Live Target Cells Research Articles

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

Abstract 1303: Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity. Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2015-1303

High-speed scanning electrochemical microscopy method for substrate kinetic determination: application to live cell imaging in human cancer.

Scanning electrochemical microscopy (SECM) is increasingly applied to study and image live cells. Quantitative analyses of biological systems, however, still remain challenging. In the presented study, single human adenocarcinoma cervical cancer cells are electrochemically investigated by means of SECM. The target cell's electrochemical response is observed over time under the influence of green tea catechins (GTC), which are suggested to offer chemopreventive and therapeutic effects on cancer. The electrochemical response of living target cells is measured experimentally and quantified in an apparent heterogeneous rate constant by using a numerical model, based on forced convection during high speed SECM imaging. The beneficial effect of GTC on cancer cells could be confirmed by SECM, and the presented study shows an alternative approach toward unraveling the mechanisms involved during inhibition of carcinogenesis.

Quantifying Single Human Cancer Cell Redox State By Scanning Electrochemical Microscopy

Scanning electrochemical microscopy (SECM) is increasingly applied to study and image live cells. Quantitative analyses of biological systems, however, still remain challenging. In the presented study, single human adenocarcinoma cervical cancer cells are electrochemically investigated by means of SECM. The target cell’s electrochemical signal is observed over time under the influence of green tea catechins (GTC), which are suggested to offer chemopreventive and therapeutic effects on cancer.1 The electrochemical response of living target cells is measured experimentally and the cell’s heterogeneous rate constant is determined by using a numerical model, based on forced convection during high speed SECM imaging. The beneficial effect of GTC on cancer cells could be confirmed by SECM and the presented study shows an alternative approach towards unravelling the mechanisms involved during inhibition of carcinogenesis. 1Connors, SK, Chornokur, G, Kumar NB (2012) Nutrition and Cancer, 64:4. Figure 1

Selective Capture and Collection of Live Target Cells Using a Photoreactive Silicon Wafer Device Modified with Antibodies via a Photocleavable Linker

A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FS-AFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody-alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.

Evidence that the Density of Self Peptide-MHC Ligands Regulates T-Cell Receptor Signaling

Noncognate or self peptide-MHC (pMHC) ligands productively interact with T-cell receptor (TCR) and are always in a large access over the cognate pMHC on the surface of antigen presenting cells. We assembled soluble cognate and noncognate pMHC class I (pMHC-I) ligands at designated ratios on various scaffolds into oligomers that mimic pMHC clustering and examined how multivalency and density of the pMHCs in model clusters influences the binding to live CD8 T cells and the kinetics of TCR signaling. Our data demonstrate that the density of self pMHC-I proteins promotes their interaction with CD8 co-receptor, which plays a critical role in recognition of a small number of cognate pMHC-I ligands. This suggests that MHC clustering on live target cells could be utilized as a sensitive mechanism to regulate T cell responsiveness.

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

Modulating Cell-Cell Communication with a High-Throughput Label-Free Cell Assay

A high-throughput label-free cell assay for modulating cell-cell communication is demonstrated with the Epic® system, a resonant waveguide grating sensor platform. Natural killer (NK) cells are known to be able to recognize abnormal cells (e.g., cancer cells and cells presenting intercellular adhesion molecule 1 [ICAM1] through cell surface receptors) and kill them. In this study, the effect of effecter cells NK92MI on two kinds of target cells, cervical cancer cells (HeLa) and Chinese hamster ovarian cells overexpressing ICAM1 (CHO-ICAM1), was examined. Living target cells' response to NK92MI cells was monitored in real time and measured as wavelength shift in picometers. The authors showed that the detectability of target cell response is affected by multiple factors: the ratio of effecter cells to target cells (E/T), the interaction time of the two types of cells, and the target cell type. For example, with the effecter cells NK92MI and the same incubation time of 16 h, a minimal E/T ratio of 1 is required to detect HeLa cell response, whereas an E/T of 0.5 is sufficient to detect CHO-ICAM1 cell response. The authors confirmed that NK92MI cell-mediated target cell cytotoxicity results in negative optical signals and is associated with apoptosis mainly through caspase pathways. Distinct optical signals could be generated with the pretreatment of the target cells with various known pharmaceutical reagents, making the assay useful for discovering new chemicals that may affect cell-cell communications.

Live-cell Microarray Surface Coatings Supporting Reverse Transduction by Adeno-Associated Viruses

High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.

Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus–Quantum Dot Conjugates

The unique spectral properties of semiconductor quantum dots (QDs) enable long-term live-cell imaging and ultrasensitive detection of viral particles, which in turn can potentially provide a practical means for detailed analysis of the underlying molecular mechanisms of virus entry. In this study, we report a general method of labeling adeno-associated virus serotype 2 (AAV2) with QDs for enhanced visualization of the intracellular behavior of viruses in living target cells. It was found that the mild conditions required for this QD conjugation reaction allowed for the retention of viral infectivity of AAV2. Furthermore, quantitative analysis of viral motility in living cells suggested that QD-labeling had no significant effect on the intracellular transport properties of AAV2 particles compared to those of conventional organic dye-labeled AAV2. Our imaging study demonstrated that QD-AAV2 was internalized mainly through a clathrin-dependent pathway and then trafficked through various endosomes. It was also observed that QD-AAV2 particles exploit the cytoskeleton network to facilitate their transport within cells, and the labeling study provided evidence that the ubiquitin-proteasome system was likely involved in the intracellular trafficking of AAV2, at least at the level of nuclear transport. Taken together, our findings reveal the potential of this QD-labeling method for monitoring the intracellular dynamics of virus-host cell interactions and interrogating the molecular mechanisms of viral infection in greater detail.

A GFP-transfected HFWT cell line, GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay.

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.

Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica

Autoantibody specific for the aquaporin-4 astrocytic water channel is restricted to serum and CSF of patients with neuromyelitis optica (NMO) and related CNS inflammatory demyelinating disorders (relapsing optic neuritis and longitudinally extensive transverse myelitis). NMO-typical lesions are distinct from MS-typical lesions. Aquaporin-4 is lost selectively at vasculocentric sites of edema/inflammation coinciding with focal deposits of immunoglobulins (Ig) G, M, and terminal complement products, with and without myelin loss. Evidence for antigen-specific autoantibody pathogenicity is lacking. We used confocal microscopy and flow cytometry to evaluate the selectivity and immunopathological consequences of Ig binding to surface epitopes of living target cells expressing aquaporin-4 fused at its cytoplasmic N-terminus with GFP. We tested serum, IgG-enriched and IgG-depleted serum fractions, and CSF from patients with NMO, neurologic control patients, and healthy subjects. We also analyzed aquaporin-4 immunoreactivity in myelinated adult mouse optic nerves and spinal cord, and plasma cell Ig isotypes in archived brain tissue from an NMO patient. Serum IgG from patients with NMO binds to the extracellular domain of aquaporin-4; it is predominantly IgG(1), and it initiates two potentially competing outcomes, aquaporin-4 endocytosis/degradation and complement activation. Serum and CSF lack aquaporin-4-specific IgM, and plasma cells in CNS lesions of NMO contain only IgG. Paranodal astrocytic endfeet highly express aquaporin-4. NMO patients' serum IgG has a selective pathologic effect on cell membranes expressing aquaporin-4. IgG targeting astrocytic processes around nodes of Ranvier could initiate demyelination.

Persistence of host-specific Bacteroides–Prevotella 16S rRNA genetic markers in environmental waters: effects of temperature and salinity

Host-specific Bacteroides-Prevotella 16S rRNA genetic markers are promising alternative indicators for identifying the sources of fecal pollution because of their high abundance in the feces of warm-blooded animals and high host specificity. However, little is known about the persistence of these genetic markers in environments after being released into environmental waters. The persistence of feces-derived four different host-specific Bacteroides-Prevotella 16S rRNA genetic makers (total, human-, cow-, and pig-specific) in environmental waters was therefore investigated at different incubation temperatures (4, 10, 20, and 30 degrees C) and salinities (0, 10, 20, and 30 ppt) and then compared with the survival of conventional fecal-indicator organisms. The host-specific genetic markers were monitored by using real-time polymerase chain reaction (PCR) assays with specific primer sets. Each host-specific genetic marker showed similar responses in non-filtered river water and seawater: They persisted longer at lower temperatures and higher salinities. In addition, these markers did not increase in all conditions tested. Decay rates for indicator organisms were lower than those for host-specific genetic markers at temperature above 10 degrees C. Furthermore, we investigated whether the PCR-detectable 16S rRNA genetic markers reflect the presence of live target cells or dead target cells in environmental waters. The result revealed that the detection of the Bacteroides-Prevotella 16S rRNA genetic markers in environmental waters mainly reflected the presence of 'viable but non-culturable' Bacteroides-Prevotella cells. These findings indicate that seasonal and geographical variations in persistence of these host-specific Bacteroides-Prevotella 16S rRNA genetic markers must be considered when we use them as alternative fecal indicators in environmental waters.

Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value

Molecular studies of rare cells, such as circulating cancer cells, require efficient pre-enrichment steps to obtain a pure population of target cells for further characterization. We have developed a two-step approach, starting with immunomagnetic enrichment, followed by specific isolation of individual, easily identifiable bead-rosetted target cells using a new semi-automated CellPick system. With this procedure, 1-50 live target cells can now be isolated. As a model system, we spiked a small number of tumor cells into millions of normal mononuclear cells (MNCs). Efficient isolation of pure target cells was obtained by use of the CellPick system, and the nature of isolated, bead-rosetted cells was verified by use of FISH. Single breast cancer cells were picked directly into an RNA preserving lysis buffer, reverse transcribed, and PCR amplified with two cDNA specific primer sets. With the isolated cells we consistently obtained both ubiquitously expressed and tumor cell specific PCR products. We also performed a successful mutation analysis of single cells using PCR and cycling temperature capillary electrophoresis (CTCE). This may have significant clinical implications in cancer and in other diseases, e.g. in characterizing micrometastatic cancer cells in blood and lymph nodes to help identifying patients who most likely will respond to therapies like tyrosine kinase inhibitors and compounds targeting specific mutations. By use of the CellPick system it is possible to specifically isolate bead-rosetted or otherwise labelled target cells from a heterogeneous cell population for further molecular characterization.

Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis

We have developed a three-colour flow cytometric method to assay the contributions of cytotoxicity and phagocytosis to antibody-dependent cell-mediated tumour cell killing. In this assay, tumour target cells are pre-labelled with CFSE, and mixed with effector cells and a tumour antigen-specific monoclonal antibody. After incubation of the cells with the antibody, effector cells are labelled with PE and dead cells with PI. Using flow cytometry, dead and phagocytosed tumour cells can be quickly and easily counted and the numbers summed to determine the total number of killed cells. One can thereby measure the phagocytic aspect of antibody-dependent cell-mediated tumour cell killing, otherwise only revealed by microscopic examination. The failure to detect phagocytosed, in addition to live and dead target cells, by standard assays may result in an underestimation of tumour cell killing and hence the potential of an antibody for immunotherapy of cancer. We illustrate the new method by analysing human monocyte-mediated cytotoxic and phagocytic cell killing of IGROV1 ovarian tumour cells by the ovarian tumour antigen-specific anti-folate binding protein monoclonal antibody, MOv18 IgE.

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells.

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors.

Functional guidance components and their cellular distribution in retinotectal co-cultures.

Recent advances in the study of the developmental processes that direct afferent axons toward their final destinations are beginning to elucidate the cellular and molecular bases for the early development of retinotectal topography. The present review focuses on two guidance components (repulsive and adhesive) and their cellular localization as revealed by a newly established retinotectal co-culture system. These co-cultures enable general outgrowth patterns to be examined when retinal ganglion cell (RGC) axons attempt to traverse cells dissociated from their target nuclei. Additionally, the recording of RGC growth cone responses to single living target cells allows a cellular localization of guidance components that is not possible by other methods. This co-culture system, therefore, complements the repertoire of cell-culture techniques used to investigate the sequence of cellular events that underlie the establishment of retinotopic development. With this new approach, time-lapse micrographs were collected when RGC growth cones from temporal or nasal regions of embryonic chick retinae encountered individual cells dissociated from optic tecta. Temporal RGC growth cones collapsed and retracted with a high probability from neuronal cells dissociated from posterior tecta, indicating that repellent components were enriched on posterior target neurons. The response to non-neuronal cells revealed a separate effect on axonal outgrowth that was less dependent upon the particular region of retina or tectum from which the cells originated: most RGC axons adhered to the edge of non-neuronal cells, without retracting. Together, the localization of repellent and adhesive components suggests a sequence of events that occurs during the early stages of tectal innervation and that results in the rudiments of retinotopic projection, and furthermore, it raises a number of experimentally approachable questions concerning the functional expression of several guidance components.

Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections

Human autologous tumour-specific cytotoxic T lymphocytes (CTL) were generated from peripheral blood on small formalin-fixed paraffin-embedded sections of a gastric cancer. The CTL killed live target cells at an effector/target ratio of 1 within 24 hours and showed the same target specificity as those induced on live cancer cells. The killing activity of the CTL lasted for more than four months in culture and was inhibited by antibodies against CD8 and MHC-class I. These results suggest that adoptive immunotherapy of tumours will be possible with CTL induced on a stable source of tumour antigen.

A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity

A flow cytometry based method has been developed to assess natural killer (NK) cell activity in both short-term (4 h) and long-term (18 h) NK assays. Target cells were either labeled with PKH-2, c′FDA or D275. Simultaneously, dead cells were identified by counter-staining with the nuclear dye propidium iodide. Using flow cytometry, only D275 in combination with propidium iodide permits the differentiation of four cell populations: live target cells, dead target cells, live effector cells, and dead effector cells. Even after the extended incubation periods (18 h) necessary for the determination of NK activity in some domestic animals these four populations remain clearly distinguishable. Comparison of results with cells of normal human individuals obtained using this D27S/propidium iodide flow cytometry assay with data derived from fluorescence microscopy or an endogenous lactate dehydrogenase release assay shows a strong correlation. Since in long-term NK assays a high proportion of dead effector cells is constantly observed this cell population frequently limits the use of the lactate dehydrogenase release assay but does not interfere with the flow cytometry assay presented here. Using this novel assay, we have demonstrated the suppressive effects of defined glycosaminoglycans on long-term porcine NK activity.