Hybrid Resistance to EL-4 Lymphoma Cells

(C57BL/6 × DBA/2)F₁ hybrid (B6D2F₁) mice resist the growth of parental-strain (B6) EL-4 lmphoma cells inoculated intraperitoneally; that is, B6D2F₁ mice survive longer than B6 mice and do not develop uscites As compared with B6 mice, B6D2F₁ mice have higher levels of natural killer (NK) activity against ⁵¹Cr-labelled EL-4 cells in their lyphiod organs. B6D2F₁ mice treated with ⁸⁹Sr lose NK activity for certain lymphoma cell targets, e.g. YAC-1, but NK(EL-4) function is usually intact. However, ⁸⁹Sr-treated mice had hybrid resistance to EL-4 cell in vivo, as determined by survival times and the development of ascites. NK(EL-4) and NK-(YAC-1) activities were stimulated by irrudiated or unirradiated EL-4 cells, Corynebacterium parvum, or polyinosinic:polycytidylic acid (pl:pC) in spleens of normal B6D2F₁ mice, but NK(EL-4) activity was depressed Within 3 days by such treatment in B6D2F₁ mice previously injected with ⁸⁹Sr. Suppressor cells for NK(EL-4) but not for NK(YAC-1) effectors were easily detected in spleens of ⁸⁹Sr-treated mice ‘challenged’ with C. parvum. Thus, agents capable of Stimulating NK cell function in normal mice may lead to suppression of that activity in mice depleted of marrow-dependent cell function by ⁸⁹Sr. Spleen cells of ⁸⁹Sr-treated B6D2F₁ mice were also unable to generate anti-EL-4 cytotoxic T lymphocytes in a cell-mediated lympholysis system; this defect appeared also in to be mediated by suppressor cells. Lymphoid cell depleted by ⁸⁹Sr-induced marrow aplasia may have two functions in host defences against tumours (especially lymphomas): they may lyse tumour cells directly and they may ‘down-regulate’ suppressor cells capable of inhibiting other ‘natural’ or ‘induced’ immune functions.     

Murine cytomegalovirus stimulates natural killer cell function but kills genetically resistant mice treated with radioactive strontium.

Treatment of C3H/St mice with 100 microCi of 89Sr weakened their genetic resistance to murine cytomegalovirus (MCMV) infection. The criteria utilized to detect increased susceptibility were: (i) survival of mice; (ii) numbers of MCMV-infected cells in the spleens and liver; and (iii) serum glutamic pyruvic transaminase levels. The natural killer (NK) cell activity of spleen cells from mice treated with 89Sr is very low. However, the NK activities of spleen cells of both normal and 89Sr-treated mice were greatly augmented 3 days after infection with MCMV. These NK cells lysed a variety of tumor cells and shared several features with conventional NK cells, but were not lysed by anti-Nk-1.2 serum (specific for NK cells) plus complement. Splenic adherent cells did not lyse tumor cells themselves but were necessary for the stimulation of NK cells by MCMV. The paradox of high NK cell function and poor survival in 89Sr-treated mice infected with MCMV was a surprise. We conclude that these augmented NK cells, of themselves, cannot account for the genetic resistance of C3H/St mice to infection with MCMV.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Natural killer activity in experimental cutaneous leishmaniasis

Studies were performed to determine the role of natural killer (NK) cell cytotoxicity in experimental cutaneous leishmaniasis. Analysis of a possible correlation between in vitro NK cell activity and in vivo susceptibility to Leishmania mexicana infection showed that there is no relationship between the degree of NK reactivity to YAC-1 lymphoma cells and in vivo leishmania growth. It was also observed that spleen lymphoid cells from mice with high NK activity did not cause an increase in isotope release by the macrophage permanent cell line J774G8-1 previously infected with the parasite and supporting its growth. Mice infected with L. mexicana manifested an increased NK activity to YAC-1 cells but not to leishmania-infected J774G8-1 tumor macrophages. The lack of effect of NK cell activity is discussed with regard to the role of NK cells in immune mechanisms to intracellular parasites.     

Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts.

Two types of host reactivities not requiring immunization in the mouse and not mediated by T lymphocytes were compared: resistance of irradiated and nonirradiated F₁ hybrids to accept parental grafts of normal or malignant hemopoietic cells (Hh system), and the natural killer cell activity against mouse lymphomas (NK system). The effects of six independent variables known to influence resistance to marrow grafts were investigated in the NK system using YAC-1 lymphoma cells as targets. The following properties were shared: (a) maturation during the fourth week of life; (b) low sensitivity to acute total body irradiation; (c) dependence on the integrity of bone marrow as demonstrated by reduced reactivity in ⁸⁹Sr-treated mice; (d) suppression by a single injection of rabbit anti-mouse bone marrow serum; (e) suppression by a single injection of the anti-macrophage agents silica and i-carrageenan; and (f) suppression by multiple injections of parental spleen cells into F₁ mice. These positive correlations are particularly significant because most of the variables have either opposing or no effect on conventional immunity. F₁ mice rendered specifically unresponsive to parental marrow grafts, could retain NK cell activity, and genetically susceptible mice could be rendered hyporeactive in terms of NK cells, indicating that the specificities of YAC-1 and Hh-1 incompatible targets were different. It is extremely unlikely that this remarkable parallelism is fortuitous. These results indicate that either a very similar, or more likely a common mechanism is operative in the two cell-mediated natural reactivities: effector cells in the NK and Hh systems do not bear B or T lymphocyte markers but are nevertheless endowed with “specificity”. They are dependent for generation in vivo (presumably by maturation or by recruitment) on the interaction with nonlymphoid accessory cells not endowed with specificity, capable of also interacting in vitro with Thy-1-positive F₁ hybrid prekiller cells specific for parental targets. Because of thymus independence in vivo and apparent restriction to target cells of the hemopoietic system, these reactivities should be effective in the regulation of hemopoiesis and surveillance over leukemogenesis.     

Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool-nonadherent cells.

Previous reports demonstrate that natural killer (NK) cells inhibit the growth of Cryptococcus neoformans in vitro, but conclusive evidence supporting the effectiveness of NK cells in host resistance to cryptococci is not available. The objective of these studies was to assess the ability of NK cells to clear C. neoformans from the lungs, livers, and spleens of infected mice. CBA/J mice were depleted of NK cells, as well as other natural effector cells, by an intraperitoneal injection of cyclophosphamide (Cy), 240 mg/kg of body weight. One day later, 7.5 X 10(7) nylon wool-nonadherent (NWN) spleen cells, either untreated or treated with anti-asialo GM1 and complement to remove NK cells, were adoptively transferred to Cy-pretreated mice. On day 2 after Cy treatment, the mice were injected intravenously with 2 X 10(4) cryptococci. At 4 and 6 days after Cy treatment, tissues were assayed for NK reactivity, using a 4-h 51Cr-release assay, and for in vivo clearance of cryptococci as reflected by mean log10 CFU per organ. We observed that Cy treatment depleted NK activity against YAC-1 targets and reduced in vivo clearance of C. neoformans from the tissues of infected mice. Additionally, Cy treatment depleted the total lung and spleen cellularity and the total number of peripheral blood lymphocytes when compared with those in normal untreated control mice. Also, spleen weights were significantly decreased in comparison with those of untreated animals 4 days after Cy treatment. Adoptive transfer of untreated NWN spleen cells into Cy-depressed mice restored the NK cell activity which correlated with enhanced clearance of cryptococci from lungs, livers, and spleens. In contrast, treatment of NWN spleen cells with anti-asialo GM1 and complement before adoptive transfer abrogated the ability of these cells to restore NK activity or reduce the numbers of cryptococci present in tissues of infected mice. Taken together, these data indicate that NK cells are the cells effective in diminishing the numbers of cryptococci in tissues of infected mice. Consequently, NK cells may play a role in first-line host resistance against C. neoformans.     

Concanavalin A-induced resistance to Listeria monocytogenes and activation of macrophages: defect in mice treated with 89Sr

⁸⁹Sr-treated mice injected with concanavalin A (Con A) 24 h prior to infection with Listeria monocytogenes (LM) could not enhance the clearance of LM from the spleen. Adoptive transfer of normal syngeneic spleen cells together with Con A rendered these animals more resistant. Spleen cells of ⁸⁹Sr-treated or age-matched control mice were stimulated with Con A for 24 h, and supernatant fluids were assessed for macrophage-activating factor (MAF), i.e. the ability to activate resident peritoneal macrophages to kill LM intracellularly in vitro. A defective MAF production by spleen cells was observed in ⁸⁹Sr-treated, 2 week-old, and athymic nude mice. Also, treatment of spleen cells with anti-Thy-1.2 antiserum plus complement inhibited MAF production. Synergism between spleen cells from ⁸⁹Sr-treated and nude mice did not occur. The cells required for MAF production were relatively resistant to gamma irradiation. Nylon wool filtration did not modify the ability of spleen cells to make MAF. ⁸⁹Sr-treated mice possess macrophages responsive to MAF derived from normal spleen cells. The data suggest that the failure of ⁸⁹Sr-treated mice to develop an anti-LM response observed in this system could be due to a defective capacity to produce protective humoral factors and/or cells in response to Con A.     

Immunomodulatory Activity of A Novel Nucleoside, 7-thia-8-oxoguanosine: I. Activation of Natural Killer Cells in Mice

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T80G)² inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T80G is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T80G at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62 %) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T80G was administered in vivo. In addition to the spleen, 7T80G activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T80G elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c, i.v., and i.p. routes all induced activition of NK cells in spleen, BM and PE. 7T80G was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T80G, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T80G showed no such synergism.     

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine: I. Activation of natural killer cells in mice.

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T8OG)2 inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T8OG is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T8OG at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62%) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T8OG was administered in vivo. In addition to the spleen, 7T8OG activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T8OG elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c., i.v., and i.p. routes all induced activation of NK cells in spleen, BM and PE. 7T8OG was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T8OG, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T8OG showed no such synergism.     

Augmented Followed by Suppressed Levels of Natural Cell-Mediated Cytotoxicity in Mice Infected with Toxoplasma gondii

The cytotoxic activity of effector cells from mice infected with Toxoplasma gondii was tested in a 4- to 5-hr (51)Cr release assay, using RL 0001000 0011100 0101010 0001000 0001000 0011100 0100010 1000001 1000001 1000001 0100010 0011100 1 and YAC-1 target cells. They showed enhanced cytotoxicity with a peak on the 3rd day postinfection followed by suppression with a peak on the 12th day. The cytotoxicity seemed to be exhibited by natural killer (NK) cells because: (i) pretreatment of the effector cells with antiasialo GM(1) or antiasialo GM(2) plus complement abolished the cytotoxic activity; (ii) the altered cytotoxicity levels were also induced in nude mice; and (iii) the activity was elicited by nonadherent-nonphagocytic cells. The alteration occurred simultaneously in various lymphoid organs with a similar profile. Neither spleen nor bone marrow cells of 12-day-postinfected mice inhibited NK activity of uninfected mice. Culture fluids of the infected mouse spleen and bone marrow cells did not affect the normal mouse NK activity. The proportion of effector cells capable of binding to target cells was constant during the infection. There was no positive correlation between NK activity and serum interferon level; i.e., interferon was detected in the serum of 12-day-postinfected mice but not in that of 3-day-postinfected or uninfected mice. Passively administered interferon or polyinosinic-polycytidylic acid could not restore the suppressed NK activity of 12-day-postinfected mice. Moreover, in vitro treatment of spleen cells from 12-day-postinfected mice with interferon failed to restore the suppressed NK activity. These results suggested that after toxoplasma infection, defective sensitivity to interferon was induced in NK precursor cells, and differentiation to functionally active NK cells might be blocked.     

Ablation of natural killer cell function by soluble cardiotoxin

A one-hour preincubation of nonadherent murine spleen cells with a soluble membrane-active cardiotoxin purified from the venom of the Thailand cobra Naja naja siamensis results in the destruction of natural killer (NK) cell activity against YAC-1 target cells in a dose-dependent manner. Prior in vivo induction of interferon production by polyinosinic/polycytidylic acid does not avert the cardiotoxin inhibition of NK function. Loss of complement-mediated lysis of cells capable of binding an NK-1.1 monoclonal antibody suggests that the cardiotoxin directly affects the integrity of the NK cell plasma membrane. Cardiotoxin which has been adsorbed to the surface of polystyrene tissue culture plates retains the ability to lyse splenic T lymphocytes, but loses the ability to interfere with NK activity, as measured either by the release of 51Cr or by the uptake of 3H-thymidine by the target lymphoma cells, suggesting that different parts of the cardiotoxin molecule are responsible for destruction of the two types of lymphocytes.     

Natural antitumor defense system of the murine liver

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1(4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Corynebacerium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver macrophages from untreated mice do not have tumoricidal activity per se but can "activate" NK cells to kill tumor cells normally resistant to NK cells.     

Restoration of prostaglandin releasing macrophage populations in lethally irradiated mice with spleen cells from bone marrow-depleted donors

Previous studies in mice severely depleted of bone marrow cells by 89Sr showed persistent monocytopenia and impaired expression of prostaglandin E2-releasing splenic macrophages (PGSM) despite the occurrence in the spleen of more than 10-fold increases in pluripotential stem cells and M phi colony-forming cells. To determine whether the observed deficits were due to a lack of precursors of blood monocytes and PGSM in the spleens of 89Sr-treated mice, radiation chimeras were established by i.v. infusion of 2 x 10(6) spleen cells from 89Sr donor CBA/J or semisyngeneic B6CB F1 hybrid mice into lethally gamma-irradiated CBA/J recipients. Blood monocyte levels were greater than normal by day 14 and PGSM induced by Corynebacterium parvum were demonstrated by day 28. These restored M phi populations expressed the donor haplotype detected in vitro with haplotype-specific monoclonal anti-H-2K plus complement. 89Sr treatment of the chimeras resulted in profound depletion of monocytes and PGSM. The data indicate that the spleen of the 89Sr-treated mouse, which is an ineffective source of circulating monocytes and PGSM, contains cells which can generate both of these populations following infusion into lethally irradiated recipients. Since the bone marrow of such recipients was capable of being repopulated, the aggregate observations suggest that functional bone marrow is obligatory for the generation of blood monocytes and PGSM populations.     

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine. II. Characterization of induced effector cells and the mechanism of induction.

In a preceding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T8OG)2. In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T8OG. Removal of macrophages from 7T8OG activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T8OG induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM1 antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T8OG induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T8OG. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T8OG resulted in effectors with reduced cytotoxicity. When injected in vivo, 7T8OG induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to alpha & beta-IFN but not to gamma-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T8OG is most likely to be mediated by alpha & beta-IFN involvement of other cytokines can not be ruled out.     

Absence of a role for natural killer cells in the control of acute infection by Toxoplasma gondii oocysts.

The active phase of primary and challenge oral infections of Toxoplasma gondii was investigated with respect to natural killer (NK) activity against YAC-1 tumour cell targets in vitro and serum interferon (IFN) titres. Primary (non-lethal) oral infection of BALB/c mice with Me49 oocysts resulted in a rapid increase of serum IFN titres, followed by augmented NK activity. NK levels became depressed, rising again by 15 days after infection to normal levels, again preceded by elevated IFN titres. In challenge infections NK was not augmented and IFN titres rose only if a high dose of oocysts was given. IFN activity was pH2-labile in all cases and considered to be due to IFN-gamma. Cold target inhibition studies indicated that T. gondii did not bind to NK cells. A bioassay for the effects of NK cells on T. gondii tachyzoites was developed and there was no evidence of killing in vitro by cells with NK function; T. gondii survived better when cultured with NK cells than when cultured alone. Studies using C57BL/6bg/bg,bg/+ and +/+ mice showed that there was no difference in mean time to death after administration of a lethal ME49 oocyst infection by mouth. Cytotoxicity against YAC-1 in both spleen and mesenteric lymph node (MLN) cell populations was highly augmented in bg/+ and +/+, but not in bg/bg mice. Genetic deficiency of NK activity had no effect on survival of mice after infection. Therefore NK has at best a minimal role to play in protection during the acute phase of Toxoplasma infection.     

Interaction between murine natural killer cells and trypanosomes of different species.

The involvement of natural killer (NK) cells in the immunological resistance of mice to murine-specific Trypanosoma musculi was evaluated. Murine NK cells were found to be unable to kill or inhibit T. musculi or to protect recipients from infection. In addition, the ability of spleen cells from normal mice and from mice on day 3 of T. musculi infection, at the time of maximum NK augmentation, to kill Trypanosoma cruzi and Trypanosoma lewisi was evaluated. Spleen cells from normal mice displayed significant killing of both T. cruzi and T. lewisi. Furthermore, augmented spleen cells from T. musculi-infected mice were considerably more effective than normal spleen cells in killing both T. cruzi and T. lewisi. The activity of NK cells toward YAC-1 tumor target cells was inhibited in a dose-dependent fashion by either live T. musculi or extracts of T. musculi, but not by extracts of rat-specific T. lewisi. The results suggest that well-adapted protozoan parasites may be nonsusceptible to the natural cell-mediated resistance mechanisms of their hosts. Their nonsusceptibility could result from the ability to elaborate substances that either inactivate NK cells or block NK cell interaction with complementary sites on the parasite surface.     

Enhanced natural killer cell activity in experimental murine encephalitozoonosis.

Spleen cells from mice infected with the protozoan parasite Encephalitozoon cuniculi demonstrated enhanced in vitro cytolysis of YAC-1 lymphoma cells. Selective cell depletion experiments showed that the dominant cell population mediating cytolysis of YAC-1 tumor cells expressed the characteristic phenotype of murine natural killer (NK) cells because (i) pretreatment of spleen cells with anti-asialo GM 1 antiserum plus complement abolished the cytotoxic activity; (ii) augmented cytolysis was found in athymic nude mice; (iii) pretreatment of spleen cells with anti-Thy 1.2 plus complement did not affect the level of cytolysis; and (iv) nylon wool removal of adherent cells did not reduce the augmented cytolysis. The augmented cytolysis peaked 7 days after infection, gradually diminished, and finally returned to control levels by 21 days postinfection. The parasite-induced augmentation of NK cell activity was dose-dependent: inoculation of 10(7) parasites gave maximum enhancement, whereas 10(5) or 10(4) parasites had an insignificant effect on spontaneous NK cell cytolysis. The augmented NK cell cytotoxicity was dependent upon viable parasites; inoculation of killed parasites failed to stimulate a significant increase in spontaneous cytolysis. An active infectious process was an important component of this process. The peak of NK activity in euthymic mice was closely correlated with the active stage of infection, and reduction of NK cell activity coincided with recovery from infection. By contrast, athymic nude mice were unable to control E. cuniculi infections yet maintained persistently elevated NK responses. The present data, along with previous reports, indicate that infection with E. cuniculi evokes transient modulation of host immune functions.     

Inhibition of natural killer cell-mediated lysis of tumor cells by normal and regenerating bone marrow

The natural killer (NK) cells which can lyse certain tumor cells during brief incubation in vitro have also been postulated to be the cells responsible for natural resistance to transplanted hemopoietic cells in vivo. To test this hypothesis, we have now measured: 1) the ability of bone marrow cells to compete with tumor cells as targets for spleen NK cells and 2) the effect of a brief incubation with spleen cells on the hemopoietic grafting potential of bone marrow cells. Firstly, when CBA/J mouse spleen cells were incubated with 51Cr-labelled YAC tumor cells together with DBA/2 mouse bone marrow cells, tumor cell lysis was reduced compared with incubation of spleen cells with tumor cells alone. Tumor cell lysis was even less when post-irradiation regenerating bone marrow was used. Secondly, C57B1/6 mouse bone marrow cells incubated with an excess of DBA/2 mouse spleen cells showed a reduced ability to produce hemopoietic spleen colonies in irradiated 129/J mice, whereas incubation with either thymus cells or fewer spleen cells produced no such effect. The results show that, when incubated with spleen cells under the conditions of a standard NK cell assay, regenerating bone marrow cells competitively inhibit the killing of YAC tumor cells and bone marrow progenitor cells are rendered ineffective in their hemopoietic colony-forming potential (CFU-s). These findings suggest that certain hemopoietic progenitor cells and YAC tumor cells can both serve as targets for NK cells, consistent with the view that the spontaneous cytolysis of tumor cells in vitro and natural resistance to bone marrow transplantation in vivo are mediated by cells of a common lineage.     

Bone marrow transplantation in the rat. Lytic functions of inflammatory leukocytes during acute graft-versus-host disease

We have isolated inflammatory leukocytes from various lymphoid and parenchymal organs after total body irradiation and bone marrow transplantation from either an allogeneic or syngeneic strain and tested their ability to perform lytic functions in vitro. No direct lytic activity (i.e. cytotoxic T lymphocytes, CTL) to relevant strain-derived target cells in the lymphoid or parenchymal target organs was seen preceding or during acute graft-versus-host disease (aGVHD). Instead, the leukocytes of the spleen and blood and the inflammatory cells of liver and lungs were efficient effector cells against recipient-derived target cells in the presence of relevant antibody (antibody dependent cellular cytotoxicity, ADCC). The NK activity against YAC-1 (natural killer, NK) target cells was first high in the spleen, but when the aGHVD appeared in the allograft marrow recipients the NK activity decreased in the spleen with a concomitant increase in the liver, but not in the other parenchymal target organs. At the same time no NK activity was seen in the syngeneic marrow graft recipients' parenchymal organs. These observations suggest functional differences in the structure of inflammation in the different target organs of aGVHD.     

Immunomodulatory Activity of A Novel Nucleoside, 7-thia-8-oxoguanosine. II. Characterization of Induced Effector Cells and the Mechanism of Induction

Abstract

In a preceeding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T80G)². In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T80G. Removal of macrophages from 7T80G activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T80G induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM₁ antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T80G induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T80G. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T80G resulted in effectors with reduced cytotoxicity.

When injected in vivo, 7T80G induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to α&β-IFN but not to γ-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T80G is most likely to be mediated by α&β-IFN involvement of other cytokines can not be ruled out.