title: Constitutive and inflammatory immunopeptidome of pancreatic β-cells.
abstract: Type 1 diabetes is characterized by the autoimmune destruction of pancreatic β-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 β-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)₂₀₆₋₂₁₄. We identified >2,000 MHC-bound peptides; 1,100 of these presented by β-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP₂₀₆₋₂₁₄ revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1₃₅₅₋₃₆₃ (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 β-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia. ndudek@unimelb.edu.au
authors.forename: Nadine L
authors.initials: NL
authors.lastname: Dudek
authors.forename: Chor Teck
authors.initials: CT
authors.lastname: Tan
authors.forename: Dhana G
authors.initials: DG
authors.lastname: Gorasia
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Croft
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Illing
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Purcell
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology / Q000378:metabolism*; D005260:Female; D005952:Glucose-6-Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006649:Histocompatibility Antigens / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018836:Inflammation Mediators / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000276:immunology / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism; D053613:Janus Kinase 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013154:Spleen / Q000276:immunology / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013950:Thymus Gland / Q000276:immunology / Q000378:metabolism
chemical_list: D001324:Autoantigens; D006649:Histocompatibility Antigens; D016056:Immunodominant Epitopes; D018836:Inflammation Mediators; D010446:Peptide Fragments; D011506:Proteins; D007371:Interferon-gamma; C507905:Jak1 protein, mouse; D053613:Janus Kinase 1; D005952:Glucose-6-Phosphatase; C117983:G6pc2 protein, mouse