Cultured Splenic B Cells Expressing a Human Bcl-2 Transgene as an Alternative Signaling Model for Primary B Cells

Tamara Roach, Sangdun Choi, Heping Han, and Xiaocui Zhu

Alliance for Cellular Signaling Laboratories

San Francisco Veterans Administration Medical Center, San Francisco CA; California Institute of Technoloogy, Pasadena, CA; University of Texas Southwestern Medical Center, Dallas, TX

Abstract

The Alliance for Cellular Signaling (AfCS) has sought to identify a suitable model cell type that can be used to study intracellular signaling networks in a detailed manner. Our laboratory examined cultured mouse splenic B lymphocytes (B cells) expressing a transgenic human bcl-2 gene (hbcl-2) and found them to be a promising model for long-term culture studies of primary B cells. The transgenic hbcl-2 B cells survived and maintained stable signaling responses for at least one week in culture, compared with wild-type C57BL/6 splenic B cells, which rapidly died. Signaling parameters measured in response to cell stimulation included intracellular calcium flux, phosphorylation of a panel of signaling proteins, chemotaxis, and changes in gene expression. Transgenic hbcl-2 B cells showed similar responses to freshly isolated wild-type B cells across these parameters. Wild-type B cell survival is notably less than that of transgenic hbcl-2 B cells, even when stimulated in culture with the prosurvival factors BAFF or anti-CD40 monoclonal antibody. Further, hbcl-2 transgenic B cells remain in a relatively quiescent state, unlike cells stimulated with BAFF or anti-CD40, which are activated. An unfortunate disadvantage of wild-type B cells that was also exhibited by the transgenic hbcl-2 B cells was their resistance to transfection or transduction. We attempted to use plasmid-based lentiviral systems that are capable of infecting many primary cells with high efficiency, but the B cells proved resistant to current methodologies. Although this trait is specifically forbidding for AfCS goals, it does not disqualify the hbcl-2 transgenic B cells from providing a stable, long-term culture system for study of splenic B cells.