Lysis Of K562 Cells Research Articles

N-linked oligosaccharides can protect target cells from the lysis mediated by NK cells but not by cytotoxic T lymphocytes: role of NKG2-A.

We have previously shown that glycophorin A (GPA), inserted by electropulsation into the membrane of K562 cells, protected them from natural killer (NK) cell-mediated cytotoxicity and the unique N-linked oligosaccharide of GPA was essential for resistance to occur. The present study demonstrates that the protection level conferred by GPA is similar to the resistance induced by HLA-Cw3 expressed by transfected K562 cells. A monoclonal antibody against NKG2-A, an NK inhibitory receptor interacting with HLA class I antigens and belonging to the C-type lectin receptor, was able to restore the ability of NK cells to lyse K562 cells expressing HLA-Cw3 at the cell membrane but not electroinserted-GPA, suggesting that the N-linked oligosaccharide of GPA cannot be a ligand for NKG2-A. GPA was then electroinserted into the membrane of two lymphoblastoid B-cell lines: one was sensitive to NK cell-mediated lysis, the other was susceptible to cytotoxic CD8+ T-lymphocyte (CTL)-mediated cytotoxicity. The electroinserted GPA protected the target cells from NK-mediated cytotoxicity, whereas it did not modify the cell susceptibility to lysis by CTL. Endoglycosidase F treatment abolished the resistance towards NK cell-mediated lysis, suggesting that N-linked glycans could inhibit mechanisms used by NK cells to exert their cytotoxic function in agreement with our previous results.

THE EFFECT OF PRAVASTATIN ON CHOLESTEROL LEVELS AND NK CELL ACTIVITY AFTER KIDNEY TRANSPLANTATION.

617 Introduction. Hyperlipidaemia is common after organ transplantation and is associated with an increased risk for cardiovascular disease. Administration of pravastatin (an HMG-CoA reductase inhibitor) early after transplantation successfully lowered cholesterol levels, while a reduced number of acute rejection episodes, accompanied by a decrease in natural killer (NK) cell activity has also been reported. If true, a consistent low NK cell activity due to pravastatin might impair tumour surveillance leading to an increased risk to develop cancer, thereby abrogating the beneficial lipid lowering effect of pravastatin. Therefore, we studied the effect of pravastatin on NK cell activity in stable renal transplant patients. Methods. From 10 cyclosporin treated patients with hypercholesterolaemia, 1 to 10 years after transplantation, we determined the number of NK cells by flowcytometry, (CD3−CD16+CD56+) and their cytotoxic activity (lysis of K562 cells) before, 6 weeks and 12 weeks after initiating pravastatin treatment. Results. Total cholesterol levels decreased from 8.7 mmol/L to 7.2 at 6 weeks and 7.1 at 12 weeks after initiation of pravastatin. LDL-levels decreased from 5.2 μmol/L to 3.6 and 3.8 at 6 and 12 weeks respectively (p<0.05), whereas HDL-levels had a tendency to increase from 1.4 to 1.6 at 6 weeks and 1.7 at 12 weeks. Although the number of NK cells remained the same, the cytotoxic activity of NK cells showed a significant (p=0.01) rise from 16.7% lysis (median, range 5.5-26.5) to 23.7% (10.0-37.7) during the first 6 weeks after administration of pravastatin. At 12 weeks NK cell activity was 19.2% (11.5-41.5). Conclusion. In contrast to previous reports on decreased NK cell activity due to pravastatin treatment early after transplantation, we can not confirm these results in stable kidney recipients. In our hands the cytotoxic activity of NK cells 12 weeks after starting pravastatin treatment was even slightly elevated. In view of the potential role of NK cells in tumour and virus surveillance these data are reassuring.

Differential effect of phototherapy on the activities of human natural killer cells and cytotoxic T cells

Exposure to ultraviolet B (UV-B) light is recognized to induce suppression of certain immune responses, particularly delayed hypersensitivity. However, its effect on cytotoxic T lymphocyte (CTL) activity, of major importance in the resistance to viruses and tumours, has not been assessed to the same extent. In this study five normal subjects, seropositive for herpes simplex virus (HSV), underwent a standard course of broadband UV-B therapy, as used in the treatment of psoriasis. They received whole-body irradiation thrice weekly for four weeks with incremental doses dependent on skin type. Blood samples were taken immediately before, at two time points during, and at the end of the therapy. An HSV-specific CTL assay was performed using autologous B cells transformed with Epstein-Barr virus as targets. No consistent modulation in CTL activity was obtained as a result of the therapy. The CTLs were separated into CD4 and CD8 subsets by positive selection and, again, no effect of irradiation on CTL activity within each of these two populations was observed. In contrast, the natural killer (NK) cell activity, assessed by the lysis of K562 cells, was significantly reduced at the first time point after the initiation of the phototherapy in all five subjects, and it continued to decline as the treatment progressed. Thus a differential effect of UV-B exposure on cytotoxic activity has been demonstrated: the HSV-specific CTL response is unchanged, while the NK response is suppressed.

Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis.

We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin. The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture. The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns. Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable. Just one spot remained unaltered in its intensity throughout the whole period of incubation. About 440 spots reveal a t1/2 shorter than 8 min. Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment. About 30 spots have a t1/2 shorter than 8 min. We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner. The system is much less effective in eliminating proteins from distant species, like yeast. Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.

Taxol Pretreatment of Tumor Targets Amplifies Natural Killer Cell Mediated Lysis

Taxol is known to polymerize and stabilize microtubules and thereby alter many cellular functions. Our studies examined the effects of taxol pretreatment of tumor targets and cytotoxic effector cells in an effort to determine whether such treatment would result in increased tumor cell lysis without affecting cytotoxic cell function. Our studies demonstrated that taxol con-centrations of 6–30 ng/ml which induced ∼50% growth inhibition and ±50% block in the G2/M phase of the K562 cell targets did not have any significant effect on the functional ability of NK cells to lyse K562 cells. Pretreatment of K562 cells with taxol (6 and 30 ng/ml) resulted in an increase in K562 cell lysis by NK cells (or NK cells stimulated with 100 units/ml of rIL-2) in 7 out of 9 donors. The amplification of NK cell-mediated lysis of tumor targets due to taxol pretreatment may provide a combination therapeutic approach which includes taxol treatment followed by rIL-2 stimulation of the immune killer cell function.

Active specific immunotherapy of renal cell carcinoma: cellular and humoral immune responses.

We investigated the effect of vaccinating renal cell carcinoma (RCC) patients with irradiated autologous or allogeneic tumor cells and Newcastle disease virus (NDV) as adjuvant on cellular and humoral antitumor immunity. By Western blot analysis, we found that vaccination induced antibody formation in 33 of 34 patients against NDV proteins but not against tumor cell related antigens. NDV proteins detected had molecular weights of 53 kDa, 55-56 kDa, and 66 kDa. ADCC by patients' isolated PBMC and patients' sera against autologous or allogeneic tumor cells was not enhanced after vaccine treatment in a nonradioactive cytotoxicity assay. Target cells infected with NDV were lysed more effectively (p < 0.05) in ADCC after vaccination than noninfected targets. Natural cellular cytotoxicity of patients' isolated PBMC was not altered during vaccine treatment. Specific lysis rates against autologous and allogeneic RCC cells not exceeded 10% (effector:target ratio 50:1). Specific lysis of K-562 cells was > 20%; a slight decrease in lysis during vaccination was not significant. Numbers of lymphocyte subsets from patients' peripheral blood analyzed by FACS revealed significant expression of CD20+ (p < 0.02) and CD39+ (p < 0.03) cell numbers by vaccine therapy. Cytokine detection in patients' sera by ELISA showed significant increases (p < 0.05) for IFN-gamma and TNF-alpha but not for IFN-alpha four h post vaccination. Thus, immunomodulation with autologous or allogeneic RCC tumor cell vaccines is mainly due to cytokine induction, whereas tumor specific humoral or cellular responses are not detectable in patients' peripheral blood.

Lysis of leukemic cells by human macrophages: inhibition by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor

Proteases are known to be involved in regulation of macrophage activation and killing. We examined the effect of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on lysis of leukemic cells by human macrophages. Monocytes, isolated by Histopaque gradients and centrifugal elutriation, were cultured for 5 days in RPMI-1640 medium with 5% AB serum, and then activated with interferon-gamma (IFN-gamma; 100 U/mL) and lipopolysaccharide (LPS) (5 ng/mL), with or without AEBSF, for 2 days. On day 7, macrophages were washed, fresh medium without AEBSF added, and target cells added for 2 days. Lytic activity against two leukemic cell lines (K562 and HL-60) was assessed by an 111indium-releasing assay. Macrophages treated with IFN-gamma + LPS lysed K562 and HL-60 cells. AEBSF (50-150 microM) blocked the killing of these leukemic cells in a concentration-dependent manner. Other protease inhibitors were not effective. AEBSF was nontoxic at the concentrations used, and did not inhibit tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion from the macrophages. The lytic activity against leukemic cells was inhibited by anti-TNF-alpha antibody, but not by anti-IL-1 beta, nor by superoxide dismutase or catalase. However, the leukemic cells were resistant to being killed by recombinant TNF-alpha alone in the absence of macrophages, indicating that TNF-alpha was required for killing, but that other factors that were inhibited by AEBSF were also required. Serum-free culture supernatant of activated macrophages had significant cytotoxic activity against leukemic cells. This cytotoxic activity was not altered by addition of AEBSF to the culture supernatant, suggesting that AEBSF affected macrophage activation, rather than inhibiting cytotoxic proteases secreted by the macrophages, or affecting the target cells themselves. Thus, a protease, which is susceptible to AEBSF, might be involved in the activation of macrophages, and might regulate the secretion of antitumor effector molecules other than TNF-alpha.

Natural Cytotoxicity toward K562 Cells by Macaque Lymphocytes from Infancy through Puberty: Effects of Early Social Challenge

The consequences of a single maternal separation experience followed by reunion at 6–7 months of age was studied in socially housed pigtail (Macaca nemestrina) and bonnet (Macaca radiata) macaques. At 15 months, these subjects were removed from their natal group and placed in same species social groups, consisting of other separated and matched control subjects. Some subjects were followed until they reached an average age of 4 years while remaining in this group. Blood samples were drawn to permit assessment of the ability of peripheral blood lymphocytes to lyse,in vitro,K562 cells. Maternal separation failed to affect lysis of K562 cells acutely, although lysis by matched control subjects appeared to be transiently reduced 2 h after removal of the adult female. A longer-term influence was noted such that lysis of targets in previously separated subjects was greater than that in matched controls. Lysis rose over time regardless of species or experimental condition. A striking internal consistency in the lysis was also noted. Lysis was highly intercorrelated (r’s > .60,p≤ .005) within subjects across time during baseline, separation, and reunion phases of the acute social challenge. In addition, there was a tendency for this correlation to hold over the longitudinal phases. Lysis of K562 targets by macaque lymphocytes would appear to possess trait-like stability; however, the range of lysis may be modified by early experiences.

Surgical and Psychological Stressors Differentially Affect Cytolytic Responses in the Rhesus Monkey

Four experiments were conducted in aged rhesus monkeys to investigate how physical and psychological stressors influence the lymphocyte cytolytic responses against two target cell lines. An initial analysis of the lytic activity of various cell subsets against K562 and RAJI target cell lines suggested that both CD3+CD8+ and several CD3− subsets were responsible for lysis of the K562 cells. The RAJI cell line, in contrast, was killed primarily by CD3− subsets. To explore the implications of this differential mediation of lysis, cytolytic activity was evaluated after a physical challenge (surgery), a psychological disturbance (social separation), andin vitroincubation of lymphocytes with cortisol. The minor surgical procedure—laparoscopic examination—resulted in a significant decrease in lymphocyte cytolytic responses against both target cells 1 week postsurgery. In contrast, psychological disturbance elicited by changes in social relations caused a dual response, differentially affecting the lysis of either K562 or RAJI cells, dependent upon the type of behavioral reaction. Incubation of lymphocytes with cortisolin vitroindicated that lysis of both targets could be affected by corticosteroids, but the high concentration required (10−6M) suggested that thein vivoinhibition of cytotoxicity may not have been mediated by adrenocortical activation. Overall, the results highlight the value of utilizing multiple target cell systems in the analysis of cytolytic activity, especially in studies using nonhuman primates.

Mycoplasma arthritidis mitogen up-regulates human NK cell activity.

While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms.

CD54/ICAM-1 Is a Costimulator of NK Cell-Mediated Cytotoxicity

The receptors and the array of cell adhesion molecules regulating MHC-unrestricted cytotoxic activity of NK cells toward tumor targets have not completely characterized. Antibody inhibition studies suggest roles for a number of cell adhesion molecules (CAMs). Recent studies suggest that CAMs can function to stabilize cell-to-cell interactions and/or to provide costimulatory signals that are crucial for T cell activation. It has been difficult to experimentally demonstrate that adhesion molecules also function as costimulators in NK cell-mediated cytotoxicity. We have developed an experimental system using cells transfected with genes encoding huICAM-1 and/or LFA-3 to investigate the function of adhesion molecules. Here we report that neither the expression of transfected ICAM-1 or LFA-3 alone nor the expression of both ICAM-1 and LFA-3, in the absence of MHC class I molecules, converts a murine cell line that is resistant to NK cell-mediated lysis into a susceptible one. We next tested the ability of ICAM-1 or LFA-3-mediated interactions to provide costimulation of NK cell cytolytic activity using a "three cell" experimental system comprising human NK cells, 51C-labeled target cells, and transfected mouse cells as a source of costimulation. The ability of NK cells to lyse K562 cells or anti-CD16-coated target cells was significantly enhanced by the addition of ICAM-1-transfected cells, whereas the addition of cells transfected with LFA-3 or irrelevant genes did not enhance lytic activity. Since the transfected huICAM-1 interacts with NK cells at sites spatially separate from the NK cell-target cell interactions, our data suggest that LFA-1-ICAM-1 or MAC-1-ICAM-1 interactions can provide remote co-stimulation, via signaling events, to induce cytotoxic activity in NK cells.

Endometriotic tissues produce immunosuppressive factors.

In order to study the possible role of the immune functions in the pathogenesis of endometriosis, effects of supernatants from endometrial and endometriotic tissue cultures on the natural killer (NK) activity were examined by determining the lymphocyte cytotoxicity toward an erythroleukemic cell line (K562). Endometrial and endometriotic tissues were obtained at the time of operation from patients with ectopic endometriosis or uterine myoma. The normal peritoneum and myoma tissues were used as controls. The supernatants obtained by culturing the ectopic endometriotic tissues for 24-48 h had significant (p < 0.01) suppressive effects on NK activity of peripheral blood lymphocytes (PBL) from a healthy woman, compared to the supernatants from the normal peritoneum and the myoma tissues. There was no significant difference between NK activities of PBL from patients with ectopic endometriosis and age-matched healthy women. However, the NK activity in patients with low NK status before surgery was significantly elevated after surgery while that in patients with normal NK status before surgery was not changed. Although PBL from the same healthy women could significantly lyse the endometrial cells, the ability was about-one-third the level at which they could lyse K562 cells. These results suggest that the immunosuppressive factors secreted by endometriotic tissues may be involved in the local implantation and development of ectopic endometriosis.

Human natural killer cells fail to kill herpes simplex virus type 1-infected term villus trophoblast cells

Summary The study was undertaken to determine if HSV infection of trophoblast cells would alter the general resistant state of trophoblast cells to MHC non-restricted cell-mediated cytotoxicity. The NK cell activity against HSV-infected human term placental trophoblast cells was investigated. In a 12-hour 51 Cr release assay, PBMC, or immunosorted peripheral blood CD56 + NK cells from healthy donors were found not to lyse HSV-1-infected trophoblast cells, while HSV-1-infected placental fibroblast cells were preferentially lysed when using the same experimental set-up. Furthermore, using cold target competition assay, no inhibition of NK cell activity against the susceptible target cell K562 was noted when using HSV-infected or uninfected trophoblast cells. Inversely, both HSV-infected and uninfected fibroblast cells demonstrated a competitive inhibition of NK cell lysis of K562 cells. The observed differences between the susceptibility of HSV-infected placental trophoblast and fibroblast cells to NK lysis could not be explained by a difference in the surface expression of HSV-antigens or transferrin receptors. Additionally, no significant differences were observed for de novo production of IFN by effector cells during the cocultivation with HSV-infected trophoblast and fibroblast cells that would explain the observed discrepancies in NK susceptibility. Consequently, these in vitro results suggest that the observed resistance of trophoblast cells to maternal cell-mediated lysis is not modified by HSV-1 infection and that the peripheral blood NK cells are not involved in the prevention of a possible vertical HSV spread through infected trophoblast cells.

Natural killer-cell function in hemodialysis patients: Effect of the dialysis membrane

Natural killer (NK) cells are specific peripheral blood lymphocytes which are involved in the lysis of malignant and virally transformed cells. In a prospective study of eight hemodialysis patients, we investigated the effects of recurrent exposure to the cuprophane (CU) membrane on the number and functional ability of NK cells, against both their classical in vitro target, K562 cell line, as well as the beta 2m/HLA negative cells that emerge during dialysis with CU membrane. The percent of NK cells, defined by the CD56 epitope, increased from 27.7 +/- 7.9% of cells at baseline to 59.2 +/- 12.0% after two weeks of dialysis with new CU membrane (P < 0.01). The ability of these cells to lyse K562 cells decreased from 28.7 +/- 16.5% at baseline to 12.5 +/- 6.2% (P < 0.001) after two weeks of dialysis with CU membrane, while their cytotoxicity against beta 2m negative cells increased during the same period from 32.5 +/- 12.4% to 61.3 +/- 23.7% (P < 0.001). These results are consistent with the observation that the cytolytic ability of NK cells is inversely related to target cell expression of HLA antigens and beta 2m expression on cell surfaces. In addition, the results of these studies confirm in vitro observations of the decrease in cytolytic activity of the NK cells when exposed to the CU membrane, and may explain the emergence of these beta 2m/HLA negative cells during dialysis with CU membrane. It is possible that these observations may also have a clinical relevance to the immune defects and increased incidence of malignancy in uremia.

Fluorescent europium chelates as target cell markers in the assessment of natural killer cell cytotoxicity

A time-resolved fluorometric assay for the detection of natural killer cell activity against target cells labelled with the fluorescent chelate europium-6,6″-bis[ N, N-bis(carboxymethyl)-aminomethyl]-4'-phenyl-2,2',6',2″-terpyridine (EuCAPT) has been developed. In the assay released EuCAPT from lysed K-562 cells is measured in the supernatant after co-incubation of the target cells with effector cells. Thus, the performance of the assay is essentially similar to the previously described EuDTPA assay and the widely used 51Cr assay. EuCAPT is released from target cells lysed by effector cells faster than 51CrO 4 2− but somewhat slower than EuDTPA. In contrast to methods based on prompt fluorometry the autofluorescence from culture medium supplemented with serum can be avoided by the use of time-resolved fluorometry. The result shows that fluorescent europium chelates provide an alternative to radioactive markers currently used for the assessment of in vitro cellular cytotoxicity.

Cefodizime stimulates subpopulations of cells mediating spontaneous or antibody-dependent cytotoxicity in patients with bacterial infections.

The influence of cefodizime (CDZ) on spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) was investigated in nine patients with conditions predisposing to infection (T-cell deficiency, humoral immune deficiency, myeloproliferative syndrome, kidney or liver damage, or chronic pulmonary disease). SCMC, using the K562 and LIK cell lines as targets, and ADCC, using the LIK cell line, were measured at baseline and after ten days of therapy with CDZ for lower respiratory tract infections. Six patients were cured; clinical outcome was not evaluable in the other three. SCMC lysis of K562 cells did not change significantly. SCMC and ADCC lysis of LIK (lymphoblastoid) cells both increased significantly. CDZ selectively stimulated a subpopulation of NK cells in this population of immunocompromised patients with lower respiratory tract infections.

Evidence for the involvement of CD56 molecules in alloantigen-specific recognition by human natural killer cells.

In recent reports we have described the generation of natural killer (NK) lines devoid of CD3/TCR structures but with apparent specificity for allogeneic target cells. Using one such NK line as an immunogen, we now report the generation of two monoclonal antibodies (mAbs), designated 2-13 and 5-38, which bind selectively to the majority of CD3-, CD16+, CD56+ lymphocytes and inhibit the lysis of specific allogeneic target cells by a panel of alloreactive NK lines. By contrast, these mAbs had no effect on classical NK cell mediated lysis of K562 cells or major histocompatibility-restricted T cell-mediated cytolysis. Immunoprecipitation of radiolabeled NK lines followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the target molecules of both mAbs have a molecular mass of approximately 180 kD. Leu 19, a well-described anti-CD56 mAb, precipitated a 180 kD protein from NK cells, and the binding of Leu 19 to NK cells was blocked by pretreatment with both 2-13 and 5-38. However, in contrast to these mAbs, Leu 19 had no effect on the cytolytic activity of allospecific NK cells. Sequential immunoprecipitation analysis revealed that all three mAbs recognized distinct molecular species of CD56. We interpret these findings as indicating that multiple isoforms of CD56 are differentially expressed on NK lines and play critical roles in the recognition/interaction of these cells with their specific allogeneic targets.

Chronic active hepatitis B. Interferon-activated natural killer-like cells against a hepatoma cell line transfected with the hepatitis B virus nucleic acid.

In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not lyse the hepatitis B virus deoxyribonucleic-acid-transfected human hepatoma cell line 2.2.15, but under the same experimental conditions they did lyse K562 cells. Peripheral blood mononuclear cells from 10 out of 16 patients with chronic active hepatitis B exhibited cytotoxic activity against 2.2.15 cells in the presence of a relatively reduced natural killer cell activity to the K562 cell target. Enhancement of the cytotoxic activity to 2.2.15 cells was statistically significant in the group of patients being treated with leukocyte alpha-interferon. The activity was not influenced by the degree of human leukocyte antigen type matching between effector and target, and was enhanced by depletion of T-cells and by in vitro interferon treatment. These results therefore support the concept of a natural killer-like cell activated by clinical administration of interferon in chronic active hepatitis B patients. This cell effector was lytic for the virus B negative HEP-G2 cells also. However, T-cells purified from a few patients failed to lyse the HEP-G2 while lysing the 2.2.15 target, thus indicating that a preferential recognition of the virus-infected target may be exerted by certain T-lymphocyte subsets. The use of the human leukocyte antigen type defined, highly differentiated, hepatitis B virus releasing 2.2.15 cell line as target for fresh lymphocytes in this cytolytic assay did not disclose cytolytic T-cells in an obvious way. Further manipulation of this system perhaps using T-cell clones may be the next step to exploit the investigative possibilities offered by the availability of the 2.2.15 cell target.

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-γ and tumor necrosis factor- α1

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) 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-γ and TNF-α. 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-γ and TNF-α. 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-γ and TNF-α 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-γ and TNF-α 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.

Lactoferrin inducible monocyte cytotoxicity defective in esterase deficient monocytes

We recently demonstrated a higher incidence of monocyte esterase deficiency in patients with lymphoproliferative neoplasia and gastrointestinal carcinoma than in the normal population. Using lactoferrin to stimulate monocyte cytotoxicity we have now compared the abilities of esterase positive and esterase deficient monocytes to lyse K562 cells. The esterase deficient monocytes were from normal subjects whose monocytes have consistently failed to show cytochemical esterase staining over 30-72 months and which lack specific monocyte isoenzymes. The esterase positive monocytes were from age and sex matched control subjects. Esterase positive monocytes responded to lactoferrin stimulation by a five-fold increase in cytotoxicity whereas deficient monocytes failed to produce any response. These results indicate a possible defect in cytotoxicity of esterase deficient monocytes and together with the epidemiological findings suggest a link between monocyte esterase deficiency and neoplasia.