To examine food allergen specific T cell responses in food anaphylaxis and EGIDs, cohorts with peanut anaphylaxis (PA), allergic eosinophilic gastroenteritis (AEG), or healthy non-atopic control (NA) subjects were recruited. Effector/memory T cell cytokine responses were measured using intracellular cytokine staining and polychromatic flow cytometry. IL-4, IL-5, IFN-gamma and TNF responses were measured in both the CD4 and CD8 T cell subsets. Antigens that were studied included peanut, Ara h1, soy, shrimp, and staphylococcal enterotoxin B (SEB). Food allergen specific T cell responses were found exclusively within the CD4 T cell compartment and were clearly demonstrable in both food allergic cohorts. Constitutive cytokine production was low for all groups. In particular, AEG subjects did not demonstrate constitutive IL-5 expression. Antigen specific CD4 T cell responses were detectable at 10 cells per 10e6. Boolean analysis of the polychromatic data was performed, to yield 5 distinct subpopulations of food allergen specific CD4 T cells: IL-5+ Th2 (IL-4+, IL-5+) and IL-5- Th2 (IL-4+, IL-5-) cells, Th0, Th1, and TNF solo producers. Peanut specific IL-5+ Th2 cells were present at a 20-fold greater frequency in AEG vs. PA (81 vs. 4 per 10e6 CD4 cells, p=0.05), whereas there were similar frequencies of IL-5- Th2 cells (67 vs. 41 per 10e6 CD4 cells). For all food responses, IL-5+ Th2 cells accounted for a significantly greater fraction of the antigen specific cells in AEG relative to PA (29% vs. 4%, p<0.0001). In PA, but not AEG, IL-5- Th2 responses to peanut were highly correlated with peanut specific IgE (r= 0.87 vs. 0.55, respectively). All subject groups elicited similar very low magnitude Th1 responses to food Ags. These findings demonstrate a significant correlation between AEG disease status and the presence of IL-5 expressing food allergen specific T cells across multiple food allergens. Given that IL-5 is a major eosinophil active cytokine, it is likely that this association is of immunopathological significance and suggests that IL-5 producing food allergen specific T cells drive the eosinophilic inflammation found in EGIDs. Additionally, these findings suggest that although the expression of IL-4 and IL-5 are linked, each is controlled in a different manner, which may have important consequences for the immunopathogenesis of allergic diseases. Current work is underway to extend these findings to gut resident T cells in patients with eosinophilic GI disease. GI biopsies will be obtained and both flow cytometry and immunohistochemisty will be used to determine if a similar association with IL-5+ Th2 cells exists. Additional experiments will identify if there are other characteristics of food allergen specific Th2 cells in these disease states that may specifically contribute to anaphylactic vs. eosinophilic inflammatory food allergy. Current work is aimed at continued development of high fidelity polychromatic flow cytometry assays to simultaneously examine multiple Th2 cytokines (IL-4, -5, -9, -13) in an allergen specific manner. This technical capacity will facilitate future interventional clinical studies designed to examine immunomodulation of allergic Th2 responses in peanut anaphylaxis and EGIDs.
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