Central to defining the repertoire of in vivo T cell responses to antigen is the selection of a limited repertoire of peptide epitopes from a pool of potential peptides generated by proteolytic degradation of antigens in antigen presenting cells. Understanding the mechanisms that define this hierarchical choice, termed immunodominance, would be greatly facilitated by cell-free systems that viably recapitulate the in vivo peptide repertoire. We report the first in vitro reconstitution of MHC class II antigen processing consisting of only four purified components that mimics cellular processing and yields physiologically processed immunodominant epitopes as defined by mass spectrometry. Purified MHC II, two lysosomal proteases cathepsin B and cathepsin H, and HLA-DM are necessary and sufficient to generate the correct repertoire of immunodominant peptides. This system was used to demonstrate that (1) the survival of MHC class II epitopes depends on its preemptive capture by MHC II prior to the exposure of antigen to lysosomal proteases, and that (2) lysosomal proteases and HLA-DM cooperatively restrict the repertoire of peptides captured by MHC class II, thereby enhancing the establishment of immunodominance. In addition, we demonstrate that small-molecule reducing agents that disrupt disulfide bonds enhance peptide binding by MHC class II.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 68/05, Dissertation Abstracts International|
|Subjects:||Molecular biology, Immunology|
|Keywords:||Antigen processing, Immunodominance, MHC class II|
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