Immune signaling reporter cell lines enable quantitative monitoring of crosstalk among cancer, innate, and adaptive immune cells in tumor microenvironment model

Background: T cell-targeted immunotherapies have led to major clinical gains, yet many patients fail to respond or develop resistance due to the immunosuppressive tumor microenvironment (TME). Increasing evidence shows that B cells and myeloid cells also influence antitumor immunity, but accessible models capable of capturing interactions among cancer cells and multiple immune cell types remain limited. To address this gap, we developed immune signaling reporter cell lines that allow real-time, quantitative monitoring of NFAT- and NF-κB-driven activation pathways. These models enable capturing dynamic interactions among multiple immune cell lineages and cancer cells relevant to immunotherapy response. Methods: Six luciferase-based reporter lines derived from T cells, B cells, or myeloid cells were engineered with NFAT or NF-κB response elements driving luciferase expression. The models retain high endogenous expression of checkpoint receptors, including PD-1, TIGIT, and GITR in T cell reporters and SIRPalpha, Siglec-10, LILRB1, and B7-1 in myeloid reporters. Reporter activation was assessed following pathway-specific stimulation: PMA and ionomycin for NFAT and TNF-alpha or T cell-conditioned media for NF-κB. The B cell NF-κB reporter with elevated basal activity was further tested with an NF-κB inhibitor. In addition, all reporters were evaluated in co-culture with primary immune and cancer cells. Results: Stimuli activating NFAT or NF-κB signaling produced strong, dose-dependent increases in luciferase activity, while pathway inhibition reduced signal as expected. Co-culture with primary immune and cancer cells generated diverse activation patterns, reflecting context-dependent signaling shaped by interactions within the TME. Conclusions: These reporter cell lines provide a scalable platform for monitoring NFAT- and NF-κB-driven immune activation across T, B, and myeloid lineages. They support sensitive, reproducible evaluation of immune responses, enable mechanistic studies of dynamic immune crosstalk, and evaluation of combinatorial immunotherapy strategies within the TME.