The Combined Action of IL-15 and IL-12 Gene Transfer Can Induce Tumor Cell Rejection Without T and NK Cell Involvement

Emma Di Carlo, Alberto Comes, Stefania Basso, Alessandro De Ambrosis, Raffaella Meazza, Piero Musiani, Karin Moelling, Adriana Albini and Silvano Ferrini
J Immunol September 15, 2000, 165 (6) 3111-3118; DOI: https://doi.org/10.4049/jimmunol.165.6.3111

Abstract

The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1β, TNF-α, and IFN-γ, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model.

The importance of MHC class I-restricted CTLs as effectors of antitumor immunity has been widely demonstrated, and several CTL-defined tumor-associated Ags, representing potential targets for a tumor-specific immunotherapy, have been identified (1, 2, 3). However, a major tumor escape mechanism that hampers the development of CTL-based immunotherapy is the lack or the loss of MHC class I expression in a significant fraction of human tumors (4, 5, 6). The loss of individual alleles can be the consequence of an in vivo selective pressure by class I-restricted CTLs (7). MHC class I defects may depend on genomic deletion or mutation of MHC or MHC-related genes in some tumor cell types (4, 6). In other tumors, a down-regulation of MHC class I molecules has been related to down-regulation of MHC class I heavy chains (8), β2-microglobulin (9), or TAP expression (10, 11). In tumors lacking MHC class I, antitumor functions may be exerted by effectors of natural immunity that can be regarded as potential tools for antitumor immunotherapy strategies (12).

Among natural immunity effectors, NK cells are known to recognize and lyse cells that lack expression of MHC class I (13, 14). In fact, the expression of MHC class I on normal cells confers protection from NK cell lysis, through NK-inhibitory signals mediated by killer-inhibitory receptors for MHC class I (15, 16). Thus, down-regulation of MHC class I expression in tumor cells results in enhanced NK susceptibility.

IL-15 is a four α-helix bundle cytokine displaying IL-2-like immunostimulatory functions (17, 18), supporting the proliferation and differentiation of T, B, and NK cells. In addition, IL-15 has been reported to control differentiation of NK cells from bone marrow precursors (19), to stimulate NK antitumor cytolytic functions (20, 21), and to act as a chemotactic stimulus for NK cells (22). The critical role of IL-15 in NK cell development and function was also evidenced in IFN-regulatory factor 1 (IRF-1) knockout mice, which lack IL-15 expression and display an NK-deficient phenotype (23). In view of these properties, IL-15 has been regarded as a suitable candidate for cancer immunotherapy (24) or gene therapy strategies (25, 26).

In a previous study, we have shown that a human MHC class I-negative tumor, engineered to secrete IL-15, displayed a reduced growth and take rate when xenotransplanted in nude mice, although tumorigenicity was not completely abrogated (27). High number of infiltrating NK cells were found at the tumor site, a finding that has been rarely observed in cytokine-transduced tumors (28), and the IL-15-mediated effects were abrogated by NK cell depletion. We speculated that the combined gene transfer of IL-15 with other NK-stimulating factor(s) could synergistically enhance the antitumor effects of NK cells recruited by IL-15 at the tumor site.

IL-12 is a heterodimeric cytokine, secreted by monocytes, macrophages, and dendritic cells, which is able to activate both T and NK cell functions. Thus, IL-12 is a potent inducer of Th1 responses and induces NK cell proliferation, cytotoxic activity, and IFN-γ production (29). Several studies have demonstrated a potent antitumor activity of IL-12 either as a recombinant cytokine or in gene transfer approaches in different syngeneic mice models (29, 30, 31). Another report showed that the combination of IL-12 and IL-15 synergistically potentiated cytokine production by NK cells in vitro (32). In addition, a combination of suboptimal doses of the two recombinant cytokines induced antitumor effects in a B16 melanoma model in syngeneic mice (33).

In this study, we have analyzed the possible cooperative antitumor effects of IL-12 and IL-15 gene transfer in a human small cell lung cancer cell line, N592, chosen as a prototype of MHC class I-negative tumor. The effects on natural immunity were studied by xenotransplantation in nude mice. Interestingly, engineering of tumor cells with both IL-15 and IL-12 resulted in a complete tumor rejection also in NK-depleted or in granulocyte-depleted nude mice, suggesting a predominant role of macrophages in this model.

Materials and Methods

Cell lines and cultures

N592 small cell lung cancer cell line was kindly provided by Dr. J. Minna, National Cancer Institute (Washington, D.C.). Cells were cultured in endotoxin-free RPMI 1640 medium (endotoxin content <0.005 EU/ml) supplemented with l-glutamine and antibiotics (all from BioWhittaker, Bergamo, Italy) and 10% FCS (endotoxin content 20 EU/ml; Seromed Biochrom, Berlin, Germany).

Plasmid vector assembly and N592 transfection

The pVkL/IL-15IRESneo plasmid vector was obtained as previously described (27, 34). The bicistronic mIL-12 cDNA, encoding for p40 and p35 murine IL-12 chains, was digested from pIRES-muIL-12 (35) with BamHI and was subcloned in pIRES1neo or pIRES1hygro plasmid vectors (Clontech Laboratories, Palo Alto, CA). The orientation of the insert was checked by further digestion with BstXI restriction enzyme.

N592 cells were transfected with 10 μg of pVkL/IL-15IRES1neo or pmuIL-12IRES1hygro plasmids or both using cationic liposomes (DOTAP; Boehringer/Roche, Milano, Italy).

Stable transfectants and clones were obtained by limiting dilution, in medium containing either G418 (500 μg/ml) or hygromicin (250 μg/ml) or both, and were then tested for IL-15 and IL-12 production.

IL-15, IL-12, GM-CSF, and IFN-γ ELISA or bioassays

As indicator cell system for determination of IL-2/IL-15 activity, we used the CTLL mouse cell line, known to proliferate in response to human IL-2 or IL-15. Cytokine activity was assessed by [3H]thymidine uptake by CTLL after 6-h pulse with 0.5 μCi at the end of 24-h period of incubation with supernatants of cell lines or of transfectants. Serial dilutions of human rIL-2 or human rIL-15 (Genzyme, Cambridge, MA) containing a known amount of IU were used as standard.

IL-15 or IL-12 ELISA was performed using a commercial available kit according to instructions provided by the manufacturer (Genzyme).

The production of murine IFN-γ and GM-CSF by nude mice splenocytes cocultured with N592pc or transfectants was analyzed by an ELISA kit (Genzyme) on 7-day supernatants.

Nude mice studies

Pathogen-free female athymic (nu/nu, CD1) mice, 6–8 wk old, were obtained from Harlan Nossan (Milano, Italy). Mice were housed under pathogen-free conditions and received autoclaved food and water.

Animals (six mice for each group) were injected s.c. with 2 × 107 N592pc, N592/neo/hygro, N592/IL-12, N592/IL-15, or N592/IL-12/IL-15 tumor cells. Cells were mycoplasma free, as assessed by ELISA (Boehringer/Roche) or 4′,6′-diamidino-2-phenylindole staining before injection. Cells were washed three times in endotoxin-free RPMI medium without FCS and one time in endotoxin-free PBS before injection. The larger and smaller diameters of the s.c. tumors were measured using a caliper at weekly intervals; these two diameters were multiplied to obtain an estimate of the tumor area. The data are displayed as mean ± SD of the areas for each group of animals at a given time point. Statistical analysis was performed using the Mann-Whitney test; p < 0.05 values were considered as significant.

NK or granulocyte depletion was performed by i.p. injection of rabbit anti-asialo GM1 antiserum (Wako Chemicals GmbH, Dussendorf, Germany) (0.2 ml of 1/20 diluted stock solution) or anti-granulocyte rat mAb (0.2 ml of a 1/50 dilution of ascites of RB6-8C5 hybridoma; kindly provided by Dr. R. L. Coffman, DNAX, Palo Alto, CA). Mice were injected at days −2, 0, +5, +10, and +17 from tumor challenge. Efficiency of depletion (>80% in case of granulocytes and >95% in case of NK cells) was confirmed by immunohistochemical analyses of the infiltrate of xenotransplanted N592/IL-12/IL-15 tumors. Control animals received normal rabbit serum or an irrelevant rat mAb.

Morphologic and immunohistochemical analysis of xenografts

Groups of three mice were euthanized 5 and 7 days after challenge. For histologic evaluation, tissues were fixed in 10% neutral buffered Formalin, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin-eosin or Giemsa.

For immunohistochemistry, acetone-fixed cryostat sections were incubated for 30 min with anti-asialo GM1 (NK cells) (Wako Chemicals GmbH); anti-Mac-1 (anti-CD11b/CD18), anti-Mac-3, and anti-Ia (all from Boehringer Mannheim); anti-polymorphonuclear leukocytes (RB6-8C5 hybridoma; provided by Dr. R. L. Coffman, (DNAX)); anti-endothelial cells (mEC-13.324) and anti-endothelial leukocyte adhesion molecule 1 (anti-ELAM-1) (provided by Dr. A. Vecchi, Negri Nord, Milano, Italy); anti-IL-1β and anti-GM-CSF (Genzyme); anti-TNF-α (Immuno Kontact, Frankfurt, Germany); anti-IFN-γ (provided by Dr. S. Landolfo, University of Turin, Turin, Italy); anti-monocyte chemoattractant protein-1 (anti-MCP-1)3 and anti-VCAM-1 (PharMingen); anti-macrophage-inflammatory protein-2 (anti-MIP-2) (Serotec, Oxford, U.K.); anti-ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-inducible NO synthase (anti-iNOS) (Transduction Laboratories, Lexington, KY) Abs. After washing, they were overlaid with biotinylated goat anti-rat, anti-hamster, and anti-rabbit, and horse anti-goat Ig (Vector Laboratories, Burlingame, CA) for 30 min. Unbound Ig was removed by washing, and the slides were incubated with ABC (avidin biotin complex) alkaline phosphatase (Dako, Glostrup, Denmark). Quantitative studies of immunohistochemically stained sections were performed independently by three pathologists in a blind fashion. For cell counts, individual cells were counted under a microscope ×400 field (×40 objective and ×10 ocular lens; 0.180 mm2 per field). Ten randomly chosen fields were counted in each sample. The expression of cytokines and iNOS was defined as absent (−), scarcely (±), moderately (+), frequently (++), and strongly (+++) present on cryostat sections tested with the corresponding Abs.

Isolation of granulocyte and macrophage populations, production of NO, and cytotoxicity

To isolate granulocytes, nude mice were injected i.p. with 1 ml of 9% sodium casein (Sigma-Aldrich, Milano, Italy) in endotoxin-free PBS, followed by a second injection 16 h later. Three hours later, peritoneal cells were recovered in 5 ml of DMEM containing 0.5 mM EDTA. To isolate macrophages, nude mice were injected i.p. with 1 ml of 2.9% aged thioglycolate (Difco, Detroit, MI) solution. After 2 days, peritoneal cells were harvested as above. Cell populations were fractioned on self-forming 90% Percoll (Pharmacia Biotech, Uppsala, Sweden) gradients by centrifugation at 60,000 × g for 20 min at 4°C, which produced a two-layer fractionation pattern. Macrophages were harvested from the upper layer of thioglycolate-induced peritoneal cell populations, while granulocytes were collected from the lower layer of casein-induced cells. Purity of each cell population was >90%, as judged by microscope examination of stained cytospin preparations.

Macrophages or granulocytes were cocultured with different N592 transfectants (2 × 104 in 1 ml of medium) at 10:1 or 20:1 ratios in 24-well plates. NO production in the supernatant was measured after 2 or 5 days by a colorimetric assay using the Griess reagent (Sigma-Aldrich) (36). Cytotoxicity against N592 transfectants, which grow in suspension, was evaluated by gently resuspending N592 and transferring them in triplicate wells of a 96-well flat-bottom plate. The amount of viable cells was evaluated by a standard MTT (Sigma-Aldrich) assay (37).

Results

Characterization of N592 transfectants secreting IL-12, IL-15, or both cytokines

N592 cells were transfected with pVkL/IL-15-IRESneo and/or pIL-12-IREShygro, either alone or in combination, and cloned after appropriate drug selection. The modified IL-15 cDNA VkL/IL-15 encodes for an IL-15 preprotein bearing the Igκ L chain signal peptide that allows enhanced secretion of biologically active IL-15, upon transfection, as compared with unmodified IL-15 cDNAs (34). The N592 clones listed in Table I⇓ were selected for further studies because they displayed similar growth kinetics in vitro and on the basis of their cytokine secretion pattern. Thus, the N592/IL-12/IL-15 clone secreted amounts of IL-15 or IL-12 similar to that produced by the clones expressing only IL-15 or IL-12. IL-15 secreted by transfectants was biologically active in sustaining both IL-2/IL-15-sensitive CTL-L proliferation and in boosting NK cytolytic activity (27).

View this table:
Table I.

Production of IL-12 and/or IL-15 by N592 cell transfectantsa

Coexpression of IL-12 and IL-15 synergistically induces antitumor effects in nude mice

The effect of cytokine engineering on tumorigenicity was evaluated by heterotopic (s.c.) implant in nude mice possessing a functional natural immunity. As shown in Fig. 1⇓A, mock-transfected N592 (N592/neo/hygro) showed a very rapid growth kinetics in 100% of injected animals, which was similar to that of unmodified N592. N592/IL-12 cells displayed only minor changes of the growth pattern, while N592/IL-15 showed a clearly reduced tumor growth rate and a slight reduction in tumorigenicity (80% tumor take). However, N592/IL-12/IL-15 produced only a transient tumor growth, followed by complete rejection in all animals tested. Consistent results were obtained in three unrelated experiments.

FIGURE 1.
FIGURE 1.

Effect of IL-15 and/or IL-12 gene transfer on the growth of the human N592 cell line implanted s.c. in nude mice. Data are expressed as average tumor size (M ± SD) in the different groups of animals, as detailed in Materials and Methods. The number of animals injected vs take rate are indicated for each experimental group. A, The tumor growth kinetics of N592/IL-12 is similar to that of N592 mock, which were transfected with empty vectors (p = NS), while N592/IL-15 display a significantly lower growth rate (p < 0.001) and N592/1L-12/IL-15 were completely rejected in 100% of animals (p < 0.001). B, N592/IL-12/IL-15 display a local bystander effect when coinjected at the same site with N592pc (p < 0.01), while weak effects (p = NS) were observed on the growth of N592pc cells injected contralaterally. The growth of N592pc alone and of N592/IL-12/IL-15, contralateral to N592pc, is shown for comparison. A total of 2 × 107 cells of either cell type was injected in each animal; thus, a total of 4 × 107 cells of N592pc + N592/IL-12/IL-15 was coinjected for the study of local bystander effects.

Next we investigated possible inhibitory effects of N592/IL-12/IL-15 on the growth of wild-type tumor cells. Coinjection of N592/IL-12/IL-15 with a tumorigenic dose of N592pc at the same site induced only a transient tumor growth, followed by complete rejection in 100% of the animals, while injection of N592pc contralaterally to N592/IL-12/IL-15 produced tumor growth with a slightly reduced kinetics (Fig. 1⇑B). Thus, a very potent local bystander effect on N592pc was produced by s.c. injection of N592/IL-12/IL-15. The weak systemic bystander effect, observed in animals injected with N592pc contralaterally to N592/IL-12/IL-15, may be related to the low levels of circulating IL-15 (15 pg/ml at day +3, undetectable at day +7). In contrast, consistent levels of IL-12 (1600 pg/ml) were found at day +3, and it was still detectable at day +7.

Effect of NK cell and granulocyte depletion on N592 transfectant tumor growth

To gain further information on the role of NK cells, N592/IL-15 and N592/IL-12/IL-15 transfectants were injected in nude mice that had been treated with anti-asialo GM1 antiserum. N592/IL-15 and N592/IL-12 showed growth kinetics similar to that of N592pc in NK-depleted nude mice, while N592/IL-12/IL-15 were still completely rejected (Fig. 2⇓). Treatment with nonimmune rabbit control Ig had virtually no effect (Fig. 2⇓B). These findings indicate that NK cells are mainly responsible for the antitumor effect of IL-15, while these cells are not necessary for the IL-15/IL-12 cooperative effect. In addition, N592/IL-12/IL-15 were also rejected in granulocyte-depleted nude mice, although a delay in the rejection process was observed in these animals (Fig. 2⇓C). Similar results were observed in nude mice depleted of both NK cells and granulocytes (data not shown), thus suggesting that macrophages play a predominant role.

FIGURE 2.
FIGURE 2.

Growth of N592pc, N592/IL-15, N592/IL-12, and N592/IL-12/IL-15 in NK (anti-asialoGM-1)-depleted nude mice (A), or in nude mice treated with irrelevant rabbit Ig (B). In C, N592/IL-12/IL-15 were injected either in granulocyte (RB6.8C5 mAb)-depleted animals or in a control group injected with an irrelevant rat mAb. A total of 2 × 107 cells of either cell type was injected in each animal.

IL-12 and IL-15 synergistically stimulate IFN-γ and GM-CSF secretion by murine splenocytes in vitro

We further analyzed whether IL-15 and IL-12 produced by transfectants could synergize in inducing cytokine production by mouse splenocytes in a coculture system. As shown in Fig. 3⇓A, after 3 days of coculture with N592/IL-12/IL-15, splenocytes released 1500 pg/ml of IFN-γ in the supernatant, vs average levels of 10 and 110 pg/ml measured in N592/IL-15 and N592/IL-12 coculture supernatants, respectively. Similar results were obtained for GM-CSF (Fig. 3⇓B). Thus, 15 and 50 pg/ml of GM-CSF were found in cocultures with N592/IL-15 and N592/IL-12, respectively, while 100 pg/ml was secreted in N592/IL-12/IL-15 cocultures. This cooperative effect of IL-15 and IL-12 was largely dependent on NK cell stimulation because splenocytes from anti-asialo GM1-depleted animals produced lower levels of IFN-γ or GM-CSF (Fig. 3⇓, A and B), as compared with splenocytes from untreated animals or from animals treated with control rabbit Ig (not shown).

FIGURE 3.
FIGURE 3.

IFN-γ (A) and GM-CSF (B) secretion in cocultures of splenocytes from intact and from NK-depleted nude mice with N592pc or with different transfectants.

Histological and immunohistochemical analysis of the rejection process

To gain further information on the mechanisms underlying the cooperative effects of transfected IL-15 and IL-12 in vivo, we performed histological and immunohistochemical analysis of the N592pc, N592/IL-12, N592/IL-15, and N592/IL-12/IL-15 tumor area in intact or NK-depleted nude mice.

When injected in nude mice, either N592/pc or N592/neo/hygro cells gave rise to a richly vascularized tumor arranged in small alveolar structures with very few infiltrating macrophages (Fig. 4⇓a). A similar growth pattern was observed in N592/IL-12 tumor growth area in which, however, microvessels were less numerous and a few necrotic areas were present (Fig. 4⇓d). The periphery of the tumor mass was moderately infiltrated by reactive cells producing proinflammatory cytokines and expressing iNOS (Table II⇓). Production of granulocyte chemotactic mediators, such as GM-CSF and particularly MIP-2, was detectable in tumor-infiltrating macrophages.

FIGURE 4.
FIGURE 4.

Histologic (a, d, g, j) and immunohistochemical (b, c, e, f, h, i, k, l) features of 7-day N592pc, N592/IL-12, and N592/IL-15 tumors, and 5-day N592/IL-12/IL-15 tumor in nude mice. N592pc cells gave rise to a well-vascularized tumor arranged in small alveolar structures (a) with no infiltrating NK cells (b) and few granulocytes (c). N592/IL-12 tumor showed a few necrotic areas (d) and a moderate peripheral infiltrate of NK cells (e) and PMNs (f). N592/IL-15 tumor showed large necrotic areas (g) with numerous NK cells (h) and several granulocytes (i). N592/IL-12/IL-15 tumor rejection area consisted in a large area of ischemic necrosis bordered by the few and injured tumor cell aggregates (j) richly infiltrated by granulocytes (k) and NK cells (l).

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Table II.

Immunohistochemical analysis of tumor growth and rejection area in nu/nu mice 7 days after s.c. injection of N592 pc and N592/IL-15 or N592/IL-12 and 5 days after s.c. injection of N592/IL-12/IL-15 cells

N592/IL-15 tumor showed large necrotic areas (Fig. 4⇑g), numerous but severely damaged microvessels, and several infiltrating reactive cells, particularly macrophages and NK cells (Fig. 4⇑h). A weak expression of GM-CSF and a strong endothelial and macrophage expression of MCP-1 were detected, while proinflammatory cytokines were almost absent (Table II⇑).

N592/IL-12/IL-15 tumor rejection area mainly consisted in a wide area of ischemic necrosis with colliquative foci and small groups of ghosts of tumor cells interspersed within numerous macrophages, granulocytes, and NK cells (Fig. 4⇑, j–l, and Table II⇑). Vascularization was scarce to absent with diffusely damaged microvessel sprouts. Production of proinflammatory cytokines was quite similar to that observed in N592/IL-12 tumor growth area, while production of GM-CSF, MIP-2, and MCP-1 was stronger.

In NK cell-depleted nude mice, N592/IL-12/IL-15 tumor rejection area was markedly infiltrated by macrophages and granulocytes with a moderate production of IL-1β, TNF-α, and MCP-1, and a strong and widely distributed expression of iNOS and MIP-2 (Fig. 5⇓, a–e, and Table II⇑). Anti-endothelial cell (anti-CD31) staining was scarce to absent with a dusty appearance, indicating the heavily injured endothelial cells (Fig. 5⇓f).

FIGURE 5.
FIGURE 5.

Histologic (a) and immunohistochemical (b–f) features of N592/IL-12/IL-15 tumor rejection area, 5 days after s.c. tumor cell injection in NK cell-depleted nude mice. An extensive necrosis of colliquative type characterized the area of tumor cell rejection (a) in which numerous macrophages (b) and granulocytes (c) were present. Infiltrating reactive cells strongly produced MIP-2 (d) and NO (e). Immunostaining with anti-endothelial cell (CD31) Ab (f) revealed a dusty appearance of positive cells, indicating the scattered heavily injured endothelial cells.

NO production and cytotoxicity of granulocyte- or macrophage-enriched populations

In the attempt to identify the cells responsible for NO production, we first isolated thioglycolate-induced macrophages or casein-elicited granulocyte fractions by density gradients and tested their ability to produce NO in coculture with N592/IL-12/IL-15. As shown in Fig. 6⇓A, only macrophage-enriched populations released significant amounts of NO in response to coculture with N592/IL-12/IL-15, while granulocyte-enriched fractions did not (Fig. 6⇓A). When macrophages and granulocytes were cocultured together with N592/IL-12/IL-15, a slight cooperative effect in NO production was observed.

FIGURE 6.
FIGURE 6.

A, Production of NO by macrophages, granulocytes, or macrophages + granulocytes after 2-day coculture with N592/IL-12/IL-15 at a 10:1 (□) or 20:1 (▪) E:T cell ratio. B, Production of NO by macrophage populations incubated with N592pc or with different N592 transfectants at a 10:1 (□) or 20:1 (▪) E:T cell ratio. C, MTT cell-colorimetric assay performed on N592pc or on different N592 transfectants recovered as nonadherent cells after 5 days of coculture with macrophage populations at a 10:1 (□) or 20:1 (▪) E:T cell ratio. Data are expressed as percentage of cytotoxicity. D, Effect of the iNOS inhibitor l-NAME on NO production and cytotoxicity by macrophages cocultured with N592/IL-12/IL-15. Data are represented as percentage of inhibition with respect to control cultures without l-NAME.

Although N592/IL-12 also induced some NO production by macrophages, induction by N592/IL-12/IL-15 had a clearly stronger effect (Fig. 6⇑B). In addition, macrophage-enriched populations markedly inhibited N592/IL-12/IL-15 growth in a 5-day MTT assay, while N592pc were not affected, and N592/IL-12 and N592/IL-15 were only partially inhibited (Fig. 6⇑C). The finding that N592/IL-15 growth was partially inhibited by macrophages, in the absence of NO production, suggested that both NO-dependent and NO-independent mechanisms of tumor cell growth inhibition were operative. This possibility was also suggested by the use of the iNOS inhibitor l-NAME, (Nω-nitro-l-arginine methyl ester) which potently inhibited NO production by macrophages at 5 mM, but only partially blocked cytotoxicity (Fig. 6⇑D) against N592/IL-12/IL-15.

Discussion

In this study, we show that the combination of IL-12 and IL-15 gene transfer in a human MHC class I-negative tumor synergistically triggers tumor rejection by natural immunity in nude mice. Although NK cells accumulate at the site of rejection of tumor cells engineered to secrete both cytokines, tumor rejection occurs also in animals that had been depleted of NK cells and of granulocytes, through the involvement of macrophages.

Although IL-12 has been proven to exert antitumor effects in a number of experimentally induced and spontaneous tumors in immunocompetent hosts (29, 30, 31), it had virtually no efficacy in our tumor gene transfer model in nude mice. In the absence of T lymphocytes, N592/IL-12 tumor growth kinetic was not affected despite an evident aspecific reactive cell infiltration and proinflammatory cytokine production at the tumor site. Furthermore, small sprouting vessels, mainly found at the periphery of tumor mass, may support tumoral cell survival, making easier tumor escape from any attempt of host natural immune response. Altogether, these observations suggest the requirement of T cells in leading to an efficient IL-12-induced antitumor reaction. Furthermore, a pivotal role of CD8+ cells has been found in the rejection of IL-12 gene-transfected cells injected in syngeneic mice (31). The involvement of CD8+ cells was frequently associated with an indirect angiogenesis inhibition by secondary cytokines (mainly IFN-γ) and third-level chemokines (inflammatory protein-10 and monokine induced by IFN-γ (MIG)) released by CD8+ themselves or induced in other leukocyte subsets (29). Hence, from here, it may be hypothesized that the failure of IL-12 immunotherapy in several clinical trials could be ascribed, besides to the advanced stage of cancer patients (bearing established and widespread tumors), to an inadequate T cell responsiveness against weakly immunogenic and well-vascularized tumors.

Although two isoforms of IL-15 mRNA are constitutively expressed in human tumors (34, 38), spontaneous secretion of IL-15 by tumor cells was not observed due to the existence of multiple posttranscriptional levels of IL-15 production control (18, 39). We previously reported that the use of a modified human IL-15 cDNA for gene transfer allowed secretion of IL-15 that was found to be biologically active on both human and murine NK and T cells (34). Injection of IL-15-engineered N592 tumor cells in nude mice resulted in an NK cell-dependent inhibition of tumor growth. In the light of the synergistic or additive effects exerted by low doses of IL-12 and IL-15 on NK and lymphokine-activated killer cytotoxicities (40), with the reciprocal up-regulation of receptor expression on mononuclear cells (33, 41), one may expect an NK cell-mediated rejection of N592 cells releasing both cytokines. In addition, it has been reported that IL-15 synergistically potentiates IL-12-induced NK cell production of several cytokines such as IFN-γ, IL-10, MIP-1α, and MIP-1β (32). Moreover, a cooperative antitumor effect, induced by subtherapeutic doses of recombinant IL-12 + IL-15 in a B16F10 melanoma model in syngeneic mice, correlated with the level of IFN-γ production (33). In agreement with these previous observations, nude mice splenocytes cocultured with N592 secreting both IL-15 and IL-12 released higher amounts of IFN-γ and GM-CSF than cocultures with N592 transfectants producing only IL-12 or IL-15. Conversely, cytokine production was clearly reduced in coculture of splenocytes isolated from NK-depleted animals, indicating that also in nude mice NK cells were mainly responsible for IL-12 + IL-15-induced IFN-γ and GM-CSF production. However, NK cell depletion of nude mice still resulted in a complete N592/IL-12/IL-15 tumor cell rejection in vivo, thus suggesting the involvement of other effector cells of natural immunity in IL-12 + IL-15-induced antitumor reaction.

Immunohistological analysis of N592/IL-12/IL-15 tumor area revealed a marked macrophage and granulocyte recruitment, associated with a strong iNOS activation and large colliquative necrotic areas. It has been reported that the cytostatic/cytotoxic effect of ex vivo macrophages from mice treated with IL-12 and/or IL-15 was dependent, at least in part, on NO production (33). Our data indicate that nude mice macrophage populations cocultured with N592/IL-12/IL-15 produced NO and inhibited the growth of these tumor cells in vitro, through both NO-dependent and NO-independent mechanisms.

In contrast, granulocytes, attracted at the tumor site by MIP-2 (murine equivalent of IL-8) (42), may also cooperate in NO production (42, 43, 44, 45), and in tumor destruction (46, 47, 48, 49, 50). Both granulocytes and macrophages could also be responsible for the blood vessel injuries observed in N592/IL-12/IL-15 tumor (29, 50). In addition, it should be outlined that human neutrophils can respond to IL-12 stimulation (51) and can also be activated by IL-15 to express IL-8 (52). Indeed, granulocytes appeared to play a cooperative role in our model because in granulocyte-depleted animals, the N592/IL-12/IL-15 rejection process was delayed.

In NK cell-depleted nude mice, it is likely that the cooperative biological effects of IL-12 and IL-15 mainly target macrophages and granulocytes, allowing the onset of a distinct natural immunity regulatory network. In this context, an increased iNOS and MIP-2 expression, detected at the tumor site, should be viewed as signs of an increased IL-12 and/or IL-15 direct macrophage and granulocyte activation (51, 52, 53, 54). Although NK-depleted nude mice showed a reduced IFN-γ production in response to IL-12 + IL-15 produced by transfectants, both in vitro and in vivo, it is likely that low amounts of macrophage-derived IFN-γ (53) may play a role, particularly in iNOS induction. Thus, low amounts of IFN-γ were found in cocultures of peritoneal macrophages with double-transfected cells (data not shown).

Different from previous studies in tumor cytokine immuno/gene therapy, which emphasize the requirement of a cross-talk between specific and nonspecific immune mechanisms to obtain a complete tumor rejection (55, 56), in this study we report evidence that the activation of macrophages by the synergistic action of IL-12 + IL-15 may lead to an effective T- and NK-independent tumor growth inhibition. These data provide new insight on the cooperative activity of IL-12 and IL-15 in the stimulation of effective natural immunity response and could be relevant for the development of gene therapy strategies against MHC class I-negative tumors escaping from specific CTL control.

Acknowledgments

We thank Dr. Douglas Noonan and Dr. Jan Schultz for their help in revising the manuscript.

Footnotes

  • ↵1 This work has been supported by grants awarded by the Italian Association for Cancer Research, Istituto Superiore di Sanità, Consiglio Nazionale delle Ricerche Target Project on Biotechnology, and Ministry of the University and Scientific and Technological Research. A.C. was supported by fellowships from Fondazione Italiana per la Ricerca sul Cancro and the Compagnia di S. Paolo.

  • ↵2 Address correspondence and reprint requests to Dr. Silvano Ferrini, Centro di Biotecnologie Avanzate, Largo Rosanna Benzi no. 10, 16132 Genova Italy. E-mail address: ferrini{at}ermes.cba.unige.it

  • ↵3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; iNOS, inducible NO synthase; l-NAME, Nω-nitro-l-arginine methyl ester; MIP, macrophage-inflammatory protein.

  • Received December 16, 1999.
  • Accepted June 21, 2000.

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The Journal of Immunology: 165 (6)
The Journal of Immunology
Vol. 165, Issue 6
15 Sep 2000
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