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

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

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