IgVH genes from different anatomical regions, with different histopathological patterns, of a rheumatoid arthritis patient suggest cyclic re-entry of mature synovial B-cells in the hypermutation process

INTRODUCTION: Although IgV genes in rheumatoid B cells have been intensively analyzed, many questions concerning antigen driven B-cell maturation and recirculation remain unanswered. It would be interesting to know whether B-cell maturation in rheumatoid tissue is different from that in secondary lymphatic organs. Moreover, it would be interesting to know whether there exists a restricted number of antigens that act on the lesions of different anatomical sites of the RA patient, and whether B cells recirculate between the different joints. METHODS: RNA and genomic DNA were prepared from tissue sections from three different anatomical sites, with different histopathologies and different onsets (left and right peroneal tendons and cubita synovial membrane), of a RA patient. Genomic DNA was amplified by seminested polymerase chain reaction (PCR), and the cDNA corresponding to the RNA was amplified by PCR using primers specific for each IgVH family. The obtained sequences were compared with their germline counterparts on the V-Base data Bank [1]. An immunohisto-chemical analysis of the infiltrate and the clinical data of local disease activity were also included. RESULTS: In the locations with longer disease duration (right peroneal tendon 5 months, left peroneal tendon 2 months) a very intense inflammatory infiltrate with germinal centers containing Ki-M4-positive follicular dendritic cells (FDC) was observed. In the location with shorter disease duration (right cubita 2 weeks) a low, diffuse and nonfollicular infiltration with marked oedema was detected. From the 55 analyzed clones seven expressed nonfunctional rearrangements (pseudogenes) with stop codons, and 48 were found to express functional genes. Among the 48 clones that expressed functional genes, there were two that had amino acid deletions on their complementarity determining region (CDR)2 - clones K194/1 and K194/111 - similar to the ones described by Wilson et al [2] and Goossens et al [3]. Two types of mixed molecules were found. Mixed molecules of the first type (k194/57, k194/67 and k194/109) are composed of rearrangements of two different IgV genes. Mixed molecules of the second type (k194/126, k194/119, k194/30 and k194/99) are composed of a IgV gene rearrangement that is fragmented by insertions of small random sequences. These insertions are different from the ones described by Wilson et al [2] since they are not duplicates or parts of IgV genes. The ratio of replacement mutations to silent mutations (R/S ratio) increased with disease duration. There was strong heterogeneity among the CDR3 segments. The amino acid sequences that belonged to the VH1-family obtained from the three anatomical regions were primarily compared with the amino acid sequences of their closest germline counterparts (Fig. 1a). One result from this comparison was the heterogeneity in the CDR3 rearrangements. Moreover, sequences k194/58 and k194/82 are clonally related (confirmed at nucleotide level). Then, the 21 amino acid sequences were compared with the most widely used germline counterpart IgHV1-18(*)01 (Fig. 1b). All of these VH1 sequences had mainly conservative mutations in the framework region (FR) and nonconservative mutations in the CDR. Also, there was an almost overall conservation of the mutational cold spots and 'structural cold spots' [4] among the 19 VH1 segments. The replacement (11 from 19 replacements resulted in a proline residue) in position 34 of CDR2 could be interpreted as an antigen-selected mutational hotspot. The comparison of the five sequences belonging to IgHV4-30-1/4-31(*)02 resulted in two types of clonal relation (Fig. 2a). The first type of clonal relation, between sequences k194/100 and k194/101 (Fig. 2b), suggests that both sequences are derived from a single progenitor cell. The second type of clonal relation is between sequences k194/23, k194/102 and k194/103 (Fig. 2c). It suggests that an unmutated progenitor cell gave rise to k194/23 (left peroneal tendon), from which k194/103 (right cubita) derived and later generated k194/102 (right cubita). DISCUSSION: The analysis of the 55IgVH sequences corroborates the findings of other groups that studied a singlelocation and RA B-cell hybridomas [5,6,7,8,9,10] and adds further information on B-cell distribution and activation in RA. First, amino acid deletions and mixed molecules could be interpreted as novel pathways to generate antibody specificities, leading, for instance, to autoreactive antibodies that could contribute to the local and systemic tissue destruction. Second, an apparently conserved mutational pattern among the 19 amino acid VH1 segments suggests that in all three RA lesions of this patient the synovial B cells are dealing with a restricted number of antigens. Third, the existence of clonally related B cells in the cubita and left peroneal tendon leaves no doubt that in this patient there is a cyclic re-entry of mutated B cells in the hypermutation process [11]. The already mutated B cells from the early RA lesions sequentially colonize new germinal centers in secondary lymphatic organs as proposed by Kepler et al [12]. These reactivated B-cells then invade new anatomical regions, leading to the perpetuation of the chronic inflammation in RA.