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,
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Dipartimento di Internistica Clinica e Sperimentale II Universita’ di Napoli, Naples, Italy;
Laboratori di Medicina Sperimentale, Fondazione "S. Maugeri," Centro Medico di Pavia, Pavia, Italy;
The Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53201;
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Laboratorio di Immunologia, Istituto Superiore di Sanita’, Rome, Italy;
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Istituto di Biologia dello Sviluppo-Consiglio Nazionale delle Ricerche, Palermo, Italy;
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Sezione Autonoma di Allergologia, Ospedale S. Martino, Genoa, Italy;
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Servizio di Immunologia, Ospedale S. Martino, Universita’ di Genoa, Genoa, Italy
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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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An important application of peptide antagonism could be the control of allergic diseases characterized by specific IgE production after contact with allergens. The ability of IgE to bind mast cells and basophils and the ensuing release of histamine and other mediators are responsible for most, if not all, of the clinical features found in allergic patients 12 . The T cell help needed for Ab production is a critical point in a developing immune response. The role of T cells in specific IgE production has been extensively assessed and provides some of the strongest evidence for T cell-mediated B cell help 13, 14 . Therefore, if T cell help can be down-regulated, it may relieve the resulting allergic response.
There has been a dramatic increase in allergic symptoms over the last few years, and the social cost for their management is heavily affecting the health care system of the developed countries 15 . Parietaria (Pellitory of the wall, family Urticaceae) accounts for most of the allergenic sensitization in the southern Mediterranean area 16 . The allergen that is the focus of these studies, Par j1, is the predominant allergen protein recovered from the pollen of Parietaria judaica. The importance of Parietaria as source of allergenic stimuli is currently under investigation in the United Kingdom, United States, and Australia in regions where the plant also flourishes 17, 18, 19, 20 . The ability to regulate T cell response could be an extremely important approach for controlling IgE production in P. judaica allergies, therefore opening a new approach to immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood was obtained from three patients (BS, DFA, DFL) sensitized to Par j1 as determined by skin prick tests and IgE measurement by radioallergosorbent test (RAST). The three patients were fully informed about the aim of the study and consented to donate blood for research purposes. PBMC were isolated by gradient density separation using Ficoll (Pharmacia, Piscataway, NJ) according to standard protocols.
Cell lines
PBMC were cultured in flat-bottom six-well plates (Costar, Cambridge, MA) suspended at 1 x 106 cells/ml in complete RPMI 1640 (supplemented with 0.2 mM L-glutamine, 100 U penicillin, 100 µg/ml streptomycin; Life Technologies, Grand Island, NY) plus 10% heat-inactivated human serum AB (HyClone, Logan, UT) and stimulated with purified Par j1 at 5 µg/ml final concentration. Purified Par j1 was a generous gift of Dr. J. Carreira, (Abello, Madrid, Spain). Irradiated autologous PBMC (3000 rad from a 60Cs source) plus Ag were used to stimulate the cells every 7 days for 2 wk. The cells were further expanded using rIL-2 20 U/ml plus purified allergen for 1 more wk. Before use in the experiments described, the specificity for Par j1 was assessed by a standard thymidine incorporation assay comparing specific proliferation to Par j1 with that to the recall Ag purified protein derivative or a different pollen Ag Lol pI (ryegrass). For the specificity assay the cell lines were divided at 2.104 cells/well in a final volume of 200 µl of complete medium (RPMI 1640 supplemented as above) in a 96-well flat-bottom microtiter plate and stimulated with Par j1 at 5 µg/ml, Lol p1 at 10 µg/ml, or purified protein derivative at 10 µg/ml. After 72 h, each well was pulsed with 1 µCi of [3H] for each well. Sixteen hours later, cells were harvested onto glass filters, and radioactivity was measured by liquid scintillation by means of a beta counter. Proliferation values of control cultures have been subtracted, and all cultures were in triplicate. The response of the lines was similar to that described for T cell clones specific for Par j1 21 . Ab-blocking studies were performed using the Ab L243 (IgG2a, anti-HLA-DR) provided as culture supernatants and used at 1:20 dilution on the basis of titration experiments.
Screening of peptides
Peptides were synthesized from various portions of the Par j1,
and an immunodominant peptide was identified by its ability to
stimulate most of the T cell clones derived from the cell lines 21 .
This peptide corresponds to residues 47–65 of the Par j1 sequence.
Several peptides were designed substituting an alanine or valine at
positions that are not likely to be DR anchor residues. The peptides
used for the experiments were chosen on the basis of their ability to
bind DR while not stimulating a large panel of clones derived from DFL,
BS, and DFA. The sequence of the wild-type peptide, as well as the
monosubstituted peptides used in these studies, is given in Table I
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Soluble DR1 was produced in Sf9 cells as described 22 . Purified soluble HLA-DR proteins (10 nM) generated by a baculovirus expression system were incubated with 1 µM FITC-labeled control peptides in the absence and presence of 1000-fold excess unlabeled control peptides or unlabeled Par j1-derived peptides in 50 µl binding mixtures for 24 h. The control peptides are influenza hemagglutinin 307–319 for DR1 and DR4, MHC class II-associated invariant chain-derived peptide (CLIP) 80–103 for DR3, and Lol pI 191–210 for DR52a. The binding conditions were 37°C in PBS, with 1 mM EDTA, 1 mM PMSF, 0.1 mM iodacetamide, and 3 mM NaN3, at pH 7.0. The binding mixtures were then resolved by native 7.5% PAGE to separate the bound from the unbound peptides. The bound FITC-labeled peptides were detected and quantitated using the FluorImager 575 (Molecular Dynamics, Sunnyvale, CA) after electrophoresis. For affinity measurements, 10 nM sDR1 proteins were incubated with 1 mM FITC-labeled influenza matrix peptide 19–31 in the presence of unlabeled wild type (wt)5 or its derivatives at a series of concentrations (10–10,000 nM) for 24 h. The binding mixtures were analyzed by native PAGE, and the emission of bound FITC-labeled peptide migrating at the position of the DR heterodimer was measured using ImageQuant software (Molecular Dynamics).
Inhibition experiments
Peptide inhibition experiments were performed as described 5
with some minor modifications. Cell lines having the best proliferation
with the wt peptide were chosen for the experiments. Specific cell
lines isolated as previously described were cultured for 1 more wk
using wt peptide. Cells were divided in 96-microtiter plates as
described. Irradiated autologous PBMC or irradiated autologous
EBV-transformed cells were incubated with suboptimal doses of wt (10
µM) and Par j1 1 µg/ml (
70 nM) for 3 h at 37°C. Cells
were washed twice with PBS to remove unbound peptide or Ag, resuspended
in complete media, and monosubstituted peptides (V6, A11, and A14) were
added at different concentrations. After 16 h, the cells were
washed twice in RPMI 1640, and T cells and complete media were added to
obtain a 1:1 ratio of APCs and T cells. After 24 h,
[3H]thymidine was added, and a standard T cell
proliferation assay was conducted as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and will be referred to as
wt. This sequence was used to design a number of altered peptides with
single alanine or valine substitutions at positions deemed unlikely to
be anchor residues. These peptides were tested on a panel of T cell
clones derived from the three donors, and those peptides incapable of
stimulating the clones were identified. Three peptides, called V6, A11,
and A14 after the single position substituted (Table I), were tested
for the necessary criteria to act as antagonists: Class II MHC binding,
inability to induce specific proliferation of the T cell, and
inhibition of T cell response to the normal Ag. Class II MHC binding properties of the peptides
Class II MHC binding of the peptides was studied by competition
experiments using empty soluble HLA-DR molecules (sDR) produced in
insect cells infected with baculovirus 24, 25 . Fig. 1 shows the results of a competition
peptide binding assay using sDR1, sDR3, sDR4, and sDR52a. The ability
of the wt and altered peptides to inhibit the binding of
well-characterized class II-binding (control) peptides to the soluble
DR molecules was tested as previously described 26 . DR1 and DR3 were
tested because they are found in the responding cell lines (line BS is
DR1,2 and lines DFA and DFL are DR3). DR4 represents a common HLA
allele, and DR52a is the product of a second class II locus associated
with DR3 and DR6. As can be seen, the wt and altered peptides can
inhibit the binding of the control peptide to sDR1, sDR3, and sDR4.
None of the peptides can inhibit the binding of the control peptide to
sDR52a.
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The ability or inability of the substituted peptides to stimulate
the T cell lines used here was measured. The results are shown in Fig. 2. For all three cell lines, the V6, A11,
and A14 substituted peptides were very poor stimulators as compared
with the wt peptide. The range of concentrations used was in the same
order as that needed to obtain stimulation with wt peptide. This
characteristic would make them suitable peptides to act as MHC blockers
or TCR antagonists when used in this concentration range. It is not
known whether at much higher doses the substituted peptides may start
to act as agonists.
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In light of these results, we tested the ability of these three
altered peptides to block the response of the T cell lines to wt
peptide and intact Par j1 protein. Inhibition experiments were
performed with minor modification to published protocols 5 . The three
T cell lines were prepulsed with suboptimal amounts of wt peptide or
Par j1 protein and then cultured with the altered peptides. When the
cultures are costimulated with the wt peptide and increasing
concentrations of two peptides, V6 and A11, the initial response is
inhibited in all three cell lines (Fig. 3). Altered peptide A14 had no influence
on the proliferation of the cell lines.
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). The effect was less
pronounced for the BS line and the A11 peptide. However, at higher
peptide concentrations, the response was inhibited to the same extent
as in the other lines. The intact protein induces a higher
proliferation rate (five- to sixfold) than does the equivalent
concentration of wt peptide (legend Fig. 2). In spite of this increased
proliferation, the two altered peptides were able to reduce the
response. However, the concentration of APL required to achieve this
inhibition was 100-fold higher than that needed for inhibiting the
response to the wt peptide.
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It is possible that the altered peptides either act as antagonists
or directly compete with the wt peptide or Par j1 Ag for MHC class II
binding. Even at approximately equimolar concentrations, the APL could
act to block the MHC class II if their affinity for the MHC was much
greater than that of wt peptide. To test this, the affinity of the
three altered peptides for HLA-DR1 was measured directly and compared
with wt (Fig. 5). The altered peptides
show a decrease in affinity compared with wt.
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To assess the DR restriction of these cell lines, we used the
anti-HLA-DR mAb L243. Proliferation experiments were conducted in
the presence of the Ab as described in Materials and
Methods. As shown in Fig. 6, the
addition of L243 to the cultures inhibits T cell responses to both Par
j1 and wt. No inhibition was detected when the anti-class I mAb
w632 (IgG2a) was added to the cultures (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While being a more realistic system, the use of T cell lines that respond to the APC derived from PBMC could introduce unexpected complications into the experimental study of a phenomenon that at its core involves class II MHC, peptide, and TCR. Thus, it should be pointed out that the ability of fixed irradiated autologous EBV lines to act as APC was determined in a number of independent experiments. The proliferative response of the T cell lines to wt peptide was impaired by the two APL (data not shown), excluding possible mechanisms due to class II recycling, peptide impairment of processing, or other effects mediated by the use of PBMC as APC 5, 29, 30 , and making it likely that the observed inhibition is due to the APL binding to class II MHC.
Peptides can act in a number of ways to inhibit immune responses (reviewed in 31 . For in vitro cultures, the two most likely mechanisms are peptide blockade of the MHC molecule, or TCR antagonism. It should be pointed out that the substituted peptides were used in a range that maximally represented a twofold excess over wt peptide. This would not be expected to favor MHC blockade. The nature of the inhibition observed here is further clarified by the results with the A14 peptide and by a more detailed analysis of the HLA-DR binding affinities of the altered peptides. The A14 peptide can bind to DR1 and -3, cannot stimulate the T cells, yet does not inhibit the T cell response to either Par j1 or wt peptide. This makes peptide competition for available HLA-DR molecules (MHC blockade) an unlikely explanation for the inhibition shown by the other two peptides. It also rules out other nonspecific effects due only to the presence of extra peptide during the stimulation. Even at equimolar concentration of agonist and antagonist, MHC blockade could occur if the APL affinity is much higher for the class II than that of the wt peptide. The APL show a lower affinity for DR1 as compared with wt peptide. This observation also rules against the occurrence of MHC blockade. To the extent that this observation can be generalized to DR3, this argument would hold for the whole system. Thus, the data from the stimulation with the wt peptide support true peptide antagonism 5 as an explanation for the observed phenomenon.
The nature of the inhibition of the response to the Par j1 protein is less clear. Even at the lowest concentration of altered peptides used, the excess of altered peptide to target was 100-fold. As mentioned above, the fact that the A14 peptide can bind to the DR alleles involved, yet not inhibit the response, does not favor an MHC blockade explanation. A number of possible explanations can be offered for why higher concentrations of altered peptides are needed to see effects equivalent to that observed with the wt peptide experiments. It could be assumed that the processing of intact protein induces the APC to function more efficiently. Factors associated with the nature of the uptake of peptide vs that of intact Ag may play a role. For example, if peptide binding is predominantly limited to surface exchange with preloaded class II, stimulation by peptides may be less efficient than by a protein that is processed by the endosomal pathway. It is also possible that other portions of Par j1 protein may act to boost the response. We have recently described T cells recognizing carbohydrate moieties of Par j1 protein that produce cytokines 32 . These T cells could partially offset the effect of the APL on the response to the peptide portion of the processed protein. While the observations with intact Ag are interesting and point to the use of altered peptides in allergic responses, more experiments are needed to further define the nature of this response and its antagonism.
The nature of the responding T cells is an interesting aspect of this system, and more detailed experimentation will be necessary to define the repertoire. Because antagonism is thought to be a clonal phenomenon, a number of tentative conclusions can be reached. The fact that the lines are antagonized by peptides corresponding to a single region of the Ag would lead to the expectation that the repertoire of responding T cells is limited in these lines. Preliminary analysis of the three lines has indeed shown a limited T cell repertoire in all three lines. However, lines from each individual display a distinct pattern of TCR V gene usage and CDR3 length (De Palma et al., manuscript in preparation). It will be necessary to link these T cells with the B cell responsible for specific IgE secretion. A major B cell epitope has been characterized on Par j1 33 . This may restrict the number of responding B cells, making their identification and the subsequent analysis of their sensitivity to activated T cells easier.
It will also be interesting to investigate the modality of antagonism in these experiments. Biochemical and functional studies will be necessary to determine to what extent altered peptides interfere with specific T cell signaling and lymphokine production. The relation between cytokine production and the inhibition of proliferation studied here will be important in this regard. The identification of the peptide residues that are involved in MHC contact and identification of TCR contact residues will further our understanding of the T cell recognition process. More importantly, studies of additional side chain substitutions at the TCR contact residues should permit the design of better antagonists.
The availability of peptides that are able to down-regulate specific T cell responses could lead to lower IgE production. The use of such peptides could avoid the risks of traditional immunotherapy, in which entire Ag is used. Specific T cells could be regulated without involvement of a further IgE secretion, because antagonist peptides can be designed that do not contain epitopes recognized by IgE. Our observation that these altered peptides bind to a number of common class II MHC alleles implies that they may be of general use. While the use of peptides as tolerizing agents in allergy has been investigated 34, 35 , this is the first report of the possible use of peptides to antagonize the response to a whole allergen. The large extent of morbidity due to allergies and the increased mortality due to allergic asthma justify a particular attention toward these diseases, and the data reported here suggest new possibilities for therapies.
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Acknowledgments |
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Footnotes |
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2 Current Address: Basel Institute for Immunology, Grenzacherstrasse 487 4005 Basel, Switzerland.
3 Current Address: Divisione di Broncologia, Ospedale "A. Cardarelli," 80131 Naples, Italy.
4 Address correspondence and reprint requests to Dr. Jack Gorski, Blood Research Institute, Post Office Box 2178, Milwaukee, WI 53201-2178. E-mail address:
5 Abbreviations used in this paper: wt, wild type; sDR, soluble HLA-DR molecules; APL, altered peptide ligands; RFU, relative fluorescence units.
Received for publication July 6, 1998. Accepted for publication November 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimers in the absence of antigenic peptide. Cell 68:465.[Medline]
/ß T Cells. J. Exp. Med. 186:899.This article has been cited by other articles:
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  T. Kinnunen, W. W. Kwok, A. Narvanen, M. Rytkonen-Nissinen, A. Immonen, S. Saarelainen, A. Taivainen, and T. Virtanen Immunomodulatory potential of heteroclitic analogs of the dominant T-cell epitope of lipocalin allergen Bos d 2 on specific T cells Int. Immunol., December 1, 2005; 17(12): 1573 - 1581. [Abstract] [Full Text] [PDF]
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 R. Nisini, G. Romagnoli, M. J. Gomez, R. La Valle, A. Torosantucci, S. Mariotti, R. Teloni, and A. Cassone Antigenic Properties and Processing Requirements of 65-Kilodalton Mannoprotein, a Major Antigen Target of Anti-Candida Human T-Cell Response, as Disclosed by Specific Human T-Cell Clones Infect. Immun., June 1, 2001; 69(6): 3728 - 3736. [Abstract] [Full Text] [PDF]
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