Lectin-induced cytotoxic activity in spleen cells from young and old mice. Age-related changes in types of effector cells, lymphokine production and response.

Concanavalin A (Con A)-induced cytotoxic activity, interferon (IFN) and interleukin-2 (IL-2) levels in cultures of spleen cells from young (2-3 months) and old (22-24 months) C57BL/6 female mice were studied. Con A-activated spleen cells from old mice attained significantly higher cytotoxic activity compared with activated spleen cells from young mice. Activated spleen cells from old and young mice showed differences in their ability to lyse different types of target cells. Both could lyse P-815 cells, neither could lyse K562 cells, and only activated cells from old mice could lyse EL-4 cells. Cytotoxic spleen cells from the old mice were more sensitive to anti-asialo-GM-1 and anti-Lyt-2.2 plus complement (C) treatment. While levels of IL-2 produced by spleen cells from young mice were higher, the addition of exogenous IL-2 had no effect on cytotoxic activity of the spleen cells from old mice. Exogenous IL-2, however, could lower cytotoxic activity of Con A-activated spleen cells from young mice. Activated spleen cells from old mice generated higher levels of IFN-gamma while the addition of an anti-IFN-gamma antibody boosted the level of cytotoxicity by Con A-activated spleen cells from young mice. These results suggest that IFN-gamma may act as a feedback inhibitory signal regulating the levels of cytotoxicity induced in spleen cells from young mice in response to Con A. The cytotoxic activity generated in Con A-activated spleen cells from old mice reflects a defect in this feed-back regulation.     

Recombinant interleukin-2 inhibits growth of human tumor xenografts in congenitally athymic mice

Administration of human recombinant interleukin-2 (RIL-2) into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of HeLa, HU 609T and T24B human carcinoma cells partially inhibited growth of the human tumor xenografts. In vitro activation of nu/nu spleen cells with human RIL-2 resulted in generation of killer cells showing in the 51Cr cytotoxicity assay similar levels of cytolysis as RIL-2-activated spleen cells from heterozygous (nu/+) mice. The RIL-2-activated (LAK) cells were cytotoxic for a variety of mouse and human tumors, reaching the peak of their cytotoxic activity after 3 days of cultivation in the RIL-2-containing medium. The cytotoxic activity of activated nu/nu spleen cells was significantly reduced by treatment with antibody against glycolipid asialo GM1, the differentiation antigen of natural killer (NK) cells. This finding suggests that in addition to the conventional, asialo GM1- LAK cells, asialo GM1+ activated NK cells participated in the cytotoxicity displayed by the IL-2-activated nu/nu killer spleen cells.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.     

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.     

Effect of protein calorie malnutrition on the levels of natural and inducible cytotoxic activities in mouse spleen cells.

Six-week-old C57B1/6 female mice were fed a normal (24% protein) or an isocaloric but protein-deficient (4% protein) diet. At different time periods after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin-A (Con-A)-induced cytotoxic activity, Con-A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and protein deficient (PD) mice. Results indicated that (a) levels of spleen NK activity increased initially in PD mice, but after 7 weeks on PD diet declined to normal and subnormal levels, (b) IL-2 generation in response to Con-A as well as IL-2 activation of NK activity were comparable in spleen cells of control and PD mice at all time points tested, (c) Con-A-induced cytotoxic activity was significantly greater in spleen cells from PD mice, the difference being greater at higher doses of Con-A, and (d) generation of alloimmune cytotoxic T cells in a MLC reaction was normal in PD mouse spleen cells until 4 weeks after the beginning of PD diet, but declined markedly thereafter. Relevance of these observations to other related findings in protein calorie malnutrition are discussed.     

Lymphokine-activated killer cells and aging in mice: significance for defining the precursor cell

The present study was undertaken to define the cell populations which mediate lymphokine-activated killer (LAK) cell activity in mice. Because old mice exhibit markedly decreased to nondetectable natural killer (NK) cell activity, this age-associated change provided an advantageous system to examine the contribution of NK and T cells to LAK activity. Spleen cells from either young (6-9 weeks) or old (20-26 months) mice were cultured with 1000 units/ml of recombinant interleukin 2 (rIL 2) for 3-5 days. The cells were then tested in a 51 Cr-release assay for their cytotoxicity against NK-resistant fresh tumor cells (MCA-102). The LAK activity exhibited by spleen cells from old mice following 5 days of culture was equivalent to that developed by spleen cells of young mice. This result was contrary to what would be anticipated if mature NK cells comprise the primary precursors of LAK activity, and required further elucidation. The Thy-1 and asialo GM1 (ASGM1) phenotypes of LAK precursor and effector cells were therefore examined by depletion techniques using the appropriate antibodies plus complement. The results using spleen cells harvested after 5 days of culture with rIL 2 showed that LAK effector cells which developed from spleen cells of both young and old mice were predominantly Thy-1+ (85.3% young; 91.8% old) and some coexpressed ASGM1. Spleen cells were treated prior to culture to study the precursor cells. Development of LAK activity by spleen cells from both young and old mice was greatly reduced by pretreatment with anti-ASGM1 plus complement. However, since spleen cells of old mice exhibit very low mature NK activity, these data suggest that the LAK precursors, at least in old mice, may be ASGM1+ NK precursor cells rather than mature ASGM1+ NK effector cells. In addition, treatment with anti-Thy-1 plus complement inhibited generation of a significant proportion of LAK activity only in the spleens of old mice, suggesting a qualitative difference in LAK precursor cells with age and supporting the heterogeneity of the cells which are capable of developing LAK activity.     

T-cell cytotoxicity and aging: differing causes of reduced response in individual mice

Specific T-cell cytotoxic responses to allogeneic and hapten-modified syngeneic cells decrease with age. In order to determine the causes of these reduced T-cell cytotoxic responses, spleen cells from individual young and senescent C57BL/6J female mice were mixed in various proportions in culture with either X-irradiated BALB/c spleen cells or trinitrophenyl-modified syngeneic cells and the resultant cytotoxic responses determined in comparison to those of spleen cells from young and old mice stimulated alone. In both allogeneic and hapten-modified syngeneic cytotoxicity, it was found that a low percentage of the aged mice suffered from decreased helper-cell activity or from increase of suppressor activity, while the majority of mice showed no synergy, positive or negative, with the cells from the young donor. Studies of interleukin-2 (IL-2) activity were performed on conditioned medium from spleen cells from mice of various ages cultured for 24 h with concanavalin A. Those preparations from senescent mice that showed reduced IL-2 activity did not contain activity suppressive or competitive to IL-2 produced by spleen cells from young mice. Limiting dilution of spleen cells from mice of various ages in the presence of semi-allogeneic stimulatory cells and subsequent assay of the resultant allogeneic cytotoxicity provided a measure of the frequency of cytotoxic units. Parallel experiments in which crude IL-2 was added to the limit dilution cultures provided a measure of the frequency of cytotoxic cell precursors. Once again in these experiments, individual senescent mice demonstrated different defects. Three different types of age-related defects were observed. Certain aged mice were devoid of detectable cytotoxic units and cytotoxic T-lymphocyte precursor at the cell dilutions used. Other senescent mice demonstrated a very low frequency of cytotoxic units (approximately 1/40 000) as compared with young mice (approximately 1/5 000), and the addition of crude IL-2 to cultures from these mice did not improve reactivity. A third group of old mice, those with a moderate age-related decrease in the frequency of cytotoxic units (approximately 1/12 000), demonstrated a cytotoxic T-lymphocyte precursor frequency in the presence of crude IL-2 which was comparable to that of young mice (approximately 1/1000).     

Identification of macromolecular insoluble cold globulin (MICG) as a new marker shared by NK and ADCC cells, but not expressed by NC cells.

We report here that cytotoxic pretreatment of spleen cells from six different strains of young adult mice with a monospecific rabbit antiserum against macromolecular insoluble cold globulin (MICG) effectively abrogates spontaneous NK activity directed towards YAC-1 tumour cells. MICG is a 225,000 molecular weight glycoprotein that is present in the plasma membrane of adult thymocytes and peripheral T cells, as well as in embryonic prothymocytes, but absent in granulocytes and B lymphocytes. The diminished NK activity in lymphocyte populations selectively depleted of MICG+ cells could not be restored by in vitro exposure to the NK boosting agents interleukin-2 (IL-2) and interferon. Lymphokine-activated spleen NK cells, generated by 48 hr preculture with IL-2 or interferon, expressed high levels of MICG surface antigen, moderate amounts of Thy 1.2 and, in striking contrast to spontaneous NK, very low to negligible amounts of AsGM1. Likewise, spontaneous NK cells in bone marrow were also shown to be both MICG+ and AsGM1+, while lymphokine-activated bone marrow NK cells remained MICG+, but lacked AsGM1. Thus, a clear distinction could be observed between spontaneous and activated NK cells with respect to differential expression of MICG and AsGM1. MICG was also detected on ADCC effector cells, whereas no surface MICG could be found on NC cells. These data are in line with the view that at least certain types of NK cells develop along a common lineage with T lymphocytes in the mouse.     

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.     

Altered Natural Killer and Natural Cytotoxic Cellular Activities in lpr Mice

Three strains of mice bearing the autosomal recessive lpr gene (MRL, C57BL/6, and C3H) that had spontaneously developed a lupus-like disease were studied sequentially for functional natural killer (NK) and natural cytotoxic (NC) cell activity. Natural killing was impaired in spleen and bone marrow cells from all the lpr strains, as well as from the congenic strain MRL--+/+, which develops a late onset lupus-like disease. The NK cell activity was found to be depleted as early as 2 months of age in all lpr strains, and decreased further with age. NK activity was augmentable by Poly I:C and interleukin 2 (IL-2), suggesting that the residual cells can respond to NK modulators. In contrast with NK cell activity, NC activity was not decreased in lpr mice but could be augmented by IL-3-rich supernatants. The spontaneous decrease in NK cell activity was associated with an increased autologous plaque-forming cell (APFC) response to bromelin-treated mouse red blood cells, which is produced primarily by B cells possessing the Ly-1 phenotype (Lyt-1+ B). When NK cell activity was increased by exogenous administration of Poly I:C, the APFC response diminished. Treatment of spleen cells with anti-asialo GM1 prior to Poly I:C treatment resulted in a decreased NK response but increased both APFC and Lyt-1+ B cells. The possible regulation of autoreactivity by NK cells is discussed.     

Interleukin-2 induced activation of natural killer cells in rats and mice: a comparative study

Effects of interleukin-2 (IL-2) on the natural killer (NK) activities of BALB/c mouse and Wistar rat spleen cells were compared. While mouse spleen cells cultured alone rapidly lost NK activity, co-culture with IL-2 resulted in a marked enhancement of NK activity. In contrast, the levels of NK activity of rat spleen cells cultured alone increased and remained high for 3 days and declined thereafter. Addition of human recombinant IL-2 or purified rat IL-2 did not influence the NK levels in rat spleen cell bulk cultures. Both IL-2 preparations were however biologically active as shown by their capacities to induced proliferation in rat spleen cells. Rat spleen cells suppressed the IL-2 activation of mouse spleen cells in a dose dependent manner, indicating a suppressor influence generated by rat spleen cells. Culture supernatants of rat spleen cells cultured with or without IL-2 for 3 or 5 days could also suppress the mouse spleen NK activation in response to IL-2. The suppressor activity could be concentrated on a 5K MW cut-off Amicon filter indicating that the molecular weight of the factor is more than 5000. These results indicate that a suppressor of IL-2 induced NK activation of mouse spleen cells is released by cultured rat spleen cells.     

Effect of feeding a diet with half of the recommended levels of all vitamins on the natural and inducible levels of cytotoxic activity in mouse spleen cells.

Groups of 6-week-old female C57Bl/6 mice were fed a normal diet with recommended levels of all vitamins or a vitamin-deficient (VD) diet containing half of the recommended level of each vitamin. At different time periods (1-11 weeks) after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin A (Con A)-induced cytotoxic activity, Con A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and VD mice. Results indicated that: (i) spleen NK activity remained normal until 2 weeks after the initiation of VD diet, fell steeply to low levels at the 4 and 5 week time points and remained depressed thereafter; (ii) IL-2- and Con A-induced levels of cytotoxic activity in spleen cells derived from VD mice declined at 4 weeks after the institution of VD diet, and then remained low throughout the study; (iii) the capacity of spleen cells from VD mice to generate IL-2 in response to Con A and cytotoxic T cells in response to allogeneic spleen cells, was normal at 1 and 4 weeks after initiation of the VD diet and was markedly depressed at the 6 and 9 week time points. These results suggest that partial combined deficiencies of dietary vitamins strongly influence assays of immune function.     

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice: natural killer cell activity in cultured spleen leukocytes concomitant with T-cell-dependent immune interferon production

The characteristics and specificities of spleen and peritoneal cytotoxic cells generated during lymphocytic choriomeningitis virus (LCMV) infection of C3H/St mice were examined. Activated natural killer (NK) cell activity was identified in fresh leukocyte populations from the 2nd to 8th days postinfection, whereas virus-specific cytotoxic T-cell activity was detected from the 6th to 14th days. When leukocytes were cultured overnight at 37 degrees C before assay, T-cell activity was still observed, but nonspecific activated NK cell-like cytotoxicity was only detected on the 6th and to a lesser degree the 8th day postinfection. Overnight culture of leukocytes taken earlier in the infection eliminated their NK cell activity. Similar activities were seen with spleen cell, plastic-adherent peritoneal cell, and nonadherent peritoneal cell populations. The virus-specific cytotoxicity observed with adherent peritoneal cells was due to contamination with cytotoxic T cells, as shown by H-2-restricted cytotoxicity and sensitivity to anti-theta antibody and complement. The nonspecific cultured day 6 effector cell from either the spleen or peritoneum displayed killing specificities and other physical properties identical to those of activated NK cells, but had sensitivities to anti-theta antibody and complement intermediate between activated day 3 NK cells and cytotoxic T cells. Culture stable NK-like cells were not found in athymic nude mice, suggesting a T-cell-dependent mechanism. Whereas LCMV spleen homogenates contained 10-fold-higher levels of interferon at day 2 than at day 6 postinfection, substantially more (nearly 20-fold) interferon was made in cultures of day 6 cells than day 2 cells. Spleen interferon was predominantly type I, whereas the culture interferon was predominantly type II, as shown by acid lability studies. Significant levels of interferon were produced by nylon-wool-passed day 6 spleen cells, and virtually all interferon production was eliminated by treatment of either day 2 or day 6 cells with antibody to theta antigen and complement, suggesting that T cells produced the interferon in vitro. Furthermore, athymic nude mice had no culture-stable NK cells 6 days postinfection, and spleen cells from them failed to produce significant levels of interferon in vitro. Addition of interferon (type I, fibroblast) to cultured C3H spleen cells affect the already elevated levels of cytotoxicity in day 6 cultures, suggesting that the NK cells in the day 6 culture were already activated. Our results suggest that T cells responding to LCMV infection secrete interferon type II which causes the continued activation of NK cells in culture. The resulting population of activated NK cells therefore appears to be relatively stable in culture and to express more theta antigen because of this T-cell dependence. Although one could mistakenly or allospecific cytotoxic T cells or cytotoxic macrophages, more careful examination shows that they are most likely activated NK cells...     

Up-regulation of natural killer cell cytotoxicity by interleukin-2: the effect of sex and parity.

PROBLEM: Peripheral blood lymphocytes (PBLs) from some, but not all, female donors showed increased cytotoxicity in response to interleukin (IL)-2. METHOD OF STUDY: The effect of IL-2 on natural killer (NK) cell cytotoxicity was compared in nulliparous females, parous females, and males. Peripheral blood lymphocytes were preincubated for 20 or 72 hr with 5 or 100 U/ml IL-2 and cytotoxicity against K562 targets was then examined. RESULTS: In the parous females, only the 72-hr preincubation with 100 U/ml IL-2 significantly increased NK cell cytotoxicity, whereas nulliparous females also showed significantly increased cytotoxicity after a 20-hr preincubation with 100 U/ml IL-2. Neither female subject group had increased activity after preincubation for 20 or 72 hr with 5 U/ml IL-2. However, male peripheral blood lymphocytes also showed a significant increase in NK cell cytotoxicity when preincubated for 72 hr with 5 U/ml IL-2. CONCLUSIONS: The effect of IL-2 on NK cell cytotoxic activity may be related to sex and the state of parity.     

Transient appearance of hepatic natural killer cells with high cytotoxicity and unique phenotype in very young mice

There were few natural killer (NK) cells in the liver in very young mice at the age of 1-2 weeks. This was because the cell yield from the liver of young mice was low. The percentage of NK cells in the liver of young mice, however, was almost comparable with that in the liver of adult mice. Lymphocytes were isolated from the liver and spleen of C57BL/6 (B6) mice, and NK cytotoxicity and phenotype were herein examined in this study. NK cytotoxicity was extremely high in the liver of very young mice. This phenomenon was seen in the liver of various normal mouse strains. In contrast, the appearance of high cytotoxicity was not seen in NK cells of the spleen, irrespective of mouse strains. The quality of NK cells in the liver of young mice was different from that in adult mice. NK cells in the liver of young mice were mainly CD69(+)Mac-1(-) Fas ligand(+), whereas those in the liver of adult mice were CD69(-)Mac-1(+) Fas ligand(-). These results revealed that the quality of hepatic NK cells changes in the process of ageing. Namely, liver NK cells in very young mice temporarily show the highest NK cytotoxicity and a unique activated phenotype. Physiological meaning of the present phenomenon was discussed.     

Natural killing of MHC class I(-) lymphoblasts by NK cells from long-term bone marrow culture requires effector cell expression of Ly49 receptors.

NK cells from long-term bone marrow culture (LTBMC) were compared with IL-2-activated splenic NK cells [short-term spleen cell culture (STSC)] with regard to expression of inhibitory Ly49 receptors and cytotoxic function. In the LTBMC, the total number of NK cells expressing either one of the Ly49 molecules A, C/I and G2 was strongly reduced (10-15% of NK1.1(+) cells) compared to the STSC (80-90% of NK1.1(+) cells). With regard to cytotoxic function, we confirmed that LTBMC-derived NK cells efficiently killed the prototype NK target YAC-1. However, against other targets, killing was more variable. First, while STSC-derived NK cells clearly distinguished MHC class I(-) from MHC class I(+) tumor cell targets, LTBMC-derived NK cells did not; they either killed both targets equally well or not at all. Secondly, LTBMC-derived NK cells were largely incapable of killing lymphoblast targets deficient in MHC class I expression. To test whether this cytotoxic defect was due to the low number of Ly49(+) NK cells in the LTBMC, we separated Ly49(+) and Ly49(-) NK cells by cell sorting and tested them individually. This experiment showed that only Ly49(+) NK cells in the LTBMC were able to kill MHC class I(-) lymphoblasts (and to distinguish them from MHC class I(+)), despite good cytotoxicity against YAC-1 cells in both populations. These data suggest that certain modes of NK cell triggering are dependent on Ly49 receptor expression. From our results, we speculate that inhibitory receptors are expressed before triggering receptors for normal self cells during NK cell development, which may be an important mechanism to preserve self tolerance during the early stages of NK cell maturation.     

Human recombinant IL-4 decreases the emergence of non-specific cytolytic cells and favours the appearance of memory cells (CD4+CD45RO+) in the IL-2-driven development of cytotoxic T lymphocytes against autologous ovarian tumour cells.

As IL-4 and IL-6 have also been reported to promote the development of T lymphocytes such as IL-2, we investigated their role in the development of specific cytotoxic T lymphocytes (CTL) against autologous ovarian tumours in mixed lymphocyte tumour cultures (MLTC). Peripheral blood lymphocytes (PBL) from five ovarian carcinoma (OC) patients were incubated with autologous OC cells at a PBL:OC cell ratio of 20:1 in IL-2 alone (50 U/ml for the first week and 200 U/ml thereafter) or with IL-4 (100 U/ml) and/or IL-6 (5 U/ml). Neither IL-4 nor IL-6 improved lymphocyte proliferation consistently. In contrast, IL-4 reduced significantly the development of LAK activity as assayed against Daudi cell line, and decreased modestly the emergence of natural killer (NK) activity as assayed against K562. This property was not shared by IL-6. The prevention of the development of non-specific cytolytic activity (LAK and NK activities) was much stronger when the MLTC was started with IL-4 in the absence of IL-2 during the first week in culture. A concomitant drop in NKH-1 expression (CD56) was observed. By inhibiting the emergence of non-specific cytotoxicity, IL-4 provided better evidence of the specific cytolytic activity directed at ovarian cells. In parallel, a significant increase in the generation of memory cells (CD4+CD45RO+) was observed with IL-4. In conclusion, in this model, IL-4 added before IL-2 decreases significantly the emergence of non-specific cytotoxic cells, and promotes the generation of memory cells. These properties may be of interest in the design of strategies aimed at obtaining tumour-specific cells for investigational and immunotherapeutic purposes.     

Activated Natural Killer Cells Suppress Myelopoiesis in Mice with Severe Combined Immunodeficiency

The in vivo effect of natural killer (NK) cell activation on aulologous myelopoiesis was studied in an environment deficient of functional Tand B cells. Administration of 3.6-bis[2-(Dimethylamino)-ethoxy]-9H-xanthen-9-one dihydrochloride) Tilorone) or recombinant interleukin-2 (rIL-2) to mice with severe combined immunodeficiency (C. B. -I7 scid/scid) resulted in an increase in YAC-1 lysis by their splenocytes as well as bone marrow cells. Recombinant IL-2 furthermore led to a fivefold increase in the cellularity of the spleen. When assayed against human NK/lymphokine-activated killer (LAK) target, K562 cell line. the IL-2-activated mouse cells exhibited no cytotoxicity across the species barrier. Both agents induced a profound suppression of myelopoietic progenitor cells as measured in a 7-day granulocyte-macrophage colony forming cell (GM-CFC) assay. We conclude that the presence of neither functional T nor B cells is necessary for NK cells to mediate inhibition cf myelopoiesis in the autologous host.     

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.     

Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12

IL-12 exerts a potent anti-tumor effect, which is possibly mediated by multiple mechanisms including activation of NK and NKT cells, induction of cytotoxic T lymphocytes, and inhibition of angiogenesis. In the present study, we characterized the cytotoxic effector cells and mechanisms responsible for the anti-metastatic effect of IL-12. Administration of IL-12 had a comparable inhibitory effect on experimental lung metastasis of B16 melanoma cells in wild-type C57BL/6 mice and RAG-2-/- mice that lack T and NKT cells, which was abolished by depletion of NK cells. Cytotoxic activity of liver and splenic mononuclear cells against B16 was induced by IL-12 administration in RAG-2-/- mice at a level comparable to that in wild-type mice, which was also abolished by depletion of NK cells. Moreover, the anti-metastatic effect of IL-12 was abrogated by perforin deficiency, but not by Fas ligand deficiency, in association with a lack of IL-12-induced cytotoxic activity of liver and splenic mononuclear cells against B16. These results suggest that perforin-dependent cytotoxicity of IL-12-activated NK cells is sufficient for the anti-metastatic effect of IL-12.