High Throughput Flow Screening Assays to Profile Cell-Mediated Killing

Abstract

Abstract While enhancing T-cell mediated killing of tumor cells is emerging as a successful therapeutic approach for a variety of cancers, improvements to these therapies are actively being sought. Traditional assays for monitoring cell-mediated killing are only capable of homogenous live/dead readouts for an entire sample. As an alternative platform for cell-mediated killing studies, IntelliCyt’s iQue Screener can identify multiple cell types in suspension and report multiple cell killing readouts in streamlined no wash assay formats. We demonstrate two example high throughput assays for cell-mediated killing using NK cells and chimeric antigen receptor (CAR) T-cells. Using the NK92 cell line as an effector cell and fluorescently encoded Jurkat cells as target cells, viability and Caspase 3 activation was determined for both Jurkat and NK92 cells in the same sample, and compounds that were generally cytotoxic to both cells could be identified. Specificity of the cell-mediated killing response was demonstrated using known signal transduction inhibitors including sunitinib, U73122, pp2, and wortmannin that would attenuate the NK cell killing activity, at a fixed target to effector cell ratio. In the CAR T-cell assay, efficacy of different CARs at targeting and killing a B-cell line (NALM-6) was profiled using multiplex readouts for cell health and secreted cytokines. Multiple cytokines including inflammatory markers and Granzyme B were quantified using bead-based ELISA on the same analysis platform. These application examples highlight the robustness and flexibility of the iQue Screener for performing multiplexed screening assays with cells and beads.