Mast cell activation test (MAT)
For the MAT, patients with peanut-sIgE ≥1.0 kUA/L were selected given previously reported lower threshold of peanut-sIgE to induce mast cell activation which can vary with intrinsic mast cell reactivity. (7) MAT was performed as previously reported. (7, 13) LAD2 cells (Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases) were primed with IL-4 and incubated for 5 days before being sensitized with patient serum. The cells were stimulated with peanut extract (ALK Abello) diluted in RPMI medium (GIBCO, Paisley, UK) at two concentrations (1000ng/mL and 10,000ng/mL), anti-IgE (1ug/mL, Sigma-Aldrich, Poole, UK) and ionomycin (1 ug/mL, Millipore). Cells were stained with CD63-allophycocyanin (Biolegend, San Diego, Calif) and surface markers IgE-PE (Biolegend), CD32-APC, FceRI-FITC (eBioscience, San Diego, Calif) before viability dye eFluor 450 (eBioscience) was added. Flow cytometry (CytoFLEX flow cytometer) was performed with FACSDiva software (BD Biosciences, San Jose, Calif) and data was analysed using FlowJoTM v10.8 Software (BD Life Sciences, Ashland OR). MATs were performed for peanut allergic and peanut sensitised but not allergic children (PS) as defined above at the three time points (12m, 36m, 7-11y), for which samples were available.