The origin and consequences of receptor editing and allelic-inclusion.

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DESCRIPTION (provided by applicant): We recently demonstrated that a surprising 10% of B lymphocytes break a fundamental principal of adaptive immunity by expressing more than one B cell receptor. Making matters worse, this occurs due to receptor editing. Further rearrangement of the immunoglobulin kappa (Ig-kappa) genes to remove autoreactive receptors instead results in expression of the second kappa allele. In the end, "allelically included" B cells are generated that express an autoreactive B cell receptor and may be a means for inappropriate and even dangerous immune responses to occur. Using the occurrence of Ig-kappa allelic-inclusion as a marker of B cells that had been subjected to receptor editing, we made some important observations. We have new preliminary data showing that one third of cells at the T2 stage of immature B cell development express both Ig-kappa alleles. In contrast, T1, T3 or mature B cells only infrequently expressed both Ig-kappa alleles. Receptor editing is defined as expression of new antibody variable genes in cells that already express an autoreactive BCR. We interpreted our new data as evidence of ongoing or recent Ig-kappa receptor editing in the T2 transitional B cells. It is known that splenic T2 B cells play a role in B cell selection and give rise to preMZ and T3 B cells that are believed to be anergic. Together we interpret these various observations to suggest that the T2 stage of immature B cell development is the nexus of primary B cell selection. This project is based on characterizing the development of B cells in the context of receptor editing and the potentially dangerous allelic-inclusion that results. A set of highly focused experiments are proposed to conclusively determine the role of T2 and all transitional B cells in receptor editing and primary B cell selection. In Specific Aim 1, we will determine if indeed receptor editing occurs in T2 or any population of transitional B cells using molecular assays. We will also explore the kinetics of receptor editing during transitional B cell development. In specific aim 2, we will analyze receptor editing and B cell selection using a modification of the classic anti-Hen Egg Lysozyme (anti-HEL) mouse model of autoreactive B cell selection. These experiments will provide an in vivo assessment of transitional B cell selection in the context of receptor editing. We are experts at powerful technology that allows expression of numerous monoclonal antibodies from any B cell isolated by flow cytometry. In Specific Aim 3 we will directly and conclusively identify the selective checkpoints in transitional B cell development by analyzing the BCR specificities in normal mice. Throughout, we will also determine the fate and autoimmune potential of allelically-included B cells. With the experiments proposed our goal is to characterize the particular B cells mediating primary immune tolerance. Characterizing these cells would provide the targets for therapy that could revolutionize treatment of autoimmune diseases.