Development and application of a multiplexable flow cytometry‐based assay to quantify cell‐mediated cytolysis

Abstract

Although target cell cytolysis has been widely employed to describe effector function of cells, cytolysis assays as commonly employed do not generate quantitative data. In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and accurate, and comparable in sensitivity with the standard 51 Cr assay. We report on the potential for this assay to generate quantitative data on the cytolytic activity of both CD8 T and NK cells; describe a relationship between the efficiency of effector cell degranulation and target cell cytolysis throughout a range of E:T ratios, and demonstrate the potential to multiplex with other platforms to obtain broader datasets for the effector phenotype of cells. Appropriate use of this assay will enhance the ability to derive quantitative and integrated correlative datasets from basic, translational, and clinical studies. ' The most commonly employed assay to measure target cell lysis is the 51 Cr release assay (1). Although the 51 Cr release assay has enjoyed wide use and has provided useful data in a large number of clinical and basic research studies, the assay suffers from certain intrinsic limitations. Specifically, (i) the use of a radioactive element ( 51 Cr) with attendant issues of licensing, isotope half-life, storage, disposal, and compliance, (ii) the requirement for metabolic labeling, since metabolically slow cells can label very poorly, and cells at different metabolic states may be differentially resistant to lysis, and (iii) despite the fact that cytolysis is reported in the context of effector:target cell ratios (E:T), the read-out from 51 Cr-based assays is not based on a per-cell measurement but rather on bulk release of radioactivity into the culture medium.

Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor–modified T cells

“…30 In brief, CFSElabeled targets were incubated at the indicated ratios with effector T cells for 4 or 16 hours. Cells were then harvested, and Countbright beads and 7-AAD were added prior to flow cytometric analysis.…”

Enhanced Function of Redirected Human T Cells Expressing Linker for Activation of T Cells That Is Resistant to Ubiquitylation

“…Flow-based cytotoxicity assays were performed as previously described (Cao et al, 2010;Zhao et al, 2010). NY-ESO-1-and CD19-coexpressing target cells were generated by lentiviral transduction Milone et al, 2009).…”

“…The CD107a mobilization assay was performed as described (Cao et al, 2010). One hundred microliters of effector and target cells was plated in 96-well round-bottom plates at a ratio of 1:1, 2:1, or 4:1 (1, 2, or 4 · 10 5 effectors + 10 5 targets).…”

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

“…The limitations of the CRA has inspired several groups to develop flow cytometry-based cytotoxicity (FCC) assays, which incorporate multi-parameter highspeed analysis at the single-cell level (Papadopoulos et al 1994;Kasatori et al 2005;Zimmermann et al 2005;Klöß et al 2007;Cao et al 2010). Flow cytometry allows for the simultaneous measurement of cytotoxic effector cell activation, cell frequency and phenotype, as well as target cell death, in the same sample.…”