CAR-T cell immunotherapy is a promising anticancer treatment that redirects the host's immune system against the cancer cells. This therapy is based on the use of autologous T cells which are genetically engineered in vitro to express a chimeric antigen receptor (CAR) capable of recognizing a tumor-specific antigen. CAR is composed of a single chain variable antibody fragment (scFv) combined with an intracellular signaling motif including CD3ζ (z) activation domain fused with one or more co- stimulatory domain (Zhang et al., 2017). Tumor antigen binding to the scFv of the CAR induces T cell activation, resulting in CAR-T proliferation, and cytotoxic activity including cytokine release or Fas-Fas ligand interactions to induce tumor cells apoptosis (Benmebarek et al., 2019).

They are already authorized due to their success particularly for anti-CD19 CAR-T (CART19) cells treatment of CD19+ B cell malignancies (Kochenderfer et al., 2010; Porter et al., 2011; Brentjens et al., 2013; Grupp et al., 2013; Maude et al., 2014) which has further contributed to the increase in the development of CAR-T cells with greater efficacy and safety profiles (Wang et al., 2021; Levine et al., 2016). In vitro studies of CAR-T are mandatory to evaluate their functionality by using cytotoxic assay for their preclinical development and to qualify the treatment after the manufacturing.

To date, 51 chromium release cytotoxicity assays have been used for decades as a gold standard assay, especially for evaluating primary cytotoxic T lymphocytes (Brunner et al., 1968). However, this assay has several drawbacks such as using radioactive components, a single time point readout limitation, and the requirement to dispose radioactive waste. Therefore, alternative methods to measure cytotoxicity of T cells are currently used to circumvent the chromium release cytotoxicity assay limitation, like the europium release assay (Blomberg et al., 1986). Nevertheless, the difficulties to label the target cells and the high europium spontaneous release have prevented widespread use of this assay (von Zons et al., 1997). Other approaches commonly used are Bioluminescence (BLI)-based cytotoxic assay which measure photons emitted from luciferase-transduced target cells with a luciferase reporter gene (Karimi et al., 2014) or flow cytometry-based assay which distinguishes target and effector cells by their properties in size and granularity and by specific cells staining with antibodies coupled to a fluorescence dye (Liu et al., 2002; Aubry et al., 1999). The main drawback here is the potential variability due to a differential uptake of the dye or a spontaneous leakage of the dye during the test (Wang et al., 1993). Recent approach uses impedance-based assays, which allows a real-time assessment of target cell number and cell size on custom designed microtiter plates. Microelectrodes embedded in the bottom of a microtiter well allow measuring the impedance of an electric current flow between electrodes upon adhesion of cells over many days (Stefanowicz-Hajduk and Ochocka, 2020). This technology can monitor continuously the target cell killing, and has already been used to increase the information on NK cell and T cell cytotoxicity (Glamann and Hansen, 2006; Peper et al., 2014). However, this assay needs to be standardized, the impedance depending on each cell type, their confluence, viability and adhesion strength (Zhu et al., 2006).

Human target cells are classically used in all laboratories to evaluate the cytotoxic function of CART19 cells, but a variable alloreactivity from one cell line to another can partly contribute to cytotoxicity independently to CAR recognition (Ambrose et al., 2021; Brentjens et al., 2003; An et al., 2016). Here we describe the use of murine EL4 and NIH/3 T3 (3T3) target cells expressing eGFP to develop a simple, sensitive, and standardizable microscopy or cytometry-based cytotoxic assay, with negligible xenoreactivity.