Abstract
In vivo, B cells are often activated by antigens that are displayed on the surface of antigen-presenting cells (APCs). Binding of membrane-associated antigens to the B cell receptor (BCR) causes rapid cytoskeleton-dependent changes in the spatial organization of the BCR and other B cell membrane proteins, leading to the formation of an immune synapse. This process has been modeled using antigens attached to artificial planar lipid bilayers or to plasma membrane sheets. As a more physiological system for studying B cell-APC interactions, we have expressed model antigens in easily transfected adherent cell lines such as Cos-7 cells. The model antigens that we have used are a transmembrane form of a single-chain anti-Igκ antibody and a transmembrane form of hen egg lysozyme that is fused to a fluorescent protein. This has allowed us to study multiple aspects of B cell immune synapse formation including cytoskeletal reorganization, BCR microcluster coalescence, BCR-mediated antigen gathering, and BCR signaling. Here, we provide protocols for expressing these model antigens on the surface of Cos-7 cells, transfecting B cells with siRNAs or with plasmids encoding fluorescent proteins, using fixed cell and live cell fluorescence microscopy to image B cell-APC interactions, and quantifying APC-induced changes in BCR spatial organization and signaling.
Jia C. Wang and Madison Bolger-Munro are the co-first authors
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Acknowledgments
This work was supported by grant MOP-68865 (to M.R.G) from the Canadian Institutes of Health Research (CIHR). J.C.W. was supported by a doctoral scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). M. B.-M. was supported by a doctoral scholarship from the University of British Columbia.
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Wang, J.C., Bolger-Munro, M., Gold, M.R. (2018). Imaging the Interactions Between B Cells and Antigen-Presenting Cells. In: Liu, C. (eds) B Cell Receptor Signaling. Methods in Molecular Biology, vol 1707. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7474-0_10
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DOI: https://doi.org/10.1007/978-1-4939-7474-0_10
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