Single cell analysis of synovial tissue B-cells.
Mononuclear cells often form highly organized lymphoid structures in the chronically inflamed synovial tissue of patients with rheumatoid arthritis (RA) within which B-cells are activated and may differentiate into effector plasma cells. The analysis of those activated B-cells and the determination of their specificity is of great importance for the understanding of the pathogenesis of RA. Here, we describe a technique that combines histological analysis of synovial tissue with a molecular analysis of the V-gene repertoire at the level of the single B-cell. Immunohistochemical staining of tissue sections allows us to identify the activated B-cells. Those cells are then isolated using a micromanipulator and the rearranged immunoglobulin (Ig) genes amplified, cloned and sequenced. The combination of the V(D)J gene segments and the pattern of somatic mutations in the V-region genes, allows us to identify clonal relationships between the isolated B cells. Once Ig genes for a heavy and a light chain have been isolated from individual B-cells, they can be used to generate recombinant antibodies. These antibodies can be used to determine the antigens which support the activation of B-cells in the inflamed synovial tissue
Kim
Hye-Jung
HJ
Memorial Sloan Kettering Cancer Center, Department of Immunology, New York, NY, USA.
Berek
Claudia
C
D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000473:pathology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000502:physiology; D030561:Databases, Nucleic Acid; D005803:Genes, Immunoglobulin; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013583:Synovial Membrane / Q000166:cytology* / Q000502:physiology