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（由适用提供）：幼稚的抗体库通常被视为由V（d）J重排和N添加创建的抗原结合位点的巨大抗原结合位点的随机抽样，尤其是在重链CDR3（CDR-H3）间隔中，该间隔位于抗原结合位点的中心。与这种随机性的推定不一致是发现氨基酸组成中明显的偏见，从而影响CDR-H3的疏水性和结构。这些观察结果使我们提出了一个过度的假设，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，自我抗原和病原体的反应。 HIV是一种病原体的一个例子，可能需要广泛保护的抗体要经历30％至40％的体细胞突变，花费数年的时间。阐明调节曲目多样性的躯体机制可能会确定其治疗性操纵的新途径。这些机制的失调也可能导致自身免疫性和疫苗衰竭。在此应用中，我们关注的是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（前BCR）检查点。前BCR的高分辨率结构分析定义了一个传感位点，其中IgHc与CDR-H3相互作用。 We will test the hypothesis that the pre-BCR uses the CDR-H3 sensitivity 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.我们将评估BCR前B细胞中的BCR表达，以及突变小鼠的骨髓和周围的B细胞发育，并将其与野生型进行比较。我们将评估凋亡和细胞周期的模式，如果我们的假设正确，则应改变这两种模式。我们将克隆并分析来自DH或VPREB1的生物和凋亡PR B细胞中的VDJCvdjcvdjcvdjcvdjcvdjc会改变小鼠，并将其与野生型进行比较。我们预测，DH序列改变在阅读框架2中通常发现的疏水氨基酸的使用将导致细胞凋亡增加和细胞分裂减少。另一方面，使用富含疏水性氨基酸的CDR-H3S的CDR-H3S的改变将促进B细胞的存活。我们将通过评估人类骨髓的早期和晚期B细胞中的CDR-H3曲目来扩展研究，以测试同样的BCR选择机制是否正在运行以限制曲目。技术创新是我们使用靶向基因的小鼠来检验以下假设：前BCR使用CDR-H3敏感性位点选择反对疏水氨基酸。我们预计这项研究将揭示出先前推测的存在，但到目前为止，是由BCR进行的未经测试的选择机制。在未来的研究中，该项目中产生的小鼠将用于探索BCR选择在调节表位识别中的作用，从而塑造对自我和非自身抗原的免疫反应。