V_H and Vκ segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus: Evidence for somatic selection

In some patients with insulin-dependent (type I) diabetes mellitus (IDDM), autoantibodies to insulin are present at diagnosis. After initiation of the treatment with not only animal but also human insulin, anti-insulin, mainly IgG, autoantibodies become a major component of the autoimmune response in virtually all IDDM patients. Their structure, however, is still relatively unknown. We analyzed the structure of the V_H and Vκ segments of three human IgG mAb derived from three IDDM patients. The sequences of V_H genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (V_HIII family), respectively. The sequence of the V_H gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (V_HI family). All three IgG mAb used VKIII segments. The VκIII gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 Vκ gene sequence was 96.5% identical to that of the kv328 gene. The V_H and/or Vκ segments of these anti-insulin IgG mAb are similar to Ig V genes expressed in the fetal, and adult normal and autoimmune B cell repertoires. The nucleotide differences displayed by the three anti-insulin IgG mAb V_H gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 × 10^-2 and 0.8 × 10^-2 difference/base in CDR and FR, respectively; p < 0.01, χ² test), and yielded a significantly higher putative replacement (R) to silent (S) mutation ratio in the CDR (12.0) than in the framework (0.2). The concentration of nucleotide differences in the CDR and their high R:S putative mutation ratios were consistent with the hypothesis that these expressed V_H genes underwent a process of somatic mutation and Ag-driven clonal selection. That such differences constituted somatic point-mutations was formally proved in IgG mAb13, by differentially targeted PCR amplification and Southern blot hybridization of the mAb13-producing cell line DNA. The putative germ-line gene that gave rise to the expressed V_H segment was cloned using genomic DNA from PMN of the same patient whose B cells were used for the generation of this mAb. Overall, in the anti-insulin IgG mAb V_H and VκIII genes, the (putative and verified) somatic point-mutations yielded 27 amino acid replacements, of which 14 nonconserved. Four of these resulted in positively charged residues, three Arg and one His. Additional two single and three tandem Arg residues were present in the H chain CDR3 of the mAb13 and mAb48, respectively. Thus, the rearrangement of Ig V genes that are commonly expressed in the human B cell repertoire, in conjunction with a process of somatic mutation and Ag-driven clonal selection can underlie the emergence of high affinity anti-insulin IgG autoantibodies in IDDM patients.