Abstract
Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule.
Highlights
Type 1 diabetes is the result of destruction of the insulin-secreting -cells of the pancreas by a process in which T-cells play a central role
Protein representing the cytoplasmic domain of IA-2 (IA-2ic), the region recognized by antibodies in type 1 diabetes, was purified by avidin affinity chromatography as a fusion protein with a biotinylated Pinpoint purification tag from bacterial extracts
Peripheral blood lymphocytes from 32 type 1 diabetic patients (9 from the Netherlands and 23 from the U.K.) and 10 healthy control subjects
Summary
Type 1 diabetes is the result of destruction of the insulin-secreting -cells of the pancreas by a process in which T-cells play a central role. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule. While antibody responses to islet cell autoantigens have been extensively characterized, relatively little is known about T-cell reactivity to islet components in the disease. More type 1 diabetic patients than healthy control subjects respond to these preparations, providing evidence of a disease-related T-cell response to the autoantigens studied. The aim of this study was to further characterize the T-cell response to IA-2 by stimulating peripheral blood lymphocytes from diabetic and control individuals with recombinant protein representing the cytoplasmic domain of IA-2, followed by expansion of activated T-cells with interleukin-2 to generate T-cell lines. Peptides recognized by T-cell lines were used to further analyze T-cell reactivity in diabetic and nondiabetic populations
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