High Throughput Flow Screening Assays to Profile Cell-Mediated Killing
Authors: Luu Kim Yen , Duensing Thomas D 
Published: October 2019

Abstract
Cell-mediated killing is an immune response that involves the activation of cells such as phagocytes, natural killer cells (NK), or antigen-specific cytotoxic T-lymphocytes to induce death to pathogenic cells. Enhancing T-cell mediated killing of tumor cells is emerging as a successful therapeutic approach for a variety of cancers. A number of exciting immuno-oncology technologies are being developed, including adoptive cell transfer approaches involving chimeric antigen receptor T- cells (CAR-T), tumor infiltrating lymphocytes (TIL) and natural killer (NK) cells. Biologics and small molecule approaches are also being developed to increase T-cell mediated killing of tumor cells by modulating molecular interactions among immune checkpoints such as PD-1 and CTLA4. While showing great promise, improvements to these therapies are actively being sought, and novel assay technologies aimed at identifying better and safer treatments are needed. Traditional assays for monitoring cell-mediated killing are only capable of homogenous live/dead readouts for an entire sample. Additionally, the gold-standard chromium release assay involves a laborious protocol and radioactive reagents. As an alternative platform for cell-mediated killing studies, IntelliCyt's iQue Screener is a high throughput suspension screening platform based on flow cytometry. The system can identify multiple cell types in suspension and report multiple cell killing readouts, including cell viability and apoptosis in streamlined no wash assay formats. Here we demonstrate two example high throughput flow assays for cell-mediated killing in assay models using NK cells and chimeric antigen receptor (CAR) T-cells. For the NK cell assay, the NK92 cell line was utilized as an effector cell and Jurkat cells were utilized as target cells. By labeling either target or effector cells with a cell encoder dye, both cell populations can be simultaneously monitored. Viability was determined by cell membrane integrity and Caspase 3 activation for both Jurkat and NK92 cells, and compounds that were generally cytotoxic to both NK92 and Jurkat cells could be identified. The 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 second assay model using CAR T-cells, the efficacy of different CARs at targeting and killing a B-cell line (NALM-6) was profiled using multiplex assays for both cell endpoints and secreted cytokine endpoints. In addition to cell encoding and live/dead analysis, the secretion of multiple cytokines including inflammatory markers and Granzyme B were evaluated 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 to provide greater contextual value for cell mediated killing studies.

Disclosures
Luu: IntelliCyt Corp: Employment. Duensing:IntelliCyt Corp: Employment.