ANALYSIS OF VH GENE REARRANGEMENTS FROM SYNOVIAL B-CELLS OF PATIENTS WITH RHEUMATOID-ARTHRITIS REVEALS INFILTRATION OF THE SYNOVIAL-MEMBRANE BY MEMORY B-CELLS

The VH gene (Variable gene segments of the heavy chain locus) repertoi re can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selec tion by analysis of somatic mutations. Using a polymerase chain reacti on (PCR) it is also possible to analyse these genes in small numbers o f cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the l ocal humoral immune response in RA. Individual B-cell aggregates of sy novial membrane of two patients with RA were isolated by micromanipula tion from microscopic slides. VH-DH-JH (variable, diversity, and joini ng segments of the heavy chain locus) rearrangements in all possible V H-JH combinations were amplified from these B cell foci, cloned and su bjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (ran ge 1.3 - 14.9%) somatic mutations and therefore were derived from anti gen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial mem brane and characterized the synovial membrane as lymphatic tissue wher e secondary immune responses to an as yet unknown antigen take place.