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Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

T cells engineered with chimeric antigen receptors (CARs) are emerging as powerful cancer immunotherapies. Remarkable efficacies have been demonstrated in treating B-cell malignancies with CAR-T cells, leading to the FDA's first approval of gene therapy. Currently, numerous clinical trials for hematological malignancies and solid tumors are underway worldwide. Production of CAR-T cells with proper qualities is essential for CAR-T success in vivo. Here we detail optimized protocols for the generation of CAR-T cells for preclinical studies using lentiviral gene transfer, expansion of CAR-T cells in culture, detection of CAR expression, and evaluation of CAR-T cellular cytotoxicity in vitro.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous "normal" cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities.

Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.

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.

The recent introduction of new technologies such as Luminex has provided alternative methods to the Complement Dependent Cytotoxicity (CDC) test for HLA specific antibody detection. In this study we compared the results obtained with CDC to those obtained using a Luminex method with the aim of evaluating the impact of this new technology on antibody screening policies in our transplant setting.A total of 1,421 sera, acquired from patients on the waiting list for a kidney transplant or following transplantation, were tested by both methodologies. CDC was performed using a whole lymphocyte population comprising a panel of 52 cells. The percentage panel reactive antibodies (PRA) and antibody specificity were evaluated using Lambda Scan Analysis software. For the Luminex method sera screening and identification of antibody specificity were carried out using the LABScreen Mixed and LABScreen PRA respectively. The overall concordance between the results obtained using the CDC and the Luminex methods was 85%. HLA antibody specificity was confirmed in 96% of the sera which tested positive using the Luminex system and serum positivity corresponded with a previous sensitisation event in these individuals. Using the Luminex method 18% of patients on the waiting list were considered and managed as sensitised as compared to 7% when testing with CDC alone. The Luminex method was able to detect a number of antibody specificities significantly more frequently than the CDC method and in addition the CDC method failed to detect some of the antibody specificities detected by the Luminex system. Based on this comparison study we have incorporated the Luminex methodology into our screening strategy.

The most traditional method used to measure the lytic activity of cytotoxic T lymphocytes or natural killer (NK) cells is the chromium release assay (CRA). No study has been reported that systematically compares the traditional gamma counting method with various benchtop microplate scintillation formats to measure chromium release. Here we investigated the utilization of microplate beta counters in comparison with the traditional gamma counting method to quantitate antigen-specific cytolysis, lymphokine-activated killer (LAK) activity, and NK activity in the CRA. Supernatants from standard CRA (n = 7) were directly transferred to a 96-well microplate containing either a solid scintillant (Lumaplate) or a liquid scintillant (flexible beta plate). Samples were quantified by using two benchtop microplate beta counters, Wallac Microbeta Trilux (Lumalux and Trilux methods, respectively) and Packard TopCount instruments (TopCount method). These results were then compared with data from an identical assay run in parallel using the traditional gamma counting method (LKB). The lytic activity for influenza virus-stimulated effectors measured in the benchtop microplate beta counters using Lumalux and Trilux methods exhibited excellent correlations with the one measured in the traditional LKB (r = 0.967 and 0.968, respectively). The TopCount method demonstrated a similar correlation (r = 0.966). Similar findings were observed for LAK and NK activity. The 96-well microplate format, specifically the dry-scintillant Lumaplates, offers several advantages over the traditional gamma counting format. Most notable are the reductions in sample volume needed and in the total sample preparation and counting time. Furthermore, this system reduces the amount of dry and mixed radioactive waste generated while using the same instrument for gamma- and beta-emitting isotopes.

The fluorescent attachment appreciably simplifies the procedure of recording the results of the lymphocytotoxic test, is compact, economic, and easily inserted in the Biolam-P-I inverted microscope. Use of fluorescent stains helps selectively stain viable and intoxicated cells, thus simplifying their estimation. The said modification may be used in HLA typing and assessment of the principal lymphocyte subpopulations in the peripheral blood.

To assess the cytotoxic activity of immune cells, we have developed a 51Cr-retention assay in which the radioactivity retained by 51Cr-labeled target cells, following coincubation with cytotoxic cells, is monitored using the automated Matrix 96 beta counter. The Matrix 96 is designed for simultaneously counting 96 samples isolated from a 96-well microplate. It uses 96 uniform and independent detectors operating on the principle of avalanche gas ionization in the Geiger-Muller mode. Samples must be dry because the detectors are of the open-window type. Therefore, samples from the 96 wells of the microplate are simultaneously harvested onto a filter using the MicroMate 196, a 96-well cell harvester, dried and quantified in the Matrix 96. Usually the 51Cr isotope is measured by the detection of gamma radiation in gamma counters. The Matrix 96, however, monitors Auger electrons, which are also emitted by 51Cr. We have shown that the retention assay can be used to monitor the cytotoxic activity of activated lymphocytes including lymphokine-activated killer cells and tumor-infiltrating lymphocytes against various tumor cell lines. This assay is most suitable for experiments in which low E/T ratios are sufficient to detect highly cytotoxic cells, such as clone screening in cloning assays or in limiting-dilution analysis assays. These assays involve processing and reading large numbers of microplates. In this case, the retention assay monitored in the Matrix 96 will improve the work flow and decrease the amount of radioactive waste.

We have tested two different personal-computer based color image analysis systems for automated reading of the microlymphocytotoxicity test (LCT) for HLA-A,B,C-typing and screening. Over 17,000 single LCT-reactions were prepared using the simultaneous double fluorescent variant of the LCT (contrast fluorescence test, CFT). All tests were read visually by experienced laboratory staff members. For image analysis, an automated scanning system was used. In a first step, reactions were recorded on a videotape recorder using a color(CCD)-video camera. In a second step, the recorded reactions were analyzed with the two different image analysis systems by specifically developed programs. Good correlation (r = 0.89) of the score values assigned by digital image analysis with the visual tray reading was obtained. Since also the other main performance characteristics of the prototype system (throughput, reliability, compatibility) were acceptable for routine application, we may conclude that digital image analysis is a feasible and very interesting new technique for automated evaluation of the LCT.

HLA molecules serve as identity markers for immunocompetent cells which respond to pathogens and transplant tissue antigens to distinguish self from non-self. The continuous discovery of new HLA polymorphisms which are associated with various diseases or are involved in graft rejection continuously increases the number of HLA antigens which need to be identified for each individual. We defined the HLA class I and HLA class II antigens with the NIH standard microcytotoxicity system and compared the results with those obtained using the commercially available automated Patimed system. Our results show that serological identification of HLA antigens with the Patimed system is reliable. It can be recommended for routine as well as for research purposes.

We have tested two different personal-computer-based color image analysis systems for automated reading of the microlymphocytotoxicity test (LCT) for HLA-A,B,C typing and screening. Over 17,000 single LCT reactions were prepared using the simultaneous double fluorescent variant of the LCT (contrast fluorescence test, CFT). All tests were read visually by experienced laboratory staff members. For digital image analysis, an automated scanning system was used. The reactions were first recorded on a videotape recorder using a color (CCD) videocamera und subsequently analyzed with the two different image analysis systems by specifically developed programs. Good correlation (r = 0.89) of the score values assigned by digital image analysis with the visual tray reading was obtained. Since also the other main performance characteristics of the prototype system were acceptable for routine application, we may conclude that digital image analysis is a feasible and very interesting new technique for automated evaluation of the LCT.

Limiting dilution analysis is a valuable technique for the quantitation and clonal analysis of immunocompetent cells. However, manual processing of the large number of samples necessary for satisfactory statistical analysis is laborious, and consequently results in inaccuracies reflected as increased standard errors. We describe the application of an automated robotic liquid handling tool to process samples in limiting dilution cytotoxicity assays. Our studies have shown that automated liquid handling is more accurate than manual methods, and that errors are limited. This results in savings of both time and resources. Furthermore, the process may be adapted for the safe, remote handling of sterile cell cultures, and human pathogens.

A microcalorimetric vessel for monitoring the initial phases of cytotoxic reactions in the micro-submicrowatt region has been designed and tested. The vessel is intended to be used with a multichannel microcalorimetric system from Thermometric, Järfälla, Sweden and can be built up stepwise in a modular way. The different functions of perfusion, stirring and addition of small amounts of soluble immune reactants can be used separately but also in combination. This is accomplished by the use of a vertical stirring mechanism and a reaction vessel of low volume, 200 microliters. The simplicity of the vessel permits an identical vessel to be used on the reference side, handled in parallel with the measuring vessel. No gas phase is present.

This communication provides information on the design and use of a counting machine that enumerates adherent cells in cytotoxicity assays. To date, investigators have relied on inverted microscopes or quantimat automatic image analysis. The former method is tedious and time consuming while the latter method is prohibitively expensive. The counting machine which consists of projection microscope attached to an electronic counter is as accurate as the inverted microscope but is both less tedious and less time consuming.

A software package has been developed to calculate, store, and statistically analyze the results of 51Cr-release assays. It is written in Applesoft BASIC, is 'user friendly', and contains error trapping routines to catch most common mistakes. The greeting program contains a master menu from which one of the following programs may be called up: (1) 51Cr-release calculation, (2) statistical analysis, or (3) file manager. These programs give the user the capability to rapidly reduce cpm data to % specific release and statistically analyze the results using randomized design analysis of variance (ANOVA) and Turkey's honestly significant difference (HSD) test.

Two major problems encountered in the application of lymphokine-activated killer (LAK) cell therapy in man are the massive culture volumes required for LAK cell induction and the paucity of LAK cells available for administration (human doses are less than or equal to 10% of effective murine LAK cell doses). We have, therefore, developed and tested a plastic porous culture device, Sclair plastic bags (E.I. DuPont De Nemours Co.), that can be utilized at virtually any volume and does not require rotation for optimal use. Normal or patient lymphocytes were cultured in the device or in plastic 16 mm wells at 1-20 X 10(6)/ml RPMI 10% human sera with 1500 pM interleukin-2 for 4 days: LAK cell activity did not decline despite high cell densities. The device was equal to the 16 mm wells in induction of normal donor and patient LAK cell activity when either autologous fresh tumor or Raji targets were used. In a non-therapeutic clinical evaluation we isolated and stored in liquid nitrogen autologous tumor cells from 11 patients with cancer. 2-6 weeks post-operatively lymphocytes and mononuclear cells from these patients and paired normal donors were obtained and LAK cells were induced in Sclair bags or standard culture wells. Autologous patient LAK cell activity and normal donor LAK cell activity against patient's tumor cells were equivalent in the Sclair culture device and culture well system. Lymphocyte recovery and [3H]thymidine incorporation were also similar. Subsequently, we developed an expansion scheme utilizing the device in which cell density was maintained at optimal levels by changing media and reducing cell concentration after 6, 10 and 14 days of culture. We were able to expand LAK cell number 5-10-fold with no loss of LAK cell activity in this time frame utilizing both normal and patient cells. In this system plasma and sera were equivalent in their capacity to support LAK cell expansion but less than 10% plasma or sera supported suboptimal activation. Thus, we have developed a practical system to augment the number of LAK cells available for human LAK cell therapy and simultaneously reduce the complexity and volume of the induction system.

A fluorometric assay avoiding the use of radioactivity has been developed for detecting cytotoxic T lymphocytes (Tc cells). The method involves labelling targets with Hoechst dye no. 33342 (H33342) which becomes brightly fluorescent on binding to DNA. Lysis of target cells by Tc cells is quantified by measuring the release of fluorescent H33342 into the supernatant of culture wells. The fluorescence is measured using an automated Microfluor reader which allows results to be obtained rapidly. The assay has been used to detect alloreactive Tc cells and H-2 restricted Tc cells against influenza virus in a short-term 6 h assay using P815 and L929 as targets with comparable results to those obtained with 51Cr labelling. In contrast, lymphocyte blasts were found to be less sensitive in 6 h fluorometric assays when compared with the 51Cr assay. In long-term overnight assays (possible because of the low spontaneous release of H33342 from targets) lymphocyte blasts gave high specific lysis and some anti-self reactivity. The cause of the anti-self reactivity may reflect fundamental differences between the H33342 and 51Cr release assays.

The construction and use of a single semiautomatic device for collecting equal aliquots of supernates after radioisotope release assays of cellular cytotoxicity are described. This device simultaneously removes an aliquot of the supernate from each of the 12 wells in one row of a microtiter plate, and transfers these aliquots directly to disposable tests tubes for insertion into a gamma-counter. Results obtained using this device were more reproducible than manual methods for supernate collection.

To conserve sera, reduce technologist time and facilitate distribution of kidneys to the most likely recipient from groups of highly sensitized patients, we studied the feasibility of using pooled sera in preliminary crossmatches. From 16 chronic renal failure patients, 275 sera were studied against fresh cell panels from 24 to 35 donors. Using the antiglobulin crossmatch technique, sera were studied individually, in consecutive pools of five and in consecutive pools of 10. Reactions of component sera were assessed on a cell-by-cell comparison with their pools of five and 10 sera. Crossmatches performed with pools of five sera and pools of 10 sera detected the same cytotoxic reactions as did their component sera in 97.6% and 97.8% of the reactions, respectively. We conclude that 10 sera can be pooled so that preliminary crossmatch testing of all positive sera of all theoretically potential allograft recipients can be accomplished with accuracy and efficiency, even in the larger regional distribution centers.

The phenanthridine dye, ethidium bromide, which is actively excluded by viable cells, undergoes a significant fluorescence enhancement at 5900 A upon binding intracellular double-stranded polyribonucleotides. A rapid and sensitive assay of antibody mediated cytotoxicity to cells grown in vitro has been developed using this phenomenon. In this communication, we describe this fluorescence probe cytotoxicity assay and a sensitive electro-optical system designed to measure the fluorescence enhancement of ethidium bromide as it intercalates with intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide and non-viable cells are presented as well as examples of this assay as it has been used to study surface membrane neoantigens of cells tranformed by the oncogenic DNA virus, SV40.

Variants of the method for determination of the natural killer cells (NK-cells), and monocytes-macrophages both in human beings and in mice have been described. Usage of agarose which permits fixing conjugates of the effector cells and a tumour target cell allows determining simultaneously the quantity of effector cells bound with the target and percentage of the cytotoxic effectors. The results in acceptable definition of the effector cells' activity with the use of various kinds of plastic ware, its treatment, diverse types of agarose, temperature and time regimen are adduced. The di-target method of testing is approved using the target cells labelled with fluorescein as well as unlabelled ones. Peculiarities of function disturbances of the NK-cells and monocytes in patients with lung cancer as well as changes in the activity of these effectors in mice CBA depending on the terms of Krebs-2 carcinoma inoculation and on repeated BCG vaccine injection were detected.