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)：天真抗体谱系通常被视为V(D)J重排和N加成产生的抗原结合位点的巨大多样性的随机样本，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符的是，在影响CDR-H3疏水性和结构的氨基酸组成中发现了明显的偏差。这些观察结果使我们得出了一个全面的假设，即对CDR-H3氨基酸含量的分类调控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。艾滋病毒是一种病原体，可能需要具有广泛保护性的抗体经历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躯体机制可能会为其治疗操作确定新的途径。这些机制的失调也可能导致自身免疫和疫苗失败。在这个应用中，我们关注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。对前bcr的高分辨结构分析确定了IgHC与cdr-H3相互作用的敏感部位。我们将测试这一假设，即Pre-BCR使用CDR-H3传感位点来选择通常由DH阅读框架2编码的疏水氨基酸。我们将使用现有的一组D-改变的小鼠和一组新的VpreB1改变的小鼠来确定CDR-H3或VpreB1序列的改变如何影响通过Pre-BCR选择检查点的结果。我们将评估Pre-BCR在Pre-B细胞中的表达，以及B细胞在突变小鼠骨髓和外周的发育，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，如果我们的假设是正确的，这两者都应该改变。我们将克隆和分析来自DH或VpreB1突变小鼠的活的和凋亡的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，改变DH序列以强制使用阅读框架2中典型的疏水氨基酸将导致更多的细胞凋亡和细胞分裂减少。另一方面，VpreB1的改变将促进使用富含疏水氨基酸的CDR-H3s提高B细胞的存活率。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谱系，将我们的研究扩展到人类，以测试是否同样的前BCR选择机制正在运作，以限制谱系。技术创新是我们使用基因靶向的小鼠来测试这一假设，即Pre-BCR使用CDR-H3传感位点来选择不同的疏水氨基酸。我们预计，这项研究将揭示存在一种先前推测的、但到目前为止尚未经过测试的由前BCR选择的机制。在未来的研究中，这个项目中产生的小鼠将被用来探索前BCR选择在调节表位识别中的作用，从而塑造对自身和非自身抗原的免疫反应。