Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing thedeath of infected or malignant host cells¹. These cells express a specializedreceptor, called the T cell receptor (TCR), which can recognize a specific antigenicpeptide bound to HLA class I molecules². Engagement of infected cells or tumorcells through their HLA class I molecule results in production of lytic molecules such asgranzymes and perforin resulting in target cell death. While it is useful to determinefrequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flowcytometry, it is also helpful to ascertain the strength of CD8 T cell responses usingcytotoxicity assays³. The most recognizable assay for assessing cytotoxicfunction is the Chromium Release Assay (CRA), which is considered a standard assay⁴. The CRA has several limitations, including exposure of cells to gammaradiation, lack of reproducibility, and a requirement for large numbers of cells. Over thepast decade, there has been interest in adopting new strategies to overcome theselimitations. Newer approaches include those that measure caspase release ⁴, BLTesterase activity ⁵ and surface expression of CD107 ⁶. Theimpedance-based assay, using the Roche xCelligence system, was examined in the presentpaper for its potential as an alternative to the CRA. Impedance or opposition to anelectric current occurs when adherent tumor cells bind to electrode plates. Tumor cellsdetach following killing and electrical impedance is reduced which can be measured by thexCelligence system. The ability to adapt the impedance-based approach to assesscell-mediated killing rests on the observation that T cells do not adhere tightly to mostsurfaces and do not appear to have much impact on impedance thus diminishing any concernof direct interference of the T cells with the measurement. Results show that theimpedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 Tcells with increased sensitivity relative to the standard CRA. Based on these results,impedance-based approaches may be good alternatives to CRAs or other approaches that aimto measure cytotoxic CD8 T cell functionality.Download video file.^{(71M, mov)}