Characterization of the T-cell response to Dau c 1, the Bet v 1-homolog in carrot.

BACKGROUND: In contrast to other Bet v 1-related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process, we sought to characterize the T-cell response to Dau c 1 and its cross-reactivity with Bet v 1. METHODS: Dau c 1-specific T-cell lines (TCL) and clones (TCC) established from PBMC of birch pollen-allergic patients with carrot allergy were analyzed for reactivity to Bet v 1, epitope specificity, allergen-induced cytokine secretion, and expression of integrins α4ß7 and α4ß1, critical for gut and lung homing, respectively. mRNA expression of GATA3 and Tbet was analyzed in sorted CD3+ CD4+ CFSElow cells proliferating upon stimulation of PBMC with Dau c 1 or Bet v 1. Dau c 1 was incubated with endolysosomal proteases, and the resulting fragments were identified by mass spectrometry. RESULTS: Among 14 distinct T-cell-activating regions, Dau c 1139-153 was recognized by 55% of the patients. Only 6 of 15 (40%) Dau c 1-specific TCL and 9 of 21 (43%) TCC reacted with Bet v 1. Bet v 1-nonreactive TCC were mainly Th1-like and showed a higher expression of the integrin ß7 and a significantly lower expression of the integrin ß1 than Bet v 1-positive TCC. A Th1-like response was also detected in Dau c 1-reactive CD3+ CD4+ CFSElow cells. Full-length Dau c 1 was still detectable after 48 h of endolysosomal degradation. Proteolytic fragments of Dau c 1 matched its T-cell-activating regions. CONCLUSION: Dau c 1 displays several characteristics of sensitizing allergens, namely a major T-cell-activating region, low susceptibility to endolysosomal degradation, and induction of a Bet v 1-independent T-cell response. These cellular insights confirm that the major carrot allergen has a special status among Bet v 1-related food allergens.

Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis.

BACKGROUND: Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen-food syndrome (PFS) and caused by cross-reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies. METHODS: As vaccine candidates, apple Mal d 1 as well as hazelnut Cor a 1 derivatives were designed by in silico backbone analyses of the respective allergens. The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens. Because Mal d 1 and Cor a 1 form cysteine-mediated aggregates, nonaggregative cysteine to serine mutants were also generated. The proteins were characterized physicochemically, immunologically, and in in vivo models with or without adjuvant. RESULTS: The structurally modified proteins showed significantly decreased IgE binding capacity. Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants. When formulated with alum, the monomeric cysteine mutants induced a similar immune response as the aggregated parental allergens, which is in contrast with data published on Bet v 1. CONCLUSION: These findings lead to the suggestion that the Bet v 1 structure has unique intrinsic properties, which could account for its high allergenicity. Obviously, these characteristics are not entirely shared with its food homologues from apple and hazelnut. Thus, it is important to tackle pollen-related food allergies from different angles for the generation of effective vaccine candidates to treat birch PFS.