Human Natural Cell-mediated Cytotoxicity against Fetal Fibroblasts

The dependence of human natural killer (NK) cell activity on antibodies was investigated. Absorption of the culture medium with target cells, trypsinization of the effector cells followed by a brief recovery, and incubation of the effector cells in serum-free nutrient medium did not affect natural killer cell activity against fetal fibroblasts. No soluble mediator in the nutrient media of effector cell--fibroblast co-cultures could be demonstrated. It was therefore concluded that antibodies are not involved in the cytolytic activity of natural killer cells. However, competition data and the activity of isolated NK cells against antibody-coated target cells suggested overlapping between the effector cells mediating natural and antibody-dependent cell-mediated cytotoxicity.     

Characterization and Possible Mechanisms of Mitogen-induced Cell-mediated Cytotoxicity

Mitogen-induced cell-mediated cytotoxicity (MICC) of human peripheral blood granulocytes (PMN) and lymphocytes (PBL) was studied by using chicken erythrocytes as targets. The possible mechanisms were elicited bidirectionally by means of the MICC inhibition test and by functional analysis of lectin. In our preliminary data it was noted that PMN-mediated cytotoxicity was more potent than that of PBL. In addition, erythrophagocytosis or lysosomal enzymes released from PMN offered little contribution to cytotoxic activity of PMN. From the inhibition study it was realized that intact cytoskeletal structures, functionally preserved membrane lectin receptors, active DNA synthesis, and environmental divalent cations were mandatory for the full expression of MICC activity by both effectors. We also identified the cytotoxic activity of PHA-P as having a unique character that is thermostable and independent of the other biological activities.     

Antibody-induced Cell-mediated Cytotoxicity in an Allogeneic Human System

In a ⁵¹Cr-release assay system it is found that serum from normal women with pregnancy-induced lymphocyte allo-antibody can cooperate with normal non-sensitized peripheral human leucocytic cells in producing cytotoxic activity against target cells from the husband. Conventional complement activation is not involved. The cooperating serum factor is found to be antibody and is located in the IgG fraction. The effector cells are lymphocytes and the activity is blocked by cytochalasin B.     

HLA Restriction of Dinitrophenyl-specific Cell-mediated Cytotoxicity in Vitro

Lymphocytes from dinitrochlorobenzene-sensitized individuals can be stimulated in vitro by autologous dinitrophenyl (DNP)-conjugated lymphocytes to produce cytotoxic T lymphocytes (CTLs). The activity of these CTLs is specific for DNP-conjugated target cells, and there is no cross-reaction with nitrosodimethylaniline or trinitrophenyl-conjugated target cells. Evidence is presented which makes it improbable that the cytotoxicity is caused by an antibody-dependent (ADCC-like) mechanism. Most of the DNP-specific cytotoxicity is restricted by the HLA-ABC antigens of the CTL donor, and there is only a low degree of lysis of DNP-conjugated allogeneic target cells not sharing HLA-ABC antigens with the donor. The CTLs did not lyse non-conjugated allogeneic target cells. When CTLs were tested against allogeneic DNP-conjugated targets sharing only one of the HLA-ABC antigens of the CTL donor, it was seen that the phenomenon of preferential restriction was pronounced; that is, only some of the antigens of the donor were restricting. A certain pattern has emerged: some antigens (e.g. A2) are good restricting antigens, some (e.g. B12) do not restrict, and some (e.g. B5) function well in one donor but not in another. The sero-logically cross-reacting antigens A2 and A28 did not restrict mutually. HLA-C antigens may in some donors function as restricting antigens.     

Effect of Histamine Receptor Blocking on Human Antibody-dependent Cell-mediated Cytotoxicity

The effect of H1 and H2 receptor-blocking agents on antibody-dependent cell-mediated cytotoxicity (ADCC) was studied. The H1 receptor-blocker clemastinum and the H2 receptor blocker cimetidine dose-dependently inhibited the antibody-dependent cytotoxic activity of normal human peripheral blood mononuclear cells on chicken erythrocytes. The inhibition cannot be explained either by a direct toxic effect on effector cells or by blocking of Fc receptors. The possible involvement of histamine receptor-bearing effector cells in human ADCC is suggested.     

Mechanisms of Cell-mediated Cytotoxicity in Mice Rejecting Xenogeneic Human Lymphoblastoid Cells

The in vivo immunization of mice with human lymphoblastoid cell line LHN13 generates direct cell-mediated cytotoxicity by spleen cells. The lytic activity appears as early as day 3 after the intraperitoneal inoculation of 7.5 × 10⁵ cells and persists at least until day 11. The killer cells do not adhere to plastic and are not retained on nylon wool columns or on Degalan heads coaled with mouse Ig plus rabbit-anti-mouse Ig The effector cells are partly inhibited by treatment with anti-Thy-1.2 serum plus complement, but this inhibition appears to be non-specific since antiserum alone or normal mouse serum plus complement have the same effects. Heat-aggregated IgG strongly inhibit cytotoxicity, indicating that the effector cells are Fc-positive and that such receptors are implicated in lysis. Altogether, these features strongly argue for an ADCC phenomenon. The involvement of antibodies is demonstrated by the fact that eluates (56°C, 30 min) from immune cells alone induce lysis in the presence of normal spleen cells as effectors The lytic activity of these eluates can be removed by specific adsorption on protein A coupled to Sepharose heads and on the human lymphoid target cells. Positive complementation between immune and non-immune spleen cells suggests that the arming process may occur in vitro during the assay, when antibodies are released by plasmacytes     

Cytosine Arabinoside Induced Changes in Natural Killer and Antibody Dependent Cellijlar Cytotoxicity Functions in Multiple Sclerosis Patients

Five multiple sclerosis patients were treated weekly with cytosine arabinoside (araC) on an escalating dose schedule. The dose was initiated at 50mg/M² and then increased once each week by 50mg/M² (unless toxicity caused delay). Dosage decisions were based on whether or not the antibody-dependent cellular-cytotoxicity (ADCC) or natural killer (NK) cytotoxicity levels had been reduced to a level more than 2 standard deviations below the control range. Cytosine arabinoside treatment was discontinued in 2 of 5 subjects at doses of 500mg/M² due to toxicity. The 3 remaining patients demonstrated sustained reductions in the percentage of FcR⁺ cells in their peripheral blood. The maximum percentage reductions from the baseline values ranged from 50% to 76%. Concomitant reductions in the NK activity at the same doses ranged from 65% to 83%. ADCC activity in all 3 patients, however, was relatively resistant to suppression. The nadirs for the ADCC activity were only 16% to 44% below the baseline minimums. AraC was shown to reduce the proportion of FcR⁺ cells and NK cytotoxic activity in preference to ADCC activity.     

Cell-mediated Cytotoxicity during Vaccinia Virus Revaccination in Man: Influence of Antibodies and Interferon

Seven healthy human adults were revaccinated with vaccinia virus. Cell-mediated cytotoxicity to vaccinia virus-infected fibroblasts was investigated on the day of vaccination and on days with peak activity. Three donors were studied until day 30 after vaccination. The addition of interferon to cytotoxicity reactions resulted in an increase in killing. This increase was not seen when antibodies were added. When a mixture of lymphocytes from a revaccinated and a non-revaccinated donor was used as effector cells, the killing observed corresponded to the killing seen with lymphocytes from the revaccinated donor, when tested alone. This finding indicates that no antibodies or other soluble mediators capable of increasing cytotoxicity are released from the lymphocytes during the cytotoxicity assay.     

Natural Cytotoxicity and Serum Blocking in Malignant Cervical Neoplasia

ABSTRACT: Peripheral blood lymphocytes from 100 patients with various stages and histological types of squamous cell carcinoma of the uterine cervix were assayed for natural killer (NK) cell activity against K562 cells using the single cell cytotoxicity assay in agarose. The effect of autologous serum on NK cell activity and a quantitation of soluble antigen-antibody complexes was also carried out. The cancer patients showed reduced NK activity compared with normal controls, the reduction increasing with tumor load. Another observation was that patients with poorly differentiated tumours showed lower levels of NK activity. Addition of autologous serum resulted in further depression of NK activity in all groups of patients. An increase in circulating immune complexes was also evident in all groups of patients. Here again increase in tumor load and poorly differentiated tumors showed the highest levels. The results point toward a possible disturbance in NK cell activity that could be further depressed by autologous serum factors.     

Natural Killer Cytotoxicity and Antibody-Dependent Cell Cytotoxicity to Herpes Simplex Virus-Infected Target Cells in Murine Pregnancy

ABSTRACT: In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C₅₇B1₆inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the ⁵¹chromium release assay for NKC and ADCC. %NKC (mean ± S.E.) to HSV-infected targets was significantly suppressed (P < 0.05) in pregnant mice, 10.3% ± 1.9, compared to that of virgin mice, 32.5% ± 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% ± 4.4 (pregnant) compared to 49.1% ± 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 + 66 PFU/ml, compared to 38 ± 11 PFU/ml in virgin mice (P < 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.     

Natural Killer Cell Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells in Human Pregnancy

ABSTRACT: Natural killer cell (NKC) cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocyte effector cells to destroy target cells in the absence and presence of antibody, respectively. Since these immune systems play a pivotal role in the body's primary lines of defense against a variety of pathogens including herpes simplex virus (HSV), a study was undertaken to evaluate the influence of pregnancy on these systems. Eleven uncomplicated gravidas were followed serially through each trimester and compared to 11 nonpregnant female controls. Mononuclear cells were acquired by Ficoll-Hypaque centrifugation of heparinized blood. Chang liver cells infected with HSV-I were utilized as target cells in a ⁵¹Cr release assay. Mean NKC values in the pregnant patients were uniformly lower than in the controls. No similar decreases in ADCC activity were observed in a comparison between the two study populations. These data support previous observations suggesting that pregnancy represents a relatively immunocompromised state. Differences apparently exist between NKC and ADCC effector cell populations with regard to the influence of pregnancy. Although these physiologic alterations in immunoregulation may help support the fetoplacental allograph, detrimental conditions may exist regarding susceptibility to various pathogens such as HSV.     

Recovery of Natural Cytotoxicity After Bone Marrow Transplantation

Reconstitution of non-MHC restricted cytotoxicity was followed in 7 and in 4 cases after auto-and allotransplantation, respectively. In autografted cases a high level of natural cytotoxicity, which exceeded the pre-transplant values, was seen at the beginning of hematological reconstitution. At that time, the high percentage of DR+ peripheral blood mononuclear cells (PBMC) was found, while the percentage of CD16+ cells was in the range of the pre-transplant values. A high level of cytotoxicity was a transient phenomenon. In all cases, 40 days after grafting the natural cytotoxicity was again in the range of the pre-transplant values. In allotransplantation the peak of cytotoxicity was seen soon after the beginning of hematological recovery and the activity decreased after 24 to 48 days after grafting.     

Recovery of Natural Cytotoxicity After Bone Marrow Transplantation

Reconstitution of non-MHC restricted cytotoxicity was followed in 7 and in 4 cases after auto-and allotransplantation, respectively. In autografted cases a high level of natural cytotoxicity, which exceeded the pre-transplant values, was seen at the beginning of hematological reconstitution. At that time, the high percentage of DR+ peripheral blood mononuclear cells (PBMC) was found, while the percentage of CD16+ cells was in the range of the pre-transplant values. A high level of cytotoxicity was a transient phenomenon. In all cases, 40 days after grafting the natural cytotoxicity was again in the range of the pre-transplant values. In allotransplantation the peak of cytotoxicity was seen soon after the beginning of hematological recovery and the activity decreased after 24 to 48 days after grafting.     

Natural Cytotoxicity to Murine Cytomegalovirus-Infected Cells Mediated by Mouse Lymphoid Cells: Role of Interferon in the Endogenous Natural Cytotoxicity Reaction

Lymphoid cells from unstimulated normal C57BL/6J mice were shown to lyse murine cytomegalovirus (MCMV)-infected syngeneic mouse embryo fibroblasts but not uninfected mouse embryo fibroblasts. This cytotoxicity by mouse effector cells was not restricted to MCMV-infected syngeneic cells since MCMV-infected xenogeneic rat heart fibroblasts were also lysed. Characterization of the effector cells mediating this cytotoxicity against MCMV-infected cells indicated that the effector cells are similar to described natural killer (NK) cells mediating lysis of tumor cells and virus-infected cells. Because of the described augmentation of NK activity by interferon, we examined the role of interferon in the NK reaction. Although low levels of virus-induced interferon were detectable in supernatants of MCMV-infected mouse embryo fibroblasts, no interferon was detectable in supernatants of MCMV-infected rat heart fibroblasts, a target significantly more sensitive to NK cytolysis than infected mouse embryo fibroblasts. We were able to augment the NK reaction against MCMV-infected cells by in vitro treatments with interferon. However, the amounts of interferon required for augmentation were significantly greater than the amounts generated by infected target cells. In vitro interferon-stimulated NK cells retained selective cytotoxic activity since they continued to remain incapable of lysing uninfected target cells. MCMV-infected rat heart fibroblasts induced more interferon and were also more susceptible to NK activity than MCMV-infected mouse embryo fibroblasts. In spite of this difference in interferon-inducing capacity, there was no augmentation of cytotoxicity of MCMV-infected mouse embryo fibroblasts when mouse splenocytes were cocultivated with both target cells. Finally, when production of interferon in the NK reaction was inhibited by the addition of actinomycin D, no reduction of NK activity was seen. Our findings suggest that native mouse NK cells can discriminate between MCMV-infected cells and uninfected cells, this ability leading to the selective lysis of the virus-infected cells. Furthermore, although we could demonstrate augmentation of NK activity by interferon, interferon activation of NK cells may not be a necessary precondition for the development of endogenous NK activity.     

Spontaneous Cell-mediated Cytotoxicity (SCMC) in Various Mammalian Species and Chickens: Selective Reaction Pattern and Different Mechanisms

Cultured cell lines derived from donors of various species as ⁷⁵Se- or ⁵¹Cr-labelled targets in microcytotoxicity assays. As in human donors, considerable spontaneous cell-mediated cytotoxicity (SCMC) was exhibited by peripheral blood lymphoid effector cells from healthy chickens, mink, swine, cattle, horses and tigers. Although all target lines tested could be lysed in SCMC, there was no 'general SCMC target' for all species of all individuals of one species. The selectivity, the kinetics, and the strength of SCMC reflected the capacity of the effector cells rather than a 'susceptibility' of the target to lysis. Classical major histocompatibility complex products, determinants specific for distinct cell types or subpopulations, or antigens associated with Epstein-Barr virus, herpes virus ateles, herpes virus papio, or Marek's disease virus were not appreciably involved in SCMC. No 'malignant phenotype' on targets was required for the cytotoxic effect. Yet undefined xenogenic, allogenic, and individual structures seem to be involved in SCMC. Depending on their expression, a target can he classified as 'species related' or as 'individual-related'. Intermediate forms are conceivable. SCMC might comprise different cytotoxic mechanisms. Antibodies are not required for lysis to occur, but serum factors including immunoglobulins can have considerable influence on SCMC.     

Kinetic Analysis of the Specificity of Human Natural Cytotoxicity

Distribution-free analysis of kinetic data for cellular cytotoxicity reactions enables the precise determination of kinetic parameters for the lysis of target cells by individual lymphocyte preparations. This report presents results obtained when this method was used to quantitate the inhibition of human natural cytotoxicity by unlabelled (cold) target cells. In agreement with previous studies, we found that the natural cytotoxicity of a given target cell can be inhibited by heterologous as well as homologous unlabelled cells; however, the strongest inhibition was usually produced when the unlabelled inhibitor cells were homologous to the labelled target cells. The general pattern of inhibition observed for the target and inhibitor cells tested in these experiments was competitive, and when inhibitor cells were homologous to the labelled cells, the observed increase in Kappm agreed with theoretical predictions based on equations derived previously. These results support earlier reports of limited but not absolute antigenic specificity by subsets of human NK cells. Moreover, while Kappm values for cytotoxicity reactions are not simply related to antigen binding, quantitative analysis of the relative inhibition of cytotoxicity by heterologous versus homologous unlabelled cells provides useful estimates of the relative affinity of the effector cell subset(s) that kill a given target cell for various NK target cells.     

Neutrophil Cytotoxicity Induced by Immune Complexes prepared with Cationized Antibodies

Here we analyse the ability of soluble imtnune complexes (IC) prepared with cationized antibodies to induce cytotoxic responses mediated by neutrophils. While eationized IC induced high levels of cytotoxicity, control IC induced very low levels of response. Inhibition of cytotoxicity by catalase but not by three haemenzyme inhibitors suggests that oxygen-dependent but myeloperoxidase-independent mechanisms are responsible for cytolysis. While the response induced by control IC was enhanced by cytochalasin B and was not modified by colchicine, that induced by cationized IC was markedly inhibited by cytochalasin B and significantly enhanced by colchicine. Cytotoxicity induced by cationized IC was completely abrogated by monoclonal antibodies to FcγRII. Using control IC, a partial inhibition was observed employing either anti-FcγRII or anti-FcγRIII monoclonal antibodies. Treatment of neutrophils with ehemotrypsine or pronase significantly enhanced cytotoxicity induced by cationized IC but not by control IC. We also found that non-specific absorptive mechanisms appear to play an important role in the binding of cationized IC, but not control IC, to the neutrophil surface. The significance of these results is discussed.     

In Vitro Modulation of Murine Natural Killer Cytotoxicity by Zinc

The in vitro effects of zinc on natural killer (NK) activity of murine spleen cells were studied. The pretreatment of splenocytes with nun-toxic concentrations of ZnSO₄ induced a decrease of lytic activity against YAC-1 and RDM-4 targets. The lytic function of non-activated and poly(I)-poly(C)-activaled NK cells was similarly inhibited When the interaction of effector cells with zinc was studied for 5 min, a significant inhibition of NK lysis was noted, which was maximal after 30 min. Zinc was undoubtedly responsible for the observed effects, since the concurrent addition of both zinc and suitable concentrations of o-phenanthroline, a Zn⁺⁺ chelating agent, made it possible to maintain a normal level of lysis. Moreover, the pretreatment of spleen cells with increased concentrations of o-phenanthroline also inhibited NK lysis, suggesting that a physiological intracellular zinc content is required to maintain an optimal lylic function of NK cells. Although the lysis was completely suppressed after the addition of 10⁻⁴ M ZnSO₄, the frequency of target-binding cells (TBC), which was assumed to represent the first stage of NK-mediated cytolysis, was only partly inhibited. The results are discussed in view of a possible action of zinc on cell membrane functions, enzyme systems, and release of lymphokines.     

In Vitro Enhancement of Natural Cytotoxicity by Tumour Necrosis Serum

Cells cytotoxic for syngeneic, allogeneic and xenogeneic cultured target cells are shown to be induced spontaneously in cultures of murine lymphoid cells. Serum containing bacillus Calmette-Guérin and endotoxin-induced tumour necrosis factor accelerates the appearance of observed natural cytotoxic cells.