Immunology and Cell Biology (2003) 81, 297–304 Research Article Knockout B lymphoma cell lines as biochemical tools to explore multiple signalling pathways MARGARET F VEALE, WENDY M DIETRICH and LYNN M CORCORAN The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia Summary Studies on B lymphocyte signalling pathways using B lymphocytes from genetically modified mice have the disadvantages of primary cell polyclonality and finite life span. B lymphoma cell lines have been generated from mice with targeted mutations in the oct-2, OBF-1, vav-1 and btk genes, as a model system that lacks these limitations and possesses additional potential for experimental manipulation. To assess their utility, activation of the B cell receptor using anti-µ, the Toll-like receptor-4 using lipopolysaccharide and the interleukin-4 receptor were assessed in these cell lines. Differential tyrosine phosphorylation of intracellular proteins was measured in the wild- type controls compared to the corresponding mutant cell lines after B cell receptor stimulation. Intracellular calcium (Ca 2+ i ) was mobilized in the control cell lines but not in the OBF-1 and Vav1-deficient cells, while Xid B cell lines (btk mutant) showed a reduced Ca 2+ mobilization. Extracellular signal-regulated kinase 1/2 phosphorylation in response to anti-µ or lipopolysaccharide stimulation was significantly reduced in Vav1-deficient cells. Interleukin-4 stimulation of wild-type cells resulted in a 2–3-fold increase in Stat-6 phosphorylation. These results indicate that the cell lines mimic the biochemical responses of the corresponding primary B cells. They therefore represent a useful model system to investigate the regulation and roles of these and other gene products in B cell signal transduction and activation. Key words: calcium signalling, lymphocyte activation, lymphoma, MAP kinase signalling, signal transduction. Introduction Differentiation of an immature B lymphocyte to a mature antibody-secreting cell is dependent upon transcriptional reg- ulators such as Oct–2 and OBF-1 (BOB-1; OCA-B), proteins that may contribute to, 1,2 but are not absolutely required for immunoglobulin gene expression. 3 In addition, knockout mouse models have shown that the transition between imma- ture and mature cells is influenced by deficiencies in a number of tyrosine phosphorylated intracellular signalling proteins that are constitutively associated with, or are recruited to the B cell receptor (BCR) after antigen engage- ment. B lymphoma cell lines have been generated from knockout mice with targeted mutations in a number of genes that influence B cell function, by crossing with mice bearing the lymphomagenic Eµ-myc transgene. 4 The B cell lymphoma cell lines generated display surface markers consistent with an immature phenotype. 4 Cell lines that are deficient in Oct–2, its coactivator OBF-1, the guanine nucleotide (GTP/ GDP) exchange factor protein Vav1, and that carry the Xid Bruton's tyrosine kinase (Btk) mutation are under study here to assess their ability to transduce a variety of immuno- logically important signals, emanating from the BCR, the lipopolysaccharide (LPS) Toll-like receptor-4 (TLR-4) and the interleukin-4 receptor (IL-4R). B cell receptor ligation induces the phosphorylation of IgM-α and IgM-β cytoplasmic domains on motifs termed immunoreceptor tyrosine-based activation motifs (ITAM), which act as binding sites for non-receptor src-family protein kinases (PTK). 5–7 Phosphorylation of the ITAM results in the recruitment of PTK Syk to the BCR, an important step in the activation of a number of down-stream signalling path- ways. 8–10 These include activation of the Phospholipase C- γ (PLC-γ) pathway by Btk or by Syk, resulting in the produc- tion of inositol-3-phosphate (IP 3 ). Inositol-3-phosphate in turn induces the subsequent release of intracellular calcium (Ca 2+ i ), and the production of diacylglycerol (DAG), which can activate protein kinase C (PKC). 11 The release of calcium and the activation of PKC can ultimately result in the activation of the mitogen activated protein (MAP) kinase pathways, including extracellular signal-regulated kinase (ERK). 12 Alternatively, BCR ligation can initiate complex formation between the adaptor Grb2 and Sos (son of sevenless), a guanidine exchange factor (GEF) for Ras. Activated GTP- bound Ras stimulates signalling via MAP kinase cascades that result in the activation of ERK. 10,12,13 Interleukin-4 is a pleiotropic cytokine that was originally identified as a growth and differentiation factor for B cells. It binds to its receptor on B cells and acts as a comitogen 14 and can induce the expression of MHC II and CD23. Interleukin- 4 stimulates transcription of immunoglobulin genes, which leads to class switching. 15 Interleukin-4 activates the Janus protein tyrosine kinases Jak1 and Jak3, which phosphorylate the receptor, resulting in the recruitment of other signalling proteins and the activation of down-stream signalling pro- teins, including the signal transducer and activator of tran- Correspondence: Dr Lynn M Corcoran, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Victoria 3050, Australia. Email: corcoran@wehi.edu.au Received 13 January 2003; accepted 7 April 2003.