Increased sensitivity of murine leukemia virus-infected tumor cells to lymphocyte-mediated cytotoxicity

The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.     

Comparison of 51Cr release and microcytotoxicity assays against human melanoma cells

Comparisons of the cytotoxic activity of mononuclear cells from venous blood of human subjects against human melanoma cell lines have been made by means of the ⁵¹CV release assay (CRA) and the microcytotoxicity assay (MCA) of Takasugi and Klein. The relative effectiveness of the two assays in the detection of killing by various sub-populations of effector cells was studied after depletion of monocytes and separation of the blood monocluear cells by SRBC receptor and Fc receptor rosetting techniques. Overall, the results showed good correlation. Most differences were the result of low values recorded with the CRA when effector cells were obtained from Hypaque-Ficoll—separated populations which had not been further fractionated. These differences were largely removed when monocytes were depleted by glass absorption and may reflect interference with the CRA by monocytes. No consistent differences were found between the assays in the detection of killing by subfractions enriched for T cells or B cells in this tumour system. The MCA appears to offer the advantage of greater sensitivity and possibly detection of a wider range of cytotoxic mechanisms but the CRA has a number of advantages of a technical nature which make it more applicable to routine assays in clinical studies.     

Effects of human sera on reactivity of lymphocytes in microcytotoxicity assays.

The effects of cancer patient and normal donor serum samples on the reactivity of patient and normal lymphocytes against both normal and malignant target cells were studied in microcytotoxicity assays. There were 17 of 140 cancer patient serum samples and 7 of 116 normal donor lymphocyte samples that selectively increased the growth of target cells in the presence of lymphocytes. This effect was most often noted with cancer patient serum against cultured tumor cells, but the effect was also noted against fibroblasts and with normal serum against both fibroblasts and tumor cells. Of 140 cancer-patient serum samples, 11 selectively decreased target cell survival in the presence of lymphocytes compared to medium and compared to other serum samples. In the absence of lymphocytes these serum samples were nontoxic. The effect was not observed with any of the normal serum samples studied. The lymphocyte-dependent serum toxicity appeared to be selectively directed against tumor target cells.     

Cellular microcytotoxicity in human tumor systems: analysis of results.

Using the tritiated-proline microcytotoxicity assay with cultured target cells, we tested a large series of melanoma, breast cancer, and bladder cancer patients for the presence of cell-mediated immunity. Specific, disease-related activity was infrequently observed, since the patients' lymphocytes exhibited selective activity against both disease-related and non-disease-related target cells. Most normal controls also demonstrated selective activity against these target cells. Neither the length of time the target cells had been cultured in vitro nor technical aspects of the assay, including the lymphocyte preparation methods, seemed to account for our results. We concluded that the experimental design of these tests may be the critical factor responsible for many of the disparate results being observed in different laboratories.     

Problems in the clinical use of the microcytotoxicity assay for measuring cell-mediated immunity to tumor cells.

Lymphocytes from control donors and from cancer patients have been tested for antitumor, cell-mediated immunity against various melanoma and breast cancer target cell cultures with a microcytotoxicity assay. Control lymphocytes inhibited growth of target cells with high frequency, particularly with cell line, as opposed to short-term, cultures. Inhibition was not found for all target cells tested at a given time with a single preparation of lymphocytes. Sequential studies over a 2-year period with lymphocytes from the same control donors showed fluctuations of inhibition against the same target cells. With serial passage, the target cells also changed in their susceptibility to destruction by control lymphocytes. Lymphocytes from melanoma patients were more inhibitory than control lymphocytes for one melanoma target cell culture but not for two other melanoma cultures. Significant inhibition by lymphocytes from melanoma patients was not seen against two cultures derived from breast cancer patients. Lymphocytes from breast cancer patients were more inhibitory than control lymphocytes for 4 of 5 breast cancer cultures and they did not inhibit two melanoma cultures. Significantly specific inhibition was seen with short-term, but not cell line, breast cultures. The over-all data show specificity for target cells of the appropriate histological type. However, the high and inconstant reactivity of control lymphocytes in this assay suggests that nonspecific inhibition of tumor target cells by patient lymphocytes is found in many experiments. It is concluded that the microcytotoxicity assay is not suitable for clinical studies, since sequential data obtained in individual patients are difficult to interpret.     

Enhancement of lymphocyte-mediated cytotoxicity after tumor resection in patients with colorectal cancer

Lymphocyte numbers and function in 37 patients with colorectal cancer were compared with those in 23 healthy controls of the same age range. No significant difference in lymphocyte count, numbers of cells forming erythrocyte rosettes or bearing surface immunoglobulin, cell-mediated cytotoxicity, or mitotic response to phytohemagglutinin (PHA) was found. Six to 12 months after tumor resection, patients showed a rise in spontaneous, antibody-induced, and PHA-induced cytotoxicities to levels significantly higher than those found in age-matched controls. This rise occurred irrespective of whether patients had received adjuvant immunotherapy with Corynebacterium parvum.     

Plasma membrane and intracellular lipid synthesis in tumor cells rendered sensitive to humoral immune killing after treatment with metabolic inhibitors.

Line 10 guinea pigs hepatoma cells are resistant to killing by antibody and guinea pig complement. Metabolic inhibitors (adriamycin and actinomycin D) that increase the sensitivity of the cells to antibody-complement (C) killing were examined for their effects on the ability of the cells to synthesize and incorporate specific lipids into plasma membrane and intracellular membrane fractions. Drug-treated cells that had been rendered sensitive to antibody-C killing were inhibited in their incorporation of newly synthesized phosphatidylcholine and cholesteryl ester into the plasma membrane, as well as incorporation of phosphatidylcholine, cardiolipin, cholesteryl ester, and triglyceride into certain intracellular organelles including mitochondria, endoplasmic reticulum, nuclear membrane, or microsomes. Drug-treated cells recultured in the absence of the drug regained their ability to resist antibody-C killing and to synthesize and incorporate lipids into plasma and intracellular membranes. These data suggested that agents modifying the sensitivity of the tumor cells to humoral immune killing have a concomitant effect on plasma membrane and intracellular lipid synthesis.     

Marked stimulation of lymphocyte-mediated attack on tumor cells by target-directed liposomes containing immune RNA.

A marked stimulation of normal guinea pig lymphocytes was obtained by incubating them with liposomes that contained both antibody to lymphocytes to provide "homing" of the vesicles and immune RNA isolated from guinea pigs immunized with syngeneic line 10 hepatocarcinoma cells. Tumor cell cytotoxicity was monitored by a 51Cr release assay. This target cell delivery of immune RNA by liposomes produced a dose-dependent stimulation up to 12 times that achieved by in vitro methods with naked immune RNA.     

Kinetics of in vivo tumor cell destruction after specific immunization

The natural cytotoxic activity in vivo can be evaluated by measuring in vivo tumor cell destruction after injecting mice with 125IUdR-labeled tumor cells, measuring their total body radioactivity and calculating the % radioactivity lost. We have studied the in vivo destruction of 125IUdR-labeled L1210 leukemic cells by B10.D2 mice previously immunized 4 times with heavily irradiated L1210 leukemic cells. Mathematical analysis of our results indicates that the radiolabel loss on day 1 is similar in normal and immunized animals, but that it stays greater over the following days in immunized animals, indicating that the difference between the two groups is not the extent of the initial cell destruction but the durability of the response. There is a good correlation between the eventual survival, the % of 125IUdR lost and the number of tumor cells present in the peritoneal cavity three hours after their injection. Such a methodology provides a very early prediction of the survival of each mouse, thus identifying animals with a poor prognosis.     

Increased protein degradation and decreased protein synthesis in skeletal muscle during cancer cachexia.

The effects of progressive cachexia on protein metabolism in skeletal muscle has been investigated in mice bearing the MAC16 adenocarcinoma which produces cachexia with tumour burdens of < 1% of the host weight. Weight loss was accompanied by loss of whole body nitrogen in proportion to the overall loss of body mass. Using L-[4-3H]phenylalanine to label proteins in gastrocnemius muscle, a significant depression (60%) in protein synthesis occurred in animals with a weight loss between 15 and 30% accompanied by an increase in protein degradation, which increased with increasing weight loss between 15 and 30%. Muscle degradation in vitro could be achieved by serum from cachectic animals, which appeared to contain a proteolysis-inducing factor. These results suggest that the increased degradation of skeletal muscle seen in this model of cachexia may be due to a circulating proteolysis-inducing factor.     

Increased susceptibility of breast cancer cells to stress mediated inhibition of protein synthesis

Protein synthesis is a tightly controlled process, and its deregulation plays an important role in tumorigenesis. Protein synthesis remains poorly understood with very few well-identified validated targets for therapeutic purposes. In this study, we use nitric oxide (NO), which suppresses protein synthesis by inactivating eukaryotic initiation factor 2-alpha (eIF2-alpha), to examine the mechanism by which low and high oxidative stress inhibits protein synthesis. In breast cancer cells, low NO stress induced heme-regulated inhibitor (HRI) activation, which facilitated gradual decline in short half-life proteins. High NO stress induced HRI and protein kinase R (PKR) activation, leading to a sharp decline in protein synthesis as accessed by a decline in short and long half-life proteins and dramatic morphologic changes. In contrast, human mammary epithelial (HME) and Ras transfected untransformed HME (MCF-10A1 neo N) cells were less susceptible to NO-induced inhibition of protein synthesis and cytostasis. Our results suggest that NO-induced cytostasis in breast cancer cells was due to PKR activation and increased phosphorylation of eIF2-alpha, whereas the reduced susceptibility of normal mammary epithelial cells to NO could be due to the inaccessibility of PKR, which is bound to inhibitor p58.     

Increased lysis of melanoma by in vivo-elicited human lymphokine-activated killer cells after addition of antiganglioside antibodies in vitro

Thirty-nine melanoma patients were treated with cyclophosphamide (350 mg/m2) followed 3 days later by 5 daily doses of interleukin 2 (3.6 million units/m2 i.v.) weekly for 2 weeks. This cycle was repeated at least twice with a 1-week interval between cycles. Natural killer and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells were measured before treatment and on the last day of each cycle by chromium release assays. Development of LAK activity of greater than 10 lytic units was correlated with a clinical response. There was no correlation between natural killer activity and clinical response. Antibody-dependent cell-mediated cytotoxicity of in vivo-induced LAK cells after the addition of mouse monoclonal antibodies (MAbs) in vitro was measured in 30 cases on the last day of each interleukin 2 cycle. Anti-GD3 MAbs MB3.6, 11C64, 6H4, and R24 increased LAK cell cytotoxicity against GD3-positive GD2 melanoma cells while anti-GD2 MAb 14.18 increased LAK cell cytotoxicity against GD3-negative GD2-positive melanoma cells. MAb 9.2.27 (IgG2a) directed against a chondroitin sulfate proteoglycan and its core protein with a molecular weight of 250,000 (p250) on human melanoma cells did not mediate antibody-dependent cell-mediated cytotoxicity. The effector cells in these antibody-dependent cell-mediated cytotoxicity. The effector cells in these antibody-dependent cell-mediated cytotoxicity reactions were Leu-19 positive. In preincubation experiments the MAbs showed superior binding to the melanoma target cells than to effector cells. Our results show that low dose interleukin 2 preceded by low dose cyclophosphamide effectively induces LAK cells in vivo. The cytotoxicity of these in vivo-activated LAK cells can be augmented in vitro by mouse MAbs against glycolipid antigens on the tumor.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. II. Is the complement receptor necessarily present on the killer cells?

The effect of various lymphocyte depletion techniques on SLMC effector cell activity against the cell line K562 could be attributed t o the effects that these procedures had on the proportions of FclC3 receptor-bearing cells in the preparations. Depletion of cytotoxic activity by removal of Fc receptor-bearing cells could be augmented by the presence of complement on the 7S-EA indicator cells used for rosette depletion. Two methods of assessing the presence of surface receptors on cytotoxic cells are presented: (1) linear regression analysis of spontaneous cytotoxicity plotted as a function of the proportions of receptor-bearing cells remaining after various depletion techniques; and (2) the use of a mathematical formula t o estimate the proportion o f cytotoxic cells with any particular receptor, based on the effects of receptor-bearing cell depletion. Linear regression analysis of cytotoxicity vs the proportions of SRBC, Fc o r C3 receptor-bearing cells demonstrated that all cytotoxic cells had the Fc receptor, but that cytotoxicity could s t i l l occur in the absence of cells with the C3 receptor. This indicated that the C3 recepor is not necessary for the demonstration of SLMC activity. The mathematical formula to predict cytotoxicity after depletion of C3 receptor-bearing lymphocytes showed, however, that a significant proportion of the Fc receptor-positive killer cells were also C3 receptor-positive. No direct evidence was found t o support a role for T cells in SLMC, although E rosette depletion resulted in lower than expected cytotoxicity i n view of the number of Fc receptor-bearing cells remaining in the preparation.