IMMUNOGLOBULIN HCDR3 IN AUTOIMMUNE DISEASE

免疫球蛋白重链的第三个互补决定区域--HCDR3-位于抗原结合位置的中心，通常决定抗体的特异性。组成HCDR3核心的DH基因片段有可能在六个阅读框中翻译，每个阅读框都有一个特征的亲水性特征-带电的、疏水的或亲水的。然而，在几乎所有物种中，最终的HCDR3谱带都富含中性的亲水序列。带电或疏水的HCDR3间期只有在特殊情况下才常见(例如致病的抗DNA抗体)。我们的假说认为，带电或疏水的HCDR3结构域是“被禁止的”，因为它们更有可能产生自我反应性抗体，从而帮助在易感生物中“触发”自身免疫性疾病。通过基因打靶，我们正在产生小鼠，其中抗体谱系已经被全局改变，以产生由带电或疏水的DH读框编码的HCDR3间隔。这些改变的谱系包含天然的带电或疏水的HCDR3序列，虽然在典型的抗体中很少见，但在一些有自身免疫倾向的小鼠品系中更常见。初步研究表明，表达带有带电HCDR3间隔的抗体的B细胞可以大量产生，但在外周是选择性的。我们认为B细胞的这种外周丢失是由于自身反应所致。携带降低自身抗体形成障碍的基因的小鼠可能会使表达具有改变的HCDR3亲水性抗体的B细胞存活下来。带有带电或疏水的HCDR3间期的抗体被认为更有可能“触发”自身免疫性疾病。为了验证这一假设，我们将把突变的DH基因座引入到携带自身免疫性疾病易感基因座的小鼠中，例如，C57BL/6携带NZM衍生的sle1、sle2和sle3基因座。这些易感基因在促进具有带电或疏水的HCDR3间期的B细胞发育中的作用将被确定。为了确定谱系的改变是否会影响小鼠对正常刺激的反应能力，将用T非依赖和T依赖的抗原以及Strep挑战小鼠。肺炎和流感病毒。最后，我们将研究带有带电或疏水的HCDR3间期抗体的常见和早期表达在自身免疫性疾病发展中的作用。这些研究应该证实或平息这样一种理论，即在抗体库中使用替代的水解酶读框可以促进自身抗体的产生。如果如我们预期的那样，答案是肯定的，那么拟议的实验将进一步帮助我们理解sle1、sle2和sle3在撤销异常抗体库的正常调节方面可能发挥的作用。

The third complementarity determining region of the immunoglobulin heavy chain-HCDR3-lies at the center of the antigen-binding site and typically determines antibody specificity. The DH gene segments that compose the core of HCDR3 are potentially translatable in six reading frames, each with a characteristic hydropathicity signature-charged, hydrophobic, or hydrophilic. However, in virtually all species the final HCDR3 repertoire is enriched for neutral, hydrophilic sequence. Charged or hydrophobic HCDR3 intervals are common only in exceptional circumstances (e.g. pathogenic anti-DNA antibodies). Our hypothesis holds that charged or hydrophobic HCDR3 domains are "forbidden" because they are more likely to generate self- reactive antibodies and thus help "trigger" autoimmune disease in susceptible organisms. Through gene targeting, we are generating mice wherein the antibody repertoire has been globally altered to produce HCDR3 intervals encoded by either charged or hydrophobic DH reading frames. These altered repertoires contain native charged or hydrophobic HCDR3 sequence that, although rare in typical antibodies, is more common in some autoimmune prone strains of mice. Preliminary studies indicate that B cells expressing antibodies with charged HCDR3 intervals can be generated in high numbers, but are selected against in the periphery. We propose that this peripheral loss of B cells is due to self-reactivity. Mice that bear genes that reduce the barrier to the formation of autoantibodies may enable B cells expressing antibodies with altered HCDR3 hydropathicity to survive. Antibodies with charged or hydrophobic HCDR3 intervals are proposed to be more likely to "trigger" autoimmune disease. In order to test this hypothesis, we will introduce mutant DH loci into mice bearing defined susceptibility loci for autoimmune disease, e.g. C57BL/6 bearing the NZM derived sle1, sle2, and sle3 loci. The role of these susceptibility loci in facilitating the development of B cells with charged or hydrophobic HCDR3 intervals will be determined. To determine whether alteration of the repertoire can affect the ability of the mice to respond to normal stimuli, the mice will be challenged with T-independent and T-dependent antigens, and with Strep. pneumoniae and influenza virus. Finally, the role of common and early expression of antibodies bearing charged or hydrophobic HCDR3 intervals on the development of autoimmune disease will be examined. These studies should either confirm or lay to rest the theory that use of alternative DH reading frames in the antibody repertoire promotes the production of autoantibodies. If, as we expect, the answer is yes; then the proposed experiments have the further potential of helping us understand the role that sle1, sle2, and sle3 may have in voiding normal regulation of abnormal antibody repertoires.