Measuring the molecular mechanisms of tumor antigen cross-presentation in dendritic cells and macrophages

Abstract

Abstract Objective Antigen cross-presentation is essential for initiation of an adaptive immune response against cancer and pathogens. During this process, dendritic cells (DCs) present peptides derived from ingested antigens in their major histocompatibility complex class I protein (MHC-I) to cytotoxic CD8+T cells. As a result, these CD8+T cells become activated, enabling them to eliminate infected or malignant cells in the human body. Aim of this study is to develop a quantitative method to measure cross-presentation of NY-ESO1, a tumor-specific antigen expressed by several cancer types. Methods DCs were activated and loaded with NY-ESO1-based antigen. To measure cross-priming, DCs were co-cultured with primary naive CD8+T cells. These T cells were transfected with mRNA coding for a specific T cell receptor recognizing an NY-ESO-1-derived epitope. After incubation, the activation of the cytotoxic T cells was measured with ELISA and flow cytometry. In addition, we followed the dynamics of bio-orthogonally tagged NY-ESO1 peptides over time for direct visualization of antigen cross-presentation in dendritic cells and Peripheral Blood Mononuclear Cells (PBMC’s). Results Cross-presentation of bio-orthogonal tagged NY-ESO1 could be followed over time in dendritic cells and PBMC's. Interestingly, in our PBMC screen, not only CD1c dendritic cells but also macrophages are highly able to cross-present ingested peptides. Conclusion This novel method allows to measure presentation of tumor antigens on MHC-I in DCs and primary cells of human blood. Since cross-presentation is crucial for elimination of virus-infected and malignant cells by the immune system, this knowledge could contribute to improvement of DC-based immune therapies.