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Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells.

ABSTRACT: OBJECTIVE:To investigate autoantigens in ?-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9. RESEARCH DESIGN AND METHODS:To purify ?-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of ?-cell tumors. The presence of antigen was monitored by measuring the interferon-? production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice. RESULTS:Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9. CONCLUSIONS:IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.

SUBMITTER: Delong T

PROVIDER: S-EPMC3161333 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells.

Delong Thomas T Baker Rocky L RL Reisdorph Nichole N Reisdorph Richard R Powell Roger L RL Armstrong Michael M Barbour Gene G Bradley Brenda B Haskins Kathryn K

Diabetes 20110706 9

<h4>Objective</h4>To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9.<h4>Research design and methods</h4>To purify β-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of β-cell tumors. The pr ...[more]

PMID: 21734016

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Project description:Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

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Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells.

Publications

Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells.

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