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 Vgene 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
Key WordsB-cells plasma cells immunoglobulin genes rheumatoid arthritis synovial tissue germinal center micromanipulation single cell PCR somatic hypermutation
- 17.Zachau. (1996) The Human Immunoglobulin k Genes. The Immunologist 4, 49–54Google Scholar
- 18.Frippiat, J. P., Williams, S. C., Tomlinson, I. M., Cook, G. P., Cherif, D., Le Paslier, D., Collins, J. E., Dunham, I., Winter, G., and Lefranc, M. P. (1995) Organisation of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4, 983–991.PubMedCrossRefGoogle Scholar