Guo J, Pichurin P, Nagayama Y, Rapoport B, McLachlan S M
Autoimmune Disease Unit, Cedars-Sinai Research Institute and UCLA School of Medicine, Los Angeles, CA, USA.
Clin Exp Immunol. 2003 Jun;132(3):408-15. doi: 10.1046/j.1365-2249.2003.02170.x.
Plasmid and adenoviral vectors have been used to generate antibodies in mice that resemble human autoantibodies to the thyrotrophin receptor. No such studies, however, have been performed for thyroid peroxidase (TPO), the major autoantigen in human thyroiditis. We constructed plasmid and adenovirus vectors for in vivo expression of TPO. BALB/c mice were immunized directly by intramuscular injection of TPO-plasmid or TPO-adenovirus, as well as by subcutaneous injection of dendritic cells (DC) infected previously with TPO-adenovirus. Intramuscular TPO-adenovirus induced the highest, and TPO-plasmid the lowest, TPO antibody titres. Mice injected with TPO-transfected DC developed intermediate levels. Antibodies generated by all three approaches had similar affinities (Kd approximately 10(-9)M) and recognized TPO expressed on the cell-surface. Their epitopes were analysed in competition assays using monoclonal human autoantibodies that define the TPO immunodominant region (IDR) recognized by patients with thyroid autoimmune disease. Surprisingly, high titre antibodies generated using adenovirus interacted with diverse TPO epitopes largely outside the IDR, whereas low titre antibodies induced by DNA-plasmid recognized restricted epitopes in the IDR. This inverse relationship between antibody titre and restriction to the IDR is likely to be due to epitope spreading following strong antigenic stimulation provided by the adenovirus vector. However, TPO antibody epitope spreading does not occur in Hashimoto's thyroiditis, despite high autoantibody levels. Consequently, these data support the concept that in human thyroid autoimmunity, factors besides titre must play a role in shaping an autoantibody epitopic profile.
质粒和腺病毒载体已被用于在小鼠体内产生类似于人类促甲状腺素受体自身抗体的抗体。然而，尚未针对人类甲状腺炎的主要自身抗原甲状腺过氧化物酶（TPO）进行此类研究。我们构建了用于TPO体内表达的质粒和腺病毒载体。通过肌肉注射TPO-质粒或TPO-腺病毒，以及皮下注射先前感染TPO-腺病毒的树突状细胞（DC），直接免疫BALB/c小鼠。肌肉注射TPO-腺病毒诱导的TPO抗体滴度最高，而TPO-质粒诱导的最低。注射TPO转染DC的小鼠产生中等水平的抗体。通过所有三种方法产生的抗体具有相似的亲和力（解离常数约为10^(-9)M），并识别细胞表面表达的TPO。使用定义甲状腺自身免疫疾病患者识别的TPO免疫显性区域（IDR）的单克隆人类自身抗体，在竞争试验中分析了它们的表位。令人惊讶的是，使用腺病毒产生的高滴度抗体与IDR之外的多种TPO表位相互作用，而DNA-质粒诱导的低滴度抗体识别IDR中的受限表位。抗体滴度与对IDR的限制性之间的这种反比关系可能是由于腺病毒载体提供的强烈抗原刺激后表位扩展所致。然而，尽管自身抗体水平很高，但在桥本甲状腺炎中不会发生TPO抗体表位扩展。因此，这些数据支持这样的概念，即在人类甲状腺自身免疫中，除滴度外的因素必须在塑造自身抗体表位谱中发挥作用。
