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.