Comparative analysis of interleukin 15 and interleukin 2 for induction of killer activity and of type 2 cytokine production by mononuclear cells from lung cancer patients.

Interleukin (IL) 15 is a novel cytokine with IL-2-like activity. In this study, we examined the effect of IL-15 on induction of non major histocompatibility complex (MHC)-restricted killer activity and of type 2 cytokine production by peripheral blood and pleural mononuclear cells (MNCs), from 34 lung cancer patients and 20 control subjects. IL-15 induced significant killer activity in blood MNCs from lung cancer patients as well as control subjects against a small-cell lung cancer cell line (SBC-3). Effective killer induction by IL-15 was observed even in blood MNCs and pleural MNCs from the site of tumour growth in advanced lung cancer patients. IL-12 had an additive effect with a suboptimal dose of IL-15 in induction of killer activity. In the case of MNCs from lung cancer patients, IL-10 production was more prominent when cells were incubated with IL-2 than with IL-15. IL-5 production was observed in MNCs from lung cancer patients stimulated with IL-2, but not with IL-15. These observations suggest that IL-15, by virtue of its lesser induction of type 2 cytokine, may be a better candidate than IL-2 for lung cancer immunotherapy.

may mediate immunosuppression (Yamamura et al. 1993: Kharkvitch et al. 1994. Production of type 2 cytokines by lung cancer has been reported (Hung et al. 1995). and immunotherapy with cytokines may alter this type 2 predominant pattern of the type 1/type 2 axis.
In this work. we studied the effect of L-15 alone or in combination with L-12 on the immune function of MNCs from lung cancer patients. in terms of expression of non-MHC-restricted killer activity and type 2 cytokine production.

MATERIALS AND METHODS
Patients with lung cancer and control patients Thirty-four patients with primary lung cancer were studied after obtaining informed consent. Of these. 24 were men and ten were women aged 36-83 years (median age 67 years). Histological examinations revealed that 17 patients had adenocarcinoma. nine had squamous cell carcinoma. five had small-cell carcinoma and three had large-cell carcinoma. Staging examination revealed that 17 patients were stage IV. ten were stage IIIB. four were stage I11A. two were stage H and one was stage I. Nine patients had malignant pleural effusion. They had received no anti-cancer therapy before this study. Twenty subjects were studied as controls. Of these. nine subjects were control patients (three males and six females) aged 22-82 years (median age 42 years). Examinations revealed no malignant lesions or autoimmune diseases in these nine patients. The other 11 control subjects (nine men and two women) were healthy volunteers who had no signs of infection. were not taking medication and were aged 22-48 years (median age 28 years). They all gave informed consent to participate in the experiments.  Figure 1 Effect of a combination of IL-15 and IL-12 on inducon of killer activity in peripheral blood MNCs from lung cancer patients. Peripheral blood MNCs (1 x 105 per well) from lung cancer patients were incubated in medium with or without 5 ng ml-' IL-15 or 5 U ml-' IL-2 in the presence or absence of a suboptimal (1 U ml-') concentraton of IL-12 (A) and 50 ng ml-' IL-15 or 500 U ml-' IL-2 in the presence or absence of an optimal (100 U ml-') concentration of IL-12 (B) for 4 days.  Isolation of peripheral blood mononuclear cells and pleural mononuclear cells and cytotoxicity assay Peripheral blood MNCs and pleural MNCs were separated from heparinized venous blood and pleural effusion. respectively. as described previously (Sone et al. 1987: Yanagawa et al. 1989). The resultant MNCs (105 per well) were incubated in CRPMI-1640.
with or without 10 U mFl or 100 U ml-1 IL-12. in the presence or absence of 5 ng ml-1 or 50 ng mlF IL-15 or 5 U mlF or 500 U mlF IL-2. at 370C under a humidified atmosphere containing 5% carbon dioxide. These concentrations of IL-2. IL-12 and IL-15 were chosen as suboptimal and optimal concentrations to augment killer activity mediated by MNCs as described previously (Nabioullin et al. 1994: Takeuchi et al. 1996. After incubation for 4 days. the culture supernatants were collected after brief centrifugations and the cell-mediated cytotoxicity was assayed against SBC-3 cells by measuring 5ICr release in a 4-h test as described previously (Sone et al. 1987).
Quantitative measurements of cytokines IL-S. IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured by enzyme immunoassay (EIA) essentially as described previously (Takeuchi et al. 1996). The sensitivity limits of all these ELAs were 20 pg ml-'.

Statistical analysis
The statistical significance of differences between groups were analysed by Student's t-test (two-tailed). Wilcoxon single-rank test (paired two groups) or Mann-Whitney U-test (unpaired two groups). Probability values of less than 0.05 were considered significant.  Figure 2 Iniucbton by IL-15 of cytolne producion by peripheral blood MNCs from lung cancer paents. Per al blood MNCs (1 x l05 per well) from iung cancer patients were incubated in meun with or without 50 ng ml-of IL-15 or 500 U ml-' of IL-2 in the presence or absence of an optimal (100 U ml-1) concntration of IL-12. After incubation for 4 days, Fe culture supematants were collected after brief centrifugations. IL-10, IL-5 and GM-CSF were measured by ElA Ckolm and bars show means ± s.e.s. Asterisks indicate signiicant differences from values in IL-1i5 (P< 0.01)

RESULTS
Effect of IL-15 on killer induction of peripheral blood MNCs and pleural MNCs from lung cancer ants We first examined the effects of 1L-2 and 11-15 on non-MHCrestricted killer induction in peripheral blood MNCs from lung cancer patients. Blood MNCs were separated from venous blood of lung cancer patients (n = 34) and control subjects (n = 20) and incubated with or without the optimal dose of 11-15 (50 ng ml-') or 1L-2 (500 U ml-') for 4 days. Then their killer activities, against a lung cancer cell line (SBC-3). were measured at an effector to target (EJM) ratio of 10. The results are shown in Table 1. MNCs (I x l0I per well), cultured in medium alone, exhibited only marginal cytotoxicity against SBC-3 cells. On the other hand, U1-15 was as effective as 11-2 at inducing killer activity on MNCs from lung cancer patients, as well as from control subjects. Next. to examine the influence of tumour progression on killer induction, the 11-15-induced killer activities of blood MNCs were evaluated in lung cancer patients without clinical distant metastases (stage I-flB) and with clinical distant metastases (stage IV). The 1L-15-induced cytotoxicity against SBC-3 cells was a little lower in cells from patients with distant metastasis (n = 17) than in those without distant metastasis (n = 17), but the difference was not statistically significant. There was no difference between the 1L-15and 1L-2-induced killer activities against SBC-3 cells of cells from stage I-MB lung cancer patients or stage IV lung cancer patients.
To investigate the effect of 1L-15 on killer induction in the tumour growing site, we examined the effect of IL-15 on killer induction by MNCs in the malignant pleural effusion from the tumour growth site. Pleural and blood MNCs were obtained simultaneously from the same patients (n = 9) and their cytotoxicities against SBC-3 cells were examined after incubation with or without the optimal dose of 1L-15 or IL-2 for 4 days. The results are shown in Table 1. Pleural and blood MNCs showed low cytotoxicity when cultured in medium alone. There was no difference in the killer activities induced by the optimal concentration of IL-15 or IL-2 of MNCs from malignant pleural effusions or from peripheral blood.
Effects of ombinaons of lL-15 orlL-2 with IL-12 on killer induction of peripheral blood MNCs of lung cancer paPtdits We examined the effects of combinations of suboptimal (5 ng ml-') and optimal (50 ng ml-') concentrations of 1L-15 on non-MHC-restricted killer induction by IL-12 in cells from lung cancer patients (n = 16). L-12 had an additive effect with suboptimal concentrations of 1L-15 and 1L-2 on induction of killer activity against SBC-3 cells ( Figure IA). In contrast, 1L-12 had no additive effect with optimal concentrations of IL-15 or 1L-2 on induction of killer activity against SBC-3 cells ( Figure IB IL-15 induction of cytokine production by peripheral blood MNCs from lung cancer patients IL-1O production was examined in 14 lung cancer patients and eight control subjects. In control subjects, the presence of 1L-12 was necessary to induce IL-10 production by IL-2-stimulated MNCs (48.5 ± 22.4 pg ml-') and IL-15-stimulated MNC ( 19.6 ± 6.4 pg ml-'). In lung cancer patients. 1L-10 production was greater when cells were incubated with 1L-2 than with 1-15 alone or in combination with 11-12 (Figure 2).
1L-5 production was examined in 11 lung cancer patients and ten control subjects. 11-15. as well as 11L-2. alone or in combination with IL-12, induced no IL-5 production in the culture supernatant of MNCs from control subjects (data not shown). As shown in Figure 2, IL-2. alone or in combination with IL-12. induced significant production of 1L-5 by MNCs from lung cancer patients. Conversely, no production was observed in culture supernatants of MNCs from lung cancer patients incubated with 1L-15 alone or in combination with IL-12 ( Figure 2). GM-CSF production was examined in 23 lung cancer patients and nine control subjects. In control subjects. there was no difference in the production of GM-CSF by 1L-2-stimulated MNCs (30.3 ± 20.3 pg ml-') and IL-15-stimulated MNCs (not detectable). In lung cancer patients. there was also no difference in the production of GM-CSF by IL-2-stimulated MNCs and 1L-15-stimulated MNCs (Figure 2).

DISCUSSION
In this study. we showed that 1L-15 is a cytokine with potential effectiveness in cancer immunotherapy based on the following findings. First, IL-15 was as effective as 1L-2 in inducing non-MHC-restricted cytotoxic activity of blood MNCs and pleural MNCs of lung cancer patients. even at advanced stages. Second. compared with IL-2. IL-15 was less effective in inducing production of type 2 cytokines such as IL-IO and 1L-5.
It is important to examine whether the presence of malignant neoplasm affects the killer induction by 11-15. Gamero et al ( 1995) reported that lymphocytes of metastatic melanoma patients express killer activity in response to 11-15. Exploring this possibility in lung cancer patients. we have demonstrated in this study that peripheral blood MNCs from lung cancer patients generated killer activity against human lung cancer cells (SBC-3) in response to 1L-15. as well as from control subjects ( Table 1). and that IL-15. like IL-2. induced killer activity in MNCs of lung cancer patients even with distant metastases (Table 1). Moreover, we found that MNCs from malignant pleural effusions. where host cells exist in contact with cancer cells. generated killer activity against human lung cancer cells (SBC-3) in response to IL-15 (Table 1). In addition, similar to our earlier report with MNCs from normal volunteers (Takeuchi et al. 1996). suboptimal concentrations of IL-15 as well as IL-2 had additive effects on IL-12-induced killer activity of MNCs from lung cancer patients against SBC-3 cells (Figure 1). IL-15 seems to be as effective as IL-2 in inducing killer activity in lung cancer patients. and combinations of lower doses of the cytokines IL-15 and 11-12 may reduce their individual adverse effects at high concentrations.
As the growth of cancer cells in situ is regulated by the cytokine network via autocrine and paracrine pathways. it is important to examine whether the exogenous cytokine affects the cytokine network in cancer patients in addition to the analysis of its ability to induce killer cell activity. In the analysis of the cytokine network, two distinct cytokine patterns generated by T lymphocytes can be considered (Mosmann et al. 1986: Romagnani et al. 1991: Salgame et al. 1991. Type 2 lymphocytes produce 11-4. IL-5 and 1L-10 and suppress the cellular immune response. whereas type 1 lymphocytes produce IL-2 and IFN-y and promote the cellular immune response (Paul and Seder. 1994). Recently. it has been established that type 2 cytokine expression is predominant at the tumour site, including lung cancer (Yamamura et al. 1993: Smith et al. 1994: Hung et al. 1995. in the tumour-infiltrating lymphocytes (Kharkevitch et al, 1994) and peripheral blood of cancer patients (Pellegrini et al. 1996). Therefore, we examined type 2 cytokine production by 1L-15-activated MNCs from lung cancer patients in this study.
Although the clinical relevance of type 2 cytokines to tumour progression is not fully elucidated in human cancer. 1L-10 is considered to be an immunosuppressive factor because of its inhibitory effect on antigen-presenting capacity (de Waal-Malefyt et al. 1991) and cytokine production (Fiorentino et al. 1991). 11-10 inhibits IFNy and TNF-a production by lymphokine-activated killer (LAK) cells (Spagnoli et al. 1993). Production of IL-10 by lung cancer cells has been reported (Smith et al. 1994;Hung et al. 1995). Moreover. Hung et al (1996) have reported that prostaglandin E, and other soluble mediators produced by lung cancer cells induce IL-10 production by blood lymphocytes and thus inhibit cell-mediated anti-tumour immune responses. 11-15. alone or in combination with IL-12, was less effective in inducing IL-10 production by MNCs from lung cancer patients compared with 1L-2 ( Figure 2) and. thus. lesser inclination to type 2 dominance in the presence of IL-15 may have a therapeutic benefit in cancer immunotherapy.
1L-5 is produced by 11-2-activated MNCs from cancer patients in vivo and in vitro (Enokihara et al. 1989;Nakamura et al. 1990: Schaafsman et al, 1991, and may cause marked eosinophilia and extravascular eosinophil degradation (van-Haelst-Pisani et al. 1991). In line with these previous reports. we observed that MNCs from lung cancer patients cultured with IL-2 alone or in combination with 1L-12 produce significant amount of 11-5 in vitro ( Figure  2). In contrast, IL-15 alone, or in combination with IL-12, induced no 11L-5 production by MNCs from lung cancer patients as well as control subjects. On the other hand. IL-15-activated and IL-2-activated MNCs from lung cancer patients showed no difference in the production of GM-CSF ( Figure 2). another possible mediator of systemic eosinophilia (Donahue et al, 1986;Schaafsman et al. 1991). These findings suggest that IL-15 therapy may cause less eosinophilia and fewer side-effects than 11L-2 therapy. The mechanism of this difference between the functions of 11L-2 and IL-15 is unknown at present, but the difference in the distribution and role of IL-1SR a-chain (Giri et al. 1995) and IL-2R a-chain may be one plausible mechanism.
In summary. we found that 1L-15 and IL-2 induced similar killer activity against SBC-3 cells: however, compared with 1L-2.  induced production of type 2 cytokines to a much lesser extent. Further studies. such as analysis of the distribution and role of the IL-lSRa-chain in comparison with those of the IL-2Ra-chain in lung cancer patients. are necessary to clarify the potential role of IL-15 in cancer immunotherapy in humans.
of Japan. The authors thank Mr Y Ohmoto and Miss K Murata for valuable comments on cytokine measurement They also thank the medical staff of this department for help and encouragement.