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

 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添加产生的抗原结合位点的巨大多样性的随机取样，特别是在位于抗原结合位点中心的重链CDR 3（CDR-H3）间隔中。与这种随机性的假设不一致的是发现影响CDR-H3的疏水性和结构的氨基酸组成中的明显偏差。这些观察结果使我们得出了一个总体假设，即CDR-H3氨基酸含量的分类调节影响特异性表位识别的可能性，这将影响对疫苗、自身抗原和病原体的应答。HIV是一种可能需要广泛保护性抗体才能经历30%至40%体细胞突变的病原体，需要数年时间才能产生。阐明调控库多样性的体细胞机制可能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。在本申请中，我们专注于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已经确定了IG MHC与CDR-H3相互作用的传感位点。我们将检验前BCR使用CDR-H3传感位点来选择通常由DH阅读框2编码的疏水氨基酸的假设。我们将使用现有的一组D-改变的小鼠和一组新的VpreB 1改变的小鼠来确定CDR-H3或VpreB 1序列的改变如何影响通过前BCR选择检查点的结果。我们将评估前B细胞中的前BCR表达，以及突变小鼠骨髓和外周中的B细胞发育，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VpreB 1改变小鼠的活的和凋亡的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序列以迫使使用通常在阅读框2中发现的疏水氨基酸将导致细胞凋亡增加和细胞分裂减少。另一方面，VpreB 1的改变将促进使用富含疏水性氨基酸的CDR-H3的B细胞的存活增加。我们将通过评估来自人骨髓的早期和晚期前B细胞中的CDR-H3库来将我们的研究扩展到人类，以测试前BCR选择的这种相同机制是否在限制库。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氨基酸的假设。我们希望这项研究将揭示以前推测的存在，但到目前为止，未经测试的前BCR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应答。

 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.