A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely us...

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Bibliographic Details
Published in:Scientific reports 2016-01, Vol.6 (1), p.19772-19772, Article 19772
Main Authors: Yamashita, Makiko, Kitano, Shigehisa, Aikawa, Hiroaki, Kuchiba, Aya, Hayashi, Mitsuhiro, Yamamoto, Noboru, Tamura, Kenji, Hamada, Akinobu
Format: Article
Language:English
Subjects:
Antibodies
Antibody-Dependent Cell Cytotoxicity - immunology
Antibody-dependent cell-mediated cytotoxicity
Antigens, Surface - metabolism
Breast Neoplasms - immunology
Breast Neoplasms - metabolism
Calcein
Cancer
Cell Line, Tumor
Cells, Cultured
Cryopreservation
Cytotoxicity
Effector cells
Female
Flow Cytometry
Humans
Immunophenotyping
Immunotherapy
Killer Cells, Natural - immunology
Killer Cells, Natural - metabolism
Leukocytes (mononuclear)
Leukocytes, Mononuclear - immunology
Leukocytes, Mononuclear - metabolism
Lysis
Peripheral blood mononuclear cells
Phenotype
Population studies
T cell receptors
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Summary:Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep19772