Characterization of B Cells in Dermatomyositis

描述(申请人提供)：在皮肌炎中，B细胞渗入肌肉组织。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性B细胞的抗原特异性，而不存在先入为主的偏见。这项建议概述了研究皮肌炎免疫生物学的分子方法。通过激光捕获显微切割，然后对重区和轻区进行聚合酶链式反应扩增和测序，将从肌肉活检标本中分离出单个B细胞。这些技术的结合将导致皮肌炎B细胞的详细形态和分子特征。一旦确定了B细胞的特征，就会产生大量的重组免疫球蛋白，自然变量重和轻的配对将被保留下来，从而可以忠实地重建这些抗体，用于抗原筛选。这些抗体将被用来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.