Natural Cell-mediated Cytotoxicity Research Articles

Natural cell-mediated cytotoxicity in vitiligo

Natural cell-mediated cytotoxicity was studied in 18 patients with vitiligo and 13 healthy age-, race-, and sex-matched control subjects. The 4-hour chromium51 (51Cr) release assay was used to determine the activity of natural killer (NK) cells in the peripheral blood against K562 and Molt-4 target cells. Patients with vitiligo had a 50%, 67%, and 60% decrease in the cytotoxic response with Molt-4 cells at effector-target ratios of 25:1, 50:1, and 100:1, respectively, in comparison with control subjects (p less than 0.001). This inhibition was consistent with an 80% decrease in the binding capacity of NK cells to Molt-4 target cells (p less than 0.005). In contrast, cytotoxic responses did not differ in patients and control subjects with K562 target cells. These results suggest that patients with vitiligo have a decreased capacity for effector cell recognition of Molt-4 target cells but not K562 target cells. Hence patients with vitiligo may have defective clones of NK cells that are incapable of initial recognition of Molt-4 target cells, a necessary prerequisite for target cell lysis. Perhaps this phenomenon occurs with other tumor cells, which would explain the association of vitiligo with certain internal malignancies.

In vitro susceptibility of different human T-cell subpopulations and resistance of large granular lymphocytes to HTLV-I infection.

T4 subpopulation of T lymphocytes is the preferential target of infection with human T leukemia/lymphoma virus of subgroup I (HTLV-I). In this study we attempt to determine whether different T-cell subsets exhibit differences in susceptibility to virus infection. T cells from cord or peripheral blood were separated according to cell densities and T-cell surface markers by Percoll gradient and Sepharose anti-Fab immunoadsorbent affinity column (IAC), respectively. Separated T-cell subpopulations were infected with HTLV-I, by means of co-cultivation with irradiated virus producer cell lines (MT-2, TK). Percentages of HTLV-I-infected cells were assayed by immunofluorescence assay (IFA), using highly specific mouse monoclonal antibody (MAb) directed against HTLV-I p19 core protein. The results showed that different T-cell subpopulations separated either by Percoll or by IAC were susceptible to HTLV-I infection with the exception of large granular lymphocytes (LGL), which exhibit high cell-mediated natural cytotoxicity (CMNC). The susceptibility to HTLV-I infection of T cells with CMNC activity was further studied on established cell clones with LGL morphology. The results showed again that these cells were resistant to the virus infection. The present studies indicate that different T-cell subpopulations, irrespective of their size and of cell-surface markers, are susceptible to HTLV-I infection, with the exception of functionally mature LGL or of immortalized LGL clones.

Natural cell-mediated cytotoxicity among a group of HIV seropositive hemophiliacs

Natural cell-mediated cytotoxicity among a group of HIV seropositive hemophiliacs

Delayed effects of low-dose radiation on cellular immunity in atomic bomb survivors residing in the United States.

Several parameters of cellular immune function were assessed among persons who survived the 1945 atomic bombs in Hiroshima and Nagasaki but who now reside in the United States. The subjects in this study were exposed to various low doses (T65D) of radiation at the time of the bomb. More than half received an estimated 0 Gy (S0 group). Of those exposed to more radiation (S+ group), nearly 90% received less than 0.50 Gy (50 rad). Lymphocytes were isolated from the peripheral blood of these individuals and were assessed for the following parameters of cellular immunity: mitogenic response to phytohemagglutinin, mitogenic response to allogeneic lymphocytes, natural cell-mediated cytotoxicity (NCMC), and interferon production. In every case, the response of the S+ group was greater than that of the S0 group, although only the difference for NCMC was statistically significant. Results of studies presently being performed on A-bomb survivors residing in Hiroshima do not confirm this difference. Therefore, it is difficult to say whether the increase in natural cytotoxicity observed among the American and not the Japanese A-bomb survivors exposed to very low doses of radiation is a hormetic effect which was modulated by post-radiation environmental conditions or a result of selective migration.

Reversible susceptibility to natural cell-mediated cytotoxicity observed in altered BHK cells resistant to HVJ (Sendai virus) infection

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) showed a high susceptibility to natural cell-mediated cytotoxicity, although BHK-R cells are not transiently or persistently infected with HVJ but contain the restricted amount of sialic acid. By repeated subcultivation of BHK-R cells in growth medium free of HVJ, the sensitivity to natural killer cytotoxicity decreased to the level of normal BHK cells with a counter increase of cellular sialic acid, and the subsequent treatment of the cells with neuraminidase caused a loss of proper sialic acid residues, once again resulting in a significant enhancement of lysis by natural killer cells. In the BHK-R cell system which exhibits a reversible resistance to the interferon action, the enhancing effect induced by interferon on target cell susceptibility to natural killer activity became more pronounced in accord with the recovery of sensitivity to the antiviral action of interferon.

Natural cell-mediated cytotoxicity to cells infected with infectious bovine rhinotracheitis virus

Cell-mediated cytotoxicity against viral-infected cells was demonstrated in a 6-hr 51Cr release assay. Peripheral blood mononuclear leukocytes from both infectious bovine rhinotracheitis virus (IBRV)-infected and noninfected cattle exhibited preferential lysis against IBRV-infected primary bovine embryonic kidney (BEK) cells compared to cells infected with pseudorabies virus and noninfected BEK cells. Addition of specific antibody to the assay did not enhance cytotoxicity. The effector cell was a nonadherent cell which was either spontaneously enriched or generated during in vitro cultivation. Maximal cytotoxic activity was detected in peripheral blood mononuclear cells cultured for 3 to 5 days. Several factors affected the magnitude of cytotoxicity during the assay: target cell type, concentration of viral inoculum, duration of effector and target cell contact. It is suggested that target cell lysis was a form of natural cell-mediated cytotoxicity mediated by a cell which has different characteristics from the typical human and murine NK cell.

Familial occurrence of breast cancer is associated with reduced natural killer cytotoxicity.

A factor in the incidence of spontaneous neoplasms in mice is the endogenous level of natural cell-mediated cytotoxicity. These immunosurveillant or host defense mechanisms are probably under the control of multiple gene products including interferons. We studied natural killer (NK) cytotoxicity using peripheral blood mononuclear cells from 59 normal individuals with either a high (17) or low (42) familial incidence of breast cancer. The K562 cell line was used as target in 51Cr release assays. Three effector: target ratios (6.2:1, 25:1, and 50:1) were studied in quadruplicate using 3, 4 and 5-h incubations. Significantly lower natural killer activity (p less than 0.002) was detected in normal individuals with high familial incidences of breast cancer compared to individuals with low incidences in each of the three separate assays (50:1). The same conclusion was reached whether the data were expressed in terms of lytic units per 10(7) blood mononuclear cells or as % specific 51Cr released. Thus, a relationship was observed between the occurrence of breast cancer in closely related family members and low natural cell-mediated cytotoxicity. This result suggests that defects in NK activity may play a role in the initiation of human breast tumors. However, prospective studies will be necessary to establish whether low NK cell activity is a risk factor for breast cancer.

Phase I evaluation of recombinant interleukin-2 in patients with advanced malignant disease.

Seventeen patients with refractory malignant tumors were treated with recombinant human interleukin-2 (IL-2) administered by weekly bolus intravenous (IV) injection in a phase I dose escalation trial. Patients received 10,000 to 1,000,000 U/m2 per injection over a course of 3 to 33 weeks. Toxicity was dose related and consisted primarily of fever, chills, nausea, and vomiting. Hypotension was observed at doses of 500,000 U/m2 or higher and in one instance was sufficiently severe to require pressors. No tumor regression was seen and all patients eventually developed progressive disease. Blood levels of cortisol, ACTH, prolactin, and growth hormone as well as the acute phase reactant C-reactive protein (CRP) increased after the administration of IL-2 in most patients. Serum IL-2 levels in excess of 250 U/mL were detected five minutes after an IV injection of 1,000,000 U/m2, after which the levels declined with a half-life of approximately 25 minutes. No alteration in lymphocyte surface phenotype or enhancement in natural cell-mediated cytotoxicity against natural killer (NK)-sensitive and resistant tumor cell lines was observed when these parameters were measured weekly just before the IL-2 injections. However, a dramatic but transient decline in circulating lymphocytes and NK activity was noted within hours of receiving IL-2. This effect was independent of fever and was not abrogated by pretreatment with ibuprofen or metyrapone. The majority of patients developed serum IgG antibodies of IL-2 detectable with a sensitive enzyme-linked immunosorbent assay (ELISA) and a nitrocellulose dot blot assay. The development of anti-IL-2 antibodies was not associated with symptoms suggestive of serum sickness, reductions in serum complement levels, or deterioration in lymphocyte tumoricidal activity. This investigation provides insight into the in vivo actions of this potent biological response modifier and will assist in the design of future studies with IL-2 administered alone or in conjunction with other treatment modalities.

Natural killer and antibody-dependent cellular cytotoxicity in cervical carcinoma patients.

Natural killer (NK) cell activity and antibody dependent cell-mediated cytotoxicity (ADCC) was measured in 62 untreated cervical carcinoma patients and 25 normal healthy women, using a short-term chromium release assay. A significant reduction in NK and ADCC activity was observed in disseminated disease than in localized disease, when compared with normal donors. The majority of the patients received radiotherapy and both NK and ADCC activity recovered after therapy. Furthermore, interferon-alpha was demonstrated to augment NK activity of peripheral blood mononuclear cells from healthy donors as well as patients. Also large granular lymphocytes separated on Percoll density gradient were the same in number in both the populations studied, although in cervical cancer there seemed to be a defect in killing activity.

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing.

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.

Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle

Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target Cell Cycle.

Phase I study of MVE-2 evaluating toxicity and biologic response modification capability.

A total of 21 patients were treated in a phase I trial using the biological response modifier MVE-2, a low molecular weight component of pyran copolymer. All patients received weekly IV MVE-2 infused over 2 h. Proteinuria, sometimes of nephrotic proportions, was the dose limiting toxicity, and was seen with increasing incidence as the cumulative dose of MVE-2 exceeded 2500 mg. Other toxicity with MVE-2 was minimal. Biologic response modification at tolerable doses was inconsistent, although several assays, particularly natural cell-mediated cytotoxicity, indicated enhanced activity at higher dosages of MVE-2. No objective tumor responses were observed. MVE-2 is not useful as a biological response modifier using our initial method of administration, since the dose limiting toxicity occurred at lower levels than were necessary to induce consistent biologic response modification. Following completion of the phase I study, we administered MVE-2 by 30-min infusion to 8 additional patients and did not detect proteinuria, in spite of large cumulative doses. It is possible that alternate schedules of MVE-2 administration could minimize proteinuria and allow the administration of dosages necessary for immunologic modification.

The monoclonal antibody CJA3 down-regulates the susceptibility of human tumor cell lines to natural cell-mediated cytotoxicity.

While developing monoclonal antibodies (MoAb) to colorectal carcinoma (CRC) cells, we noted that one MoAb, termed CJA3, down-regulated natural cell-mediated cytotoxicity (NCMC) against CRC cell lines SW480 and SW620. The MoAb CJA3 was developed by immunizing a BALB/c mouse with fresh human colorectal adenocarcinoma cells. The antigen recognized by the MoAb CJA3 was expressed on several solid tumor cell lines and on one of the six lymphoreticular cell lines tested, but was not detected on normal peripheral blood lymphocytes (PBL). SDS-PAGE analysis of the antigen immunoprecipitated by the MoAb CJA3 from the CRC cell lines SW480 and SW620 and from the melanoma cell line MALME-3M revealed a component with a m.w. of 150,000. Preincubation of CRC cell lines SW480 and SW620 with the MoAb CJA3 for 16 hr reduced their susceptibility to NCMC by about 50%. Kinetic experiments showed that prolongation of the incubation of target cells with the MoAb CJA3 resulted in a time-dependent increase in the amount of MoAb bound. Maximum binding of the MoAb CJA3 was reached after 4 hr of incubation. The increase in antigen expression chronologically paralleled the decrease in NCMC target cell sensitivity, suggesting that the membrane alterations induced by the MoAb CJA3 were important for NCMC against these two cell lines.

Indomethacin abrogates the suppression by cyclosporin A of lectin-dependent cell-mediated cytotoxicity to HEp-2 cells

The effects of cyclosporin A, prostaglandin E1 and indomethacin were studied on lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC activity by human peripheral blood lymphocytes was evaluated by detachment from the monolayer of [3H]thymidine-prelabelled HEp-2 cells in a 24-h assay at 50:1 effector:target cell ratio in the presence of 25 μg/ml concanavalin A. Under these conditions, but without concanavalin A, considerable natural cell-mediated cytotoxicity was not elicited although LDCC was significantly augmented in the presence of concanavalin A. Addition of both cyclosporin A (0.1, 1.0 or 10 μg/ml) and prostaglandin E1 (10−8, 10−7 or 10−6 M) dose-dependently suppressed LDCC activity. Indomethacin (0.1, 1.0 or 10 μg/ml) did not in itself influence LDCC although suppression of LDCC by cyclosporin A, but not prostaglandin E1, was abrogated in the presence of indomethacin. Similar to indomethacin, acetyl salicylic acid also reversed the inhibition of LDCC by cyclosporin A. In parallel experiments, cyclosporin A elicited a more than two-fold increase of prostaglandin E production under LDCC assay conditions as measured by radioimmunoassay. Contrary to LDCC, depression of concanavalin A induced blastogenesis by cyclosporin A was not influenced by indomethacin, suggesting that the inhibition by cyclosporin A of LDCC and concanavalin A-induced blastogenesis proceed via different mechanisms.

Decreased natural cytotoxicity in mice with high incidence of mammary adenocarcinoma

In an attempt to gather evidence relevant to the question of whether natural killer (NK) cells play a role in resisting the development of primary tumors, we compared natural cell-mediated cytotoxicity in two substrains of C 3H mice. Animals of the C 3 H OuJ substrain are at high risk for the formation of mammary adenocarcinomas, while C 3 Heb FeJ mice have a low incidence of such tumors. Natural cytotoxicity of splenic mononuclear cells was lower in the high-risk substrain, suggesting that a lesion in NK cell activity may be involved in murine mammary tumorigenesis. This difference was observed in animals between 5 and 37 weeks of age. There was no significant difference in the number of splenic large granular lymphocytes between the substrains. A significant difference in the ability of splenic lymphocytes from the two substrains to bind to the target cells was noted. Since the binding capacity of lymphocytes was greater in mice with reduced NK cell activity, the lesion in cytotoxicity may exist at a postbinding step in the lytic sequence. It is felt that the C 3H mouse may provide a useful model for studying the role of NK cells in controlling primary tumors.

Natural Killer Activity and Antibody-Dependent Cell-Mediated Cytotoxicity in Multiple Myeloma

Natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) were studied by routine methods in 11 patients with untreated malignant monoclonal gammopathy. NK and/or ADCC activity was clearly reduced in three patients with advanced disease. Moreover, sera from some myeloma patients impaired the ADCC and NK activity. A large quantity of purified monoclonal IgG from one patient appeared to inhibit NK and ADCC activity as did high concentrations of pooled polyclonal immunoglobulin from healthy persons. In two of these 11 patients, other malignancies were diagnosed prior to chemotherapy. One of these patients, who had nonsecretory myeloma, had marked impairment of NK and ADCC activity; the other, with IgG myeloma, had normal NK and ADCC activity.

Contrasting effects of RNA and protein synthesis blocking on natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

In this study, earlier observations concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against 3H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.

The relationship of clinical status and therapeutic modality to natural killer cell activity in human breast cancer.

The role of clinical status and therapeutic intervention on natural cell-mediated cytotoxicity in breast cancer was ascertained by monitoring natural killer (NK) cell activity in peripheral blood samples. Patients with localized disease on chemotherapy showed significant reductions in NK activity concomitant with reduced lymphocyte numbers, when compared to untreated patients (18.1% versus 32.7%, P less than 0.005). Lymphocyte counts were included in a calculation of the absolute proportion of NK activity that incorporates a correction factor for the leukopenia that occurs as a result of cytotoxic therapy and disease progression. This calculation more accurately reflects the significant reduction of NK activity that occurs in patients with localized and systemic disease on chemotherapy when compared to untreated patients with no current evidence of disease (10.3% and 14.9% versus 30.7%, respectively; P less than 0.001). Different chemotherapeutic regimens were found to selectively affect NK cell function. The levels of both actual and absolute NK activity were significantly reduced in patients receiving 5-fluorouracil and L-phenylalanine mustard; cyclophosphamide, methotrexate, and 5-fluorouracil; and vincristine, Adriamycin (doxorubicin), and 5-fluorouracil, whereas only the levels of absolute NK activity were significantly reduced in patients receiving mitomycin, Megace (megestrol acetate), and Adriamycin when compared to untreated cancer patients. In contrast, tamoxifen-treated patients demonstrated levels of actual and absolute NK activity observed with untreated cancer patients. Patients receiving tamoxifen showed significantly elevated NK activity when compared to patients on all other chemotherapies. These results indicate that monitoring NK cell function may be useful in assessing the immunosuppressive effects of chemotherapeutic intervention.

Concentration-dependent augmentation of anti-K562 activity following short-term culture of null cell-enriched human blood lymphocyte populations

The increase in natural cell-mediated cytotoxicity against K562 cells following short-term culture (e.g. 12 hours) of null cells was dependent upon the concentration of cells during incubation. The increased lytic activity by cells cultured at higher concentrations was associated with increased numbers of target-binding cells. It occurred with a variety of serum supplements in the medium. The augmentation of natural killer activity required close cellular proximity or contact and was not associated with detectable increased release of interferon or interleukin 2, although the participation of these molecules cannot be excluded. The lower recovery of lytic activity from cells incubated at lower concentrations was not associated with increased PGE synthesis.

An assessment of natural cell-mediated cytotoxicity in patients with malignant lymphoma

Fifty per cent of untreated malignant lymphoma patients were shown to have profoundly reduced levels of peripheral blood lymphocyte-natural cell mediated cytotoxicity (PBL-NCMC) when tested against the leukaemic cell-line K562; assessment of NCMC in unfractionated blood from a large number of these patients showed a comparable reduction in activity. High levels of NCMC were observed in some patients with stage III/IV non-Hodgkin's lymphoma (NHL), while Hodgkin's disease (HD) patients over 40 yr of age had normal levels. In both NHL and HD patients there was no correlation between NCMC and absolute lymphocyte count or lymphoma histology. Human interferon was seen to boost NCMC in the majority of NHL patients, but most HD patients were non-responders. Almost all patients tested had normal number of target binding lymphocytes, and most had numbers of HNK-1 + cells within the control range. However, on exposure to IFN, the number of target binding lymphocytes increased in over half of the patients tested, with some patients showing an increase in HNK-1 + cells, in the majority of cases without enhancement of NCMC.