Presentation of immunodominant peptides is strongly dependent on cathepsin resistance and preliminary cleavage of antigens

Abstract

An understanding of the basis for immunogenicity (the ability of a foreign substance to induce an adaptive immune response) is critical for advancing our knowledge of infectious or autoimmune diseases, as well as contributing to the design of vaccines and biologics. A key step in the initiation of an adaptive immune response is the presentation of pathogen-derived peptides by major histocompatibility class II (MHCII) molecules on the surface of antigen-presenting cells (APC) such as a dendritic cell (DC) to CD4⁺ T cells. Peptides that are superior at eliciting strong T-cell responses are termed immunodominant. The generation and selection of peptides occurs within a system of endosomal compartments that form when a DC takes up the antigen. These compartments become increasingly more acidic and reducing as proteolytic enzymes, MHCII and a peptide binding modulator human leukocyte antigen DM (HLA-DM, DM) are trafficked through to facilitate the degradation of proteins and the loading of peptides onto the MHCII. Although the general aspects of the antigen processing and presentation mechanisms are understood, important details of the endosomal machinery have yet to be determined. These studies focus on the elucidation of the pathways of antigen processing, the contribution of proteolytic cleavage to immunodominance, and the activity of four proteases in the cysteine family of cathepsins. Our findings strongly implied the existence of multiple, parallel pathways within antigen processing. These include the ability of MHCII to capture the native form of hemagglutinin (HA) from influenza A/New Caledonia 20/99 (H1N1), our model antigen, and the production of unique peptidomes through cleavage alone or through cleavage and capture by MHCII, in the presence or absence of DM, at varying pH. I demonstrate that one determinant for immunodominance is peptide resistance to cleavage by cathepsins, although it does not hold true for every immunodominant peptide. I establish that the cleavage of a protein and the generation of peptides available for binding to MHCII is strongly influenced by the pH and reducing potential of the cleavage environment as well as the flexibility of microdomains of the protein itself. Collectively, these results show that the cleavage events of antigen processing are pivotal to the generation of a broad and diverse peptidome available for binding to MHCII, and presentation to T-cells, that may lead to a more robust adaptive immune response.