Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MS(E) to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MS(E) confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.     

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS.Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.     

The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens

Background

Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.

Methodology

Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.

Conclusion

The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.     

Molecular basis of allergic cross-reactivity between group 1 major allergens from birch and apple

Patients allergic to birch pollen often also react with fruits and vegetables, such as apple. The major cause of cross-reactivity between birch and apple is biochemical and immunological similarity between the major allergens, Bet v 1 and Mal d 1, as demonstrated by serological and cellular immunoassays. In addition, birch pollen-specific therapeutic allergy vaccination has been shown to improve allergic symptoms caused by oral ingestion of apple. Detailed analysis of molecular surface areas based on the crystal structure of Bet v 1, and primary sequence alignment, identify potential epitopes for cross-reactive antibodies. Two or more conserved patches are identified when comparing Bet v 1 and Mal d 1, thus providing a molecular model for serological cross-reactivity involving more than one IgE-binding epitope. A minimum of two epitopes would be necessary for cross-linking of receptor bound IgE in functional histamine release assays and skin test. Individual amino acid substitutions, as occurring in isoallergenic variation, may, however, have a dramatic effect on epitope integrity if critical residues are affected. Thus, one area large enough to accommodate antibody-binding epitopes shared by all known Mal d 1 isoallergens and variants is identified, as well as areas shared by Bet v 1 and individual Mal d 1 isoallergens or variants. The occurrence of limited epitope coincidence between Bet v 1 and Mal d 1 is in agreement with the observation that some, but not all, birch pollen allergic patients react with apple, and that the epitope repertoire recognised by the IgE of the individual patients determines the degree of cross-reactivity.     

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.     

Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins

Pollen of the European white birch is a major source of spring pollinosis in Europe. Pollen-allergy diagnosis and treatment by specific immunotherapy commonly rely on extracts of natural origin. To gain insight into the protein content and its variability, we evaluated the profile of allergenic and non-allergenic proteins in extracts of pollen from different origins by MS-based proteomics. Aqueous extracts prepared from commercially available Swedish birch pollen, pollen collected from Austrian trees and a commercial skin prick extract were analyzed by 1-DE, 2-DE, immunoblotting and mass spectrometry, resulting in a complete inventory of extractable, disease-relevant pollen proteins. A main focus of this study was on the isoform distribution of Bet v 1, the major allergen of birch pollen. Using a combination of intact mass determination and peptide sequencing, five isoforms (a, b, d, f and j) were unequivocally identified in Swedish and Austrian birch pollen extracts, while the skin prick extract contained only isoforms a, b and d. Using the same methods as for Bet v 1, divergencies in the sequence of birch profilin (Bet v 2), a plant panallergen, were solved. The molecular characterization of pollen extracts is relevant for standardization and development of new reagents for specific immunotherapy.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

Source of Bet v 1 loaded inhalable particles from birch revealed

 Allergenic proteins present in pollen grains, when inhaled, interact with the airways to cause an attack of asthma in susceptible humans. In one system, grass pollen grains rupture osmotically in rainfall, releasing allergen-containing inhalable particles into the atmosphere. In contrast, birch tree pollen grains do not rupture under these conditions, yet the major allergen, Bet v 1, has been detected in the atmosphere in inhalable particles of unknown origin. It is possible that Bet v 1 may diffuse from intact settled pollen grains and the allergenic material may again become airborne, interacting with settled fine particles from other sources prior to resuspension. This study investigates the mechanism for the release of birch pollen allergen-containing inhalable particles from pollen grains. We propose the hypothesis that (1) airborne birch pollen grains settle on nearby leaf surfaces; (2) then, following light rainfall, the grains germinate and, (3) later, pollen tubes burst, releasing inhalable particles carrying Bet v 1 into the atmospheric aerosol.   We used microscopic analyses of pollen behaviour following anther opening, a Burkard volumetric trap for pollen counts and a high volume air sampler with a two-stage cascade impactor for quantitative immunochemical analyses of Bet v 1. On dry days of high birch pollen count (48 grains/m³, 1.5 ng/m³ of Bet v 1), we found that the surfaces of birch leaves became coated with pollen. This ”pollen rain” is a source of secondary emission of allergens into the atmosphere. We observed that following light rainfall (<1 mm per day), about 80% of the birch pollen grains germinated, producing pollen tubes, especially in the sticky surface secretions of leaf glands. These pollen tubes may grow up to 300 μm in length prior to rupturing, each releasing about 400 starch granules coated with allergen molecules that may, after drying, be dispersed into the aerosol. On these days following light rainfall, the highest atmospheric levels of Bet v 1 (1.18 ng/m³) are associated with inhalable particles. Following heavy rainfall, both pollen and inhalable particles are washed from the atmosphere. Immunoprinting studies show that Bet v 1 is associated with starch granules rather than the smaller orbicules. Bet v 1 is present in the atmosphere in large particles, i.e. in particular pollen grains and in inhalable particles, i.e. in particular starch granules. Received: 28 May 1997 / Revision accepted: 18 August 1997     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.     

Nitration of the pollen allergen bet v 1.0101 enhances the presentation of bet v 1-derived peptides by HLA-DR on human dendritic cells

Nitration of pollen derived allergens can occur by NO(2) and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.     

Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.     

Bet v 1 from Birch Pollen Is a Lipocalin-like Protein Acting as Allergen Only When Devoid of Iron by Promoting Th2 Lymphocytes

It is hypothesized that allergens are at the borderline of self and non-self and, through as yet elusive circumstances, mount a Th2 response for allergic sensitization. The major birch pollen allergen Bet v 1 is considered the prototype for the PR-10 protein family causing respiratory allergy. Here, we give structural evidence that Bet v 1 is a lipocalin-like protein with a striking structural resemblance to human lipocalin 2. Lipocalin 2 is highly expressed in the lung where it exerts immunoregulatory functions dependent on being loaded with siderophore-bound iron (holo-form) or not (apo-form). We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated K_d values of 66 nm surpassed affinities to known ligands nearly by a power of 10. Moreover, we give functional evidence of the immunomodulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments and second structure information of Fag s 1: Fagales allergen from <Emphasis Type="Italic">Fagus sylvatica</Emphasis>

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the ¹H, ¹⁵N and ¹³C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.     

Post-Messinian evolutionary relationships across the Sicilian channel: Mitochondrial and nuclear markers link a new green toad from Sicily to African relatives

Background

The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.

Results

Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.

Conclusion

The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.