Novel plate-based detection method for T cell activation/proliferation, migration, and cytotoxicity assay using image cytometry

Abstract

Abstract The field of cancer immunotherapy has grew significantly due to numerous successful trials in cancer vaccines, antibody-based treatments, CAR T cell therapy, and other immunotherapies. Specifically for T cell-based assays, they have been traditionally analyzed using ELISA or ELISpot to detect specific cytokines concentration for measuring T cell responses. Other methods such as T cell activation/proliferation, migration, and cytotoxicity assays have employed flow cytometry or microscopy to understand the functionality of T cells. In the recent years, image cytometry has been used to develop robust assays to monitor T cell immune response, which is highly critical for immuno-oncology research. In this work, we demonstrate the use of Celigo Image Cytometer to perform high-throughput in vitro T cell-based functional assays. Three direct cell counting methods are developed for label-free T cell activation and proliferation measurement, T cell migration assay, as well as T cell-mediated cytotoxicity. First, primary T cells are seeded into a 96-well plate and stimulated with antibodies or co-culture with cancer cells. The cells are directly counted in the well over 48 hours in bright field images. Next, T cells are seeded in a transwell with chemokines in the bottom wells. As the T cells migrate through and drop to the bottom, the cells are directly counted using bright field or fluorescence with Hoechst staining. Finally, T cell-mediated cytotoxicity assay is performed by co-culturing CD8 T cells with calcein- or fluorescent protein-labeled cancer cells. As target cancer cells are killed, the fluorescence dissipates, and by counting the changes in the number of “live” target cells, the cytotoxicity percentages are measured.