Characterization of B Cells in Dermatomyositis

描述（由申请人提供）：在皮肌炎中，B细胞浸润肌肉组织。这项建议的重点将首次允许检查直接来自皮肌炎患者肌肉组织的浸润B细胞的抗原特异性，而没有先入为主的偏见。该提案概述了皮肌炎免疫生物学研究的分子方法。通过激光捕获显微切割，然后进行PCR扩增和重链和轻链可变结构域测序，从肌肉活检标本中分离单个B细胞。这些技术的结合将导致详细的地形和分子特征的B细胞在皮肌炎。一旦表征了B细胞，将产生大量重组免疫球蛋白，并且将保留天然可变重链和轻链配对，从而允许这些抗体的忠实重建用于抗原筛选。这些抗体将被用来鉴定皮肌炎中驱动体液反应的自身抗原。

DESCRIPTION (provided by applicant): In dermatomyositis, B cells infiltrate the muscle tissue. The focus of this proposal will allow for the first time, examination of the antigen specificity of infiltrating B cells derived directly from muscle tissue of patients with dermatomyositis without preconceived bias. This proposal outlines molecular methods for studying the immunobiology of dermatomyositis. Single B cells will be isolated from muscle biopsy specimens by laser capture microdissection followed by PCR amplification and sequencing of the heavy and light variable domains. The combination of these techniques will lead to detailed topographic and molecular characterization of the B cells in dermatomyositis. Once the B cells have been characterized, large amounts of recombinant immunoglobulin will be generated, and the natural variable heavy and light pairing will be preserved allowing faithful reconstruction of these antibodies for antigen screening. These antibodies will be used to identify autoantigens driving the humoral response in dermatomyositis.