No abstract

Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.

BackgroundParasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.Methodology/Principal FindingsA complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.Conclusion/SignificanceThis method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.

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 humanized 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.

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose–response curve, and correlated (R2 = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format.