Tumour cells engineered to secrete interleukin-15 augment anti-tumour immune responses in vivo

We examined the effect of interleukin-15 (IL-15) gene transfer into tumour cells on the host's anti-tumour response. In BALB/c mice IL-15 producing Meth-A cells (Meth-A/IL-15) underwent complete rejection, in a response characterized by massive infiltration of CD4+ T-cells and neutrophils. In contrast, Meth-A cells transfected with vector alone (Meth-A/Neo) grew rapidly. Moreover, rechallenged parental cells also were rejected in association with CD8+ T-cell infiltration. However, in nude mice there was no drastic difference between Meth-A/IL-15 and Meth-A/Neo cells. These results demonstrate that IL-15-secreting tumour cells can stimulate local and systemic T-cell-dependent immunity and therefore may have a potential role in cancer therapy. © 1999 Cancer Research Campaign

Interleukin (IL)-15 is a novel cytokine that uses βand γ-chains of the IL-2 receptor (R) for signal transduction and shares biologic activities with IL-2 Grabstein et al, 1994).  has been reported to activate and stimulate the proliferation of T-and B-lymphocytes (Armitage et al, 1995;Mori et al, 1996). It also stimulates the proliferation of natural killer (NK) cells (Carson et al, 1996) and facilitates the induction of cytolytic effector cells including cytotoxic T-lymphocyte (CTL) and lymphokine-activated killer (LAK) cells (Gamero et al, 1995;Lewko et al, 1995;Munger et al, 1995). These properties suggest that IL-15, like IL-2, may be of value in cancer treatment. Unlike IL-2, however, IL-15 is produced by a wide variety of tissues and cells that include activated monocytes/macrophages, skeletal muscle, kidney, epithelial cells, activated endothelial cells and placenta Bamford et al, 1996). Moreover, the IL-15R employs a unique α-chain which has a higher affinity and broader tissue distribution than the IL-2R α-chain (Andeson et al, 1995;Giri et al, 1995). These differences in the expression pattern of IL-15 and its receptor compared to the IL-2/IL-2R system suggest unique in vivo roles for IL-15.
Transfection of various cytokine genes [IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF) and macrophage inflammatory protein (MIP)-1α (Colombo and Forni, 1994;Hirose et al, 1995;Lebel-Binay, et al, 1995;Zitvogel et al, 1995)] into tumour cells results in inhibition of tumour growth. This effect is mediated through the infiltration of T-lymphocytes, macrophages and/or polymorphonuclear leucocytes into the tumour sites. These studies have shown the primary effects of cytokine release as well as effects seen with the induction of secondary cytokines (Colombo and Forni, 1994). However, there are no studies that have examined transfection of the IL-15 gene into animal tumour cells, and its effects on the anti-tumour immune response in vivo.
In this study we transfected tumour cells with an IL-15 expression vector and examined the response to the IL-15-secreting tumour cells in both BALB/c and BALB/c nu/nu mice with defective T-lymphocyte-mediated immunity.

RT-PCR and primers
RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) were carried out as previously described with some modifications (Iizuka et al, 1995). Briefly, cells (5 × 10 6 ) were solubilized in 1 mL of TRIzol reagent (Life Technologies, Grand Island, NY, USA) and total cellular RNA was isolated according to the manufacturer's instructions. One microlitre of total RNA (1 µg) was added to 19 µl of RT-mixture (Takara, Ohtsu, Japan). After mixing, the samples were incubated at 30°C for 10 min, 55°C for 30 min, 95°C for 5 min and 4°C for at least 5 min. PCRmixture (80 µl) (Takara) containing 100 nM primers was added to the RT products. PCR cycling parameters were as follows for the isolation of human IL-15 (hIl-15) cDNA and for the selection of hIL-15 mRNA-expressing clones: denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min. PCR amplification of the IL-15 and β-actin genes utilized 40 and 28 cycles respectively.
The expected sizes of the PCR products were 347, 296 and 1046 bp for human-IL-15, murine IL-15 and β-actin respectively. The PCR products were subjected to 1% agarose gel electrophoresis and visualized by staining with ethidium bromide.

Human IL-15 cDNA expression vector
A human IL-15 cDNA was isolated by RT-PCR using total RNA purified from lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The PCR fragment was cloned into a HindIII/XbaI site of the pGEM-4Z plasmid (Promega, Madison, WI, USA), and the nucleotide sequence was confirmed by sequence analysis. The same fragment was then recloned into a HindIII/XbaI site of the pRc/CMV eukaryotic expression vector (Invitrogen, San Diego, CA, USA).

Tumour cells and transfection
The Meth-A cell line, a methylcholanthrene-induced fibrosarcoma of BALB/c origin (DeLeo et al, 1977), was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U ml -1 penicillin G and 100 g ml -1 streptomycin. Subconfluent cultures in 100-mm petri dishes were transfected with 5 µg of hIL-15 expression plasmid or vector alone using the lipofectAMINE reagent (Life Technologies) according to the manufacturer's instructions. G418 (200 µg ml -1 ) (Life Technologies) was added to the cells 48 h later. G418-resistant clones were isolated and expanded in culture medium containing 100 µg ml -1 of G418.

Measurement of IL-15 production by bioassay
Each transfectant as well as the parental Meth-A cells (1 × 10 6 cells ml -1 ) were cultured in serum-free RPMI-1640 without G418 for 24 h. The culture supernatants were collected by centrifugation for 5 min at 400 g and were concentrated using Centricon concentrators (10 000) (Grace, Danvers, MA, USA) to tenfold enrichment prior to bioassay. IL-15-dependent CTLL-2 cells (4000 cells per well) in 96-well flat microtitre plates were incubated with culture medium supplemented with 5 × 10 -5 M 2-mercaptoethanol in the presence of various concentrations of hIL-15 (Genzyme, Cambridge, MA, USA) or culture supernatant with or without anti-hIL-15 monoclonal antibody (mAb; M111, Genzyme) at various concentrations. After a 20 h incubation 50 µg of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) (Chemikon International, Temecula, CA, USA) were added to each well, and the reaction was allowed to incubate for an additional 4 h at 37°C. Isopropanol with 0.04 N hydrochloric acid (100 µl) was then added. Colour development at a wavelength of 540 nm was monitored by an enzyme-linked immunorsorbent assay (ELISA) reader (SLT Labinstruments, Austria). The sensitivity of this bioassay was 50 pg ml -1 .

Measurement of IL-15 production by ELISA
IL-15 levels were measured using ELISA (BioSource, Camarillo, CA, USA). In each assay a standard curve using recombinant cytokine was constructed and each sample assayed in duplicate. The sensitivity of the assay was 37 pg ml -1 .

Animal studies
Seven-week-old female BALB/c mice and male BALB/c nu/nu mice were purchased from Japan SCL (Hamamatsu, Japan). Injections of Meth-A cells were carried out with freshly prepared suspensions at a concentration of 2 × 10 7 cells ml -1 . The total number of tumour cells injected per animal was 2 × 10 6 . All injections were subcutaneous (s.c.) in the right lower abdominal quadrant via a 27-gauge needle. Tumour volumes were measured in mm 3 with a vernier caliper and determined according to the following formula: a × b 2 ÷ 2, where a is the larger and b the smaller of the two dimensions. All animal experiments were conducted in accordance with UKCCCR Guidelines on Animal Welfare (Workman et al, 1988).

Rechallenge with Meth-A or colon 26
Thirty days after disappearance of the initial Meth-A/IL-15 implantation, 20 mice were injected with 2 × 10 6 parental Meth-A cells in the previously uninjected side (left) of the lower abdominal quadrant. Colon-26 cells, a murine colon adenocarcinoma derived from BALB/c mice (Corbett et al, 1975), were also injected into both non-immunized and immunized mice (BALB/c mice that had rejected Meth-A/IL-15 cells).

Antibody treatment in vivo
In vivo neutralization of produced hIL-15 in tumorigenesis studies was accomplished by intraperitoneal injection with a mouse antih IL-15 mAb (M111, Genzyme) at 50 µg per 0.2 ml per injection on days 0 (the day of tumour cell injection), 3, 7, 10, 14 and 17 after tumour cell injection. This antibody at approximately 5-10 µg ml -1 neutralized approximately 80% of bioactivity induced by 5 ng ml -1 IL-15 in CTLL-2 cells. Normal mouse IgG (50 µg per 0.2 ml per injection; Caltag Laboratories, Burlingame, CA, USA) was injected into untreated mice on the same schedule.

Histologic evaluation and immunohistochemical staining
On day 7 and 14 after inoculation, the tumours were dissected, fixed in 10% neutral-buffered formalin and embedded in paraffin.

Statistical analysis
Statistical analysis used the Student's t-test. A value of P < 0.05 was considered statistically significant. Results are presented as mean ± s.e.m.

Expression of IL-15 in transfectants
Eighteen independent G-418-resistant colonies were isolated and expanded. RNA was isolated and RT-PCR performed. Three IL-15 levels, measured by the parallel use of an ELISA kit, were 25 pg, 31 pg and 29 pg for clone 2, 14 and 16 respectively. Moreover, the production of IL-15 by these three clones was stable for more than 1 year, because the fluctuations of this production at different times were < 15% in each clone. IL-15 production could not be detected in the supernatant of the parental cells or in transfectants by vector alone. We selected clones 16 (named Meth-A/IL-15), 2 (Meth-A/2), 14 (Meth-A/14) and 20 (Meth-A/Neo) for further examination.

Inhibition of tumour growth in vivo
The transfection of IL-15 or vector alone did not alter the growth properties of Meth-A cells in vitro as analysed by doubling time or morphology (data not shown). Tumorigenicity of Meth-A/IL-15 and Meth-A/Neo cells was examined by s.c. injection into BALB/c and BALB/c nu/nu mice in three independent experiments. In BALB/c mice Meth-A/IL-15 cells initially grew, then gradually regressed and were completely rejected within 21 days (n = 10), while Meth-A/Neo cells grew progressively until they caused the death of the animals (n = 10) ( Figure 3A). There was no difference in the growth patterns of the three IL-15-secreting clones (n = 5), Meth-A/IL-15, Meth-A/2 and Meth-A/14, of which production of IL-15 was 42 pg (10 6 cells 24 h -1 ), 35 pg and 53 pg respectively ( Figure 3C). Moreover, the growth of Meth-A/IL-15 (n = 5) was augmented by the injection of anti-hIL-15 mAb ( Figure 3D). In nude mice, both Meth-A/IL-15 cells (n = 10) and Meth-A/Neo cells (n = 10) grew progressively and were not rejected, although tumour growth was relatively inhibited in the case of Meth-A/IL-15 cells ( Figure 3B).

Histology at the site of tumour cell injection
Histological analysis of the injected site was performed at 7 and 14 days following the injection of tumour cells in order to characterize the host cellular responses augmented by IL-15 production.
In BALB/c mice, massive infiltration of mononuclear cells and polymorphonuclear neutrophils was noted on day 7 after the implantation of Meth-A/IL-15 cells ( Figure 4A). Moreover, on day 14, Meth-A/IL-15 cells were scattered and infiltrating cells were increased ( Figure 4C). In contrast, in the case of Meth-A/Neo cells, few mononuclear infiltrates were observed ( Figure 4B, D). In BALB/c nude mice, few mononuclear cells and neutrophils were observed at the injection sites of both Meth-A/IL-15 cells and Meth-A/Neo cells ( Figure 4E, F). Immunohistochemical analysis of tumour implantation sites in BALB/c mice 7 days after s.c. injection of Meth-A/IL-15 cells revealed a infiltrate of CD4 + T cell ( Figure 5A) and CD11b + cell (not shown) in the Meth-A/IL-15 tumour. CD8 + T-cells ( Figure  5B) were virtually absent.

Rechallenging with parental Meth-A and colon-26 cells
We next examined whether the primary rejection of IL-15 transfectants leads to protective immunity. Thirty days after disappearance of the initial Meth-A/IL-15 implantation, 20 mice were injected with 2 × 10 6 parental Meth-A cells in the previously uninjected side (left) of the lower abdominal quadrant. In all mice, tumours were rejected by 21 days, though transient tumour growth was observed up to the 3rd day after injection ( Figure 6A). Interestingly, the remarkable infiltration of mononuclear cells without neutrophils at the tumour site was seen 7 days after implantation ( Figure 6B). Immunohistochemical analysis of rechallenged sites 7 days after s.c. injection of parental Meth-A cells revealed an infiltrate of CD8 + T-cells ( Figure 5D) and CD11b + cells (not shown) in the Meth-A/IL-15 tumour. CD4 + T-cells ( Figure 5C) were virtually absent.
To demonstrate the specificity of the protective immunity described above, Colon-26 cells, a murine colon adenocarcinoma derived from BALB/c mice, were injected into both nonimmunized (n = 10) and immunized mice (n = 10). There was no significant difference in the growth of implanted Colon-26 cells between non-immunized mice and immunized mice (Figure 7).

DISCUSSION
It is well established that transfected tumour cells, which secrete a broad variety of cytokines, are rejected due to the cytokineinduced increase in the host's anti-tumour defense (Colombo and Forni, 1994). These effects provide new experimental approaches for cancer immunotherapy. Namely, tumour cell-targeted cytokine gene therapy is achieved by the transfer and expression of appropriate genes in tumour cells in vitro followed by analysis of their growth characteristics in vivo. Here we report that such tumour rejection can be obtained by local IL-15 production. Different mechanisms have been demonstrated in cells transfected with different cytokines. IFN-γ, IL-2, IL-6 and IL-12 transfectants have induced mainly specific CTL activity (Colombo and Forni, 1994;Zitvogel et al, 1995), while TNF-α, IL-4, MIP-1α and IL-8 induced non-specific anti-tumour activity and granulocyte infiltration (Colombo and Forni, 1994;Hirose et al, 1995). A new cytokine, IL-15, has shown many functional similarities to IL-2, such as stimulating proliferation of T-and B-lymphocytes and NK cells Armitage et al, 1995;Munger et al, 1995;Mori et al, 1996). These properties suggest that IL-15, like IL-2, may have a role in cancer treatment. However, differences in the expression pattern of IL-15 and its receptor compared to the IL-2/IL-2R system also suggest unique in vivo roles for IL-15 (Andeson et al, 1995;Giri et al, 1995;Bamford et al, 1996). The present study is the first demonstration of the modification of host immune responses due to the implantation of syngeneic tumour cells engineered to secrete IL-15.
This study provides two new observations. First, we demonstrated that hIL-15-transfected tumour cells were rejected completely in syngeneic mice, but not in nude mice. This rejection was accompanied by predominantly a mononuclear (CD4 + Tlymphocyte) and neutrophilic infiltration. It is possible that hIL-15 expressed by the tumour cells may be recognized as foreign and function as a tumour-rejection antigen for murine T-cells. However, we observed that the growth of Meth-A/IL-15 was augmented by anti-IL-15 injection. This result may support the direct effects of IL-15 on T-lymphocytes. Second, rechallenged parental cells were also rejected completely. This was associated with a predominantly mononuclear cell (CD8 + T-lymphocyte) infiltration, but not a neutrophilic infiltration.
In spite of these demonstrated anti-tumour effects of IL-15 in vivo, our understanding of the underlying mechanism is rudimentary. IL-15 has been reported to activate and stimulate proliferation of T-and B-lymphocytes in vitro Armitage et al, 1995;Mori et al, 1996). It stimulates the proliferation of NKcells and acts as a co-stimulator with IL-12 to facilitate the synthesis of IFN-γ and TNF-α (Carson et al, 1996). IL-15 also facilitates the induction of cytolytic effecter cells, including CTL and LAK cells Gamero et al, 1995;Lewko  In-vivo growth of initial-challenged or rechallenged parental Colon-26 cells. After disappearance of the initial Meth-A/IL-15 implantation, 5 × 10 5 Colon-26 cells were injected into the opposite (left) lower abdominal quadrant (n = 10). At the same time, Colon-26 cells were injected into nonimmunized mice (n = 10). Tumour volumes are mean. Each s.e.m. was < 10%* Munger et al, 1995). In general, it is well-established that tumour rejection induced by local secretion of cytokines is mediated by a dual mechanism involving specific and non-specific anti-tumour effectors. With regard to the specific effects, our results indicate that IL-15 stimulates in vivo the sensitization of memory T-lymphocytes which are responsible for the specific long-lasting anti-tumour protection. Specifically, BALB/c mice that rejected Meth-A/IL-15 cells were protected against parental Meth-A tumours associated with CD8 + T-lymphocytic infiltration, but not against Colon-26 tumours. Direct effects of IL-15 on T-lymphocytes seems likely since the IL-15 stimulation of Tlymphocytes may directly utilize the trimolecular IL-15 receptor complex Grabstein et al, 1994;Andeson et al, 1995). Interestingly, massive neutrophilic infiltration was observed in tumour sites of BALB/c mice that rejected Meth-A/IL-15 cells, though it has been reported that IL-15 does not attract neutrophils (Wilkinson et al, 1995). These reports suggest that the neutrophilic infiltration may be due to a cascade of cytokines and soluble mediators, which were produced by activated T lymphocytes and other cells induced by the IL-15 secretion. Cavallo et al (1992) have reported that rejection of IL-2-transfected tumours is associated with neutrophil infiltration, and this neutrophildominated rejection generates a long-lasting, tumour-specific, Tlymphocyte-mediated immune memory. The role of neutrophils remains to be characterized in our studies.
The amounts of IL-15 secreted may be important. Meazza et al (1997) showed the poor efficiency of IL-15 natural signal peptides. They described that substitution of the sequence encoding natural signal peptide(s) with the one from IgVκ chain in the IL-15 cDNA resulted in a significantly higher secretion of biologically active IL-15 (15-to 30-fold) upon cDNA transfection. Although the amount of IL-15 produced by our transduced cells was relatively low, the in vitro effects were obtained.
In conclusion, these studies demonstrate that IL-15-transfected cancer cells can elicit an anti-tumour immune response and suggest that IL-15 may have a role as a component of tumour vaccines. Further studies are needed to understand the role of lymphocytes and neutrophils in the response to IL-15.