Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

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.     

Synergistic effects of phorbol ester and interferon-alpha: target cell class I HLA antigen expression and resistance to natural killer and lymphokine-activated killer cell-mediated cytolysis

This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-alpha (IFN-alpha) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-alpha or phorbol ester (PDBu). Addition of PDBu together with IFN-alpha had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-alpha and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-alpha made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-alpha. In contrast to PDBu, 4 alpha PDD, a biologically inactive phorbol analogue, alone or combination with IFN-alpha, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class I antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-alpha clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-alpha decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.     

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.     

IFN increases class I MHC antigen expression on adenovirus-infected human cells without inducing resistance to natural killer cell killing.

It has been previously shown that unstimulated NK cells cannot preferentially lyse adenovirus serotypes 2 and 5-infected human cells. In this study, the ability of IFN to promote the selective NK cell-mediated lysis of adenovirus-infected human cells was determined. The relationship between target cell susceptibility to NK cell-mediated killing and class I Ag expression was also analyzed through the use of adenovirus serotype 2 and 5 mutants that do not make the adenovirus early region 3 19-kDa class I binding protein. IFN induced the selective lysis of adenovirus serotype 2 and 5-infected human cells by activating NK cells (IFN-alpha) and protecting uninfected, but not adenovirus-infected cells, from NK cell-mediated lysis (IFN-gamma). IFN-gamma increased the expression of class I Ag on the surface of cells infected with the adenovirus early region 3 deletion mutants, dl327 or dl801, to a level equal to or greater than that expressed on uninfected cells. Despite the increased expression of class I Ag, IFN-gamma could not protect these adenovirus-infected cells from NK cell-mediated lysis. Thus, dl327 or dl801 infection prevented IFN-gamma's induction of cytolytic resistance to NK cell-mediated killing but left IFN-gamma's induction of class I Ag intact. Surface class I Ag levels were substantially higher on IFN-gamma-treated, dl327-, and dl801-infected cells in comparison to cells infected with wild type adenovirus serotype 5. Again, higher target cell levels of class I Ag did not correlate with increased resistance to NK cell-mediated lysis because there was equivalent NK cell-mediated killing of IFN-gamma-treated adenovirus serotype 5-, dl327-, or dl801-infected cells. Thus, IFN-gamma only protects uninfected cells from NK cell-mediated killing, irrespective of target class I Ag levels, and thereby concentrates NK lytic activity on just adenovirus-infected cells. These data demonstrate that IFN-gamma's ability to protect target cells from NK cell-mediated cytolysis is unrelated to IFN-gamma's induction of surface class I MHC Ag.     

Mechanisms involved in NK resistance induced by interferon-gamma.

Human tumor cell lines were treated with interferon-gamma (IFN-gamma) and then used as target cells in NK assays to measure their ability to form conjugates and stimulate the production of NK cytotoxic factors (NKCF) and to determine their susceptibility to NKCF lysis. K562 and cell lines RS1, RS3, RS7, CAC, and CAP2, obtained from solid brain tumors, were used as targets, and peripheral blood lymphocytes (PBL) from normal donors were used as effector cells. IFN-gamma-treated cell lines had a decreased susceptibility to NKCF lysis and a decreased ability to induce the release of these factors without affecting target-effector cell binding. These results were not due to changes in HLA class I antigen expression, given that the level of HLA class I antigens on the tumor cell lines was not affected, the only exception being K562. In an attempt to further clarify the possible influence of HLA class I expression on K562, IFN-gamma-pretreated K562 cells were separated into HLA class I positive and HLA class I negative subsets for the NK assays. The results showed that both populations behaved similarly upon target-effector conjugate formation, whereas the HLA class I positive population showed a reduced susceptibility to lysis by NK cells and NKCF. Thus, these results establish that NK resistance induced by IFN-gamma is mediated by blocking the target cell's ability to activate NK cell triggering and release of NKCF and by blocking its susceptibility to lysis by these factors. This analysis helps to clarify not only the NK process but also the controversial regulatory effect of IFN in NK lysis.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection

Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-gamma production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-alpha or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-alpha-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-alpha stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-alpha. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-alpha stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-alpha activates NK cells; this impairment might affect IFN-alpha responsiveness in hepatitis C virus infection.     

Activated endothelial cells resist lymphokine-activated killer cell-mediated injury. Possible role of induced cytokines in limiting capillary leak during IL-2 therapy

We previously demonstrated that IL-2 promotes the adhesion of NK cells to endothelial cells (EC) and that EC are readily lysed by lymphokine-activated killer (LAK) cells in vitro, suggesting that cell mediated endothelial injury may contribute to the capillary leak syndrome observed in patients treated with IL-2. In this investigation, we sought to determine the effects of EC activation on the in vitro susceptibility of EC to LAK cell-mediated cytolysis. Despite increased binding of CD16+ lymphocytes to TNF-activated EC monolayers, prior exposure of EC to any of several IL-2-inducible cytokines including TNF-alpha, IL-1 beta, and IFN-gamma not only failed to render the EC more vulnerable to cytolysis but increased their resistance to LAK cells in 111Indium release cytolysis assays. This decrement in susceptibility to cytolysis resulting from prior exposure to cytokines preceded any detectable increase in HLA class I or II Ag expression. In cold target competition experiments with LAK cell effectors and radiolabeled K562 target cells, TNF-primed EC were no more competitive than unstimulated EC, and in assays with unstimulated PBMC effectors, the addition of unlabeled TNF-activated EC actually increased the cytolysis of the radiolabeled tumor cells. The effects of various cytokines and lymphocyte preparations on EC permeability were also evaluated. In these experiments, saphenous vein EC were cultured on porous filter disks, exposed to cytokines or lymphocytes, and the diffusion of 125I-BSA through the filters was then measured. Exposure to IL-2, IFN-gamma, or TNF-alpha did not increase the diffusion of the BSA through the EC-coated filters, whereas LAK cells markedly increased their permeability. Consistent with the results of the cytolysis assays, pretreatment of the EC with TNF, IL-1, or IFN-gamma diminished the LAK cell-induced increase in BSA diffusion. These results suggest that although circulating IL-2-inducible cytokines such as TNF and IFN-gamma may activate EC in vivo and contribute to lymphocyte margination and lymphopenia, they may not be directly responsible for the IL-2-induced capillary leak syndrome and may actually protect EC from LAK cell-mediated injury.     

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.     

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Interferon and butyrate treatment leads to a decreased sensitivity of NK target cells to lysis by homologous but not by heterologous effector cells

Human K-562 and HHMS cells were pretreated with human recombinant interferon (IFN)-gamma and used as targets in NK assays against human and murine effector cells. A protective effect against NK lysis was observed only in the homologous assay, whereas no change or even a slight increase in NK sensitivity against heterologous effector cells was found. In cold target inhibition experiments IFN-treatment of K-562 cells led to a decrease in their capacity to act as competitors in the homologous NK assay, leaving their inhibitory capacity unaltered in the heterologous assay. In accordance with results observed using human NK targets, murine YAC-1 cells treated with mouse recombinant IFN-gamma did not lose their susceptibility to human NK cells. However, they were markedly less susceptible to lysis mediated by murine effectors. Butyrate, another compound causing decreased sensitivity of K-562 cells for human natural killing, also failed to reduce the susceptibility against murine NK cells. The results indicate that the NK-resistant tumor target phenotype caused by IFN or differentiation-inducing agents can only be detected by homologous but not by heterologous effector cells. This suggests that major differences exist between the inter- and intraspecies NK killing mechanisms.     

Characteristics and mechanism of IFN-gamma-induced protection of human tumor cells from lysis by lymphokine-activated killer cells

IFN-gamma has been shown to reduce the sensitivity of tumor cells to lysis by NK cells. The close relationship between NK cells and lymphokine-activated killer (LAK) cells has prompted us to investigate whether IFN-gamma pre-treatment also affects the sensitivity of tumor cells to lysis by LAK. We have shown previously that IFN-gamma can induce a significant reduction in the sensitivity of both cultured and fresh (surgically obtained) human tumor cells to lysis by LAK. Herein we show that changes in the sensitivity to LAK lysis of cultured human tumor cells can be induced by as little as 1 to 10 U/ml of IFN-gamma; a dose well within the range that can be achieved in vivo. Protection is induced within hours after treatment with IFN-gamma and is dependent on the continued presence of IFN-gamma. Tumor cells cultured in IFN-gamma for several days remain less sensitive to lysis and do not become refractory to IFN-gamma-mediated protection. In the absence of IFN-gamma, treated tumor cells regain "normal" sensitivity to lysis within 48 to 72 h. We have also investigated the mechanisms by which IFN-gamma reduces tumor cell sensitivity to LAK lysis using cold target competition, monolayer depletion, direct binding, and kinetic assays. IFN-gamma pre-treatment does not alter the kinetics of tumor cell lysis by LAK. Our data are most compatible with a model in which IFN-gamma reduces the ability of a subpopulation of tumor cells to induce the LAK effector cell to initiate lysis. These results are closely parallel to observations made on the IFN-mediated protection of targets from NK lysis and support the notion that NK- and LAK-mediated lysis are closely related. These results may have significance in vivo because high levels of IFN-gamma may be present at the tumor site or may be induced after therapeutic immunomodulation.     

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.     

Factors determining the ability of cytokine-activated killer cells to lyse human ovarian carcinoma targets

Lysis of human ovarian carcinoma cells by natural killer (NK) cells, interferon-alpha activated NK cells (alpha-NK) and lymphokine-activated killers cells (LAK) was studied using both fresh tumor cells and a cell line (HEY) as targets. A clonogenic assay to measure cell kill was more sensitive than a 4-h 51Cr release assay. Both assays showed that single cells were more effectively lysed than were tumor clumps (spheroids). Freshly isolated tumor cells studied in the 51Cr release assay appeared equally susceptible to lysis by LAK cells whether in the form of clumps or single cells, but NK and alpha-NK effectors appeared much less effective in lysing susceptible target cells when they were in clumps. Tumor cells from some patients showed marked resistance to lysis by NK and alpha-NK cells in fractions enriched for clonogenic cells, even when tested in a single cell-suspension, whereas LAK cells were always cytolytic. These data suggest that intrinsic resistance of ovarian carcinoma to lysis by LAKs is unlikely to explain failure of LAK + IL-2 therapy to eradicate tumor in vivo.     

Properties and characterization of a rat spleen cell-derived factor that induces resistance to natural killer cell lysis in YAC lymphoma cells

Supernatants of Con A-stimulated rat spleen cell cultures contain a factor that induces relative resistance to NK cell-mediated cytotoxicity in the YAC cell line, a line that is otherwise highly susceptible to murine NK cell-mediated lysis. This NK-lysis resistance-inducing factor (LRIF) has a Mr of 12,600 Da, as determined by gel filtration chromatography, and an isoelectric pH of 4.8. NK-LRIF is heat labile and is de-activated by treatment with proteolytic enzymes. Unlike immune-IFN (IFN-gamma), NK-LRIF is not inactivated by pH 2 treatment, and antibodies capable of neutralizing IFN-alpha and IFN-gamma do not abrogate the effect of NK-LRIF. Highly purified IL-2 preparations lack NK-LRIF activity. NK-LRIF does not induce a general resistance to lysis in YAC cells, because control and NK-LRIF-treated YAC cells were equally susceptible to alloimmune cytotoxic T cells. YAC cells treated with NK-LRIF showed a marked enhancement (5- to 10-fold) in the expression of class I MHC Ag. This observation supports the proposition that the NK susceptibility of target cells could be inversely related to the expression of class I MHC Ag.     

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.     

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of human lymphokine-activated killer cells by IFN-alpha and IFN-gamma

By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.