Jug r 2-reactive CD41 T cells have a dominant immune role in walnut allergy

Background: Allergic reactions to walnut can be life-threatening. Although IgE epitopes of walnut have been studied, CD4(+) T cell-specific epitopes for walnut remain uncharacterized. In particular, the relationship of both phenotype and frequency of walnut-specific T cells to the disease have not been examined. Objectives: We sought to provide a thorough phenotypic analysis for walnut-reactive T cells in allergic and nonallergic subjects, particularly the relationship of phenotypes and frequencies of walnut-specific T cells with the disease. Methods: The CD154 upregulation assay was used to examine CD4(+) T-cell reactivity toward the walnut allergens Jug r 1, Jug r 2, and Jug r 3. A tetramer-guided epitope mapping approach was used to identify HLA-restricted CD4(+) T-cell epitopes in Jug r 2. Direct ex vivo staining with peptide-major histocompatibility complex class II tetramers enabled comparison of the frequency and phenotype of Jug r 2-specific CD4(+) T cells between allergic and nonallergic subjects. Jug r 2-specific T-cell clones were also generated, and mRNA transcription factor levels were assessed by using quantitative RT-PCR. Intracellular cytokine staining assays were performed for further phenotypic analyses. Results: Jug r 2 was identified as the major allergen that elicited CD4(+) T-cell responses. Multiple Jug r 2 T-cell epitopes were identified. The majority of these T cells in allergic subjects have a CCR4(+) phenotype. A subset of these T cells express CCR4(+) CCR6(+) irrespective of the asthmatic status of the allergic subjects. Intracellular cytokine staining confirmed these T(H)2-, T(H)2/T(H)17-, and T(H)17-like heterogenic profiles. Jug r 2-specific T-cell clones from allergic subjects mainly expressed GATA3, nonetheless, a portion of T-cell clones both GATA3 and RAR-related orphan receptor C (RORC) or RORC alone, confirming the presence of T(H)2, T(H)2/T(H)17, and T(H)17 cells. Conclusions: Jug r 2-specific responses dominate walnut T-cell responses in patients with walnut allergy. Jug r 2 central memory CD4(+) cells and terminal effector T cells were detected in peripheral blood, with the central memory phenotype as the most prevalent phenotype. In addition to conventional T(H)2 cells, T(H)2/T(H)17 and T(H)17 cells were also detected in nonasthmatic and asthmatic patients with walnut allergy. Understanding this T-cell heterogeneity might render better understanding of the disease manifestation.