No abstract

SUMMARYThe human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (V H ) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of V H genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the V H composition of peripheral blood B cells was analysed among four RA patients and four age-and sexmatched healthy controls. Usage of individual V H genes (eight V H 3 and three V H 4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these V H genes (estimated by differential hybridization with motifspecific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of V H 3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged V H 4 genes was also observed in these patients. These data suggest that although usage of individual V H genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.

The acquisition of somatic mutations in the rearranged immunoglobulin V regions in B cells occurs within the tightly regulated microenvironment of a germinal centre. The precise mechanism responsible for turning on the mutational process is unknown. To dissect the role of different components of the germinal centre in this mechanism, we have used in vitro cultures of normal human IgD+ peripheral blood B lymphocytes co-cultured with activated CD4+ T cells, or with resting CD4+ T cells, or with CD40 ligand and IL-4. We observed that if the cultures included activated CD4+ T cells, then up to 100% of VH transcripts on day 14 were somatically mutated. Transcripts were found to carry from one to 36 substitutions (median five). In contrast, in the absence of activated T cells, transcripts contained only background levels of somatic mutation irrespective of the presence of resting T cells or CD40 ligand and IL-4. Cell-cell contact was required for mutation because mutations were not detected when B cells were separated from activated T cells by a membrane.