only one component Ag. Removal of the contribution of a single Ag to the synergistic processes in disease induction may be the most significant impact of this therapy, and such an effect may also prove significant in human disease.
In comparison with MBP Ac1–11[4Y], the MOG 41–60 peptide
demonstrated an inferior capacity to treat MOG plus MBP EAE when
administered alone at a dose of 250 nmol. There are several potential
explanations for the difference between the therapeutic potentials of
these two peptides. It is possible that the MOG-specific T cell
component contributes substantially less to the generation of the
disease pathology than does the MBP-induced component, making treatment
of the combination disease less potent with this Ag. Alternatively, 250
nmol of MOG 41–60 might not be a sufficiently high dose to achieve the
treatment effect observed with 250 nmol of MBP Ac1–11[4Y]. It has
been shown that effectiveness of MBP peptide therapy is highly
dependent on the dose of peptide used for each injection, and that the
strong agonist peptide MBP Ac1–11[4Y] is at least 100-fold more
potent than MBP Ac1–11, both as a stimulator of in vitro proliferation
and as a therapeutic in vivo (19, 22, 41). In support of this
hypothesis, the LN proliferative response to MBP Ac1–11[4Y] was
clearly better than the response to MOG 41–60 in MOG plus
MBP-immunized mice (Fig. 4).
A marmoset model of MOG-induced EAE was recently described (47), in which disease onset could be delayed by administration of soluble MOG, using a treatment schedule very similar to the studies reported in this work. Despite the protective effect of MOG administration, a striking finding in the marmoset model was the onset of a very severe, and even fatal, episode of EAE within 1 wk of therapy completion. This exacerbation was not observed in control animals. The MOG therapy-treated animals showed elevated Ab titers to several different peptides of MOG. We have not observed such a disease exacerbation following peptide treatment in either the SJL or the (PL/J x SJL)F1 murine models of MOG-induced disease. While it is true that mice with MOG plus MBP-induced disease did exhibit a rise in daily MCS after MOG peptide treatments were withheld (generally within 3 to 15 days after the last peptide injection), these disease relapses were never observed to be more severe than ongoing disease in the PBS-treated control group. It is possible that treatment with the MOG protein is more likely to boost titers of pathogenic autoantibodies than is administration of T cell epitope-containing peptides.
In summary, we describe a new model of EAE induced with a combination of the myelin proteins MBP and MOG, in which clinical severity is greater than predicted by immunization with either Ag alone. This disease can be treated postinduction with a combination of MBP and MOG peptides, which individually have been shown to be therapeutic for EAE induced with either MBP or MOG, respectively. Both peptides appear to be necessary for optimal therapy of disease when administered at a limiting dose. However, a single MBP peptide administered at a higher dose also provides significant reductions in disease severity. This is the first demonstration that parenteral administration of a T cell epitope peptide can suppress disease symptoms in an EAE model in which it is clear that reactivity to more than one myelin component plays a role in generation of the disease pathology. These results support the hypothesis that a human autoimmune disease, such as MS, may be down-regulated by administration of peptides derived from a single myelin protein, despite the presence of an immune response to multiple myelin proteins.
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Acknowledgments |
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Footnotes |
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2 Current address: Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080.
3 Current address: Health Effects Institute, 955 Massachusetts Avenue, Cambridge, MA 02139.
4 Current address: Dyax Corporation, One Kendall Square, Building 600, Cambridge, MA 01239.
5 Current address: Point Therapeutics, Inc., 75 Kneeland Street, Boston, MA 02111.
6 Address correspondence and reprint requests to Dr. Mary Pat Happ, ImmuLogic Pharmaceutical Corp., 610 Lincoln Street, Waltham, MA 02154. E-mail address:
7 Abbreviations used in this paper: MS, multiple sclerosis; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; gpMBP, guinea pig myelin basic protein; LN, lymph node; MBP, myelin basic protein; MCS, mean clinical score; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; thr-MOG, truncated human recombinant myelin oligodendrocyte glycoprotein.
Received for publication April 14, 1997. Accepted for publication March 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  J. Karman, H. H. Chu, D. O. Co, C. M. Seroogy, M. Sandor, and Z. Fabry Dendritic Cells Amplify T Cell-Mediated Immune Responses in the Central Nervous System J. Immunol., December 1, 2006; 177(11): 7750 - 7760. [Abstract] [Full Text] [PDF]
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A. Lobell, R. Weissert, S. Eltayeb, K. L. de Graaf, J. Wefer, M. K. Storch, H. Lassmann, H. Wigzell, and T. Olsson Suppressive DNA Vaccination in Myelin Oligodendrocyte Glycoprotein Peptide-Induced Experimental Autoimmune Encephalomyelitis Involves a T1-Biased Immune Response J. Immunol., February 15, 2003; 170(4): 1806 - 1813. [Abstract] [Full Text] [PDF]
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E. Lovegrove, G. J. Pettigrew, E. M. Bolton, and J. A. Bradley Epitope Mapping of the Indirect T Cell Response to Allogeneic Class I MHC: Sequences Shared by Donor and Recipient MHC May Prime T Cells That Provide Help for Alloantibody Production J. Immunol., October 15, 2001; 167(8): 4338 - 4344. [Abstract] [Full Text] [PDF]
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H. I. McFarland, A. A. Lobito, M. M. Johnson, G. R. Palardy, C. S. K. Yee, E. K. Jordan, J. A. Frank, N. Tresser, C. P. Genain, J. P. Mueller, et al. Effective Antigen-Specific Immunotherapy in the Marmoset Model of Multiple Sclerosis J. Immunol., February 1, 2001; 166(3): 2116 - 2121. [Abstract] [Full Text] [PDF]
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 K. L. Legge, B. Min, J. J. Bell, J. C. Caprio, L. Li, R. K. Gregg, and H. Zaghouani Coupling of Peripheral Tolerance to Endogenous Interleukin 10 Promotes Effective Modulation of Myelin-Activated T Cells and Ameliorates Experimental Allergic Encephalomyelitis J. Exp. Med., June 19, 2000; 191(12): 2039 - 2052. [Abstract] [Full Text] [PDF]
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K. Falk, O. Rotzschke, L. Santambrogio, M. E. Dorf, C. Brosnan, and J. L. Strominger Induction and Suppression of an Autoimmune Disease by Oligomerized T Cell Epitopes: Enhanced in Vivo Potency of Encephalitogenic Peptides J. Exp. Med., February 21, 2000; 191(4): 717 - 730. [Abstract] [Full Text] [PDF]
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