Mast cell activation in type I allergy is triggered when an allergen binds to IgE, resulting in the crosslinking of IgE-bound receptors on mast cells (Galli and Tsai, 2012). Measuring serum allergen-specific IgE antibody levels is a common diagnostic approach in clinical settings (Ando et al., 2008). Typically, serum IgE levels are quantified using labeled anti-IgE secondary antibodies, which bind to IgE bound to solid-phase allergens. However, this method cannot assess IgE-induced crosslinking, potentially overestimating clinically irrelevant IgE binding (Okamoto-Uchida et al., 2016).

To address this limitation, we established a stable cell line, RS-ATL8, by introducing a luciferase reporter gene under the transcriptional control of NF-AT into a rat mast cell line (RBL-SX38) transfected with human FcεRI genes (α, β, γ) (Nakamura et al., 2010). These cells are sensitized with minute amounts of human serum IgE and are activated by IgE crosslinking when exposed to specific allergens, thereby enabling sensitive quantification of activation through luciferase assays (Nakamura et al., 2012). This method, named “IgE Crosslinking-induced Luciferase Expression (EXiLE),” has been widely adopted by researchers globally (Falcone et al., 2015; Nakamura et al., 2013; Kamemura et al., 2014).

However, we encountered an issue: human FcεRIβ gene expression was lost both in RS-ATL8 cells, and their parental RBL-SX38 cells (Ali et al., 2019). Initially, these cells expressed rat endogenous FcεRI genes (α, β, γ subunits), and human FcεRI receptors are likely expressed on the cell surface as hybrid compositions with rat receptor subunits. The absence of the human β subunit may adversely affect human FcεRI receptor function in RS-ATL8 cells in two ways. First, the β subunit is crucial for the surface expression of the FcεRIα subunit on mast cells (Donnadieu et al., 2000). Second, the β subunit amplifies signal transduction (Lin et al., 1996). The lack of signal amplification caused by the absence of β subunit is a major concern.

Considering that only 12 out of 19 amino acid residues (63.2%) in the transmembrane domain of the human and rat FcεRIα subunits are identical, it is possible that the human α subunit may not interact as effectively with the rat non-α subunits within the cell membrane.

Therefore, this study aimed to design novel chimeric receptors to create cell lines with higher α chain expression and increased sensitivity without depending on the human β subunit. We aim to advance the development of the next-generation EXiLE method.