Alternative splicing and differential expression of the islet autoantigen IGRP between pancreas and thymus contributes to immunogenicity of pancreatic islets but not diabetogenicity in humans
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Thymic expression of self-antigens during T-lymphocyte development is believed to be crucial for preventing autoimmunity. It has been suggested that G6PC2, the gene encoding islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), is differentially spliced between pancreatic beta cells and the thymus. This may contribute to incomplete elimination of IGRP-specific T lymphocytes in the thymus, predisposing individuals to type 1 diabetes. We tested whether specific splice variation in islets vs thymus correlates with loss of tolerance to IGRP in type 1 diabetes.
Expression of G6PC2 splice variants was compared among thymus, purified medullary thymic epithelial cells and pancreatic islets by RT-PCR. Differential immunogenicity of IGRP splice variants was tested in patients and healthy individuals for autoantibodies and specific cytotoxic T lymphocytes using radiobinding assays and HLA class I multimers, respectively.
Previously reported G6PC2 splice variants, including full-length G6PC2, were confirmed, albeit that they occurred in both pancreas and thymus, rather than islets alone. Yet, their expression levels were profoundly greater in islets than in thymus. Moreover, three novel G6PC2 variants were discovered that occur in islets only, leading to protein truncations, frame shifts and neo-sequences prone to immunogenicity. However, autoantibodies to novel or known IGRP splice variants did not differ between patients and healthy individuals, and similar frequencies of IGRP-specific cytotoxic T lymphocytes could be detected in both patients with type 1 diabetes and healthy individuals.
We propose that post-transcriptional variation of tissue-specific self-proteins may affect negative thymic selection, although this need not necessarily lead to disease.
KeywordsAlternative splicing Autoantibodies CD8 T cell HLA multimers IGRP Immunopathogenesis Islet autoantigen Thymic selection Tolerance
Cytotoxic T lymphocyte
Islet-specific glucose-6-phosphatase catalytic subunit-related protein
Medullary thymic epithelial cells
Peripheral blood mononuclear cell
We are grateful to P. Goubert and E. Quartier (Brussels Free University and University Hospital, Brussels, Belgium) for technical assistance in performing the radiobinding assays, and to the Belgian Diabetes Registry for providing sera from well-characterised patients with recent-onset type 1 diabetes and controls (list of active BDR members on www.bdronline.be).
This work was funded by grants from the Leiden University Medical Center, the Dutch Diabetes Research Foundation, The Juvenile Diabetes Research Foundation, the 7th Framework Program of the European Commission (FP-7 project no. 241833), the Belgian Fund for Scientific Research (FWO-Vlaanderen projects G.0311.07 and G.0868.11), the research council (OZR) of the Brussels Free University and the Willy Gepts Funds (University Hospital Brussels).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
VMdJ and JRFA contributed to the conception and design, acquisition and interpretation of data and writing and revision of the manuscript. ARvdS, KV and FKG contributed to experimental design, acquisition and interpretation of data and revision of the manuscript. AAVS, MAE, BB and FJTS were responsible for data acquisition and revision of the manuscript. BOR was responsible for the conception of the study, analysis and interpretation of the data, and writing and revision of the manuscript. All authors approved the final version of the article.
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