Immunomodulatory effects of corticosteroids on natural killer and antibody-dependent cellular cytotoxic activities of human lymphocytes

The in vitro effect of prednisolone (PRD) on NK and ADCC activities of human lymphocytes was investigated. PRD at concentrations ranging from 7.5 X 10(-3) to 1 X 10(-5) M significantly inhibited NK activity, while concentrations of 7.5 X 10(-3) to 1 X 10(-4) M inhibited ADCC activities of PBL when added directly to the mixture of effector and target cells. Lymphocytes pre-cultured for 24 hr with PRD at concentrations ranging from 1 X 10(-4) M to 1 X 10(-6) M showed significant suppression of their NK activity. Inhibition was proportional to the concentration of the drug, and was observed at as early as 1 hr of incubation at various effector to target cell ratios with several targets. PRD also inhibited NK and ADCC activities of purified T cells, non-T cells, and NK-enriched effector cells. In target-binding assays, PRD decreased the target-binding capacity of effector lymphocytes in a dose-dependent manner. PRD-induced inhibition could be reversed by incubating lymphocytes for 1 hr with interferon or IL 2. Pretreatment of targets with PRD for 4 hr did not affect cytotoxic activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells because lymphocytes treated with PRD showed normal spontaneous 51Cr release, and their viability after 24 hr of pre-culture with PRD was comparable to that of untreated control cells. These results demonstrate that PRD has significant immunomodulatory effects on human NK and ADCC activities that may be of clinical relevance.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Synergistic effect of IL-4 and IFN-gamma on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.     

Inhibition of mouse natural killer cytotoxicity by heparin

The effect of heparin on mouse natural killer (NK) cytotoxicity was investigated. Heparin greatly inhibited NK activity at a concentration of more than 10 units/ml. Inhibition of NK cytotoxicity was observed only when heparin was present in the reaction mixture of the cytotoxicity assay. The results of kinetic study of NK inhibition and target-effector binding assay proposed the possibility that heparin inhibits NK cytotoxicity after the binding of effector cells to target cells. Dextran sulfate, the heparin analog, which has potent negative charge also had an inhibitory effect on NK activity. Fractionation of heparin on Sephadex A-25 column revealed the parallelism of the negative charge and the inhibitory effect of heparin on NK cytotoxicity. These results demonstrated that polyanion could modulate NK cytotoxicity.     

Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid

The effect of eicosapentaenoic acid (EPA) on natural killer (NK) cell activity of human lymphocytes was examined. The addition of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG) emulsified with purified phosphatidylcholine from krill to a cytotoxicity assay system resulted in a marked depression of NK activity. The inhibition was proportional to the concentration of EPA-TG emulsion, and was observed as early as the first one hour of incubation at various effector to target cell ratios. Pretreatment of effector cells with EPA-TG emulsion resulted in significant suppression of their NK activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells or decreased target cell binding. These results indicate that EPA is a potent inhibitor of NK activity in vitro.     

Inhibition of human natural killer (NK) activity and antibody dependent cellular cytotoxicity (ADCC) by lipomodulin, a phospholipase inhibitory protein

A highly purified preparation of lipomodulin, a phospholipase-inhibitory protein from rabbit neutrophils treated with glucocorticoids, inhibited NK and antibody-dependent cellular cytotoxicity (ADCC) activities of human peripheral blood lymphocytes in a dose-dependent manner. The presence of lipomodulin during the early period of the cytotoxicity assay was necessary to obtain maximal inhibition. The inhibition of NK or ADCC activity by lipomodulin was greater when effector cells were treated with lipomodulin than when target cells were incubated with lipomodulin. As lipomodulin did not block binding of effector cells to target cells, our results suggest that lipomodulin inhibits the cytolytic phase of NK and ADCC activities after binding to target cells, and imply that phospholipase(s) may be involved in NK and ADCC activities.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.     

Inhibition of human natural killer activity by lysosomotropic agents

We have examined the effect of three lysosomotropic amines on human NK cell activity. Dansylcadaverine (DCA), diphenylamine (DPA), and lidocaine (LID) inhibited NK activity of nylon wool-purified and large granular lymphocyte (LGL)-enriched cell preparations. Cadaverine (CAD), an analog of DCA that does not affect lysosomal function, had no effect on NK activity. Binding of the K562 target cells to effector cells, as assessed in a single cell assay, was not inhibited by DCA, DPA, or LID. Cytotoxicity was inhibited by DCA and DPA only when these drugs were added within 5 min after the initiation of NK assays. In contrast, LID inhibited NK activity even when added 60 min after the addition of effector cells to target cells. All three amines that inhibited NK activity also reduced the intracellular concentration of the lysosomal enzyme beta-glucuronidase without affecting the activity of the cytoplasmic enzyme lactate dehydrogenase. Kinetic analysis revealed that LID inhibited both the maximum velocity (Vmax) of the cytotoxicity reaction as well as the affinity constant (Km); whereas DCA and DPA only inhibited Vmax.     

Role of interferon in human natural killer activity against target cells infected with HSV-1

Human natural killer (NK) cells show high cytotoxic activity against target cells infected with herpes simplex virus type 1 (HSV-1). Substantial amounts of interferon (IFN) were generated in co-cultures of NK effector cells and infected target cells; however, the cytotoxic activity seen against a specific infected cell target did not correlate with the amount of IFN induced. The production of IFN increased steadily from 4 to 18 hr of co-culture, as did NK activity; however, IFN production peaked 4 hr later than NK activity. Pretreatment of NK effector cells with exogenous IFN increased cytotoxic activity against all targets tested, but the differential pattern of reactivity against cells infected with wild type and mutant viruses was unaltered. When effector cells were treated with the RNA synthesis inhibitor actinomycin D before co-culture with virus-infected targets, IFN production was markedly reduced, without a concomitant reduction in cytotoxicity. Similarly, the addition of anti-IFN antiserum to co-cultures greatly decreased the available IFN present, but had no effect on NK activity. We conclude that the induction of cytotoxic activity in co-cultures of NK effector cells and HSV-1-infected target cells is independent of the induction of IFN.     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.     

Rat natural killer cells synthesize fibronectin. Possible involvement in the cytotoxic function

The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.     

Role of interferon in natural kill of HSV-1-infected fibroblasts

The production of interferon during natural killer (NK) assays against HSV-1-infected fibroblasts (NK(HSV-1)) was studied to determine whether this interferon was responsible for inducing the preferential lysis of herpes-virus-infected target cells over uninfected target cells. The interferon produced during NK(HSV-1) assays was analyzed and found to have the properties of HU-IFN-alpha. Little or no IFN was produced during NK assays against uninfected fibroblasts (NK(FS)) or K562 (NK(K562)) cells. Although the appearance of interferon in the culture supernatants seemed to parallel the development of cytotoxicity during NK(HSV-1) assays, the levels of cytotoxicity and IFN generated did not correlate, arguing against a strict quantitative dependence of cytotoxicity upon IFN production. NK(K562) and NK(FS) cytotoxicity developed with little or no production of IFN. When IFN-pretreated effector cells were used, there was still a preferential lysis of infected over uninfected target cells. This preferential lysis by IFN-treated effector cells of infected over uninfected targets was seen as early as 2 hr into the assay. Anti-IFN antibodies added to the NK assays, although neutralizing all the IFN produced during the assays, had no effect on NK(FS) or NK(K562) cytotoxic activity and caused a slightly reduction of NK(HSV-1) activity only in one of three experiments. We conclude that although IFN is generated during NK(HSV-1) assays, this IFN cannot solely account for the increased lysis of infected over uninfected cells and that NK(HSV-1) activity is in some other way dependent on the virus infection.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Propagation of mouse cytotoxic clones with characteristics of natural killer (NK) cells

Splenocytes obtained from normal mice (BALB/c nude, BALB/c, C₃H, C57Bl/6) and from mice bearing lung or pulmonary carcinomas were propagated for 1–12 months in the presence of crude or mitogen-depleted T-cell growth factor (TCGF). Clones from several TCGF-propagated lymphoid cell lines were established by limiting dilution or the soft agar techniques. All the cultured lines and the majority of the clonal populations derived from them exhibited strong cytotoxic activity in vitro (⁵¹Cr release assay) toward a variety of syngeneic and allogeneic tumor target cells, both freshly obtained and passaged in culture, and both lymphoid and solid in origin, and including targets usually resistant to fresh NK cells. Considerable cytotoxic activity was also observed with several rat and human cultured tumor lines. Only low cytotoxic activity was detected against normal lymphoid mouse cells. Cloned populations generally exhibited more restricted target cytotoxicity than the parental cultured lines, and the pattern of reactivity varied among the clones. Of the clones tested for surface markers, all were positive for Thy 1.2, T200, and asialo GM1 and had strong binding to peanut agglutinin (PNA), all had undetectable receptors for IgG or IgM, and some were positive for Lyt 2. The cytotoxic activity was augmented by pretreatment of the effector cells with interferon and inhibited by the presence of mannose or galactose during the assay. Several clones were capable of mediating antibody-dependent cellular cytotoxicity and lectin-induced cellular cytotoxicity (LICC), and produced relatively large quantities of interferon and lymphotoxinlike material. The findings indicated continuous culturing in TCGF of previously antigen-nonstimulated mouse lymphocytes selects for the growth of at least two distinct populations with activated NK activity, one reacting preferentially with lymphoid tumor target cells (designated CNK-L), and the second reacting effectively with both lymphoid and solid tumor targets (designated CNK-SL). Both populations have several features of both T lymphocytes and NK cells.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

The cytotoxic activity of human natural killer cells requires an intact secretory apparatus

We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations.     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.