An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells

J Immunol Methods. 2014 Mar;405:192-8. doi: 10.1016/j.jim.2014.01.012. Epub 2014 Jan 29.

Abstract

The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

Keywords: Chromium release assay; Impedance; Real time; T-cell cytotoxicity assay; xCELLigence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apoptosis / immunology*
  • Cell Adhesion / immunology
  • Cell Line, Tumor
  • Cell Survival / immunology
  • Cells, Cultured
  • Chromium Radioisotopes / metabolism
  • Coculture Techniques
  • Cytotoxicity Tests, Immunologic / methods*
  • Cytotoxicity, Immunologic / immunology*
  • Electric Impedance
  • HLA-A2 Antigen / immunology
  • Humans
  • Influenza A virus / immunology
  • Killer Cells, Natural / immunology
  • Leukocytes, Mononuclear / immunology
  • Peptide Fragments / immunology
  • Reproducibility of Results
  • T-Lymphocytes / immunology*
  • Time Factors
  • Viral Matrix Proteins / immunology

Substances

  • Chromium Radioisotopes
  • HLA-A*02 antigen
  • HLA-A2 Antigen
  • Peptide Fragments
  • Viral Matrix Proteins