VH and V kappa segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus. Evidence for somatic selection.

Abstract

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 VH and V kappa segments of three human IgG mAb derived from three IDDM patients. The sequences of VH genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (VHIII family), respectively. The sequence of the VH gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (VHI family). All three IgG mAb used V kappa III segments. The V kappa III gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 V kappa gene sequence was 96.5% identical to that of the kv328 gene. The VH and/or V kappa 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 VH gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 x 10(-2) and 0.8 x 10(-2) difference/base in CDR and FR, respectively; p < 0.01, chi 2 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 VH 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 VH 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 VH and V kappa III 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Intrinsic B cell TLR-BCR linked coengagement induces class-switched, hypermutated, neutralizing antibody responses in absence of T cells

“…V1-72DJ H -Cγ3 and V1-72DJ H -Cγ2b transcripts with identical CDR3 and J H sequences allowed for identification of a few dominant and intermediate NP-specific B cell clones together with several small clones and many B cell microclones in NP-LPS-immunized Tcrβ −/− Tcrδ −/− mice. The long IgH CDR3s of these transcripts were evocative of the long IgH CDR3s of the antibodies elicited by naturally occurring antigens in humans (62)(63)(64)(65). In Tcrβ −/− Tcrδ −/− mice, the NP-specific B cell clonal and intraclonal diversification, which led to emergence of high-affinity antibodies, likely reflected a stepwise SHM and clonal selection process similar to that underpinning the maturation of the T-dependent anti-NP V1-72DJ H -Cγ1 response elicited by NP-CGG in C57BL/6 mice.…”

A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

“…To identify individual huB and huT cell clones and analyze huB or huT cell clonal diversity, huB cell V H DJ H Cμ or huT cell VβDJβCβ tran scripts (up to 250,000 sequences) of healthy humans and THX mice were analyzed by Illumina MiSeq amplicon sequencing and segregated based on the same huV H or huVβ gene segment, the same and unique huIgH or huTCRβ CDR3 together with the same huJ H or huVβ sequence [87][88][89][90][91] . Each discrete clone was depicted as an individual rectangle or square (unique color), whose area reflects huB or huT cell clone size, as inferred from the sum of identical huV H DJ H Cμ or huVβDJβCβ transcripts (TreeMaps, Microsoft Excel v16.83 and IMGT/HighVQUEST statistic data).…”

DNA repair mechanisms that promote insertion-deletion events during immunoglobulin gene diversification

“…In addition to indels, many bnAbs have unusual features that are shared with poly- or autoreactive antibodies, such as long complementarity-determining region 3 (CDR3) regions ( 15 , 16 ). Long CDR3s of the Ig heavy ( IgH ) chain have been shown to play a critical role in providing structural correlates of antigen binding in human polyreactive and specific antibodies ( 17 , 18 ). B cells with long CDR3 regions are normally negatively selected by tolerance mechanisms ( 19 ).…”