ORIGINAL RESEARCH article
Front. Immunol.
Sec. T Cell Biology
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1558620
This article is part of the Research TopicAdvancing T Cell Biology: Novel Insights into Epitope Recognition and Immune Response DynamicsView all articles
Highly sensitive live-cell imaging-based cytotoxicity assay enables functional validation of rare epitope-specific CTLs
Provisionally accepted
- 1German Cancer Research Center (DKFZ), Heidelberg, Germany
- 2German Center for Infection Research, partner site Heidelberg, Heidelberg, Baden-Württemberg, Germany
- 3Faculty of Biosciences, Heidelberg University, Heidelberg, Baden-Württemberg, Germany
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Many immunotherapeutic approaches aim at inducing epitope-specific T cell cytotoxicity. However, identification and especially functional validation of suitable epitopes by in vitro cytotoxicity assays can be challenging, particularly if the amount of available epitope-specific cytotoxic T cells (CTLs) is limited.Here we present a highly sensitive image-based cytotoxicity assay that allows functional analysis of rare epitope-specific T cells. The live-cell imaging-based setup combines transient red labeling of target cells with a green caspase 3/7 probe, which allows reliable measurement of the fraction of apoptotic target cells. Time course analysis enables monitoring of subtle differences. This very flexible assay can be applied to either assess the killing of target cells with endogenous epitope presentation or target cells that were artificially loaded with the epitope of interest. Analysis of assay sensitivity demonstrated that cytotoxicity mediated by as few as 0.1% epitope-specific CTLs in a T cell culture can still be detected. The epitope-specificity of the assay was additionally validated by specific upregulation of PD-1 and LAG-3 on epitope-specific T cells, as well as the epitope-specific induction of interferon-γ release. Finally, the assay was successfully applied to functionally validate human papillomavirus (HPV)16 epitopes, by detecting epitope-specific killing of established patient-derived tumor cell lines by rare T cell populations expanded from peripheral blood. Overall, this cytotoxicity assay setup provides a straightforward approach to assess the cytotoxic capacity of rare epitope-specific T cells and enables the analysis of T cell responses against endogenously presented epitopes.
Keywords: T cells, Cytotoxicity, Epitopes, live-cell imaging, rare CTL populations
Received: 10 Jan 2025; Accepted: 14 Apr 2025.
Copyright: © 2025 Wellach and Riemer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Angelika B Riemer, German Cancer Research Center (DKFZ), Heidelberg, Germany
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