Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2

Ananieva-Jordanova, Rossitza; Evans, Michele K.; Nakamatsu, Takashi; Premawardhana, Lakdasa; Sanders, Jane; Powell, Michael J.; Chen, S.; McGrath, Vivienne; Belton, C.; Arnold, Clare; Baker, Stuart; Betterle, Corrado; Zanchetta, R.; Smith, Bernard Rees; Furmaniak, Jadwiga

doi:10.1016/j.jaut.2005.03.003

Cited by 8 publications

(6 citation statements)

References 34 publications

(47 reference statements)

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“…Although the JM and PTP determinants are separated by more than 200 residues in the primary structure of IA-2, these regions may be closely aligned in the 3-dimensional structure, such that B-cell recognition of JM domain epitopes may facilitate presentation of determinants within the PTP domain. Evidence for close structural relationships between the JM and central PTP domain epitopes comes from competitive binding studies of IA-2 antibodies in which mouse monoclonal antibodies directed to JM domain epitopes were shown to block IA-2 binding of the human monoclonal IA-2 autoantibody M13 (42), now known to bind epitopes in the 831-860 region of IA-2 (23).…”

Section: Discussionmentioning

confidence: 99%

HLA-DR4–Associated T and B Cell Responses to Specific Determinants on the IA-2 Autoantigen in Type 1 Diabetes

McLaughlin

Gulati

Richardson

et al. 2014

The Journal of Immunology

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Autoantibodies to IA-2 in Type 1 diabetes are associated with HLA-DR4, suggesting influences of HLA-DR4 restricted T-cells on IA-2-specific B-cell responses. The aim of this study was to investigate possible T-B-cell collaboration by determining whether autoantibodies to IA-2 epitopes are associated with T-cell responses to IA-2 peptides presented by DR4. T-cells secreting the cytokines interferon-γ and interleukin-10 in response to seven peptides known to elicit T-cell responses in Type 1 diabetes were quantified by cytokine ELISPOT in HLA-typed patients characterised for antibodies to IA-2 epitopes. T-cell responses were detected to all peptides tested, but only interleukin-10 responses to 841-860 and 853-872 peptides were associated with DR4. Phenotyping by RT-PCR of FACS-sorted CD45ROhi T-cells secreting interleukin-10 in response to these two peptides indicated that these expressed GATA-3 or T-bet, but not FoxP3, consistent with these being Th2 or Th1 memory T-cells rather than of regulatory phenotype. T-cell responses to the same two peptides were also associated with specific antibodies; those to 841-860 peptide with antibodies to juxtamembrane epitopes, which appear early in pre-diabetes, and those to peptide 853-872 with antibodies to an epitope located in the 831-862 central region of the IA-2 tyrosine phosphatase domain. Antibodies to juxtamembrane and central region constructs were both DR4-associated. This study identifies a region of focus for B- and T-cell responses to IA-2 in HLA-DR4 diabetic patients that may explain HLA- associations of IA-2 autoantibodies and this region may provide a target for future immune intervention to prevent disease.

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Section: Discussionmentioning

confidence: 99%

HLA-DR4–Associated T and B Cell Responses to Specific Determinants on the IA-2 Autoantigen in Type 1 Diabetes

McLaughlin

Gulati

Richardson

et al. 2014

The Journal of Immunology

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“…This finding suggests either that alanine substitution of the lysine at residue 609 disturbs the conformation of the 625–627 region, for example by disrupting ionic interactions, or that the JM region is folded such that residue 609 is brought in close proximity to the 625–627 region and participates directly in antibody binding. Mouse monoclonal antibodies to the IA-2 JM domain having similar binding characteristics to those seen in human type 1 diabetes [ 14 , 15 ], also displayed sensitivity to amino acid substitutions within noncontiguous regions of the JM domain [ 16 ]. Thus, four different mouse monoclonal antibodies to the IA-2 JM domain were all inhibited by alanine substitutions in the cluster 4 amino acids 615, 635 and 636, with each antibody being also affected by different amino acid substitutions elsewhere in the JM domain, similar to the observations with the patient sera.…”

Section: Discussionmentioning

confidence: 99%

Influence of HLA-DR and -DQ alleles on autoantibody recognition of distinct epitopes within the juxtamembrane domain of the IA-2 autoantigen in type 1 diabetes

et al. 2015

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Aims/hypothesisInsulinoma-associated protein 2 (IA-2) is a major target of autoimmunity in type 1 diabetes. When first detected, IA-2-autoantibodies commonly bind epitopes in the juxtamembrane (JM) domain of IA-2 and antibody responses subsequently spread to the tyrosine phosphatase domain. Definition of structures of epitopes in the JM domain, and genetic requirements for autoimmunity to these epitopes, is important for our understanding of initiation and progression of autoimmunity. The aims of this study were to investigate the contribution of individual amino acids in the IA-2 JM domain to antibody binding to these epitopes and the role of HLA genotypes in determining epitope specificity.MethodsRegions of the JM domain recognised by autoantibodies were identified by peptide competition and inhibitory effects of alanine substitutions of residues within the JM region. Antibody binding was determined by radioligand binding assays using sera from patients genotyped for HLA-DRB1 and -DQB1 alleles.ResultsPatients were categorised into two distinct groups of JM antibody reactivity according to peptide inhibition. Inhibition by substitutions of individual amino acids within the JM domain differed between patients, indicating heterogeneity in epitope recognition. Cluster analysis defined six groups of residues having similar inhibitory effects on antibody binding, with three clusters showing differences in patients affected or unaffected by peptide. One cluster demonstrated significant differences in antibody binding between HLA-DRB1*04 and HLA-DRB1*07 patients and within DRB1*04 individuals; antibody recognition of a second cluster depended on expression of HLA-DQB1*0302.Conclusions/interpretationThe results identify amino acids contributing to distinct epitopes on IA-2, with both HLA-DR and HLA-DQ alleles influencing epitope specificity.

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“…We have previously described the isolation and characterization of one human monoclonal autoantibody (hMAb) to IA‐2 (M13) 2 and three hMAbs to GAD 65 (3C3, 4B4, and 5B3) 3 . Respective hybridoma cell lines have been stably expressing high levels of these monoclonal antibodies for several years.…”

Section: Methodsmentioning

confidence: 99%

“…Respective hybridoma cell lines have been stably expressing high levels of these monoclonal antibodies for several years. IA‐2 hMAb M13 is of IgG1 subclass combined with kappa light chain and binds IA‐2 with an affinity of 2 × 10 8 L/mol at 2–8°C 2 . GAD 65 hMAb 3C3 (IgG1/κ) has binding affinity for GAD 65 of 1.3 × 10 10 L/mol; 4B4 GAD 65 hMAb (IgG1/λ) binds GAD 65 with an affinity of 2.2 × 10 9 L/mol; and the GAD 65 binding affinity of 5B3 (IgG1/λ) is 5.8 × 10 9 L/mol 3 .…”

Section: Methodsmentioning

confidence: 99%

“…GAD 65 hMAb 3C3 (IgG1/κ) has binding affinity for GAD 65 of 1.3 × 10 10 L/mol; 4B4 GAD 65 hMAb (IgG1/λ) binds GAD 65 with an affinity of 2.2 × 10 9 L/mol; and the GAD 65 binding affinity of 5B3 (IgG1/λ) is 5.8 × 10 9 L/mol 3 . The IgG preparations of different hMAbs were purified from culture supernatants by Protein A column chromatography (MabSelect™, GE Healthcare UK Ltd, Chalfont St Giles, UK) to >95% homogeneity as assessed by SDS‐PAGE and Coomassie blue staining 2,3 . All four antibodies are available in gram quantities.…”

Section: Methodsmentioning

confidence: 99%

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Preparation and Testing of Diabetes Autoantibody Controls

Brooking¹,

Powell²,

Amoroso³

et al. 2008

Annals of the New York Academy of Sciences

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Stocks of the WHO islet cell antibody, GAD(65) antibody, and IA-2 antibody standard (NIBSC 97/550) are now very limited. We have therefore made and tested a series of control preparations in which human monoclonal autoantibodies to IA-2 and to GAD(65) were diluted in antibody-negative human serum to different concentrations. Three different diabetes autoantibody controls (DAC 1-3) were made as was a negative control preparation. Aliquots containing 1 mL of autoantibodies in serum were freeze-dried. After reconstitution (with 1 mL of water) the controls were tested by (125)I-IA-2 immunoprecipitation assay (IPA), (125)I-GAD(65) IPA, GAD(65) Ab ELISA and IA-2 Ab ELISA (kits from RSR Ltd.) and for ICA by immunofluorescence test (IFT). DAC1 is particularly suitable as a control for the (125)I IA-2 IPA; DAC2 is suitable for the (125)I-GAD(65) IPA, GAD(65) Ab ELISA, and IA-2 Ab ELISA; and DAC3 is suitable for the ICA IFT. Freeze-dried preparations showed good stability at 37 degrees C. Reconstituted liquid preparations were stable when stored at 4 degrees C and at 37 degrees C. Availability of an essentially unlimited supply of these reagents should be useful in establishing reproducible and comparable measurements of diabetes autoantibodies in different laboratories using different assays.

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Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2

Cited by 8 publications

References 34 publications

HLA-DR4–Associated T and B Cell Responses to Specific Determinants on the IA-2 Autoantigen in Type 1 Diabetes

HLA-DR4–Associated T and B Cell Responses to Specific Determinants on the IA-2 Autoantigen in Type 1 Diabetes

Influence of HLA-DR and -DQ alleles on autoantibody recognition of distinct epitopes within the juxtamembrane domain of the IA-2 autoantigen in type 1 diabetes

Preparation and Testing of Diabetes Autoantibody Controls

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