Developing a Robust and High Throughput Assay for Screening Innate Immune Training Molecules

Abstract Innate immune training molecules have several potential applications in the biomedical field. We developed an assay to screen for new by generating HiBiT tagged TNF cells THP1 monocytic cell line through CRISPR knock-in. The tag, 11 amino acid long peptide, was fused C-terminus produce luminescence upon addition of LgBiT substrate. To optimize assay, we first titrated different concentrations multiple TLR ligands including pIC, p(dAdT), PGN, FLGN, LPS, R848, and P3C. P3C induced strongest signal noise (S/N) ratio pool TNF-HiBiT cells. From pool, then single cloned characterized them optimum response. clones D3 D6 had significantly higher S/N compared pools, especially response PGN. a screen, used previously known such as Syk kinase inhibitor, beta-Glucan Rutaecarpine, train clones. found that lower acute activation exhibited more robust phenotype signal. therefore optimal protocol assess trained immunity is use activating (0.1, 0.1, 0.0001 ng/mL P3C, LPS PGN) This may be due saturation TNF-HiBit reporter when are challenged with ligand (10, 100, 10 PGN). In brief, optimized screening novel molecules. work supported Intramural Research Program NIAID, NIH Supported grants from Office AIDS at NIH.