Characteristics of natural killer cells (NK cells) and features of the cytotoxic test in Syrian hamsters

The cytotoxic test in vitro with the use of xenogeneic target cells of human myeloma, strain K-562, labeled with 51Cr has demonstrated natural cytotoxicity of lymphoid cells from noninbred Syrian hamsters. This cytotoxicity occurs at the cost of non-adherent splenocytes. NK may be isolated over the gradient density of ficoll (1.078), selective for large granular lymphocytes. To detect the maximal lytic activity of NK from Syrian hamsters in the cytotoxic test in vitro, they should be brought into 10-12 hour contact with sensitive target cells K-562. In Syrian hamsters, the highest natural cytotoxicity is shown by the cells of the blood and spleen. In the bone marrow and thymus, it is little pronounced and is virtually absent from the peripheral lymph nodes.     

Sensitization of mouse splenocytes to normal tissue antigens and natural killer activity. I. The effect of immunization with normal syngeneic tissues

Immunization of C3HA mice with homogenized syngeneous liver led to sensibilization of splenocytes towards liver antigens and to the increase in natural killer cell activity towards K-562 cells. The dynamics of sensibilization and cytotoxicity in different time intervals after immunization has led to a conclusion about a correlation between the two above phenomena.     

Effect of a monoclonal anti-large granular lymphocyte antibody on the human NK activity

A monoclonal hybridoma antibody of IgGIa subclass was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human LGL cells. This hybridoma antibody, termed NK-8, was reactive by indirect immunofluorescence with 33% of peripheral blood LGL cells and 70% of LGL forming conjugates with K-562 cells. Monocytes, granulocytes, and other lymphocytes were nonreactive. In iodinated protein A binding assays NK-8 was nonreactive with all kinds of leukemia and lymphoma lines tested and showed activity only against LGL cells. NK-8 inhibited the LGL-mediated cytotoxicity against K-562 cells by 50 to 60% without complement and inhibited the K-562 induced interferon production from the LGL population. However, the spontaneous cytotoxicity against human skin fibroblasts was augmented if the effector cells were pretreated with NK-8.     

Specific effect of anti-transferrin antibodies on natural killer cells directed against tumor cells. Evidence for the transferrin receptor being one of the target structures recognized by NK cells

Treatment of PBL or Percoll-isolated LGL with anti-transferrin antibodies plus complement reduced their natural killing activity against K-562 cells between 30 and 70%. The same antibodies inhibited natural cytotoxicity when added directly to the assay. Similar depletion or inhibition of NK cytotoxicity was observed when using HeLa cells as targets. The decrease or inhibition by transferrin antibodies was less marked when IFN-treated PBL or LGL as effector cells were used. The inhibition of anti-transferrin antibodies seems to be located at the level of the effector cell population. When PBL but not target K-562 cells were pretreated with anti-transferrin antibodies and were washed before use in the assay, cytotoxicity was decreased by 50%. In addition, about 80% of the LGL positively selected on anti-transferrin plates stained with Leu-11. Furthermore, no reduction by anti-transferrin antibodies plus complement treatment of PBL or LGL, or inhibition by antibodies alone, was observed when the cells were tested against HSV-1-infected cells. Membrane extracts from LGL inhibited NK cytotoxicity against K-562 or HeLa cells. Moreover, the inhibitory component of this extract was removed by anti-transferrin IgG but not by control IgG. These results are in agreement with the recent hypothesis that NK cells recognize the transferrin receptor in tumor target cells, because both the transferrin receptor and anti-transferrin antibodies may share a similar structure that interacts with the NK cells.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Activity of human natural killer cells against target cells possessing different sensitivity to interferon]

The cytotoxic activity of peripheral blood natural killers (NK) against target cells (TC) J-96 and L-929 with high sensitivity to interferon (IFN) action, J-41 and MCB resistant to IFN action and line K-562 labelled by H3-uridine was studied in 14 hrs cytotoxic test. It has been shown that human TC J-96 didn't differ from the J-41 in their sensitivity to NK cytotoxicity and they are strongly resistant to NK than TC K-562. The murine TC L-929 as the human TC didn't differ from the MCB in their sensitivity to NK lysis and had also the same sensitivity to NK as the K-562 cells.     

Cytotoxic properties of the splenocytes of C3HA mice following treatment with exogenous RNA

The influence of fractions of exogenous RNA, isolated from spleens of C3HA mice and of rats, both intact (control--cRNA) and immunized with homogenate of normal syngenic, allogenic and xenogenic tissues (immune--immRNA), on the cytotoxic properties of splenocytes of C3HA intact mice was compared in the in vitro cytotoxic experiments. The splenocytes treated with different RNA fractions were used as effector-cells. In vitro cultivated MGXXIIa cells of strain specific C3HA mice hepatoma, and K562 cells of human erythroleukemia, both labeled with 3H-uridine, served as target cells. Thus, it is only the cytoplasmic fraction of immRNA isolated from the spleens of rats immunized with tissue antigens of C3HA mice that induced a more pronounced stimulation of cytotoxic activity of splenocytes.     

Natural killer cell-mediated antitumor reactivity of rhesus monkeys

We have analyzed natural killer (NK) cell-mediated antitumor activity or peripheral blood mononuclear cells of rhesus monkeys. All monkeys displayed significant NK cell cytolytic activity against the human tumor cell lines K-562, Daudi and CEM in a short-term (3 h) 51Cr-release assay. Similar to NK cells described in other species, the cytotoxic cells of monkeys were relatively nonadherent to nylon wool columns, exhibited low density after separation on discontinuous Percoll density gradients, and displayed large granular lymphocyte (LGL) morphology. Analysis of the mechanism of NK cell cytotoxicity of rhesus monkeys demonstrated that on the average, 7.1% (range: 3.1-13.2%) of lymphocytes bound to K-562 tumor, and that approximately 14.8% (range: 7.9-26.3%) of these tumor-binding cells (TBC) were cytolytically active. Examination of TBC on cytocentrifuge slides indicated that the majority of binders displayed LGL morphology. The cytotoxic reaction mediated by monkey NK cells exhibited Michaelis-Menten kinetics pattern; the maximum rate of lysis (Vmax) of K-562 was found to be 1-2 X 10(4) following 3 h of incubation. Using similar culture conditions, the recycling capacity of NK cells of this species was estimated at 2-6 times. Finally, it was observed that the NK cell activity of most monkeys could be potentiated following in vitro exposure to the biological response modifier, interleukin-2.     

Spontaneous cell-mediated cytotoxicity in patients with transitional cell carcinoma of the urinary bladder

Summary Lymphocytes isolated from the blood of TCC patients, like those of control patients, were capable of mediating spontaneous cell-mediated cytotoxicity against K-562 cells. When this natural cytotoxicity was analyzed with regard to the effector cell type it was found that in TCC patients the SLMC was mostly displayed by E-rosetting T lymphocytes, whereas compared with controls, a significant decline in the SLMC of the non-T lymphocytes was observed. The SLMC of the T lymphocytes derived from TCC patients was further demonstrated on a T leukemia target cell (Peer). When the SLMC on K-562 and on Peer target cells was compared, a specificity difference was observed between TCC and the control patients' effector cells. The SLMC activity of the TCC patients' T cells was not abolished after depletion of Fc receptor-positive cells or following treatment with monoclonal antibodies OKT 8 or OKT 4 and complement (C'). These NK-like cells are therefore distinguished from cytotoxic T lymphocytes and NK 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.     

Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts.

This study was undertaken to characterize the phenotype and function of lymphocytes derived from endomyocardial biopsies in heart transplant patients. To this aim, tissue infiltrating lymphocytes were derived from seven heart transplant patients and were analyzed for the expression of a panel of markers, including CD3, CD4, CD8, CD16, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta T cell receptor, and for their ability to lyse a series of targets, including NK-sensitive K-562 targets, NK-resistant Raji targets, donor related, and unrelated normal splenocytes. Our data show that the majority of cultured lymphocytes expressed the CD3+ phenotype and the alpha/beta T cell receptor. The CD4 and CD8 molecules were heterogeneously expressed among T cell lines tested. Concerning cytotoxic related markers, a significant percentage of cells were CD56+. The evaluation of CD45 isoforms showed that both "naive" and "memory" cells were present among heart TIL. Cytotoxic in vitro studies demonstrated that all our T cell lines showed an efficient cytotoxic machinery when tested against NK-sensitive targets. A marked lysis of donor-related splenocytes was demonstrated in all patients tested. To investigate the role of CD3 and HLA class I molecules in the cytotoxic mechanisms taking place in human heart allograft rejection mechanisms, TIL were assessed for their lytic activity against different targets in the presence of anti-CD3 and anti-HLA class I monoclonal antibodies (mAbs). Although donor-specific cytotoxicity was considerably inhibited by the anti-CD3 mAb, no inhibitory effect was displayed by this antibody on TIL-mediated cytotoxicity against donor-unrelated splenocytes. Anti-HLA class I mAb was able to inhibit both allospecific and nonallospecific cytotoxicity. These data suggest that different types of cytotoxic cells may be propagated from biopsy specimens of heart transplant patients.     

Ovine endometrial cells fail to lyse K-562 target cells: a preliminary investigation

The ability of unfractionated and fractionated endometrial cells to lyse K-562 target cells was investigated within ewes during the late luteal phase of the estrous cycle (Days 12 to 14) and pregnancy (Days 16 and 19). In separate experiments, lymphokine activated killer (LAK) cells and endometrial cells, both designated as effector cells, were co-cultured with chromium-51 (51Cr)-labeled K-562 target cells in varying effector: target cell ratios. At 22 h, lytic activity was assessed by the release of 51Cr into the culture medium. The LAK cells exhibited lytic activity in a ratio-dependent manner, whereas the unfractionated and fractionated endometrial cells failed to lyse the target cells. For ratios combined, the rate of cytotoxicity for unfractionated endometrial cells recovered from ewes in the cyclic and pregnant (Days 16 and 19 combined) groups was 13.9 and 5.4%, respectively. Although the findings are preliminary, they indicate that ovine endometrial cells recovered during the late luteal phase and early pregnancy failed to exhibit natural killer activity upon K-562 target cells.     

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines--relationship between structure and biological activity

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3相似文献   

Inhibition of human natural killer cell activity by platelet-derived growth factor

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.     

Human natural cell-mediated cytotoxicity against fetal fibroblasts. II. Isolation and target cell specificity of the effector cells.

Human peripheral Wood lymphocytes were depleted of natural killer cells cytotoxic against human fetal fibroblasts by allowing them to attack the fibroblast targets grown on plastic beads followed by gravity sedimentation under conditions in which single cells floated but the attacker cells sedimented with the carrier beads. The attacker cells could be released from the bead-grown targets and shown to be greatly enriched in natural cytotoxic activity. The effector cells depleted by fibroblast adsorption were also depleted of cytotoxic activity against other monolayer targets whereas suspension grown lymphoma and leukemia cells (MOLT-4, RAJI, and K-562) were killed as effectively as by non-depleted effector cells. In competition assays other monolayer cells inhibited the natural cytotoxicity against fetal fibroblasts but the suspension-grown cells were unable to compete. The results suggested that different effector cell populations were probably involved when monolayer vs suspension targets were used in assays for human natural cell-mediated cytotoxicity. The separation was not, however, functionally complete since in competition assays with suspension-grown target cells also monolayer cells were able to compete. Preliminary morphological characterization of the natural killer cells against fetal fibroblasts is also presented.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

In vitro cytotoxicity of melleolide antibiotics: structural and mechanistic aspects

Melleolide sesquiterpene aryl esters are secondary products of the mushroom genus Armillaria. We compared the cytotoxicity of eleven melleolides—five thereof are new natural products—against four human cancer cell lines. Armillaridin, 4-O-methylarmillaridin, and dehydroarmillylorsellinate were most active, at IC₅₀ = 3.0, 4.1 and 5.0 μM, respectively, against Jurkat T cells for the former two compounds, and K-562 cells for the latter. Dehydroarmillylorsellinate did not inhibit respiration and RNA-synthesis of K-562 cells at 5 μM. However, replication of DNA dropped to 35% after 120 min at this concentration, and translational activity also decreased.     

Regulation of the growth of Epstein-Barr virus-infected B cells: temporal profile of the in vitro development of three distinct cytotoxic cells

The time course of the appearance of cytotoxic cells was examined in cocultures of E-rosetting (E+) cells and EBV-infected non-T cells (4:1 ratio) from the blood of VCA-positive healthy adults. Classical HNK-1+ NK cells were present at the initiation of the cultures and they produced 76 +/- 2% specific 51Cr-release from K-562 cells, but they did not effectively lyse the NK-resistant Daudi cells, nor did they kill autologous EBV-induced lymphoblasts (LCLEBV). The NK activity decreased during the first week in culture to 40 +/- 7% cytotoxicity. At the same time, nonspecific cytotoxic cells capable of killing Daudi as well as K562 developed and persisted into the third week in culture when it declined. This later nonspecific cytotoxicity was mediated by 4F2+, T8-, HNK-1- activated E+ cells. After 10 days in culture, killing of autologous LCLEBV increased continuously, from 4 +/- 3% at Day 10 to 38 +/- 4% by Day 22. The cytotoxicity to LCLEBV was mediated by classical T8+ CTL, and it was antigen specific and at least partially HLA Class I restricted. The regression of BEBV growth that occurs in E+/BEBV cocultures coincides with the development of this CTL-mediated cytotoxicity.     

Segregation of the NK-sensitive phenotype in human x mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human X mouse interspecies somatic cell hybrids. Highly NK-sensitive (NKs) human tumor cells were fused with NK resistant (NKr) mouse fibroblasts (LMTK-) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NKs erythroleukemia cells (K-562) or NKs retinoblastoma cells (Y-79) with LMTK- displayed an intermediate NK-sensitive phenotype. One Y-79 X LMTK- hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NKr RAJI cells generated NK-resistant hybrids when fused with LMTK- fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 X LMTK-) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.     

Immunomodulation of human leukocytes by staphylococcal enterotoxin A: augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.