Barbara Bohle PhD Department of Pathophysiology Center for Physiology and Pathophysiology Medical University of Vienna Waehringer Guertel 18-20 AKH-3Q A-1090 Wien Austria
Patients with birch pollen allergy frequently develop hypersensitivity reactions to certain foods, e.g. apples, celery, carrots and hazelnuts. These reactions are mainly caused by IgE-antibodies specific for the major birch pollen allergen, Bet v 1, which cross-react with homologous proteins in these foods. Analyzing the T-cell response to Bet v 1-related food allergens revealed that these dietary proteins contain several distinct T-cell epitopes and activate Bet v 1-specific T cells to proliferate and produce cytokines. Several of these cross-reactive T-cell epitopes were not destroyed by simulated gastrointestinal digestion of food allergens and stimulated Bet v 1-specific T cells despite nonreactivity with IgE antibodies. Similarly, cooked food allergens did not elicit IgE-mediated symptoms (oral allergy syndromes) but caused T-cell-mediated late-phase reactions (deterioration of atopic eczema) in birch pollen-allergic patients with atopic dermatitis because thermal processing affected their conformational structure and not the primary amino acid sequence. Thus, T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE-cross-reactivity in vitro and in vivo. We speculate that symptom-free consumption of pollen-related food allergens may have implications for the pollen-specific immune response of allergic individuals.
Food allergy represents an important manifestation of atopic allergy. Primary food allergy mainly affects young children whereas adults frequently develop food allergy as a consequence of an inhalant sensitization. Pollen-related food allergy has become the most frequent form of food allergy in adolescent and adult individuals in Europe. A typical example is the ‘birch-fruit-vegetable-syndrome’. More than 70% of birch pollen-allergic individuals develop allergic reactions to stone-fruits, nuts or certain vegetables. In the majority of patients these reactions are confined to the oropharynx and summarized as ‘oral allergy syndrome’ (OAS) (1). Nevertheless, systemic IgE-mediated reactions such as urticaria, asthma or anaphylactic shock occur occasionally (2, 3). The major birch pollen allergen, Bet v 1, is the most relevant sensitizing protein causing this type of food allergy (4–8) but minor allergens such as Bet v 2, Bet v 5 and Bet v 6 have also been shown to be involved (6–10). Whereas these allergens are recognized by 10–32% of birch pollen-allergic patients, more than 95% display IgE against Bet v 1. The major birch pollen allergen belongs to the pathogenesis-related (PR) protein family 10 (11). Other members of this protein family are present in various foods, such as fruits of Rosaceae (e.g. Mal d 1 in apple, Pru a 1 in cherry, Pyr c 1 in pear), vegetables of Apiaceae (e.g. Api g 1 in celery, Dau c 1 in carrot), hazelnut (Cor a 1), soybean (Gly m 4), mungbean (Vig r 1) and peanut (Ara h 8) (9, 12–21). These proteins share a high degree of amino acid sequence similarity with the major birch pollen allergen resulting in a similar tertiary structure (22). Thus, Bet v 1-specific IgE antibodies can bind to these dietary proteins which may cause immediate hypersensitivity reactions upon consumption of the respective foods. The IgE-cross-reactivity between Bet v 1 and food homologues correlates roughly with the similarity of their primary protein structures. Hence, Bet v 1-specific IgE antibodies react preferentially with allergens in Rosaceae fruits sharing between 56% and 59% of amino acid similarity with Bet v 1 and less frequently with homologues in vegetables of the Apiaceae family sharing 37–41% (23, 24). Analyzing the IgE binding of 50 Bet v 1-positive patients to different food allergens we observed that 99% reacted with Mal d 1, 93% with Cor a 1, 59% with Api g 1 and 38% with Dau c 1. Vice versa, patients with birch pollen-related food allergy are predominantly sensitized to Bet v 1-homologues and less frequently recognize other allergens contained in both sources, e.g. profilins (14, 25, 26).
In accordance with the continuously increasing prevalence of birch pollinosis during the past 15 years, birch pollen-related food allergy has become an important disorder perennially impairing the quality of life of allergic patients including the time period outside the pollen season (27). However, at present no efficient therapy for this type of food allergy is available. One reason for this lack is obviously the still limited knowledge about mechanisms operative in this allergic disorder. By adjusting the experimental methodology which has previously been successfully applied for allergens from sources other than food, we characterized the T-cell response underlying pollen-associated food allergy (28–31). Our experimental approach is based on the isolation and expansion of allergen-specific T-cell lines (TCL) and clones (TCC) from the peripheral blood of humans which then serve as perfect tools to analyze the phenotype of specific T cells, their cytokine response to specific stimulation as well as their epitope recognition and MHC restriction (Fig. 1). Up to now, T-cell responses to three relevant Bet v 1 homologues from different botanical families, namely Mal d 1 from apple, Api g 1 from celery and Cor a 1 from hazelnuts were analyzed in detail (32–34). The majority of food-specific TCC belonged to the Th2 subset, which produced high amounts of IL-4 but only little or no IFN-γ upon allergen-specific stimulation. By stimulating food-specific TCL and TCC with a panel of synthetic 12-mer peptides overlapping for nine residues and representing the entire amino acid sequence of each allergen, T-cell epitopes in Mal d 1, Api g 1 and Cor a 1 were identified. Similar to the major birch pollen allergen, its dietary homologues contained multiple T-cell-reactive determinants (35). These T-cell epitopes were located in parts corresponding to relevant T-cell-activating regions of Bet v 1 (Fig. 2). Accordingly, the majority of food-reactive T-cell cultures also responded to stimulation with Bet v 1 demonstrating that these T-cell epitopes were indeed cross-reacting. Interestingly, most T-cell cultures induced with Mal d 1- and Api g 1 proliferated by far more pronounced in response to Bet v 1 than to the food proteins. These cultures also lost their reactivity with the food allergens after a few expansion cycles in vitro but remained highly Bet v 1-reactive. These results strongly indicated that T cells specific for these food allergens were in fact pollen-specific, cross-reactive clonotypes. Thus, these findings at the T-cell level corroborate the concept that sensitization to PR-10 like food allergens develops as consequence of primary inhalant sensitization to the major birch pollen allergen.
Food allergens are potent activators of pollen-specific T cells
To evaluate whether pollen-related food allergens were able to activate pollen-specific T cells, Bet v 1-specific TCL and TCC recognizing different T-cell epitopes were stimulated with Mal d 1 (apple), Api g 1 (celery) or Cor a 1 (hazelnut) (32–34). Most cultures responded with proliferation and cytokine secretion showing that Bet v 1-related food allergens activated Bet v 1-specific T cells with diverse specificities. To elucidate whether particular epitopes of Bet v 1 were predominantly involved in T-cell reactivity with each food homologue, more than 50 Bet v 1-specific TCL with known epitope specificity from different birch pollen-allergic patients were screened for reactivity with Mal d 1 or Api g 1 (36). Epitope recognition patterns of food allergen-reactive and nonreactive TCL were compared and the percentage of peptide-reactive cultures was calculated for each group. Cross-reactivity between Bet v 1 and Mal d 1 was predominantly restricted to one single T-cell epitope, Bet v 1142−−153. This finding was surprising because both allergens share 71% of amino acid sequence similarity (12). On the other hand, T-cell cross-reactivity between Bet v 1 and Api g 1 was associated with several different epitopes spreading the entire amino acid sequence of the pollen allergen although both allergens share only 61% of sequence similarity (15). It has to be emphasized that all individuals (100%) studied had an OAS to apple whereas only around 35% of these patients experienced immediate IgE-mediated reactions to celery. Hence, the degree of amino acid sequence similarity between Bet v 1 and its dietary homologues correlates roughly with the occurrence of food allergy but not with the magnitude of cross-reactivity at the T-cell level. These findings support the necessity for the characterization of the T-cell response including the identification of T-cell epitopes in allergens to understand immunological mechanisms underlying different manifestations of atopic diseases.
Analyzing the epitope recognition pattern in Bet v 1-specific TCL generated from a large number (n = 57) of different birch pollen-allergic patients, one immunodominant T-cell epitope of Bet v 1, recognized by 61% of the individuals, was identified (35). This peptide which comprises amino acid residues 142–156 is located in the C-terminal part of the major birch pollen allergen, a region highly conserved among different PR-10 like proteins. When TCL and TCC specific for this immunodominant epitope were stimulated with PR-10 like proteins from apple, cherry, pear, peach, hazelnut, celery, carrot or soybean, the majority of cultures responded to more than one food allergen by proliferation and production of IL-4 and IL-5 (35). Hence, Th2 cells specific for the most frequently recognized T-cell epitope of Bet v 1 can be easily activated by various birch pollen-related foods to proliferate and synthesize cytokines.
Gastrointestinal digestion does not destroy the T-cell-activating capacity of pollen-related food allergens
It has been previously shown that PR-10-like proteins undergo rapid gastric degradation leading to the immediate loss of their IgE-binding capacity (37, 38). In contrast to the IgE-binding sites of these proteins, which are conformational epitopes depending on the tertiary protein structure (22, 39), T-cell epitopes are short, linear peptides (32–34). However, such peptides may survive gastrointestinal degradation and remain stimulatory for T lymphocytes (40). To evaluate this hypothesis, Mal d 1 (apple), Api g 1 (celery) and Cor a 1 (hazelnut) were incubated with pepsin followed by trypsin to simulate gastrointestinal degradation. Thereafter, digested allergens were tested for IgE binding, their capacity to induce mediator release and T-cell activation in peripheral blood mononuclear cells (PBMC) as well as in Bet v 1-specific TCL and TCC. Although allergens had completely lost their capacity to bind IgE and to induce mediator release after incubation with enzymes their products still induced proliferation in PBMC from allergic and nonallergic individuals. Enzymatically digested Mal d 1 and Cor a 1 also activated Bet v 1-specific TCL and TCC to proliferate and to synthesize similar cytokines comparable with stimulation with entire food allergens or Bet v 1 (Fig. 3). Different allergen fragments created by enzymatic degradation were identified and found to match relevant T-cell epitopes of Bet v 1. TCC specific for these Bet v 1 epitopes responded to digested Mal d 1 and Cor a 1, confirming that their proteolytic fragments indeed contained epitopes cross-reactive with pollen-specific T cells. Taken together, exposure of PR-10 like food proteins to gastrointestinal proteases does completely abolish their ability to bind IgE but not their capability to stimulate Bet v 1-specific T cells. These findings provide a biochemical and immunological mechanism underlying the clinical observation that ingestion of birch pollen-related foods can trigger the worsening of atopic eczema in patients with cutaneous T-cell responses to Bet v 1 (41). After passing stomach and duodenum, fragments of pollen-related dietary allergens remain that can activate Bet v 1-specific T cells to upregulate homing receptors, migrate to the skin and induce effector reactions. These T-cell-mediated late reactions in the skin seem to be elicited even independently of an IgE-mediated mechanism, similar to late phase reactions which have been shown in the lung of cat-asthmatic patients after subcutaneous administration of peptides from the major cat allergen, Fel d 1 (42). In accordance with the Fel d 1-peptides, none of the fragments of gastrointestinal-digested Bet v 1-related food allergens was able to bind IgE or to induce basophil degranulation (40).
T-cell cross-reactivity occurs independently of IgE cross-reactivity
PR-10 like food allergens are considered to be easily destroyed by thermal processing as birch pollen-allergic individuals can generally consume cooked birch pollen-related foods without difficulty. Therefore, allergologists and guide books recommend heating of these aliments to make them safe for consumption (5, 21, 43–45). However, thermal processing of proteins generally causes conformational changes – which in the case of an allergen may result in diminished IgE-binding capacity – but does not affect the primary protein structure and thus, linear T-cell epitopes. Therefore, we speculated that cooked birch pollen-related food will not induce immediate reactions but may still cause T-cell-mediated symptoms. To prove this hypothesis, Bet v 1-homologues from apple (Mal d 1), celery (Api 1) and carrot (Dau c 1) were exposed to increasing temperatures. Thermal processing changed the conformational protein structure resulting in abrogated IgE-binding and mediator-releasing capacity after incubation at >80°C for a few minutes (36). However, allergens cooked for 60 min at 95°C still activated PBMC and Bet v 1-specific T-cell cultures to proliferate and produce Th2 cytokines comparably to nonheated proteins (Fig. 4). To substantiate these results in vivo and with natural allergens, Bet v 1-monosensitized patients with OASs and atopic dermatitis were tested in double-blind placebo-controlled food challenges (DBPCFC) with fresh and cooked apple, celery and carrot, respectively (36). The fact that cooked foods did not induce an OAS in any of the patients confirmed that heated Bet v 1-related allergens in their natural food matrix lose the ability to induce IgE-mediated immediate reactions in vivo. On the other hand, ingestion of cooked foods triggered a comparable worsening of atopic eczema as fresh foods. As already mentioned, late eczematous reactions in birch pollen-allergic patients observed after consumption of fresh pollen-related foods have been associated with an increased expression of cutaneous lymphocyte antigen of peripheral blood T cells and the presence of Bet v 1-reactive T cells in the isolated skin lesions (41). Our recent in vitro and in vivo results extend these previous findings by showing that (i) cooked birch pollen-related foods induce similar T-cell activation and T-cell-mediated symptoms as fresh aliments and (ii) T-cell-mediated reactions occur also in the absence of IgE-binding and IgE-mediated reactions. Therefore, pollen-related foods must be considered as important stimuli for pollen-specific T cells even in patients without immediate clinical symptoms of food allergy.
New treatment strategies for food allergy
Food allergy in adolescents and adults seems to develop as a consequence of primary sensitization to aero-allergens and subsequent immunological cross-reactivity with homologous proteins in foods. This hypothesis is supported by our findings that atopic adults who severely reacted after consumption of millet-containing dishes had been previously exposed to millet dust (46). The majority of birch pollen-allergic patients also experience allergic reactions to foods after having developed pollinosis and Bet v 1 contains most relevant B and T-cell epitopes of its homologous food allergens which is not the case vice versa. Provided that Bet v 1 is the sensitizing allergen for food allergy, one would expect that successful specific immunotherapy (SIT) of pollen allergy should also abolish allergic reactions to foods. As this issue is still controversial (47–51), we conducted a clinical trial analyzing the clinical and immunological effects of sublingual immunotherapy (SLIT) with birch pollen on concomitant allergy to apple. We speculated that the administration of pollen extract directly at the site of food-induced allergic symptoms, i.e. the OAS, may increase the therapeutic efficacy. Furthermore, SLIT is a safe and convenient alternative to subcutaneous specific immunotherapy that has been demonstrated to improve significantly birch pollen-induced rhinitis and asthma (52–54). After 1 year of SLIT, patients showed improvement in nasal challenges with birch pollen. However, similar to reports on subcutaneous treatment with birch pollen, not all patients improved allergic reactions to apples as determined in DBPCFC (47, 49–51). In addition, we pioneered in monitoring the T-cell response to Bet v 1 and Mal d 1 in each individual revealing that only the pollen-specific T-cell response was significantly down regulated by SLIT. From these data we deduce that food-specific clonotypes exist, which escape the induction of peripheral tolerance during pollen-SIT. In line with this hypothesis, we have previously observed that a minority of Mal d 1- or Api g 1-specific TCC did not cross-react with Bet v 1 (32, 33). Furthermore, analyzing the T-cell response to the Bet v 1-homologue in hazelnuts revealed that several TCC specific for the most frequently recognized T-cell epitope of Cor a 1 did respond to stimulation neither with the major birch pollen nor the hazel pollen allergen (34). Down-regulation of the allergen-specific T-cell response has been shown to be an important component of successful SIT (55). The existence of exclusively food-reactive T cells that are not modulated by vaccination with pollen allergens may provide an immunologic explanation for the noneffectiveness of pollen-therapy on associated food allergy. Therefore, our current and future projects include studies to develop approaches for food allergy treatment, which target all food-specific T lymphocytes. As food extracts are difficult to produce and standardize, we will evaluate the use of recombinant food allergens and/or ‘hypoallergenic’ variants thereof. Recently, a mutated Mal d 1 molecule lacking most important IgE-binding sites but still containing relevant T-cell epitopes was created (56).
Only studying the T-cell response to typical aero-allergen-associated food allergens revealed that pollen-related foods have an important impact on pollen-allergy (illustrated in Fig. 5). After consumption, pollen-related food allergens stimulate pollen-specific T cells to exert effector functions. This capability is neither abolished by gastrointestinal digestion nor by cooking of the respective foods, two treatments which abolish immediate IgE-mediated reactions. Thus, T-cell cross-reactivity between pollen and related food allergens is strikingly different at the B- and T-cell level and relevant T-cell-mediated clinical reactions can occur independently from IgE-mediated symptoms. Accordingly, patients who do not suffer from food allergy consume the respective aliments and provide antigens for pollen-specific T lymphocytes. This ingestion may cause ‘visible’ clinical symptoms, e.g. the deterioration of atopic eczema, but may also solely result in a ‘hidden’ boost of pollen-specific immune responses. It is known that inhalation of pollen allergens during the pollen season induces local and systemic IgE synthesis and CD4+ memory T cells (57–59). However, outside the pollen season specific serum IgE levels are typically maintained at increased levels although no allergens are encountered. The consumption of birch pollen-related foods activates pollen-specific Th2 cells in vivo also outside the pollen season, thus providing a continuous activation necessary for long-term maintenance of pollen-specific Th2 cells (60, 61). Moreover, perennially activated pollen-specific Th2 cells continuously synthesize high amounts of IL-4, which together with small concentrations of non-degraded, IgE-binding pollen-related food allergens absorbed via the oral and/or gastrointestinal mucosa, could then be sufficient to provoke IgE synthesis in B cells outside the pollen season (62, 63). We conclude that ingestion of pollen-related food allergens could maintain perennially increased allergen-specific IgE levels in patients suffering from seasonal allergic disorders. These new aspects regarding the induction and maintenance of pollen and associated food allergy are important for the development of efficient vaccines for prevention and therapy of these allergic disorders. Finally, the view that pollen-related food allergens are only nonsensitizing, easily degradable and thus ‘irrelevant secondary’ allergens should be reconsidered, in particular for pollen-allergic patients with chronic atopic dermatitis.
The author wishes to thank Christof Ebner, Beatrice Jahn-Schmid, Astrid Radakovics, Eva Maria Schimek, Renate Steiner and Bettina Zwölfer. This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF, SFB-F1807-B04) and Biomay, Austria.