Cor a 1-reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen-associated food allergy to hazelnut

Background: IgE- and T-cell cross-reactivity contribute to the birch pollen-food syndrome. Objectives: We performed a comprehensive analysis of T-cell cross-reactivity in primary cell cultures, facilitating the identification of allergen-specific T-cell subpopulations from individual patients. Methods: Patients with birch pollen allergy and associated food allergy to hazelnuts, carrots, or both were analyzed for IgE cross-reactivity, T-cell responses, and T-cell cross-reactivity to recombinant Bet v 1.0101 (Bet v 1; birch), Cor a 1.0401 (Cor a 1; hazelnut), and Dau c 1.0104 (Dau c 1; carrot). A novel flow cytometry-based method using a 2-step staining process with fluorescent dyes was established to identify subpopulations of cross-reactive T cells. Results: IgE-binding inhibition tests of individual sera revealed that the vast majority of Cor a 1-reactive IgE was cross-reactive to Bet v 1, whereas Bet v 1-reactive IgE was only partially inhibited by preincubation with Cor a 1. Primary stimulation of T cells with Bet v 1 or Cor a 1 resulted in a significant increase in specific responses to Cor a 1 or Bet v 1 after secondary stimulation, respectively, indicating T-cell cross-reactivity between birch and hazelnut allergens in all patients of the study cohort. Preactivation with Dau c 1 induced less pronounced effects. A novel flow cytometry-based proliferation assay identified a predominant Cor a 1/Bet v 1-cross-reactive T-cell subpopulation within highly Bet v 1/Cor a 1-responsive T cells. Conclusion: Analysis of primary allergen-specific T cells combined with flow cytometry-based proliferation assays facilitates investigation of allergen-specific T-cell subpopulations in subjects and might be helpful to evaluate the effect of birchspecific immunotherapy on pollen-associated food allergies.