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@ARTICLE{Wellach:301580,
      author       = {K. Wellach$^*$ and A. Riemer$^*$},
      title        = {{H}ighly sensitive live-cell imaging-based cytotoxicity
                      assay enables functional validation of rare epitope-specific
                      {CTL}s.},
      journal      = {Frontiers in immunology},
      volume       = {16},
      issn         = {1664-3224},
      address      = {Lausanne},
      publisher    = {Frontiers Media},
      reportid     = {DKFZ-2025-01088},
      pages        = {1558620},
      year         = {2025},
      note         = {#EA:D410#LA:D410#},
      abstract     = {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.},
      keywords     = {Humans / T-Lymphocytes, Cytotoxic: immunology /
                      T-Lymphocytes, Cytotoxic: metabolism / Epitopes,
                      T-Lymphocyte: immunology / Cytotoxicity, Immunologic /
                      Lymphocyte Activation Gene 3 Protein / Cytotoxicity Tests,
                      Immunologic: methods / Cell Line, Tumor / Interferon-gamma:
                      metabolism / Programmed Cell Death 1 Receptor: metabolism /
                      Programmed Cell Death 1 Receptor: immunology / Apoptosis /
                      Caspase 3: metabolism / T cells (Other) / cytotoxicity
                      (Other) / epitopes (Other) / live-cell imaging (Other) /
                      rare CTL populations (Other) / Epitopes, T-Lymphocyte (NLM
                      Chemicals) / Lymphocyte Activation Gene 3 Protein (NLM
                      Chemicals) / Interferon-gamma (NLM Chemicals) / Lag3
                      protein, human (NLM Chemicals) / Programmed Cell Death 1
                      Receptor (NLM Chemicals) / Caspase 3 (NLM Chemicals)},
      cin          = {D410},
      ddc          = {610},
      cid          = {I:(DE-He78)D410-20160331},
      pnm          = {314 - Immunologie und Krebs (POF4-314)},
      pid          = {G:(DE-HGF)POF4-314},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:40406125},
      pmc          = {pmc:PMC12095279},
      doi          = {10.3389/fimmu.2025.1558620},
      url          = {https://inrepo02.dkfz.de/record/301580},
}