Constitutive and Inflammatory Immunopeptidome of Pancreatic β-Cells

  1. Anthony W. Purcell1
  1. 1Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia
  2. 2Department of Immunology and Microbiology, The University of Melbourne, Victoria, Australia
  1. Corresponding author: Anthony W. Purcell, apurcell{at}unimelb.edu.au, or Nadine L. Dudek, ndudek{at}unimelb.edu.au.

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 Kd-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 Kd (∼25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed Kd-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.

Footnotes

  • Received September 22, 2011.
  • Accepted May 17, 2012.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

| Table of Contents

This Article

  1. Diabetes vol. 61 no. 11 3018-3025
  1. Supplementary Data
  2. All Versions of this Article:
    1. db11-1333v1
    2. 61/11/3018 most recent