Characterization of human Fc[epsilon]RI[alpha] chain expression and gene copy number in humanized rat basophilic leukaemia

• Published in: PloS one Vol. 14; no. 8; p. e0221034
• Main Authors: Ali, Eman Ali, Kalli, Marina, Wan, Daniel, Nakamura, Ryosuke, Onion, David, Alanine, Daniel G. W, Alcocer, Marcos J. C, Falcone, Franco H
• Format: Journal Article
• Language: English
• Published: Public Library of Science 20.08.2019
• Subjects: Antibodies; Cell lines; Fluorescence; Gene expression; Genes; Genetic aspects; Immunoglobulin E; Journalistic ethics; Leukemia; Luciferase; Messenger RNA; Monoclonal antibodies; RNA
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0221034

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (Fc[epsilon]RI.sub.H). 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 Fc[epsilon]RI.sub.H 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 Fc[epsilon]RI.sub.H chain in humanized RBL cell lines. We compared surface levels of Fc[epsilon]RI[alpha].sub.H by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. Fc[epsilon]RI[alpha].sub.H copy numbers were assessed by qPCR, and the sequence verified. Transfection with Fc[epsilon]RI[gamma].sub.H cDNA was assessed for its ability to increase Fc[epsilon]RI[alpha].sub.H 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 Fc[epsilon]RI[alpha].sub.H expression, respectively. This was neither related to Fc[epsilon]RI.sub.H gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, Fc[epsilon]RI[alpha].sub.H surface expression appeared to correlate with the co-expression of Fc[epsilon]RI[gamma].sub.H . Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFc[epsilon]RI gamma, which constitutively expresses Fc[epsilon]RI[gamma].sub.H, increased Fc[epsilon]RI[alpha].sub.H chain expression levels. Levels of Fc[epsilon]RI[alpha].sub.H 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.