Fluorochromasia lymphocytotoxicity assay for detection of natural killer cell activity using fluorescein-activated cell sorter

A new fluorochromasia lymphocytotoxicity assay using a fluorescein-activated cell sorter (FACS) was developed for detecting natural killer cell (NK) activity. Carboxy-fluorescein-diacetate (C-FDA) labeled K 562 cell was used as the target cell. An optimal labeling condition is incubation with 25 micrograms/ml of C-FDA for 1 hour to separate target cells from effector cells by FACS. These labeled cells were cocultured with peripheral blood mononuclear cells (PBMC) as effector cells or with heat-activated PBMC as control cells. At various effector/target cell ratios, the number of C-FDA positive cells determined by FACS showed good reproducibility (coefficient of variation ranged from 0.9 to 9.1%), and an incubation period of 4 hours was sufficient for lysis. At the end of the lysis period, the number of target cells cultured with effector cells (A) or with control cells (B) was determined by FACS. Percent NK activity was calculated according to the following formula: (1-A/B) x 100. An adequate correlation between NK activity assayed by the 51Cr-method (x) and C-FDA (y) on the same cell population simultaneously was obtained (r = 0.89, the regression curve was y = 0.86 x + 3.74). C-FDA assay using FACS appears to be a good alternative to the 51Cr assay in the detection of NK activity.     

Natural killing of cytomegalovirus-infected fibroblasts by human mononuclear leucocytes.

The ability of human peripheral blood mononuclear (MN) cells to lyse uninfected and cytomegalovirus (CMV) infected human fibroblasts was determined in a 51Cr-release assay. Maximal release was obtained with 6-day infected fibroblasts incubated with MN cells for 24 hr. A linear relationship existed between E/T ratios of 12.5:1 to 100:1 and lysis of CMV-infected targets. Donor immune status had no effect on the magnitude of killing of infected or uninfected targets. Killing was mediated by non-B, predominantly non-T, Fc receptor-bearing cells. Preincubation of effector cells with interferon enhanced killing of both CMV-infected and uninfected fibroblasts, but infected targets were more effectively killed. These results indicated a possible role for natural killer cells in recovery from CMV infection.     

Sensitivity of B16 melanoma sublines to lymphokine-activated killer cells as determined by 51Cr-release and clonogenic assays

The aim of this study was to compare the differential sensitivities of B16 melanoma sublines to LAK cells by means of the standard 51Cr release assay and a clonogenic assay, which measures both cell survival and proliferation. LAK cells, generated after 4 days incubation with 150 international units (IU)/ml of interleukin-2 (IL-2), showed both cytolytic and anti-proliferative activities against B16 targets. Using an 18 h 51Cr release assay, murine LAK cells showed the highest cytolytic activity against B16 parental cells compared to B16-F1, B16-F10, B16-FLR and B16-BL6 sublines at effector/target (E/T) ratios ranging from 6/1 to 100/1. Purified adherent LAK (A-LAK) cells showed greater cytolytic activity against B16 parental cells and other B16 sublines compared to LAK cells, but otherwise the pattern of reactivity was similar. Using a clonogenic assay, the surviving fraction of B16 parental cells co-cultivated with LAK cells decreased to 0 at an E/T ratio of 50/1, while a 400/1 ratio was required to achieve a similar reduction of B16-F1, B16-F10, B16-FLR, and B16-BL6 sublines. No differences in subline sensitivity were seen with the 51Cr release assay, but these were observed using the clonogenic assay. An inverse linear relationship existed between % surviving fraction, as determined by the clonogenic assay, and cytolytic activity, as determined by the 51Cr release assay. Our data indicate that the clonogenic assay can detect differences in target cell sensitivity that otherwise are undetectable by the standard 51Cr release assay. The clonogenic assay may prove useful in delineating the long-term anti-adherent and anti-proliferative properties of effector cells from their cytolytic activity.     

In vitro induction of tumor-specific immunity. VI: analysis of specificity of immune response by cellular competitive inhibition: limitations and advantages of the technique.

The cellular competitive inhibition 51Cr-release assay makes two distinct contributions to the in vitro study of cell-mediated immunity. It allows target cells which are not amenable to isotopic labelling to be investigated for their antigenic specificity, and it provides a means, complementary to the direct cytotoxicity assay, of estimating qualitative and quantitative differences in antigen expression on intact normal and neoplastic cells. Various parameters of a micro-51Cr-release inhibition assay have been studied, and it was found that the assay conditions markedly influenced both the sensitivity and specificity. It is concluded that optimal assay conditions for specificity include: 1) moderate levels of lysis on the linear part of the CL/T titration curve, 2) avoidance of prolonged assay times, and 3) low ratios of blocker to target cells. When tumor cells with large cell volumes are used as competitive inhibitor (blocker) cells, non-specific blocking will occur; limits have been defined for this particular micro-inhibition assay which, in general, exclude these effects.     

Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release.

The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes (CTL) generated in vitro in mixed leukocyte cultures (MLC), were incubated for various periods of time at 37 degrees C with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37 degrees C, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51Cr-release assay involving EDTA addition, gave comparable results to the cloning inhibition assay. These results raise the possibility that the events leading to 51Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability.     

Stable induction of a 51K cellular protein in neuronal cells surviving herpes simplex virus type 1 infection

A series of survivor cell lines derived by infection of B103 rat neuroma cells with active wild-type herpes simplex virus type 1 (HSV-1) (M. Levine, A. L. Goldin, and J. C. Glorioso, J. Virol. 35, 203-210 (1980)) has been isolated. The survivor cells produced no infectious virus, yet they continued to react with HSV-1 antiserum for over 100 cell generations following the initial infection. The reactivity of the survivor cells with HSV-1 antiserum is characterized as being due to expression of a 51K protein. The 51K protein reacted with antiserum prepared against HSV-1 virions and was not detectable in the parental B103 cells. A protein of the same molecular weight was seen in productively infected B103 and HEL cells. The protein detected in the survivor cells comigrated with that seen in the infected cells on two-dimensional gel electrophoresis, indicating that they represent similar proteins. Despite the presence of the 51K protein reactive with HSV-1 antiserum, the survivor cells contain no detectable HSV-1 DNA sequences. They do contain DNA sequences which cross-hybridize with HSV-1 DNA, but similar cross-hybridizing sequences were also present in the parental B103 cells. No hybridizing polysomal, polyadenylated RNA species were present in the survivor cells that were not present in the parental B103 cells when probed with the cross-hybridizing HSV-1 restriction fragments. Therefore, the 51K protein evidently represents a cellular protein induced by the HSV-1 infection.     

Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays

Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive (51)chromium ((51)Cr) release assay is a "gold standard" for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland-Altman statistical method was applied to measure true agreement for all CAM-(51)Cr, CFSE-(51)Cr, DiO-(51)Cr and MTG-(51)Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard (51)Cr release assay. Considering linear relationships between data obtained with four fluorochromes and (51)Cr release assay as well as linear regression analysis with R(2)=0.9393 value for CAM-(51)Cr pair, we found the CAM assay to be the most closely related to the (51)Cr assay.     

Radioisotopic 15Cr-Leukocyte adherence inhibition (LAI) assay. I. Demonstration of anti-tumor immunity in patients with breast carcinoma

a simplified radioisotopic leukocyte adherence inhibition assay (⁵¹Cr-LAI assay) was used to determine tumor-directed immune responses in patients with cancer of the breast. Essential steps in development of this assay are the standardization of conditions for optimal ⁵¹Cr uptake by peripheral blood lymphocytes (PBL) and the inclusion of autologous or normal AB serum in the incubation media. A dextrose salt mixture (GNK) was found to enhance intracellular uptake of ⁵¹Cr significantly (8-fold) without affecting viability of the cells or without causing selective loss of lymphocyte subpopulations. The presence of 10% autologous or normal AB serum prevented non-specific LAI responses to unrelated tumor antigens.In a study of 46 preoperative patients with suspected breast cancer, clear and accurate prediction of the presence of cancer was achieved with this new assay. All patients with localized breast cancer showed significant adherence inhibition in response to allogeneic breast tumor extracts whereas normal control women and patients with benign disease did not respond. Neither patients with cancer nor those with benign breast diseases reacted to extracts of benign breast tissue antigens. LAI reactivities appeared to be directed selectively against tumor-associated antigens (TAA) and reflect specific anti-tumor immunity.This short term (4 h) ⁵¹Cr-LAI assay provides reproducible and specific results analogous to those using tube-LAI assay. The test has the advantages of being accurate, sensitive and free from technical bias.     

A novel technique to measure total IgM and IgG in vitro haemolysin production by mouse spleen cells,using 51Cr-labelled sheep red blood cells

A quantitative method for measuring in vitro production of IgM and IgG haemolysis is described. Immune mouse spleen cells, ⁵¹Cr-labelled sheep red blood cells, guinea pig complement and—where applicable—rabbit anti-mouse gammaglobulin serum are incubated in the fluid phase at 37°C, and the degree of chromium release measured in the supernatant. The assay gives reproducible results which compare well with the numbers of plaque-forming cells obtained in the conventional plaque-forming assay.     

Activation of human NKCC by moderate exercise: increased frequency of NK cells with enhanced capability of effector--target lytic interactions.

In the present study we examined the mechanism of human natural killer cellular cytotoxicity (NKCC) augmentation by 5 min of moderate exercise and its interrelationship to in vitro interferon (IFN) activation. Cytotoxicity was measured by employing both a single-cell cytotoxic assay and a standard 3-hr chromium-51 (51Cr) release assay. The former was used to assess changes at the single NK cell--target cell level and the latter to assess changes in overall lytic capacity of a given population of NK cells. Several findings were obtained: (1) moderate exercise augmented NKCC in vivo by recruiting a 'new' population of active cytotoxic NK cells. (2) This 'new' population of active cells probably was derived from cells which can bind targets but are non-cytotoxic. (3) In a standard 51Cr-release assay, additional augmentation of these exercise-activated cells occurred in vitro following exposure to interferon. (4) This additional increase in cytotoxicity produced no alteration in the frequency of killer cells as viewed at the single cell level. (5) Thus interferon's capacity to increase further the overall lytic ability of exercise-activated NK cells was not due to its activation of an additional subset of pre-NK cells, but due to its increasing the capacity of effector--target lytic interactions (recycling) of the same set of NK and pre-NK cells.     

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.     

~(51)Cr释放法检测杀伤性T细胞的细胞毒作用和评价

本文报道了国产铬酸钠(Na_2~(51)CrO_4)标记P815细胞的条件,和用~(51)Cr释放法检测杀伤性T细胞(CTL)的细胞毒作用。~(51)Cr标记P815细胞的适宜条件是:2×10~5个P815细胞加入100μCi~(51)Cr,37℃恒温下温育2小时。效应细胞与靶细胞作用的条件是:效靶比100:1,作用时间4.5小时。C57小鼠脾脏CTL的特异杀伤作用在P815细胞致敏后第7天开始出现,第11天达高峰,以后开始下降。环磷酰胺50mg/kg可明显抑制小鼠CTL的细胞毒作用。     

Induction of specific cytotoxic lymphocytes in mice vaccinated with Brucella abortus RB51

A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional 51Cr release assay for detection of Brucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated that Brucella abortus strain RB51 vaccination of mice induced specific CTLs against both strain RB51- and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3+ CD4+ T cells secreted the highest level of gamma interferon (IFN-gamma) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3+ CD8+ T cells secreted low levels of IFN-gamma but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis. These findings indicate that B. abortus strain RB51 vaccination of mice induces specific CTLs and suggest that CD3+ CD4+ and CD3+ CD8+ T cells play a synergistic role in the anti-Brucella activity.     

Automated fluorometric assay for T cell cytotoxicity

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.     

A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of transplanted pig organs. The purpose of this study was to establish a suitable in vitro method that would allow for screening and comparison of various agents and methods potentially useful in the prevention of hyperacute rejection. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG08472), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytotoxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conventional 51Chromium (51Cr)-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotoxicity assay performed in Terasaki trays. Using the microcytotoxicity assay, a 1-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studied, PK15 cells were the most sensitive to cytotoxic injury, followed by AG cells and the primary endothelial culture. Good correlation between the 51Cr-release and the two-color fluorescence method was documented. There was good agreement between the results obtained using the 2-chambered slide method and the microcytotoxicity assay, as there was between the 1- and the 2-step assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluorescence assay is suitable for the rapid and inexpensive screening of therapeutic interventions that might be useful in the prevention of hyperacute xenograft rejection, and that PK15 cells are suitable for use in this assay.     

A fluorescence NK assay using flow cytometry

A flow cytometric NK assay was developed in which the K562 targets were labelled with the fluorogenic substrate, carboxyfluorescein diacetate (c'FDA). This new assay compared favourably with results obtained using the conventional 51Cr-release assay. c'FDA was not toxic to target cells and did not inhibit lysis. The assay permits the evaluation of various aspects of NK activity such as the activity of NK-enriched, IFN-alpha-activated, and ALG-inhibited populations. The assay can be used in place of 51Cr-release, and has the advantages of being able to directly monitor target cell lysis of reducing overall assay time, and the avoidance of radioisotope usage.     

A rapid and sensitive fluorometric microassay for determining cell mediated cytotoxicity to adherent growing cell lines.

In order to measure cell mediated cytotoxicity to adherent growing cell lines in vitro more rapidly and conveniently, a fluorometric microassay was developed and results were compared with those obtained by the 51Cr release assay. The fluorometric method is based on the hydrolysis of the fluorochrome 4-methylumbelliferyl heptanoate (MUH) by intracellular esterases of viable cells. Melanoma cell monolayers were incubated with lymphokine activated killer (LAK) cells for 4 h at various effector: target (E:T) cell ratios (E:T = 16, 8, 4, 2:1). Thereafter surviving adherent melanoma cells were stained with MUH for 30 min and fluorescence was measured directly in a 96 well plate reader. For the calculation of LAK cell cytotoxicity fluorescence values were corrected for the number of nonspecifically detached tumor cells during the washes and the number of nonspecifically adherent LAK cells. Using identical target and effector cell preparations both assays showed a nearly proportional increase of percentage cytotoxicity with rising numbers of lymphocytes. Compared with the 51Cr release assay, however, higher cytotoxicity values were obtained with the fluorometric MUH microassay: 57% with MUH versus 26% with 51Cr and 39% versus 14% for cell lines StML-11 and SKMel-28, respectively (E:T ratio = 16:1). The higher cytotoxicity rates obtained with the fluorometric MUH microassay were not due to the additional 30 min staining with MUH or due to nonspecific hydrolysis of MUH by extracellular esterases released from damaged cells, as could be shown by a series of experiments. In conclusion, a simple and rapid fluorometric microassay has been developed showing reliable reproducibility and a higher sensitivity compared with the 51Cr release assay for the determination of cellular cytotoxicity to adherent growing cell lines, avoiding hazardous radioactive labels.     

Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma.

Natural killer (NK) and lymphokine-activated killer (LAK) cell activities were measured in peripheral blood obtained from healthy women to compare a standard 51Cr release assay with a nonradioactive europium (Eu3+) release assay based on time-resolved fluorescence. The two types of cytotoxicity assays were first compared in paired determinations performed on 28 samples of peripheral blood mononuclear cells obtained from healthy women who had normal pap smears or no biopsy evidence of cervical squamous intraepithelial lesions (SIL). Target cells (NK-sensitive K562 and NK-resistant Raji cell lines) were labeled with Eu3+ only, 51Cr only, or both labels and compared in cytotoxicity assays using fresh or interleukin 2 (IL-2)-activated effector cells. Spontaneous release in the Eu3+ release assay was comparable to that observed in the 51Cr release assay, but maximum Eu3+ release always exceeded that of 51Cr. In 4-h assays, specific release of Eu3+ from target cells was more rapid than that of 51Cr, consistently resulting in 30 to 40% higher levels of activity. However, a significant linear correlation (P < 0.001) was observed between cytotoxicity levels based on measurements of Eu3+ and 51Cr release in 4-h assays. The Eu3+ release assay was then used to measure NK and LAK activities in the peripheral blood of women with cervical SIL or cervical squamous cell carcinoma (SCC). Mean NK activity of women with advanced SIL (121 lytic units [LU]) or SCC (93 LU) was found to be similar to that of controls (101 LU) or patients with normal cervical biopsies (90 LU), as was the ability to generate IL-2-stimulated NK activity. However, LAK activity during 18 h of incubation in the presence of IL-2 was reduced in patients with cervical SCC (P < 0.05) compared with that in normal controls. Results of 51Cr assays performed in parallel with patient samples gave comparable results. Advantages of EU3+ release assays for routine evaluation of cytotoxicity are discussed.     

Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence.

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay.     

Application of Non-Radioactive Europium(EU3+) Release Assay to a Measurement of Human Natural Killer Activity of Healthy and Patient Populations

Europium (Eu³⁺) release assay is a non-radioactive method for a measurement of cytotoxicity of lymphocytes and has several advantages compared with a conventional ⁵¹Cr release assay. However, the Eu³⁺ release assay has not been applied to a natural killer (NK) activity measurement of a large number of the human population mainly due to a lack of comparability with the ⁵¹Cr release assay. With some modifications of the procedures and careful manipulation of cells, constant and reproducible results were obtained by the Eu³⁺ release assay. NK activity of several individuals was measured by the Eu³⁺ release assay and was compared with data obtained by ⁵¹Cr release assay performed simultaneously. The obtained values by the two methods were almost identical. We applied the Eu³⁺ method to measure NK activity of a large number of individuals, including 68 apparently healthy donors and 36 autoimmune and 21 cancer patients. Some of these diseases are known to show abnormal NK activity. The obtained cytotoxicities were mostly consistent with the previously reported data obtained by the ⁵¹Cr release assay. These results indicated that the Eu³⁺ release assay could be used as an alternative method for a measurement of human NK activity of mass population including patients.