Abstract
We have reported that the IL-2 Luc assay can detect the effects of chemicals on IL-2 promoter activity by using a dual reporter cell line, 2H4 cells that measure IL-2 promoter-driven luciferase activity (IL2LA) and GAPDH promoter-driven luciferase activity (GAPLA). Since the IL-2 Luc assay cannot detect immunosuppressive drugs that are antimitotic towards rapidly proliferating cells, we attempted to establish a new assay to detect these chemicals by taking advantage of the dual reporter cell properties of 2H4 cells. We first determined the optimal incubation time with drugs and the seeding cell density, and confirmed that the change in GAPLA and IL2LA levels reflects the change in cell count and IL-2 production of 2H4 cells after drug treatment. We designed the IL-2 luciferase lymphotoxicity test (IL-2 Luc LTT) to detect the antimitotic effects of chemicals by modifying the protocol and criteria of the IL-2 Luc assay. To determine the performance of the IL-2 Luc LTT and that of the combination of the IL-2 Luc LTT and the IL-2 Luc assay, we examined 46 drugs: 19 immunosuppressive drugs with different mechanisms of action, 12 anti-cancer drugs, and 15 non-immunosuppressive drugs. The performances of the IL-2 Luc LTT, the IL-2 Luc assay and their combination were 43.3%, 61.3%, and 93.3%, respectively, for sensitivity, 84.6%, 53.3%, and 50.0%, respectively, for specificity, and 55.8%, 58.7%, and 79.5%, respectively, for accuracy. These results demonstrated that the combination of these two assays is promising for the detection of immunosuppressive drugs with different mechanisms of action.
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This study was supported by Grants-in-Aid from the Ministry of Economy, Trade and Industry (METI), Japan, the Ministry of Health, Labour and Welfare (MHLW), Japan, and the Japanese Society for Alternatives to Animal Experiments (JSAAE).
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This study was supported by Grants-in-Aid from the Ministry of Economy, Trade and Industry (METI), Japan, the Ministry of Health, Labour and Welfare (MHLW), Japan, and the Japanese Society for Alternatives to Animal Experiments (JSAAE), Japan.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by YK, HT, RA, TT, CF, and SA. YK and HT were equally contributed. The first draft of the manuscript was written by SA and all authors commented on the manuscript. All authors read and approved the final manuscript.
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Kimura, Y., Terui, H., Fujimura, C. et al. Optimization of the IL-2 Luc assay for immunosuppressive drugs: a novel in vitro immunotoxicity test with high sensitivity and predictivity. Arch Toxicol 95, 2755–2768 (2021). https://doi.org/10.1007/s00204-021-03101-4
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DOI: https://doi.org/10.1007/s00204-021-03101-4