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Naturally processed T cell–activating peptides of the major birch pollen allergen
Naturally processed T cell–activating peptides of the major birch pollen allergen Sonja Mutschlechner, PhD,a,b Matthias Egger, PhD,a Peter Briza, PhD,a Michael Wallner, PhD,a Peter Lackner, PhD,a Anette Karle, PhD,c Anne B. Vogt, PhD,c Gottfried F. Fischer, MD,d Barbara Bohle, PhD,b* and Fatima Ferreira, PhDa* Salzburg and Vienna, Austria, and Basel, Switzerland Background: Although antigen processing and presentation of allergens to CD41T lymphocytes are key events in the pathophysiology of allergic disorders, they still remain poorly understood. Objective: To investigate allergen processing and presentation by dendritic cells using the major birch pollen allergen Bet v 1 as a model. Methods: Endolysosomal extracts of dendritic cells derived from patients with birch pollen allergy were used to digest Bet v 1. Dendritic cells were pulsed with Bet v 1, and peptides were eluted from MHC class II molecules. Peptides obtained by either approach were sequenced by tandem mass spectrometry. Bet v 1–specific T-cell cultures were stimulated with HLA-DR– eluted Bet v 1–derived peptides. Bet v 1–specific T-cell lines were generated from each patient and analyzed for epitope recognition. Results: A high proportion of Bet v 1 remained intact for a long period of endolysosomal degradation. The peptides that appeared early in the degradation process contained frequently recognized T-cell epitopes. Bet v 1–derived peptides eluted from MHC class II molecules corresponded to those generated by endolysosomal degradation, matched known T-cell epitopes, and showed T cell–activating capacity. The Bet v 1–specific T-cell line of each individual harbored T cells reactive with peptides located within the MHC class
From athe Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg; bthe Christian Doppler Laboratory for Immunomodulation, Department of Pathophysiology, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna; cF. Hoffmann-La Roche Ltd, Immunosafety, Non-Clinical Safety, Basel; and dthe Clinical Department for Blood Group Serology, University Clinic for Blood Group Serology and Transfusion Medicine, Vienna. *These authors are cosenior authors of this article. ¨ sterreichische Nationalbank, the Christian Doppler Supported by P10150 of the O Laboratory for Allergy Diagnosis and Therapy, and the Christian Doppler Laboratory for Immunomodulation, Austria. Disclosure of potential conflict of interest: F. Ferreira has received research support from Biomay AG, the Austrian Science Fund, the Christian Doppler Research Association, and the Austrian National Bank and has provided legal consultation services or expert witness testimony relevant to Indoor Biotechnologies and AllergenOnline Database. B. Bohle has received research support from the Austrian Science Fund and the Christian Doppler Laboratory. The rest of the authors have declared that they have no conflict of interest. Received for publication July 10, 2009; revised October 6, 2009; accepted for publication October 29, 2009. Available online February 4, 2010. Reprint requests: Barbara Bohle, PhD, Institute of Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria, E-mail: barbara.bohle@ meduniwien.ac.at; Fatima Ferreira, PhD, Department of Molecular Biology, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria. E-mail: fatima. [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2009.10.052
II–eluted Bet v 1–derived sequences demonstrating their occurrence in vivo. Conclusion: We report for the first time how epitopes of allergens are generated and selected for presentation to T lymphocytes. The limited susceptibility of Bet v 1 to endolysosomal processing might contribute to its high allergenic potential. (J Allergy Clin Immunol 2010;125:711-8.) Key words: Birch pollen allergy, Bet v 1, antigen processing, antigen presentation, dendritic cells, T cells, T-cell epitopes
IgE-mediated disorders such as allergic rhinitis and asthma have tremendously increased during the recent decades. Understanding the immune mechanisms underlying allergic disorders is important to develop new strategies for their prevention and treatment. CD41 TH lymphocytes play a pivotal role in the sensitization and maintenance of type I allergy. Allergic diseases result from an aberrant T-cell response to allergens dominated by long-lived TH2 cells.1,2 Among other cytokines, allergen-specific TH2 cells secrete high amounts of IL4 and IL-13, which induce the production of allergen-specific IgE antibodies that mediate immediate allergic symptoms.3 In addition to this indirect involvement in immediate reactions, allergen-specific TH2 cells have been demonstrated to be directly involved in clinical late-phase reactions in target organs such as the lung and skin.4,5 Allergen-specific CD41 T lymphocytes are activated by peptides generated by proteolytic degradation of allergens in endolysosomal compartments of antigen-presenting cells (APCs). The T-cell receptor recognizes the peptides in the context of MHC class II molecules exposed on the cell surface of APCs.6 The most specialized APCs are dendritic cells (DCs).7 They are capable of priming naive T lymphocytes because they efficiently take up allergens by phagocytosis or pinocytosis or via receptors expressed on their surface (eg, FceRI) and express a large number of MHC class II molecules and different costimulatory molecules.8-11 Until now, T cell–activating regions in allergens have been identified on the basis of proliferative and cytokine responses to short overlapping peptides spanning the entire amino acid (aa) sequence of the molecule.12,13 Although this approach identifies the core epitopes recognized by the T-cell receptor, the actual sequences of naturally processed MHC class II–bound peptides cannot be predicted. Such peptides have been demonstrated to display a high degree of heterogeneity in both length and site of their terminal truncations.14,15 Furthermore, the aa residues flanking the core epitope recognized by the T-cell receptor have been reported to enhance or hinder T-cell activation, respectively.16-19 711
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Abbreviations used aa: Amino acid APC: Antigen-presenting cell cpm: Counts per minute DC: Dendritic cell SI: Stimulation index TCC: T-cell clone TCL: T-cell line
Because naturally processed MHC class II–bound peptides derived from allergens have not been identified so far, we investigated such peptides presented by DCs from patients with allergy. We used Bet v 1, the clinically highly relevant major birch pollen allergen, which has been extensively characterized by our group. In addition to dissecting its IgE-binding properties,20,21 we have identified Bet v 1–T cell epitopes by stimulation of allergenspecific T cells isolated from the peripheral blood of a large number of patients with birch pollen allergy with synthetic overlapping 12mer peptides.22,23 HLA-DR molecules were shown to be highly relevant restriction elements for Bet v 1–specific T lymphocytes.24-26 We isolated HLA-DR–bound peptides from Bet v 1–pulsed DCs derived from individuals with birch pollen allergy. Moreover, Bet v 1 was incubated with microsomal proteases isolated from these DCs. Bet v 1–derived fragments obtained by either approach were sequenced by tandem mass spectrometry. The T cell–activating properties of HLA-DR–eluted peptide sequences were confirmed by using Bet v 1–specific T-cell clones (TCCs). To verify the in vivo relevance of naturally processed Bet v 1 peptides, we analyzed the peripheral blood of each patient with birch pollen allergy for the presence of T cells reactive with epitopes matching the eluted peptide sequences.
METHODS Patients with birch pollen allergy and allergen All patients with birch pollen allergy included in this study were sensitized to Bet v 1, had rhinoconjunctivitis in spring, and showed specific IgE CAP/ RAST class >3 (Pharmacia Diagnostics, Uppsala, Sweden) and positive skin prick reactions (wheal diameter >5 mm) to birch pollen. All patients gave written consent before enrolment in the study, which was approved by the local Medical Ethical Committee of Vienna. Molecular HLA typing for HLADRB1 alleles of the patients was performed according to methods previously referenced.27 Recombinant Bet v 1 was purchased from Biomay (Vienna, Austria).
Endolysosomal degradation assays Monocyte-derived (md) DCs were generated from CD141 cells purified from PBMCs as described.28 DCs were homogenized in 10 mmol/L TRIS acetate pH7 containing sucrose (250 mmol/L), and microsomes were isolated by ultracentrifugation.29 Bet v 1 (5 mg) was digested with microsomal enzymes (7 mg) in 100 mmol/L citrate buffer pH 4.8 containing dithiothreitol (2 mmol/ L) at 378C. Reactions were stopped by heat denaturation and analyzed by SDS-PAGE and mass spectrometry using an ESI-QTOF mass spectrometer fitted with a capillary reversed phase HPLC (Waters, Milford, Mass).
Allergen-specific T-cell cultures Allergen-specific T-cell lines (TCLs) were generated by stimulating PBMCs (1.5 3 106) with Bet v 1 (10 mg/mL) as described.23 TCLs were stimulated with varying concentrations of Bet v 1 (2.5-10 mg/mL) or peptide
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(5 mg/mL) in the presence of 5 3 104 irradiated (60 Gy) autologous PBMCs for 48 hours in duplicate. Proliferation was assessed by [3H] thymidine incorporation. The stimulation index (SI) was calculated as ratio between counts per minute (cpm) of TCL plus PBMC plus allergen and cpm of TCL plus PBMC only. Cryopreserved TCCs specific for different Bet v 1 epitopes were thawed, expanded, and stimulated with Bet v 1 (5 mg/mL) or the indicated peptides (0.6-2.5 mmol/L) in the presence of irradiated autologous PBMCs. Supernatants were harvested after 24 hours. Cytokine levels were assessed by ELISA as described.23 Cytokine levels in controls were subtracted from those determined in unstimulated cultures.
Isolation of peptides from HLA-DR molecules The monoclonal anti–HLA-DR antibody L243 (ATCC HB-55) was purified by ammonium sulfate precipitation and protein G affinity chromatography. Antibody specificity was checked by flow cytometry (data not shown). Dendritic cells (65-100 3 106) were stimulated with Bet v 1 (200 mg/mL) plus LPS (1 mg/mL) (Sigma, St Louis, Mo) on day 5 and lysed in 10 mmol/L TRIS/HCl pH 7.6 containing 0.6% CHAPS and Complete Protease Inhibitor (Roche Diagnostics, Mannheim, Germany) for 30 minutes on ice. After ultracentrifugation, protein G beads (GE Healthcare, Vienna, Austria; 200 mL in 200 mL PBS) were added to the supernatant for 2 hours on ice. After centrifugation, L243 antibody (4 mg) was added to the supernatant and incubated for 4 hours at 48C. Protein G beads (200 mL in 200 mL PBS) were added for 16 hours. After centrifugation, the pellet was washed with PBS, resuspended in 300 mL 10% trifluoroacetic acid, incubated at room temperature for 5 minutes, and ultrafiltrated through a Microcon-10 filter unit (Millipore, Molsheim, France). The eluate was immediately analyzed by tandem mass spectrometry.
Mass spectrometry analysis of HLA-DR–eluted peptides Peptides from patient 4 were eluted and identified by mass spectrometry as described.30 For patients 1 to 3, peptides (20 mL) were separated by capillary rpHPLC (Waters) directly coupled to a Q-Tof Global Ultima mass spectrometer (Waters) by using a Nanoflow spray head (precolumn Nanoease Symmetry300 trap column, separating column Nanoease Atlantis dC18; Waters). Peptides were eluted with an acetonitrile gradient (solvent A 0.1% vol/vol formic acid/5% vol/vol acetonitrile; solvent B 0.1% vol/vol formic acid/95% vol/ vol acetonitrile; 5% to 45% B in 90 minutes). Data acquisition and instrument control were done with MassLynx software V4.1 (Waters). Singly, doubly, and triply charged ions were selected for fragmentation by collision with argon (collision energy settings, 25-50 kV). Survey and fragment spectra were analyzed by using the software PLGS version 2.2.5 (Waters) with automatic and manual validation with an in-house Bet v 1 database. As control for this analytical system, mdDCs pulsed with the major mugwort pollen allergen were used. No Bet v 1–derived peptides were detected (data not shown).
RESULTS Endolysosomal processing of the major birch pollen allergen Microsomal proteases were isolated from DCs of 2 donors with birch pollen allergy and incubated with Bet v 1. At defined intervals, allergen degradation was monitored by SDS-PAGE (see this article’s Fig E1 in the Online Repository at www.jacionli ne.org), and proteolytic fragments were sequenced by tandem mass spectrometry (Fig 1). Intact Bet v 1 was detectable for 24 hours. Taking all time points, 113 different fragments were detected in a total of 368 sequenced peptides. After 0.5 hours, first peptide clusters derived from the N-terminus Bet v 11-20, the central region Bet v 184-97, and the C-terminus Bet v 1146-157 were identified. After 1 hour, peptides originating from the region Bet v 1104-115 were detected. Some of these initially identified
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FIG 1. Time kinetics of endolysosomal processing of Bet v 1. After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases isolated from DCs, generated peptides were sequenced by mass spectrometry. Frequently recognized T-cell epitopes are framed in the amino acid sequence of Bet v 1, shown on top.
peptide clusters were detectable until 24 hours of degradation. Peptide clusters in the regions Bet v 122-37, Bet v 133-55, Bet v 156-65, and Bet v 169-80 did not appear before 3 hours of proteolytic cleavage. Remarkably, early appearing peptide clusters matched frequently recognized T cell–activating regions such as Bet v 14-15, Bet v 113-24, Bet v 119-30, Bet v 179-90, Bet v 197-108, Bet v 1112-123, and Bet v 1142-153 previously identified in Bet v 1–specific TCL derived from 58 donors with birch pollen allergy.23
Bet v 1–derived naturally processed peptides First, the optimum incubation period for appearance of allergen-derived peptides on the surface of Bet v 1–pulsed DCs was evaluated. DCs were incubated with Bet v 1 for 6, 11, 24, and 48 hours; washed; irradiated; and used to stimulate autologous Bet v 1–specific T-cell cultures. DCs pulsed for 11 hours induced the most pronounced proliferative responses (Fig 2). Next, HLA-DR– bound peptides were eluted from DCs of 4 patients with birch pollen allergy pulsed with Bet v 1 for 11 hours and analyzed by
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FIG 2. Optimizing the period for allergen pulsing of DCs. DCs from an individual with birch pollen allergy were incubated with Bet v 1 (5 mg/0.5 3 106 cells) for 6, 11, 24, and 48 hours, respectively, and used to stimulate a Bet v 1–specific T-cell clone. T-cell proliferation (SI) is shown.
tandem mass spectrometry. In total, 1606 different peptides with a mean length of 16.4 aa residues (ranging from 6 to 35 aa residues) were detected. Database sequence searches revealed that these peptides derived from more than 650 different proteins of mainly human origin (S. Mutschlechner et al, December 2009). Within this peptide pool, 27 Bet v 1–derived peptides were identified (Fig 3, B). Naturally processed Bet v 1–derived peptides ranged from 14 to 28 aa residues (mean, 18 aa residues). Each patient presented nested sets of peptides originating from 1 or 2 regions of the Bet v 1 sequence that shared a similar core motif but varied in the length of their N-terminal and C-terminal flanking regions. In 2 of 4 patients, peptides were detected from the C-terminal region Bet v 1132-159, which harbors the immunodominant T cell– activating epitope23 (Fig 3, A). DCs from 2 patients presented N-terminal peptides located within Bet v 112-32, and peptides covering the region Bet v 169-96 were detected in 1 individual. The sequences of all naturally presented peptides are summarized in this article’s Table E1 in the Online Repository at www.jacionline.org.
HLA-DR phenotypes of patients with birch pollen allergy All donors used for the isolation of HLA-DR–associated peptides from Bet v 1–pulsed DCs were HLA-DRB1–typed at high resolution. DCs generated from patient 1 (HLADRB1*0701, *1501) presented peptides located within Bet v 1140-159. DCs from patient 2 (HLA-DRB1*0701) presented peptides located within Bet v 169-96. DCs from patient 3 (HLADRB1*0101, *0301) presented peptides located within Bet v 118-32, and DCs from patient 4 (HLA-DRB1*1501, *1601) presented peptides located within 2 different regions, Bet v 112-29 and Bet v 1132-159. T cell–activating properties of Bet v 1–derived naturally processed peptides To confirm that naturally processed Bet v 1 peptides were able to activate T cells, peptides consisting of 18 aa residues, which was the average length of HLA-DR–eluted Bet v 1 peptides, were synthesized and used to stimulate Bet v 1–specific TCCs with known epitope specificity. Peptide FKYNYSVIEGGPIGDTLE representing the naturally processed Bet v 169-96 was incubated with TCC218 specific for Bet v 179-90. Peptide GETLLRAVESYLLAHSDA representing the 18mer shared by the nested
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peptide sets derived from the C-terminus isolated from patient 1 and patient 4 was incubated with TCC266, TCC334, TCC14VR, and TCC4R, all specific for Bet v 1142-153 and obtained from 3 different patients. Proliferative and cytokine responses to the respective 18mer and 12mer peptides (each 2.5 mmol/L) and Bet v 1 are shown in Fig 4. TCC218 specific for FKYNYSVIEGGP proliferated equally well in response to the 12mer peptide, to FKYNYSVIEGGPIGDTLE, or to Bet v 1 (Fig 4, A). As in response to the full-length allergen, TCC218 produced IFN-g, no IL-4, and marginal amounts of IL-5 in response to both peptides (Fig 4, B). Similarly, TCC266, TCC334, TCC14VR, and TCC4R specific for TLLRAVESYLLA proliferated equally well in response to the 12mer peptide, to GETLLRAVESYLLAHSDA, or to Bet v 1. TCC266, TCC14VR, and TCC4R secreted both IL-5 and IFN-g in response to either stimulus. Despite strong proliferation, TCC334 produced marginal amounts of the tested cytokines. In all clones, similar responses were found over a broad range of equimolar concentrations of both peptides (data not shown).
In vivo relevance of Bet v 1–derived naturally processed peptides To reveal a link between the presentation of HLA-eluted Bet v 1–derived peptides and their in vivo recognition by T cells, we generated Bet v 1–specific TCLs from the 4 patients with birch pollen allergy whose DCs had been used in the pulsing experiments. Each TCL was analyzed for epitope recognition by determining proliferative responses to 50 overlapping 12mer peptides covering the aa sequence of Bet v 1. Each TCL proliferated in response to 12mer peptides matching the sequences of naturally processed Bet v 1 peptides eluted from DCs of the respective individual. Three lines harbored T cells specific for Bet v 1 epitopes located in peptide sequences not contained in the pool of HLADR–eluted peptides (Fig 3, B). We also analyzed the allergen-induced production of IL-4 and IFN-g of 19 TCCs previously obtained from 11 different patients with birch pollen allergy and reactive with epitopes located within the naturally processed peptides Bet v 112-32, Bet v 169-96, and Bet v 1132-159 (Table E1). Three of 5 clones specific for epitopes within Bet v 112-32 belonged to the TH2 subset. One clone was designated TH0 and 1 clone TH1. Similar data were found for clones specific for epitopes within Bet v 169-96 (6/8 TH2, 1/8 TH0, and 1/8 TH1) and Bet v 1132-159 (4/6 TH2, 2/6 TH0, and 0/6 TH1).
DISCUSSION For the first time, antigen processing and presentation of an allergen are reported. The major birch pollen allergen was subjected to endolysosomal degradation, and HLA-DR–bound peptides were eluted from Bet v 1–pulsed DCs from patients with birch pollen allergy. Bet v 1–derived peptides represented 1.6% of all eluted peptides, displayed an average length of 18 aa residues, and occurred as nested clusters sharing a common core with varying flanking regions. These features are characteristic for HLA-DR–presented peptides.15,31 The core sequences of the HLA-DR–eluted Bet v 1–derived peptides generally corresponded to frequently recognized T cell–activating regions and overlapped with the peptides created by endolysosomal degradation of Bet v 1 (Fig 1). Finally, the naturally processed sequences
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FIG 3. Naturally processed Bet v 1–derived peptides. Peptides eluted from purified HLA-DR:peptide complexes isolated from DCs of 4 patients with Bet v 1 allergy were sequenced by mass spectrometry. A, Amino acid sequence of Bet v 1. Frequently recognized T-cell epitopes are framed. B, HLA-DR–eluted peptides are shown in black; 12mer peptides inducing proliferative responses in Bet v 1–specific TCLs are shown in gray.
matched those identified by another study using DCs from 10 healthy donors (Karle et al, December 2009). From DCs of 2 of 4 patients with birch pollen allergy, nested peptide sets containing the immunodominant T-cell epitope Bet v 1142-153 were eluted.23 TH cell epitope immunodominance has been shown to be frequently associated with mobile loops within the 3-dimensional structure of antigens because proteases tend to cleave at exposed sites that exhibit a higher degree of local disorder.32 Bet v 1142-153 is located within the C-terminal a-helix of the native allergen and is N-terminally flanked by a mobile loop. These structural determinants are illustrated in Fig 5. Structural analysis revealed that this loop contains 1 possible cleavage site that is solvent-exposed on the surface of Bet v 1, Asp125His126. Regarding the sequences of the HLA-DR–eluted peptide clusters, we propose that processing of the immunodominant T-cell epitope starts at Asp125-His126. The resulting C-terminal fragment is loaded onto HLA-DR molecules and further trimmed on both ends, leaving the core Bet v 1142-153 intact for recognition by a large number of different T-cell receptors.23 The kinetics of endolysosomal degradation of Bet v 1 revealed that peptide clusters generated early on encompassed frequently recognized T-cell epitopes, whereas those appearing at later time points contained epitopes rarely involved in T-cell activation (Fig 1). Of note, significant amounts of Bet v 1 remained intact for 24 hours of endolysosomal proteolysis. Complete degradation of proteins within 1 hour has been associated with low immunogenicity, whereas partial resistance to proteolysis for more than 5 hours has been correlated with the capacity to induce efficient T-cell priming and antibody responses.33 By comparison, Bet v 1 shows limited susceptibility to endolysosomal degradation, quasi forming a depot of intact protein that allows continuous generation of proteolytic fragments over a long period. This property may contribute to the high allergenic potential of the major birch pollen allergen. Our data may also explain the efficient T-cell priming to Bet v 1142-153. First, this epitope is located in
a region flanked by a loop that is accessible to endolysosomal proteases in the native fold of Bet v 1. Second, it is early and continuously generated from a depot of intact Bet v 1 and thus persistently supplied for loading onto MHC class II molecules. Finally, this peptide binds to diverse HLA-DR molecules encoded by alleles common in the Caucasian population.26 Two 18mer peptides representing the HLA-DR–eluted peptides derived from Bet v 169-96 and Bet v 1140-157 induced proliferative and cytokine responses in Bet v 1–specific TCCs independently of their TH subset (TH2, TH1, or TH0; Fig 4). To investigate a functional relevance of regions flanking the core epitope recognized by the T-cell receptor, we compared the T-cell responses to Bet v 169-96 and Bet v 1140-157 containing longer flanks with the respective 12mer peptides containing the core epitope. All TCCs specific for Bet v 1142-153 responded equally to both peptide variants even though they possessed different T-cell receptors. These data suggest that flanking regions do not affect the activation of T lymphocytes specific for the immunodominant epitope of Bet v 1. Similar results were observed for a clone specific for Bet v 179-90, another frequently recognized epitope. Finally, we found T cells recognizing peptides located within the HLA-DR–eluted Bet v 1–derived sequences in allergeninduced TCLs of each tested individual, demonstrating that analogous peptides had been presented in vivo (Fig. 3). Some TCLs reacted with peptides that had not been identified in the pool of HLA-DR–eluted peptides. We cannot exclude that Bet v 1–derived peptides were lost during elution and purification steps. Alternatively, these peptides may have been presented by HLA molecules other than HLA-DR or by APCs other than DCs, such as B cells or monocytes, which were present in the T-cell mapping experiments. Investigating Bet v 1–reactive TCCs from patients with birch pollen allergy specific for epitopes covered by the naturally processed peptides revealed that the majority (>68%) produced high amounts of IL-4 (Table E1). These data
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FIG 4. T cell–activating properties of naturally processed Bet v 1–derived peptides. Bet v 1–reactive TCCs were stimulated with 2.5 mmol/L of the epitope-containing 12mer peptide (black bars), an 18mer peptide representing the HLA-DR–eluted peptides (white bars), and Bet v 1 (gray bars). TCC218 was stimulated with FKYNYSVIEGGP and FKYNYSVIEGGPIGDTLE. TCC266, TCC334, TCC14VR, and TCC4R were stimulated with TLLRAVESYLLA and GETLLRAVESYLLAHSDA. A, Proliferative responses (SI) are shown. B, Cytokines (pg/mL) were measured by ELISA.
support the importance of the HLA-DR–eluted Bet v 1 peptides in the activation of disease-relevant TH2 cells in vivo. Because these peptides originated from allergen-processing by DCs capable of priming naive T lymphocytes, we suggest the involvement of these epitopes in the sensitization process of birch pollen allergy. On the basis of the finding that T cells from individuals with and without birch pollen allergy recognize the same epitopes in Bet v 1 but differ in their cytokine profiles,34 we conclude that the naturally processed Bet v 1 peptides identified in this study are relevant for the immune recognition of Bet v 1 in vivo, but per se do not determine the quality of the T-cell response to this important allergen. The development of a TH2-dominated response rather depends on other variables, such as the cytokine milieu present during T-cell receptor activation.35
In summary, we have identified immunologically relevant, allergen-derived MHC class II–presented peptides processed by DCs from individuals with allergy. The naturally processed peptides match previous results from epitope mapping studies using synthetic peptides and allergen-specific TCL. Therefore, the presented results confirm the reliability of the conventional approach for the identification of T-cell epitopes of allergens. Thus, this study is important for the development of new T-cell–targeting approaches for prevention and treatment of allergies. We thank Beatrice Jahn-Schmid and Winfried Pickl for reading the manuscript critically and for fruitful discussions. We thank Ingrid Fae´ for HLA-typing.
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FIG 5. Structural determinants of the immunodominant T-cell epitope Bet v 1142-153 (TLLRAVESYLLA). A, Zigzags in the aa sequence of Bet v 1 represent a-helices, arrows indicate b-sheets, and bars represent loops. Structure assignments were taken from Protein Data Bank file 1BV1. B, The loop (green) flanking the C-terminal a-helix (golden) containing Bet v 1142-153 is shown in the 3-dimensional structure. C, Surface representation of the loop (green) flanking the C-terminal a-helix (golden) containing Bet v 1142-153. Peptide C-N atoms in the loop region are yellow and blue. The arrow indicates the only solvent-accessible peptide bond. Molecular graphics in B and C were created with UCSF Chimera (PMID: 15264254).
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11. Novak N, Tepel C, Koch S, Brix K, Bieber T, Kraft S. Evidence for a differential expression of the FcepsilonRIgamma chain in dendritic cells of atopic and nonatopic donors. J Clin Invest 2003;111:1047-56. 12. Bohle B. T-cell epitopes of food allergens. Clin Rev Allergy Immunol 2006;30:97-108. 13. Woodfolk JA. T-cell responses to allergens. J Allergy Clin Immunol 2007;119: 280-94; quiz 95-96. 14. Chicz RM, Urban RG, Lane WS, Gorga JC, Stern LJ, Vignali DA, et al. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHCrelated molecules and are heterogeneous in size. Nature 1992;358:764-8. 15. Lippolis JD, White FM, Marto JA, Luckey CJ, Bullock TN, Shabanowitz J, et al. Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets. J Immunol 2002;169:5089-97. 16. Malcherek G, Gnau V, Stevanovic S, Rammensee HG, Jung G, Melms A. Analysis of allele-specific contact sites of natural HLA-DR17 ligands. J Immunol 1994;153: 1141-9. 17. Vignali DA, Strominger JL. Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor. J Exp Med 1994;179:1945-56. 18. Carson RT, Vignali KM, Woodland DL, Vignali DA. T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage. Immunity 1997;7:387-99. 19. Moudgil KD, Sercarz EE, Grewal IS. Modulation of the immunogenicity of antigenic determinants by their flanking residues. Immunol Today 1998;19:217-20. 20. Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, Grimm R, et al. Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. J Exp Med 1996;183:599-609. 21. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl AJ, et al. Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J 1998;12:231-42. 22. Ebner C, Szepfalusi Z, Ferreira F, Jilek A, Valenta R, Parronchi P, et al. Identification of multiple T cell epitopes on Bet v I, the major birch pollen allergen, using specific T cell clones and overlapping peptides. J Immunol 1993;150:1047-54. 23. Jahn-Schmid B, Radakovics A, Luttkopf D, Scheurer S, Vieths S, Ebner C, et al. Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens. J Allergy Clin Immunol 2005;116:213-9.
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24. Friedl-Hajek R, Spangfort MD, Schou C, Breiteneder H, Yssel H, Joost van Neerven RJ. Identification of a highly promiscuous and an HLA allele-specific T-cell epitope in the birch major allergen Bet v 1: HLA restriction, epitope mapping and TCR sequence comparisons. Clin Exp Allergy 1999;29:478-87. 25. Breiteneder H, Scheiner O, Hajek R, Hulla W, Huttinger R, Fischer G, et al. Diversity of TCRAV and TCRBV sequences used by human T-cell clones specific for a minimal epitope of Bet v 1, the major birch pollen allergen. Immunogenetics 1995; 42:53-8. 26. Van Overtvelt L, Wambre E, Maillere B, von Hofe E, Louise A, Balazuc AM, et al. Assessment of Bet v 1-specific CD49 1 T cell responses in allergic and nonallergic individuals using MHC class II peptide tetramers. J Immunol 2008;180: 4514-22. 27. Jahn-Schmid B, Fischer GF, Bohle B, Fae I, Gadermaier G, Dedic A, et al. Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01. J Allergy Clin Immunol 2005;115:399-404. 28. Gerstmayr M, Ilk N, Schabussova I, Jahn-Schmid B, Egelseer EM, Sleytr UB, et al. A novel approach to specific allergy treatment: the recombinant allergen-S-layer fusion protein rSbsC-Bet v 1 matures dendritic cells that prime Th0/Th1 and IL-10-producing regulatory T cells. J Immunol 2007;179:7270-5.
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29. Delamarre L, Pack M, Chang H, Mellman I, Trombetta ES. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 2005;307: 1630-4. 30. Rohn TA, Reitz A, Paschen A, Nguyen XD, Schadendorf D, Vogt AB, et al. A novel strategy for the discovery of MHC class II-restricted tumor antigens: identification of a melanotransferrin helper T-cell epitope. Cancer Res 2005;65: 10068-78. 31. Rammensee HG. Chemistry of peptides associated with MHC class I and class II molecules. Curr Opin Immunol 1995;7:85-96. 32. Carmicle S, Dai G, Steede NK, Landry SJ. Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s. J Biol Chem 2002;277:155-60. 33. Delamarre L, Couture R, Mellman I, Trombetta ES. Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis. J Exp Med 2006;203:2049-55. 34. Ebner C, Schenk S, Najafian N, Siemann U, Steiner R, Fischer GW, et al. Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients. J Immunol 1995;154:1932-40. 35. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 2000;1:311-6.
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FIG E1. Time kinetics of endolysosomal processing of Bet v 1. A, After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases, 10 mL of the digestion reactions corresponding to 2.5 mg Bet v 1 were analyzed by SDS-PAGE and Coomassie staining. M, Marker (kd); B, Bet v 1 in digestion buffer without microsomal fractions. B, Grayscaled and inverted bands corresponding to intact Bet v 1 were quantified as integrated measures of intensity and size using the software Adobe Photoshop CS3. Protein degradation is shown as function of time.
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TABLE E1. Allergen-induced production of IL-4 and IFN-g of 19 Bet v 1–specific T-cell clones obtained from 11 patients with birch pollen allergy and reactive with epitopes located within the naturally processed peptides Bet v 112-32, Bet v 169-96, and Bet v 1132-159 Naturally processed peptides
T-cell clone
T-cell–stimulatory peptide
IL-4 (pg/mL)
IFN-g (pg/mL)
Bet v 112-32 VIPAARLFKAFILDGDN IPAARLFKAFILDGDNL LFKAFILDGDNLFPK FKAFILDGDNLFPK Bet v 169-96
K20 M2 M9 S3 W35 C195 G17 G19 J17 J10 M3 M5 S218
FILDGDNLF FILDGDNLF FILDGDNLF ARLFKAFILDGD FILDGDNLF EVDHTNFKYNYS NYSVIEGGPIGD FKYNYSVIE VIEGGPIGD VIEGGPIGD FKYNYSVIE FKYNYSVIE FKYNYSVIE
361 <9.0 <9.0 1300 170 310 338 154 <9.0 15.0 1954 218 66.3
25.3 <9.5 941 70.2 <9.5 <9.5 <9.5 18.1 51.4 36.4 <9.5 <9.5 <9.5
TH2 TH0 TH1 TH2 TH2 TH2 TH2 TH2 TH1 TH0 TH2 TH2 TH2
H17 R4 SA10 SU1 S266 S334
TLLRAVESYLLA TLLRAVESYLLA TLLRAVESYLLA RAVESYLLAHSD TLLRAVESYLLA TLLRAVESYLLA
464 726 <9.0 528 1000 434
<9.5 <9.5 <9.5 <9.5 448 <9.5
TH2 TH2 TH0 TH2 TH0 TH2
DRVDEVDHTNFKYNYS FKYNYSVIEGGPIGDTLE
Bet v 1132-159 GETLLRAVESYLLAHSDAYN GETLLRAVESYLLAHSD GETLLRAVESYLLAHS QVKASKEMGETLLRAVESYLLAHSDA QVKASKEMGETLLRAVESYLLAHSDAYN ASKEMGETLLRAVESYLLAHSDA ASKEMGETLLRAVESYLLAHSDAYN KEMGETLLRAVESYLLAHSDA EMGETLLRAVESYLLAHSDA MGETLLRAVESYLLAHS MGETLLRAVESYLLAHSD MGETLLRAVESYLLAHSDA GETLLRAVESYLLAH GETLLRAVESYLLAHSDA ETLLRAVESYLLAH ETLLRAVESYLLAHS ETLLRAVESYLLAHSD ETLLRAVESYLLAHSDA TLLRAVESYLLAHSD
*Clones were assigned to TH subsets as follows: TH2, IL-4/IFN-g >5; TH1, IL-4/IFN-g <0.2; TH0, IL-4/IFN-g 5 0.2 to 5.
TH subset*
From athe Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg; bthe Christian Doppler Laboratory for Immunomodulation, Department of Pathophysiology, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna; cF. Hoffmann-La Roche Ltd, Immunosafety, Non-Clinical Safety, Basel; and dthe Clinical Department for Blood Group Serology, University Clinic for Blood Group Serology and Transfusion Medicine, Vienna. *These authors are cosenior authors of this article. ¨ sterreichische Nationalbank, the Christian Doppler Supported by P10150 of the O Laboratory for Allergy Diagnosis and Therapy, and the Christian Doppler Laboratory for Immunomodulation, Austria. Disclosure of potential conflict of interest: F. Ferreira has received research support from Biomay AG, the Austrian Science Fund, the Christian Doppler Research Association, and the Austrian National Bank and has provided legal consultation services or expert witness testimony relevant to Indoor Biotechnologies and AllergenOnline Database. B. Bohle has received research support from the Austrian Science Fund and the Christian Doppler Laboratory. The rest of the authors have declared that they have no conflict of interest. Received for publication July 10, 2009; revised October 6, 2009; accepted for publication October 29, 2009. Available online February 4, 2010. Reprint requests: Barbara Bohle, PhD, Institute of Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria, E-mail: barbara.bohle@ meduniwien.ac.at; Fatima Ferreira, PhD, Department of Molecular Biology, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria. E-mail: fatima. [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2009.10.052
II–eluted Bet v 1–derived sequences demonstrating their occurrence in vivo. Conclusion: We report for the first time how epitopes of allergens are generated and selected for presentation to T lymphocytes. The limited susceptibility of Bet v 1 to endolysosomal processing might contribute to its high allergenic potential. (J Allergy Clin Immunol 2010;125:711-8.) Key words: Birch pollen allergy, Bet v 1, antigen processing, antigen presentation, dendritic cells, T cells, T-cell epitopes
IgE-mediated disorders such as allergic rhinitis and asthma have tremendously increased during the recent decades. Understanding the immune mechanisms underlying allergic disorders is important to develop new strategies for their prevention and treatment. CD41 TH lymphocytes play a pivotal role in the sensitization and maintenance of type I allergy. Allergic diseases result from an aberrant T-cell response to allergens dominated by long-lived TH2 cells.1,2 Among other cytokines, allergen-specific TH2 cells secrete high amounts of IL4 and IL-13, which induce the production of allergen-specific IgE antibodies that mediate immediate allergic symptoms.3 In addition to this indirect involvement in immediate reactions, allergen-specific TH2 cells have been demonstrated to be directly involved in clinical late-phase reactions in target organs such as the lung and skin.4,5 Allergen-specific CD41 T lymphocytes are activated by peptides generated by proteolytic degradation of allergens in endolysosomal compartments of antigen-presenting cells (APCs). The T-cell receptor recognizes the peptides in the context of MHC class II molecules exposed on the cell surface of APCs.6 The most specialized APCs are dendritic cells (DCs).7 They are capable of priming naive T lymphocytes because they efficiently take up allergens by phagocytosis or pinocytosis or via receptors expressed on their surface (eg, FceRI) and express a large number of MHC class II molecules and different costimulatory molecules.8-11 Until now, T cell–activating regions in allergens have been identified on the basis of proliferative and cytokine responses to short overlapping peptides spanning the entire amino acid (aa) sequence of the molecule.12,13 Although this approach identifies the core epitopes recognized by the T-cell receptor, the actual sequences of naturally processed MHC class II–bound peptides cannot be predicted. Such peptides have been demonstrated to display a high degree of heterogeneity in both length and site of their terminal truncations.14,15 Furthermore, the aa residues flanking the core epitope recognized by the T-cell receptor have been reported to enhance or hinder T-cell activation, respectively.16-19 711
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Abbreviations used aa: Amino acid APC: Antigen-presenting cell cpm: Counts per minute DC: Dendritic cell SI: Stimulation index TCC: T-cell clone TCL: T-cell line
Because naturally processed MHC class II–bound peptides derived from allergens have not been identified so far, we investigated such peptides presented by DCs from patients with allergy. We used Bet v 1, the clinically highly relevant major birch pollen allergen, which has been extensively characterized by our group. In addition to dissecting its IgE-binding properties,20,21 we have identified Bet v 1–T cell epitopes by stimulation of allergenspecific T cells isolated from the peripheral blood of a large number of patients with birch pollen allergy with synthetic overlapping 12mer peptides.22,23 HLA-DR molecules were shown to be highly relevant restriction elements for Bet v 1–specific T lymphocytes.24-26 We isolated HLA-DR–bound peptides from Bet v 1–pulsed DCs derived from individuals with birch pollen allergy. Moreover, Bet v 1 was incubated with microsomal proteases isolated from these DCs. Bet v 1–derived fragments obtained by either approach were sequenced by tandem mass spectrometry. The T cell–activating properties of HLA-DR–eluted peptide sequences were confirmed by using Bet v 1–specific T-cell clones (TCCs). To verify the in vivo relevance of naturally processed Bet v 1 peptides, we analyzed the peripheral blood of each patient with birch pollen allergy for the presence of T cells reactive with epitopes matching the eluted peptide sequences.
METHODS Patients with birch pollen allergy and allergen All patients with birch pollen allergy included in this study were sensitized to Bet v 1, had rhinoconjunctivitis in spring, and showed specific IgE CAP/ RAST class >3 (Pharmacia Diagnostics, Uppsala, Sweden) and positive skin prick reactions (wheal diameter >5 mm) to birch pollen. All patients gave written consent before enrolment in the study, which was approved by the local Medical Ethical Committee of Vienna. Molecular HLA typing for HLADRB1 alleles of the patients was performed according to methods previously referenced.27 Recombinant Bet v 1 was purchased from Biomay (Vienna, Austria).
Endolysosomal degradation assays Monocyte-derived (md) DCs were generated from CD141 cells purified from PBMCs as described.28 DCs were homogenized in 10 mmol/L TRIS acetate pH7 containing sucrose (250 mmol/L), and microsomes were isolated by ultracentrifugation.29 Bet v 1 (5 mg) was digested with microsomal enzymes (7 mg) in 100 mmol/L citrate buffer pH 4.8 containing dithiothreitol (2 mmol/ L) at 378C. Reactions were stopped by heat denaturation and analyzed by SDS-PAGE and mass spectrometry using an ESI-QTOF mass spectrometer fitted with a capillary reversed phase HPLC (Waters, Milford, Mass).
Allergen-specific T-cell cultures Allergen-specific T-cell lines (TCLs) were generated by stimulating PBMCs (1.5 3 106) with Bet v 1 (10 mg/mL) as described.23 TCLs were stimulated with varying concentrations of Bet v 1 (2.5-10 mg/mL) or peptide
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(5 mg/mL) in the presence of 5 3 104 irradiated (60 Gy) autologous PBMCs for 48 hours in duplicate. Proliferation was assessed by [3H] thymidine incorporation. The stimulation index (SI) was calculated as ratio between counts per minute (cpm) of TCL plus PBMC plus allergen and cpm of TCL plus PBMC only. Cryopreserved TCCs specific for different Bet v 1 epitopes were thawed, expanded, and stimulated with Bet v 1 (5 mg/mL) or the indicated peptides (0.6-2.5 mmol/L) in the presence of irradiated autologous PBMCs. Supernatants were harvested after 24 hours. Cytokine levels were assessed by ELISA as described.23 Cytokine levels in controls were subtracted from those determined in unstimulated cultures.
Isolation of peptides from HLA-DR molecules The monoclonal anti–HLA-DR antibody L243 (ATCC HB-55) was purified by ammonium sulfate precipitation and protein G affinity chromatography. Antibody specificity was checked by flow cytometry (data not shown). Dendritic cells (65-100 3 106) were stimulated with Bet v 1 (200 mg/mL) plus LPS (1 mg/mL) (Sigma, St Louis, Mo) on day 5 and lysed in 10 mmol/L TRIS/HCl pH 7.6 containing 0.6% CHAPS and Complete Protease Inhibitor (Roche Diagnostics, Mannheim, Germany) for 30 minutes on ice. After ultracentrifugation, protein G beads (GE Healthcare, Vienna, Austria; 200 mL in 200 mL PBS) were added to the supernatant for 2 hours on ice. After centrifugation, L243 antibody (4 mg) was added to the supernatant and incubated for 4 hours at 48C. Protein G beads (200 mL in 200 mL PBS) were added for 16 hours. After centrifugation, the pellet was washed with PBS, resuspended in 300 mL 10% trifluoroacetic acid, incubated at room temperature for 5 minutes, and ultrafiltrated through a Microcon-10 filter unit (Millipore, Molsheim, France). The eluate was immediately analyzed by tandem mass spectrometry.
Mass spectrometry analysis of HLA-DR–eluted peptides Peptides from patient 4 were eluted and identified by mass spectrometry as described.30 For patients 1 to 3, peptides (20 mL) were separated by capillary rpHPLC (Waters) directly coupled to a Q-Tof Global Ultima mass spectrometer (Waters) by using a Nanoflow spray head (precolumn Nanoease Symmetry300 trap column, separating column Nanoease Atlantis dC18; Waters). Peptides were eluted with an acetonitrile gradient (solvent A 0.1% vol/vol formic acid/5% vol/vol acetonitrile; solvent B 0.1% vol/vol formic acid/95% vol/ vol acetonitrile; 5% to 45% B in 90 minutes). Data acquisition and instrument control were done with MassLynx software V4.1 (Waters). Singly, doubly, and triply charged ions were selected for fragmentation by collision with argon (collision energy settings, 25-50 kV). Survey and fragment spectra were analyzed by using the software PLGS version 2.2.5 (Waters) with automatic and manual validation with an in-house Bet v 1 database. As control for this analytical system, mdDCs pulsed with the major mugwort pollen allergen were used. No Bet v 1–derived peptides were detected (data not shown).
RESULTS Endolysosomal processing of the major birch pollen allergen Microsomal proteases were isolated from DCs of 2 donors with birch pollen allergy and incubated with Bet v 1. At defined intervals, allergen degradation was monitored by SDS-PAGE (see this article’s Fig E1 in the Online Repository at www.jacionli ne.org), and proteolytic fragments were sequenced by tandem mass spectrometry (Fig 1). Intact Bet v 1 was detectable for 24 hours. Taking all time points, 113 different fragments were detected in a total of 368 sequenced peptides. After 0.5 hours, first peptide clusters derived from the N-terminus Bet v 11-20, the central region Bet v 184-97, and the C-terminus Bet v 1146-157 were identified. After 1 hour, peptides originating from the region Bet v 1104-115 were detected. Some of these initially identified
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FIG 1. Time kinetics of endolysosomal processing of Bet v 1. After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases isolated from DCs, generated peptides were sequenced by mass spectrometry. Frequently recognized T-cell epitopes are framed in the amino acid sequence of Bet v 1, shown on top.
peptide clusters were detectable until 24 hours of degradation. Peptide clusters in the regions Bet v 122-37, Bet v 133-55, Bet v 156-65, and Bet v 169-80 did not appear before 3 hours of proteolytic cleavage. Remarkably, early appearing peptide clusters matched frequently recognized T cell–activating regions such as Bet v 14-15, Bet v 113-24, Bet v 119-30, Bet v 179-90, Bet v 197-108, Bet v 1112-123, and Bet v 1142-153 previously identified in Bet v 1–specific TCL derived from 58 donors with birch pollen allergy.23
Bet v 1–derived naturally processed peptides First, the optimum incubation period for appearance of allergen-derived peptides on the surface of Bet v 1–pulsed DCs was evaluated. DCs were incubated with Bet v 1 for 6, 11, 24, and 48 hours; washed; irradiated; and used to stimulate autologous Bet v 1–specific T-cell cultures. DCs pulsed for 11 hours induced the most pronounced proliferative responses (Fig 2). Next, HLA-DR– bound peptides were eluted from DCs of 4 patients with birch pollen allergy pulsed with Bet v 1 for 11 hours and analyzed by
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FIG 2. Optimizing the period for allergen pulsing of DCs. DCs from an individual with birch pollen allergy were incubated with Bet v 1 (5 mg/0.5 3 106 cells) for 6, 11, 24, and 48 hours, respectively, and used to stimulate a Bet v 1–specific T-cell clone. T-cell proliferation (SI) is shown.
tandem mass spectrometry. In total, 1606 different peptides with a mean length of 16.4 aa residues (ranging from 6 to 35 aa residues) were detected. Database sequence searches revealed that these peptides derived from more than 650 different proteins of mainly human origin (S. Mutschlechner et al, December 2009). Within this peptide pool, 27 Bet v 1–derived peptides were identified (Fig 3, B). Naturally processed Bet v 1–derived peptides ranged from 14 to 28 aa residues (mean, 18 aa residues). Each patient presented nested sets of peptides originating from 1 or 2 regions of the Bet v 1 sequence that shared a similar core motif but varied in the length of their N-terminal and C-terminal flanking regions. In 2 of 4 patients, peptides were detected from the C-terminal region Bet v 1132-159, which harbors the immunodominant T cell– activating epitope23 (Fig 3, A). DCs from 2 patients presented N-terminal peptides located within Bet v 112-32, and peptides covering the region Bet v 169-96 were detected in 1 individual. The sequences of all naturally presented peptides are summarized in this article’s Table E1 in the Online Repository at www.jacionline.org.
HLA-DR phenotypes of patients with birch pollen allergy All donors used for the isolation of HLA-DR–associated peptides from Bet v 1–pulsed DCs were HLA-DRB1–typed at high resolution. DCs generated from patient 1 (HLADRB1*0701, *1501) presented peptides located within Bet v 1140-159. DCs from patient 2 (HLA-DRB1*0701) presented peptides located within Bet v 169-96. DCs from patient 3 (HLADRB1*0101, *0301) presented peptides located within Bet v 118-32, and DCs from patient 4 (HLA-DRB1*1501, *1601) presented peptides located within 2 different regions, Bet v 112-29 and Bet v 1132-159. T cell–activating properties of Bet v 1–derived naturally processed peptides To confirm that naturally processed Bet v 1 peptides were able to activate T cells, peptides consisting of 18 aa residues, which was the average length of HLA-DR–eluted Bet v 1 peptides, were synthesized and used to stimulate Bet v 1–specific TCCs with known epitope specificity. Peptide FKYNYSVIEGGPIGDTLE representing the naturally processed Bet v 169-96 was incubated with TCC218 specific for Bet v 179-90. Peptide GETLLRAVESYLLAHSDA representing the 18mer shared by the nested
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peptide sets derived from the C-terminus isolated from patient 1 and patient 4 was incubated with TCC266, TCC334, TCC14VR, and TCC4R, all specific for Bet v 1142-153 and obtained from 3 different patients. Proliferative and cytokine responses to the respective 18mer and 12mer peptides (each 2.5 mmol/L) and Bet v 1 are shown in Fig 4. TCC218 specific for FKYNYSVIEGGP proliferated equally well in response to the 12mer peptide, to FKYNYSVIEGGPIGDTLE, or to Bet v 1 (Fig 4, A). As in response to the full-length allergen, TCC218 produced IFN-g, no IL-4, and marginal amounts of IL-5 in response to both peptides (Fig 4, B). Similarly, TCC266, TCC334, TCC14VR, and TCC4R specific for TLLRAVESYLLA proliferated equally well in response to the 12mer peptide, to GETLLRAVESYLLAHSDA, or to Bet v 1. TCC266, TCC14VR, and TCC4R secreted both IL-5 and IFN-g in response to either stimulus. Despite strong proliferation, TCC334 produced marginal amounts of the tested cytokines. In all clones, similar responses were found over a broad range of equimolar concentrations of both peptides (data not shown).
In vivo relevance of Bet v 1–derived naturally processed peptides To reveal a link between the presentation of HLA-eluted Bet v 1–derived peptides and their in vivo recognition by T cells, we generated Bet v 1–specific TCLs from the 4 patients with birch pollen allergy whose DCs had been used in the pulsing experiments. Each TCL was analyzed for epitope recognition by determining proliferative responses to 50 overlapping 12mer peptides covering the aa sequence of Bet v 1. Each TCL proliferated in response to 12mer peptides matching the sequences of naturally processed Bet v 1 peptides eluted from DCs of the respective individual. Three lines harbored T cells specific for Bet v 1 epitopes located in peptide sequences not contained in the pool of HLADR–eluted peptides (Fig 3, B). We also analyzed the allergen-induced production of IL-4 and IFN-g of 19 TCCs previously obtained from 11 different patients with birch pollen allergy and reactive with epitopes located within the naturally processed peptides Bet v 112-32, Bet v 169-96, and Bet v 1132-159 (Table E1). Three of 5 clones specific for epitopes within Bet v 112-32 belonged to the TH2 subset. One clone was designated TH0 and 1 clone TH1. Similar data were found for clones specific for epitopes within Bet v 169-96 (6/8 TH2, 1/8 TH0, and 1/8 TH1) and Bet v 1132-159 (4/6 TH2, 2/6 TH0, and 0/6 TH1).
DISCUSSION For the first time, antigen processing and presentation of an allergen are reported. The major birch pollen allergen was subjected to endolysosomal degradation, and HLA-DR–bound peptides were eluted from Bet v 1–pulsed DCs from patients with birch pollen allergy. Bet v 1–derived peptides represented 1.6% of all eluted peptides, displayed an average length of 18 aa residues, and occurred as nested clusters sharing a common core with varying flanking regions. These features are characteristic for HLA-DR–presented peptides.15,31 The core sequences of the HLA-DR–eluted Bet v 1–derived peptides generally corresponded to frequently recognized T cell–activating regions and overlapped with the peptides created by endolysosomal degradation of Bet v 1 (Fig 1). Finally, the naturally processed sequences
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FIG 3. Naturally processed Bet v 1–derived peptides. Peptides eluted from purified HLA-DR:peptide complexes isolated from DCs of 4 patients with Bet v 1 allergy were sequenced by mass spectrometry. A, Amino acid sequence of Bet v 1. Frequently recognized T-cell epitopes are framed. B, HLA-DR–eluted peptides are shown in black; 12mer peptides inducing proliferative responses in Bet v 1–specific TCLs are shown in gray.
matched those identified by another study using DCs from 10 healthy donors (Karle et al, December 2009). From DCs of 2 of 4 patients with birch pollen allergy, nested peptide sets containing the immunodominant T-cell epitope Bet v 1142-153 were eluted.23 TH cell epitope immunodominance has been shown to be frequently associated with mobile loops within the 3-dimensional structure of antigens because proteases tend to cleave at exposed sites that exhibit a higher degree of local disorder.32 Bet v 1142-153 is located within the C-terminal a-helix of the native allergen and is N-terminally flanked by a mobile loop. These structural determinants are illustrated in Fig 5. Structural analysis revealed that this loop contains 1 possible cleavage site that is solvent-exposed on the surface of Bet v 1, Asp125His126. Regarding the sequences of the HLA-DR–eluted peptide clusters, we propose that processing of the immunodominant T-cell epitope starts at Asp125-His126. The resulting C-terminal fragment is loaded onto HLA-DR molecules and further trimmed on both ends, leaving the core Bet v 1142-153 intact for recognition by a large number of different T-cell receptors.23 The kinetics of endolysosomal degradation of Bet v 1 revealed that peptide clusters generated early on encompassed frequently recognized T-cell epitopes, whereas those appearing at later time points contained epitopes rarely involved in T-cell activation (Fig 1). Of note, significant amounts of Bet v 1 remained intact for 24 hours of endolysosomal proteolysis. Complete degradation of proteins within 1 hour has been associated with low immunogenicity, whereas partial resistance to proteolysis for more than 5 hours has been correlated with the capacity to induce efficient T-cell priming and antibody responses.33 By comparison, Bet v 1 shows limited susceptibility to endolysosomal degradation, quasi forming a depot of intact protein that allows continuous generation of proteolytic fragments over a long period. This property may contribute to the high allergenic potential of the major birch pollen allergen. Our data may also explain the efficient T-cell priming to Bet v 1142-153. First, this epitope is located in
a region flanked by a loop that is accessible to endolysosomal proteases in the native fold of Bet v 1. Second, it is early and continuously generated from a depot of intact Bet v 1 and thus persistently supplied for loading onto MHC class II molecules. Finally, this peptide binds to diverse HLA-DR molecules encoded by alleles common in the Caucasian population.26 Two 18mer peptides representing the HLA-DR–eluted peptides derived from Bet v 169-96 and Bet v 1140-157 induced proliferative and cytokine responses in Bet v 1–specific TCCs independently of their TH subset (TH2, TH1, or TH0; Fig 4). To investigate a functional relevance of regions flanking the core epitope recognized by the T-cell receptor, we compared the T-cell responses to Bet v 169-96 and Bet v 1140-157 containing longer flanks with the respective 12mer peptides containing the core epitope. All TCCs specific for Bet v 1142-153 responded equally to both peptide variants even though they possessed different T-cell receptors. These data suggest that flanking regions do not affect the activation of T lymphocytes specific for the immunodominant epitope of Bet v 1. Similar results were observed for a clone specific for Bet v 179-90, another frequently recognized epitope. Finally, we found T cells recognizing peptides located within the HLA-DR–eluted Bet v 1–derived sequences in allergeninduced TCLs of each tested individual, demonstrating that analogous peptides had been presented in vivo (Fig. 3). Some TCLs reacted with peptides that had not been identified in the pool of HLA-DR–eluted peptides. We cannot exclude that Bet v 1–derived peptides were lost during elution and purification steps. Alternatively, these peptides may have been presented by HLA molecules other than HLA-DR or by APCs other than DCs, such as B cells or monocytes, which were present in the T-cell mapping experiments. Investigating Bet v 1–reactive TCCs from patients with birch pollen allergy specific for epitopes covered by the naturally processed peptides revealed that the majority (>68%) produced high amounts of IL-4 (Table E1). These data
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FIG 4. T cell–activating properties of naturally processed Bet v 1–derived peptides. Bet v 1–reactive TCCs were stimulated with 2.5 mmol/L of the epitope-containing 12mer peptide (black bars), an 18mer peptide representing the HLA-DR–eluted peptides (white bars), and Bet v 1 (gray bars). TCC218 was stimulated with FKYNYSVIEGGP and FKYNYSVIEGGPIGDTLE. TCC266, TCC334, TCC14VR, and TCC4R were stimulated with TLLRAVESYLLA and GETLLRAVESYLLAHSDA. A, Proliferative responses (SI) are shown. B, Cytokines (pg/mL) were measured by ELISA.
support the importance of the HLA-DR–eluted Bet v 1 peptides in the activation of disease-relevant TH2 cells in vivo. Because these peptides originated from allergen-processing by DCs capable of priming naive T lymphocytes, we suggest the involvement of these epitopes in the sensitization process of birch pollen allergy. On the basis of the finding that T cells from individuals with and without birch pollen allergy recognize the same epitopes in Bet v 1 but differ in their cytokine profiles,34 we conclude that the naturally processed Bet v 1 peptides identified in this study are relevant for the immune recognition of Bet v 1 in vivo, but per se do not determine the quality of the T-cell response to this important allergen. The development of a TH2-dominated response rather depends on other variables, such as the cytokine milieu present during T-cell receptor activation.35
In summary, we have identified immunologically relevant, allergen-derived MHC class II–presented peptides processed by DCs from individuals with allergy. The naturally processed peptides match previous results from epitope mapping studies using synthetic peptides and allergen-specific TCL. Therefore, the presented results confirm the reliability of the conventional approach for the identification of T-cell epitopes of allergens. Thus, this study is important for the development of new T-cell–targeting approaches for prevention and treatment of allergies. We thank Beatrice Jahn-Schmid and Winfried Pickl for reading the manuscript critically and for fruitful discussions. We thank Ingrid Fae´ for HLA-typing.
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FIG 5. Structural determinants of the immunodominant T-cell epitope Bet v 1142-153 (TLLRAVESYLLA). A, Zigzags in the aa sequence of Bet v 1 represent a-helices, arrows indicate b-sheets, and bars represent loops. Structure assignments were taken from Protein Data Bank file 1BV1. B, The loop (green) flanking the C-terminal a-helix (golden) containing Bet v 1142-153 is shown in the 3-dimensional structure. C, Surface representation of the loop (green) flanking the C-terminal a-helix (golden) containing Bet v 1142-153. Peptide C-N atoms in the loop region are yellow and blue. The arrow indicates the only solvent-accessible peptide bond. Molecular graphics in B and C were created with UCSF Chimera (PMID: 15264254).
Clinical implications: The characterization of naturally processed allergen-derived peptides is important for the development of T cell–targeting allergen-specific vaccines for prevention and treatment of type I allergy. REFERENCES 1. Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, et al. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med 2004;199:1567-75. 2. Bohle B, Schwihla H, Hu HZ, Friedl-Hajek R, Sowka S, Ferreira F, et al. Longlived Th2 clones specific for seasonal and perennial allergens can be detected in blood and skin by their TCR-hypervariable regions. J Immunol 1998;160:2022-7. 3. Christensen LH, Holm J, Lund G, Riise E, Lund K. Several distinct properties of the IgE repertoire determine effector cell degranulation in response to allergen challenge. J Allergy Clin Immunol 2008;122:298-304. 4. Haselden BM, Kay AB, Larche M. Immunoglobulin E-independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions. J Exp Med 1999;189:1885-94. 5. Bohle B, Zwolfer B, Heratizadeh A, Jahn-Schmid B, Antonia YD, Alter M, et al. Cooking birch pollen-related food: divergent consequences for IgE- and T cellmediated reactivity in vitro and in vivo. J Allergy Clin Immunol 2006;118:242-9. 6. Germain RN. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 1994;76:287-99. 7. Steinman RM, Hemmi H. Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol 2006;311:17-58. 8. Savina A, Amigorena S. Phagocytosis and antigen presentation in dendritic cells. Immunol Rev 2007;219:143-56. 9. Maurer D, Ebner C, Reininger B, Fiebiger E, Kraft D, Kinet JP, et al. The high affinity IgE receptor (Fc epsilon RI) mediates IgE-dependent allergen presentation. J Immunol 1995;154:6285-90. 10. Maurer D, Fiebiger E, Reininger B, Ebner C, Petzelbauer P, Shi GP, et al. Fc epsilon receptor I on dendritic cells delivers IgE-bound multivalent antigens into a cathepsin S-dependent pathway of MHC class II presentation. J Immunol 1998; 161:2731-9.
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24. Friedl-Hajek R, Spangfort MD, Schou C, Breiteneder H, Yssel H, Joost van Neerven RJ. Identification of a highly promiscuous and an HLA allele-specific T-cell epitope in the birch major allergen Bet v 1: HLA restriction, epitope mapping and TCR sequence comparisons. Clin Exp Allergy 1999;29:478-87. 25. Breiteneder H, Scheiner O, Hajek R, Hulla W, Huttinger R, Fischer G, et al. Diversity of TCRAV and TCRBV sequences used by human T-cell clones specific for a minimal epitope of Bet v 1, the major birch pollen allergen. Immunogenetics 1995; 42:53-8. 26. Van Overtvelt L, Wambre E, Maillere B, von Hofe E, Louise A, Balazuc AM, et al. Assessment of Bet v 1-specific CD49 1 T cell responses in allergic and nonallergic individuals using MHC class II peptide tetramers. J Immunol 2008;180: 4514-22. 27. Jahn-Schmid B, Fischer GF, Bohle B, Fae I, Gadermaier G, Dedic A, et al. Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01. J Allergy Clin Immunol 2005;115:399-404. 28. Gerstmayr M, Ilk N, Schabussova I, Jahn-Schmid B, Egelseer EM, Sleytr UB, et al. A novel approach to specific allergy treatment: the recombinant allergen-S-layer fusion protein rSbsC-Bet v 1 matures dendritic cells that prime Th0/Th1 and IL-10-producing regulatory T cells. J Immunol 2007;179:7270-5.
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29. Delamarre L, Pack M, Chang H, Mellman I, Trombetta ES. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 2005;307: 1630-4. 30. Rohn TA, Reitz A, Paschen A, Nguyen XD, Schadendorf D, Vogt AB, et al. A novel strategy for the discovery of MHC class II-restricted tumor antigens: identification of a melanotransferrin helper T-cell epitope. Cancer Res 2005;65: 10068-78. 31. Rammensee HG. Chemistry of peptides associated with MHC class I and class II molecules. Curr Opin Immunol 1995;7:85-96. 32. Carmicle S, Dai G, Steede NK, Landry SJ. Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s. J Biol Chem 2002;277:155-60. 33. Delamarre L, Couture R, Mellman I, Trombetta ES. Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis. J Exp Med 2006;203:2049-55. 34. Ebner C, Schenk S, Najafian N, Siemann U, Steiner R, Fischer GW, et al. Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients. J Immunol 1995;154:1932-40. 35. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 2000;1:311-6.
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FIG E1. Time kinetics of endolysosomal processing of Bet v 1. A, After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases, 10 mL of the digestion reactions corresponding to 2.5 mg Bet v 1 were analyzed by SDS-PAGE and Coomassie staining. M, Marker (kd); B, Bet v 1 in digestion buffer without microsomal fractions. B, Grayscaled and inverted bands corresponding to intact Bet v 1 were quantified as integrated measures of intensity and size using the software Adobe Photoshop CS3. Protein degradation is shown as function of time.
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TABLE E1. Allergen-induced production of IL-4 and IFN-g of 19 Bet v 1–specific T-cell clones obtained from 11 patients with birch pollen allergy and reactive with epitopes located within the naturally processed peptides Bet v 112-32, Bet v 169-96, and Bet v 1132-159 Naturally processed peptides
T-cell clone
T-cell–stimulatory peptide
IL-4 (pg/mL)
IFN-g (pg/mL)
Bet v 112-32 VIPAARLFKAFILDGDN IPAARLFKAFILDGDNL LFKAFILDGDNLFPK FKAFILDGDNLFPK Bet v 169-96
K20 M2 M9 S3 W35 C195 G17 G19 J17 J10 M3 M5 S218
FILDGDNLF FILDGDNLF FILDGDNLF ARLFKAFILDGD FILDGDNLF EVDHTNFKYNYS NYSVIEGGPIGD FKYNYSVIE VIEGGPIGD VIEGGPIGD FKYNYSVIE FKYNYSVIE FKYNYSVIE
361 <9.0 <9.0 1300 170 310 338 154 <9.0 15.0 1954 218 66.3
25.3 <9.5 941 70.2 <9.5 <9.5 <9.5 18.1 51.4 36.4 <9.5 <9.5 <9.5
TH2 TH0 TH1 TH2 TH2 TH2 TH2 TH2 TH1 TH0 TH2 TH2 TH2
H17 R4 SA10 SU1 S266 S334
TLLRAVESYLLA TLLRAVESYLLA TLLRAVESYLLA RAVESYLLAHSD TLLRAVESYLLA TLLRAVESYLLA
464 726 <9.0 528 1000 434
<9.5 <9.5 <9.5 <9.5 448 <9.5
TH2 TH2 TH0 TH2 TH0 TH2
DRVDEVDHTNFKYNYS FKYNYSVIEGGPIGDTLE
Bet v 1132-159 GETLLRAVESYLLAHSDAYN GETLLRAVESYLLAHSD GETLLRAVESYLLAHS QVKASKEMGETLLRAVESYLLAHSDA QVKASKEMGETLLRAVESYLLAHSDAYN ASKEMGETLLRAVESYLLAHSDA ASKEMGETLLRAVESYLLAHSDAYN KEMGETLLRAVESYLLAHSDA EMGETLLRAVESYLLAHSDA MGETLLRAVESYLLAHS MGETLLRAVESYLLAHSD MGETLLRAVESYLLAHSDA GETLLRAVESYLLAH GETLLRAVESYLLAHSDA ETLLRAVESYLLAH ETLLRAVESYLLAHS ETLLRAVESYLLAHSD ETLLRAVESYLLAHSDA TLLRAVESYLLAHSD
*Clones were assigned to TH subsets as follows: TH2, IL-4/IFN-g >5; TH1, IL-4/IFN-g <0.2; TH0, IL-4/IFN-g 5 0.2 to 5.
TH subset*