A Luciferase-Based Co-Culture System for Convenient Monitoring of CD8⁺ T Cell Cytotoxicity in Tumor Immunotherapy

Current methods for assessing CD8 + T cell cytotoxicity in tumor immunotherapy, such as Annexin V/PI staining or lactate dehydrogenase assays, often involve laborious protocols requiring complex workflows. Here, we developed a luciferase-based co-culture system that enables rapid and convenient evaluation of CD8 + T cell-mediated tumor killing through direct supernatant analysis. CD8 + T cells (>85% purity) were isolated from OT-1 mouse spleens and activated with SIINFEKL peptide and IL-2. B16F10 melanoma cells were stably transfected with firefly luciferase (B16F10-FLuc) to allow monitoring of tumor cell viability. In co-culture assays, luminescence signals from supernatants peaked at 3–5 hours and correlated strongly with T cell cytotoxicity. Pretreatment of tumor cells with Atractylenolide I (ATT-I), an MHC-I enhancer, amplified luminescence, validating the system’s sensitivity to immune modulation. This method eliminates cell lysis and centrifugation steps while maintaining high reproducibility. Our platform offers a scalable tool for screening immunotherapeutic agents (e.g., checkpoint inhibitors) and dissecting T cell-tumor interaction mechanisms.