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. ? ? ?
