GAS-Luc2 reporter cell lines exhibit enhanced performance compared to the industry-standard interferon-gamma ELISA for immune activation studies and CAR-T evaluation

Background: Interferon-gamma (IFN-γ) is a key cytokine involved in the activation of cellular immunity and the promotion of anti-tumor responses, and it serves as a critical biomarker for evaluating the efficacy of cancer immunotherapies. In CAR-T cell therapy research and development, testing for IFN-gamma (IFN-γ) expression is a crucial aspect, particularly in assessing the potency and safety profile of the CAR-T cells. Although enzyme-linked immunosorbent assay (ELISA) is commonly used to detect IFN-γ, it has limited sensitivity in detecting low levels of the cytokine during the early stages of immune activation. In addition, ELISA is not well-suited for accurately quantifying paracrine IFN-γ signaling in three-dimensional (3-D) co-culture models.

Methods: To overcome these limitations and meet the growing need for effective immune activation monitoring in immunotherapy research, we developed several IFN-γ reporter cancer cell lines. These were engineered with a gamma-interferon activation site (GAS) upstream of a luciferase reporter gene. Upon activation of IFN-γ signaling, the cells express luciferase, enabling direct and quantifiable readout of immune activation. Three of the cell lines were selected through comprehensive protein expression profiling to ensure endogenous expression of immune checkpoint ligands such as PD-L1, CD155, and B7-H3, expanding their utility for immune checkpoint studies. Additional one was derived from the commonly used monocyte cell line THP-1, which recapitulates the function of monocyte and can be further differentiated into macrophage. 

Results: To validate the system, reporter cells were exposed to graded doses of IFN-γ, treated with conditioned media from activated primary T cells, or co-cultured with IFN-γ-producing primary immune cells. They were also tested in both 2-D and 3-D co-culture formats alongside ELISA for comparison. Upon IFN-γ stimulation, the reporter cells exhibited a dose-dependent increase in bioluminescence intensity of approximately 100- to 250-fold. Conditioned media from T cells induced a 50- to 100-fold signal increase, while co-cultures with primary T or NK cells in the presence of immune checkpoint inhibitors produced 3- to 12-fold signal enhancements. Importantly, the reporter cells generated robust bioluminescent signals in both 2-D and 3-D systems, even at IFN-γ concentrations undetectable by ELISA, underscoring their superior sensitivity and adaptability. Testing for INF-γ expressions of Car-T cells were also conducted and compared between ELISA and reporter cell system in this study. 

Conclusions: These reporter cell lines offer a sensitive, reliable, and user-friendly platform for early immune activation assessment and represent a valuable tool for evaluating cancer immunotherapy candidates.