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001     301580
005     20250601020839.0
024 7 _ |a 10.3389/fimmu.2025.1558620
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041 _ _ |a English
082 _ _ |a 610
100 1 _ |a Wellach, Kathrin
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245 _ _ |a Highly sensitive live-cell imaging-based cytotoxicity assay enables functional validation of rare epitope-specific CTLs.
260 _ _ |a Lausanne
|c 2025
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520 _ _ |a Many immunotherapeutic approaches aim to induce epitope-specific T-cell cytotoxicity. However, the identification-and especially the functional validation-of suitable epitopes by in vitro cytotoxicity assays can be challenging, particularly when the number of available epitope-specific cytotoxic T cells (CTLs) is limited. Here, we present a highly sensitive image-based cytotoxicity assay that allows the 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, allowing reliable measurement of the fraction of apoptotic target cells. Time-course analysis enables the monitoring of subtle differences. This highly flexible assay can be applied to assess the killing of either target cells with endogenous epitope presentation or those 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.
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650 _ 7 |a T cells
|2 Other
650 _ 7 |a cytotoxicity
|2 Other
650 _ 7 |a epitopes
|2 Other
650 _ 7 |a live-cell imaging
|2 Other
650 _ 7 |a rare CTL populations
|2 Other
650 _ 7 |a Epitopes, T-Lymphocyte
|2 NLM Chemicals
650 _ 7 |a Lymphocyte Activation Gene 3 Protein
|2 NLM Chemicals
650 _ 7 |a Interferon-gamma
|0 82115-62-6
|2 NLM Chemicals
650 _ 7 |a Lag3 protein, human
|2 NLM Chemicals
650 _ 7 |a Programmed Cell Death 1 Receptor
|2 NLM Chemicals
650 _ 7 |a Caspase 3
|0 EC 3.4.22.-
|2 NLM Chemicals
650 _ 2 |a Humans
|2 MeSH
650 _ 2 |a T-Lymphocytes, Cytotoxic: immunology
|2 MeSH
650 _ 2 |a T-Lymphocytes, Cytotoxic: metabolism
|2 MeSH
650 _ 2 |a Epitopes, T-Lymphocyte: immunology
|2 MeSH
650 _ 2 |a Cytotoxicity, Immunologic
|2 MeSH
650 _ 2 |a Lymphocyte Activation Gene 3 Protein
|2 MeSH
650 _ 2 |a Cytotoxicity Tests, Immunologic: methods
|2 MeSH
650 _ 2 |a Cell Line, Tumor
|2 MeSH
650 _ 2 |a Interferon-gamma: metabolism
|2 MeSH
650 _ 2 |a Programmed Cell Death 1 Receptor: metabolism
|2 MeSH
650 _ 2 |a Programmed Cell Death 1 Receptor: immunology
|2 MeSH
650 _ 2 |a Apoptosis
|2 MeSH
650 _ 2 |a Caspase 3: metabolism
|2 MeSH
700 1 _ |a Riemer, Angelika
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773 _ _ |a 10.3389/fimmu.2025.1558620
|g Vol. 16, p. 1558620
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|x 1664-3224
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