Cellular stratification of atopic inflammatory circuits

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Innate immune cells play an important role in food allergy with antigen-presenting cells (APC) priming the initial T cell response, while mast cells (or basophils) being major effector cells in allergic reactions. In the 1st Funding Period (FP), we developed a Basophil Activation Test (BAT) for routine testing of the study participants. We determined a strong diagnostic power of the CDSENS, the antigen concentration evoking activation of 50% basophil response, to predict food allergy from the BAT assay. CDSENS correlated strongly with the oral food challenge threshold dose, indicating that the assessment of the reaction threshold during desensitization is relevant. With our BAT, we also assess phosphorylation of S6 in plasmacytoid and classical dendritic cells (pDC and cDC), which displayed significant differences between tolerant and allergic individuals.To assess the induction of phosphorylation patterns, activation and inhibition markers during basophil activation, we utilized mass cytometry. We observed a significant different induction of CD300a, CD33 and CD32b between tolerant and allergic donors, suggesting these as candidates as novel biomarkers in food allergy. Finally, we assessed the activation of APC three days following allergen stimulation, thereby identifying atopic polarization profiles of several APC subsets in allergic donors, especially regarding the expression changes of regulatory receptors (CD103, PDL1, CTLA4) that may play a role in desensitization. We will continue these now well-established assays in the extension studies in the 2nd FP to complete the growing data body. Furthermore, we want to determine whether these signatures are present in other type 2 disorders. The mass cytometric BAT assay will be developed to a conventional flow cytometry assay. In addition, we will build upon our insights into APC polarization by food allergens and will assess whether different mediators, miRNAs or short chain fatty acids modify APC phenotypes during allergen stimulation (in collaboration with B5 and C2), and how these different APC subsets can functionally affect T cell priming. In this assay system, we will make use of a Pan-HLA-DR-peptide called PADRE, that allows for broad activation of naive as well as memory CD4+ T cells. Altogether, our data will clarify the role of innate immune cells in allergy induction, maintenance and tolerization.