Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.
The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase-like molecules IA-2 and phogrin are major research goals in type 1 diabetes. In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens. Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells. For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens. Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content. These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.
Peakman
M
M
Department of Immunology, Guy's King's and St. Thomas' School of Medicine, London, United Kingdom. mark.peakman@kcl.ac.uk
Tree
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Endl
J
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van Endert
P
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Atkinson
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Roep
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D000818:Animals; D001324:Autoantigens / Q000276:immunology*; D016367:Baculoviridae; D002460:Cell Line; D002999:Clone Cells; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis* / Q000276:immunology*; D004731:Endotoxins / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007328:Insulin / Q000276:immunology / Q000494:pharmacology; D007515:Islets of Langerhans / Q000276:immunology*; D007527:Isoenzymes / Q000276:immunology*; D008213:Lymphocyte Activation / Q000187:drug effects; D011384:Proinsulin / Q000276:immunology*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011994:Recombinant Proteins / Q000276:immunology; D012307:Risk Factors; D012680:Sensitivity and Specificity; D018411:Spodoptera; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D013745:Tetanus Toxoid / Q000276:immunology; D014162:Transfection
D001324:Autoantigens; D004731:Endotoxins; D007328:Insulin; D007527:Isoenzymes; D011994:Recombinant Proteins; D013745:Tetanus Toxoid; D011384:Proinsulin; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2