Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

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.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro

To study cell-to-cell interactions in microgravity we examined the functional activity of natural killer cells on board of the ISS. NK cells are the effector cells with direct cytotoxic activity to oncogenic, virus-infected cells and cells with modified differentiation. Ground-based experiments have shown that the examination of target cell lysis after incubation with NK cells is a simple and informative model for studying the influence of microgravity. NK cytotoxicity was measured as the value of non-degradeted labeled myeloblasts (K-562) after 24 hrs exposure with human lymphocytes in suspension. A special device was developed for space flight experiments. Human cultured lymphocytes and labeled K-562 cells were loaded into special syringes and delivered to the Russian segment of the ISS. Cosmonauts prepared co-cultured suspensions during the first day of microgravity, exposed them at 37 degrees C for 24 hrs and then separated H3-labeled cells on special filters. The results of ISS-8 mission showed that human NK cells in vitro remain lysis activity toward target cells in microgravity. The basal level of NK cytotoxicity was low and we did not found significant differences between "control" and "flight" values. Interferon production during the interaction between immune and target cells (ratio 10:1) in microgravity did not differ compared with ground-based control experiments. Ground exposure of the same lymphocyte samples with K-562 cells to 24 hrs clinorotation also did not lead to significant differences. These experiments paved the way for understanding the cell interaction mechanisms in space flight and the obtained results suggest that microgravity does not disrupt the interaction of NK cells with tumor cells.     

A function-associated molecule on rat natural killer cells identified by anti-idiotypic monoclonal antibodies.

Monoclonal antibodies were generated against idiotopes on an NK target antigen-specific IgM monoclonal antibody (mab). This mab (18C2) was originally produced against (NC-37) human EBV-transformed B cells. The 18C2 mab inhibits natural killer cell lysis of NC-37 and other target cells by preventing conjugate formation. Anti-18C2(id) mabs were tested for binding to effector cells and screened by ELISA, flow cytometry, and by inhibition of NK cytotoxicity. Two of the anti-18C2(id) (anti-id) mabs (12H1.C5 and 6D9.B11) were chosen for further study. The idiotypic specificity of these anti-id mabs was confirmed by testing their binding to 18C2 hybridoma cells in the presence of homologous and heterologous "cold" inhibitor mabs. Experiments were also conducted to determine the functional properties of these mabs. Anti-18C2(id) mab 12H1.C5 inhibited the cytotoxic activity of rat splenic NK (nylon wool nonadherent cells, NWNA) and rat ALAK cells. Flow cytometric (FCM) analysis of the binding of the anti-18C2(id) mabs demonstrated that mab 12H1.C5 bound 75.43% rat NWNA spleen cells, 43.74% rat ALAK cells, and 74.33% rat CRC- cells. Anti-id mab 6D9.B11 bound 45.20% NWNA cells, 70.45% rat ALAK cells, and 55.86% CRC- cells. Two-color FCM analysis demonstrated that the anti-id mabs not only bound to the same molecule on NK cells, but also these mabs bound to the same molecule as 5C6, an anti-NK cell mab. Biochemical analysis of the antigen recognized by mab 12H1.C5 was determined by Western blotting. The determinant on NWNA cells recognized by mab 12H1.C5 had an M(r) of 40 kDa and appeared to be identical to that recognized by mab 5C6. The same experiment using a transformed rat RNK-16 (CRC-) cell extract and Western blot analysis, demonstrated an M(r) of 42 and 48 kDa in the presence of mabs 5C6 and 12H1.C5. Monoclonal antibody 5C6 was previously shown to recognize a vimentin-like function-associated molecule on NK cell membranes. The anti-id mabs were also shown to have cross-reactivity with the intermediate filament vimentin as determined by Western blot analysis.     

Induction of alloreactive cytotoxic T cells by acute virus infection of mice

Alloreactive cytotoxic T lymphocytes (CTL) distinct from virus-specific CTL and activated natural killer (NK) cells were generated during acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice. The alloreactive CTL shared similar antigenic markers (Thy-1.2+, Lyt-2.2+, and asialo GM1-) with the virus-specific CTL that appeared at the same time 7 days postinfection, but had different target specificities. These alloreactive CTL lysed allogeneic but not syngeneic or xenogeneic targets. These were distinct from activated NK cells, which lysed all target cell types, peaked 3 days postinfection, and had a phenotype of asialo GM1+, Thy-1 +/-, Lyt-2.2-. Cold target competition studies indicated that there were several subsets of alloreactive T cells with distinct specificities, and that these alloreactive T cells were not subsets of the virus-specific T cells. Similar types of alloreactive CTL were induced at much lower levels in C3H/St mice. This may indicate that the generation of this "aberrant" T cell activity is under genetic control. Hence, the LCMV infection of C57BL/6J mice induces several cytotoxic effector populations including alloreactive CTL, activated NK cells, and virus-specific CTL. Virus infections therefore have the ability not only to polyclonally stimulate B cells, as previously described, but also to stimulate CTL.     

Presence of T cell-associated surface antigens on murine NK cells.

The expression of T cell-associated surface antigens on natural killer (NK) spleen cells of C57BL/6 mice was evaluated by cytotoxic depletion experiments with alloantisera prepared against the Thy 1, Ly 1, Ly 2, Ly 5, Ly 6, and NK 1 antigens. The NK activity of these nonimmunized spleen cells for YAC-1 leukemia cells was dramatically reduced by antisera to the Ly 5 and NK 1 antigens. Variable results were obtained with anti-Ly 6 sera--certain pools of this antiserum decreased the NK activity, whereas other pools showed only negligible effects. The NK activity of the same cell suspensions was not affected by antisera to the Thy 1, Ly 1, and Ly 2 antigens. In parallel tests the T cell-associated cell surface antigens of alloimmune T killer cells were similarly evaluated by cytotoxic depletion experiments. In this case, the activity of these cells was consistently diminished by antisera to the Thy 1, Ly 2, Ly 5, and Ly 6 antigens, but not by antisera to the Ly 1 and NK 1 antigens. On this basis it was concluded that the NK cells expressed a restricted subset of T cell-associated alloantigens and therefore may have been derived from the T cell lineage of lymphocytes.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.     

In vitro modulation of natural killer cell activity in non-Hodgkin's lymphoma patients after therapy

Summary The depressed natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC) and NK cytotoxic factor cytotoxicity in untreated non-Hodgkin's lymphoma patients were found to be elevated after chemotherapy. In vitro treatment of the effector NK cells with interferon could augment the NK activity in normal subjects and treated patients to a comparable degree. Chemotherapy mainly affected the post-binding events in the NK cytotoxic process by causing an increase in the active killing potential of the NK cells. This study provides a better understanding of changes in the NK cytotoxic mechanism in non-Hodgkin's lymphoma patients and the role of interferon in this process.B. A. Mehta is a recipient of the Lady Tata Memorial Trust, India, Senior Scholarship     

The "lazy" NK cells of Chediak-Higashi syndrome

Natural killer (NK) function, measured in a short-term (4-hr) 51Cr-release assay, is profoundly depressed in circulating PBL of donors with Chediak-Higashi syndrome (CHS). In this study, we demonstrate that CHS NK cells can express relatively normal lytic function after prolonged exposure in vitro to high levels of activating as well as cytotoxic stimuli. After activation with the human cloned interferon (B1) for 24 hr, CHS NK cells have lytic activity comparable to unactivated normals in a 4-hr 51Cr-release assay. In addition, after 5 days of activation with mitomycin C-treated B cell lines, CHS NK cells have levels of activity similar to those of activated normals but are defective in generating cytotoxic cells capable of lysing the stimulator B cell. Even though CHS NK cells are defective in a 4-hr 51Cr-release assay, after 16 hr they enhance their killing capability 200 to 400-fold. In fact, after 16 hr of interaction with K562 target cells, CHS NK cells are capable of releasing NK soluble cytotoxic factors. These results are consistent with the hypothesis that CHS NK cells have all the necessary cellular structures and molecules required for them to function as lytic effector cells, but their lack of cytotoxic function is due to a relative refractoriness in initiating the post-binding lytic mechanism.     

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.     

Detection of function-associated molecules on rat NK cells and their role in target cell lysis.

Anti-effector cell mAb 5C6.10.4 (5C6) inhibits cytotoxic activity of fish nonspecific cytotoxic cells (NCC). We now show that 5C6 also inhibits mammalian NK cell activity using fresh and cultured (CRC) leukemic rat NK cells. The inhibitory activity of 5C6 was caused by blocking of conjugate formation between NK cells and YAC-1 targets. Binding studies done by flow cytometry (FCM) showed that mAb 5C6 specifically bound to 8% of unfractionated rat spleen cells. Enrichment by nylon-wool fractionation produced 27.2% specific binding, along with a 3.4-fold enrichment in cytotoxic activity. Tissue distribution studies revealed that the highest number of cells recognized by mAb 5C6 were found in NWNA spleen cells (28.7%), followed by liver (18.9%) and peripheral blood (13.9). Two-color FCM showed that although all 3.2.3 mAb-positive cells were also stained with mAb 5C6, a small percentage of 3.2.3. negative noncytotoxic NWNA spleen T cells were 5C6 positive. Redirected lysis experiments demonstrated that anti-effector mAb-producing myeloma cells could be killed by CRC and NWNA spleen cells. In addition, mAb 5C6 produced specific inhibition of redirected lysis of each myeloma target. Experiments were also conducted to determine the signaling capability of the FAM complex. Binding of the anti-FAM mAbs to NWNA rat spleen cells caused a rapid increase in cytosolic free calcium of approximately 472 nM. Western blot analysis of CRC cell lysates showed that the molecules recognized by anti-FAM mAbs have molecular weights of 38 and 42 kDa. These studies indicate that the anti-effector mAbs recognize a functionally relevant molecule on rat NK cells that is involved in the first steps of cytolysis, i.e., antigen recognition, and which also triggers the activation of signal-transducing events in these cells.     

Fucose-activated killer (FAK) cells: anomalous killers with augmented cytotoxic activity

The effects of monosaccharides on various lymphocyte functions have provided useful probes for the study of cell-cell interactions. In this report, we show that a monosaccharide, alpha-L-fucose, significantly enhances the cytolytic capacity of MLC-induced or preincubated effector cells. The increase in activity was seen against cytotoxic T lymphocyte (CTL) targets (:relevant PHA blasts), natural killer cell (NK) targets (:K562), and natural cytotoxic cell (NC) targets (:MA-160). In addition, traditionally NK-insensitive targets (Raji cells, irrelevant and autologous PHA blasts) were lysed after preincubation of effector cells with fucose. Conversely, ADCC activity was not significantly increased with fucose induction. The addition of fucose directly to assay cultures did not enhance NK or CTL activity, whereas other sugars, such as alpha-methyl-D-mannoside and D-fructose, were inhibitory. The proportion of target-binding cells was not affected by preincubation with fucose, but the percentage of lytic conjugates was doubled. Significant augmentation of NK activity could be observed within 24 hr of incubation with alpha-L-fucose. Conversely, when fucose was added more than 24 hr after initiation of the culture, the increase in cytolytic activity was not observed. Parallel to the increase in cytolytic activity, after preincubation with alpha-L-fucose, an increase in the expression of a newly defined human NC cell marker, HNC-1A3, was observed. The HNC-1A3+ cells were not the major subpopulation responsible for fucose-induced activity, as ascertained by the use of positively sorted cells. The populations expressing antigens defined by the antibodies OKT8 and Leu-7 showed no quantitative change. The treatment of cells with OKM1 and complement (C) before culture eliminated fucose-enhanced killing, whereas similar treatment with OKT8 and C had no significant effect. The induction of fucose-activated killers (FAK) does not result in higher concentrations of interferon (IFN) in culture supernatants, in contrast to poly I:C, which induced both higher cytolytic activity and high titers of IFN. In addition, the induction of FAK was not sensitive to 100 ng/ml of cyclosporin A, suggesting that IL 2 did not play a major role in fucose activation of killing. These results provide strong evidence that alpha-L-fucose is capable of augmenting nonspecific activity by acting on OKM1+ precursors of cytotoxic cells and influencing a postbinding event.     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

An analysis of the natural killer cell defect in beige mice

Although C57BL/6 bg^J/bg^J mice exhibit very low or undetectable levels of endogenous natural killer cell activity, such activity can be induced by the administration of BCG or tilorone hydrochloride to these animals. This cytotoxic activity has been shown to be due to NK cells by the criteria of nylon-wool nonadherence and of effector cell sensitivity to treatment with either anti-asialo GM₁ serum or high concentrations of anti-Thy 1.2 serum, in the presence of complement. Even after the administration of inducing agents, however, beige mice continue to display significantly less NK activity than do their heterozygous littermates. In an attempt to ascertain what cell might be defective in responding to induction, we utilized an in vitro system in which the induction of NK activity by poly I:C in a nylon-wool nonadherent population is dependent upon plastic-adherent cells. We found that adherent cells from either beige or heterozygous mice were indistinguishable in their ability to restore the NK response of nylon-wool-nonadherent spleen cells stimulated with poly I:C. This was true when either beige or heterozygous mice were used as the source of responder cells. Thus, it appears that the defect in responsiveness to inducing agents may reside in the beige NK cell itself.     

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.