Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers

Wendy W. Unger, Jurjen Velthuis, Joana R. F. Abreu, Sandra Laban, Edwin Quinten, Michel G. D. Kester, Sine Reker Hadrup, Arnold H. Bakker, Gaby Duinkerken, Arend Mulder, Kees L. M. C. Franken, Robert Hilbrands, Bart Keymeulen, Mark Peakman, Ferry Ossendorp, Jan Wouter Drijfhout, Ton N. Schumacher, Bart O. Roep

Research output: Contribution to journalJournal articleResearchpeer-review


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 languageEnglish
JournalJournal of Autoimmunity
Issue number3
Pages (from-to)151-159
Number of pages9
Publication statusPublished - 2011
Externally publishedYes


  • 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
  • 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


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