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

H. Ikematsu, Y. Ichiyoshi, E. W. Schettino, M. Nakamura, Paolo Casali

Research output: Contribution to journalArticle

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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 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(H)III 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(H)I family). All three IgG mAb used VκIII 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 x 10-2 and 0.8 x 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.

Original languageEnglish (US)
Pages (from-to)1430-1441
Number of pages12
JournalJournal of Immunology
Volume152
Issue number3
StatePublished - Jan 1 1994
Externally publishedYes

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Type 1 Diabetes Mellitus
Autoantibodies
Insulin
Genes
Immunoglobulin G
Immunoglobulin Genes
B-Lymphocytes
Point Mutation
Germ Cells
anti-IgG
Mutation
Nucleotides
DNA
Southern Blotting
Autoimmunity
Amino Acids

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

V(H) and Vκ segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus : Evidence for somatic selection. / Ikematsu, H.; Ichiyoshi, Y.; Schettino, E. W.; Nakamura, M.; Casali, Paolo.

In: Journal of Immunology, Vol. 152, No. 3, 01.01.1994, p. 1430-1441.

Research output: Contribution to journalArticle

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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 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(H)III 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(H)I family). All three IgG mAb used VκIII 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 x 10-2 and 0.8 x 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.",
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T1 - V(H) and Vκ segment structure of anti-insulin IgG autoantibodies in patients with insulin-dependent diabetes mellitus

T2 - Evidence for somatic selection

AU - Ikematsu, H.

AU - Ichiyoshi, Y.

AU - Schettino, E. W.

AU - Nakamura, M.

AU - Casali, Paolo

PY - 1994/1/1

Y1 - 1994/1/1

N2 - 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(H)III 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(H)I family). All three IgG mAb used VκIII 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 x 10-2 and 0.8 x 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.

AB - 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(H)III 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(H)I family). All three IgG mAb used VκIII 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 x 10-2 and 0.8 x 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.

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