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

Abstract

Background & Aim The field of cancer immunotherapy has grown significantly due to numerous successful trials in antibody-based treatments, Chimeric Antigen Receptor (CAR) T cell therapy, and cancer vaccines. Traditionally, ELISA or ELISpot are standard assays used to detect specific cytokines concentration for measuring T cell response. In addition to the standard assays, other methods such as T cell activation/proliferation, migration, and cytotoxicity assays are performed to understand the functionality of immune responses. In general, these functional assays are performed using flow cytometry or qualitative analysis by microscopy. In the recent years, image cytometry has been employed to develop robust assays to monitor immune response; the ability to perform high-throughput screening assays for monitoring T cell immune response is highly critical for cancer vaccine research. The Celigo Image Cytometer can perform whole well bright field (BF) and fluorescence (FL) image acquisition and analysis for a 96- or 384-well plate in less than 5 min for BF and 9 min for FL. Furthermore, directly counting cells within each well allows for more accurate representation of T cell function without perturbation. Methods, Results & Conclusion In this work, we demonstrate the use of Celigo Image Cytometer to perform in vitro cell-based assays to monitor immune response for cancer vaccine research. Three novel methods are developed: label-free T cell activation and proliferation measurement, T cell migration assay, and a T cell-mediated cytotoxicity assay using direct cell counting. For T cell activation and proliferation, 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 using the image cytometer in bright field images. To measure T cell migration, T cells are seeded in a transwell plate with chemokines in the bottom well. As the T cells migrate through the transwell and drop to the bottom of the wells, the cells can be directly counted using bright field or fluorescence imaging 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.