A cell free antigen processing system provides insights to the mechanism of immunodominance

T cell-mediated responses to protein antigen involve recognition of selected antigenic peptides bound to Major Histocompatibility Complex (MHC) molecules. Proteolytic digestion of protein antigens during antigen processing generates many peptide fragments that can potentially bind to MHC molecules. However, only few selected antigenic epitopes induce T cell responses, defined as "immunodominant". To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of four purified components: antigen, MHC class II, Cathepsins, and HLA-DM (DM). Strikingly, this minimalist system identified the physiologically selected epitopes of two model antigens. By utilizing the cell-free assay, we demonstrate that role of both cathepsins and DM is crucial during immunodominant epitope selections. Most peptides being sensitive to DM and cathepsins were efficiently eliminated first. However, immunodominant epitopes either sensitive to DM but resistant to cathepsin digestion, or DM-resistant but cathepsin-sensitive were successfully selected. Especially, we observed that survival of DM-resistant and cathepsin-sensitive epitopes might require binding of a large protein to MHC class II prior to proteolytic digestion and trimming of antigens into short epitope determinants. In addition, the cell-free assay provides a novel approach for identifying the immunodominant epitopes from protein antigens. Two different novel antigens, Hemagglutinin (HA1) of the A/Vietnam/1203/2004 (H5N1) virus, and a recombinant form of Plasmodium falciparum liver-stage antigen 1 (LSA-NRC) were used to evaluate the assay. HLA-DR1 restricted epitopes from both proteins were identified by mass spectrometry and were tested for their ability to activate T cells in HLA-DR1 transgenic mice. We observed that the identified epitopes were indeed immunodominant as CD4+ T cells from mice immunized with the proteins proliferated and produced cytokines upon re-challenge with the identified epitopes at levels comparable to the whole protein. Establishment of this cell-free antigen processing system provides a powerful tool for gaining insights into antigen recognition by CD4+ T cells and for identification of I cell epitopes for specific MHC class II alleles.