A guide to studying tumour-T-cell interactions using in vitro coculture assays.

T cells are central mediators of antitumour immunity and are the cellular basis of many successful cancer immunotherapies, including immune checkpoint blockade, T-cell receptor (TCR)-engineered therapies and chimeric antigen receptor (CAR) T cells. However, clinical responses remain variable, and many tumours evade or resist T-cell-mediated control. Understanding how T cells recognise tumour cells, deliver cytotoxic signals, produce inflammatory cytokines and shape tumour cell fate is therefore essential for improving modern immunotherapies. In vitro tumour T-cell coculture systems provide a powerful experimental framework for dissecting these processes under controlled conditions. They allow investigators to manipulate antigen specificity, HLA/MHC restriction, tumour genotype, T-cell activation state, cytokine exposure and drug-mediated perturbation in ways that are difficult to achieve in vivo. However, these systems require careful design, because assay outcomes are strongly influenced by tumour model, antigen expression, effector-to-target ratio, assay duration and readout. This guide provides a practical framework for studying tumour T-cell interactions in vitro, with a focus on CD8+ T-cell-mediated tumour recognition and killing. It discusses commonly used mouse and human antigen-specific models, how antigen expression and presentation can be experimentally controlled, T-cell activation and coculture conditions, cytotoxicity assays, immune synapse imaging, competition assays, cytokine-mediated bystander killing, transcriptional profiling, CRISPR perturbation strategies, therapeutic testing and patient-derived models. The aim is to help researchers choose the most appropriate model, experimental setup and readout for the biological question being asked and to avoid common interpretive pitfalls when studying tumour immune sensitivity, resistance and therapeutic response.

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Authors

Khoshdoozmasouleh N; Kearney CJ

Published

Jan 1, 2026

Journal or venue

The FEBS journal

DOI

10.1111/febs.70765

Paper type

journal article

Source

Pubmed