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000301580 041__ $$aEnglish
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000301580 1001_ $$0P:(DE-He78)c59ffaab8ba99ade9e79149ac033f804$$aWellach, Kathrin$$b0$$eFirst author$$udkfz
000301580 245__ $$aHighly sensitive live-cell imaging-based cytotoxicity assay enables functional validation of rare epitope-specific CTLs.
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000301580 520__ $$aMany 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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000301580 650_7 $$2Other$$aT cells
000301580 650_7 $$2Other$$acytotoxicity
000301580 650_7 $$2Other$$aepitopes
000301580 650_7 $$2Other$$alive-cell imaging
000301580 650_7 $$2Other$$arare CTL populations
000301580 650_7 $$2NLM Chemicals$$aEpitopes, T-Lymphocyte
000301580 650_7 $$2NLM Chemicals$$aLymphocyte Activation Gene 3 Protein
000301580 650_7 $$082115-62-6$$2NLM Chemicals$$aInterferon-gamma
000301580 650_7 $$2NLM Chemicals$$aLag3 protein, human
000301580 650_7 $$2NLM Chemicals$$aProgrammed Cell Death 1 Receptor
000301580 650_7 $$0EC 3.4.22.-$$2NLM Chemicals$$aCaspase 3
000301580 650_2 $$2MeSH$$aHumans
000301580 650_2 $$2MeSH$$aT-Lymphocytes, Cytotoxic: immunology
000301580 650_2 $$2MeSH$$aT-Lymphocytes, Cytotoxic: metabolism
000301580 650_2 $$2MeSH$$aEpitopes, T-Lymphocyte: immunology
000301580 650_2 $$2MeSH$$aCytotoxicity, Immunologic
000301580 650_2 $$2MeSH$$aLymphocyte Activation Gene 3 Protein
000301580 650_2 $$2MeSH$$aCytotoxicity Tests, Immunologic: methods
000301580 650_2 $$2MeSH$$aCell Line, Tumor
000301580 650_2 $$2MeSH$$aInterferon-gamma: metabolism
000301580 650_2 $$2MeSH$$aProgrammed Cell Death 1 Receptor: metabolism
000301580 650_2 $$2MeSH$$aProgrammed Cell Death 1 Receptor: immunology
000301580 650_2 $$2MeSH$$aApoptosis
000301580 650_2 $$2MeSH$$aCaspase 3: metabolism
000301580 7001_ $$0P:(DE-He78)3743a1b712edca2ffa829b7096d7037e$$aRiemer, Angelika$$b1$$eLast author$$udkfz
000301580 773__ $$0PERI:(DE-600)2606827-8$$a10.3389/fimmu.2025.1558620$$gVol. 16, p. 1558620$$p1558620$$tFrontiers in immunology$$v16$$x1664-3224$$y2025
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