In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats

The present study was performed to further evaluate the possible in vivo involvement of natural killer (NK) cells in host resistance against tumors. Selective depression of NK activity in Wistar Furth rats was induced by i.p. or i.v. injection of rabbit anti-asialo GM1. This antiserum has previously been shown to produce a decrease in NK activity and a parallel increase in tumor growth in mice. In the present study, rats treated with this antibody showed a parallel decrease in NK activity and in the frequency of large granular lymphocytes (LGL) in the spleen and peripheral blood, indicating that the antiserum-induced depression of NK activity in these sites was probably caused by an elimination of most effector cells. To further determine the possible role of rat LGL in tumor rejection in vivo, we studied LGL involvement in the rapid clearance of radiolabeled tumor cells from the lungs, an assay previously shown to correlate well with in vitro NK activity. Animals treated with anti-asialo GM1 antiserum were found to have a substantial decrease in the in vivo rate of clearance of tumor cells from the lungs. Furthermore, the adoptive transfer of a highly enriched population of LGL into NK-depressed animals 2 hr before tumor challenge, partially restored their cytotoxic activity against established cell lines in vitro and their ability to eliminate radiolabeled cells from the lungs. These results provide direct support for the hypothesis that NK cells are involved in in vivo resistance to tumors, particularly in the elimination of potentially metastatic tumor cells from the circulation and capillary beds.     

Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

Natural killer cell regulation of murine embryonic pulmonary fibroblast survival in vivo

We examined the role of the natural killer (NK) cell in controlling the survival of embryonic pulmonary fibroblasts in vivo. In vitro, both primary embryonic fibroblasts and an embryonic fibroblast line (10T1/2) were lysed by syngeneic C3H/HeN splenocytes threefold more efficiently than primary adult fibroblasts. The membrane phenotype of the effector cells was typical of NK cells. It was asialo GM1+, Lyt2.1-, Lyt 1.1-, Thy 1.2-. The cytotoxicity of the effector cell could be enhanced by IFN-alpha/beta but was deficient in the C3H/HeJ bg/bg mutant. Iododeoxyuridine (131I-dUrd)-labeled embryonic fibroblasts were injected intravenously into syngeneic mice with either enhanced or deficient NK function and their survival in the lung was quantitated. Enhanced fibroblast survival was detected in the NK deficient C3H/HeJ beige (bg/bg) mutant strain compared to its normal littermate C3H/HeJ (bg/+). A second method of NK depletion by pretreatment with rabbit anti-asialo GM1 antiserum also produced a striking increase in fibroblast survival. Poly(I:C) significantly enhanced the elimination of pulmonary fibroblasts from the lung between 4 and 24 hr after injection. Poly(I:C) did not enhance clearance of pulmonary fibroblasts in the C3H/HeJ (bg/bg) mutant, but did so in the normal littermate C3H/HeJ (bg/+). In conclusion, we have shown that the survival of embryonic pulmonary fibroblasts was inversely correlated with in vivo NK activity suggesting a possible role for this cytotoxic cell in the control of fibroblast growth in vivo.     

Synergistic defense system by cooperative natural effectors against metastasis of B16 melanoma cells in H-2-associated control: different behavior of H-2+ and H-2- cells in metastatic processes

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.     

The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.     

Augmentation of NK cell activity in tissue specific sites by liposomes incorporating MTP-PE

Liposomes incorporating a variety of immunomodulators have been shown to activate macrophages and monocytes for tumoricidal activity both in vivo and in vitro. We report that in addition to the activation of macrophages, the i.v. injection of liposomes (multilamellar vesicles) that have encapsulated muramyl tripeptide-phosphatidylethanolamine (MTP-PE) could also augment interstitial natural killer (NK) cell activity in the lung and the liver. In contrast, liposomes incorporating MTP-PE were unable to augment NK cell activity in the spleen, peripheral blood, or peritoneal cavity (after i.p. injection). In addition, liposomes did not augment splenic NK cell activity in vitro. This suggests that the augmentation of NK cell activity in the lungs and liver was not due to direct effects of the liposomes but may have been a secondary effect mediated by a monokine. The augmentation of pulmonary NK cell activity was paralleled by the nonspecific immunoprophylaxis of experimental pulmonary metastases. The augmented NK cell activity, as well as the enhanced nonspecific immunoprophylactic activity, was reduced by pretreatment of the mice with anti-asialo GM1 antiserum. Thus, the augmentation of organ-associated NK cell activity by liposomes incorporating MTP/PE plays a major role in the host's increased resistance to the formation of experimental metastases.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Murine natural killer cells limit coxsackievirus B3 replication

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.     

Intraepithelial leukocytes contain a unique subpopulation of NK-like cytotoxic cells active in the defense of gut epithelium to enteric murine coronavirus

Initially the intraepithelial leukocytes (IEL) of specific pathogen free (SPF) mice were compared with those of mice held without isolation and were found to differ markedly in total number and distribution of cell surface antigens. The IEL from SPF mice expressed significantly less Thy-1, Lyt-1, and Lyt-2 antigens than their conventional counterparts. The local cell-mediated immune response of mucosal lymphocytes to an enteric murine coronavirus (MHV-Y) was studied in inbred strains of naive SPF mice. A potent in vitro cytotoxic activity was demonstrated by mucosal leukocytes, especially IEL, and spleen cells for MHV-Y-infected syngeneic and allogeneic target cells. The cytotoxicity was not restricted by the major histocompatibility complex. Targets infected with Pichinde virus, an enveloped nonenterotropic virus, were not lysed by these cells. The phenotype of the IEL effector cell was asialo GM1+, Thy-1-, Lyt-1-, Lyt-2-. This cell represents a small subpopulation of the total IEL. After the in vivo administration of anti-asialo GM1 sera, the virus-specific cytotoxic function of the IEL was markedly diminished in in vitro assays, and there was enhanced persistence of virus in gut tissues in vivo. The IEL effector population is defined as a natural killer-like cell that appears to be active in the defense of the gut epithelium to a murine enteric coronavirus.     

Preparation of monoclonal antibodies against a glycolipid asialo GM1

Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo GM1 were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo GM1 adsorbed to naked Salmonella. All the MAbs reacted only with asialo GM1 when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo GM1 antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo GM1 and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

A subpopulation of allospecific cytotoxic T-cell precursors with phenotypic characteristics of natural killer cells

We have proposed that natural killer (NK) cells are germ-line V-gene encoded prothymocytes specific for either self or non-self histocompatibility antigens. This hypothesis predicts that at least some precursors of allospecific cytotoxic T cells (allo-CTL) are NK cells. To test this we examined the effect of depleting NK cells and/or T cells (by complement lysis with anti-asialo GM1 and/or anti-Thy 1) on the development of allo-CTL induced during mixed lymphocyte culture (MLC). Removal of Thy 1+ cells from MLC responder populations prevented development of allo-CTL. This was partially reversed by addition of concanavalin A-conditioned medium (Con A-CM) to the MLC at day 0. Removal of asialo GM1+ cells eliminated NK activity measured at day 0, but failed to prevent development of allo-CTL of otherwise intact responder cells. However, removal of asialo GM1+ cells did prevent the Con A-CM dependent development of allo-CTL by responder cells depleted of Thy 1+ cells. These findings indicate that a subpopulation of allo-CTL precursors has the phenotypic characteristics of NK cells: absence or low density of Thy 1, and susceptibility to complement lysis by anti-asialo GM1.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Characterization of natural killer cells and their precursors in the murine bone marrow

We have fractionated murine bone marrow cells according to their density on bovine serum albumin (BSA) gradient and studied (a) the NK activity against YAC-1 targets, (b) the proportion of asialo GM1+ lymphocytes, (c) and the presence of large granular lymphocytes (LGL) in the different fractions (A, B, C, D). The NK activity was found mainly in the C fraction, but the proportion of asialo GM1+ cells was the same in every fraction. No LGLs were found in the bone marrow. Cells from the various fractions were also transplanted into irradiated recipients. Seven days later the highest NK activity was found in the spleens of mice injected with cells from the A + B fractions indicating that the immediate precursors for NK cells reside in the low density fractions of the BSA gradient. Mice transplanted with C or D fractions needed longer time to develop normal NK levels. The treatment of bone marrow cells before transplantation with anti-asialo GM1+ complement did not inhibit the development of NK activity, so it can be concluded that the precursor for NK is asialo GM1-.     

Infection of DBA/2 or C3H/HeJ mice by intraperitoneal injection of vaccinia virus elicits activated macrophages,cytolytic and cytostatic for S91-melanoma tumor cells

Summary Murine peritoneal macrophages harvested 3–4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5–6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Ia^d antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(–); asialo GM1(–)] and not natural killer cells [asialo GM1(+); Thy 1.2(±)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells, to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.     

Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.