Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand

Blomberg, Kaj; Hautala, Rasmus; Lovgren, Janina; Mukkala, Veli-Matti; Lindqvist, Christer; Åkerman, Karl E.O.

doi:10.1016/0022-1759(96)00063-4

Cited by 99 publications

(70 citation statements)

References 26 publications

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“…NK cell activity was quantified by measuring the intense fluorescence of the Europium TDA chelates using a time-resolved fluorometer (VICTOR from Wallac Oy, PerkinElmer Life Sciences, Waltham, MA, USA) as previously published. 21 Real-time cytotoxicity assay…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients

Schlegel

Feuchtinger

Nitschke-Gérard

et al. 2015

Bone Marrow Transplant

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Natural killer (NK) cell activity has been shown to have potential activity against Ewing's sarcoma (EWS) especially in tumors with low HLA I expression and high NKG2D expression. Two patients with metastatic relapsed and primary metastatic stage IV EWS who had received two courses of high dose chemotherapy with autologous stem cell rescue were transplanted from a haploidentical parental stem cell donor. Patients are alive in ongoing CR for 10.2 and 3.4 years now. Post transplant local second and first relapses were treated successfully in both patients. In vivo IL-2 stimulation not only increased the number and activity of effector cells in one patient but was also associated with severe GvHD. In vitro studies demonstrated high NK cell activity against K562 and relevant activity against EWS cell line A673 post transplant. NK activity was enhanced by cytokine prestimulation as well as by EWS targeting anti-GD2 Ab. Haploidentical hematopoietic stem cell transplantation (HSCT) might contribute to long-term survival by NK cellmediated effect exerted by donor-derived NK cells. Local tumor recurrence was manageable in both high-risk patients indicating systemic immune control preventing subsequent metastasizing. The efficacy of haploidentical HSCT, cytokine application and tumor targeting antibodies for the use of Ab-dependent cellular cytotoxicity needs evaluation in clinical trials.

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Section: Cytotoxicity Assaymentioning

confidence: 99%

Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients

Schlegel

Feuchtinger

Nitschke-Gérard

et al. 2015

Bone Marrow Transplant

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“…Fluorescence-based EuTDA cytotoxicity assay As described (Conaghan et al, 2008), a fluorescent probe, BATDA (Blomberg et al, 1996) (Perkin Elmer, Boston, MA, USA), was used to label target cells. PBMCs, NK cells, monocytes and granulocytes were used as effectors with varying effector : target cell ratios and concentrations of antibodies.…”

Section: Isolation Of Immune Effector Cellsmentioning

confidence: 99%

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

Ashraf

Umaña²,

Mössner³

et al. 2009

Br J Cancer

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BACKGROUND: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. METHODS: The antibody was modified using EBNA cells cotransfected with b-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. RESULTS: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. CONCLUSION: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

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“…The adhesion assay was based on loading the neutrophils with a fluorescenceenhancing ligand 2,2':6',2''-terpyridine-6,6''diacarboxylic acid (TDA) [23]. In its esterified form, bis(acetoxymethyl) 2,2':6',2''-terpyridine-6,6''diacarboxylate (BATDA), the ligand diffuses readily through the cell membrane of viable cells.…”

Section: Neutrophil Adhesion Assaymentioning

confidence: 99%

Systemic suppression of human peripheral blood phagocytic leukocytes after whole-body UVB irradiation

Leino

Saarinen

et al. 1999

Journal of Leukocyte Biology

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We examined systemic effects of wholebody UVB irradiation on human peripheral blood phagocytes. We found that 24 h after a single erythemal dose of UVB radiation two phagocyte functions, adhesion and phagocytosis, were reduced by 50%. This functional suppression was accompanied by a significant decrease in the expression of complement receptors (CR1 and CR3) and IgG Fc receptors (FcRII and FcRIII). The greatest reduction (47%) was observed in CR3, which is important for both adhesion and phagocytosis. A kinetic analysis showed that both CR1 and CR3 levels started to decrease 15 min after the UVB exposure, reaching the lowest levels at 4.5-and 24-h time points, respectively. The down-modulation of CRs after whole-body UVB exposure was not due to a defective receptor synthesis or translocation from internal stores to plasma membrane because the maximal CR levels in stimulated cells were not affected by UVB. No change in the serum soluble ICAM-1 was detected after UVB, which rules out CD11b epitope masking by sICAM-1. UVB did not release low-receptor-density myeloid progenitor cells from storage pools into circulation. Interleukin 10, a mediator of UVB-induced immunosuppression, was unable to modulate CR expression in vitro. When seven suberythemal whole-body UVB exposures were given repeatedly within 2 weeks, a significant decrease in CR expression was seen, which was greatest after three irradiations. Our data suggest that an exposure to UVB has systemic effects in humans which, possibly due to the down-modulation of preexisting cell-surface receptors, suppress some important functions of circulating phagocytic cells. J. Leukoc. Biol. 65: 573-582; 1999.

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Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand

Cited by 99 publications

References 26 publications

Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients

Favorable NK cell activity after haploidentical hematopoietic stem cell transplantation in stage IV relapsed Ewing’s sarcoma patients

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

Systemic suppression of human peripheral blood phagocytic leukocytes after whole-body UVB irradiation

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