The role of Immunoglobulin CDRH3 in Autoimmune Disease

描述(申请人提供)：系统性红斑狼疮(SLE)是一种常见的多系统自身免疫性疾病，估计影响超过50万美国人。我们的目标是更好地了解通常阻止导致疾病的自我反应性抗体表达的机制，以及更好地了解这些机制在哪里以及如何失败。出于伦理方面的考虑，研究人类疾病的根本原因往往是不可能的。数十年的医学研究证明，小鼠模型作为一种确定根本原因以及治疗和预防的新途径的手段是有用的。在C57BL/6中，Sle1、Sle2和/或Sle3/5 NZM2410狼疮基因座的存在促进了自身抗体驱动的疾病的发展，这种疾病与人类SLE的许多特征相同。我们的假设是，在易感人群中，未能适当地调节抗体的一个特定部分CDR-H3，会促进抗DNA抗体的产生，并引发疾病。CDR-H3很重要，因为它位于抗原结合部位的最中心。CDR-H3是通过VDJ加入而从头创建的。我们认为，水解酶的序列对CDR-H3含量具有显性影响，通常限制CDR-H3的主要机制是通过阅读框架保守水解酶序列。我们认为，自身免疫是由于未能调节不受欢迎的阅读框中氨基酸的使用所致。这些氨基酸也可以通过N加成或体细胞突变来引入。我们假设，通过允许携带不受欢迎的CDR-H3的B细胞存活增加，sle1、sle2和sle3/5基因对B细胞的调节和谱系的发育产生不利影响。为了验证这些假设，我们将在C57BL/6基因组中培育三个不同的等位基因，每个等位基因仅限于一个DH基因片段。第一个DH等位基因将用精氨酸和其他来自倒排阅读框1的正电荷氨基酸取代通常由首选阅读框1通过缺失编码的酪氨酸和甘氨酸。第二个将包含一个单一的、正常的水解酶，作为失去水解酶基因其余部分的对照。第三种是通过取代疏水阅读框2中的密码子来控制酪氨酸和甘氨酸含量的丢失。如果我们的假设是正确的，那么在DH阅读框1中富含精氨酸将增强不受欢迎的CDR-H3序列的表达。这种高表达将超过CDR-H3含量正常控制的阈值，加速包括抗DNA在内的自我反应性抗体的表达。在Sle1、Sle2和Sle3/5存在的情况下，含有突变的DH等位基因的小鼠将表现出B细胞和谱系发育模式的改变，对抗原的免疫反应发生改变，抗dsDNA抗体的Ig G表达增强，并导致自身免疫性疾病的加速表达。

DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is a common multisystem autoimmune disease that is estimated to affect more than 500,000 Americans. Our goal is to gain a better understanding of the mechanisms that normally prevent expression of the self-reactive antibodies that cause the disease as well as to better understand where and how these mechanisms fail. Research into fundamental causes of disease in humans is often impossible due to ethical considerations. Decades of medical research attest to the usefulness of mouse models as a means to identify root causes as well as new avenues for treatment and prevention. The presence of the sle1, sle2, and/or sle3/5 NZM2410 lupus loci in C57BL/6 promotes development of an autoantibody driven disorder that shares many of the features of human SLE. It is our hypothesis that failure to properly regulate one specific part of the antibody, CDR-H3, in susceptible individuals facilitates production of anti-DNA antibodies and triggers disease. CDR-H3 is important because it lies at the very center of the antigen binding site. CDR-H3 is created de novo by VDJ joining. We propose that the sequence of the DH has a dominant effect on CDR-H3 content and that a primary mechanism normally constraining CDR-H3 is conservation of DH sequence by reading frame. We propose that autoimmunity results from failure to regulate use of the amino acids that can be found in disfavored reading frames. These amino acids can also be introduced by N addition or somatic mutation. We hypothesize that sle1, sle2, and sle3/5 genes adversely affect regulation of B cell and repertoire development by permitting increased survival of B cells bearing disfavored CDR-H3. To test these hypotheses we will breed three different DH alleles, each limited to a single DH gene segment, into the C57BL/6 genome. The first DH allele will substitute arginine and other positively charged amino acids from inverted reading frame 1 for the tyrosine and glycine that are normally encoded by the preferred reading frame 1 by deletion. The second will contain a single, normal DH as a control for the loss of the rest of the DH locus. The third will control for the loss of tyrosine and glycine content by substituting codons from hydrophobic reading frame 2. If our hypotheses are correct, enrichment for arginine in DH reading frame 1 will enhance expression of disfavored CDR-H3 sequence. This heightened expression will exceed the threshold for normal control of CDR-H3 content, accelerating expression of self-reactive antibodies, including anti-DNA. In the presence of sle1, sle2, and sle3/5, mice containing altered DH alleles will display altered patterns of B cell and repertoire development, evidence altered immune responses to antigen, demonstrate enhanced expression of IgG anti-dsDNA antibodies, and lead to accelerated expression of autoimmune disease.