Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response.

Abstract

SummaryAlthough much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (KFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG KFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG KF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this KF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM KF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed ILF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with Ll's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG KF must have been driven by the Fc antigen.

Analysis of Vκ genes in rheumatoid arthritis (RA) synovial B lymphocytes provides evidence for both polyclonal activation and antigen-driven selection

“…These observations support our previous studies of V H gene expression within IgM-and IgGproducing synovial hybridomas and studies of synovial RF by other investigators [3,4,34]. The current data disagree with a previous investigation in which a cDNA library established from the synovium of an RA patient was used [20].…”

The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

“…Calculation of the inherent Rf and R:S mutation ratio values for the CDRs and FRs of 12 human germline V L genes [35][36][37][38][39][40][41][42][43][44][45][46] (Table 2), selected by criteria similar to those used for the V H -gene sampling, revealed Rf CDR values that were higher than the expected 0.7452 Rf value (t = 11.50, p = 9.01×10 −8 ) and the respective Rf FR values (t = 10.2, p = 3.0 × 10 −7 ), but lower values overall than those calculated for the V H -segment CDRs (t = 5.45, p = 3.0 × 10 −6 ). The V L -gene Rf FR values did not significantly differ either from the expected value or from the values calculated for the 24 V H genes.…”

Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody

“…The nucleotide sequence of the Kimll.4 VA gene, Humlal 114, suggests that it was derived from the VAI subgroup germline gene, HumlvLl [24] (Fig. 2a).…”