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The pre-BCR CDR-H3 sensing site and H chain selection

BCR前CDR-H3传感位点和H链选择

基本信息

批准号：
9089913
负责人：
Harry William Schroeder
金额：
$ 18.38万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9089913
关键词：
Affect Amino Acids Antibodies Antibody Diversity Antibody Formation Antibody Repertoire Antigens Antithymoglobulin Apoptosis Apoptotic Autoantigens Autoimmune Diseases Autoimmunity B-Cell Development B-Lymphocyte Subsets B-Lymphocytes Binding Binding Sites Bone Marrow Bone Marrow Transplantation CCL21 gene Cell Cycle Cell Death Cell division Complex Computer software Development Epitopes Failure Future Gene Targeting Genes Goals HIV Health Human Hydrophobicity Immune Immune response Immunoglobulins Inbred BALB C Mice Individual Infection Life Mature B-Lymphocyte Membrane Modeling Mus Mutant Strains Mice Mutate Nucleotides Outcome Pathway interactions Pattern Positioning Attribute Prevalence Production Proteins Reading Frames Receptor Signaling Regulation Resolution Risk Role Sampling Shapes Signal Pathway Signal Transduction Site Somatic Mutation Sorting - Cell Movement Staging Structure Testing Therapeutic Transcript Translations Tyrosine V(D)J Recombination Vaccines antigen binding base disorder risk embryonic stem cell in vivo innovation mutant neutralizing antibody pathogen pre-B cell receptor prevent response

项目摘要

DESCRIPTION (provided by applicant): The naïve antibody repertoire is often viewed as a random sampling of the enormous diversity of antigen binding sites created by V(D)J rearrangement and N addition, especially in the heavy chain CDR3 (CDR-H3) interval that lies at the center of the antigen binding site. Inconsistent with this presumption of randomness is the finding of clear biases in amino acid composition that influence the hydrophobicity and structure of CDR- H3. These observations have led us to the over-all hypothesis that categorical regulation of CDR-H3 amino acid content affects the likelihood of specific epitope recognition, which will influence responses to vaccines, self-antigens and pathogens. HIV is an example of a pathogen that may require broadly protective antibodies to undergo 30% to 40% somatic mutation, taking years to produce. Elucidation of the somatic mechanisms that regulate the diversity of the repertoire is likely to identify new pathways for its therapeutic manipulation. Dysregulation of these mechanisms may also contribute to autoimmunity and vaccine failure. In this application we focus on one of the earliest stages where the amino acid composition of CDR-H3 appears to be constrained; the pre-B cell receptor (pre-BCR) checkpoint. High resolution structural analysis of the pre-BCR has defined a sensing site where the Ig HC interacts with CDR-H3. We will test the hypothesis that the pre- BCR uses the CDR-H3 sensing site to select against hydrophobic amino acids typically encoded by DH reading frame 2. We will use an existing panel of D-altered mice and a new panel of VpreB1 altered mice to determine how alteration of CDR-H3 or VpreB1 sequence affects the outcome of passage through the pre-BCR selection checkpoint. We will evaluate pre BCR expression in pre-B cells, and B cell development in the bone marrow and periphery of mutant mice and compare them to wild type. We will evaluate patterns of apoptosis and cell cycle, both of which should be altered if our hypothesis is correct. We will clone and analyze VDJCtranscripts from living and apoptotic pr B cells from the DH or VpreB1 altered mice and compare them to wild type. We predict that alteration of DH sequence to force use of hydrophobic amino acids typically found in reading frame 2 will result in increased apoptosis and diminished cell division. On the other hand, alteration of VpreB1 will promote increased survival of B cells using CDR-H3s enriched for hydrophobic amino acids. We will extend our studies to humans by evaluating the CDR-H3 repertoire in early and late pre-B cells from human bone marrow to test if this same mechanism of pre-BCR selection is operating to constrain the repertoire. The technical innovation is our use of gene-targeted mice to test the hypothesis that the pre-BCR uses the CDR-H3 sensing site to select against hydrophobic amino acids. We expect that this study will reveal the existence of a previously speculated but, until now, untested mechanism of selection by the pre-BCR. In future studies, mice generated in this project will be used to explore the role of pre-BCR selection in regulating epitope recognition and thus shaping the immune response to both self and non-self antigens.

描述（由申请人提供）：初始抗体库通常被视为由 V(D)J 重排和 N 添加产生的巨大多样性抗原结合位点的随机抽样，特别是位于抗原结合位点中心的重链 CDR3 (CDR-H3) 区间。与这种随机性假设不一致的是，我们发现影响 CDR-H3 疏水性和结构的氨基酸组成存在明显偏差。这些观察结果使我们得出一个总体假设，即 CDR-H3 氨基酸含量的分类调节会影响特定表位识别的可能性，从而影响对疫苗、自身抗原和病原体的反应。 HIV 是一种病原体，可能需要广泛的保护性抗体才能发生 30% 至 40% 的体细胞突变，需要数年时间才能产生。阐明调节曲目多样性的体细胞机制可能会为其治疗操作找到新的途径。这些机制的失调也可能导致自身免疫和疫苗失败。在此应用中，我们重点关注 CDR-H3 的氨基酸组成似乎受到限制的最早阶段之一；前 B 细胞受体 (pre-BCR) 检查点。前 BCR 的高分辨率结构分析确定了 Ig HC 与 CDR-H3 相互作用的传感位点。我们将测试前 BCR 使用 CDR-H3 传感位点来选择通常由 DH 阅读框 2 编码的疏水性氨基酸的假设。我们将使用一组现有的 D 改变小鼠和一组新的 VpreB1 改变小鼠来确定 CDR-H3 或 VpreB1 序列的改变如何影响通过前 BCR 选择检查点的结果。我们将评估突变小鼠前 B 细胞中的前 BCR 表达以及骨髓和外周 B 细胞的发育，并将其与野生型进行比较。我们将评估细胞凋亡和细胞周期的模式，如果我们的假设正确，这两者都应该改变。我们将克隆并分析 DH 或 VpreB1 改变小鼠的活体和凋亡 pr B 细胞的 VDJC 转录本，并将其与野生型进行比较。我们预测，改变 DH 序列以强制使用通常在阅读框 2 中发现的疏水性氨基酸将导致细胞凋亡增加和细胞分裂减少。另一方面，VpreB1 的改变将促进使用富含疏水性氨基酸的 CDR-H3 的 B 细胞的存活率增加。我们将通过评估人骨髓早期和晚期前 B 细胞中的 CDR-H3 库，将我们的研究扩展到人类，以测试这种相同的前 BCR 选择机制是否正在运行以限制库。技术创新是我们使用基因靶向小鼠来测试前 BCR 使用 CDR-H3 传感位点来选择疏水性氨基酸的假设。我们预计这项研究将揭示之前推测但迄今为止未经测试的 BCR 前选择机制的存在。在未来的研究中，该项目中产生的小鼠将用于探索前 BCR 选择在调节表位识别中的作用，从而形成对自身和非自身抗原的免疫反应。