Abstract
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.
Original language | English |
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Journal | Journal of Autoimmunity |
Volume | 37 |
Issue number | 3 |
Pages (from-to) | 151-159 |
Number of pages | 9 |
ISSN | 0896-8411 |
DOIs | |
Publication status | Published - 2011 |
Externally published | Yes |
Keywords
- type 1 diabetes Diabetes Mellitus, Insulin-Dependent (MeSH) endocrine disease/pancreas, immune system disease, metabolic disease etiology
- Primates Mammalia Vertebrata Chordata Animalia (Animals, Chordates, Humans, Mammals, Primates, Vertebrates) - Hominidae [86215] human common
- epitopes
- HLA-binding peptides
- preproinsulin 61116-24-3
- proteasome 140879-24-9 EC 3.4.25.1
- 02506, Cytology - Animal
- 02508, Cytology - Human
- 10060, Biochemistry studies - General
- 10802, Enzymes - General and comparative studies: coenzymes
- 13002, Metabolism - General metabolism and metabolic pathways
- 13020, Metabolism - Metabolic disorders
- 15002, Blood - Blood and lymph studies
- 15004, Blood - Blood cell studies
- 17002, Endocrine - General
- 17008, Endocrine - Pancreas
- 34502, Immunology - General and methods
- 34508, Immunology - Immunopathology, tissue immunology
- Human Medicine, Medical Sciences
- beta-cell endocrine system
- blood blood and lymphatics
- cytotoxic CD8 T-cell immune system, blood and lymphatics
- immune cell immune system
- islet grafting therapeutic and prophylactic techniques, clinical techniques
- quantum dot-fluorochrome labeling laboratory techniques
- UV-exchange method laboratory techniques
- Biochemistry and Molecular Biophysics
- Clinical Endocrinology
- Clinical Immunology
- Metabolism