In the last funding period, our laboratory generated intriguing new data which suggests that the loss of basal signaling through the B cell receptor (BCR) in immature B cells can lead to the induction of RAG proteins and new light chain (LC) rearrangements, as well as evidence for a striking back-differentiation of cells to an earlier stage in development. We have demonstrated this through inducible Cre-mediated deletion of the BCR in immature B cells, by inhibiting basal signaling using pharmacologic agents such as PI-3K inhibitors, and by crosslinking the BCR of immature B cells with antigen. These data, together with evidence from other systems, have led us to propose a model describing the signals that control the expression of RAG proteins and drive receptor editing in developing pre-B and immature B cells. We hypothesize that developing cells at the pre-B stage initiate LC rearrangements, which then continue until the B cell produces a functional BCR at sufficiently high levels on the cell surface to signal for a shut-off of RAG gene expression. This model suggests that a critical determinant of LC editing is the level of basal signaling from slgM. We have also recently tested a model system where B cells carry receptors specific for hen egg lysozyme (HEL), in which a knock-in anti-HEL LC is capable of receptor editing. Our initial experiments suggest that editing to membrane-bound antigens is extremely efficient, with normal numbers of edited cells produced and selected into the periphery. Using BrdU labeling, we found that it takes no longer than 6 hours for B cells to edit away from high-level reactivity with membrane HEL. These data provide important evidence for the efficiency of editing during B cell development, and provide us with a novel in vivo model to further explore the mechanisms that maintain allelic exclusion and tolerance centrally. In this application we propose to test a series of novel hypotheses that flow from these findings. First, we propose that the basal signaling important for maintaining LC allelic exclusion initiates from basal tyrosine phosphorylation of IgA, an important BCR-associated protein, and will test this using mice with targeted and conditional alleles of IgA. Second, we propose that the basal BCR signal important for suppressing RAG involves a signaling pathway that includes Lyn, Btk, PI-3K, PLC?2 and protein kinase C-beta (FKC|3). This hypothesis will be tested using pharmacologic agents and signaling inhibitors, and loss-of-function transgenic mouse models. Third, we hypothesize that there are key transcription factors expressed at the early immature B stage that function to suppress editing and maintain the B cell program, and we will identify these factors using microarray approaches. We believe that these experiments will lead to a better understanding of the mechanisms that regulate receptor editing and the signals important for maintaining developmental stage of pre-B and immature B cells, and are likely to provide important new insights into the initiation of autoimmunity.
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