Figure 6. Exchange of IgE on human basophils.

(a) Left panel: 2-h treatment with 25 μM E2_79 removes surface IgE from primary human basophils, but does not alter surface FcɛRIα levels within 24 h. Middle panel: E2_79-treated basophils can be reloaded with JW8-IgE and tracked by JW8's mouse-λ-light chain (λ-LC). Right panel: biotinylated WT and IgE-R419N-Fc_3–4 variants bind E2_79-treated basophils. (b) Experimental design for the IgE exchange. In brief, IgE is removed from primary basophils using E2_79. JW8-IgE is then reloaded on basophils to generate a traceable starting IgE population. (c) Experimental validation shows distinct populations of basophils with JW8-IgE (Q1), JW8-IgE and biotinylated IgE-Fc_3–4 (Q2), biotinylated IgE-Fc_3–4 alone (Q4) and E2_79-treated cells without labelled IgE (Q3; displaying merged dot plots from each sample). (d) All panels show starting JW8-reloaded population in Q1 before treatment, and cells after treatment. Left panel: overnight treatment with high-concentration omalizumab (25 μM) is sufficient to remove the majority of JW8-IgE. Middle panel: overnight treatment of cells with omalizumab (25 μM) and IgE-Fc_3–4 (1 μg ml⁻¹ or ∼18 nM) results in depletion of JW8-IgE, but no exchange for IgE-Fc_3–4. Right panel: overnight treatment of cells with omalizumab (25 μM) and IgE-R419N-Fc_3–4 (1 μg ml⁻¹ or ∼18 nM) results in depletion of JW8-IgE, and IgE exchange to IgE-R419N-Fc_3–4. (Representative dot plots shown. N=3 at 10 μg ml⁻¹ IgE-Fc doses and controls, and N=3 at 1 μg ml⁻¹ IgE-Fc doses).