GAS-Luc2 reporter cell lines enable sensitive detection of interferon-gamma signaling for immune activation and CAR-T evaluation across 2D and 3D systems

Background: Interferon-gamma (IFN-gamma) is a key cytokine regulating cellular immune activation and is widely used as a functional biomarker in immunotherapy development. Standard ELISA method has limited sensitivity for early, low-level IFN-gamma detection and does not effectively capture paracrine signaling in three-dimensional (3-D) models. To address these limitations, we developed IFN-gamma-responsive luciferase reporter cell lines driven by a gamma-interferon activation site (GAS) promoter. Methods: Three cancer cell lines with high endogenous expression of checkpoint ligands (PD-L1, CD155, B7-H3) were engineered with a GAS-Luc2 construct. An additional GAS-Luc2 reporter cell line was generated from THP-1 cells for monocyte- and macrophage-related applications. Reporter activity was evaluated after stimulation with recombinant IFN-gamma, T cell-conditioned media, and co-culture with primary T or NK cells. Assays were performed in 2-D and 3-D systems and compared with IFN-gamma ELISA. CAR-T cells were also evaluated using both methods. Results: Reporter cells showed strong dose-dependent luciferase expression, with 100- to 250-fold increases after recombinant IFN-gamma treatment and 50- to 100-fold induction with T-cell conditioned media. Co-cultures with primary immune cells produced 3- to 12-fold activation. Reporter signals remained robust in 3-D models and detected IFN-gamma levels below the ELISA detection limit. In CAR-T assays, reporters identified early and low-level cytokine signaling not measurable by ELISA. Conclusions: GAS-Luc2 reporter cell lines provide a sensitive, quantitative, and scalable platform for monitoring IFN-gamma signaling. Their superior performance over ELISA, especially for early activation and 3-D applications, supports their utility in immunotherapy evaluation, immune checkpoint studies, and CAR-T functional assessment.