The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 microgram/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously described, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained more melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE2 (di-M-PGE2) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE2 or di-M-PGE2 plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE2.     

Augmentation of natural killer activity, induction of IFN and development tumor immunity during the successful treatment of established murine renal cancer using flavone acetic acid and IL-2

The investigational drug flavone acetic acid (FAA) has been previously shown to systemically augment NK activity in vivo in normal mice within 24 h of i.p. or i.v. administration. The current study investigates the ability of FAA, and/or rIL-2, to augment NK activity and antitumor responses in mice bearing murine renal cancer (Renca). The results demonstrate that FAA potently augments NK activity in the blood, spleen, and liver of Renca-bearing mice and that the administration of rIL-2 in addition to FAA results in a further augmentation of NK activity over that observed with FAA alone. Renca-bearing mice treated with FAA (200 to 250 mg/kg) plus rIL-2 exhibited a significantly increased incidence of long term survivors (59%) over that observed following treatment with FAA (0%) or rIL-2 (5%) alone. Therapeutic synergy between FAA and rIL-2 was observed against primary tumors, minimal residual disease, and experimental-induced pulmonary metastases. Mice cured of Renca by FAA plus rIL-2 treatment were largely resistant to rechallenge with Renca suggesting a role for T lymphocytes. The augmentation of NK activity and the therapeutic effects of FAA coincided with the rapid induction of high titers of serum IFN of the alpha/beta type within 4 h of FAA administration. Subsequent studies demonstrated that the contribution of FAA could be partially replaced by the administration of several doses of human rIFN-alpha A/D Bg1 before the initiation of rIL-2 administration. The observed synergistic antitumor effects of FAA plus rIL-2 coincided with the augmentation of NK activity, induction of IFN-alpha/beta, and induction of long lasting tumor immunity. Overall, these results suggest that this approach may obviate the need for adoptive immunotherapy in association with rIL-2 administration for at least some tumor types.     

Strain difference in mouse natural killer activity augmented by mouse interferon and poly I.C

The level of natural killer (NK) activity was found to vary considerably among several mouse strains. In vivo and in vitro, interferon (IFN) and IFN inducers have been shown to augment mouse NK activity. C3H/He mice showed high NK activity, DDD/1 and A/J mice low NK activity, and C57BL/6, BALB/c and DBA/2 mice intermediate NK activity after injection with polyinosinic polycytidylic acid (poly I. C.). The same NK activity correlation was observed in nontreated mice, but the NK activities were lower compared with the poly I. C.-injected mice. Moreover, the DDD/1 and A/J mice showed almost no augmentation of NK activity on injection with poly I.C. In vivo, C3H/He, BALB/c and C57BL/6 mice injected with IFN showed augmented NK activity, but DDD/1 mice showed no such reaction. In vitro, C3H/He, BALB/c and C57BL/6 mouse spleen cells treated with IFN also showed augmented NK activity, but DDD/1 mouse spleen cells showed almost none. F1 hybrids between high (C3H/He) and low (DDD/1) NK-activity strains showed high NK activity. Thus, activity is dominant over low activity. The segregation of (DDD/1 X C3H/He) Fl X DDD/1 back-cross mice suggested that the strain differences in NK activity are under polygenic control.     

Dose dependent inhibition of B-16 melanoma growth in vivo by a synthetic analogue of PGE2

Daily intratumor administration of 16,16-dimethyl-PGE2-methyl ester in two different dosages inhibited tumor growth in C57Bl/6J mice bearing subcutaneous B-16 melanomas. The larger dose (20 microgram/day/mouse) produced a 68% decrease in tumor volume, a 69% decrease in tumor weight and a 60% decrease in the number of cells in mitotic phase. The smaller dose (10microgram/day/mouse) was one fifth less effective than the 20microgram dose but produced similar changes. Histological examination of tumors revealed no significant differences either in the inflammatory cell population or the amount of necrosis in the control and di-M-PGE2-treated tumors.     

Modulation of IL 2-dependent growth of mouse NK cells by interferon and T lymphocytes

The regulatory role of interferon (IFN) on the growth of mouse natural killer (NK) cells in the presence of interleukin 2 (IL 2) was analyzed by the limiting dilution assay. Pretreatment for 5 hr with IFN (600 U/ml) was able to augment the frequency of proliferating cells and NK effector cells when spleen cells of BALB/c nu/+ and BALB/c nu/nu were cultured for 7 days in the presence of IL 2. When IFN was present during the 7-day culture period, we again found an increase in proliferative and cytotoxic frequencies in cultures of spleen cells from nude mice, but in contrast, found a decrease in these frequencies in cultures of spleen cells from euthymic mice. Addition of irradiated (3000 R) spleen or thymus feeder cells from euthymic mice to the nu/nu cultures caused an inhibitory activity of IFN also on nu/nu cells. These data indicate that IFN can have both positive and negative regulatory effects on the in vitro growth and differentiation of mouse NK cells and that the inhibitory effects are mediated via T lymphocytes.     

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge-132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat- or acid-treatment, suggesting that the induced IFN is of gamma-nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge-132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge-132, neither induction of IFN nor augmentation of NK activity occurred, and X-ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge-132 are mediated by the induced IFN.     

Effect of interferon on the inhibition of malignant cell growth by human peripheral leukocytes. III. Effect of systemic administration

Human leukocyte interferon preparation (HuIFN-alpha LE) was given to the patients with cancer or with chronic hepatitis. Spontaneous tumor cell growth inhibition by human peripheral lymphocytes (STGI) and NK activity were enhanced by the systemic administration of HuIFN-alpha LE, although there were differences in the kinetics between the two activities after one time administration or by the repeated administration. This suggests that IFN acts indirectly on the tumor cells by the medium of normal lymphocytes or NK cells, and that tumor cell growth inhibition is different from NK activity.     

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

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

Studies on the mechanism of low natural killer cell activity in infant and aged mice

NK activity in mice is high between about 6 and 10 weeks of age. In contrast, infant mice and mice older than 12-14 weeks of age usually have quite low or undetectable NK activity. Studies were performed to analyze the mechanisms underlying this characteristic age-related regulation of NK activity. Spleen cells from infant mice did not develop appreciable NK activity upon incubation for 12-18 h with either interferon (IFN) or interleukin-2 (IL-2). Analysis of the frequency of IL-2-dependent progenitors of NK cells, in a limiting dilution assay, also indicated that the spleens of infant mice are deficient in precursors of NK cells. In contrast, spleen cells from old mice (30 weeks old) developed substantial levels of NK activity upon incubation with either IFN or IL-2, and they showed a frequency of IL-2-dependent progenitors of effector cells that was similar to that of young mice. Both infant and old mice had plastic-adherent suppressor cells in their spleens, which could strongly inhibit NK activity. In addition, both infant and old mouse spleen cells contained nonadherent suppressor cells, which had a higher density on Percoll gradients than NK cells. Thus, several factors appear to contribute to the age-related regulation of NK activity in mice.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

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.     

Natural killer (NK) cells as a responder to interleukin 2 (IL 2). II. IL 2-induced interferon gamma production

In the accompanying paper, we showed that natural killer (NK) cells were a major population in the naive spleens of normal mice that responded directly to a T cell growth factor, interleukin 2 (IL 2), and clonally replicated without other stimulating agents. The cloned cells growing in IL 2 showed a potent NK activity against several NK targets without addition of an NK-activating agent, interferon (IFN). In the present study, therefore, we examined whether these cloned NK cells on their own produced IFN. It was found that all NK clones growing in IL 2 produced IFN in the culture fluids. The titers of IFN produced in the IL 2-containing media correlated well with the number of growing cells. With the culture in the absence of IL 2, neither cell growth nor IFN production could be detected. Addition of Con A into the culture in the IL 2-free media showed no IFN production. The antiserum neutralizing IFN alpha and IFN beta failed to significantly neutralize IFN produced by NK clones. Treatment with either a pH of 2.0 or antiserum neutralizing mouse IFN gamma resulted in a marked reduction of IL 2-induced NK IFN, indicating that a major part of IFN produced was IFN gamma. These results indicate that IL 2 stimulates NK clones to proliferate, accompanied by IFN gamma production. The results also show that an NK clone, when stimulated with Sendai virus, produced a type 1 IFN (IFN alpha and/or IFN beta), suggesting that murine NK cells can produce both type 1 (alpha and/or beta) and type 2 (gamma) IFN, depending on inducers.     

Intratumoral hu14.18-IL-2 (IC) Induces Local and Systemic Antitumor Effects That Involve Both Activated T and NK Cells As Well As Enhanced IC Retention

hu14.18-IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18-IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18-IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E(+) NKp46(+) NK cells and NKG2A/C/E(+) CD8(+) T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

Elevated natural killer cell-mediated cytotoxicity, plasma interferon, and tumor cell rejection in mice persistently infected with lymphocytic choriomeningitis virus

To assess the effects of chronic virus infection on NK cells, the related phenomena of interferon (IFN) production, NK cell activation, and resistance to tumor implants were studied in mice persistently infected with lymphocytic choriomeningitis virus (LCMV). NK cells from these LCMV-carrier mice displayed augmented killing of the NK-sensitive YAC-1 target cell. They did not lyse the more resistant targets L-929 and P815, whereas NK cells from acutely infected mice efficiently lysed all three cell types. The plasma from LCMV-carrier mice contained an antiviral substance identified as IFN type I, based on species specificity, virus nonspecificity, resistance to pH 2, and sensitivity to antibody to type I IFN. IFN titers in plasma from LCMV-carrier mice were 32 to 64 U/ml, about 20-fold less than those in acutely infected mice. Both the IFN and NK cell levels continuously remained elevated in the LCMV carrier mice up to at least 6 months of age. IFN is known to activate NK cells and to induce their blastogenesis in vivo. As determined by centrifugal elutriation, large NK blast-size cells were isolated from the spleens of acutely infected mice, but not from either normal or LCMV-carrier mice, suggesting augmented NK cell-mediated lysis in the absence of enhanced proliferation. Poly inosinic-cytidylic acid induced high levels of NK cell-mediated cytotoxicity and blastogenesis in both control and LCMV-carrier mice, but IFN was induced to lower levels in carriers as compared with controls. Coincidental with augmented NK cell activity, the LCMV-carrier mice rejected intravenously injected 125IUdR-labeled tumor cells more efficiently than did normal mice. Thus, LCMV carrier mice have low levels of type I IFN, moderately augmented NK cell activity lasting for at least 6 months, and increased resistance to tumor cell implants. This indicates that augmented NK cell-mediated cytotoxicity can be maintained in vivo over prolonged periods of time in the presence of chronic low-level IFN stimulation.     

Changes in number and density of large granular lymphocytes upon in vivo augmentation of mouse natural killer activity

NK activity of mice as well as humans and rats has been clearly associated with large granular lymphocytes (LGL). To better understand the effects of interferon (IFN) and IFN inducers on natural killer (NK) cells, we have compared the LGL in the spleens of normal and boosted mice. Cells were fractionated by centrifugation on discontinuous Percoll density gradients, and each fraction was tested for NK activity against YAC-1 targets and for the presence of LGL. In vivo treatment with C. parvum (0.7 mg/mouse, i.p., day-3), MVE-2 (25 mg/kg, i.p., day-3), poly I:C (4 mg/kg, i.p., day-3), or IFN (10(5) U/mouse, i.p., day-1) resulted in a marked augmentation and a change of distribution of cytotoxic activity. Most of the NK activity of boosted spleen cells was associated with lower density fractions 1 and 2, whereas active normal spleen cells had somewhat higher density (fractions 2 and 3). In parallel to their increased reactivity, the boosted spleens had a marked increase in the percentage of LGL, particularly in fractions 1 and 2. The augmented activity appeared to be mediated by the LGL, because treatment with anti-asialo GM1 or anti-Thy-1.2 plus complement reduced NK as well as the number of LGL. These results indicate that IFN-mediated boosting of NK activity in the spleen is due to an increase in the lower density LGL, as well as to an increase in the function of preexisting NK cells.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.     

Distinct requirements for IFNs and STAT1 in NK cell function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.     

Interleukin 3 enhanced interleukin 2-dependent maturation of NK progenitor cells in bone marrow from mice with severe combined immunodeficiency

Human recombinant interleukin 2 (IL 2) and highly purified murine interleukin 3 (IL 3) were tested for their ability to generate NK activity in bone marrow cells from mice with severe combined immunodeficiency. IL 2 alone could dose dependently induce NK activity in marrow cells as determined by cytotoxicity against YAC-1 target cells. It was demonstrated that IL 3 had dual effects on the generation of NK activity in this culture system. The addition of IL 3 resulted in inhibition of NK cell activity seen at high concentrations of IL 2. In contrast, when IL 3 was added together with low concentrations of IL 2, the generation of NK cells as judged by cytotoxicity assay as well as the appearance of cells with NK phenotypes was markedly augmented. In some experiments, mice were treated with 5-fluorouracil (5-FU) to eliminate relatively differentiated NK precursors from bone marrow cells. It was noted that the residual immature marrow cells from 5-FU-treated mice showed little NK activity even after the culture with high concentrations of IL 2. Importantly, IL 3 could induce the generation of NK activity from 5-FU-treated marrow cells in the presence of IL 2. Kinetic studies indicated that NK activity was appreciably generated from 5-FU-treated marrow cells when preincubated with IL 3 at least for 12 hr and subsequently cultured with IL 2. The cells bearing IL 2 receptors appeared in 5-FU-treated marrow cells, even though cultured only with IL 3, which implied that IL 3 could support the development of very primitive NK cells from IL 2-unresponsive to IL 2-responsive states. These results suggested that IL 3 might play a crucial role for the IL 2-induced generation of NK cells in bone marrow through promoting the expression of IL 2R on NK progenitor cells.