Appropriate stimulation of resting B lymphocytes by T cells or their products (lymphokines) results in a large increase in the synthesis of the secretory form of IgM but not the membrane form. An assay has been developed utilizing a murine B cell tumor, (BCL1), for characterizing lymphokines which affect this step. The assay is simple in that BCL1 cells are cultured with or without B cell differentiation factors (BCDFu) and the supernatant assayed for IgM. A factor produced by T cell lines and tumors which is distinct from previously described lymphokines such as B cell growth factor or T cell growth factor, is active in this assay.
The aim of this proposal will be to purify and characterize this factor immunologically and biochemically, and to begin studies on cellular receptors for the factor. Preliminary experiments have been performed to optimize both the BCDFu assay and conditions for the production of BCDFu from EL-4 tumor cells. We propose to continue purification of BCDFu using two strategies: (a) by physical and chemical techniques which have been used successfully in the purification of other lymphokines. These include gel filtration, hydrophobic chromotography, chromatofocusing, high pressure liquid chromatography, and SDS gel electrophoresis. (b) by attempting to make monoclonal anti-BCDFu antibodies. As biochemical purification proceeds, fractions will be used to immunize rats for hybridization. Hybridoma anti-BCDFu will then be used to purify the factor. Partially purified factor will be used to develop a radiolabel binding assay for BCDFu receptors on which will then be used to assess BCDFu receptors normal B cells in various states. Finally, we will use the receptor binding assay to screen for anti-receptor hybridomas, which will be generated against BCDFu-responsive cells. These studies should provide insights into the molecular characteristics of a lymphokine which induces IgM secretion and its interaction with B cells.
| Birkeland, M L; Simpson, L; Isakson, P C et al. (1987) T-independent and T-dependent steps in the murine B cell response to antiimmunoglobulin. J Exp Med 166:506-19 |
| Isakson, P C (1986) Antiimmunoglobulin-treated B cells respond to a B cell differentiation factor for IgG1. J Exp Med 164:303-8 |
| Simpson, L G; Isakson, P C (1985) Phorbol esters specifically inhibit induction of immunoglobulin secretion in a murine B cell leukemia. J Immunol 134:2759-66 |
