Prelabeled Target Cells Research Articles

Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays

In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., 51Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness.

In vitro effects of leukocyte interferon on the cytotoxic potential of human monocytes.

The in vitro cytotoxicity of human monocytes was enhanced by leukocyte interferon (IFN-alpha). In contrast to lymphokine activation which is obtained gradually under long-term contact with monocytes, maximal activation of effector cells was obtained within 4 hours of culture with IFN-alpha prior to medium exchange and target cell addition. The degree of activation was clearly dose-dependent, reaching a plateau level of cytolysis at 400 U/ml of IFN-alpha. In cytostasis no such plateau was demonstrable. IFN-alpha enhanced the level of cytolysis mediated by human monocytes without inducing any change in the kinetics of lysis, regardless of whether IFN-alpha was used for activation of monocytes prior to or simultaneously with the cytolysis assay. The direct effect of IFN-alpha on K-562 target cells was sparse. The incorporation of thymidine was modestly depressed in the presence of IFN-alpha. Exposure of K-562 cells to IFN-alpha did not have any effect on the spontaneous isotope leakage from prelabelled target cells. The exposure of K-562 cells to IFN-alpha prior to the start of co-culture with monocytes did not change the level of cytolysis reached, either with non-activated or with IFN-alpha activated monocytes, and irrespective of the in vitro age of the monocytes. The effect of IFN-alpha thus seems to be mediated via the effector cell population and not via target cell modulation.

Synergistic Effect of Cortisol and HIV-1 Envelope Peptide on the NK Activities of Normal Lymphocytes

In order to examine the potential role of stress hormones and circulating HIV-1-derived products in the progression of HIV infections, we developed an in vitro model system that investigates the effects of cortisol and HIV soluble gene products on the natural killer cell activity of normal lymphocytes. The system employs a 4-h 51Cr release assay and K562- and LAV-infected 8E5/LAV target cells. Direct addition of cortisol at 0.05, 0.1, and 0.2 μg/ml or the HIV recombinant peptide, env-gag, at 1, 10, and 50 ng/ml separately to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on NK cell activity against either target. However, cortisol or env-gag at concentrations that did not produce any inhibitory effect on NK activity when used separately, manifested significant inhibitory effects when added in combination. Suppression was evident at concentrations as low as 1 ng/ml of env-gag and 0.05 μg/ml of cortisol and was observed at different effector:target cell ratios. Suppression was not caused by nonspecific toxicity of cortisol or HIV peptides when added in combination to the effector cells nor was due to decreased susceptibility of targets to lysis by effector cells. A non-HIV viral antigen (Rubeola virus) and another HIV-1 envelope-derived sequence (env 578-608 aa) were used as controls separately or in combination with cortisol and did not produce significant inhibition thus demonstrating the specificity of env-gag-induced inhibition. The synergistic inhibitory effect of cortisol-and HIV-derived soluble products in patients with HIV infections are consistent with a model that proposes that stress and circulating HIV-1-derived products may be involved in the progression of HIV infections.

Selective inhibitory effects of stress hormones on natural killer (NK) cell activity of lymphocytes from AIDS patients.

To examine the potential role of stress hormones in the progression of HIV infections, we developed an in vitro model system that investigates the effects of cortisol, adrenocorticotropin-releasing hormone (ACTH) and beta-endorphin on the natural killer cell activity of lymphocytes from normal subjects and AIDS patients. The system employs a 4 hr 51Cr release assay and K562 target cells. Direct addition of cortisol (0.05, 0.1, and 0.2 microgram/ml) or ACTH (10(-6) to 10(-8) M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of normal lymphocytes. Direct addition of beta-endorphin (10(-13) to 10(-17) M) to the mixture of effector and prelabeled target cells did not produce any significant immunoregulatory effects on the NK cell activity of lymphocytes from normal or AIDS subjects. However, cortisol and ACTH significantly inhibited the NK activity of lymphocytes from AIDS patients. The selective inhibitory effects of cortisol and ACTH in patients with HIV infections are consistent with a model which proposes that stress related neurohormones and/or neuropeptides may be involved in the progression of HIV infections.

ENHANCED MACROPHAGE CYTOTOXICITY AGAINST TUMOR CELLS TREATED WITH PHOTODYNAMIC THERAPY

In vitro treatment of human lung adenocarcinoma cells A549 with photofrin-based photodynamic therapy (PDT) resulted in the potentiation of macrophage-mediated killing of these cells assayed either by measuring 3H-thymidine release from the prelabeled target cells, or by determining the survival of A549 cells based on colony formation. The effector cells in these experiments were human promyelocytic leukemia cells HL60 induced to differentiate into macrophages. Very similar results were obtained with the murine squamous carcinoma SCCVII cells treated with PDT and subsequently admixed with mouse peritoneal macrophages. This effect increased with PDT dose and reached its maximum (i.e. complete or nearly complete release of the radioactive label) with the photodynamic treatment that was lethal to 40-50% of cells. In contrast, the PDT treatment of normal mouse kidney cells resulted in only a very limited enhancement of their cytolysis by mouse peritoneal macrophages. The exposure of A549 cells to X-ray irradiation had not affected the macrophage-mediated killing of these cells. The PDT survival curves of A549 cells cultured either alone or with the effector cells showed that the presence of macrophages even at very low effector: target cells ratios enhanced the PDT response of tumor cells. The enhancement ratio of 3.6 (at S = 0.01) was achieved with the effector: target cell ratio 2.5:1, which was the highest ratio tested with this assay. It is suggested that macrophages may recognize potentially repairable damage induced by PDT in tumor cells (presumably lipid fragments exposed in damaged cellular membranes), which helps them to identify the affected cells as their targets.

Chromium and Tritiated Thymidine Releases from Target Cells Are Differential Events in Human Monocyte/Macrophage-Mediated Cytotoxicity

Cytotoxic properties of human activated macrophages were investigated by measuring both 51Cr and [3H]TdR releases from prelabeled target cells. The kinetic study of macrophage-mediated cytotoxicity indicated that 51Cr and [3H]TdR releases showed a distinct time course. Next, when [3H]TdR release was measured as a parameter for cytotoxicity, pretreatment of activated macrophages with either actinomycin D or emetine remarkably decreased their cytotoxicity in a dose-dependent manner. In contrast, the pretreatment with these inhibitors had little effect on 51 Cr release. Furthermore, the addition of Zn2+ in the assay medium caused the decrease in [3H]TdR release but not 51 Cr release from the target cells. Taken together, our findings indicate that 51Cr and [3H]TdR releases from the target cells are induced by activated macrophages through different cytotoxic mechanisms; the former does not require RNA and protein syntheses in macrophages during coculture, whereas the latter requires them. Our findings also suggest that macrophage-derived nuclease may play an important role for inducing [3H]TdR release from the target cells.

Cytotoxicity assays using cryopreserved target cells pre-labeled with the fluorescent marker europium

We describe a method for standardizing cytotoxicity assays by the use of cryopreserved, fluorescently labeled target cells. The cells are labeled in batches with the fluorogenic element europium (Eu) and frozen in multiple aliquots. Replicate aliquots can be thawed on different days and used for cytotoxicity assays. Thawed pre-labeled cells from the same batch are killed reproducibly, allowing cytotoxicity assays to be standardized. The target cells need only be thawed, washed, counted for viability and used. The availability of pre-labeled, cryopreserved targets facilitates the assessment of cytotoxic activity of individual donors at different times. The method also simplifies the use of large panels of target cell types. By using this procedure it is not necessary to maintain active cultures of target cells, nor is it necessary to use and dispose of radioisotopes. These pre-labeled target cells can be used in various assays where the result is a destroyed target cell such as cell-mediated cytotoxicity, complement mediated cytotoxicity, or antibody-dependent cellular cytotoxicity.

Microassay for photometric quantitation of macrophage mediated tumor cytotoxicity using an automated densitometer

A simple and reproducible microassay for the quantitation of macrophage mediated cytotoxicity is described. The method is based on the measurement of absorbance at 630 nm of residual Giemsa stained target cells and effector macrophages using an automated densitometer. Applying this novel method, it was possible to demonstrate time dependent growth characteristics of C3H/MCA and BHK/Py target cell lines. Using C3HeB/FeJ or C3H/HeJ murine effector macrophages and a syngeneic transformed fibroblast target cells (C3H/MCA or 3T12), the method was further applied to demonstrate: (1) dose related activation of macrophages by lipopolysaccharide (LPS) and by macrophage activating factor (MAF); (2) synergistic augmentation of MAF-mediated macrophage cytotoxicity by LPS; (3) unresponsiveness of C3H/HeJ macrophages to LPS; and (4) increased cytotoxicity with increasing effector: target cell ratios. Guinea pig peritoneal macrophages were also shown to produce enhanced LPS or MAF-mediated cytotoxicity for C3H/MCA or BHK/Py target cells. The novel method was shown to compare favorably with results obtained by cytotoxic release of [ 3H]thymidine from prelabeled target cells. The advantages of the method are: (1) the elimination of the need for radioactive materials; (2) the ability to perform quantitation directly in microtiter plates; (3) the relative ease and rapidity in which experiments may be performed and quantitated; (4) its sensitivity and reproducibility; and (5) the ability to simultaneously quantitate and observe the biological events either microscopically or macroscopically.

Macrophage activation for tumor cytotoxicity: reactivity of peritoneal and bone marrow macrophages.

Peritoneal macrophages recovered from mice infected with intracellular parasites such as Mycobacterium bovis, strain BCG,Toxoplasma gondii or Listeria monocitogenes exert non specific cytotoxic activity against tumor cells in vitro (1 ,2).Macrophage cytotoxicity is selectively expressed against neoplastic cells.Tumor cells,derived from viral or chemical carcinogen-induced or spontaneous syngeneic and non syngeneic tumors,were found to be quantitatively more susceptible to macrophage cytophatic effects than were non neoplastic cells.Tumor cell killing requires cell to cell contact;soluble cytotoxic factors released by macrophages or by macrophage-tumor cell interactions have not been described.A cinemicrographic analysis revealed that macrophage-tumor cell interactions are characterized by repeated and relatively short (2 hr.) contacts inducing permanent tumor cytostasis and degenerative morphological changes (3).The development of a cytotoxicity assay based on radioisotope release facilitated quantitative analysis of macrophage tumoricidal activity (4).Tumor cell death was evaluated as release of tritiated thymidine from prelabeled target cells into supernatant culture fluids BCG-activated macrophages induced significant release of 3HTdR from tumor cells by 24 hours;however,optimal measurement of tumor cytotoxicity (50% total counts) was observed after 48–72 hours.Macrophages from uninfected mice or from oil-, starch- or thioglycollate induced peritoneal exudates had little effect on labeled tumor mono-layers.

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysiś by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.

Effect of chemotherapeutic agents on natural and BCG-stimulated macrophage cytotoxicity in mice

Spontaneous or Bacillus Calmette Guérin (BCG)-stimulated macrophage cytotoxicity was investigated in the peritoneal cavity of BALB/c × DBA/2 mice treated with various chemotherapeutic agents. The SV40-transformed mKSA TU5 (TU5) line and the mFS6 sarcoma were used as targets. Cytolytic activity of macrophages from individual mice (4–6 per experimental group) was measured as release of [ 3H methyl] thymidine from prelabelled target cells. Treatment with azathioprine (a single injection of 100–400 mg/kg or 5 daily doses of 10–80 mg/kg s.c.) and dimethyltriazenoimidazole carboxamide (a single injection of 45–180 mg/kg or 5 daily doses of 3–30 mg/kg i.v.) resulted in marked dose-dependent inhibition of spontaneous macrophage cytotoxicity against TU5 tumor cells 2 days later. By 7 days after drug treatment spontaneous cytotoxicity of macrophages had returned to normal levels. In contrast adriamycin (a single dose of 5–10 mg/kg or 5 daily doses of 0.6–2.5 mg/kg i.v.) and cyclophosphamide (a single dose of 50–200 mg/kg or 5 daily doses of 6–25 mg/kg i.v.) did not affect spontaneous cytolytic activity of murine macrophages. BCG-stimulated macrophage cytotoxicity was investigated using mFS6 target cells which were relatively resistant to the spontaneous levels of macrophage-mediated cytolysis; the same pattern of effects was observed, azathioprine and dimethyltriazenoimidazole carboxamide inhibiting cytolytic activity levels in contrast to adriamycin and cyclophosphamide. These results are discussed in terms of the known effects of these agents on other functions of mononuclear phagocytes and on other immune reactivities.

Cytolytic and cytostatic activity on tumor cells of circulating human monocytes

AbstractMonocytes from the peripheral blood of normal adult human donors were found to have appreciable levels of cytotoxic activity against murine and human tumor‐cell lines. Adherent cells ( > 90% monocytes) were obtained from the peripheral blood of 29 normal healthy volunteers either by adherence on plastic and scraping with a rubber policeman or by adherence on microexudate‐coated plastic and exposure to ethylene diamine tetra‐acetic acid. Cytolytic capacity was tested by incubating effector cells for 72 h with murine and human tumor cell lines prelabelled with tritiated thy‐midine. Cytostasis was evaluated by inhibition of [125I]iododeoxyuridine (125IdUrd) uptake. Tumor target cells employed were: a murine SV40‐trans‐formed kidney line (TU5), a murine chemically‐induced sarcoma (1023), a human breast‐cancer‐derived cell line (G11) and a human lung‐cancer‐derived cell line (CaLu). Monocyte preparations at attacker to target cell ratios of 1:1 to 40:1, showed significant cytolytic and cytostatic activity against tumor target cells. Tumor cells showed different susceptibility to cytolytic activity, whereas comparable levels of cytostasls were observed with the various targets: TU5 and G11 tumor cells were more susceptible than 1023 and CaLu target cells to the cytolytic capacity of human monocytes and TU5 was used in most subsequent experiments. Peak isotope release from prelabelled target cells was observed after 72 h of incubation, whereas peak inhibition of [125I]dUrd uptake occurred after 24 h of culture. The cytotoxic capacity of monocytes isolated by either of the two methods mentioned above was similar. The monocytes had higher cytotoxic activity than unseparated mononuclear cells, and non‐adherent cells showed minimal cytotoxic effects. Cytotoxicity by natural killer cells did not appear to have a major role in these assays, since adherent cells did not lyse K562 cells in a 4‐h 51Cr release assay. Treatment with anti‐human T‐cell serum and complement did not inhibit the cytotoxic capacity of the monocyte preparations, whereas exposure to silica particles significantly inhibited the cytotoxic activity. Monocyte‐mediated cytotoxicity on tumor cells was expressed in the presence of both fetal bovine serum and human AB serum.

Cytolytic activity of circulating human monocytes on transformed and untransformed human fibroblasts

Monocytes from the peripheral blood of 10 normal adult human donors were tested for their cytolytic activity on untransformed or SV40-transformed, early passage, human fibroblasts. Two pairs of human fibroblast lines (2303 and SV40-2303, 2931 and SV40-2931) were used and lysis was assessed in terms of release of [3H]thymidine from prelabelled target cells over a 72-h incubation period. High levels of cytolytic activity were consistently observed on transformed fibroblasts at attacker to target cell (A:T) ratios ranging from 5:1 to 40:1. Low, but significant, lysis of untransformed cells was observed only with two donors at the highest (40:1) A:T ratio tested. With all donors, the cytolytic activity on transformed lines was significantly greater than on untransformed fibroblasts. Thus natural human monocyte-mediated cytotoxicity is preferentially expressed on transformed, as compared to untransformed, cells although sparing of the latter is not absolute.

680 RESISTANCE OF CULTURED CHORIOCARCINOMA CELLS TO CYTOTOXIC LYMPHOCYTES

Cultured choriocarcinoma cells (BeWo) exist which share many of the morphologic and biosynthetic properties of normal human trophoblasts.In an attempt to develop a model for the immunologic relationship between mother and fetus, we mixed BeWo cells with mitogen-activated cytotoxic lymphocytes in vitro. Microscopic observations revealed that BeWo cells exhibited a marked resistance to the cytolytic effects of the activated lymphocyte in spite of 24 hour exposure and intimate cell to cell contact. The control target cells, a line of human hepatoma cells, were readily destroyed. A quantitative cytotoxicity assay was devised which involved measuring the residual radioactivity of 3-H-Thymidine prelabeled target cells following exposure to activated lymphocytes. Employing this assay, we confirmed the morphologic results and showed that BeWo cells were resistant to the cytotoxic effects of lymphocytes activated by phytohemagglutinin, pokeweed, and allogeneic cells in mixed lymphocyte cultures. Moreover, BeWo were resistant to injury over a wide range of killer to target cell ratios. Significant killing of the BeWo cells occurred only after prolonged exposure (48 and 72 hours) to the activated lymphocytes. Exposing the target cells briefly to trypsin augmented the killing of control cells but not BeWo. These observations suggest that one mechanism which may assist the fetus (or a choriocarcinoma) in its immunologic survival is the intrinsic resistance of trophoblast to lymphocyte mediated cytotoxicity.

A Cytotoxic Proteinase Isolated from Human Lymphocytes

A proteinase active at physiologic pH was isolated from unstimulated human peripheral blood lymphocytes with gel filtration and affinity chromatography. The proteinase with a molecular mass of approximately 30,000 daltons was completely inhibited by diisopropylfluorophosphate (DFP) and soybean trypsin inhibitor (STI). Incubation of the lymphocyte enzyme with 3H-proline labeled T24 human bladder carcinoma cells resulted in significant cytoxicity of the target cells. Cytotoxicity was only observed with much higher concentrations of trypsin. Similar results were obtained with a 51Cr release assay. This investigation demonstrates that unstimulated human peripheral blood lymphocytes contain a cytotoxic proteinase capable of killing pre-labeled target cells. The proteinase may be involved in lymphocyte-mediated cytotoxicity.

Macrophages isolated from regressing Moloney sarcomas are more cytotoxic than those recovered from progressing sarcomas.

THERE is increasing evidence that many tumours contain appreciable numbers of macrophages1–3, and that these inflammatory cells can be cytotoxic for various neoplastic cells in vitro4,5. Thus at first it seems paradoxical that many tumours are progressive and lethal in spite of a high content of macrophages. But to kill a malignant cell, these inflammatory cells must be brought to an ill-defined state of ‘activation’6,7. It is therefore conceivable that the macrophages in progressing tumours are less activated, and thus less cytotoxic, than those in spontaneously regressing neoplasms. To test this hypothesis we isolated macrophages from either regressing or progressing Moloney sarcomas and tested them in vitro for their capacity to release chromium-51 (51Cr) from prelabelled target cells. The former were usually significantly more cytotoxic than the latter.

Detection of cytotoxic lymphoid spleen cells from STU-mice with Moloney sarcoma by a 3H-proline microcytotoxicity assay.

A microcytoxicity assay with 3H-proline prelabeled target cells was used for the detection of sensitized lymphoid spleen cells from STU inbred mice inoculated with Moloney sarcoma virus (MSV-M) or ascitic MSV-M tumor cells. The target cell line was derived from ascitic MSV-M tumor cells. With regard to the specificity of the assay nonimmune slpeen cells displayed no or only a weak cytotoxicity against these cells, and this was also the case when 3H-proline-labeled secondary cultures of syngeneic mouse embryo cells were exposed to both sensitized and nonimmune spleen cells. The time-course pattern of the development of cytotoxic lymphoid spleen cells in STU mice inoculated intramuscularly either with MSV-M or ascitic MSV-M tumor cells was studied. At the stages of tumor development, peak tumor size, and tumor regression the lymphoid spleen cell preparations were found to have relatively strong cytotoxic activity independent of whether the tumor was induced by MSV-M inoculation or tumor cell transplantation. However, in the latter case effector cells appeared earlier and were demonstrable for a longer period than in MSV-M-inoculated mice. Anti-theta serum treatment of lymphoid spleen cells taken at the stage of peak tumor size abrogated the cytotoxic activity or diminished it considerably indicating a T-lymphocyte response.

Interaction of BCG-activated macrophages with neoplastic and nonneoplastic cell lines in vitro : quantitation of the cytotoxic reaction by release of tritiated thymidine from prelabeled target cells.

Peritoneal cells from mice infected ip with Mycobacterium bovis, strain BCG, were cytotoxic to syngeneic tumor cells in vitro. Cytotoxicity was estimated by measurement of release of tritiated-thymidine (3-H-TDR) from prelabeled target cells. The cell responsible for tumor cytotoxicity was the macrophage. Macrophages from uninfected mice or from oil-, starch-, or thioglycollate-induced peritoneal exudates had little effect on labeled tumor monolayers. Tumoricidal macrophages were present at 3-7 days and persisted through 6 weeks after a single BCG injection. Two neoplastic/nonneoplastic cell-line pairs, all four of the cell lines derived from a cloned syngeneic embryo cell line, were used as target cells for BCG-activated macrophages. Both tumor cell lines released significantly more 3-H-TDR than did the two nonneoplastic lines. In a mixed neoplastic/nonneoplastic target cell population, BCG-activated macrophages selectively destroyed the neoplastic cells; nonneoplastic cells were not affected as "innocent bystanders".

Differential Cytotoxicity of Tumorigenic and Nontumorigenic Strain-2 Guinea Pig Cells as Mediated by Syngeneic Phytohemagglutinin-Stimulated Peritoneal Exudate Cells

Phytohemagglutinin (PHA)-stimulated peritoneal exudate (PE) cells from strain-2 guinea pigs were more cytotoxic in culture to syngeneic tumorigenic cells than to syngeneic nontumorigenic cells. Cytotoxicity was measured by the release of 3H-thymidine from prelabeled target cells. Tumor-producing guinea pig fetal cells transformed in culture by chemical carcinogen released up to 70 percent of their label in the presence of PHA-stimulated PE cells. Non-tumor-producing cells, regardless of their morphologic characteristics, were less affected by PHA-stimulated PE cells. Nontumorigenic cultures included untreated early passage and long-term cultures previously treated with a carcinogenic or noncarcinogenic chemical. Differential cytotoxicity to tumorigenic cultures were best distinguished from nontumorigenic cultures when 0.5-1 times 10-6 PE cells and 50-100 mug PHA were incubated with 1 times 10-4 3H-thymidine-labeled target cells for 48 hours.