GAS-Luc2 Reporter Cell Lines to Supersede the Industrial Standard of Interferon-Gamma ELISA for ex vivo Immune Activation Studies and Drug Screening

Background

Interferon-gamma (IFN-γ) is a cytokine that plays a major role in activating cellular immunity and promoting anti-tumor immune response. Monitoring the level of IFN-γ has become the industrial standard for evaluation of the efficacy of immunotherapy drugs. However, widely used enzyme-linked immunosorbent assay (ELISA) is not only costly and time-consuming, but also often fails to detect the low level IFN-γ in the early-stage immune activation. Moreover, it is difficult to correctly quantify the paracrine signaling of IFN-γ secreted by T cells in recently popularized 3D co-culture models using ELISA. A novel approach to fulfill the rapidly expanding need of assessing immunotherapy drug candidates and more effective monitoring of immune activation for cancer immunotherapy studies is urgently needed. 

Methods

We engineered three reporter cancer cell lines that contain a gamma interferon activation site (GAS)-response element upstream of luciferase gene. In response to IFN-γ receptor binding and subsequent signaling activation, these cells express luciferase, which can be readily detected and quantified to evaluate the level of immune activation. The cell lines were selected for their endogenous expression of immune checkpoint ligands, PD-L1, CD155, or B7-H3, based on a comprehensive protein profiling. The established reporter cells were stimulated with various concentrations of IFN-γ, incubated with conditioned media from primary T cells, or co-cultured with IFN-γ-producing immune cells in the presence of immune checkpoint inhibitors followed by a luciferase assay to evaluate the system. Then the reporter cells were 2D or 3D co-cultured with primary T cells after which the cells were harvested for luciferase assay and the conditioned media were collected for IFN-γ ELISA for direct comparison of the two assays. 

Results

Our data showed that the bioluminescence intensity from the reporter cells increased approximately 100-250 folds in a dose-dependent manner in response to IFN-γ stimulation. It increased approximately 50-100 folds in response to primary T cell-conditioned media stimulation. In co-culture assays with primary T cells or NK cells in the presence of corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated 3-12-fold increase in bioluminescence intensity. In comparison to ELISA in 2D and 3D co-culture systems, the reporter cells produced robust luminescence signal even when IFN-γ concentrations fell below the assay range of conventional ELISA.

Conclusions

These GAS-Luc2 reporter cell lines demonstrated exceptional assay sensitivity and adaptability in detection of IFN-γ in 2D and 3D cultures.  They provide an excellent tool for early-stage monitoring of immune activation and convenient and sensitive evaluation of immunotherapy drugs.