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

BCR前CDR-H3传感位点和H链选择

• 批准号: 8987028
• 负责人: Harry William Schroeder
• 金额: $ 22.05万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8987028
• 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; 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; public health relevance; response

 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，特别是在重链 CDR3 (CDR-H3) 区间与这种随机性假设不一致的是影响 CDR-H3 疏水性和结构的氨基酸组成的明显偏差。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，这将影响对疫苗、自身抗原和病原体的反应，HIV 是可能需要广泛治疗的病原体的一个例子。保护性抗体会发生 30% 至 40% 的体细胞突变，需要数年时间才能产生，阐明调节库多样性的体细胞机制可能会找到治疗操纵这些机制的新途径。也可能导致自身免疫和疫苗失败。在本应用中，我们重点关注 CDR-H3 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。对前 BCR 的分析确定了 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编码的疏水性氨基酸的假设。 DH 阅读框 2。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预 BCR 选择检查点的结果。评估前 B 细胞中的前 BCR 表达以及突变小鼠骨髓和外周血中的 B 细胞发育，并将它们与野生型进行比较。我们将评估细胞凋亡和细胞周期的模式，这两者都应该是我们的。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，并将它们与野生型进行比较，我们预测 DH 序列的改变会强制使用通常发现的疏水性氨基酸。另一方面，使用富含疏水性氨基酸的 CDR-H3，VpreB1 的改变将促进 B 细胞的存活率增加。我们将通过评估来自人类骨髓的早期和晚期前 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该库。我们使用技术创新。我们期望这项研究将揭示之前推测但迄今为止未经测试的选择机制的存在。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。