Tertiary Lymphoid Structures

Corsiero, Elisa, Jagemann, Lucas, Bombardieri, Michele, Pitzalis, Costantino, Elisa Corsiero, Lucas Jagemann, Michele Bombardieri, Costantino Pitzalis

Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS+ synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.