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Background Although T cell-targeted immunotherapies have achieved significant clinical success, a considerable proportion of patients either do not respond or eventually relapse, largely due to the immunosuppressive characteristics of the tumor microenvironment. Growing evidence indicates that additional immune cell types, including B cells and myeloid cells, play vital roles in regulating the effectiveness of cancer immunotherapies. Despite this, widely accessible immunological models that accurately recapitulate the complex, multidirectional interactions between cancer cells and both adaptive and innate immune cells remain limited.Methods To address this need, we developed six luciferase-based immune activation reporter cell lines derived from T cells, B cells, or myeloid cells. These lines were engineered to express luciferase under the control of either the nuclear factor of activated T cells (NFAT) or nuclear factor kappa B (NF-κB) signaling pathways. In addition, the cell lines endogenously express high levels of relevant immune checkpoint receptors, including PD-1, TIGIT, and/or GITR for T cells, and SIRPα, Siglec-10, LILRB1, and/or B7-1 for myeloid cells, facilitating their use in immune checkpoint research.Results To validate the reporter functionality, NFAT-luciferase T cell lines were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, while NF-κB-luciferase myeloid lines were activated using tumor necrosis factor-alpha (TNF-α) or T cell-conditioned media. A B cell-derived NF-κB reporter line (BDCM-NFκB-Luc2), characterized by high basal luciferase activity, was treated with an NF-κB pathway inhibitor. These reporter lines were further evaluated in co-culture with primary immune and/or cancer cells to investigate immune cell interactions. Luciferase assays were used for rapid and quantitative assessment of reporter activity under all experimental conditions. Results demonstrated that stimuli activating NFAT or NF-κB pathways led to dose-dependent increases in luciferase expression, while pathway inhibition caused corresponding decreases. Notably, co-culture with various other immune and cancer cell types elevated luminescence signals in different levels indicating that interactions with other cell types in tumor microenvironment lead to varying degrees of signaling activation.Conclusions These newly developed immune reporter cell lines provide a robust and scalable ex vivo platform for the evaluation of cancer immunotherapies. They enable sensitive, reproducible monitoring of the dynamic interplay between cancer cells and components of the innate and adaptive immune systems, offering a powerful tool for assessing combinatorial immune responses within the tumor microenvironment.