High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines

Abstract

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…HTFC can be performed using living cells, fixed cells, suspension cells, or even adherent cells and bead based assays. Examples for applications are Ab development , toxicology and apoptosis studies , analysis of protein phosphorylation , identification of stem cell expanding compounds , identification of immune enhancing or immune inhibiting compounds , monitoring of chemotherapy side effects , and characterization and cytotoxicity monitoring of engineered T cells . In addition, secreted molecules such as chemokines or cytokines as well as gene expression can be quantified using barcoded bead based multiplex assays .…”

Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators of T Cell Activation

“…This assay could be adapted to measure T cell activation and tumor cell killing simultaneously by barcoding target cells with a cell tracing dye prior to co-culture. A similar assay with CAR-T and tumor cell co-culture was recently reported [22]. Identification of a cluster of 3 kinase inhibitors with similar phenotypic signatures as a well-known myelofibrosis drug ruxolitinib (Jak1/2 inhibitor).…”

A “No‐Touch” Antibody‐Staining Method of Adherent Cells for High‐Throughput Flow Cytometry in 384‐Well Microplate Format for Cell‐Based Drug Library Screening

“…We have adapted a protocol from Kaur and Esau, in which the potential of EDTA as a nonenzymatic cell detachment agent in “no‐touch” HTFC was demonstrated . Using EDTA instead of enzymatic detachment agents bypasses the need of indispensible wash steps to wash away the fetal calf serum and to neutralize the enzymatic activity to decrease cell toxicity and potential antigen loss . Bypassing washing steps not only contributes to the HT format of the protocol, but it also contributes to the cell retrieval by preventing cell loss, which is especially a problem when working with small cell numbers.…”

“…A limited number of studies reports the preparation of adherent cells for HTFC . The published protocols either involve one or more washing steps or use trypsin/EDTA to generate single‐cell suspensions . Washing steps may cause loss of cells, thereby hampering HT screening protocols, whereas trypsin is reported to potentially cause loss of surface antigen expression .…”