The role of ADAM10 in B cell development and humoral immunity

描述(申请人提供)：去整合素和金属蛋白酶(ADAMS)对膜结合受体的切割可以调节淋巴细胞的发育和功能。具体地说，对B细胞特异性ADAM10基因敲除小鼠的研究表明，ADAM10对边缘区B细胞的分化至关重要。后来的研究确定，ADAM10是Notch2信号的蛋白水解性激活所必需的。Notch信号的改变与许多B细胞恶性肿瘤有关，包括B细胞慢性淋巴细胞白血病、霍奇金淋巴瘤和多发性骨髓瘤。因此，ADAM10可能成为治疗B细胞发育异常的靶点。为了确定ADAM10在早期B细胞发育中的作用，我们将使用MB1-cre小鼠模型。与ADAM10FLOX/FLOXCD19-cre+不同，该模型将允许从骨髓内的B细胞前体有效地删除ADAM10。免疫组织化学和流式细胞仪方法将用于确定ADAM10缺失继发于哪些B细胞亚群受到影响。最近对ADAM10FLOX/FLOXCD19-cre+小鼠的研究也表明，这些小鼠不能形成正常的生发中心。与野生型小鼠相比，这些小鼠的生发中心更少、更小。我们建议对生发中心B细胞的凋亡和增殖进行研究，以进一步了解ADAM10在生发中心形成中的作用。此外，虽然在ADAM10基因敲除小鼠的脾和骨髓中观察到的抗原特异性抗体分泌细胞(ASCs)的总数与野生型没有区别，但ADAM10flx/FloxCD19-cre+显著减少了分泌高亲和力抗体的ASCs的水平。与这些发现一致的是，ADAM10FLOX/FLOXCD19-cre+小鼠的总抗原特异性IgG抗体水平正常。然而，与野生型小鼠相比，基因敲除小鼠产生的高亲和力抗体明显较少。为了进一步研究ADAM10在体细胞超突变中的作用，我们将用NP-KLH免疫小鼠，并对小鼠的生发中心B细胞和浆细胞进行分选。用套式聚合酶链式反应扩增出186.2个V区。扩增产物将被克隆，然后进行测序。然后将对序列进行突变分析。我们还将检测ADAM10基因敲除小鼠的回忆反应，以进一步了解ADAM10在记忆B细胞生成中的作用。这项工作对治疗B细胞恶性肿瘤、自身免疫性疾病、过敏和哮喘具有重要意义。 公共卫生相关性：我们建议研究ADAM10在B细胞发育、生发中心形成和抗体产生中的作用。B细胞发育异常与B细胞恶性肿瘤有关，而生发中心形成和抗体产生异常与过敏、哮喘和自身免疫性疾病有关。因此，这些研究可以提供关键信息，可能增强我们治疗B细胞恶性肿瘤、自身免疫性疾病、过敏和哮喘的能力，从而改善临床结果。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.