A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry

Muraglia, Glenn M. La; O'Neil, Michael J.; Madariaga, Maria Lucia; Michel, Sebastian; Mordecai, K.S.; Allan, James S.; Madsen, Joren C.; Hanekamp, I.; Preffer, Frederic I.

doi:10.1016/j.jim.2015.10.005

Cited by 6 publications

(5 citation statements)

References 26 publications

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“…Measurement of target cell lysis so far characterized CTL function. Methods for such measurements include the former gold standard method, the 51 Cr release assay [Brunner et al, ; La Muraglia et al, ]. However, there is considerable interest in the field toward understanding the molecular mechanisms on the T‐cell site in ex vivo experiments.…”

Section: Discussionmentioning

confidence: 99%

“…In this respect, methods using flow cytometry have been established in the 1990's. The advantage of flow cytometry is, that the phenotypes of effector and target cells can be analyzed in parallel at the single cell level ([La Muraglia et al, ] and reviewed in [Zaritskaya et al, ]). It allows measurement of different stages of cytotoxicity and apoptosis, including CD107a degranulation, Annexin‐V and PI staining or detection of active caspase 3.…”

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confidence: 99%

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Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

2017

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The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

2017

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“…In the event of abnormalities within cytotoxic mechanisms or prolonged duration of the immunological synapses leading to an extended effector function timeframe, this could escalate the release of cytokines and chemokines, thus increasing inflammation, compromising the efficacy of the therapeutic response, and potentially causing a relapse of antigen-free malignancy (10,122,124,129,(138)(139)(140). Methods for ex vivo examination of cytotoxic efficiency of CAR T cells are described in Table 2.…”

Section: Markers Of the Immune Systemmentioning

confidence: 99%

Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives

Levstek,

Janžič,

Ihan

et al. 2024

Front. Immunol.

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Chimeric antigen receptor (CAR) T cell therapy holds enormous potential for the treatment of hematologic malignancies. Despite its benefits, it is still used as a second line of therapy, mainly because of its severe side effects and patient unresponsiveness. Numerous researchers worldwide have attempted to identify effective predictive biomarkers for early prediction of treatment outcomes and adverse effects in CAR T cell therapy, albeit so far only with limited success. This review provides a comprehensive overview of the current state of predictive biomarkers. Although existing predictive metrics correlate to some extent with treatment outcomes, they fail to encapsulate the complexity of the immune system dynamics. The aim of this review is to identify six major groups of predictive biomarkers and propose their use in developing improved and efficient prediction models. These groups include changes in mitochondrial dynamics, endothelial activation, central nervous system impairment, immune system markers, extracellular vesicles, and the inhibitory tumor microenvironment. A comprehensive understanding of the multiple factors that influence therapeutic efficacy has the potential to significantly improve the course of CAR T cell therapy and patient care, thereby making this advanced immunotherapy more appealing and the course of therapy more convenient and favorable for patients.

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“…Besides conduction of multiparametric evaluation of cells by using multi-purpose staining in flow cytometric assays, conjugated cells in addition to effector and target cells can be imaged clearly at single cell level (40,41). Moreover, assays measuring cell-mediated cytotoxicity in flow cytometry are being used and developed (accelerated) for other T-cells (γδ T cell) than NK or CTL (42,43).…”

Section: Future Expectations: Multi-color Flow Cytometric Assay Multmentioning

confidence: 99%

Cell-Mediated Cytotoxicity Assays

Özdemir¹

2019

Asthma Allergy Immunol

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Cell-mediated cytotoxicity measurements can be divided in two methods, depending on whether radioactive material is used or not. Natural Killer cell-mediated cytotoxicity is routinely measured with a short-term assay by labeling the target cells with radioactive chromium (51Cr). The advantages of this method are (1) highly sensitivity, (2) easy execution, (3) low spontaneous release, and (4) nontoxic markers. The disadvantages include short half-life of the label and handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/enzymatic assays are highly sensitive and easy to use but unfortunately works only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of the dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false positive results leading to a decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.

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A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry

Cited by 6 publications

References 26 publications

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives

Cell-Mediated Cytotoxicity Assays

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A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry

Cited by 6 publications

References 26 publications

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8+ T-cells

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8+ T-cells

Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives

Cell-Mediated Cytotoxicity Assays

Contact Info

Product

Resources

About

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells