Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Vincenzo Casolaro,Eman Ali Ali,Daniel G W Alanine,Ryosuke Nakamura,Daniel Wan,David Onion,Marcos J C Alcocer,Franco H Falcone,Marina Kalli

doi:10.1371/journal.pone.0221034

Vincenzo Casolaro, Eman Ali Ali + Show 7 more

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https://doi.org/10.1371/journal.pone.0221034

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Abstract

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay–a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703–21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

Highlights

Humanized rat basophilic leukaemia (RBL) cell lines derived from the parental RBL-2H3 cell line [1,2] are increasingly used for detection of allergen-specific Immunoglobulin E (IgE) in human blood samples [3]
The same CRA-1 anti-FcεRIαH APC-labelled antibody, we found SX-38 and RS-ATL8 cells to have approximately 450,000–500,000 FcεRIαH molecules per cell (Fig 2 and Table 2), which is similar to the maximal number of receptors described on human peripheral blood basophils[21], while RBL 703/21 and NFAT-DsRed cells appeared to have ~52,000 and ~16,000 human α-chains per cell, respectively
We aimed to determine whether the differences in surface expression of the transgenic FcεRIαH chain could be due to differences in gene copy numbers

Summary

Introduction

Humanized rat basophilic leukaemia (RBL) cell lines derived from the parental RBL-2H3 cell line [1,2] are increasingly used for detection of allergen-specific Immunoglobulin E (IgE) in human blood samples [3]. The most recent generation of transgenic cell lines has further modified these humanized RBL lines to include sensitive and easy to measure reporter genes, such as firefly luciferase (RS-ATL8; [11]) or red fluorescent protein (NFAT-DsRed; [12]). These reporter cell lines have a series of advantages over the older generation, which we have described in detail in a recent review [3]

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