Adherent Target Cells Research Articles

Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.

High-throughput Quantification of Antibody-Dependent Phagocytosis using Live-Cell Analysis

Abstract The uptake and clearance of tumor cells can be promoted with monoclonal antibodies (mAbs) via antibody-dependent phagocytosis (ADCP) or through the blockade of “don’t-eat-me” signals, such as CD47. These mechanisms hold immunotherapeutic promise and are studied extensively in drug development. High-throughput assays were conducted using pHrodo®, a pH-sensitive fluorophore, that enables live-cell imaging and kinetic quantification of phagocytosis. pHrodo® labeled target cells were incubated with mAbs and added to effector cells in 96-well plates. Images were acquired using the Incucyte® Live-Cell Analysis System and fluorescence automatically quantified with integrated software. In line with known pro-phagocytic effects, anti-CD47 promoted phagocytosis of CCRF-CEM tumor cells by blocking CD47 “don’t-eat-me” signals. Clinical anti-CD20 mAbs Truxima® and Rituximab both promoted ADCP of Ramos target cells by primary macrophages. In addition, the ADCP response of different Rituximab isotypes were compared. Anti-CD20-IgG1 mAb and Fc mutated anti-CD20-IgG1fut (non-fucosylated) exhibited concentration-dependent responses (EC50 values of 25.1 ng/mL and 42.8 ng/mL, respectively), however anti-CD20-IgG1NQ (non-glycosylated) showed no response. ADCP was also examined in adherent target cells with varied HER2 profiles. Anti-HER2 (Trastuzumab) induced an ADCP response in HER2-positive AU565 cells but not in HER2-low MDA-MB-231 cells, consistent with established correlations between HER2 expression and ADCP response. These data exemplify that live-cell analysis is a powerful approach that enables functional quantification of ADCP and is amenable to antibody screening for therapeutic candidates.

Accelerating ADCP screening using the iQue® Advanced Flow Cytometry Platform

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an immune process stimulated by monoclonal antibodies (mAbs) that promotes specific recruitment of phagocytes to eliminate targeted cancer cells. In vitro functional assays are used extensively in drug development to characterize the effects of mAbs on a range of mechanisms of action, including ADCP. We describe a high throughput assay for ADCP quantification via co-localization analysis of labeled target cells with CD14 positive effector cells. Target cells were incubated in 96 or 384 well plates with clinically relevant mAbs prior to addition of PBMCs or isolated immune cells (e.g. monocytes or macrophages). Cells were analyzed using the iQue®3 Advanced Flow Cytometer and inbuilt software. Various effector:target (E:T) cell ratios were used to validate the assay. Maximal ADCP of non-adherent Ramos target cells by PBMCs in the presence of Truxima was 43 ± 2% with an E:T ratio of 20:1, reducing to 19 ± 6% with a 5:1 ratio. Further pharmacological data was obtained for adherent AU565 cells, comparing the response between anti-HER2 mAb isotypes. The potency of response to anti-HER2-IgG1 mAb (Trastuzumab) was five times greater than to another native human isotype, anti-HER2-IgA2, with EC50 values of 6.6 ng/mL and 35 ng/mL, respectively. These data are in line with previous characterizations of isotype effector functions which showed increased ADCP functionality with IgG1 compared to IgA2 mAbs. The iQue® platform provides a solution for rapid profiling of ADCP effector function in both adherent and suspension target cell models. This accelerates antibody discovery by highlighting potential efficacy and toxicity of antibody drug candidates, leading to greater productivity and insight.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

Chip-based platform for dynamic analysis of NK cell cytolysis mediated by a triplebody.

Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis.

Characterization of Bispecific T-cell Engager (BiTE®) Antibodies with a High-Capacity T-cell Dependent Cellular Cytotoxicity (TDCC) Assay

The Bispecific T-cell Engager (BiTE®) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors.

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Evaluation of immunosuppressive function of regulatory T cells using a novel in vitro cytotoxicity assay.

Naturally occurring regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance due to their intrinsic immunosuppressive activity. Currently, a number of human clinical trials are being conducted to investigate the roles of Tregs in treating various immune-mediated disorders. Traditionally, the suppressive activity of Tregs is measured using either a thymidine incorporation assay, which is a radioactive assay; or CFSE based flow cytometry assay, which requires a relatively large number of cells. Consequently, there is an increasing need to develop novel alternative bioassays that can characterize various aspects of the immunosuppressive function of Tregs in vitro. In this study, using murine clonal CD8+ T cells specific for an islet antigen as responder T cells, we first established a novel, sensitive and quantitative in vitro luminescence based cell viability assay to measure cytotoxicity. Then we used this assay to measure if Tregs could inhibit the cytotoxicity of CD8 effector T cells. This assay does not involve the use of radioisotopes and only needs relatively low number of Tregs. Since normally Tregs only constitute 5-10% of peripheral CD4+ T cells, this advantage is noteworthy compared with other methods. With the assay we developed, we demonstrated that regulatory T cells (Tregs) could inhibit the antigen-specific killing of an adherent target cell monolayer by the CD8+ cytotoxic T cells. We observed more inhibition when Tregs and CD8 killer T cells were incubated during the in vitro activation (stimulation) stage of the cytotoxic T lymphocytes (CTL) than when they were added later at the start of the effector phase. Interestingly, Tregs from B6 mice demonstrated higher suppression of CD8+ T cell killing than Tregs from NOD mice. Moreover, IL-2/anti-IL-2 mAb complexes induced expansion of Tregs in vivo, as well as enhancing the Treg’s suppressive activity per cell.Therefore, this novel non-radioactive, luminescence based cytotoxicity assay mediated by clonal islet antigen-specific CD8 T cells can be used to measure, characterize, and quantitate the immunosuppressive activity of natural Tregs, representing a useful approach to characterize the functions of Tregs in the setting of autoimmune diseases and to elucidate the mechanisms for Treg cell-mediated immunoregulation.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-4-51) contains supplementary material, which is available to authorized users.

Measuring potency of bispecific effector/target-linking antibodies for inducing cytotoxicity of tumor cells using an impedance-based assay platform (P2094)

Abstract Cancer patients mount weak tumor-specific T-cell responses due to immune escape mechanisms of tumors. Although cytotoxic T-cells are potent killers, activation requires antigen processing, specific recognition, and co-stimulation. Bispecific antibody-derived proteins that bind both CD3 and tumor antigens can bypass these requirements by directly linking cytotoxic T cells to tumor cells and have been shown clinically to enhance T cell-mediated tumor cell killing. Our goal was to assess the relative potency of anti-CD3/anti-tumor antigen full-length IgG1 bispecific antibodies. Assays based on electrical impedance are well suited for tumor cell killing assays because the adherent target cells generate an impedance signal, while the non-adherent effector cells do not. Human tumor cells were plated with CD8+ T cells and bispecific antibodies, and cytotoxicity was recorded in real time at intervals during culture. Tumor cell killing was found to be dependent on the bispecific antibody concentration, with EC50 values of 27-40 pM. In addition, killing was dependent on the number of CD8 T cells added, with an optimal effector:target cell ratio of 4:1. Compared to a well established but labor-intensive FACS-based assay performed in parallel, the results of the impedance assay agreed closely. Moreover, impedance-based cell killing assays have the advantages of real-time, label-free readings as well as simple assay development and performance.

Abstract 5523: Measuring the Fc effector function of therapeutic antibody in antibody-dependent cell-mediated cytotoxicity using a bioluminescent cell-based reporter bioassay.

Abstract Primary peripheral blood mononuclear cells (PBMCs) or the sub-population of natural killer (NK) cells are used to quantify antibody drug biological activity in antibody-dependent cell-mediated cytotoxicity (ADCC) in classic ADCC bioassays. However, cell acquisition and preparation is labor intensive and those ADCC bioassays have high inherent variability, resulting primarily from the primary effector cell source. This variability has been challenging for acceptance of classic ADCC bioassays for use in lot release potency determination and stability-testing for antibody drugs. Here, we developed a cell-based bioassay that can quantify potency of Fc effector function of target cell-bound monoclonal antibody in pathway activation in effector cells that leads to ADCC. For this, Jurkat T-cell lines that stably express NFAT-luciferase reporter and either human FcγRIIIa(V158) or FcγRIIIa (F158) were generated to replace primary PBMCs or NK cells as effector cells in ADCC bioassay while retaining measurement of pathway activation mediated by Fc effector functionality of antibody drugs. Cells were developed in thaw-and-use format, to minimize assay variability due to cell culture and handling. The resultant bioluminescent reporter-based bioassay outperforms classic ADCC bioassays in several key ways: it has good assay precision (low variability), high accuracy, and is simple, robust and specific. In addition, we have demonstrated that the ADCC reporter bioassay is able to quantify potency of rituximab, trastuzumab, and cetuximab, which are on-market monoclonal antibody drugs for cancer using a variety of suspension or adherent target cells, and that this bioassay is stability-indicating for these antibody drugs after heat-treatment. These performance characteristics demonstrate suitability of the ADCC reporter bioassay for lot release potency determination and stability monitoring of antibody drugs. Furthermore, new antibody drugs with enhanced binding affinity to Fc receptors and improved ADCC efficiency are being developed to address improvement of drug efficacy in patient populations. Many of these ‘enhanced ADCC’ antibodies differ structurally in the N-glycan moiety. By testing blended mixtures of intact and glycan-modified antibody drugs in the ADCC reporter bioassay, we have demonstrated the linear correlation between relative antibody biological activity and the percentage of Fc N-glycan modification. The ability of this bioassay to quantifiably differentiate such differences in Fc effector functionality due to N-glycan moiety structural differences demonstrates its suitability as a tool for biosuperior antibody drug research and development. Citation Format: Zhi-Jie Jey Cheng, Denise Garvin, Rich Moravec, Aileen Paguio, Frank Fan, Teresa Surowy. Measuring the Fc effector function of therapeutic antibody in antibody-dependent cell-mediated cytotoxicity using a bioluminescent cell-based reporter bioassay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5523. doi:10.1158/1538-7445.AM2013-5523

Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions

This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.

Evaluation of NK cell function by flowcytometric measurement and impedance based assay using real-time cell electronic sensing system.

Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1α β, IFN-γ, and TNF-α, and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions.

Kaposi's Sarcoma Associated Herpesvirus Entry into Target Cells.

Herpesvirus infection of target cells is a complex process involving multiple host cell surface molecules (receptors) and multiple viral envelope glycoproteins. Kaposi’s sarcoma associated herpesvirus (KSHV or HHV-8) infects a variety of in vivo target cells such as endothelial cells, B cells, monocytes, epithelial cells, and keratinocytes. KSHV also infects a diversity of in vitro target cells and establishes in vitro latency in many of these cell types. KSHV interactions with the host cell surface molecules and its mode of entry in the various target cells are critical for the understanding of KSHV pathogenesis. KSHV is the first herpesvirus shown to interact with adherent target cell integrins and this interaction initiates the host cell pre-existing signal pathways that are utilized for successful infection. This chapter discusses the various aspects of the early stage of KSHV infection of target cells, receptors used and issues that need to be clarified, and future directions. The various signaling events triggered by KSHV infection and the potential role of signaling events in the different stages of infection are summarized providing the framework and starting point for further detailed studies essential to fully comprehend the pathogenesis of KSHV.

MHC-restricted cytotoxic T-lymphocyte assay: An improved method based on normal and SV40-immortalized rabbit epidermal target cells

Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous 51Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.

RhoA-GTPase Facilitates Entry of Kaposi's Sarcoma-Associated Herpesvirus into Adherent Target Cells in a Src-Dependent Manner

Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8) binds to adherent target cell surface heparan sulfate molecules via its envelope glycoproteins gB and gpK8.1A, to integrins via gB, to the transporter CD98/xCT complex, and possibly to another molecule(s). This is followed by virus entry overlapping with the induction of preexisting host cell signal pathways, such as focal adhesion kinase, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, protein kinase C-zeta, and extracellular signal-regulated kinase 1/2. Here, using hemagglutinin-tagged plasmids expressing wild-type, dominant-positive, and dominant-negative forms of RhoA in HEK (human embryonic kidney) 293 cells, we investigated the role of RhoA-GTPase in virus entry. The dominant-negative form of RhoA GTPase and treatment of target cells with Clostridium difficile toxin B (CdTxB), a specific inactivator of Rho-GTPases, significantly blocked KSHV entry. KSHV infection induced closely similar levels of FAK and PI3-K in all three cell types. In contrast, very strong Src activation was observed in KSHV-infected dominant-positive RhoA cells compared to wild-type cells, and only moderate Src activation was seen in dominant-negative cells. Inhibition of Src activation by CdTxB and reduction of RhoA activation by Src inhibitors suggest that KSHV-induced Src is involved in RhoA activation, which in turn is involved in a feedback-sustained activation of Src. Since the decreased entry in RhoA dominant-negative cells may be due to inefficient signaling downstream of RhoA, we examined the induction of RhoA-activated Dia-2, which is also known to induce Src. Dia-2 coimmunoprecipitated with activated Src, which was inhibited by Src inhibitors, in the infected cells. Together with the reduced virus entry in RhoA dominant-negative cells, these results suggest that activated RhoA-dependent Dia-2 probably functions as a link between RhoA and Src in KSHV-infected cells, mediating the sustained Src activation, and that KSHV-induced Src and RhoA play roles in facilitating entry into adherent target cells.

A novel fluorescent sensitive assay for detection of differential T cell mediated lysis of multiple adherent target cells

There are few studies that have investigated T cell mediated lysis of adherent cells. We have developed a novel, rapid and sensitive fluorescent dye-swap assay that allows efficient detection of adherent target cell lysis. The assay allows simultaneous use of multiple differentially sensitised targets and facilitates concomitant surface or intracellular effector cell phenotypic analysis.

Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays

A microelectronic sensor-based platform, the RT-CES (real time electronic sensing) system, is introduced for label free assessment of natural killer (NK) cell-mediated cytotoxic activity. The RT-CES system was used to dynamically and quantitatively monitor NK-mediated cytotoxic activity towards 8 different adherent target cell lines, including cancer cell lines commonly used in laboratories. The cytotoxic activity monitored by RT-CES system was compared with standard techniques such as MTT measurement and shows good correlation and sensitivity. To test the specificity of the assay, pharmacological agents that inhibit NK cell degranulation and cytotoxic activity were employed and were shown to selectively and dose-dependently inhibit NK-mediated cytotoxic activity toward target cells. In summary, the RT-CES system offers fully automated measurement of cytotoxic activity in real time, which enables large-scale screening of chemical compounds or genes responsible for the regulation of NK-mediated cytotoxic activity.

Screening of high cytotoxic tumor killer cells using a sensitive adherent target detachment assay

Screening of high cytotoxic tumor killer cells is of great importance in adoptive immunotherapy. Here, we describe a more sensitive assay, as compared to traditional 51Cr- or Calcein-release assay, to measure cytolytic activity of killer cells. This adherent target detachment (ATD) assay is carried out in microwells of Terasaki tissue culture trays using adherent tumor cells as targets. Target tumor cells are seeded at the concentration of 300–400 cells/well and incubated overnight to allow for the adhesion of cells to the plastic surface of the wells. Effector cells were added at various effector: target (E:T) ratios, and incubated for 24 h. During incubation, dead target cells became nonadherent and together with the added effector cells were removed by washing. The remaining viable adherent target cells were stained with acrydine orange contained in the quencher and optically counted by microcomputer. A notable dose-dependent killing on target tumor cells was reproducibly obtained by tested effector cells. Cytotoxic activities of most effector cells were significantly higher in the 24-h ATD assay than those of concordant 4-h target cell lysis test. Chloroquine (chq) inhibition test in the 24-h ATD assay showed negative or weak inhibition on cytotoxicity against adherent targets. Linear regression analysis also manifested the lack of a close correlation between 4- and 24-h target cell lysis tests, indicating these two assays measured different killing activities of the same effector cells. Because of its high sensitivity and reproducibility, this semiautomatic 24-h assay with computerized fluorescence measurement system could serve as a sensitive screening assay to select high cytotoxic tumor killer cells in adoptive immunotherapy.

Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules.

Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.