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

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

Abstract

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRI 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 human-ized RBL cell lines. However, no studies so far have addressed the levels of FcεRI 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 FcεRI H chain in humanized RBL cell lines. We compared surface levels of FcεRIα H by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIα H copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγ H cDNA was assessed for its ability to increase FcεRIα 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 FcεRIα H expression, respectively. This was neither related to FcεRI H gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIα H surface expression appeared to correlate with the co-expression of FcεRIγ H. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγ H , increased FcεRIα H chain expression levels. Levels of FcεRIα 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.

Citation

Ali Ali, E., Kalli, M., Wan, D., Nakamura, R., Onion, D., Alanine, D. G., Alcocer, M. J., & Falcone, F. H. (2019). Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines. PLoS ONE, 14(8), Article e0221034. https://doi.org/10.1371/journal.+pone.0221034

• Journal Article Type: Article
• Acceptance Date: Jul 29, 2019
• Online Publication Date: Aug 17, 2019
• Publication Date: Aug 20, 2019
• Deposit Date: Sep 10, 2019
• Publicly Available Date: Sep 11, 2019
• Journal: Plos ONE
• Electronic ISSN: 1932-6203
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 14
• Issue: 8
• Article Number: e0221034
• DOI: https://doi.org/10.1371/journal.+pone.0221034
• Public URL: https://nottingham-repository.worktribe.com/output/2596729
• Publisher URL: https://fg.bmj.com/content/early/2019/08/16/flgastro-2019-101277
• Contract Date: Sep 10, 2019