1094 Luciferase reporter cell lines with endogenous T cell or myeloid checkpoint expression enable monitoring of multi-directional crosstalk between immune and cancer cells in tumor microenvironment

<h3>Background</h3> Despite the huge success of immunotherapies targeting T cells, a substantial proportion of patients experience resistance or relapse due to the immunosuppressive nature of tumor microenvironment. Increasing number of studies have indicated that other immune cells, such as B cells and myeloid cells, also play a critical role in determining the overall efficacy of cancer immunotherapeutic. However, there is a definite shortage of broadly accessible immunological models effectively representing the complex multi-directional interaction amongst cancer cells, adaptive immune cells, and innate immune cells. <h3>Methods</h3> To address this need, we selected six human cell lines of T cell, B cell, or myeloid cell origin with high endogenous expression of T cell checkpoint (PD-1, TIGIT, and/or GITR) or myeloid checkpoint (SIRPα, Siglec-10, LILRB1, and/or B7-1) and engineered them to express luciferase reporter protein in response to nuclear factor of activated T cells gene (NFAT) or nuclear factor kappa B (NF-κB) signaling activation, respectively. To assess the robustness of luciferase expression, the established NFAT luciferase cell lines were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin and NF-κB luciferase cell lines were stimulated with tumor necrosis factor alpha (TNF-α) or T cell-conditioned media. One of the NF-κB cell lines of B cell origin with high basal luciferase expression (BDCM-NFκB-Luc2) was treated with a NF-κB signaling inhibitor to observe the change in luciferase expression. The reporter cells were then applied in various co-culture conditions with primary immune cells and/or cancer cells to evaluate the immune crosstalk among different cell types. All treatments and co-cultures were followed by a luciferase assay for convenient quantification of luciferase expression. <h3>Results</h3> The data showed that PMA and ionomycin activation increased luminescence intensity by 20–1,000 folds from NFAT reporter cell lines. TNF-α stimulation also increased luminescence intensity by 20–800 folds in a dose-dependent manner from all NF-κB reporter cell lines but from BDCM-NFκB-Luc2. Incubation with T cell-conditioned media resulted in 10–170-fold increase in luminescence intensity from all NF-κB reporter cell lines except for BDCM-NFκB-Luc2. Treatment of BDCM-NFκB-Luc2 cells with a NF-κB signaling inhibitor yielded 20-fold decrease in luminescence intensity. Further co-culture of the reporter cell lines with various immune cells and/or cancer cells significantly increased luminescence signal intensity by up to 200 folds. <h3>Conclusions</h3> These novel luciferase reporter cell lines with endogenous T cell or myeloid checkpoint expression offer an excellent ex vivo model for studying the complex interactions among cancer cells, innate immune cells, and adaptive immune cells in tumor microenvironment.