Structural analysis of the V(H)-D-J(H) segments of human polyreactive IgG mAb: Evidence for somatic selection

H. Ikematsu, M. T. Kasaian, E. W. Schettino, P. Casali

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Abstract

Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the V(H) regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound with various efficiencies a number of different self and foreign Ag. Gene cloning experiments showed that the V(H) region sequences were unique to each IgG mAb. The H chain complementary determining region (CDR3) of two IgG (mAb10 and mAb426.4.2F20) displayed an identical stretch of five amino acids (RFLEW), but the other three IgG mAb CDR3 were divergent in both length and composition. The V(H) gene sequences of two IgG, mAb426.4.2F20 and mAb410.7.F91, were 99% identical to those of the germ line V(H)4.11 and V(H)4.21 genes, respectively. Those of the remaining three IgG mAb displayed a number of differences (93.6 to 95.9% identity) when compared with the germ line V(H)4.18, V(H)4.11, and hv1263 gene sequences. These and the V(H)4.21 gene have been found to encode polyreactive IgM and IgA and, in mutated configuration monoreactive high affinity autoantibodies and antibodies induced by foreign Ag. When compared with the respective framework region, the CDR of three IgG mAb V(H) segment sequences displayed a significantly higher: 1) frequency of total nucleotide differences (6.1 x 10-2 vs 4.5 x 10-2 difference/base); 2) frequency of putative nucleotide changes yielding amino acid replacements (5.6 x 10-2 vs 1.4 x 10-2 replacement change/base); and 3) ratio of overall putative replacement to silent (R:S) mutations (11.0 vs 0.4). Thus, the distribution and nature of the nucleotide differences were consistent with a process of somatic mutation and Ag- dependent clonal selection. This was formally proved in IgG mAb426.12.3F1.4 and IgG mAb10 by differentially targeted polymerase chain reaction amplification and cloning and sequencing of the germ line genes that gave rise to the expressed V(H) segments, using DNA from polymorphonuclear cells of the same subjects whose B cells were used for the generation of these IgG mAb. Somatic mutations might have been responsible for bringing about polyreactivity in originally monoreactive antibodies or, more likely, they accumulated in originally polyreactive antibodies, which after undergoing a process of Ag selection, retained polyreactivity and may have or may have not acquired a higher affinity for the selecting Ag.

Original languageEnglish (US)
Pages (from-to)3604-3616
Number of pages13
JournalJournal of Immunology
Volume151
Issue number7
StatePublished - Jan 1 1993

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ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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