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

We report a strategy for high through-put sequence analyses of large MHC cl ass Ii-bound peptide repertoires which combines automated electrospray ioni zation tandem mass-spectrometry with computer-assisted interpretation of th e tandem mass spectra using the algorithm SEQUEST. This powerful approach d iscerned 128 peptide sequences displayed by the murine MHC class II molecul e I-A(b) in activated B cells and macrophages, including a surprisingly lar ge number of peptides derived from self cytosolic proteins. Mice lacking th e chaperone molecule H-2M were used to generate T cells specific for select ed self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are effic iently presented by splenic and thymic dendritic cells, but less so by rest ing B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class Ii-bound self peptides derive d from cytosolic proteins exists in vivo.