Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1)

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.     

Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.     

Activation of natural killer (NK) T cells during murine cytomegalovirus infection enhances the antiviral response mediated by NK cells

NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and alpha-galactosylceramide (alpha-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jalpha281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by alpha-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-gamma), have revealed that the antiviral effects of alpha-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of alpha-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-gamma.     

Enhancement of susceptibility of HSV-1-infected cells to natural killer lysis by interferon

The effect of interferon (IFN) on the natural killer (NK) activity of human PBL against HSV-1-infected HeLa cells was studied. Human PBL from several individuals did not consistently show a preferential lysis of HSV-1-, vaccinia-, or adenovirus type 5-infected cells with respect to uninfected HeLa cells. Treatment with IFN of effector PBL increased their lytic activity but did not alter the degree of preference on the lysis of the target cells shown by untreated PBL. Pretreatment with IFN of HSV-1-infected HeLa cells increased their susceptibility to lysis 5- to 10-fold. In contrast, identical pretreatment of the uninfected, adenovirus type 5- or vaccinia virus-infected HeLa cells before the assay decreased their susceptibility to NK lysis. This effect was not likely to be due to a block of the viral replication because other inhibitors like mitomycin C did not have the same effect. All target cells induced IFN synthesis in effector PBL cells. A similar level of IFN was induced by HSV-1-infected or uninfected HeLa cells. Pretreatment with IFN of HSV-1-infected, but not uninfected, HeLa cells induced 5 to 10 times more IFN by PBL, in good correlation with the increase in lytic activity. PBL treated with IFN, however, in conditions to give maximal stimulation of NK activity, presented the same preferential lysis of HSV-1-infected HeLa cells and synthesized similar levels of IFN as untreated PBL. In addition, HSV-1-infected HeLa cells were killed through different target structures than uninfected cells. Taken together, our results indicate an effect of IFN at the level of the NK target structures in HSV-1-infected HeLa cells by increasing either their number or, more likely, their affinity for NK cells independent of the effect of IFN in the effector cells or as an antiviral agent.     

Oncogenicity of human papillomavirus- or adenovirus-transformed cells correlates with resistance to lysis by natural killer cells.

The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity.     

The isolation of natural killer (NK)-resistant variants of the K562 cell line by mutagenesis and selection with antibodies which inhibit NK cell-mediated lysis

Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.     

Establishment of hybridoma cells with natural killer(NK)-like activity against syngenic tumor cells.

The 4D1D4 hybridoma cells were derived from the fusion of spleen cells from BALB/c nude mice with NS-1 mouse myeloma cells. The surface phenotypes of 4D1D4 hybridoma cells were Thy-1.2+, L3T4 (CD4)-, Lyt-2 (CD8)-, Asialo GM1+ and p-55 interleukin-2 (IL-2) receptor (CD25)-. This phenotypic pattern was consistent with the surface phenotype of NK cells. The 4D1D4 cells showed the definite killer activity against a syngenic tumor cell line, RL male-1, but not against an allogenic YAC-1 line. The killer activity of the 4D1D4 cells was not affected by the addition of exogenous IL-2. It was, therefore, suggested that 4D1D4 cells might be representative of resting NK cells with expression of no functional IL-2 receptors. The hybridoma technology might be useful for establishment of the cloned NK cells.     

Activation of natural killer (NK) cells in vivo with H-2 and non-H-2 alloantigens

Intraperitoneal inoculation of allogeneic lymphoid cells rapidly activates cytotoxic cells in the peritoneum which are nonadherent and express the NK-1, asialo-GM₁, and Thy-1 antigens. Allogeneic spleen cells were very efficient at activating these natural killer (NK) cells, while allogeneic thymocytes were much less effective. Heat-killed allogeneic cells or sonicates also could augment NK activity. — Incompatibility atH-2K, H-2I-A, orH- 2D readily evoked NK cell activity, whileH-2S- andH-2I-E/C-associated disparities did not. Non-H- 2 differences also stimulated NK activity and augmentation was particularly evident inMls-disparate combinations. Thus, the same alloantigens which efficiently activate T cells also activate NK cells.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Natural killer cell-mediated lysis of herpes simplex virus-infected fibroblasts: inability to detect soluble factors that contribute to lysis

We investigated the role of soluble factors in natural killer (NK) cell-mediated lysis of herpes simplex virus (HSV)-infected cells. Supernatants generated by incubating human peripheral blood mononuclear cells with HSV-infected human fibroblasts contained tumor necrosis factor (TNF) and lysed uninfected U937 cells, but not HSV-infected fibroblasts. U937 cells, but not HSV-infected fibroblasts, were lysed when exposed to recombinant TNF (rTNF) for 18 hr. NK cell-mediated lysis of HSV-infected fibroblasts was not inhibited by addition of anti-TNF or anti-lymphotoxin (LT) antibodies to cytotoxicity assays. Thus, a role for soluble factors, and in particular TNF and LT, in NK cell-mediated lysis of HSV-infected cells could not be demonstrated.     

Role of histamine in natural killer cell-mediated resistance against tumor cells

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.     

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.     

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.     

Specific inhibition of natural killer (NK) activity against different alloantigens

Allogeneic lymphocyte cytotoxicity (ALC), i. e., rapid rejection of i. v. injected allogeneic lymphocytes in unprimed hosts, is an example of NK activity. Apparently anomalous rejection patterns, such as acceptance of F₁ hybrid cells by parental hosts and rejection of parental cells by F₁ hybrid hosts in many strain combinations, would fit the hypothesis that the effector cells in ALC recognize the absence of certain self-molecules (passwords) rather than the presence of nonself determinants. However, cold target inhibition studies showed that ALC displays allospecificity: when a mixture of radiolabeled AO and DA cells were injected i. v. into euthymic or athymic PVG rats, adding a surplus of cold DA cells reduced killing only of labeled DA cells and vice versa. Furthermore, semiallogeneic cold target cells were ineffective in inhibiting elimination of fully allogeneic cells, which supports the argument against a modification of the hypothesis that self-determinants inhibit a postbinding stage of lysis. Finally, (DA × AO)F₁ cells injected into (DA × PVG)F₁ hosts were rapidly rejected, despite the fact that donor and host shared expressed DA determinants. In sum, our results show that a hypothesis based on inhibition of killing by self-determinants can only be sustained with extensive modifications, and favor the alternative mechanism that the effector cells positively recognize the presence of allospecific determinants on the target cell surface.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Suppression of natural killer (NK) cell activity of spleen cells by thymocytes

The in vitro influence of thymus cells on natural killer cell activity of spleen cells against prelabeled target cells (YAC-I and RL♂I) has been studied in syngeneic as well as in allogeneic murine models. In mixing experiments to demonstrate suppression, total thymocytes have been found to have no effect on NK activity of syngeneic or allogeneic spleen cells. Among several thymocyte fractions separated by velocity sedimentation, a relatively faster sedimenting fraction showed remarkable suppression of NK activity by spleen cells against two target cells. The suppressive effect of this particular fraction on NK activity was demonstrated to be proportional to the cell dose. The suppressive function was resistant to irradiation at 1000 or 2000 rad administered in vitro and was not restricted by the major histocompatibility complex. Moreover, the thymocyte fraction which induced suppression was not sensitive to NK-mediated cytolysi? by syngeneic spleen cells. The suppression of NK cytolysis in vitro by certain subpopulations of thymocytes as observed in the present studies may be consistent with a role for the thymus in regulating NK activity in vivo.     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

Propagation of mouse cytotoxic clones with characteristics of natural killer (NK) cells

Splenocytes obtained from normal mice (BALB/c nude, BALB/c, C₃H, C57Bl/6) and from mice bearing lung or pulmonary carcinomas were propagated for 1–12 months in the presence of crude or mitogen-depleted T-cell growth factor (TCGF). Clones from several TCGF-propagated lymphoid cell lines were established by limiting dilution or the soft agar techniques. All the cultured lines and the majority of the clonal populations derived from them exhibited strong cytotoxic activity in vitro (⁵¹Cr release assay) toward a variety of syngeneic and allogeneic tumor target cells, both freshly obtained and passaged in culture, and both lymphoid and solid in origin, and including targets usually resistant to fresh NK cells. Considerable cytotoxic activity was also observed with several rat and human cultured tumor lines. Only low cytotoxic activity was detected against normal lymphoid mouse cells. Cloned populations generally exhibited more restricted target cytotoxicity than the parental cultured lines, and the pattern of reactivity varied among the clones. Of the clones tested for surface markers, all were positive for Thy 1.2, T200, and asialo GM1 and had strong binding to peanut agglutinin (PNA), all had undetectable receptors for IgG or IgM, and some were positive for Lyt 2. The cytotoxic activity was augmented by pretreatment of the effector cells with interferon and inhibited by the presence of mannose or galactose during the assay. Several clones were capable of mediating antibody-dependent cellular cytotoxicity and lectin-induced cellular cytotoxicity (LICC), and produced relatively large quantities of interferon and lymphotoxinlike material. The findings indicated continuous culturing in TCGF of previously antigen-nonstimulated mouse lymphocytes selects for the growth of at least two distinct populations with activated NK activity, one reacting preferentially with lymphoid tumor target cells (designated CNK-L), and the second reacting effectively with both lymphoid and solid tumor targets (designated CNK-SL). Both populations have several features of both T lymphocytes and NK cells.     

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.