In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses

• Published in: European journal of immunology 2001-05, Vol.31 (5), p.1485-1494
• Main Authors: Dongre, Ashok R., Kovats, Susan, deRoos, Paul, McCormack, Ashley L., Nakagawa, Terry, Paharkova‐Vatchkova, Vladislava, Eng, Jimmy, Caldwell, Harlan, Yates III, John R., Rudensky, Alexander Y.
• Format: Article
• Language: English
• Subjects: Algorithms; Amino Acid Sequence; Animals; Antigen Presentation - immunology; Antigen-Presenting Cells - immunology; Antigen-Presenting Cells - metabolism; Autoantigens - chemistry; Autoantigens - immunology; Autoantigens - metabolism; Automation - methods; B-Lymphocytes - immunology; B-Lymphocytes - metabolism; Chromatography, High Pressure Liquid; Cytosol - chemistry; Cytosolic peptide; Dendritic Cells - immunology; Dendritic Cells - metabolism; Epithelial Cells - immunology; Epithelial Cells - metabolism; histocompatibility antigen H-2; Histocompatibility Antigens Class II - immunology; Histocompatibility Antigens Class II - metabolism; Hybridomas - immunology; Immune Tolerance - immunology; Macrophages - immunology; Macrophages - metabolism; MHC class II presentation; Mice; Molecular Sequence Data; Protein Structure, Tertiary; Proteins - chemistry; Proteins - immunology; Proteins - metabolism; Spectrometry, Mass, Electrospray Ionization - methods; T-Lymphocytes - immunology; Tandem mass‐spectrometry; Thymus Gland - cytology; Thymus Gland - immunology
• ISSN: 0014-2980; 1521-4141
• DOI: 10.1002/1521-4141(200105)31:5<1485::AID-IMMU1485>3.0.CO;2-A

We report a strategy for high through‐put sequence analyses of large MHC class II‐bound peptide repertoires which combines automated electrospray ionization tandem mass‐spectrometry with computer‐assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I‐Abin activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H‐2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen‐presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II‐bound self peptidesderived from cytosolic proteins exists in vivo.