Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity

Nat Protoc. 2021 Mar;16(3):1331-1342. doi: 10.1038/s41596-020-00467-0. Epub 2021 Feb 15.


The antitumor efficacy of genetically engineered 'living drugs', including chimeric antigen receptor and T-cell receptor T cells, is influenced by their activation, proliferation, inhibition, and exhaustion. A sensitive and reproducible cytotoxicity assay that collectively reflects these functions is an essential requirement for translation of these cellular therapeutic agents. Here, we compare various in vitro cytotoxicity assays (including chromium release, bioluminescence, impedance, and flow cytometry) with respect to their experimental setup, appropriate uses, advantages, and disadvantages, and measures to overcome their limitations. We also highlight the US Food and Drug Administration (FDA) directives for a potency assay for release of clinical cell therapy products. In addition, we discuss advanced assays of repeated antigen exposure and simultaneous testing of combinations of immune effector cells, immunomodulatory antibodies, and targets with variable antigen expression. This review article should help to equip investigators with the necessary knowledge to select appropriate cytotoxicity assays to test the efficacy of immunotherapeutic agents alone or in combination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Review

MeSH terms

  • Animals
  • Cytotoxicity Tests, Immunologic / methods*
  • Flow Cytometry / methods
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Receptors, Antigen, T-Cell / metabolism
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes / immunology
  • Xenograft Model Antitumor Assays / methods


  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen