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An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells

Published on Mar 1, 2014in Journal of Immunological Methods 2.19
· DOI :10.1016/j.jim.2014.01.012
Janet Kerstin Peper6
Estimated H-index: 6
(University of Tübingen),
Heiko Schuster13
Estimated H-index: 13
(University of Tübingen)
+ 3 AuthorsStefan Stevanovic71
Estimated H-index: 71
(University of Tübingen)
Abstract
Abstract The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6 h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24 h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.
  • References (13)
  • Citations (27)
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References13
Newest
Published on Jan 1, 2013in Journal of Clinical Oncology 26.36
Vanya Icheva1
Estimated H-index: 1
,
Simone Kayser7
Estimated H-index: 7
+ 12 AuthorsMichaela Nathrath21
Estimated H-index: 21
Purpose Reactivation of Epstein-Barr virus (EBV) after allogeneic stem-cell transplantation (SCT) can lead to severe life-threatening infections and trigger post-transplantation lymphoproliferative disease (PTLD). Since EBV-specific T cells could prevent PTLD, cellular immunotherapy has been a promising treatment option. However, generation of antigen-specific T-cell populations has been difficult within a short time frame. Patients and Methods To improve availability in urgent clinical conditio...
133 Citations Source Cite
Published on Aug 8, 2012in Journal of Visualized Experiments 1.18
Courtney L. Erskine10
Estimated H-index: 10
(Mayo Clinic),
Andrea M. Henle2
Estimated H-index: 2
(Mayo Clinic),
Keith L. Knutson44
Estimated H-index: 44
(Mayo Clinic)
Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to det...
15 Citations Source Cite
Published on Jan 1, 2009in Cellular Immunology 3.00
Esin Aktas14
Estimated H-index: 14
(Istanbul University),
Umut Can Kucuksezer10
Estimated H-index: 10
(Istanbul University)
+ 2 AuthorsGunnur Deniz18
Estimated H-index: 18
(Istanbul University)
NK cells play important roles in innate immunity against tumors and infections of the host. Studies show that CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8+ T cells. In our study, the relationship between the expression of CD107a, cytokine secretion and cytotoxic activity in CD56+ NK, CD8+ T cells and lymphocytes has been determined after various stimuli. Effector cells from PBMCs of healthy subjects were isolated and K562 cell line was used as target of cytotoxicity....
188 Citations Source Cite
Published on Oct 1, 2006in Assay and Drug Development Technologies 1.59
Joakim Jakob Glamann1
Estimated H-index: 1
,
Anker Jon Hansen1
Estimated H-index: 1
Measurement of electrical impedance is a relatively new real-time and label-free method for monitoring cell adhesive properties. Impedance measurements are performed in tissue culture wells in which the bottom is equipped with gold electrodes. The extent of electrode coverage by living cells as well as the strength of the bond between the cell membrane and the electrode surface determines the impedance, which in real-time cell electrical sensing (RT-CES™, ACEA Biosciences, San Diego, CA) is meas...
41 Citations Source Cite
Published on Jan 1, 2006in Journal of Investigative Dermatology 6.45
Mads Hald Andersen32
Estimated H-index: 32
,
David Schrama34
Estimated H-index: 34
+ 1 AuthorsJürgen C. Becker77
Estimated H-index: 77
The immune system is a complex arrangement of cells and molecules that preserve the integrity of the organism by elimination of all elements judged dangerous. Within the immune system, a humoral and a cellular as well as an innate and an adaptive arm can be differentiated. The key players of adaptive cellular immune responses are T lymphocytes in general and, for the effector function, cytotoxic T lymphocytes (CTLs) in particular. T lymphocytes arise in the bone marrow and migrate to the thymus ...
210 Citations Source Cite
Published on Jun 1, 2004in Journal of Immunological Methods 2.19
Andrea Weren1
Estimated H-index: 1
(Otto-von-Guericke University Magdeburg),
Bernd Bonnekoh16
Estimated H-index: 16
(Otto-von-Guericke University Magdeburg)
+ 2 AuthorsAndreas Ambach9
Estimated H-index: 9
(Otto-von-Guericke University Magdeburg)
Abstract CD8 hi+ cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8 hi+ CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in...
16 Citations Source Cite
Published on Apr 1, 2004in Blood 15.13
Inge Jedema20
Estimated H-index: 20
,
Nicole van der Werff2
Estimated H-index: 2
+ 2 AuthorsJ.H. Frederik Falkenburg39
Estimated H-index: 39
For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-indu...
126 Citations Source Cite
Published on Jan 1, 1993in Journal of Immunological Methods 2.19
R.F. Hussain4
Estimated H-index: 4
(Royal London Hospital),
A.M.E. Nouri10
Estimated H-index: 10
(Royal London Hospital),
R. T. D. Oliver17
Estimated H-index: 17
(Royal London Hospital)
Abstract Using in vitro established tumour cell lines attempts were made to assess the suitability of tetrazolium salt reduction (MTT) assay to replace the conventional radioactive base techniques for measuring cell proliferation and cell killing. The optimum conditions for MTT loading time, concentration of MTT and the time for colour development were found to be 4 h, 5 mg/ml and 30 min respectively, conditions which were used for subsequent experiments. Analysis of the correlation between incr...
250 Citations Source Cite
David N. Garboczi29
Estimated H-index: 29
(Howard Hughes Medical Institute),
Deborah T. Hung37
Estimated H-index: 37
(Howard Hughes Medical Institute),
Don C. Wiley97
Estimated H-index: 97
(Howard Hughes Medical Institute)
Abstract The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecul...
536 Citations Source Cite
Published on Jan 1, 1992in Journal of Immunological Methods 2.19
Andreas Suhrbier48
Estimated H-index: 48
(QIMR Berghofer Medical Research Institute),
Annette Fernan3
Estimated H-index: 3
(QIMR Berghofer Medical Research Institute)
+ 2 AuthorsDenis J. Moss50
Estimated H-index: 50
(QIMR Berghofer Medical Research Institute)
Abstract Granules released by cytotoxic T cells (CTL), during recognition and killing of target cells, contain granule enzyme A. This serine protease has an esterase activity, which is easily measured using the substrate benzyloxycarbonyl- L -lysine thiobenzyl ester (BLT). BLT activity, routinely used as an assay for granule release, provides an alternative to the standard chromium release assay as a measure of CTL-mediated killing. The two methods were highly comparable when either exogenous sy...
23 Citations Source Cite
Cited By27
Newest
Published on May 1, 2019in Gastroenterology 20.77
Anthony T. Tan16
Estimated H-index: 16
(National University of Singapore),
Ninghan Yang (Genome Institute of Singapore)+ 18 AuthorsDiana Low11
Estimated H-index: 11
(Agency for Science, Technology and Research)
Background & Aims Hepatocellular carcinoma (HCC) is often associated with hepatitis B virus (HBV) infection. Cells of most HBV-related HCCs contain HBV-DNA fragments that do not encode entire HBV antigens. We investigated whether these integrated HBV-DNA fragments encode epitopes that are recognized by T cells and whether their presence in HCCs can be used to select HBV-specific T-cell receptors (TCRs) for immunotherapy. Methods HCC cells negative for HBV antigens, based on immunohistochemistry,...
Source Cite
Published on Apr 1, 2019in Nature Medicine 32.62
Jun Wang6
Estimated H-index: 6
(Yale University),
Jingwei Sun3
Estimated H-index: 3
(Yale University)
+ 16 AuthorsXu Zhou6
Estimated H-index: 6
(Yale University)
Overexpression of the B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some patients with cancer, and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genome-scale T cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating my...
1 Citations Source Cite
Published on Feb 1, 2019in British Journal of Cancer 5.92
Marie Christine Wulff Westergaard5
Estimated H-index: 5
(University of Copenhagen),
Rikke Andersen12
Estimated H-index: 12
(University of Copenhagen)
+ 9 AuthorsMichal Bassani-Sternberg13
Estimated H-index: 13
(Ludwig Institute for Cancer Research)
Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens. Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality...
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Published on Jan 1, 2019in European Journal of Pharmaceutical Sciences 3.47
Marcin Płaczek4
Estimated H-index: 4
(Gdańsk Medical University),
Dorota Wątróbska-Świetlikowska2
Estimated H-index: 2
(Gdańsk Medical University)
+ 3 AuthorsMalgorzata Sznitowska17
Estimated H-index: 17
(Gdańsk Medical University)
Abstract Lecithin and isolated phospholipids (mainly phosphatidylcholine) have been used for years as pharmaceutical excipients in parenteral formulations: submicron emulsions, liposomes and mixed micelles. Under development are also other lecithin-based drug delivery systems, e.g. aqueous lecithin dispersions (WLDs). The aim of the study was to investigate the properties and potential cytotoxicity of 7 different phospholipid-based dispersions intended for parenteral administration: emulsions, l...
2 Citations Source Cite
Published on Dec 1, 2018in Scientific Reports 4.12
Frank Fasbender3
Estimated H-index: 3
(Technical University of Dortmund),
Carsten Watzl31
Estimated H-index: 31
(Technical University of Dortmund)
The use of impedance-based label free cell analysis is increasingly popular and has many different applications. Here, we report that a real-time cell analyzer (RTCA) can be used to study the stimulation of Natural Killer (NK) cells. Engagement of NK cells via plate-bound antibodies directed against different activating surface receptors could be measured in real time using the label-free detection of impedance. The change in impedance was dependent on early signal transduction events in the NK ...
3 Citations Source Cite
Published on Nov 1, 2018in Breast Cancer Research and Treatment 3.60
Yismeilin R. Feliz-Mosquea1
Estimated H-index: 1
(Wake Forest University),
Ashley A. Christensen1
Estimated H-index: 1
(Wake Forest University)
+ 13 AuthorsRajarshi Guha35
Estimated H-index: 35
(National Institutes of Health)
Background A perennial challenge in systemic cytotoxic cancer therapy is to eradicate primary tumors and metastatic disease while sparing normal tissue from off-target effects of chemotherapy. Anthracyclines such as doxorubicin are effective chemotherapeutic agents for which dosing is limited by development of cardiotoxicity. Our published evidence shows that targeting CD47 enhances radiation-induced growth delay of tumors while remarkably protecting soft tissues. The protection of cell viabilit...
3 Citations Source Cite
Published on Aug 1, 2018
High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell–dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be...
Source Cite
Published on May 1, 2018in Cytotherapy 3.99
Charlotte de Wolf4
Estimated H-index: 4
(Utrecht University),
Marja van de Bovenkamp2
Estimated H-index: 2
,
Marcel C Hoefnagel2
Estimated H-index: 2
Abstract The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and...
4 Citations Source Cite
Published on Apr 19, 2018in JCI insight
Benjamin Y. Jin1
Estimated H-index: 1
(National Institutes of Health),
Tracy E. Campbell4
Estimated H-index: 4
(National Institutes of Health)
+ 7 AuthorsChristian S. Hinrichs24
Estimated H-index: 24
(National Institutes of Health)
5 Citations Source Cite
Published on Mar 2, 2018in PLOS ONE 2.77
Fabio Cerignoli6
Estimated H-index: 6
,
Yama A. Abassi16
Estimated H-index: 16
+ 6 AuthorsBiao Xi11
Estimated H-index: 11
4 Citations Source Cite