The goal of this project is to define the pathways by which LPS and IFN control the expression of k light chains and CD40 during B cell development. This grant focuses on a single model system, the B cell lymphoma 70Z/3, to dissect the pathways used by LPS and IFN to activate kappa and CD40 transcription. Both ligands are important to interactions in the immune system, IFN as a cytokine used by T cells and LPS as an important agent of human pathology. The expression of these two genes is also regulated as B cells enter a critical stage in their development. 70Z/3 cells provide the opportunity to examine the initiation of these key developmental steps in a homogenous population of cells. We have isolated a series of mutants from these cells whose kappa genes fail to respond to LPS or to IFN. At least some of these mutants are not defective in their activation of CD40, suggesting that the pathways for activation of kappa and CD40 may not be completely overlapping.
Aim 1. To identify the lesion in four mutants of 70Z/3 with a defect in their response to LPS or IFN. We will test the hypothesis that LPS and IFN activate kappa expression by different pathways. This will be accomplished by detailed analysis of the mutant phenotypes.
Aim 2. To define the signals that activate CD40 expression in 70Z/3 cells. We will test the hypothesis that LPS and IFN activate CD40 expression by different pathways than those used to activate kappa expression in 70Z/3 cells. We will examine the expression of CD40 after LPS or IFN treatment of mutants selected to have a defect in kappa expression after activation by these inducers.
Aim 3. To determine whether the signaling pathway used by LPS and IFN overlap with the signals generated by stimulation of CD40. We will test the hypothesis that the LPS, IFN and CD40 signalling pathways are different in 70Z/3 cells. We will render wild type or mutant 70Z/3 cells CD40+ by treatment with either LPS or IFN. Mutants with known defects in particular aspects of LPS or IFN signalling will be examined to determine whether the defect compromises CD40 signalling. Study of these mutants and of the wild type 70Z/3 cells will allow us to combine genetics, biochemistry and molecular biology to define precisely the pathway of activation used by LPS and IFN to stimulate kappa or CD40 expression. We think that the responses we define in 70Z/3 cells will allow better understanding of the same responses in normal B cells.
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