Research paper
Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis

https://doi.org/10.1016/j.jim.2007.04.009Get rights and content

Abstract

We have developed a three-colour flow cytometric method to assay the contributions of cytotoxicity and phagocytosis to antibody-dependent cell-mediated tumour cell killing. In this assay, tumour target cells are pre-labelled with CFSE, and mixed with effector cells and a tumour antigen-specific monoclonal antibody. After incubation of the cells with the antibody, effector cells are labelled with PE and dead cells with PI. Using flow cytometry, dead and phagocytosed tumour cells can be quickly and easily counted and the numbers summed to determine the total number of killed cells. One can thereby measure the phagocytic aspect of antibody-dependent cell-mediated tumour cell killing, otherwise only revealed by microscopic examination. The failure to detect phagocytosed, in addition to live and dead target cells, by standard assays may result in an underestimation of tumour cell killing and hence the potential of an antibody for immunotherapy of cancer. We illustrate the new method by analysing human monocyte-mediated cytotoxic and phagocytic cell killing of IGROV1 ovarian tumour cells by the ovarian tumour antigen-specific anti-folate binding protein monoclonal antibody, MOv18 IgE.

Introduction

In previous work we demonstrated that IgE might be more effective than IgG for the immunotherapy of solid tumours (Gould et al., 1999, Karagiannis et al., 2003). In the course of this work we sought to understand the mechanisms by which IgE effector cells mediate the killing of tumour cells. We employed a monoclonal antibody (MOv18), directed against an ovarian tumour-specific-antigen, folate binding protein (FBP), monocytes as effector cells and the ovarian tumour cell line, IGROV1, which expresses FBP, as target cells (Benard et al., 1985, Campbell et al., 1991, Coney et al., 1991, Tomassetti et al., 1993, Toffoli et al., 1997).

IgE has two cell-bound receptors, the high-affinity receptor, FcεRI, and the low-affinity receptor, CD23 (Gould et al., 2003). The former is homologous to IgG receptors, which mediate antibody-dependent cellular cytotoxicity (ADCC) of tumour cells (Ravetch and Kinet, 1991, Clynes et al., 1998, Clynes et al., 2000, Dyall et al., 1999, Carter, 2006). The low-affinity IgE receptor, CD23, is a member of the C-type lectin superfamily, and has no counterpart among IgG receptors (Gould et al., 2003). CD23, expressed in monocytes, mediates the phagocytosis of hapten-coated red cells (Yokota et al., 1992). We have shown for the first time, by fluorescence microscopy, that IgE receptors on monocytes can also mediate phagocytosis of tumour cells (Karagiannis et al., 2003). We therefore sought to develop an assay to separately quantify IgE antibody monocyte-mediated tumour cell killing by cytotoxicity (ADCC) and phagocytosis (ADCP). Here, we describe a new three-colour cytometric assay that achieves this purpose.

Standard cytotoxicity (ADCC) assays, such as Chromium-51 (51Cr) release (Brunner et al., 1968) and the lactate dehydrogenase (LDH) release (Korzeniewski and Callewaert, 1983), rely on the loss of membrane integrity, associated with cell death, to release an intracellular substance (in some cases, pre-loaded) into the medium, where it can be measured. In the event of phagocytosis, however, these markers are instead released into phagocytes, where they go undetected. The three-colour flow cytometric assay we describe overcomes this problem. It employs three fluorophores to quantify phagocytosis in addition to cytotoxicity. It is based on the same principle as the fluorocytometric (FCM) assay described by Derby et al. (2001), in which the third fluorophore gives information about the mechanism of cytotoxicity, distinguishing the contributions of lytic granule- and Fas Ligand/TNF-mediated apoptosis.

The assay we describe involves labelling of tumour cells with CFSE (Carboxy-fluorescein diacetate, succinimidyl ester), a fluorescent dye normally used to monitor cell proliferation, prior to incubation of antibody with the tumour and effector cells. Following incubation, effector cells are distinguished by Phycoerythrin (PE) labelling of an antigen absent on target cells. Dead cells are labelled with Propidium Iodide (PI). Cell death is then assessed by flow cytometry, whereby five populations can be clearly distinguished: live targets (CFSE+, PE, PI), dead targets (CFSE+, PI, PI+), live effectors (CFSE, PE+, PI), dead effectors (CFSE, PE+, PI+) and phagocytosed target cells (CFSE+, PE+, PI). The principle of this assay is shown schematically in Fig. 1. We have applied this assay to determine the contribution of cytotoxicity and phagocytosis to MOv18 IgE antibody killing of IGROV1 ovarian tumour target cells by monocytic effector cells, including cells of the U937 cell line and primary peripheral blood monocytes.

Section snippets

Antibodies and reagents

Chimeric antibodies MOv18 IgE against the FBP and NIP IgE specific for the hapten 4-hydroxy-3-nitro-phenacetyl (NIP) were prepared as before (Gould et al., 1999, Neuberger et al., 1985). We used anti-CD89-PE (BD Biosciences, Oxford, UK) and PE-conjugated mouse IgG isotype-matched control antibody (Dako, Glostrup, Denmark). Propidium Iodide (PI), CFSE dye, tissue culture media, and reagents were from Invitrogen (Paisley, UK). Flow cytometric evaluations of fixed (dead) cells were performed using

Flow cytometric ADCC and ADCP assay set up

Voltage and compensation adjustments were made using a series of control tubes performed for each effector or target cell population (Fig. 3). These included live unstained effectors, live PE+ effectors, live CD89-PE-, and PI-stained effectors (Fig. 3A). IGROV1 target cell population controls included live CFSE+ and live CFSE+ with and without CD89-PE, live CFSE+ and PI-stained, or killed (fixed and permeabilised) CFSE+ and PI-stained targets (Fig. 3B).

Calculation of ADCC and ADCP

Twenty thousand events were acquired from

Discussion

We describe here a new flow cytometric method for mechanistic studies of antibody-dependent cell-mediated tumour cell killing, applied to a realistic system. This assay allows the measurement of ADCP, ADCC and total cell killing. IGROV1 tumour target cells are pre-labelled with the green fluorescent dye, CFSE. The CFSE+ target cells are incubated with human U937 or primary monocytic effector cells and anti-FBP MOv18 IgE for tumour cell killing. The monocytes are labelled with PE and dead cells

Acknowledgements

This study was supported by the King's College London Medical School, a Marie-Curie Fellowship and the Association for International Cancer Research, United Kingdom. We are grateful to Dr. Natalie McCloskey and Dr. James Hunt for providing technical expertise and advice, Ms. Kate Kirwan for expert assistance with the figures and Prof. Jean-Pierre Kinet, Dr. Marie-Hélène Jouvin, and Dr. Silvana Canevari for the generous provision of materials and advice.

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