Abstract
The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mutated memory B cells with a high proportion of members of the VH4 family. In order to characterize further the VH4 response in one patient, B cell-rich areas from different sections of synovial membrane (SM) were identified by CD20 staining, isolated by microdissection and pooled, in order to analyse highly enriched B cells without selection by in vitro culture procedures. From DNA of about 5 x 10(3) B cells rearranged VH genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged VH4 gene products revealed that seven were potentially functional, and all were mutated with 84-96% homology to known germ-line (gl) genes and VH4 gl genes amplified from the patient's genomic DNA. Analysis of the complementarity determining region (CDR) 3 revealed that two products represented members of one B cell clone which differed by five nucleotide changes. Three of the five mutations encoded amino acid replacements in CDRs indicating antigen-driven expansion of one specific clone. Additional analyses of 25 members of three B cell clones from isolated aggregates showing intraclonal diversity in one of three clones provided further evidence that antigen selection takes place in the SM. Overall, the pattern of mutations and the replacement to silent (R:S) ratios were diverse, with six products indicating antigen selection by their high R:S ratios in CDRs. Although DNA analysis does not allow a characterization of antibody specificities, we can conclude from our analysis does not allow a characterization of antibody specificities, we can conclude from our analysis of antibody-encoding genes that selection by antigen and expansion of specific clones occur in the SM against the background of polyclonal activation.