title: Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.
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 (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/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.
authors.affiliation: Department of Immunohematology & Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
authors.forename: Wendy W
authors.initials: WW
authors.lastname: Unger
authors.forename: Jurjen
authors.initials: J
authors.lastname: Velthuis
authors.forename: Joana R F
authors.initials: JR
authors.lastname: Abreu
authors.forename: Sandra
authors.initials: S
authors.lastname: Laban
authors.forename: Edwin
authors.initials: E
authors.lastname: Quinten
authors.forename: Michel G D
authors.initials: MG
authors.lastname: Kester
authors.forename: Sine
authors.initials: S
authors.lastname: Reker-Hadrup
authors.forename: Arnold H
authors.initials: AH
authors.lastname: Bakker
authors.forename: Gaby
authors.initials: G
authors.lastname: Duinkerken
authors.forename: Arend
authors.initials: A
authors.lastname: Mulder
authors.forename: Kees L M C
authors.initials: KL
authors.lastname: Franken
authors.forename: Robert
authors.initials: R
authors.lastname: Hilbrands
authors.forename: Bart
authors.initials: B
authors.lastname: Keymeulen
authors.forename: Mark
authors.initials: M
authors.lastname: Peakman
authors.forename: Ferry
authors.initials: F
authors.lastname: Ossendorp
authors.forename: Jan Wouter
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Ton N
authors.initials: TN
authors.lastname: Schumacher
authors.forename: Bart O
authors.initials: BO
authors.lastname: Roep
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D015551:Autoimmunity; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D020650:Combinatorial Chemistry Techniques; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006084:Graft Rejection / Q000175:diagnosis / Q000276:immunology / Q000378:metabolism; D015234:HLA-A Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015793:HLA-B7 Antigen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000637:transplantation; D016381:Islets of Langerhans Transplantation / Q000276:immunology; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D045663:Quantum Dots
chemical_list: D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; D015793:HLA-B7 Antigen; D007328:Insulin; D010455:Peptides; D011498:Protein Precursors; C013737:preproinsulin; D046988:Proteasome Endopeptidase Complex