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Department of Allergy and Clinical Immunology, Imperial College School of Medicine, National Heart and Lung Institute, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in cultures. Thus, allergen-derived peptides induce tolerance to
subsequent peptide injection in the target organ (the lung), reduce
late-phase cutaneous responsiveness to whole allergen, and alter in
vitro T cell reactivity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies in mice have shown that the development of T cell
tolerance in vivo is preceded by transient T cell activation. Webb et
al. (2) reported that the clonal elimination of
V
6+ cells responding to a superantigen was
preceded by marked expansion of these cells, whereas Vidard et al.
(3) found that before the establishment of specific T cell
tolerance to OVA, T cells displayed transient responsiveness and
synthesized IL-2 upon antigenic stimulation in vitro. Similar findings
were reported by Hoyne et al. (4) using an immunodominant
peptide derived from house dust mites. In this model, a strong,
transient T cell CD4+ response in vitro preceded
the development of tolerance in vivo.
Peripheral T cell tolerance can also be induced in both naive and primed mice by s.c. injection of peptides from the major cat allergen, Fel d 1 (5). We previously demonstrated that short Fel d 1-derived peptides directly initiated T cell-dependent, late asthmatic reactions (LARs),3 without the requirement for an early IgE/mast cell-dependent response, in cat-allergic asthmatics (6). LARs occurred only in those individuals expressing appropriate HLA restriction elements for one or more of the injected peptides. In the present study, we have tested the hypothesis that in humans, as in mouse models of tolerance, allergen-derived peptides induce hyporesponsiveness to rechallenge with peptides either clinically or in terms of in vitro T cell responses.
It has also been proposed that allergen-specific immunotherapy, as with other forms of immune modulation, may involve mechanisms by which nonresponsiveness induced to one epitope of a molecule confers nonresponsiveness either to other epitopes within the whole molecule (intramolecular epitope suppression) (7), adjacent molecules (bystander tolerance), or is passed to the next generation of regulatory T cells (infectious tolerance) (8). For this reason, we speculated that in vivo administration of Fel d 1 peptides would inhibit the late-phase allergic reaction, and in vitro T cell reactivity, induced by whole cat allergen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cat-allergic asthmatics were recruited, diagnosed, and assessed as described in detail elsewhere (6). The study received prior approval from the Ethics Committee of the Royal Brompton and Harefield Hospitals National Health Service Trust, (London, U.K.), and written witnessed informed consent was obtained. Twenty-four subjects were recruited in three groups of eight subjects into an open dose-ranging study. Group 1 received 1 µg peptide, group 2 received 2.5 µg peptide, and group 3 received 5 µg peptide. On study day 1 (control day), subjects had venous blood withdrawn for proliferation and cytokine assays and then received an intradermal (ID) injection of whole cat dander extract (30 BU; ALK-Abello, Horsholm, Denmark) into the volar surface of the left forearm. The cutaneous reaction was recorded after 15 and 360 min, as previously described (9). Forced expiratory volume in 1 s (FEV1) was measured at 5, 15, 30, and 60 min, and hourly thereafter for an additional 5 h. At least 2 wk later, subjects received Fel d 1-derived peptides via the ID route. Clinical monitoring of skin and lung reactions was the same as study day 1. Two weeks after peptide challenge, the eight subjects in group 3 (5 µg peptide) had a second ID challenge with whole cat dander, and blood was withdrawn for proliferation and cytokine assays. Clinical monitoring was performed as on day 1. To assess the development of peptide-specific bronchial hyporesponsiveness, the seven subjects (one from group 1, two from group 2, and four from group 3) who developed a LAR on initial peptide challenge during the pilot study were rechallenged with the same dose of Fel d 1-derived peptides between 2 and 14 wk later. A positive late-phase asthmatic reaction was defined as before, i.e., a fall of >20% in FEV1 from baseline (6). The magnitude, as well as the frequency, of the LARs was dose dependent. Thus, the LARs observed with 5 µg produced a proportionately greater decrease in the FEV1, and a more sustained reaction, than the 1 and 2.5 µg doses. The decrease in the FEV1 in the 5 µg group was significantly greater than either the 2.5 µg group (p = 0.031), or the 1 µg group (p = 0.015), or the control day (p = 0.008). Clinical monitoring was performed as described (6). A further seven subjects who developed a LAR following the first administration of 5 µg of the 12 peptides received a second injection of the same dose between 3 days and 68 wk later as part of a separate study.
Peptide synthesis and validation
Sixteen overlapping peptides from chains 1 and 2 of Fel d 1 were synthesized by F-moc chemistry at the Advanced Biotechnology Center, Imperial College School of Medicine (London, U.K.). The sequences were: chain 1, 1) EICPAVKRDVDLFLTGT, 2) LFLTGTPDEYVEQVAQY, 3) EQVAQYKALPVVLENA, 4) KALPVVLENARILKNCV, 5) RILKNCVDAKMTEEDKE, 6) KMTEEDKENALSLLDK, 7) KENALSVLDKIYTSPL; chain 2, 8) VKMAETCPIFYDVFFA, 9) CPIFYDVFFAVANGNEL, 10) GNELLLKLSLTKVNAT, 11) LTKVNATEPERTAMKK, 12) TAMKKIQDCYVENGLI, 13) CYVENGLISRVLDGLV, 14) SRVLDGLVMTTISSSK, 15) ISSSKDCMGEAVQNTV, 16) AVQNTVEDLKLNTLGR. Peptides 8, 9, 10, and 14 were relatively insoluble due to a high proportion of hydrophobic residues, and were consequently excluded from the injected, in vivo, preparation. The remaining 12 peptides were mixed and used for in vivo studies. They produced less than 2% histamine release from basophil-enriched PBMC, in vitro, over the concentration range 0.02–100 µg/ml. In contrast, whole cat extract (0.02–200 µg/ml) induced up to 40% release of the total histamine, in a dose-response fashion, as previously reported (6).
Primary PBMC proliferation assays
These were performed as previously described (6). Stimulation indices (used to allow pre-post comparisons) were calculated by comparing the Ag-containing wells with those containing medium alone.
Cytokine assays
PBMCs were cultured at 5 x 105
cells/well in the presence of individual peptides (100 µg/ml), whole
cat dander (100 µg/ml), or purified protein derivative (PPD; 10
µg/ml), for 6 days before the collection of culture supernatants.
Supernatants were frozen at -20°C before analysis. IL-4, IL-13, and
IFN- were analyzed in duplicate by ELISA (Pelikine, CLB, Amsterdam,
The Netherlands). The sensitivity of the assays was 0.4 pg
ml-1 (IL-4), 1.5 pg ml-1
(IL-13), and 2 pg ml-1 (IFN-).
Statistical analyses
LARs. FEV1 data were summarized over time for each subject for the control day and the 2 peptide days. Areas under each curve for the FEV1 measured over 6 h were calculated using the trapezoidal rule. Differences in the areas under each curve between the control day and peptide day 1, peptide days 1 and 2, and peptide day 2 and control day were analyzed by paired t test.
Allergen-induced cutaneous reaction. Differences in the magnitude of the allergen-induced early- and late-phase skin reactions measured as described (9) were analyzed using a nonparametric Wilcoxon signed rank test.
Proliferation data. Data were tested for normality using a modified Shapiro-Wilks test. Because significant deviations from normality were found, differences between pre and post were calculated and the changes tested with a nonparametric Wilcoxon signed rank test.
Cytokine production. Data were tested for normality using a modified Shapiro-Wilks test. No significant differences from normality were found. Differences between pre and post were calculated and the changes tested with a paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven subjects who developed a LAR following ID challenge of Fel d
1 peptides were subsequently challenged on a second occasion, 2–14 wk
later, and the results compared with the control day. The first
challenge was associated with a significant (p
= 0.012) decrease in FEV1 compared with control
(Fig. 1
). The second ID challenge did not
induce a LAR (second challenge vs control day, p =
0.82). The differences between the two Fel d 1 peptide challenges were
statistically significant (p = 0.003).
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). There was then a "window" between
2 and 8 wk, when there was marked bronchial hyporesponsiveness to a
second peptide challenge with no evidence of a LAR and patients were
asymptomatic. This was then followed by a gradual return in bronchial
responsiveness to peptide challenge over a 32- to 40-wk interval, at
which time the magnitude of the second LAR was similar to the first
LAR. The data in Fig. 2 were analyzed using S-Plus (Mathsoft,
Cambridge, MA) and modeled using a smoothing spline with
cross-validation to determine the penalty.
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The effect of a single injection of Fel d 1 peptides on the
allergen-induced early- and late-phase cutaneous reaction to whole cat
dander was evaluated in the eight subjects who received 5 µg peptide
(Fig. 3). No difference was observed in
the magnitude of the early (15-min) reaction. There was a significant
(p = 0.014) decrease in the cross-sectional
area of the cutaneous late-phase (6-h) reaction, with all subjects
showing reduced reactivity. The development of a peptide-induced LAR
was not a requirement for subsequent attenuation of the late-phase skin
reaction because four of eight subjects did not experience a LAR
following the first injection of Fel d 1 peptides.
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The effect of a single administration of Fel d 1 peptides on
proliferation and IL-4, IL-13, and IFN- production induced by the
individual injected peptides is shown in Fig. 4. When pre- and postpeptide challenge
samples (PBMC) were compared, responses to all peptides appeared to be
reduced, although in some cases this did not achieve statistical
significance. There were statistically significant decreases in
proliferation (peptides 1, 11, 12, 13, 15, and 16), IL-4 (peptides 4,
6, and 12), IL-13 (peptides 1, 2, 3, 4, 5, 15, and 16), and IFN-
concentrations (peptides 1, 2, 5, 6, and 7). In contrast, there were no
significant differences in either proliferation, IL-4, IL-13, or
IFN- (pre- vs postchallenge) to the recall Ag PPD. When the data
from the individual peptides were pooled, there were significant
differences (pre- vs postinjection) for proliferation (2.03 (1.68
- 4.56) vs 1.22 (1.04 - 1.52), p = 0.007), IL-4
(8.99 (± 1.33) vs 7.35 (± 1.25), p = 0.002), IL-13
(38.47 (± 5.67) vs 22.39 (± 6.30), p = 0.015), and
IFN- (33.35 (± 10.31) vs 8.94 (± 4.58), p =
0.021). The data are expressed as median with interquartile range
(proliferative response) and mean ± SE (cytokine production).
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). There were
significant decreases in proliferation (p =
0.023) and IL-13 (p = 0.032), and trends for a
reduction in IL-4 (p = 0.052) and IFN-
(p = 0.084) release. Again, no significant
differences were observed (pre- vs postpeptide challenge) in
PPD-induced proliferation and cytokine production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an earlier study, Norman and colleagues (10) attempted to treat cat-allergic subjects by s.c. injection of two peptides (termed IPC1 and IPC2), which spanned a large proportion of chain 1 of Fel d 1. However, IPC1 and IPC2 were 27 residues long and associated with immediate and late allergic symptoms that occurred between 10 min and 6 h after s.c. injections. In contrast, the Fel d 1 peptides in the present and previous study (6) were of relatively small size (16/17 residues) and linear configuration to enable them to be presented to T cells in the absence of Ag processing and without the ability to cross-link allergen-specific IgE.
The mechanisms involved in the inhibition of the LARs, observed on the
second injection of Fel d 1 peptide (Fig. 1
), may be similar to those
described for classical immunological tolerance. Thus, the peptides may
have induced anergy due to absence of costimulation (11),
activation-induced cell death (12), a switch from a Th2 to
a Th1 cytokine profile (13, 14, 15), the induction of
regulatory/suppressor T cells (16, 17, 18), or combinations of
these mechanisms. The very gradual return to baseline bronchial
responsiveness to Fel d 1 peptide challenge over several months (Fig. 2) could support a role for any of these mechanisms.
Akdis and colleagues (19), using either whole bee venom or
peptides from phospholipase A2 (the major bee
allergen), have shown that successful immunotherapy, in bee-sensitive
individuals, is associated with the elaboration of IL-10
(20) (C. Akdis and K. Blaser, personal communication). It
has been suggested that the IL-10 induced during venom immunotherapy
gives rise to anergic Ag-specific T cells, and that these changes could
be partially reversed in vitro by the addition of exogenous IL-2 or
IL-15 (19). It is currently unclear whether the
Ag-specific hyporesponsiveness seen in the venom model is due to
classical anergy or the influence of IL-10-secreting regulatory T
cells. Clonal deletion as an overall mechanism is unlikely from our
findings, as we provide evidence that Fel d 1 peptides can suppress the
subsequent in vivo response to whole allergen (Figs. 3 and 5).
To investigate the possibility of a peptide epitope-induced switch from
a Th2 to a Th1 cytokine profile, we assessed the primary proliferative
responses and cytokine production of PBMCs to both the individual
peptides (Fig. 4) and whole cat dander (Fig. 5). We found that a single
ID injection of 5 µg Fel d 1 peptides was associated with a reduction
in both proliferation and production of IL-4, IL-13, and IFN-. Thus,
it appeared that an increase in IFN- responses (observed after
successful immunotherapy with whole allergen extracts)
(13, 14, 15) had not occurred after administration of
peptides, at least under the conditions of our experiments. However,
down-regulation of both Th1 and Th2 cytokines after peptide
administration has been previously described in both murine
(5) and human models of allergic disease
(21).
We also assessed in vitro responses to whole allergen (Fig. 5). Again,
there was a reduction in all the in vitro parameters measured,
suggesting that the induction of linked/intramolecular epitope
suppression had occurred. This may also contribute to the inhibition of
the late-phase cutaneous response to whole allergen (Fig. 3). It has
been previously shown in animal models that regulatory
CD4+ T cells exert their suppressive effect by
linked recognition of epitopes on the same or neighboring APCs
(intramolecular epitope suppression) (7), adjacent
molecules (bystander tolerance), or is passed to the next generation of
regulatory T cells (infectious tolerance) (8). This has
implications for the design of peptide-based immunotherapy, as this
phenomenon means that fewer peptides may need to be administered to
induce immunosuppression to the whole allergen. This hypothesis is
further strengthened by our previous observation that at least one T
cell epitope from Fel d 1 displays promiscuous MHC-binding
characteristics (6). Additionally, we have demonstrated
that tolerance to the whole allergen can be induced both in vivo (Fig. 3) and in vitro (Fig. 5), using peptides spanning only a proportion of
the whole allergen. Studies are currently underway to identify critical
binding residues in the four noninjected peptides, as this may allow
the replacement of one or more hydrophobic residues with alternative
hydrophilic residues, thus rendering the peptide soluble. A peptide
mixture spanning the whole of chain 1 and chain 2 of Fel d 1 may be
more tolerogenic than the 12 peptides used in the present study,
especially as these would presumably bind more HLA molecules.
The 12 Fel d 1 peptides, like FC1P, did not release histamine from blood basophils of cat-allergic subjects. These observations, taken together with the absence of peptide-induced early asthmatic reactions (even on the second challenge), suggest 1) peptide-specific IgE was not induced as a result of Fel d 1 peptide administration, and 2) Fel d 1 peptides did not cross-link preformed IgE, thereby leading to mediator release by mast cells and basophils.
It is possible that the inability of a second injection of peptides to induce a LAR was due to the induction of peptide-specific Abs (non-IgE isotype), which sequestered the peptide immediately following administration. However, this is unlikely because T cell peptides 1) inhibited the late, but not the early skin reaction, and 2) had predominant T cell effects, i.e., reduced proliferative and cytokine responses in vitro.
Asthmatics who received Fel d 1 peptides, but did not develop LARs,
were still protected against subsequent challenge with whole allergen,
as shown by inhibition of the late-phase cutaneous reaction (Fig. 3).
Therefore, reactivity, in terms of the development of bronchospasm, was
not essential for the development of hyporesponsiveness. Consequently,
in a therapeutic setting, it should be possible to achieve
hyposensitization or tolerance, without the unwanted LARs, by gradually
increasing the dose of peptide and ensuring an optimal interval between
the doses. In support of this concept, we have recently shown that a
total of 41.1 µg of a similar Fel d 1 peptide could be administered
to cat asthmatics without the development of LARs, by gradual up-dosing
at 14-day intervals (22). Recently, two groups attempted
to treat patients with multiple sclerosis by administration of an
altered peptide ligand derived from the sequence of myelin basic
protein (23, 24). Both studies were halted, as many
subjects developed disease exacerbations. In both situations, the dose
of peptide administered on a repeated basis was 5–50 mg. Our
experience with peptide-induced LARs may provide an explanation for the
induction of exacerbations in multiple sclerosis patients and point the
way for safer and clinically effective dose ranging for future
peptide-based clinical interventions.
In conclusion, we have demonstrated that multiple, short, overlapping
peptides containing T cell epitopes can induce both peptide- and whole
allergen-specific hyporesponsiveness. This clinical hyporesponsiveness
was associated with a marked reduction in the T cell effector response
to both the peptides and the whole allergen compatible with the
induction or expansion of a population of allergen-specific regulatory
cells. Further studies are in progress, involving measurements of IL-10
and TGF- and the identification of T cell clones or lines,
postpeptide therapy, which have a regulatory cell phenotype, to resolve
these issues. Our data also provide further evidence that
allergen-derived peptides may, in time, provide a useful alternative to
specific immunotherapy in the treatment of atopic allergic disease and
other conditions in which T cells contribute to pathogenesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. Barry Kay, Department of Allergy and Clinical Immunology, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London, SW3 6LY U.K. E-mail address: a.b.kay{at}ic.ac.uk
3 Abbreviations used in this paper: LAR, late asthmatic reaction; FEV1, forced expiratory volume in 1 s; ID, intradermal; PPD, purified protein derivative.
Received for publication April 10, 2001. Accepted for publication June 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion in specific allergen-stimulated T cell cultures. J. Immunol. 154:4187.[Abstract]
but not IL-4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669. (TGF-)-dependent inhibition of T helper cell 2 (Th2)-induced autoimmunity by self-major histocompatibility complex (MHC) class II-specific, regulatory CD4+ T cell lines. J. Exp. Med. 185:1769.This article has been cited by other articles:
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  J. D. Campbell, K. F. Buckland, S. J. McMillan, J. Kearley, W. L.G. Oldfield, L. J. Stern, H. Gronlund, M. van Hage, C. J. Reynolds, R. J. Boyton, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression J. Exp. Med., July 6, 2009; 206(7): 1535 - 1547. [Abstract] [Full Text] [PDF]
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 J. Edlmayr, K. Niespodziana, B. Linhart, M. Focke-Tejkl, K. Westritschnig, S. Scheiblhofer, A. Stoecklinger, M. Kneidinger, P. Valent, R. Campana, et al. A Combination Vaccine for Allergy and Rhinovirus Infections Based on Rhinovirus-Derived Surface Protein VP1 and a Nonallergenic Peptide of the Major Timothy Grass Pollen Allergen Phl p 1 J. Immunol., May 15, 2009; 182(10): 6298 - 6306. [Abstract] [Full Text] [PDF]
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A. Camarca, R. P. Anderson, G. Mamone, O. Fierro, A. Facchiano, S. Costantini, D. Zanzi, J. Sidney, S. Auricchio, A. Sette, et al. Intestinal T Cell Responses to Gluten Peptides Are Largely Heterogeneous: Implications for a Peptide-Based Therapy in Celiac Disease J. Immunol., April 1, 2009; 182(7): 4158 - 4166. [Abstract] [Full Text] [PDF]
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 F R. Ali, A B. Kay, and M. Larche Airway hyperresponsiveness and bronchial mucosal inflammation in T cell peptide-induced asthmatic reactions in atopic subjects Thorax, September 1, 2007; 62(9): 750 - 757. [Abstract] [Full Text] [PDF]
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T. Nagato, H. Kobayashi, M. Yanai, K. Sato, N. Aoki, K. Oikawa, S. Kimura, Y. Abe, E. Celis, Y. Harabuchi, et al. Functional Analysis of Birch Pollen Allergen Bet v 1-Specific Regulatory T Cells J. Immunol., January 15, 2007; 178(2): 1189 - 1198. [Abstract] [Full Text] [PDF]
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 M. Muller, J. Grunewald, C. Olgart Hoglund, B. Dahlen, A. Eklund, and H. Stridh Altered apoptosis in bronchoalveolar lavage lymphocytes after allergen exposure of atopic asthmatic subjects Eur. Respir. J., September 1, 2006; 28(3): 513 - 522. [Abstract] [Full Text] [PDF]
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A. Immonen, S. Farci, A. Taivainen, J. Partanen, S. Pouvelle-Moratille, A. Narvanen, T. Kinnunen, S. Saarelainen, M. Rytkonen-Nissinen, B. Maillere, et al. T Cell Epitope-Containing Peptides of the Major Dog Allergen Can f 1 as Candidates for Allergen Immunotherapy J. Immunol., September 15, 2005; 175(6): 3614 - 3620. [Abstract] [Full Text] [PDF]
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D S Robinson Regulation: the art of control? Regulatory T cells and asthma and allergy Thorax, August 1, 2004; 59(8): 640 - 643. [Full Text] [PDF]
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 M. Larche, A. B. Kay, and L.-P. Boulet Peptide Therapy and Asthma Am. J. Respir. Crit. Care Med., June 15, 2004; 169(12): 1331 - 1332. [Full Text] [PDF]
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A. J. Reefer, R. M. Carneiro, N. J. Custis, T. A. E. Platts-Mills, S.-S. J. Sung, J. Hammer, and J. A. Woodfolk A Role for IL-10-Mediated HLA-DR7-Restricted T Cell-Dependent Events in Development of the Modified Th2 Response to Cat Allergen J. Immunol., March 1, 2004; 172(5): 2763 - 2772. [Abstract] [Full Text] [PDF]
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F. R. Ali, W. L. G. Oldfield, N. Higashi, M. Larche, and A. B. Kay Late Asthmatic Reactions Induced by Inhalation of Allergen-derived T Cell Peptides Am. J. Respir. Crit. Care Med., January 1, 2004; 169(1): 20 - 26. [Abstract] [Full Text] [PDF]
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A. Sundstedt, E. J. O'Neill, K. S. Nicolson, and D. C. Wraith Role for IL-10 in Suppression Mediated by Peptide-Induced Regulatory T Cells In Vivo J. Immunol., February 1, 2003; 170(3): 1240 - 1248. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and the pooled results were compared
with the control day (
). The points represent the mean
(±SEM).
