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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 Schuster12
Estimated H-index: 12
(University of Tübingen)
+ 3 AuthorsStefan Stevanovic69
Estimated H-index: 69
(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)
  • Cited By (24)
Cite
References13
Published on Feb 1, 1968in Immunology 3.36
K. T. Brunner3
Estimated H-index: 3
,
J. Mauel3
Estimated H-index: 3
+ 1 AuthorsB. Chapuis3
Estimated H-index: 3
The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the g...
1,103 Citations
Published on Feb 1, 1986in Journal of Immunological Methods 2.19
Kaj Blomberg13
Estimated H-index: 13
,
Christer Granberg3
Estimated H-index: 3
+ 1 AuthorsTimo Lövgren45
Estimated H-index: 45
Abstract The use of a new marker for labelling cells used as targets for natural killer cells is described. The human erythroleukaemic cell line K-562 was used as target. The cells were labelled with europium diethylenetri-aminopentaacetate (EuDTPA) chelates. The detection of the released marker is based on time-resolved fluorometry. The results obtained show that the method is sensitive, specific and rapid. The high specific activity of the marker and the sensitivity of the detection apparatus ...
123 Citations Source Cite
Published on Jan 1, 2006in Journal of Investigative Dermatology 6.45
Mads Hald Andersen35
Estimated H-index: 35
,
David Schrama33
Estimated H-index: 33
+ 1 AuthorsJürgen C. Becker75
Estimated H-index: 75
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 ...
208 Citations Source Cite
Published on Jan 1, 2009in Cellular Immunology 3.00
Esin Aktas13
Estimated H-index: 13
(Istanbul University),
Umut Can Kucuksezer10
Estimated H-index: 10
(Istanbul University)
+ 2 AuthorsGunnur Deniz18
Estimated H-index: 18
(Istanbul University)
Abstract 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 c...
178 Citations Source Cite
David N. Garboczi30
Estimated H-index: 30
(Howard Hughes Medical Institute),
Deborah T. Hung33
Estimated H-index: 33
(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...
527 Citations Source Cite
Published on Apr 1, 2004in Blood 15.13
Inge Jedema12
Estimated H-index: 12
,
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...
122 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. Knutson45
Estimated H-index: 45
(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...
14 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 Bonnekoh23
Estimated H-index: 23
(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 Jan 1, 1992in Journal of Immunological Methods 2.19
Andreas Suhrbier46
Estimated H-index: 46
(QIMR Berghofer Medical Research Institute),
Annette Fernan2
Estimated H-index: 2
(QIMR Berghofer Medical Research Institute)
+ 2 AuthorsDenis J. Moss52
Estimated H-index: 52
(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
Published on Aug 1, 2012in Nature Medicine 32.62
Steffen Walter23
Estimated H-index: 23
,
Toni Weinschenk22
Estimated H-index: 22
+ 36 AuthorsRegina Mendrzyk3
Estimated H-index: 3
In this issue, Walter et al. report the results of two clinical trials of a new therapeutic vaccine, IMA901, for the treatment of renal cell carcinoma (RCC). IMA901 consists of ten tumor-associated peptides identified as naturally presented T cell epitopes in RCC, and the authors show longer overall survival in subjects with immune responses to multiple vaccine peptides and identify serum and cellular biomarkers that may help predict overall survival in future studies of the vaccine.
524 Citations Source Cite
Cited By24
Published on Jun 1, 2015in Bone Marrow Transplantation 4.50
Ursula Jördis Eva Seidel4
Estimated H-index: 4
,
A-M Lang1
Estimated H-index: 1
+ 8 AuthorsRupert Handgretinger54
Estimated H-index: 54
Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients
5 Citations Source Cite
Published on May 1, 2015in International Journal of Pharmaceutics 3.86
Urszula Posadowska5
Estimated H-index: 5
(AGH University of Science and Technology),
Martin Parizek7
Estimated H-index: 7
(Academy of Sciences of the Czech Republic)
+ 4 AuthorsElzbieta Pamula15
Estimated H-index: 15
(AGH University of Science and Technology)
Systemic administration of bisphosphonates, e.g. sodium alendronate (Aln) is characterized by extremely low bioavailability and high toxicity. To omit aforementioned drawbacks an injectable system for the intra-bone delivery of Aln based on Aln-loaded nanoparticles (NPs-Aln) suspended in a hydrogel matrix (gellan gum, GG) was developed. Aln was encapsulated in poly(lactide-co-glycolide) (PLGA 85:15) by solid–oil–water emulsification. Drug release tests showed that within 25 days all the encapsul...
17 Citations Source Cite
Published on Oct 1, 2015in Cancer Immunology, Immunotherapy 4.22
Janet Kerstin Peper6
Estimated H-index: 6
(University of Tübingen),
Stefan Stevanovic69
Estimated H-index: 69
(University of Tübingen)
The breakthrough development of immune checkpoint inhibitors as clinically effective novel therapies demonstrates the potential of cancer immunotherapy. The identification of suitable targets for specific immunotherapy, however, remains a challenging task. Most peptides previously used for vaccination in clinical trials were able to elicit strong immunological responses but failed with regard to clinical benefit. This might, at least partly, be caused by an inadequate peptide selection, usually ...
6 Citations Source Cite
Published on Apr 16, 2015in Biosensors
Dan Kho1
Estimated H-index: 1
,
Christa MacDonald4
Estimated H-index: 4
+ 5 AuthorsEuan S. Graham7
Estimated H-index: 7
The xCELLigence technology is a real-time cellular biosensor, which measures the net adhesion of cells to high-density gold electrode arrays printed on custom-designed E-plates. The strength of cellular adhesion is influenced by a myriad of factors that include cell type, cell viability, growth, migration, spreading and proliferation. We therefore hypothesised that xCELLigence biosensor technology would provide a valuable platform for the measurement of drug responses in a multitude of different...
41 Citations Source Cite
Published on Dec 2, 2014in Frontiers in Immunology 5.51
Ursula Jördis Eva Seidel1
Estimated H-index: 1
(Boston Children's Hospital),
Fabian Vogt2
Estimated H-index: 2
(University of Tübingen)
+ 3 AuthorsPeter Lang61
Estimated H-index: 61
(German Cancer Research Center)
γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT) strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells...
16 Citations Source Cite
Published on Oct 1, 2014in Journal of Medical Primatology 0.43
Andrew G. MacLean14
Estimated H-index: 14
(Tulane University),
Edith Walker1
Estimated H-index: 1
(Tulane University)
+ 5 AuthorsStephen E. Braun3
Estimated H-index: 3
(Tulane University)
Background To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. Methods We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors....
11 Citations Source Cite
Published on May 1, 2015in Cancer immunology research 9.19
Alexander J. Davenport6
Estimated H-index: 6
(University of Melbourne),
Misty R. Jenkins16
Estimated H-index: 16
(University of Melbourne)
+ 7 AuthorsPaul Neeson19
Estimated H-index: 19
(University of Melbourne)
Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which ...
19 Citations Source Cite
Published on Dec 1, 2014in Cancer Research 9.13
David R. Soto-Pantoja17
Estimated H-index: 17
(National Institutes of Health),
Masaki Terabe34
Estimated H-index: 34
+ 5 AuthorsDavid D. Roberts63
Estimated H-index: 63
(National Institutes of Health)
While significant advances in radiotherapy have increased its effectiveness in many cancer settings, general strategies to widen the therapeutic window between normal tissue toxicity and malignant tumor destruction would still offer great value. CD47 blockade has been found to confer radioprotection to normal tissues while enhancing tumor radiosensitivity. Here we report that CD47 blockade directly enhances tumor immunosurveillance by CD8+ T cells. Combining CD47 blockade with irradiation did no...
44 Citations Source Cite
Published on Mar 1, 2018in Cytometry Part B-clinical Cytometry 2.76
P. Anoop Chandran1
Estimated H-index: 1
(German Cancer Research Center),
Karoline Laske1
Estimated H-index: 1
(German Cancer Research Center)
+ 5 AuthorsCécile Gouttefangeas27
Estimated H-index: 27
(German Cancer Research Center)
Background: Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA-peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen-specific T cell responses after immunotherapy. Methods: Param...
2 Citations Source Cite
Published on Oct 2, 2016in OncoImmunology 5.50
Michael S. Magee12
Estimated H-index: 12
(Thomas Jefferson University),
Crystal L. Kraft3
Estimated H-index: 3
(Thomas Jefferson University)
+ 5 AuthorsAdam E. Snook17
Estimated H-index: 17
(Thomas Jefferson University)
ABSTRACTAdoptive T-cell therapy (ACT) is an emerging paradigm in which T cells are genetically modified to target cancer-associated antigens and eradicate tumors. However, challenges treating epithelial cancers with ACT reflect antigen targets that are not tumor-specific, permitting immune damage to normal tissues, and preclinical testing in artificial xenogeneic models, preventing prediction of toxicities in patients. In that context, mucosa-restricted antigens expressed by cancers exploit anat...
11 Citations Source Cite
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