Ig H and L Chain Variable Region Gene Sequence Analyses of Twelve Synovial Tissue-Derived B Cell Lines Producing IgA, IgG, and IgM Rheumatoid Factors Structure/Function Comparisons of Antigenic Specificity, V Gene Sequence, and IG Isotype

Abstract

In the present study, the complete sequences of the Ig H and L chain variable region genes of twelve RF⁺ B cell lines from two patients with RA were analyzed. Seven of the RF-producing B cells used V_H3 family genes, four used V_H4 genes, and one a V_H1 gene. All but two of the cell lines expressing V_H3 genes utilized different family members; among the V_H4-expressing cells, a more restricted pattern was noted. V_k gene use was restricted to the V_k I and III families; V_λ gene use was more diverse, involving five different families. Computer comparisons of the expressed V_H genes with their presumed germline progenitors indicated significant differences in every instance; eight of the corresponding V_L genes also were significantly different. In many cases, assignment of the germline D segment(s) incorporated into the rearranged V_H genes was impossible. These differences from the germline gene segments indicated the extensive changes induced by rearrangement, enzymatic activities, and somatic mutation.

In hopes of defining a structural reason for the disparate antigen specificities of these cells, the CDR3 amino acid sequences of the multi-vs. the mono-reactive RF-producers were compared. Although CDR3 length was not appreciably different between these two sets of mAb, a greater than two-fold increase in charged amino acids was found in the H chain CDR3 of the multireactive RF. This relationship did not exist for the L chain CDR3.

Thus, these sequence data indicate the use of a broad base of Ig V gene segments that have undergone extensive diversification. Based on the localization of R substitutions in the CDR of most of the V genes studied, the diversification appears to be antigen driven and selected. The significance of these findings for the evolution of these B cell clones into isotype-switched producers that are heterogeneous for antigen specificity (mono-vs. multi-reactivity) is discussed.

Key Words:

• V genes
• nucleic acid sequence
• rheumatoid factor
• synovial tissue
• antigenic specificity
• isotype