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

¹ Graduate School, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, P.R. China
² School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, P.R. China

^* dongzhixin1998@163.com
^** ljl180826@163.com
^*** wuhl@sumhs.edu.cn

Abstract

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.