CyTOF-based Whole Blood Assay to Explore the Allergen Specific Signaling Pathways in Basophil

Abstract

Peanut allergy is the most common cause of anaphylaxis in childhood. This IgE-mediated allergic reaction is initiated by basophils or mast cells upon exposure and cross-linking of membrane bound IgE by allergens. The subtle molecular and cellular mechanisms that define the difference between peanut allergy and asymptomatic sensitization and during oral immunotherapy are not fully understood. We established a mass cytometry (CyTOF) panel to study differences in allergen specific signaling pathways upon peanut stimulation in human whole blood. In an initial pilot peanut-allergic (n=9) and peanut-sensitized non-allergic children (n=3) were recruited. Fresh blood samples were stimulated with 10pg/ml-100 ng peanut extract, PMA/Ino, anti-FceRI or media control for 3 or 15 minutes. Barcoded samples were pooled and stained with a panel of surface markers and anti-phosphorylation antibodies for p38, erk, mTOR and akt. The samples were acquired on a CyTOF2 and analysis were performed using Cytobank. Positive peanut specific IgE ( 0.30 kUa/L) was detected in peanut-allergic patients but negative sIgE in peanut-sensitized nonallergic. Basophils from peanut-allergic patients showed higher phosphorylation of erk and p38 in dosage-depended manner at 3 minutes whereas phosphorylation of akt and mTOR significantly increased at 15 minutes. Phosphorylation was significantly lower to absent in the sensitized non-allergic patients. We applied the phospho-CyTOF to address the signaling profile after peanut stimulation. This application may allow interesting insights in the kinetics of desensitization during oral immunotherapy.