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*
Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611; and
The Ben May Institute for Cancer Research and the Committee for Immunology, University of Chicago, Chicago, IL 60637
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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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We have been examining the functional role of epitope spreading using relapsing-remitting EAE (R-EAE) in the SJL mouse, a CD4+ Th1-mediated demyelinating disease which serves as a model of MS. The R-EAE system is useful for multiple reasons: 1) disease can be actively induced in a peptide-specific manner; 2) numerous encephalitogenic epitopes on multiple myelin proteins including proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG) have been identified; and 3) the relative encephalitogenic dominance (hierarchy) of these epitopes in R-EAE induction has been characterized in SJL (1, 11, 12) and (SWR x SJL)F1 (13) mice. PLP139–151 is the dominant encephalitogenic epitope in the SJL mouse based on priming for active disease (10 µg/mouse), PLP178–191 is secondary (50 µg/mouse), and MBP84–104 (200 µg/mouse plus pertussis toxin) is only weakly encephalitogenic. In R-EAE induced by the immunodominant PLP139–151 epitope, recovery from the acute clinical episode is accompanied by the expansion of PLP178–191-specific T cells (i.e., intramolecular epitope spreading). In contrast, SJL mice remitting from acute R-EAE induced with the weakly encephalitogenic MBP84–104 epitope develop PLP139–151-specific T cell responses concomitant with disease relapse (i.e., intermolecular epitope spreading) (11). Yu et al. (13) have described a similar pattern of epitope spreading in PLP139–151-induced R-EAE in (SWR x SJL)F1 mice.
Diversification of autoimmune responses thus appears to be a common sequelae to myelin destruction in murine R-EAE. However, it remains unclear whether T cells specific for endogenous myelin epitopes play a significant pathologic role in tissue damage during the relapsing clinical episodes, or whether this is simply an epiphenomenon without pathologic consequences. Previous studies showing that T cells specific for relapse-associated determinants purified from mice primed with PLP139–151 adoptively transfer EAE to naive recipients suggest, but do not prove, that these T cells are pathologic (11, 13).
The current study explores the immunopathological consequences and peptide dominance of epitope spreading in R-EAE induced in SJL mice by both the highly encephalitogenic PLP139–151 epitope and the weakly encephalitogenic MBP84–104 epitope. Using a number of functional criteria, the results provide strong evidence that responses to relapse-associated endogenous myelin epitopes drive the pathologic progression of R-EAE. We show that splenocytes from mice with MBP84–104-R-EAE, activated in vitro with PLP139–151, can serially transfer R-EAE to naive mice demonstrating that these T cells have encephalitogenic potential. Second, we show that T cells specific for the relapse-associated epitopes are demonstrable in the CNS of mice during disease remission. Third, induction of peptide-specific tolerance to the relapse-associated epitope alone, either before disease induction or after the acute phase, blocks disease progression as assessed by a decreased relapse rate. Last, in vivo blockade of the B7-1/CD28 costimulatory pathway with anti-B7-1 F(ab) during remission in R-EAE blocks epitope spreading and clinical relapses when administered either following the acute disease episode or following the first relapse. The pattern of spreading correlates with encephalitogenic dominance and the precursor frequency of T cells in the draining lymph nodes of primed mice specific for the various encephalitogenic epitopes. Collectively, these results indicate that clinically relevant epitope spreading takes place in a hierarchical order of peptide dominance and plays a critical role in disease progression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice, 6–7 wk old, were purchased from Harlan Laboratories (Indianapolis, IN). All mice were housed in the Northwestern animal care facility and were maintained on standard laboratory food and water ad libitum. Paralyzed mice were afforded easier access to food and water.
Peptides
PLP139–151 (HSLGKWLGHPDKF), PLP178–191 (NTWTTCQSIAFPSK), MBP84–104 (VHFFKNIVTPRTPPPSQGKGR), and OVA323–339 (RGAENIEAHAAVAQSI) were synthesized using a synergy peptide synthesizer (Applied Biosystems, Foster City, CA). Purity (>97%) of these peptides was confirmed by mass spectroscopy at the University of North Carolina Peptide Synthesis Core.
I-As peptide binding assay
Peptide binding to cell-surface class II was measured using a competition assay (14, 15). Next, 10 µM of a biotinylated influenza hemagglutinin (HA) indicator peptide (BHA307–319; PKYVKQNTLKLAT) was incubated with 3 x 105 I-As-expressing DAS.15 fibroblasts in the presence or absence of titrated amounts of a competitor peptide for 1 h at pH 7.0, 37°C, in PBS containing 5% FBS. The cells were washed thoroughly in PBS plus 5% FBS and subsequently stained with fluoresceinated-avidin D (Vector Laboratories, Burlingame, CA), followed by biotinylated anti-avidin D (Vector Laboratories), and again with fluoresceinated-avidin D. Fluorescence was analyzed on a FACScan flow cytometer (Becton Dickinson, San Diego, CA) by gating for equivalent forward scatter on the live cell population. The fluorescent signal was in a range that is linear with respect to fluorescein density. The relative binding affinity of a given peptide to I-As class II molecules was determined by assessing the concentration of the peptide required to reduce the fluorescence intensity of binding of the biotin-labeled HA peptide alone to DAS.15 cells by 50%. The BHA307–319 indicator peptide bound specifically to MHC class II as the peptide did not bind to the nontransfected parent fibroblast line DAP/3 (data not shown).
Induction of and clinical scoring of R-EAE induced by active immunization with PLP139–151 and MBP84–104
For PLP139–151-induced R-EAE, each mouse received 100 µl of a CFA emulsion containing 200 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) and 40 µg of PLP139–151 distributed s.c. over three spots on the dorsal flanks on day 0. For MBP84–104-induced R-EAE, the adjuvant emulsion contained 200 µg of MBP84–104 distributed over three sites on the dorsal flanks on both days 0 and 7 and 400 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) i.v. in 0.5 ml of PBS on days 7 and 10. Clinical scores were assessed on a 0–5 scale as follows: 1, limp tail or hind limb weakness (legs fall through the cage top); 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, total hind limb paralysis; and 5, moribund. A relapse was defined as an increase in at least one clinical grade sustained for at least two consecutive days after the animal had previous improved at least a full clinical score and had stabilized.
Induction of R-EAE by adoptive transfer with in vitro-activated MBP84–104-specific lymph node T cells
Mice were immunized s.c. with 100 µl of an IFA emulsion containing 200 µg of M. tuberculosis H37Ra (Difco) and 200 µg of MBP84–104 distributed over three spots on the flank. Seven to 10 days after immunization, the inguinal, brachial, and axillary lymph nodes were removed from sensitized donors, and single-cell suspensions were prepared. The cells were adjusted to 8 x 106/ml in T75 flasks in complete DMEM containing 50 µg/ml MBP84–104 in a total volume of 30 ml/flask and incubated at 37°C in a humidified atmosphere containing 5% CO2. Four days later, the cells were washed twice with buffered saline solution, counted, and 50 x 106 viable cells transferred i.p. to naive SJL mice.
Serial transfer of PLP139–151- or MBP84–104-specific splenic T cells from mice with MBP84–104-induced R-EAE
Forty-eight days after active immunization with MBP84–104, mice were sacrificed and spleens were removed. Spleen cells were cultured with 50 µg/ml PLP139–151 or MBP84–104 as described above. After 4 days of culture, 4–6 x 107 PLP139–151-activated splenocytes or 3–5 x 107 MBP84–104-activated splenocytes were transferred i.p. into naive SJL recipients.
Isolation of CNS-infiltrating mononuclear cells
Mice were anesthetized with methoxyflurane and perfused through the left ventricle with 60 ml of PBS. Spinal cords were extruded by flushing the vertebral canal with PBS and rinsed in PBS. Tissues were forced through 100-mesh stainless screens to give a single-cell suspension. The spinal cord homogenate was resuspended in 30% Percoll (Pharmacia, Piscataway, NJ), divided into tubes (equivalent to 4–5 spinal cords per tube), and underlaid with 70% Percoll. The gradients were centrifuged at 500 x g for 20 min at 24°C. CNS mononuclear cells were collected from the 30:70% interface, washed, and incubated on plastic dishes at 37°C in complete DMEM containing 10% FBS for 2 h. Nonadherent cells were eluted, counted, and used in T cell proliferation assays.
In vitro T cell proliferation assays
T cell proliferative responses were assessed by incorporation of
[3H]thymidine. A total of 5 x
105 viable splenocytes recovered from mice with
ongoing R-EAE were cultured in triplicate in 96-well flat-bottom
microculture plates (Falcon Labware, Oxnard, CA) in 0.2 ml of DMEM
(Life Technologies, Grand Island, NY) supplemented with 5% FBS (Sigma,
St. Louis, MO), 2 mM L-glutamine (Sigma), 100 µg/ml
streptomycin, and 100 U/ml penicillin (Sigma). Alternately,
CNS-infiltrating nonadherent mononuclear cells were plated at 5 x
104 per well with 5 x
105 irradiated naive splenocytes as APCs in
complete DMEM. A variety of peptide concentrations were tested. Plates
were pulsed with 1 µCi [3H]TdR after 72
h of culture and harvested for scintillation counting 24 h
thereafter. The results are expressed as 
cpm = mean cpm of
Ag-containing cultures - mean cpm of control cultures, and as
stimulation index = mean cpm of Ag-containing cultures/mean cpm of
control cultures.
Precursor frequency analyses
Precursor frequencies of PLP139–151-,
PLP178–191-, and
MBP84–104-specific T cells were determined using
CD4+ T cells purified using MACS columns
(Miltenyi Biotec, Auburn, CA) from the draining lymph nodes (pooled
axillary, brachial, and inguinal) of SJL mice primed 7 days previously
with 25 µM of the individual peptides. Frequencies were determined
both by limiting dilution analysis (LDA) using
[3H]TdR incorporation as a readout and by
determining the numbers of IFN-
-producing ELISPOTs.
For LDA, T cells were plated in U-bottomed microtiter plates (Nunc,
Naperville, IL), using 36 replicates per 2-fold dilution, at
concentrations ranging from 2.5 x 105 to
1.95 x 103 cells/well. Peptide (10
µg/well) was added to 27 wells, and PBS was added to the remaining 9
wells along with 4 x 105 irradiated spleen
cells in all wells as APCs. Cultures were incubated for 72 h at
37°C, pulsed with [3H]TdR, and harvested
24 h thereafter. Wells were scored positive if the cpm were
> 3 SD above the mean of the control wells. Minimal estimates of the
peptide-specific cell frequency were calculated by analysis of the
Poisson distribution relationship between the number of responder cells
added to the cultures and the percentage of replicate wells that failed
to show significant proliferation. Frequency calculations were
performed by computer using 
2 minimization.
This analysis gives a minimal frequency estimate and the 95%
confidence limits.
For ELISPOT, T cells were cultured in 96-well flat-bottom,
nitrocellulose-coated microculture plates (Whatman Polyfiltronics,
Clifton, NJ) according to the method of Forsthuber et al.
(16). The plates were precoated overnight at 4°C with
100 µl of anti-IFN- (R46A2; PharMingen, San Diego, CA) at 4
µg/ml. Plates were then washed four times with sterile PBS, and the
wells were blocked with DMEM-1% BSA for 1 h at room temperature.
T cells were plated in triplicate at numbers ranging from 2.5 x
105 to 1.95 x 103
cells/well in HL-1 serum-free media (BioWhittaker, Walkersville, MD)
supplemented with 1% L-glutamine ± 10 µg/well of
the appropriate peptide and 4 x 105 naive
SJL spleen cells as APCs. Cultures were incubated for 24 h at
37°C, washed three times with PBS, and then washed four times with
PBS-0.05% Tween 20. Next, 2 µg/ml of biotinylated anti-IFN-
(XMG1.2; PharMingen, San Diego, CA) diluted in PBS/Tween 20/1% BSA was
added, and the plates were incubated at 4°C overnight. The plates
were, washed four times with PBS/Tween 20 and 0.1
ml of anti-biotin Ab (Vector Laboratories) at a 1:1000 dilution in
PBS/Tween 20/1% BSA was added and the plates incubated at room
temperature for 2 h. Plates were then washed with PBS four times
and developed in nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl
phosphate substrate solution (Sigma). The developing reaction was
quenched after 30–45 min using distilled water. The plates were air
dried and spots enumerated under a dissecting microscope.
Delayed-type hypersensitivity (DTH)
DTH responses were quantitated using a 24-h ear swelling assay. Prechallenge ear thickness was determined using a Mitutoyo model 7326 engineer’s micrometer (Schlesinger’s Tools, Brooklyn, NY). DTH responses were elicited by injecting 10 µg of peptide (in 10 µl of saline) into the dorsal surface of the ear using a 100 µl Hamilton syringe fitted with a 30-gauge needle. Twenty-four hours after ear challenge, the increase in ear thickness over prechallenge measurements was determined. Results are expressed in units of 10-4 inches ± SEM. Ear swelling responses were the result of mononuclear cell infiltration and showed typical DTH kinetics (i.e., minimal swelling at 4 h, maximal swelling at 24–48 h).
In vivo Ab treatment
The following mAbs were employed: hamster control Ig (Parsi12) and the anti-CD80 (B7-1) mAb 16-10A1. Abs were produced in an Acusyst Jr. Bioreactor (Endotronics, Coon Rapids, MN), and F(ab) of anti-B7-1 mAb 16-10A1 were produced and purified as previously described (17, 18). Mice were treated following recovery from the initial paralytic episode of active PLP139–151-induced R-EAE (20–25 days postimmunization) and/or following recovery of control mice from the first relapse (45–50 days postimmunization). Ab was administered i.p. every other day for five treatments of 25 µg (total of 125 µg), and mice were monitored for development of clinical relapses for an additional 40–60 days.
Tolerance induction
Peripheral tolerance induction using peptide-coupled splenocytes
was performed as previously described (19). Briefly,
erythrocyte-free (Tris-NH4Cl-treated) splenocytes
were coupled with PLP139–151,
PLP178–191, or MBP84–104
using an ethylene carbodiimide procedure. Saline-washed SJL/J
splenocytes were centrifuged in 50-ml tubes and resuspended to a final
concentration of 5 x 108 cells/ml in saline
containing 2.0 mg/ml peptide, pH 7.0. Control cells were prepared
either without peptide in the reaction mixture (sham-coupled) or
coupled with OVA323–339. The coupling reaction
was initiated by the addition of 0.5 ml of freshly prepared ethylene
carbodiimide (200 mg/ml saline; Calbiochem-Behring, La Jolla, CA) per
ml of cell suspension. Following a 1-h incubation with shaking, cells
were washed three times with balanced salt solution and maintained at
4°C until use. Coupling efficiency has previously been determined to
be 
30%, yielding 24–55 µg of peptide/5 x
107 splenocytes (19). Tolerance was
induced by the i.v. injection of 5 x 107
peptide- or sham-coupled splenocytes into syngeneic recipient
mice.
Statistical analyses
Comparison of the percentage of animals showing clinical
relapses between any two groups of mice was done by
2 using Fisher’s exact probability.
Comparisons of the mean day of onset of relapse, mean peak disease
severity, and DTH responses between any two groups of mice were
analyzed by the Student’s t test. Values of
p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serial transfer of disease by T cells specific for relapse-associated epitopes
SJL mice with MBP84–104-R-EAE experience a
severe clinical relapse associated with the activation of
PLP139–151-specific T cells (11).
Using adoptive MBP84–104, we first asked if
these PLP139–151-specific T cells were
encephalitogenic as assessed by their ability to transfer clinical
R-EAE to naive recipient mice. As seen in Fig. 1
, splenic T cells recovered 48 days
after adoptive MBP84–104-specific EAE (during
the initial clinical relapse) were capable of transferring disease
following in vitro activation with either the initiating
MBP84–104 epitope (3 of 3 mice affected with a
mean day of onset of 11.7 and mean maximal severity of 3.3) or with the
relapse-associated PLP139–151 epitope (9 of 10
mice affected with a mean day of onset of 10.4 and mean maximal
severity of 3.3). Peptide restimulation was required for successful
serial transfer. Thus, following a relatively mild course of acute
R-EAE initiated by MBP84–104-specific T cells,
PLP139–151-specific T cells that arise
concomitant with the initial severe disease relapse have potent
encephalitogenic activity. These results expand our earlier observation
that PLP178–191-specific T cells recovered from
mice in remission from severe acute R-EAE initiated by active priming
with PLP139–151/CFA could serially transfer
R-EAE (11).
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If T cells specific for endogenous myelin epitopes play a major
pathologic role in relapsing disease, they should be demonstrable in
the CNS of mice before and during clinical relapses. We recovered
plastic nonadherent mononuclear cells from the spinal cords of mice
remitting from active (day 18 postpriming)
PLP139–151- and adoptive (day 25 posttransfer)
MBP84–104-specific R-EAE. The cells were rested
for 7 days in 2 U/ml rIL-2 and then cultured with irradiated splenic
APCs from naive mice ± 50 µM of
PLP139–151, PLP178–191,
or MBP84–104. As seen in Fig. 2, CNS-infiltrating T cells recovered
from mice remitting from the acute phase of
PLP139–151-induced R-EAE respond to both the
initiating PLP139–151 peptide and to the
relapse-associated PLP178–191 epitope, but not
to MBP84–104. Similarly, CNS-infiltrating T
cells from mice in remission from adoptive
MBP84–104-specific R-EAE responded very strongly
to both the initiating MBP84–104 peptide and to
the relapse PLP139–151 epitope. Interestingly,
CNS T cells from these mice also responded, although less vigorously,
to a secondary PLP epitope, PLP178–191, at
this time.
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To directly determine whether newly recruited
PLP139–151-specific T cells play a significant
role in relapsing EAE initiated by the adoptive transfer of
MBP84–104-specific T cells, peptide-specific
tolerance was employed. We examined the effects of tolerance to either
the initiating and/or relapse-associated epitopes on disease initiation
and progression in MBP84–104- and
PLP139–151-induced R-EAE. As seen in Fig. 3A, pretolerization with the
disease inducing MBP84–104 epitope 7 days before
induction of active EAE with MBP84–104/CFA
totally protected the mice from acute and relapsing disease.
Pretolerization with PLP139–151 had no
significant effect on acute clinical disease, but profoundly suppressed
disease progression reducing the relapse rate from 0.83 to 0.14
(p = 0.026), indicating a pathologic role for
PLP139–151-specific T cells in disease relapse.
This was confirmed by the finding that tolerance induction during the
remission period following recovery from acute
MBP84–104-specific adoptive EAE with either the
relapse-associated PLP139–151 epitope alone or a
combination of PLP139–151 plus
MBP84–104 totally suppressed expression of
disease relapses from a level of 7 of 10 (70%) in controls to 0 of 10
(0%, p = 0.003). Relapses were delayed, but the rate
was not significantly reduced (p = 0.179), in
mice tolerized with the disease-inducing
MBP84–104 epitope alone (Fig. 3B).
The mean peak clinical score for mice displaying relapses was also not
significantly different (1.33 in
MBP84–104-tolerized compared with 1.66 in the
sham-tolerized group).
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A), while pretolerization
with the PLP178–191 had no effect on acute
disease, but reduced the primary relapse rate from 1.0 to 0.25
(p = 0.007). These results again suggest that
the response to the relapse-associated
PLP178–191 epitope are primarily responsible for
the primary disease relapse. In confirmation, induction of tolerance
following recovery from acute PLP139–151-induced
EAE (Fig. 4B) with either the relapse-associated
PLP178–191 epitope alone or with a combination
of PLP178–191 plus
PLP139–151 (RR = 0.2, p =
0.023), but not with PLP139–151 alone (RR =
0.5, p = 0.349), significantly reduced the relapse rate
when compared with sham-tolerized controls (RR = 0.8). The
decrease in the clinical symptoms during relapse correlated with
inhibition of the PLP178–191-specific DTH
response (Fig. 4C). Thus, in R-EAE in which the animals have
undergone either a relatively mild acute disease (transfer of
MBP84–104-specific T cells in the absence of
CFA; Fig. 3
) or a relatively severe acute disease (active
PLP139–151-induced EAE; Fig. 4), T cells
specific for the relapse-associated epitope play a major pathologic
role in mediating disease relapses even though T cell specific for the
initiating epitope persist in the periphery. Interestingly,
tolerance to the disease initiating MBP84–104
epitope during remission from MBP84–104-induced
adoptive R-EAE was more effective than
PLP139–151-specific tolerance during remission
from active PLP139–151-induced R-EAE. This may
relate to the large number (5 x 107) of
MBP84–104-specific T cells required to initiate
adoptive disease.
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We have previously reported that treatment of mice with
anti-B7-1 F(ab) after the acute phase of
PLP139–151-induced R-EAE blocks relapses and
activation of T cells specific for the relapse-associated
PLP178–191 epitope (12). The
efficacy of anti-B7-1 F(ab) treatment in inhibiting disease
progression and T cell responses to relapse-associated epitopes upon
administration following recovery from the acute stage of disease was
compared with later treatment following recovery from the primary
relapse according to the protocol shown in Fig. 5A. As seen in Fig. 5B, SJL mice treated with anti-B7-1 F(ab)s at the time
of remission from the acute phase of active
PLP139–151-induced R-EAE displayed significantly
fewer clinical relapses (group B, 3 of 18 relapses) over the ensuing 25
days than hamster Ig-treated controls (group A, 17 of 22 relapses,
p = 0.0003). This confirmed our earlier studies that
blockade of B7-1-mediated costimulation during disease remission was a
potent way to ameliorate ongoing disease. In a second experiment, on
day 50, after the hamster Ig-treated controls had recovered from the
first relapse, mice in both the hamster Ig-treated control and the
anti-B7-1 F(ab)-treated groups were further divided into two groups
of equal clinical score and relapse history and treated again with
either hamster control Ig or anti-B7-1 F(ab)s. This resulted in
four treatment groups: C, received the hamster control Ig at the first
and second remission; D, received control Ig at the first remission and
anti-B7-1 F(ab)s only during the second remission; E, received the
anti-B7-1 F(ab)s only at the first remission and hamster control Ig
at the second remission; and F, received the anti-B7-1 F(ab)s
at both the first and second remissions. As can be seen in Fig. 5C, mice that received anti-B7-1 F(ab) treatment at the
first and/or second remission (groups D, E, and F) exhibited
significantly (p 
0.03) fewer secondary
relapses (1 of 15, 1 of 13, 1 of 13) during the ensuing 30 days
compared with the control group, which received hamster control Ig
during both the first and second remission (group C, 7 of 13 relapses).
Thus, the ameliorating effect of anti-B7-1 blockade is long-lasting
and effective even when given late in disease following recovery from
the primary relapse.
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Epitope spreading follows a hierarchical order of relative immunodominance of myelin peptides
Our past experience in induction of R-EAE in the SJL mouse has
indicated that PLP139–151 is the most
immunodominant epitope in mouse myelin (20) and that
disease can be initiated by a single s.c. immunization with 10 µg of
peptide (Table I).
PLP178–191 is also highly encephalitogenic, but
requires at least 50 µg/mouse to initiate disease. In contrast,
MBP84–104 is weakly encephalitogenic in that two
immunizations of 200 µg/mouse plus i.v. administration of pertussis
toxin are required to initiate active disease. Current and past
(11, 13) data suggest that clinically relevant epitope
spreading follows this hierarchical order in that responses proceed
from the most to the least dominant encephalitogenic myelin
determinants.
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, the relative encephalitogenicity of these three
myelin peptides is likely not due to their relative ability to bind to
MHC class II as all three peptides bound well to
I-As, with MBP84–104
exhibiting the highest affinity. In contrast, the precursor frequency
of T cells in the draining lymph nodes of SJL mice primed 7 days
previously with equimolar amounts of the three peptides directly
correlates with their relative encephalitogenicity and the order in
which responses arise during ongoing R-EAE. As determined by LDA, the
precursor frequency of PLP139–151-specific T
cells in the draining lymph nodes was 1/50,000, two to three times
the frequency of T cells specific for PLP178–191
and MBP84–104. Interestingly, the T cell
precursor frequency for each peptide was 5- to 10-fold higher when
determined by an IFN- ELISPOT assay.
PLP139–151-specific Th1 frequency was 1/5000,
two to six times the frequency of T cells specific for
PLP178–191 and
MBP84–104. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-EAE has proven to be a useful model in which to examine the initiation and development of epitope spreading during a progressive autoimmune disease. It has long been recognized that T cell specificities change during the course of R-EAE (3, 26, 27, 28, 29). The advantages of using R-EAE to study the pathogenesis of epitope spreading include: the ease of disease induction using defined myelin peptide epitopes; knowledge of encephalitogenic epitopes on a variety of myelin proteins in several inbred mouse strains (30, 31, 32, 33, 34); and knowledge of the relative encephalitogenic dominance of these myelin epitopes (1, 11, 13, 35). It has recently been demonstrated in PLP139–151-induced R-EAE in both SJL (11, 36) and (SJL x SWR)F1 mice (13) that clinical relapses are associated with the development of T cell responses to newly emerging epitopes on the same (i.e., intramolecular epitope spreading to distinct PLP epitopes) and/or different myelin epitopes (i.e., intermolecular epitope spreading to MBP epitopes). Moreover, these studies have clearly shown that the development of T cell reactivity to these endogenous myelin epitopes correlates with the extent of myelin destruction occurring during acute clinical disease (11). More recent evidence suggests that a similar pattern of epitope focusing and spreading may occur during the transition from isolated monosymptomatic demyelinating syndromes (a group of distinct clinical disorders with variable rates of progression to MS) to clinically defined MS (37). Thus, elucidation of the cellular and molecular mechanisms driving the epitope spreading process are critical to the design of efficient therapies for treating chronic inflammatory autoimmune diseases.
Because the mere existence of self-reactive CD4+
T cells does not necessarily initiate pathology in humans or in animal
models of autoimmunity, the development of immune reactivity to
additional autoepitopes during disease progression in R-EAE may or may
not play an inciting and/or regulatory role in chronic disease
pathogenesis. In the current study, we used a number of functional
criteria to determine the potential pathologic role of epitope
spreading in R-EAE disease progression in SJL mice. The results
indicate that T cells specific for spread epitopes play a major
pathologic role in mediating disease relapses. Clearly,
CD4+ T cells specific for relapse-associated
epitopes have encephalitogenic potential as they can serially transfer
R-EAE to naive recipients (Fig. 1). In addition, T cells specific for
relapse-associated epitopes are easily demonstrable in the CNS
immediately before and during the primary disease relapse (Fig. 2).
More significantly, induction of peptide-specific tolerance to
relapse-associated epitopes during remission from acute disease in both
MBP84–104- (Fig. 3) and
PLP139–151-induced R-EAE (Fig. 4) blocks disease
progression as assessed by the observation that tolerant animals
display a significantly reduced relapse rates. Interestingly, tolerance
to the relapse-associated epitope by the i.v. injection of
peptide-pulsed, ethylene carbodiimide-fixed splenocytes could also be
induced before disease initiation, in which case the animals underwent
a normal acute clinical disease course, but failed to exhibit the
primary relapses (Figs. 3A and 4A). This
indicates that tolerance induced by peptide-coupled splenocytes is
exquisitely Ag specific and does not cause immune deviation or
TGF-
-mediated bystander suppression as do tolerance protocols
previously employed to determine the role of T cell responses to
endogenous epitopes in R-EAE relapses (13). In a more
clinically relevant context, our results indicate that if the
specificity of T cells responsible for ensuing disease relapse is
known, peptide-specific peripheral tolerance is a powerful potential
method for treating ongoing autoimmune diseases because this approach
is effective in blocking disease relapses in animals in remission from
acute disease (Figs. 3B and 4B). The current data
thus provide direct and strong evidence that T cells specific for
endogenous myelin epitopes play the major pathologic role in the
relapsing phase of disease when compared with prior studies, which used
tolerance to a crude mouse spinal cord homogenate or to the intact PLP
molecule on fixed APCs (3, 11) or high doses of peptides
in IFA (13).
These findings have important implications for the design and use of
specific forms of immunotherapy for the treatment of autoimmune
diseases. Clearly peptide-specific tolerance is an effective method for
arresting ongoing disease progression provided the identity of the next
epitope in the spreading cascade is known and therapy is initiated
before the relapse (Figs. 3 and 4). Interestingly, disease progression
is blocked in the mice tolerized to the relapse-associated epitopes
although they retain significant peripheral T cell reactivity to the
disease-initiating epitope as assessed by DTH reactivity (Fig. 4C) and the ability of splenic T cells to transfer disease
following peptide reactivation in vitro (Fig. 2). This may indicate
that peripheral T cells specific for the disease-initiating epitope in
animals have down-regulated critical homing receptors required for
transmigration across the blood-brain barrier and/or they may be under
the control of a specific regulatory T population that prevents their
reactivation in situ. Alternately, the peptide-specific cells retained
in the periphery may have lower overall avidity than those cells that
originally trafficked to the CNS during acute disease. If epitope
spreading contributes to disease pathology in MS and other chronic
human autoimmune diseases, peptide-specific disease therapy will have
to be individualized for every patient due to the myriad of potential
organ-specific autoepitopes and extensive MHC diversity. However, the
efficient ability to inhibit ongoing R-EAE by blockade of the B7-1/CD28
costimulatory pathway using anti-B7-1 F(ab), even following the
initial clinical relapse (Fig. 5), provides a powerful alternative that
requires no prior knowledge of the identity of the relapse-associated
epitopes. Preliminary experiments indicate that short-term B7-1
blockade during disease remission results in long-term unresponsiveness
in T cells specific for relapse-associated epitopes (C. L.
Vanderlugt et al., manuscript in preparation) similar to the previously
reported ability of CTLA4-Ig to induce long-term, donor-specific
tolerance to transplantation Ags (38).
The results also indicate that epitope spreading in SJL mice occurs in
predictable hierarchical order similar to published results in EAE in
(SJL x SWR)F1 mice (13). The
temporal sequence of anti-myelin peptide responses did not
correlate with the affinity of the individual peptides for binding to
the I-As molecule, but correlated well with the
precursor frequency of T cells in the SJL repertoire specific for the
individual peptides (PLP139–151 >
PLP178–191 > MBP84–104),
which was directly related to the encephalitogenic dominance of these
determinants (Table I). Interestingly, precursor frequencies of
peptide-specific CD4+ T cells were considerably
higher when assessed by IFN- ELISPOT in comparison to conventional
LDA paralleling recent data for CD8+ CTLs
(39, 40). During the natural course of R-EAE, it is also
possible that the sequence of epitope spreading may be in part dictated
by the efficiency of the various peptides to be processed by relevant
APCs in the CNS or the periphery. The immunodominance of the
PLP139–151 response in the SJL mouse is quite
remarkable. In our experience, following acute myelin damage in
R-EAE initiated by either MBP84–104 (
Figs. 1–3)
or by PLP178–191, and in (SJL x
B10.PL)F1 mice with R-EAE initiated by
MBPAc1–11 (data not shown), spreading always
first involves T cell reactivity to PLP139–151.
PLP139–151 responses also arise within 2–3 wk
after the onset of myelin destruction in SJL mice infected with
Theiler’s murine encephalomyelitis virus (9) with
autoimmune responses to other encephalitogenic epitopes (including
PLP178–191, PLP56–70,
MOG92–106, and MBP84–104)
arising progressively later in the chronic disease.
While many questions remain about the initiation of the epitope spreading cascade and its significance in the pathogenesis and regulation of human autoimmune diseases, the results clearly indicate that clinically relevant epitope spreading in the relapsing-remitting SJL mouse model of MS follows a predictable hierarchical order related to the precursor frequency of myelin epitope-specific CD4+ T cells and is functionally important in disease progression. Moreover, these results indicate that peptide-specific tolerance is an effective therapy for an ongoing autoimmune disease. Because determining the specificity and hierarchical order of epitope spreading in human disease is not currently feasible, our results suggest that Ag-specific therapies for ongoing treatment of ongoing autoimmune disease may require peripheral tolerance induction strategies using whole tissue extracts (3), mixtures of encephalitogenic proteins/peptides (41), or costimulatory blockade (12).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; R-EAE, relapsing EAE; DTH, delayed-type hypersensitivity; MS, multiple sclerosis; PLP, proteolipid protein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; LDA, limiting dilution analysis.
Received for publication July 12, 1999. Accepted for publication November 1, 1999.
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