Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry

J Immunol Methods. 2022 Jul:506:113290. doi: 10.1016/j.jim.2022.113290. Epub 2022 May 27.


Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.

Keywords: Cancer; Imaging cytometry; Immuno-oncology; Nur77-GFP; T cell activation; T cell-mediated cytotoxicity.

Publication types

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

MeSH terms

  • Cytotoxicity, Immunologic
  • Image Cytometry
  • Immunity, Cellular
  • Lymphocyte Activation*
  • Receptors, Antigen, T-Cell
  • T-Lymphocytes, Cytotoxic*


  • Receptors, Antigen, T-Cell