Related Experiment Video
Updated: Sep 21, 2025
Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells
Published on: March 7, 2025
Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry
Monica K Chanda1, Claire E Shudde2, Taylor L Piper2
1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, United States of America; Cancer Biology Graduate Program, University of Michigan Medical School, Ann Arbor, MI 48109, United States of America.
Abstract:
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.
Insights
A new imaging cytometry platform quantifies T cell activation and cancer cell killing. This system uses the Nur77-GFP reporter to assess T cell-mediated cancer immunotherapy drug efficacy.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Cancer immunotherapies aim to enhance T cell-mediated tumor cell killing.
- Evaluating T cell responses in co-cultures with cancer cells is crucial for drug development.
- Existing methods may not fully capture the dynamic interplay between T cell activation and cancer cell death.
Purpose of the Study:
- To develop and validate an imaging cytometry platform for simultaneous quantification of T cell activation and cancer cell killing.
- To assess the utility of the Nur77-GFP reporter system in T cell-cancer co-culture models.
- To evaluate the platform's ability to test experimental therapeutics and heterogeneous lymphocyte populations.
Main Methods:
- Development of an imaging cytometry platform utilizing the Nur77-GFP reporter system.
- Co-culture of T cells with a cancer cell line expressing a T cell receptor (TCR)-specific antigen.
- Quantification of activated cytotoxic CD8+ T cells (CTLs) and their capacity to induce cancer cell death.
- Assessment of T cell responses in heterogeneous lymphocyte populations and in the presence of a SRC family kinase inhibitor.
Main Results:
- Demonstrated simultaneous quantification of T cell activation and cancer cell killing using imaging cytometry.
- Validated the Nur77-GFP reporter system as a direct readout of TCR-induced T cell activation.
- Successfully evaluated the impact of a SRC family kinase inhibitor on CTL activity and cancer cell viability.
- Showcased the platform's capability to analyze antigen-specific T cell responses within mixed lymphocyte populations.
Conclusions:
- The developed imaging cytometry platform provides a robust method for assessing T cell-mediated cancer cell death.
- The Nur77-GFP reporter system is effective for evaluating diverse treatment conditions in T cell-cancer co-cultures.
- This platform facilitates rapid assessment of immuno-oncology drug candidates and T cell-based therapeutics.
