IMMUNOPATHOLOGY OF BETA CELL AUTOANTIGENS

神经内分泌蛋白谷氨酸脱羧酶65(GAD65)和IA-2是超过90%的1型糖尿病患者的靶抗原，但它们在诱发疾病中的作用尚不清楚。这两种蛋白的自身抗体识别天然分子表面的构象表位，在临床发病前几年就已经存在，GAD65特异性T细胞可能在非肥胖糖尿病(NOD)小鼠的糖尿病发病机制中发挥作用。16个人类单抗识别的GAD65蛋白的构象自身反应表位的高分辨率图谱，加上二维和三维结构预测算法以及与已知晶体结构的同源性，已经产生了GAD65二聚体的模型。自体反应表位聚集在表面的亲水性斑块中。有几个表位在DR*0401单倍型的主要T细胞表位中间或附近有接触残基，这表明GAD65通过自身反应性B细胞表面Ig的摄取和呈递可能会影响T细胞表位的呈递。初步实验表明，自身抗体结合可以导致T细胞杂交瘤相邻表位呈递能力提高3倍。还为IA-2生成了一个三维模型，并突出了该分子特有的可能的自体反应表位区域。第一个目标是利用三维结构完成GAD65中自身反应性B细胞表位的精细定位，剖析IA-2中的自身反应性表位，并研究人类疾病表位识别的时间进程。第二个目的是验证一种假设，即自身反应性B细胞的GAD65和IA-2表位特异性影响DR4和DQ8限制性T细胞杂交瘤的抗原提呈。第三个目的是验证这样一个假设，即诱导强大的Th1和CTL主导对GAD65、IA-2和/或第三种自身抗原--小鼠胰岛素B链--的反应，可以导致小鼠的β细胞破坏和糖尿病。我们的长期目标是确定导致人类胰腺β细胞破坏的慢性自身免疫的途径，确定靶抗原在诱导和归巢自身免疫中的作用，并最终利用机制知识设计诊断和预防方法。

The neuroendocrine proteins glutamic acid decarboxylase 65 (GAD65) and IA-2 are target antigens in greater than 90 percent of individuals, who develop Type 1 diabetes but their role in induction of disease is unclear. Autoantibodies to both proteins, recognizing conformational epitopes on the surface of the native molecules, are present up to several years before clinical onset of disease, and GAD65 specific T cells may play a role in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse. High resolution mapping of conformational autoreactive epitopes in the GAD65 protein, recognized by 16 human monoclonal antibodies, together with 2- and 3-dimensional structure prediction algorithms and homology with known crystal structures, has resulted in a model of the GAD65 dimer. The autoreactive epitopes cluster in hydrophilic patches on the surface. Several epitopes have contact residues in the midst of or adjacent to the main T-cell epitopes identified for the DR*0401 haplotype, suggesting the possibility that uptake and presentation of GAD65 via surface Ig on autoreactive B cells can influence the presentation of T-cell epitopes, Preliminary experiments show that autoantibody binding can result in a 3-fold enhancement of the presentation of an adjacent epitope to a T- cell hybridoma. A 3-dimensional model has also been generated for IA-2 and highlights the possible autoreactive epitope areas specific for this molecule. The first aim proposes to use the 3-dimensional structures to complete the fine mapping of autoreactive B cell epitopes in GAD65, dissect the autoreactive epitopes in IA-2, and to study the temporal progression of epitope recognition of human disease. The second aim tests the hypothesis that GAD65 and IA-2 epitope specificity of autoreactive B-cells affects the presentation of antigen to DR4 and DQ8 restricted T-cell hybridomas. The third aim proposes to test the hypothesis that induction of strong Th1 and CTL dominated responses to GAD65, IA-2, and/or a third autoantigen, mouse insulin B-chain, can result in beta-cell destruction and diabetes in the mouse. Our long term goal is to define the pathways to chronic autoimmunity that result in pancreatic beta-cell destruction in man, identify the role of target antigens in induction and homing of autoimmunity, and eventually using the knowledge of mechanisms to design diagnostic and preventive methods.