B cells are involved in many autoimmune diseases. CD19 regulates B cell functions in vivo that are important in autoimmunity. Extensive studies in vitro have identified multiple signaling pathways, linked to specific CD19 cytoplasmic tyrosines, that may mediate CD19 function but their relative importance in vivo is unknown. We have developed a system for studying CD19 tyrosine-based signaling in vivo. Mice that are deficient in CD19 lack peritoneal B 1 and marginal zone B cells and respond poorly to T-independent antigens. We reconstituted these functions with an unmutated CD19 transgene, but a construct with mutation of all cytoplasmic tyrosines completely failed to do so, demonstrating the dependence of these B cell functions on CD19 tyrosine signaling. To determine the relevance in vivo of the multiple pathways that have been linked to particular tyrosines and the molecules that bind them in vitro, we produced four transgenes each containing mutations of distinct pairs of homologous CD19 tyrosines. Founder lines for each have been crossed onto the CD19-/- background. We initially studied the role of Y482 and Y513, which bind Lyn and phosphatidylinositol 3-kinase (PI3K), and found an absolute requirement for these in all in vivo CD19 functions tested. The likely mechanism is the abrogation of phosphorylation of all CD19 tyrosines by these mutations. This finding establishes that targeting particular CD19 tyrosines can modulate B cell responses in vivo. We propose to: 1) Identify the CD19 structures that control phosphorylation of the molecule, as these would be targets for global control of CD19 function, 2) Determine the role of binding of cytoplasmic molecules to particular CD19 tyrosines, as these mediate CD19 function, 3) Determine which downstream pathways are controlled by particular CD19 tyrosines in vivo, as these would be identified as regulating important B cell functions, and 4) Determine the role of CD19 tyrosine-based signaling in a mouse model of lupus, to test the hypothesis that targeting specific CD19 tyrosines could be used for therapeutic manipulation of B cells in autoimmune disease.
