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Natural killer (NK) cell stimulatory factor increases the cytotoxic activity of NK cells from both healthy donors and human immunodeficiency virus-infected patients.

Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL- 12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and IL-2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5- NK cells and does not confer cytotoxic activity to CD16-CD5+ T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumor-derived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV- infected patients with NKSF and/or IL-2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon gamma in response to NKSF and/or IL-2, although at levels 5- or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/IL-12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.


Summary
Natural killer cell stimulatory factor (NKSF), or interleukin 12 (ID12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and ID2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5 -NK cells and does not confer cytotoxic activity to CD16-CD5 + T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumorderived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV-infected patients with NKSF and/or Ib2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon 3' in response to NKSF and/or II.-2, although at levels 5-or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/ID12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.
N 'atural killer cell stimulatory factor (NKSF) 1 is a heterodimeric lymphokine composed of a heavy (p40) and a light chain (p35) covalently linked (1,2). The NKSF p40 chain belongs to the recently defined cytokine receptor family of proteins and has structural homology with the cellular receptor for II.-6 (3). NKSF has also recently been described as cytotoxic lymphocyte maturation factor (CLMF); the name I1.-12 has been proposed for this lymphokine (4,5).
NKSF was originally purified from the cell-free supernarant fluid of phorbol diester-activated human B lymphoblastoid cell lines. The two genes coding for the p40 and p35 chains of NKSF have been molecularly cloned; simultaneous transfection of mammalian cells with the two genes is necessary for the production of biologically active NKSF (2,5). However, B lymphoblastoid-cell lines produce an excess of free P40 chain, in addition to the biologically active heterodimer (1,4).
NKSF exerts a variety of biological functions on human T and NK cells and possibly on other cell types. In particular, it has been shown to: (a) induce IFN-3' production from both T and NK cells and synergize in this effect with I1.-2, mitogens, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens (1, 2, 6); (b) exert a comitogenic effect on fresh resting T cells together with lectins or phorbol diesters (1, 2); (c) mediate a direct mitogenic effect on activated T or NK cell blasts (5,7, and our unpublished results); (d) en-hance the cytotoxic activity of resting peripheral blood NK cells (1,2); (e) synergize with Ib2 in the generation of LAK cells (4).
NKSF, independently or synergistically with other stimuli, enhances the in vitro responsiveness of both T and NK cells, acting as an immunostimulating lymphokine at the level of proliferation, cytotoxic activity, and production of lymphokines. If NKSF is produced by normal B cells upon antigenic stimulation, it could play an important regulatory role in T-B cell interaction, synergizing with IL-2 and antigens to stimulate T cells during an immune response.
PBL from HIV-infected patients have often a reduced NK cell cytotoxic activity (8)(9)(10)(11) and the ability to produce IFN-y (12)(13)(14)(15)(16). These reduced functions of PBL from HIV-infected patients can be explained only in part by a specific depletion of the lymphocyte subsets mediating these functions (16)(17)(18)(19) and suggest that T and NK cells in the patients are functionally anergic. Identification of lymphokines with stimulating activity on the lymphocytes of HIV-infected patients has important practical implications. NKSF, alone or in combination with other stimuli, might represent a possible stimulant of several functions of lymphocytes of HIV-infected patients.
In this study, we analyze the enhancing effect of NKSF on the cytotoxic activity of PBL from healthy donors and HIV-infected patients. NKSF, at concentrations as low as 10 pg/ml, rapidly (<4 h) and efficiently enhanced the cytotoxic activity of peripheral blood NK cells against a variety of target cells. The depressed cytotoxic activity of PBL from HIVinfected patients is efficiently enhanced by NKSF against both tumor-derived and virus-infected target cells to levels comparable with those mediated by PBL from control healthy donors. NKSF also induced IFN-3' production from the PBL of HIV-infected patients, although at lower levels than from PBL from healthy donors.

Materials and Methods
Patients. 29 consenting adult patients, positive for HIV antibodies and at various stages of the disease, were enrolled in this study. HIV serology was determined using commercially available assays. All patients were repeatedly positive for HIV antibodies using HIV ELISA, and confirmed by immunofluorescence and Western blot analysis. Patient classification was according to their absolute CD4 + cell counts (<200/mm 3, between 200 and 500/mm 3, higher than 500/ram3). CD4 + and CD8 + lymphocyte subsets were enumerated by immunofluorescence. Healthy HIV-seronegative donors, matched for age and sex, were included as controls. At the time of the study, none of the patients were receiving lymphokine therapy.
l~pheral Blood Lymphocytes. Peripheral blood was obtained from consenting adult healthy donors and patients using heparin as anticoagulant. PBMC were separated on Ficoll-Hypaque density gradient (Lymphoprep; Nygaard and Co., Oslo, Norway). PBL were obtained from PBMC after partial depletion of monocytes by adherence to plastic surfaces (45 min, 37~ Positive and negative selection of PBL subsets reacting with mAbs was performed using antiglobulin indirect rosetting and gradient separation, as described (20). Cell preparations obtained by this method are >95% pure. 790 The mAbs used in this study were B36.1 (IgG2b, anti-CDS) and B73.1 (IgG1, anti-CD16).
To test the effect of lymphokines on cytotoxic activity, PBMC, PBL, or PBL subsets were incubated (5 x 10 ~ cells/ml in RPMI 1640/10% FCS 37~ for the indicated times in the presence or absence of NKSF or other lymphokines. In some experiments, cellfree supernatant fluids were collected from 18-h cultures and analyzed for IFN-3, content by ILIA (21).
Cytokines and Cytoleine Assays. Human rlL-2 (107 U/mg) was kindly provided by Dr. T. Taguchi, (Osaka University and Takeda Chemical Industry, Inc., Osaka, Japan). Four different preparations of NKSF were used in this study. Pure natural (n)NKSF (4 x 106 U/ml) was purified from the cell-free superuatant fluid of this phorbol diester-stimulated RPMI 8866 B lymphoblastoid cell line according to Kobayashi et al. (1). In some experiments, a semipurified nNKSF preparation was used, obtained after a two-step purification utilizing hydroxylapatite and Mono Q ion exchange chromatography. The IFN-3,-inducing and NK cell-enhancing activities of this semipurified preparation were completely blocked by neutralizing polyclonal or monoclonal antibodies prepared against the recombinant 40-kD chain of NKSF (2, and our unpublished results). Two preparations of rNKSF were used: crude supernatant fluid from transfected COS cells (250 U/ml) or purified cytokine from transfected CHO cells (107 U/mg) (2).
IFN-'y was measured by ILIA as previously described (21). NKSF biological activity was measured in an IFN-3, induction assay (1, 6). Briefly, PBL (106 cells/200 /~l/well) were incubated (18 h, 37~ with NKSF dilutions in round-bottomed 96-well microtiter plates (Costar, Cambridge, MA). After incubation, triplicate 50-/~1 samples of the call-free supernatants were collected for each well and IFN-~/was measured by ILIA. 1 U of NKSF activity is defined as the amount required to induce half-maximal IPN-'y production. To eliminate interexperimental variability in the determination of NKSF units, a laboratory standard of pure nNKSF was used. 1 U NKSF correspond to 3.6 pM of this standard nNKSF preparation (1).
Human embryonic foreskin fibroblasts (FS4 strain; kindly provided byJ. Vilcek, New York Medical Center, New York, NY) were maintained in Eagle's Modified MEM (Flow Laboratories, Inc., McLean, VA) supplemented with 10% heat-inactivated FCS and double vitamins. FS4 cells were used at passage 16-24. Monolayers of FS4 cells in 75-cm 2 flasks were infected with human CMV strain AD-169 at a multiplicity of infection of "~0.1. When 90% of the cells exhibited cytopathic effect, the monolayers were trypsinized, and the cells were washed and frozen at -120~ until use. Cryopreserved CMV-infected targets were thawed and labeled with NaeSlCrO4 (50/~Ci 106 cells) for 1 h at 37~ 5% CO2 with gentle shaking every 15 min. Plates were centrifuged at 100 g for 5 min and then incubated for 18 h at 37~ 5% COz. All determinations were in triplicate.
Percent specific SlCr release was calculated using the formula: percent specific SlCr release = 100x [(cpm experimental -cpm spontaneous)/(cpm maximum -cpm spontaneous)], where spontaneous release was that obtained from target cells incubated with medium alone, and maximum release was that obtained from target cells incubated with 1% Triton X-100. NK Cell Cytotoxicity Enhancement by NK Cell Stimulatory Factor induced by optimal doses of NKSF (Fig. 2). However, in the presence of 100 or 1,000 U/ml of IL-2, doses of NKSF as low as 0.1 U/ml were able to increase NK cytotoxicity as efficiently as 2.5 U/ml of NKSF alone (Fig. 2).  Figure 3).   comparable with those observed with untreated PBL from control healthy donors, although still significantly lower (t = 4.06, p < 0.001) than those mediated by NKSF-treated PBL from healthy donors. Killing of CMV-FS4 was significantly increased after preincubation of PBL from HIVinfected patients with 1 U/ml of NKSF (t = 4.08, p < 0.01). The dose of 1 U/ml of NKSF was as efficient as 100 U/ml of IL-2 in enhancing the cytotoxicity from HIV-infected patients against K562 as well as CMV-FS4 target cells. Treatment of PBL with both NKSF and I1:2 resulted in a higher, but less than additive, enhancement of cytotoxicity when compared with treatments with each cytokine alone (Table 1). When K562 target cells were used, there was no significant difference in response between patients with CD4 + cell counts <200/ram 3, and those having CD4 § cell counts between 200 and 500/ram 3. The number of patients tested, using CMV-FS4 target cells, was too low to evaluate the differences among the three groups.

100-A CD16+/-Effector Cells
The ability of PBL from nine healthy subjects, and 12 patients already tested for cytotoxicity, to produce IFN-'y in response to NKSF and I1:2 was compared. Stimulation with 100 U/ml of II:2 or with 1 U/ml of NKSF for 18 h resulted in an IFN-'y production from PBL of HIV-infected patients that was 5-to 10-fold lower than that from PBL of control healthy donors (Table 2). Although simultaneous stimulation of PBL from HIV-infected patients with NKSF and I1:2 induced a mean titer of 153 U/ml, this amount was approximately sixfold lower than that observed after stimulation of PBL from healthy subjects ( Table 2). Patients with CD4 § cell counts >500/mm 3 produced higher amounts of IFN-',/ than those with lower CD4 + cell counts. In the three patients' groups tested, simultaneous stimulation with NKSF and IL-2 induced higher IFN-3' production than that induced by each cytokine alone (

Discussion
IFN-ol and IFN43 were the first cytokines shown to enhance NK cell-mediated cytotoxic activity (23). Later, I1:2 and TNF were also shown to be stimulatory (24)(25)(26). IFNand I1:2-induced enhancement of cytotoxicity occurs with fast kinetics (a few hours to 1-2 d), and in the absence of cell division (25). In these conditions, IFN and I1:2 increase the cytotoxic activity of the NK cell subset, mostly CD16+CD56+CD3 -lymphocytes, and do not induce cytotoxic activity in the majority of CD3 § T cells, which do not mediate spontaneous cytotoxicity (25).
Similarly to IFN-a and I1:2, NKSF is a potent stimulator of NK cell cytotoxic functions of PBL, acting with a rapid kinetics (<4 h) and at extremely low concentrations. Our data show that in an 18-h incubation period, NKSF increases the cytotoxic activity of CD16+CD5 -NK cells, but does not confer this function to CD16-CD5 + T cells. As we previously reported for natural NKSF (1), and confirm now using pure recombinant cytokine, NKSF efficiently enhances NK cell-mediated cytotoxicity at concentrations <1 pM, whereas similar effects are observed with I1:2 or IFN-ol only when these cytokines are used at concentrations at least 100-fold higher. Because NKSF synergizes with I1:2 to induce IFN-'y production from both T and NK cells (1,6), it was of interest to assay whether the two lymphokines synergize in enhancing cytotoxic activity. PBL stimulated with optimal concentrations of both 11:2 and NKSF mediated only marginally higher cytotoxicity than PBL stimulated with NKSF alone. However, in the presence of 100 or 1,000 U/ml of I1:2, a maximal stimulation of cytotoxic activity was obtained with concentrations of NKSF (0.1 U/ml or 0.35 pM) that by themselves induce only a modest enhancement.
As previously observed after stimulation with IFN-a or II:2 (20,25), only the cytotoxicity of CD16 § CD5-NK cells is enhanced by NKSF and no cytotoxic ability is induced in the CD5 § T cells at the time points tested. Gubler et al. Infection with HIV is associated with a wide spectrum of clinical stages ranging from asymptomatic illness to fullblown AIDS. In AIDS patients, a multitude of profound defects in the various compartments of the immune system have been described (27). Some of the dysfunctions include a depression in CD4 § T lymphocytes number and function, and a decrease in monocyte functions, NK cell cytotoxic activity, lymphoproliferative responses to mitogens and aUoantigens, MHC class II antigen expression, and polyclonal B cell activation (27). In vitro, the production of lymphokines such as IFN-ol, IFN-3,, and I1:2 is diminished (12)(13)(14)(15)(16)28). The nature and role of humoral and cellular immune response mechanisms against HIV have not been fully characterized. It is increasingly evident that in vivo progression of AIDS is intimately related with abnormal functioning of the immune system. Tremendous efforts are being made to develop new effective immunomodulatory agents that, in association with antiretroviral drugs, may be of beneficial therapy. Several groups have previously used I1:2 alone in HIV-infected patients with varying results, and other groups are currently studying the effects of AZT and 11:2 in individuals infected with HIV (29-32). However, I1:2 at doses between 1 and 12 x 106 U/m 2 can cause serious side effects. One approach to improving the efficacy of I1:2 therapy while reducing its toxicity is to use a combination of cytokines or a combination of a cytokine with antiretroviral drugs.
Conflicting data have been reported concerning NK cell cytotoxic activity in patients with AIDS (33), although most studies have reported NK cell defects, the pathogenesis of which is still unknown. We have previously reported data on the susceptibility of clonal and polyclonal populations of NK cells to HIV infection, suggesting that loss of NK cells and reduced NK cell functions in patients with AIDS occur via two different mechanisms (22). In the present study, we evaluated the in vitro effect of NKSF and IL-2 on NK cell-mediated activity and on IFN-3, production by PBL from HIV-infected patients. Since the mechanisms involved in killing of virus-infected targets are different from those involved in lysis of tumor cells, we used K562 cells and CMV-infected fibroblasts, CMV-FS4, as target cells. Our results indicate that HIV-infected patients showed a deficiency in NK cell-mediated cytotoxicity against both target cells, which is in agreement with other studies (33). We did not find a clear correlation between the level of cytotoxicity and the absolute number of CD4 § T lymphocytes. Using NKSF at a concentration of 1 U/ml, our data revealed an increase in NK cell-mediated cytotoxicity in all HIV-infected patients tested. A similar effect was achieved with 100 U/ml of Ib2. Of interest, NKSF restores NK activity of HIV-infected patients to a level similar to that observed with healthy subjects. NKSFdependent enhancement of NK cell cytotoxicity in normal donors and in patients was completely blocked by two different neutralizing mAbs to rNKSF (not shown). Depletion of CD16 + NK cells from NKSF-stimulated PBL of patients or controls reduced the cytotoxicity against K562 and CMV-FS4 to baseline levels (not shown).
When Ib2 and NKSF were used separately, the levels of IFN-'y production from 12 HIV-infected patients tested were 5-to 10-fold lower than those of healthy donors; stimulation with both cytokines synergistically induced IFN-3' at levels higher than those observed in healthy donors with each cytokine alone. IFN-3, production in patients with AIDS correlated with the absolute CD4 § cell number.
Clinical trials using IFN-3, in patients with AIDS (34, 35) have been initiated on the basis of the observation that although the patients have a defect in their ability to produce IFN-'r in response to different inducers, their PBMC were responsive to IFN-3, in vitro (12,(36)(37)(38). Heagy et al. (35) reported a phase I/II study in which 21 AIDS patients were treated with IFN-3,. In six patients, a decrease in p24 core antigen was observed, suggesting an antiviral effect of IFN-y. In patients with high CD4 + cell counts, a transient regression of the malignant lesions associated with Kaposi sarcomas was also observed. Murphy et al. (34) reported that IFN-3,treated patients have fewer nonopportunistic infections than the IL-2-treated ones.
It is clear that the treatment of a syndrome with multiple immunological dysfunctions such as AIDS would be facilitated by the combined use of multiple cytokines and antiviral agents that would offer the possibility to correct or compensate some of the dysfunctions while exerting a direct antiviral effect. NKSF is one such potential candidate cytokine because of its multiple immunopotentiating effects, its activity at much lower concentrations than other cytokines, and, particularly important, its ability to synergize with other physiological stimuli on immune cells, such as IL-2 or antigenic stimulation.