Jug r 2-reactive CD4(+) T-cells have a dominant immune role in walnut allergy

BACKGROUND: Allergic reactions to walnut can be life threatening. While IgE epitopes of walnut have been studied, CD4(+) T-cell specific epitopes for walnut remain uncharacterized. Particularly, 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 non-allergic subjects. Particularly, the relationship of phenotypes and frequencies of walnut specific T-cells with the disease. METHODS: CD154 up-regulation assay was used to examine CD4(+) T-cell reactivity towards walnut allergens.Jug r 1, Jug r 2 and Jug r 3. Tetramer-Guided epitope mapping approach was utilized to identify HLA-restricted CD4(+) T-cells epitopes in Jug r 2. Direct ex vivo staining with peptide-major histocompatibility complex class II (pMHC-II) tetramers enabled the comparison of frequency and phenotype of Jug r 2-specific CD4(+) T-cells between allergic and non-allergic subjects. Jug r 2-specific T-cell-clones were also generated and mRNA transcription factor levels were assessed by RT qPCR. Intracellular cytokine staining (ICS) assays were performed for further phenotypical 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(+) T(CM) (central memory) phenotype. A subset of these T-cells express CCR4(+)CCR6(+) irrespectively of the asthmatic status of the allergic subjects. ICS 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 expressed either GATA3 and RORC, or RORC, 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 subjects 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 non-asthmatic and asthmatic subjects with walnut allergy. Understanding this T-cell heterogeneity may render better understanding of the disease manifestation.