Constitutive and In fl ammatory Immunopeptidome of Pancreatic b-Cells

Type 1 diabetes is characterized by the autoimmune destruction of pancreatic b-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 b-cells, interferon-g (IFN-g)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global LC-MS/MS analysis, the targeted approach of multiplereaction monitoring was used to quantitate the immunodominant K-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 b-cells grown under normal conditions or after exposure to IFN-g. 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-g treatment compared with the simultaneous presentation of the endogenously processed K-restricted peptide Janus kinase-1355–363 (;15,000 copies/cell). We have successfully sequenced peptides from NIT-1 b-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.