Characterization of B Cells in Dermatomyositis

描述（由申请人提供）：皮肌炎中，B细胞浸润肌肉组织。本提案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性B细胞的抗原特异性，而没有先入为主的偏见。本文概述了研究皮肌炎免疫生物学的分子方法。单个B细胞将通过激光捕获显微解剖从肌肉活检标本中分离出来，随后进行PCR扩增和重、轻变量域测序。这些技术的结合将导致详细的地形和分子表征的B细胞皮肌炎。一旦B细胞被鉴定出来，就会产生大量的重组免疫球蛋白，并保留自然可变的重、轻配对，从而忠实地重建这些抗体，用于抗原筛选。这些抗体将用于识别驱动皮肌炎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.