EVIDENCE FOR A SELECTED HUMORAL IMMUNE-RESPONSE ENCODED BY V(H)4 FAMILY GENES IN THE SYNOVIAL-MEMBRANE OF A PATIENT WITH RHEUMATOID-ARTHRITIS (RA)

The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mu tated memory B cells with a high proportion of members of the V(H)4 fa mily. In order to characterize further the V(H)4 response in one patie nt, B cell-rich areas from different sections of synovial membrane (SM ) were identified by CD20 staining, isolated by microdissection and po oled, 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 rear ranged V-H genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged V(H)4 gene prod ucts revealed that seven were potentially functional, and all were mut ated with 84-96% homology to known germ-line (gl) genes and V(H)4 gl g enes amplified from the patient's genomic DNA. Analysis of the complem entarity determining region (CDR) 3 revealed that two products represe nted members of one B cell clone which differed by five nucleotide cha nges. Three of the five mutations encoded amino acid replacements in C DRs indicating antigen-driven expansion of one specific clone. Additio nal analyses of 25 members of three B cell clones from isolated aggreg ates showing intraclonal diversity in one of three clones provided fur ther evidence that antigen selection takes place in the SM. Overall, t he pattern of mutations and the replacement to silent (R:S) ratios wer e diverse, with six products indicating antigen selection by their hig h R:S ratios in CDRs. Although DNA analysis does not allow a character ization of antibody specificities, we can conclude from our analysis o f antibody-encoding es that selection by antigen and expansion of spec ific clones occur in the SM against the background of polyclonal activ ation.