Cleavage of membrane-bound receptors by disintegrin and metalloproteinases (ADAMs) can regulate lymphocyte development and function. Specifically, the study of B cell-specific ADAM10 knock out mice, ADAM10flox/floxCD19-cre+, revealed that ADAM10 is critical for marginal zone B cell differentiation. Subsequent studies determined that ADAM10 is required for the proteolytic activation of Notch2 signaling. Alterations in Notch signaling have been implicated in numerous B cell malignancies, including B cell chronic lymphocytic leukemia, Hodgkin's lymphoma and multiple myeloma. Thus, ADAM10 may be a target for the treatment of aberrant B cell development. In order to determine the role of ADAM10 in early B cell development, we will use an mb1-cre mouse model. In contrast to ADAM10flox/floxCD19-cre+, this model will allow efficient deletion of ADAM10 from B cell precursors within the bone marrow. Immunohistochemistry and flow cytometric approaches will be used to determine which B cell subsets are affected secondary to ADAM10 deletion. Recent studies with ADAM10flox/floxCD19-cre+ mice also demonstrated that these mice fail to form normal germinal centers. These mice have fewer and smaller germinal centers when compare to wild type mice. We propose to study germinal center B cell apoptosis and proliferation in order to further understand the role of ADAM10 in germinal center formation. Furthermore, although the total number of antigen specific antibody- secreting cells (ASCs) observed in ADAM10 knockout mice within the spleen and bone marrow does not differ from wild type, ADAM10flox/floxCD19-cre+ have dramatically reduced levels of ASCs that secrete high-affinity antibodies. Consistent with these findings, ADAM10flox/floxCD19-cre+ mice have normal levels of total antigen- specific IgG antibodies. However, knockout generated significantly less high affinity antibodies than wild type mice. In order to further study the role of ADAM10 in somatic hypermutation, mice will be immunized with NP- KLH in alum and germinal center B cell and plasma cells will be sorted. DNA will be isolated and 186.2 V regions will be amplified by nested PCR. PCR products will be cloned and then sequenced. Sequences will then be analyzed for mutations. We will also examine the recall response in ADAM10 knockout mice in order to further understand the role of ADAM10 in memory B cell generation. This work has important implications for the treatment B cell malignancies, autoimmune disease and allergy and asthma.
We propose to study the role of ADAM10 in B cell development, germinal center formation and antibody production. Aberrant B cell development is associated with B cell malignancies, while altered germinal center formation and antibody production are involved in allergy, asthma and autoimmune diseases. These studies could, therefore, provide critical information that may enhance our ability to treat B cell malignancies, autoimmune disease and allergy and asthma, and thus improve clinical outcomes.