Introduction

Splenic B lymphocytes (B cells) from C57BL/6 (B6) mice were one of the original models for cell signaling studies used by the Alliance for Cellular Signaling (AfCS). While freshly isolated wild-type B6 splenic B cells are a suitable model for studying proximal membrane signaling, they rapidly die when cultured in the absence of proliferative and/or survival stimuli. Therefore, in cultured B cells, long-term signaling responses are measured against a background of cell death. The rapid death of cultured B cells also precludes using RNA interference (RNAi) or antisense oligodeoxynucleotides to manipulate signaling protein expression (1-4).

The AfCS therefore sought to define conditions that allow the sustained survival of B cells in culture and that might ultimately permit transfection or transduction of these cells for the manipulation of cell signaling. Specifically, we compared the survival in culture of fresh wild-type B cells with the survival of transgenic B cells expressing the human bcl-2 gene (hbcl-2). For the studies presented here, we used Em-bcl-2-22 mice, created by Suzanne Cory and her colleagues, in which hbcl-2 is expressed under the control of the Em enhancer (5). Overexpression of the human bcl-2 gene in mouse B cells prolongs their survival both in vivo and in vitro in the absence of stimulation (5-9). We compared the survival and functional properties of these hbcl-2 transgenic B cells with those of wild-type B cells grown in BAFF (also known as BlyS/TALL-1/zTNF-4) or with anti-CD40 antibody (a substitute for CD40 ligand). BAFF and anti-CD40 are two members of the tumor necrosis factor (TNF) superfamily that stimulate receptors on B cells known to enhance B cell survival (10-13).

This report describes the survival, phenotype, and signaling responses of cultured hbcl-2 transgenic B cells, compared with freshly isolated or stimulated (BAFF or anti-CD40) cultured wild-type B cells. We found that hbcl-2 transgenic B cell survival was approximately 80% after six days in culture (with serum), significantly greater than wild-type B cells, whether unstimulated or stimulated with either BAFF or anti-CD40. In addition, cultured hbcl-2 transgenic B cells retained a characteristic mature B cell surface phenotype, as determined by expression of IgM, B220, CD23, CXCR4, and CXCR5. In hbcl-2 transgenic B cells cultured for six days, signaling through the B cell receptor (BCR) was intact (as assessed by calcium transients, signaling protein phosphorylation, and upregulation of the activation markers CD86 and CD69) and was comparable to that of fresh B cells. Chemotactic responses to SDF-1a or BLC by cultured hbcl-2 transgenic B cells were also similar to those of fresh B cells, confirming the expression of functional chemokine receptors CXCR4 and CXCR5. The mRNA expression profiles for hbcl-2 transgenic B cells produced by DNA microarray analyses were relatively stable over time in culture and revealed a reproducible response to stimulation of the BCR even after three or six days. B cell proliferation in response to BCR ligation, CD40 ligation, or LPS stimulation revealed that both hbcl-2 transgenic and wild-type B cells were stimulated to synthesize DNA, as assessed by ³H-thymidine incorporation, albeit hbcl-2 transgenic B cells showed lower levels of incorporation and reduced increases in cell numbers when counted at two days poststimulation. Disappointingly, cultured hbcl-2 transgenic B cells were relatively refractive to transfection or transduction, including lentiviral-mediated transduction. Lentivirus vector systems have recently been used to implement gene silencing mediated by RNA interference (RNAi). Lentivirus transduction of sequences driving the transcription of small hairpin RNAs (shRNAs) processed into small interfering RNA (siRNA) duplexes has been used to functionally silence genes in activated T cells (14, 15). The inability to efficiently transduce or transfect B cells currently precludes the use of vector-based RNAi or antisense to manipulate signaling protein expression in these cells. Despite this shortcoming, we conclude that cultured hbcl-2 transgenic B cells provide a good model system to study primary B cell signaling responses due to their prolonged survival in culture and their similar surface phenotype and signaling responses to wild-type B cells.