Journal
JOURNAL OF AUTOIMMUNITY
Volume 37, Issue 3, Pages 151-159Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jaut.2011.05.012
Keywords
Preproinsulin; CD8 T-cell; Type 1 diabetes; Autoreactive; Immunotherapy
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Funding
- JDRF [7-2005-877]
- Dutch Diabetes Research Foundation DFN
- UK Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre
- NHS Foundation Trust in partnership with King's College London
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Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI4-13/B7, PPI29-38/A2, PPI76-84/A3 and PPI79-88/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes. (C) 2011 Elsevier Ltd. All rights reserved.
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