The role of ADAM10 in B cell development and humoral immunity

性状（由申请方提供）：去整合素和金属蛋白酶（亚当斯）对膜结合受体的切割可调节淋巴细胞发育和功能。具体而言，对B细胞特异性ADAM 10敲除小鼠（ADAM 10 flox/CD 19-cre+）的研究表明，ADAM 10对边缘区B细胞分化至关重要。随后的研究确定，ADAM 10是Notch 2信号传导的蛋白水解激活所必需的。Notch信号传导的改变与许多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，ADAM 10可能是治疗异常B细胞发育的靶点。为了确定ADAM 10在早期B细胞发育中的作用，我们将使用mb 1-cre小鼠模型。与ADAM 10 flox/CD 19-cre+相反，该模型将允许从骨髓内的B细胞前体中有效地删除ADAM 10。将使用免疫组织化学和流式细胞术方法来确定哪些B细胞亚群继发于ADAM 10缺失。最近对ADAM 10 flox/CD 19-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，这些小鼠具有更少和更小的衰老中心。我们拟通过对成熟中心B细胞凋亡和增殖的研究，进一步了解ADAM 10在成熟中心形成中的作用。此外，尽管在ADAM 10敲除小鼠的脾脏和骨髓中观察到的抗原特异性抗体分泌细胞（ASC）的总数与野生型没有差异，但ADAM 10 flox/ADAM 19-cre+具有显著降低的分泌高亲和力抗体的ASC水平。与这些发现一致，ADAM 10 flox/CD 19-cre+小鼠具有正常水平的总抗原特异性IgG抗体。然而，敲除产生的高亲和力抗体显著低于野生型小鼠。为了进一步研究ADAM 10在体细胞超突变中的作用，将NP-KLH在明矾中免疫小鼠，并将其接种于衰老中心B细胞，分选浆细胞。将分离DNA，并通过巢式PCR扩增186.2 V区域。PCR产物将被克隆，然后测序。然后将分析序列的突变。我们还将检查在ADAM 10基因敲除小鼠的回忆反应，以进一步了解ADAM 10在记忆B细胞生成中的作用。本研究对治疗B细胞恶性肿瘤、自身免疫性疾病、变态反应和哮喘具有重要意义。 公共卫生相关性：我们建议研究ADAM 10在B细胞发育、生殖中心形成和抗体产生中的作用。异常的B细胞发育与B细胞恶性肿瘤有关，而改变的生发中心形成和抗体产生与过敏、哮喘和自身免疫性疾病有关。因此，这些研究可以提供重要的信息，可以提高我们治疗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.