Characteristics of the NK cells in Syrian hamsters carrying tumors with varying metastatic and resistance-suppressing activity

Experiments in vivo and in vitro were made to study the effects of HETR-MLN-8 and HETR tumor cells differing in metastatic ability and inhibition of the natural host resistance to tumor on cytotoxic activity of NK from Syrian hamsters. Marked inhibition of cytotoxicity and ability for interferon activation was detected in NK isolated from tumors (as compared with blood), with that inhibition being far more pronounced in highly malignant HETR-MLN-8 tumors. This may indicate a direct inhibitory action of the tumor or its products on NK cytotoxicity. The in-vitro competition inhibition test yielded results showing that HETR-MLN-8 cells capable of in-vivo inhibition of the natural host resistance to the tumor also display much more demonstrable ability for in-vitro inhibition of NK cytotoxicity as compared to HETR cells.     

Susceptibility of cells with different stage of malignancy to cytostatic action of resident peritoneal macrophages of Syrian hamsters

Syrian hamster non-activated resident peritoneal cells (PC) and peritoneal macrophages (Mph) was demonstrated. The in vivo selection of highly tumourigenic and highly metastatic variants of this strain correlated with their resistance to CSA PC and Mph in four cell variants out of five examined. The highly tumourigenic Syrian hamster embryo cells in vitro transformed by Rous sarcoma virus were highly resistant to CSA PC without selection in vivo. The resistance of highly malignant cells to CSA PC appeared to be unrelated to their ability to produce immunosuppressing prostaglandins of E type.     

Regulation of human natural killing. II. Protective effect of interferon on NK cells from suppression by PGE2

Activation of human natural killer (NK) cells in vitro with interferon (IFN) and poly I:C results in a partial loss of sensitivity of these cells to suppression by PGE2. The acquired resistance to suppression can be induced with the large granular lymphocytes (LGL) in the absence of monocytes. With K562, HSB, and CEM used as NK target cells, the IFN-induced resistance to suppression by PGE2 is observed with all three target cells. Furthermore, ADCC activity of IFN-activated cells against tumor (SB-TNP) and erythroid (CRC-TNP) target cells is also less susceptible to suppression by PGE2. The dual effect of IFN on NK cells is prompt; the augmentation of NK activity and the acquired resistance to suppression by PGE2 can be seen after 3 hr of treatment with IFN. Both of these characteristics seem to be quite stable for at least 24 hr. Spleen cells from mice (CBA, C3H, and BALB/c nude) treated in vivo with poly I:C also acquire partial resistance to suppression by PGE2. Our data therefore suggest that IFN-stimulated NK cells are protected from suppression by PGE2. Biologically, the IFN-induced protective effect may be beneficial to host resistance to neoplasia.     

Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

Phosphodiesterase 4A confers resistance to PGE2‐mediated suppression in CD25+/CD54+ NK cells

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25⁺/CD54⁺ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25⁺/CD54⁺ NK cells for adoptive cell therapy should be considered.     

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.     

Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells in vitro, but its physiological role in tumor surveillance remains unknown. Here, we report that TRAIL is constitutively expressed on murine natural killer (NK) cells in the liver and plays a substantial role in suppressing tumor metastasis. Freshly isolated NK cells, but not natural killer T cells or ordinary T cells, from the liver expressed cell surface TRAIL, which was responsible for spontaneous cytotoxicity against TRAIL-sensitive tumor cells in vitro along with perforin and Fas ligand (FasL). Administration of neutralizing monoclonal antibody against TRAIL significantly increased experimental liver metastases of several TRAIL-sensitive tumor cell lines. Such an anti-metastatic effect of TRAIL was not observed in NK cell-depleted mice or interferon-gamma-deficient mice, the latter of which lacked TRAIL on liver NK cells. These findings provide the first evidence for the physiological function of TRAIL as a tumor suppressor.     

Indomethacin therapy abrogates the prostaglandin-mediated suppression of natural killer activity in tumor-bearing mice and prevents tumor metastasis

We have shown earlier that a decline in splenic natural killer (NK) activity during the development of transplanted or spontaneous tumors in mice results from an inactivation of NK lineage cells, mediated by prostaglandins (primarily PGE2) secreted by NK suppressor cells of the monocyte-macrophage lineage. In the present study we have used a C3H mouse mammary carcinoma model to examine whether this mechanism of NK suppression is conducive to tumor metastasis in vivo and whether a reversal of this suppression by a chronic indomethacin therapy can prevent metastatic spread from the primary tumor site. Three mammary tumor lines, all derived in our laboratory from a spontaneous C3H mammary tumor were employed: T-58 (uncloned parental line, having weak lung metastasizing ability from the subcutaneous site), C3 (a clone of T-58, showing high metastatic ability), and C10 (a nonmetastatic clone of T-58). Although the degree of NK susceptibility of these lines varied inversely with their metastatic potential, none was NK resistant. A chronic administration of indomethacin in the drinking water (14 micrograms/ml) to mice beginning on Day 4 after subcutaneous transplantation of 10(6) tumor cells resulted in a significant reduction in the growth rate of primary tumors in all hosts and led to a complete or nearly complete abrogation of lung metastasis in T-58- or C3-transplanted hosts examined at 1 month after tumor transplantation; C10-transplanted mice showed no metastasis in the control or the treated group. Concomitantly, there was a substantial restoration of splenic NK activity in all indomethacin-treated hosts. Plastic-adherent cells (greater than 95% macrophages) isolated from tumors growing in control mice, when coincubated for 20 hr with normal splenic effector cells caused a suppression of NK activity, reversible in the presence of indomethacin (10(-5) M) in vitro. Similar cells recovered from the residual primary tumors in indomethacin-treated mice had no suppressor ability. Chemically pure PGE2 (at concentrations of 0.5 to 1 X 10(-6) M, but not 0.25 X 10(-6) to 10(-8) M) also caused a suppression of NK activity of normal splenic effector cells, when added during the 4-hr 51Cr-release assay or allowed to interact with effector cells alone for a 20-hr incubation period; a removal of the cell-free PGE2 in the latter case prior to the NK assay did not relieve the suppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cationic lipid gene transfer of an IL-2 transgene leads to activation of natural killer cells in a SCID mouse human tumor xenograft

Natural killer (NK) cells play an important role in combating infectious and malignant diseases and interleukin-2 (IL-2) has been shown to promote proliferation and activation of NK cells in vitro and in vivo. Here we investigate the effects of local cationic lipid-mediated IL-2 gene transfer on intratumoral accumulation and activation of NK cells in a SCID mouse tumor model. UM449 human melanoma tumors in SCID mice received intratumoral injections of DMRIE/DOPE admixed with VR1103, a DNA plasmid encoding the gene for human IL-2. Dissagregated tumor cells were tested for IL-2 secretion and were characterized using antibodies to asGM1, MAC-1, and F4/80 antigens. Granzyme A, a proteolytic serine esterase, was also measured in tumor cell lysates. IL-2 secretion from tumors injected with VR1103:DMRIE/DOPE peaked at 48 h after injection and fell to baseline levels on day 8. Intratumoral granzyme A activity was significantly increased in tumors injected with IL-2 plasmid:DMRIE/DOPE complexes, but not by an irrelevant plasmid DNA:DMRIE/DOPE control. Importantly, the growth of UM449 tumors was slowed in VR1103:DMRIE/DOPE-injected tumors. These results indicate that local cationic lipid-mediated gene transfer of IL-2 induces activation of intratumoral NK cells and slows tumor growth.     

Inhibition of human NK cell and LAK cell cytotoxicity and differentiation by PGE2

Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression.     

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

Immunosuppression by seminal plasma from fertile and infertile men: Inhibition of natural killer cell function correlates with seminal PG concentration

Human seminal plasma has uniquely high concentrations of PGE and 19-hydroxy PGE but the function of these PGs has not been elucidated. PGs of the E series have been shown to be paracrine and autocrine regulators of the function of immune cells and high levels of PGE have been shown consistently to suppress function in such cells. Human seminal plasma has a potent immunosuppressive effect and evidence is accumulating that this is largely due to PG components. In this study the effects of human seminal plasma on the killing activity of natural killer (NK) cells as judged by 51Cr release from K562 cells have been studied in groups of fertile and infertile men. Although there was no significant difference in the PGE, 19-hydroxy PGE or the NK cell inhibitory activity in the two groups, the inhibition of NK cell activity was closely correlated with the PGE and the 19-OH PGE content of the seminal plasma in the fertile group. This finding is further evidence that the major contribution to the immunosuppressive properties of human semen is provide by the high concentration of PGs of the E series in this fluid.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Migration activity of peritoneal exudate cells in Syrian hamsters in normal conditions and during suppression of their natural resistance to tumors

Migration activity (MA) of peritoneal exudate cells (PEC) was studied in Syrian hamsters in normalcy and under intraperitoneal injection into the animals of inactivated normal and tumor cells including those capable of inhibiting the natural animals' resistance to tumor. No significant individual differences were found in MA of PEC of intact hamsters. MA of PEC of hamsters treated with inactivated normal or spontaneously transformed in vitro cells of hamster embryo did not differ from the means of MA in the control. MA of PEC of hamsters treated with inactivated tumor cells was found to be appreciably enhanced. The ability of inactivated tumor cells to induce the enhancement of MA of PEC correlates with their ability to suppress natural tumor resistance.     

Human cytotoxic T lymphocytes (CTL) induced by phytohemagglutinin: conditions for CTL generation and effect of interferon

The present study demonstrates that human peripheral blood mononuclear cells (PMC) can be stimulated in vitro to become cytotoxic T lymphocytes (CTL) by PHA. A significant cytotoxic activity of PMC was detected 48 hr after the culture initiation in the presence of 5 micrograms/ml of PHA and the peak level of the activity was obtained by culturing PMC for 72 hr. The cytotoxic cells require the presence of PHA as a cell agglutinin for the expression of their cytotoxic activity. The effector cells mediating the activity were identified as T lymphocytes by E-rosette fractionation of PMC. In this system, removal of carbonyl iron phagocytosed or attached cells from PMC did not abrogate CTL generation of PMC. In addition, human alpha-interferon did not augment CTL generation or expression of their activity. Although the target cells employed were sensitive to natural killer (NK) cells, the effector cells induced by PHA did not seem to have any relation to the NK cells. The present study may provide a useful tool to analyze for precursors of killer T cells.     

Immunological surveillance against DNA-virus-transformed cells: correlations between natural killer cell cytolytic competence and tumor susceptibility of athymic rodents.

Adenovirus type 2 (Ad2)-transformed hamster and rat cells are susceptible to lysis by natural killer (NK) cells from the host of origin and are nontumorigenic in immunocompetent hamsters and rats, respectively. These NK-cell-susceptible, virus-transformed cells are, however, highly tumorigenic in athymic (nude) mice--animals with intact NK-cell responses. In vitro lysis of these xenogeneic, Ad2-transformed cells by nude-mouse NK cells was found to be defective. In contrast, Ad2-transformed hamster and rat cells were highly susceptible to lysis by nude-rat NK cells. Furthermore, xenogeneic, Ad2-transformed hamster cells were nontumorigenic in nude rats unless the NK-cell responses of the challenged animals were compromised. The results of the nude-rat studies show that thymus-dependent, cytotoxic T-lymphocyte-mediated, host cellular immune responses are not essential for rejection of xenogeneic cells transformed by nononcogenic Ad2. The data suggest instead that immunologically nonspecific host cellular immune responses, such as those mediated by NK cells, are sufficient for rejection of Ad2-transformed cells. These results indicate that biologically important differences exist in the NK-cell-mediated defenses mounted by nude mice and nude rats against transformed cells that may account for the different patterns of tumor induction by various neoplastic cell types in these athymic animals.     

Squamous cell carcinoma cells differentially stimulate NK cell effector functions: the role of IL-18

Tumor cells stimulate natural killer (NK) cell effector functions, but the regulation of cytokine secretion and cytolysis is incompletely understood. We tested whether oral and pharyngeal squamous cell carcinoma cell lines differentially stimulated NK cell interferon-gamma (IFN-gamma) secretion and cytolysis using a clone of the NK-92-transformed human NK cell line, NK92.35. SCC-4 and SCC-25 cells, but not FaDu or Cal 27 cells, stimulated robust NK92.35 IFN-gamma secretion. All four carcinoma cell lines were lysed by NK92.35 cells. These findings indicate that carcinoma cells differentially stimulate NK cell IFN-gamma secretion and cytolysis. In Transwell experiments, a combination of SCC-4 or SCC-25 cell soluble factors and contact with FaDu cells synergistically stimulated NK92.35 cell IFN-gamma secretion. Stimulatory SCC-4 cells constitutively secreted IL-18, a cytokine that potently augments IFN-gamma secretion by T cells and NK cells. In contrast, poorly stimulatory FaDu cells produced little or no IL-18, but synergized with recombinant IL-18 to stimulate NK92.35 IFN-gamma secretion. mAb to IL-18 or IL-18 receptor diminished SCC-4-stimulated IFN-gamma secretion by NK92.35 cells and by nontransformed NK cells. Thus, IL-18 was necessary for optimal carcinoma stimulation of NK cell IFN-gamma secretion. In vivo, oral and upper aerodigestive tract epithelia and carcinomas produced IL-18, but one squamous cell carcinoma had heterogeneous IL-18 expression. Thus IL-18 production can account for squamous cell carcinoma differential stimulation of NK cell effector functions in vitro and may be important for stimulation of NK cells in vivo.     

Effect of interleukin 2 on the inhibition of human natural killer activity by monolayer cells

We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL-2), when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition caused by malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2-activated killer cells toward malignant target cells involves weaker inhibition of activated killer cells by malignant cells.     

Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity

Background

Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.

Results

UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.

Conclusion

Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.