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Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, and Departments of
Pharmacology and Pathophysiology and
Pharmaceutics, Faculty of Pharmacy, Utrecht University, Utrecht, The Netherlands
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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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Previously it has been shown that allergen immunotherapy, by s.c. administration of increasing doses of allergen, can inhibit clinical symptoms and allergen-specific Th2 cytokine production upon challenge with the allergen (8, 9, 10). Although this classic form of immunotherapy is beneficial for treatment of rhinitis and insect venom allergy, it is less effective in allergic asthma (11). Furthermore, it has been reported that s.c. administration of allergens can induce severe systemic reactions, due to cross-linking of allergen-specific IgE on mast cells (12, 13). To prevent this cross-linking of IgE during immunotherapy, interest has focused on immunotherapy using small synthetic peptides of immunodominant allergen-derived T cell epitopes. It has been shown in a murine model that s.c. pretreatment with Fel d I peptide can prevent immediate hypersensitivity and airway hyperresponsiveness (14). Moreover, in the first clinical immunotherapy studies using peptides of immunodominant T cell epitopes of the major bee and cat allergens, amelioration of airway symptoms in humans (15, 16), which coincided with a reduced Th2 cytokine production in vitro, has been described (15, 17). However, Fel d I peptide therapy provoked side effects in 65% of the patients directly after injection with peptide (16). Recently, we showed in an experimental model of allergic asthma that s.c. treatment of OVA-sensitized mice with the immunodominant OVA323–339 epitope deteriorated both airway function and airway inflammation upon exposure to OVA (18). These findings indicate that immunotherapy using allergen-derived peptides, after allergen sensitization had already occurred, has the potential to activate allergen-specific Th2 cells, leading to an unfavorable enhanced Th2 cell response upon challenge with the allergen.
An alternative and safer approach for peptide immunotherapy could be the development of peptide analogues of the wild-type (WT)3 epitope bearing T cell modulatory capacities. Several studies have shown that stimulation of T cells with T cell epitope-derived peptide analogues can result in changes in the effector function of T cells, e.g., dissociation of proliferation and cytokine production, shifts in cytokine profile, or induction of anergy (19, 20, 21). There is ample evidence that these changes are caused by an altered interaction between the MHC/peptide-TCR complex, which affects the signaling through the TCR (22). Although the exact mode of action is largely unclear and the prediction of modulatory peptide analogues is still poor, the use of Th1-skewing or anergy-inducing peptide analogues for immunotherapy in allergic diseases has a great potential. Besides the prevention of cross-linking of IgE on mast cells, peptide therapy with Th1-skewing or anergy-inducing peptides could also prevent the induction of adverse side effects which can occur after WT peptide therapy.
In this paper, we have defined peptide analogues of an allergen-derived immunodominant epitope that modified allergen-specific Th2 cells in vitro as well as a Th2-dominated allergic response in vivo. We used a murine model of allergic asthma in which sensitization of BALB/c mice with OVA before OVA challenge resulted in airway hyperresponsiveness, eosinophilia, OVA-specific IgE, and production of Th2 cytokines upon OVA restimulation in vitro (23, 24). The immunodominant epitope of OVA, OVA323–339, is recognized by a population of OVA-specific Th2 cells, which produces large amounts of IL-5 upon stimulation with OVA323–339 in vitro and which plays an important role in the development of airway symptoms in vivo (18).
Our present data show that peptide immunotherapy with a Th1-skewing peptide analogue inhibited allergen-specific Th2 responses and airway inflammation, whereas a Th2-skewing peptide analogue, like the WT peptide, aggravated the ongoing allergic immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OVA323–339 WT peptide (ISQAVHAAHAEINEAGR) was obtained from Isogen Bioscience (Maarn, The Netherlands). Peptide analogues of OVA323–339 were synthesized as single alanine substitutions of all nonalanines. As control, peptide HA126–138 (HNTNGVTAASSHE) was used. Peptide analogues and control peptides were synthesized by automatic multiple peptide synthesis (25). For use in the MHC-peptide binding assay, marker peptide HA126–138 was biotinylated during synthesis. Peptides were analyzed, purified via reversed-phase HPLC, and checked by fast atom bombardment mass spectrometry.
Animals
Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Specified pathogen-free male BALB/c mice (6–8 wk old) were obtained from the Central Animal Laboratory (Utrecht, The Netherlands) and were housed in macrolon cages and provided with OVA-free food and water ad libitum. OVA323–339 TCR transgenic DO11.10 (26) mice were a gift from Prof. L. Adorini (Roche Milano Ricerche, Milan, Italy)
MHC class II binding assay
MHC class II binding assays were performed as described before
(27). Briefly, murine MHC class II
I-Ad molecules were purified from A20 hybridoma
cell lysates through affinity chromatography using the MKD6 Ab
(28). Purified MHC molecules were dissolved in PBS
containing 0.1% azide and 1% n-
-octyl glucopyranoside
(Sigma, St. Louis, MO). A total of 3 µM purified MHC class II
I-Ad molecules were incubated with 200 nM of
biotinylated HA126–138 and a dose range of
competitor peptide (WT or analogue; 0.5–250 µM) at pH 5 for 48
h at room temperature in the presence of a protease inhibitor mix
(final concentration 4.3 µM PMSF, 0.33 µM
N-
-p-tosyl-L-lysine
chloromethyl ketone, 0.35 µM
L-p-tosylamina-2-phenylethyl
chloromethyl ketone, 10 µM N-ethyl maleimide, 2.6 µM
ethylene diamine-tetra-acetic acid, 13 µM 1.10 phenanthroline, and
0.73 µM pepstatin A). Samples were analyzed by SDS-PAGE under
nonreducing conditions and blotted onto nitrocellulose (Hybond-ECL,
Amersham, Bucks, U.K.). Biotinylated peptide was visualized
through enhanced chemiluminescence (Western blot ECL kit, Amersham),
and IC50 values for the binding of biotinylated
HA126–138 for each peptide were determined.
Cells and culture medium
OVA323–339-specific DO11.10 T cells were
obtained by negative selection. DO11.10 splenocytes were incubated for
1 h with Dynabeads (Dynal, Oslo, Norway) coupled to Abs to MHC
class II I-Ad (MK-D6; Ref. 28), B220
(RA3–6B2; Ref. 29), and Fc
RII/III (24G2; Ref.
30) at 4°C. Negative selection was performed by two
passes over a magnetic particle concentrator (Dynal). Cells
obtained after depletion were shown to be >90%
OVA323–339-specific T cells as demonstrated by
FACS analysis using the clonotype-specific Ab KJ1.26 (31).
Viability of the cells was >95%. T cells were cultured in Iscove’s
medium supplemented with 10% FCS, 2 nM L-glutamine, 100 IU
penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME
(Iscove’s+).
Activation of DO11.10 T cells
To investigate the primary response of T cells toward the different peptides, 1 x 105 freshly isolated DO11.10 T cells were cultured with 2 x 105 irradiated BALB/c splenocytes (APC; 3000 rad) and a dose range of peptide (0.01–10 µg/ml) in 96-well plates. After 24, 48, 72, and 120 h supernatant was collected for cytokine analysis. At the same time points proliferation was determined in parallel cultures by pulsing the cells for another 16 h with [3H]thymidine. Cells were harvested on fiberglass filters, and [3H]thymidine incorporation was determined by liquid scintillation spectroscopy.
To study the capacity of the peptides to skew naive cells toward Th1 or Th2 cells, 4 x 106 freshly isolated DO11.10 T cells were cultured with 10 µg/ml peptide (WT or analogue) in the presence of 4 x 106 APC in a total volume of 2 ml. After overnight culture, cells were washed, dead cells were removed by Ficoll gradient (Lympholyte-M, Cedarlane Laboratories, Hornby, Ontario, Canada), and the remaining cells were cultured in Iscove’s+ for either 5 or 7 days. Cells were restimulated in 96-well plates (1 x 105 T cells + 2 x 105 APC/well) with a dose range (0.01–10 µg/ml) of WT peptide. Cytokine production and proliferation were determined after 24, 48, and 72 h of culture.
To determine the modulatory effects of the peptide analogues on
differentiated T cells, Th2 cells were generated. DO11.10 T cells were
cultured at 4 x 106 per well with 4 x
106 APC and 1 µg/ml WT peptide in a total
volume of 2 ml. After 7 days cells were washed, and dead cells were
removed by Ficoll gradient. Cells were restimulated with APC and WT
peptide under the same conditions used for the initial stimulation.
After the third cycle of stimulation, T cells displayed a Th2 phenotype
(high IL-4, IL-5, and intermediate IFN- production). Subsequently,
these Th2 cells were restimulated in 96-well plates (1 x
105 T cells + 2 x 105
APC/well) with a dose range (0.01–10 µg/ml) of WT peptide or peptide
analogue, and cytokine production was determined after 48 and 72 h
of culture.
Cytokine analysis
IL-2 production was measured using the IL-2-dependent CTLL2
clone (32). A total of 1 x 104
CTLL2 cells were cultured with 100 µl supernatant collected from the
DO11.10 T cell cultures. After 24 h of culture, cells were pulsed
for 16 h with [3H]thymidine, and
proliferation was determined. Levels of IL-4, IL-5, IL-10, and IFN-
in DO11.10 culture supernatants were determined by capture ELISA
(PharMingen, San Diego, CA) as described by the manufacturer. The
detection limits of the ELISAs were 16 pg/ml for IL-4 and IL-5, and 100
pg/ml for IL-10 and IFN-.
Flow cytometric analysis of T cell activation
For flow cytometric analysis of T cell activation, 1 x
105 freshly isolated DO11.10 T cells were
cultured in the presence of 2 x 105
irradiated BALB/c splenocytes (APC) and 1, 5, or 10 µg/ml peptide in
96-well plates. After 24 h incubation, cells were collected and
incubated with PBS buffer containing 5% rat serum and 1:500 diluted
supernatant of 24G2 hybridoma cells (FcRII/III). Subsequently,
cells were stained with annexin-V-FITC, Abs against Thy1.2-PE (clone
30-H12; 1:200), CD69-FITC (clone H1.2F3; 1:200), CD80-FITC (clone 1G10;
1:100), CD86-FITC (clone GL1; 1:100), or the relevant isotype controls
(PharMingen). Dead cells were excluded using propidium iodide. Cells
were analyzed on a FACS scan apparatus using Cell Quest (Becton
Dickinson, San Jose, CA).
Preparation of peptide-liposomes
Liposomes were prepared as described by ’t Hart et al. (33) with minor modifications. Briefly, phospholipids and cholesterol (egg-yolk phosphatidyl choline (EPC): egg-yolk phospatidyl glycerol (EPG): cholesterol = 10:1:4 molar ratio) were dissolved in a mixture of chloroform and methanol (3:1 v/v) in a round-bottom flask. The solvent was removed through rotary evaporation at 40–50°C under reduced pressure for 1–2 h. Lipid films were subsequently flushed with nitrogen for at least 20 min. The lipids were hydrated with a small volume of the peptide solution (25 mg/ml in PBS) by vigorously shaking at room temperature (initial phospholipid concentrations were >100 mM). Glass beads were added for optimal dispersion of the lipid film. Subsequently, the dispersions were diluted with small aliquots of PBS. The external phase was removed via three rounds of ultracentrifugation (20,000 x g for 30 min at room temperature). Final liposome pellets were dispersed in PBS. By preparing the liposomes in this way, high-encapsulation efficiencies of the peptides could be achieved (routinely, 40–50% encapsulation as assayed via HPLC analysis).
Disease induction and treatment protocol
Active sensitization was performed without adjuvant by 7 i.p. injections of 10 µg OVA (grade V, Sigma) in 0.5 ml saline on alternate days (23). Peptide treatment was performed 14 days after the last sensitization by s.c. injection of 150 µl liposomes containing 300 µg of peptide. Starting six days after liposome administration, mice were exposed to OVA (2 mg/ml) or saline aerosol challenge for 5 min on 8 consecutive days. Aerosols were performed in a Plexiglas exposure chamber coupled to a Jet nebulizer (Pari IS-2 Jet nebulizer, Pari Respiratory Equipment, Richmond, VA; particle size 2–3 microns) driven by compressed air at a flow rate of 6 L/min. Aerosols were given in groups of a maximum of six animals.
Determination of OVA-specific Igs
Serum samples were taken 24 h after the last challenge, and OVA-specific IgG1, IgG2a, and IgE were determined by ELISA as described before (18, 34). The detection limits of the ELISA were 0.005 U/ml for IgG1, 0.05 U/ml for IgG2a, and 0.5 U/ml for IgE.
Analysis of bronchoalveolar lavage (BAL) cells
After blood collection, a cannula was placed in the trachea, and lungs were lavaged five times with 1 ml aliquots of pyrogen-free saline warmed to 37°C. The lavage cells were washed with cold PBS, and the cells were resuspended in 150 µl cold PBS. A Burker-Türk chamber was used to count the total number of BAL cells. For differential BAL cell counts, cytospin preparations were made and stained with Diff-Quick (Merz & Dade, Düdingen, Switzerland). Per cytospin, 400 cells were counted and differentiated into mononuclear cells, lymphocytes, neutrophils, and eosinophils by standard morphology.
Cytokine analysis of lung-draining lymph node (LN) cells
Twenty-four hours after the last aerosol, lung-draining LN were
collected, and single cell suspensions were made. Cells (2 x
105 cells/well in 96-well plates) were cultured
in Iscove’s+ in the presence of OVA (10 µg/ml), medium, or control
Ag hen egg lysozyme (Sigma; 10 µg/ml). As a positive control, cells
were cultured with immobilized CD3 Ab (CD3; clone 17A2, 50 µg/ml;
Ref. 24). After 120 h of culture, supernatants were
collected for cytokine analysis.
Statistics
Unless stated otherwise, data are expressed as mean ± SEM and evaluated using two-way ANOVA and then a Dunnett test for comparison between two groups. A p value <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twelve peptide analogues were synthesized based on single alanine
substitutions of all nonalanine residues in peptide
OVA323–339 (WT peptide). First, peptide
analogues were tested for their ability to induce proliferation and
cytokine production in freshly isolated DO11.10 T cells. Peptide
analogues 323I-A, 324S-A, 325Q-A, 327V-A, 334I-A, 338G-A, and 339R-A
induced T cell proliferation comparable to that of WT peptide (Table I
). Peptides 331H-A and 333E-A did not
induce proliferation (Table I), and higher concentrations (up to 500
µg/ml) could not restore proliferation (data not shown). Peptides
328H-A and 335N-A induced proliferation, but were 20–50 times less
potent than WT peptide. In contrast, peptide 336E-A had a
superagonistic effect and was significantly more potent than WT peptide
in inducing proliferation (Table I).
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, IL-4, and IL-5 production
(Table I
). Peptide analogues that induced proliferation comparable to
that of WT peptide also induced comparable levels of IL-2, IFN-,
IL-4, and IL-5 (Table I). Corresponding with the lack of proliferation,
cytokine production was undetectable after stimulation with peptides
331H-A, and 333E-A. The partial agonistic peptide 328H-A could only
induce detectable levels of IL-2 and IFN-, which were significantly
reduced compared with levels after WT peptide incubation, whereas
partial agonistic peptide 335N-A induced significantly reduced levels
of IL-2, IFN-, IL-4, and IL-5. Interestingly, the superagonistic
peptide 336E-A was more potent than the WT peptide for inducing IL-2
and IFN-, but both IL-4 and IL-5 levels were comparable with those
from WT peptide stimulation. This indicates that the 336E-A peptide was
not only more potent in activating DO11.10 T cells, but also induced a
shift in the cytokine profile. Proliferation and cytokine data are
shown for incubation with respectively, 0.1 and 1 µg/ml peptide. The
characteristics of the peptides did not change in the entire dose range
(0.01–10 µg/ml) tested (data not shown). Based on these data,
peptide analogues that induced changes in T cell proliferation and
cytokine production compared with results achieved with WT peptide
(peptides analogues 328H-A, 331H-A, 333E-A, 335N-A, and 336E-A) were
selected for further study.
To determine whether the observed modulatory effects on T cell
activation were due to altered MHC binding affinity of the peptide
analogues rather than altered TCR interaction, the selected peptide
analogues 328H-A, 331H-A, 333E-A, 335N-A, and 336E-A were tested for
relative MHC class II I-Ad binding affinity. All
peptide analogues, except peptide 328H-A, had a similar MHC binding
affinity to that of WT peptide (IC50 < 1–5
µM; Table II). Because 328H-A had a
strongly decreased affinity for MHC class II, the final panel of
peptide analogues for further study consisted of WT peptide, 331H-A,
333E-A, 335N-A, and 336E-A.
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To study T cell activation, freshly isolated DO11.10 T cells were cultured overnight with 10 µg/ml peptide (WT or analogue) and APC. T cells were stained with anti-Thy1.2-PE and analyzed for CD69, CD80, CD86, and annexin-V expression using FITC-labeled mAbs.
Compared with a nonrelated control peptide (Fig. 1, dotted-line histograms), incubation
with WT peptide resulted in a strong expression of CD69 on T cells.
Furthermore, up-regulation of B7.2 (CD86) was seen, whereas B7.1 (CD80)
levels remained low. Analysis of the two peptide analogues that did not
induce proliferation or cytokine production (Table I) showed that
incubation with peptide 331H-A induced an up-regulation of CD69, but
B7.1 and B7.2 expression remained low, whereas incubation with peptide
333E-A did not result in any up-regulation of CD69, B7.1, or B7.2 (Fig. 1). Incubation with the partial agonistic peptide 335N-A and the
superagonistic peptide 336E-A led to up-regulation of CD69 and B7.2
comparable to that found after incubation with WT peptide. Similar
results were obtained after incubation with 1 or 5 µg/ml peptide.
Furthermore, none of the tested peptides induced apoptosis as measured
by annexin-V/propidium iodide double staining (data not shown).
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In subsequent experiments, we tested the ability of the peptide
analogues to skew freshly isolated DO11.10 T cells toward a Th1 or Th2
phenotype. T cells were preincubated with 10 µg/ml peptide (WT or
analogue) and APC, expanded, and subsequently restimulated with WT
peptide. T cells preincubated with medium, 331H-A, or 333E-A showed a
comparable dose-dependent proliferation (Fig. 2A) and IL-2 production (Fig. 2B) upon restimulation with WT peptide. Preincubation of T
cells with WT peptide, 335N-A, or 336E-A significantly augmented the
proliferation and IL-2 production induced by the WT peptide upon
restimulation. Whereas WT peptide and 336E-A preincubation led to a
comparable 50- to 100-fold increased response upon WT peptide
restimulation, peptide 335N-A preincubation resulted in a >1000-fold
increased response upon WT stimulation (Fig. 2, A and
B).
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, IL-4, IL-5, and IL-10 production were observed
upon restimulation with WT peptide. Cells that were preincubated with
medium, 331H-A, or 333E-A showed a cytokine profile upon WT peptide
restimulation similar to that found in freshly isolated DO11.10 T cells
(intermediate levels of IFN- and IL-5 and low levels of IL-4; Fig. 3). Preincubation with both WT peptide
and partial agonistic peptide 335N-A profoundly increased the
production of Th2-associated cytokines IL-4 (10-fold) and IL-5
(3-fold), whereas IFN- production was only doubled (Fig. 3).
Interestingly, preincubation with the superagonistic peptide 336E-A
resulted in a Th1-like cytokine profile. Upon WT peptide restimulation,
both IL-4 and IL-5 production were low and comparable to levels
produced by naive cells, whereas IFN- production was significantly
increased. Data are shown for restimulation with 1 µg/ml WT peptide,
but the characteristics were essentially the same for the entire dose
range (0.1–10 µg/ml) tested (data not shown).
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Next we studied the effects of the peptide analogues on polarized
Th2 cells. Th2 cells were generated by culturing DO11.10 T cells with
WT peptide and APC for three stimulation cycles. After the third cycle,
T cells displayed a Th2 phenotype (high IL-4 and IL-5, and intermediate
IFN- production). Restimulation of such Th2 cells with WT peptide
resulted in production of high levels of IL-4 and IL-5, and
intermediate levels of IFN- (Fig. 4).
Restimulation with peptide analogues 331H-A and 333E-A did not result
in any detectable cytokine production. Interestingly, partial agonistic
peptide 335N-A induced high levels of IL-4 and IL-5 (comparable to
levels found after WT peptide stimulation), but failed to induce
IFN- production. In contrast, superagonistic peptide 336E-A did not
induce IL-4 and only induced small amounts of IL-5, whereas IFN-
production was significantly increased (Fig. 4). These data show that
the WT peptide and the partial agonistic peptide 335N-A favor a further
skewing toward a Th2 cytokine profile, whereas superagonistic peptide
336E-A can modulate already polarized Th2 cells toward a Th1 profile.
Data are shown for cell lines obtained after three restimulations with
1 µg/ml peptide, but the characteristics were essentially the same
for cell lines that were obtained after restimulations with 5 or 10
µg/ml (data not shown).
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To study the effect of peptide administration in a Th2-mediated disease process, we used a murine model of allergic asthma. Because it was expected that, compared with protein, the half lives of peptides were rather low, we compared three different peptide administration protocols.
Previously we showed that two s.c. administrations of 150 µg WT
peptide (with a 3-day interval) in OVA-sensitized mice led to a
significant increase in airway hyperresponsiveness and eosinophilia
upon challenge with OVA (18). Now we compared this former
immunotherapy protocol with a single high dose (300 µg) of WT
peptide, or one dose of liposomes containing 300 µg WT peptide, which
can act as a depot. Six days after the treatment, mice were challenged
with OVA, and infiltration of eosinophils in the airways was determined
by BAL. Comparison of the different treatment protocols showed clearly
that 2 x 150 µg WT peptide augmented infiltration of
eosinophils more than a single high dose of 300 µg (Fig. 5). However, a single dose of 300 µg WT
peptide incorporated in liposomes showed the strongest aggravation of
airway inflammation (Fig. 5). Although treatment with the WT peptide
induced a nondesirable aggravation of the disease, the liposome-peptide
formula appeared to be the most efficient method for peptide
administration. Therefore, we selected the liposome-peptide formula for
immunotherapy.
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To determine the effect of the peptide analogues in vivo, we incorporated the peptide analogues in liposomes and administered the liposomes s.c. in OVA-sensitized mice. Six days after treatment, mice were challenged with OVA or saline, and airway eosinophilia, OVA-specific Igs, and OVA-specific T cell responses were measured.
To analyze inflammatory cells in the BAL fluid, BAL was performed
24 h after the last aerosol. In all treatment groups, OVA
challenge induced a significant increase of total numbers of cells
compared with the saline-challenged groups (Table III). The increase in total number of
cells was largely due to the increase of eosinophils, because no
significant changes in neutrophils or mononuclear cells were found
(Table III). As shown previously, WT peptide treatment resulted in a
significant increase in eosinophils in OVA-challenged mice compared
with those induced after vehicle treatment (Table III; Fig. 6). Treatment with the Th2-skewing
partial agonistic peptide 335N-A also resulted in a significant
increase in eosinophil infiltration (Table III; Fig. 6). Interestingly,
treatment with the Th1-skewing superagonistic peptide 336E-A led to a
significant reduction of eosinophil infiltration compared with
vehicle-treated mice (Table III; Fig. 6). Treatment with 331H-A or
333N-A had no effect on eosinophil infiltration in OVA-challenged
mice.
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). The production of
OVA-specific IgG1 and IgE in these mice indicated that already during
the sensitization period OVA-specific Th2 cells and B cells were
induced. OVA challenge of sensitized mice induced a significant rise of
OVA-specific IgG1 and IgE in all treatment groups (Table IV) compared
with corresponding saline-challenged groups. OVA-specific IgG2a was
slightly, but not significantly, increased after OVA challenge compared
with the results of saline challenge. None of the peptides used for
immunotherapy did affect the levels of OVA-specific IgG1, IgG2a, or IgE
(Table IV).
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CD3 (data not shown). In contrast, LN
cell cultures from OVA-challenged mice showed substantial levels of
IL-4, IL-5, and IL-10 after in vitro OVA restimulation (Fig. 7). Compared with vehicle treatment,
treatment with WT peptide, 331H-A, 333E-A, and 335N-A did not affect
OVA-specific IL-4, IL-5, or IL-10 production. Remarkably, treatment
with the Th1-skewing peptide 336E-A resulted in a significant reduction
of IL-4 and IL-5, but not of IL-10 production (Fig. 7). Interestingly,
in none of the groups was IFN- production detectable after in vitro
restimulation with OVA, but CD3 induced similar levels of IFN- in
all groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the definition of modulatory peptide analogues we used different in
vitro assays. Based on changes in proliferation, cytokine production,
or Th1/2 skewing capacities, four modulatory peptide analogues were
selected for in vivo studies. Importantly, the MHC class II binding
affinity of these peptide analogues was comparable with that of the
OVA323–339 WT peptide. Using
OVA323–339-specific DO11.10 T cells, we showed
that the agonistic WT peptide was a strong inducer of Th2 cytokines and
favored skewing toward a Th2 phenotype. Peptide analogue 331H-A
appeared to be a partial agonist, inducing T cell activation as
measured by CD69 up-regulation, whereas no proliferation, cytokine
production, or anergy was induced in the DO11.10 T cells. Stimulation
of DO11.10 T cells with peptide 333E-A did not induce any T cell
activation. Also, more conserved substitutions (like 333E-D) failed to
induce T cell activation (data not shown), suggesting that position
333E is a primary TCR contact residue in which no substitutions are
tolerated. Peptide analogue 335N-A was a partial agonist, inducing
cytokines similar to those induced by WT peptide (albeit at lower
levels). Comparable with the WT peptide, 335N-A also skewed toward a
Th2 phenotype. In contrast, peptide analogue 336E-A was a superagonist
and strongly promoted skewing toward a Th1 phenotype. More importantly,
peptide analogue 336E-A even induced a Th1 cytokine profile in an
already polarized Th2 population, leading to an enhanced IFN-
production and an almost complete absence of IL-4 and IL-5 production.
Although there is a clear shift toward a Th1 profile in this Th2-skewed
population, we cannot dissect whether the changes in cytokine pattern
resulted from an inhibition of IL-4 and IL-5 in fully polarized Th2
cells, or from an increased IFN- production by and proliferation of
less polarized cells.
How peptide analogues induce T cell modulatory effects is still poorly
understood (35, 36). It has been reported that changes in
the MHC class II binding affinity of peptide analogues may lead to
altered T cell activation. Several studies have suggested that peptide
analogues with low affinity for MHC induce IL-4 production and Th2
development, whereas peptide analogues with high affinity promote Th1
development (35, 36). However, our data show that this is
no general rule because the partial agonistic peptide 328H-A, which has
decreased MHC binding affinity compared with the WT peptide, induced
Th1 (IL-2 and IFN-), but not Th2 cytokines (Table I). Alternatively,
modification of TCR contact residues within the peptide has been
suggested to affect the dissociation of the TCR from the peptide MHC
complex and, consequently, to change the early biochemical events
leading to T cell activation (37, 38). In vitro studies
have indicated that weak TCR-mediated signaling preferentially induces
Th2 differentiation, or anergy, whereas strong TCR-mediated signaling
induces Th1 responses (20, 39). To distinguish between
modulatory effects due to changes in MHC binding affinity and altered
TCR affinity, we selected only peptide analogues with MHC binding
affinity comparable with WT peptide. Our findings suggest that peptide
analogues 331H-A and 333E-A have a very low affinity, 335N-A has an
intermediate affinity, and 336E-A a high affinity for the
OVA323–339-specific DO11.10 TCR.
Previously it has been reported that position 331H in the OVA323–339 peptide is an important TCR contact residue for I-Ad-restricted T cell recognition (40). Our data indicate, besides the 331H TCR contact residue, the presence of at least three other TCR contact residues at positions 333E, 335N, and 336E. Recently, Scott et al. (41) showed, after crystallization of MHC class II I-Ad with OVA323–339, that residues 323–333 were involved in MHC binding and that residue 331H protruded toward the TCR. Although 323–333 is probably the most preferable peptide core sequence for MHC binding, they suggested that I-Ad can bind OVA323–339 in two alternative alignments due to the minimal side-chain requirements for peptide binding by I-Ad (41). In these two alternative alignments involving residues 325–335 and 328–338, residues 333E and 336E protrude from the MHC toward the TCR, like 331H in the crystallized alignment (41). This may explain why modifications of residues 335 and 336 which, according to the most abundantly present alignment, are not involved in MHC or TCR binding can have such a clear effect on T cell activation. Although we cannot exclude that the two other described alignments are important for recognition of OVA323–339 by the DO11.10 TCR, our data suggest that alignment 328–338, containing all four TCR contact residues as defined in this study, would be the most favorable conformation recognized by the DO11.10 TCR.
To study the possibilities of peptide analogue therapy in the experimental asthma model, we treated mice s.c. with peptide analogues after OVA sensitization. Subsequently, mice were challenged with OVA. Our data show that treatment with peptide analogues that were very poor T cell activators (331H-A and 333E-A) had no effect on airway eosinophilia and cytokine production. In contrast, administration of the Th2-skewing WT peptide and the Th2-skewing partial agonistic 335N-A peptide dramatically increased airway eosinophilia upon OVA challenge, indicating that the Th2 response in the lungs was augmented. Treatment with the Th1-skewing 336E-A peptide resulted in a significant decrease in eosinophilia and OVA-specific IL-4 and IL-5 production by the lung-draining LN cells.
In all experimental groups, OVA-specific IgE was significantly
increased after OVA challenge compared with the results of saline
challenge, but was not affected by peptide therapy, indicating that
there is no clear correlation between serum levels of OVA-specific IgE
and the degree of airway inflammation. These findings correspond with
our previous study (18) and the studies by Korsgren et al.
(42) and Mehlhop et al. (43), who
demonstrated that airway inflammation can even occur in B
cell-deficient and IgE-knockout mice. Although peptide analogue therapy
did not clearly affect B cell responses, our data show that T cell
responses were modulated. Treatment with Th2-skewing peptides enhanced
the Th2-associated allergic response, whereas a Th1-skewing peptide
analogue inhibited OVA-specific Th2 cells in vivo. Because the
Th1-skewing peptide 336E-A had the capacity to inhibit IL-4 and IL-5
production in polarized Th2 cells in vitro, the observed reduction of
OVA-specific Th2 cytokines in vivo after treatment with this peptide
analogue may be due to a peptide-induced Th2
Th1 cytokine shift in
OVA-specific Th2 cells in vivo. We and others have shown that treatment
with the Th1-associated cytokine IFN- before and during challenge
significantly reduced airway hyperresponsiveness and eosinophil
infiltration in murine asthma models (44, 45). These
findings suggest that IFN- plays an important role in the
down-regulation of a Th2-mediated allergic response. However, although
peptide 336E-A induced high levels of IFN- in vitro, after peptide
therapy, no IFN- production by lung-draining LN cells or
IFN--induced IgG2a could be detected. Alternatively, it is possible
that the reduction of Th2 cytokines in vivo was due to the induction of
anergy or activation-induced cell death. It has recently been reported
that anergy followed by activation-induced cell death can be induced in
effector T cells by stimulation with high doses of Ag and by antigenic
stimulation that is prolonged beyond an optimal time (46).
Increasing the affinity of the MHC peptide complex for the TCR may
result in overstimulation of these Th2 cells and eventually may lead to
anergy or apoptosis. A third possibility could be the induction of a
regulatory T cell population, consisting of either a novel T cell
subset or the modulated Th2 cells. Previously it has been shown that
IL-10 is a very powerful regulatory cytokine (47) and that
successful immunotherapy in humans is associated with increased
production of IL-10 by allergen-specific T cells (48).
Although our data do not show an increase in OVA-specific IL-10
production, it is possible that the altered IL-4/IL-10 and IL-5/IL-10
ratios after Th1 peptide analogue therapy positively contributed to the
reduction of the allergen-specific Th2 response.
In summary, we have shown that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate an allergen-specific polarized Th2 response in vitro. More importantly, we demonstrated for the first time that an in vitro-defined Th1-skewing peptide analogue can modulate an ongoing allergen-specific Th2 response in vivo, thereby inhibiting airway inflammation. Furthermore, the efficacy of peptide immunotherapy was clearly correlated with Th1- or Th2-skewing characteristics of the therapeutic peptide as defined in vitro. These findings indicate that the design of Th1-skewing peptide analogues, instead of using WT peptides, may improve peptide immunotherapy and may contribute to the development of successful and safe allergen-specific immunotherapy for allergic patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. H. M. Wauben, Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80165, 3508 TD Utrecht, The Netherlands. E-mail address:
3 Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; LN, lymph node.
Received for publication August 6, 1999. Accepted for publication October 20, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by T cells. Proc. Natl. Acad. Sci. USA 92:9510. on immunoglobulin E, cellular infiltration, and airway responsiveness in a murine model of allergic asthma. Am. J. Respir. Cell Mol. Biol. 19:826. decreases IgE production and normalizes airway function in a murine model of allergen sensitization. J. Immunol. 152:2546.[Abstract]
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, WT; +, 331H-A; 
,
333E-A; 
, 335N-A; 
, 336E-A
