Tumor-Infiltrating Lymphocyte Secretion of IL-6 Antagonizes Tumor-Derived TGF-β1 and Restores the Lymphokine-Activated Killing Activity¹ Free

The cytokines present within the tumor microenvironment affect tumor growth and survival (1). TGF-β1 has been detected in tissue specimens from a variety of tumor types (2). TGF-β1, a potent inhibitor of immune function, allows tumors to escape immune surveillance (3, 4). TGF-β1 production by malignant tumors is essential for tumor progression and is one of the most important immunosuppressive cytokines secreted by tumors (5, 6). There are other mechanisms that enable tumors to evade host immune surveillance, including: 1) down-regulation of MHC class I Ag (7, 8) due to defective expression of proteosome multicatalytic complex subunits, low m.w. proteins 2 and 7, or peptide transporters associated with Ag processing 1 and 2 (9); 2) over-expression of nonclassical HLA-G (10) and classical HLA-Cw7 molecules, which interact with killing inhibitory receptors (11); 3) resistance to cytotoxic molecules such as granzyme B and perforin (12, 13); 4) expression of soluble, stress-induced ligands (MHC class I chain A and chain B) (14); 5) defective death receptor signaling (Fas ligand and TRAIL) (15); 6) decreased tumor Ag (16); and 7) secretion of immunosuppressive factors such as IL-4 and IL-10 (17).

In some malignant tumors, tumor-infiltrating lymphocytes (TIL)⁴ secrete IL-6, a multifunctional cytokine that acts in the immune system (18, 19). IL-6 almost completely antagonizes the immunosuppressive effects of TGF-β1 on T cell proliferation in eyes with endotoxin-induced uveitis (20). Combined IL-2 and IL-6 gene therapy, transferred intratumorly by liposomes to mice bearing B16F10 melanoma, significantly enhances the CTL and NK activity of splenocytes and TIL (21). However, the ability of IL-6 to counteract the effects of TGF-β1 and restore NK capability is still unknown.

Canine transmissible venereal tumor (CTVT) is an excellent animal model (22) with which to study tumor-host interactions. CTVT is a canine-specific round cell neoplasm that can be transplanted across MHC barriers within species (23). Experimentally transplanted CTVT exhibits a predictable growth pattern that includes a progressive growth (P phase) and a spontaneous regression (R phase) (24). Different types of TIL are present during both P and R phases (23). The number and size of TIL subpopulations vary with CTVT growth phase. TIL may be associated with CTVT regression (7, 23, 24). CTVT expresses extremely low numbers of MHC I molecules during P phase, much like malignant tumors in humans (6). At ∼12 wk after inoculation, MHC I expression suddenly increases 30–40% as R phase commences (7). The proportion and number of TIL increased significantly during R phase (23). Still, 60–70% of R phase CTVT cells were devoid of MHC I molecules. During R phase, cells other than T cells, such as cells with NK activity, have significant cytotoxic effects.

In this study, we determined the effect of TGF-β1 on LAK activity during tumor progression and assessed the effect of the interaction between IL-6 and TGF-β1 on LAK activity during tumor regression. We demonstrated that TGF-β1 suppressed TIL killing activity. TIL isolated from regressing CTVT secreted high concentrations of IL-6, antagonizing the anti-LAK activity of tumor TGF-β1.

Spontaneous CTVT on the external genitals of two male dogs was used for the original experimental transplantation. Each of three male and three female beagles were injected s.c. with 7.5 × 10⁷ freshly prepared, viable tumor cells at each of 10 sites on their backs. Tumor dimensions were measured with calipers once a week and the tumor volume was estimated as π × length × width × thickness/4 (cm³) (7). Tumor growth stages were classified according to the tumor growth curve. A tumor increasing in volume was classified as P phase and a tumor decreasing in volume was classified as R phase.

Every 2–3 wk post-inoculation, tumor tissue samples were surgically excised from experimental dogs. We followed the methods previously described to isolate CTVT tumor cells and TIL (7). Briefly, 10 g of aseptic tumor tissue was minced in 90 ml HBSS (Life Technologies, Grand Island, NY). To obtain a single cell suspension, samples were mechanically crushed with stainless steel mesh and filtered once through two pieces of gauze (pore size: 190 μm). Then, 8 ml of the cell suspension was overlaid on 4 ml 42% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient and centrifuged at 820 × g and 4°C, for 25 min. After centrifugation, CTVT cells deposited at the interface were harvested and washed three times with DMEM (Life Technologies) supplemented with 10% FCS. TIL deposited at the bottom of tube were collected carefully and washed three times with RPMI 1640 (Life Technologies) supplemented with 10% FCS. The purified CTVT cells and TIL were stained with Hemacolor (Merck, Darmstadt, Germany) to confirm their purities. Freshly isolated, CTVT cells (1 × 10⁶ cells/ml) were cultured at 37°C for 72 h, the supernatants were collected for future use. The same method was used to obtain supernatants of TIL. We collected R phase TIL supernatants from cultures after 24, 48, and 72 h to evaluate TIL secretion of IL-6 over time. The CTVT supernatants were collected from ten P phase and six R phase tumors. TIL supernatants were collected from six P phase and six R phase tumors. Each supernatant collected was tested individually and used in the following experiments.

Total RNA was prepared from the CTVT mass, purified CTVT cells and TIL using Microto-Midi Total RNA Purification System (Invitrogen, Leek, The Netherlands). The TGF-β sense primer was designed as 5′-TTC CTG CTC CTC ATG GCC AC-3′ and antisense primer was 5′-GCA GGA GCG CAC GAT CAT GT-3′. Cycle conditions were 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min for 34 cycles. The PCR products were separated on 2% agarose gel. The PCR products were sequenced and the TGF-β sequence was confirmed with a basic local alignment search tool (BLAST), a public computer program. Canine PBMC from healthy beagles were isolated by layering 4 ml PBS diluted canine peripheral blood on 3 ml Ficoll-Paque, and centrifuging the solution at 450 × g for 25 min at 4°C. The isolated PBMC were stimulated with 2 μg/ml Con A (Sigma-Aldrich, Steinheim, Germany) for 4 h, and the RNA was isolated as the positive control for TGF-β.

Purified CTVT cells (1 × 10⁶) were fixed and permeabilized with a cell permeabilization kit (Caltag Laboratories, Burlingame, CA) and then incubated with TGF-β mAb, MCA797 (Serotec, Oxford, U.K.) for 60 min at 4°C. Cells were washed and incubated with FITC-conjugated F(ab′)₂ goat anti-mouse IgG (Serotec) for 30 min. The intensity of the positive intracellular immunofluorescence of 10⁴ viable cells was measured with a FACSCaliber flow cytometer (BD Biosciences, Mountain View, CA). The concentration of TGF-β1 was determined with CellQuest software (BD Biosciences).

The concentration of TGF-β1 in each supernatant was measured with an ELISA using the TGF-β1 Emax ImmunoAssay system (internal standard provided; Promega, Madison, WI) according to the manufacturer’s instructions. Each CTVT supernatant sample was divided into two parts. To assess the total TGF-β concentration, one part of each sample was treated with 1 N HCl to lower the pH to 2 for 15 min. Then the sample was neutralized at pH 7.6 with 1 N NaOH. The other part of each sample was used directly, without 1 N HCl treatment, to determine the concentration of active TGF-β. The concentration of IL-6 in each TIL supernatant sample was determined with ELISA, using mAb M621B (Endogen, Woburn, MA). All measurements were done in triplicate.

A total of 1 × 10⁶ P phase and 1 × 10⁶ R phase TIL were homogenized, separately, in homogenization buffer (10% sucrose, 0.004% Pefabloc SC, and 0.5 M Tris-HCl in Mili-Q H₂O) and centrifuged at 15,000 rpm for 10 min. The protein concentration of each supernatant was measured with a modified Lowry protein assay reagent kit (Pierce, Rockford, IL). Proteins were electrophoresed on a 12.5% SDS-PAGE and transferred by capillary diffusion to two sheets of nitrocellulose paper (Amersham Pharmacia Biotech) in transfer buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 0.5 mM 2-ME). Blots were blocked with 5% skim milk and incubated with 5 μg/ml rabbit anti-human IL-6 polyclonal Ab (Endogen) in Tween 20 plus TBS (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20, pH 7.4) for 60 min. Blots were extensively washed in the same buffer and incubated with HRP-conjugated goat anti-rabbit IgG F(ab′)₂ (1:1000) for 60 min. They were visualized with an ECL detector (Pierce) according to manufacturer’s instructions.

LAK and TIL natural killing activity was assessed with the morphometric cytotoxicity assay (MCA) developed by Geldhof et al. (25). The MCA estimates adherent, target cell lysis by measuring the holes in a confluent monolayer of cells. Each hole represents a group of the cells that were killed. Dead cells detach from the plastic surface on which the monolayer was spread.

Adhered, canine thyroid adenocarcinoma cells (CTAC) (5 × 10⁵/ml) were cultured in a 24-well plate for 24 h to obtain a cell monolayer that was 90% confluent.

Canine LAK cells and purified TIL were used as effector cells. LAK cells were prepared by culturing isolated PBMC with recombinant IL-2, 500 U IL-2 per 1 × 10⁶ PBMC, for 5 days. In addition, 2-ME (Sigma-Aldrich), 5 × 10⁴ mM per 1 × 10⁶ PBMC, was added. On day 3, another 500 U of recombinant IL-2 was added to each culture.

LAK and TIL were reacted with the CTAC monolayer for 16 h and washed once with PBS (pH 7.4). The monolayer was stained with 1 ml Coomassie blue solution (0.2% R250 Coomassie brilliant blue; Sigma-Aldrich) in acetic acid/methanol/water (10/45/45:v/v/v). After 15 min, the plate was thoroughly rinsed with distilled water and air-dried for storage and analysis. Most dead CTAC, their remains, TIL, and LAK cells were lost during rinsing. Images of five different fields (three images per field) of each stained monolayer sample were taken with a light microscope and analyzed by Image-Pro version 4.5 (Silver Spring, MD). After digital contrast enhancement, the gray level image was converted into a binary image. The ratio of the cleared area to the confluent area provided a measure of cell lysis. Clearance percentage is the cleared, cell-free area as a percentage of the total area of the microscope field.

Isolated TIL (1 × 10⁶) and LAK (1 × 10⁶) were stained for 60 min at 4°C with mAbs MCA1774, MCA1998S, MCA1999S, and MCA1781S (all Serotec) against CD3, CD4, CD8, and CD21, respectively. The cells were washed and incubated with FITC-conjugated F(ab′)₂ goat anti-mouse IgG for 30 min. They were washed again and resuspended with fluorescent assay buffer (1% BSA and 0.02% sodium azide in PBS, pH 7.2) containing 5 μg/ml propidium iodide. PBMC from healthy beagles were the positive control (n = 6). The intensity of the positive surface immunofluorescence of 10⁴ viable cells was measured with a FACSCaliber flow cytometer. Dead cells, identified by red propidium iodide fluorescence, were gated out. The proportions of cells that expressed CD3, CD4, CD8, or CD21 were determined with CellQuest software.

There were two types of experiments in this study. The first type evaluated the inhibition of LAK cell killing activity by TGF-β in CTVT P and R phase supernatants. Treatments included: 1) CTAC plus LAK; 2) CTAC plus LAK and TGF-β polyclonal Ab T9429 (Sigma-Aldrich); 3) CTAC plus LAK and CTVT P phase supernatant; 4) CTAC plus LAK and CTVT P phase supernatant plus TGF-β polyclonal Ab; 5) CTAC plus LAK and CTVT R phase supernatant; 6) CTAC plus LAK and CTVT R phase supernatant plus TGF-β polyclonal Ab; 7) CTAC alone (negative control). The second type assessed the inhibition of TIL killing activity by CTVT supernatants. TIL were cultured for 24 h before being mixed with CTAC to eliminate the effect of the in vivo tumor microenvironment. The experimental design and treatments were the same as those listed earlier except that the effector cells were TIL instead of LAK cells.

To explore the mechanism by which TIL supernatant inhibited TGF-β activity, we conducted an experiment with the following treatments: 1) CTAC plus LAK and CTVT supernatants plus TIL P phase supernatant; 2) CTAC plus LAK and CTVT supernatants plus TIL P phase supernatant and IL-6 polyclonal Ab p-620; 3) CTAC plus LAK and CTVT supernatants plus TIL R phase supernatant; 4) CTAC plus LAK and CTVT supernatants plus TIL R phase supernatant and IL-6 polyclonal Ab.

Recombinant human TGF-β1 (PeproTech, London, U.K.) and recombinant human IL-6 (IL-6; PeproTech) were substituted for CTVT supernatants and TIL supernatants, respectively, in the LAK natural killing assay. Based on ELISA measurements of tumor TGF-β and TIL IL-6, the TGF-β concentrations used in this experiment were 0, 0.5, and 5 ng/ml and the concentrations of IL-6 were 0, 1, 5, 10, and 20 ng/ml.

Experiments were performed in triplicate and repeated at least six times. Results are expressed as means ± SE. The statistical significance of differences between mean values was estimated using the Student t test. Values of p < 0.05 were considered significant.

CTVT masses and purified CTVT cells expressed TGF-β mRNA, but TIL did not (Fig. 1). The TGF-β1 sequence of the PCR product was confirmed by analysis with BLAST.

Flow cytometry was used to determine the level of intracellular TGF-β expression in isolated CTVT cells. Both P and R phase CTVT cells contained TGF-β. The percentages of R phase (27.37 ± 8.2) and P phase (28.45 ± 7.86) CTVT cells that expressed TGF-β were not significantly different (Fig. 2,A). Furthermore, using ELISA, we found the latent and active forms of TGF-β1 were expressed in cultured P and R phase cells. The amounts of active and total TGF-β1 in P and R phase CTVT were not significantly different (Fig. 2 B).

LAK cells killed CTAC efficiently (Fig. 3, Ab and B). Addition of P or R phase CTVT supernatants to the LAK cell/CTAC coculture significantly decreased LAK cell killing activity (Fig. 3,B). When TGF-β polyclonal Ab was added, it blocked the effect of TGF-β and the killing activity of LAK cells returned to normal levels (Fig. 3,B). It was possible that Ab-dependent cell-mediated cytotoxicity was induced by TGF-β polyclonal Ab. To determine whether Ab-dependent cell-mediated cytotoxicity played a significant role in cell lysis in our system, we added only TGF-β polyclonal Ab to LAK/CTAC coculture. The result was negative (Fig. 3,B). P and R phase TIL killed the CTAC effectively (Fig. 4). CTVT supernatants containing active TGF-β significantly inhibited the killing activity of TIL. When TGF-β polyclonal Ab was added to the TIL/CTAC coculture, the killing activity of TIL was restored (Fig. 4).

Most TIL (90 ± 3.2%) were non-T and non-B cells (Fig. 5,A). LAK were comprised of non-T and non-B cells (66.19 ± 4.24), monocytes (18.23 ± 2.65), T cells (13.34 ± 1.34), and B cells (2.24 ± 0.71) (Fig. 5 B).

IL-6 protein expression in R phase TIL was much higher than it was in P phase (Fig. 6).

IL-6 expression by R phase TIL was significantly higher than that by P phase TIL (p < 0.01) (Fig. 7,A). During in vitro culture, TIL secretion of IL-6 decreased gradually (Fig. 7 B).

R phase, but not P phase TIL supernatants significantly increased the killing activity of LAK cells inhibited by TGF-β1 (Fig. 8; p < 0.05). When we added IL-6 polyclonal Ab, to R phase TIL supernatants, the inhibitory effect of CTVT supernatants on LAK killing activity was restored. To determine whether IL-6 polyclonal Ab induced Ab-dependent cell-mediated cytotoxicity, IL-6 polyclonal Ab was added to the LAK/CTAC coculture. The result was negative. In addition, we added recombinant human IL-6 to LAK cells to determine whether IL-6 directly affected LAK cell activation. The killing activity of treated LAK cells was not enhanced by recombinant human IL-6 (Fig. 8).

Exogenous TGF-β1 significantly inhibited LAK killing activity, but the addition of exogenous IL-6 blocked this effect. At concentrations of 10 ng/ml and higher, IL-6 significantly increased the cytotoxicity of LAK cells in both the 0.5 ng/ml and 5 ng/ml TGF-β1 treatments (p < 0.01). At concentrations lower than 5 ng/ml, IL-6 was unable to counter the effects of TGF-β1 (Fig. 9).

In most studies, freshly isolated TIL were unable to kill autologous tumors and appeared to be immunologically inert or suppressed (26, 27). However, TIL can be stimulated in vitro to re-express cytolytic activity against autologous tumors (1). This suggests that factors within the tumor microenvironment are responsible for the observed immunological impotence of freshly isolated TIL. These observations have fostered attempts to identify factors in tumors that can influence tumor immunity and tumor progression. The cytokines expressed in tumor microenvironments are thought to be important mediators of both the host immune response and tumor survival (19).

TGF-β is often secreted by tumor cells. It inhibits T and B cell proliferation and T cell and NK cell cytolytic activity, enabling the tumor to escape host immune response (28). Our results demonstrated that CTVT cells express TGF-β mRNA and proteins. Before their secretion by a cell, TGF-β proteins undergo a number of intracellular processing steps. The most important step appears to be the proteolytic digestion of the precursor by the endopeptidase furin (29). The latent TGF-β complex is unable to bind to TGF-β receptors unless the biologically active, mature TGF-β is dissociated from the latency-associated peptide (30). Because TGF-β receptors are expressed ubiquitously, the key event that regulates TGF-β1 biological activity in vivo is its activation by release from the latent complex. We found that P and R phase CTVT secreted similar levels of TGF-β1 and that the latent form was always the major protein in the supernatants from both phases. Interestingly, TGF-β1 from both P and R phase CTVT effectively inhibited LAK killing activity. Addition of TGF-β1 specific Abs restored LAK killing activity. Importantly, CTVT supernatants also inhibited the killing activity of P and R phase TIL. Again, addition of TGF-β1 specific Abs restored TIL killing activity. Thus, TGF-β1 clearly plays a major important role in helping tumors evade host immune responses. Other types of tumors, such as human non-small cell lung cancer and ovarian cancers, express high levels of TGF-β mRNA and suppress the development of TIL T cell cytolytic activity against autologous tumor cells (19). During R phase, TGF-β1 concentrations remained high and >60% of CTVT cells were still MHC negative. This leads to a question whose answer is of great importance: How, and by what mechanism, does the host overcome the inhibitory effects of TGF-β1?

We found that IL-6 was present in the TIL supernatants. Although IL-6 was present in both P and R phase TIL supernatants, its concentration was significantly higher in R phase TIL supernatant. IL-6 is a pleiotropic cytokine that is produced by a variety of cells and acts on a wide range of tissues. Depending on the target, IL-6 can inhibit growth, induce growth, or induce differentiation (31, 32). IL-6 can directly up-regulate the functions of NK cells including proliferation, cytotoxicity, expression of surface activation Ag and adhesion molecules, and anti-metastatic activity (33). IL-6 almost completely antagonizes the immunosuppressive effects of TGF-β1 on T cell proliferation (20). Combined liposome-mediated intratumoral cotransfer of IL-2/IL-6 genes markedly increased NK activity of TIL (21). However, before the present study, there had been no research on whether IL-6 could antagonize TGF-β1 inhibition of LAK activity in a tumor. We demonstrated that only R phase TIL IL-6 was capable of counteracting TGF-β1 inhibition of LAK cell killing activity. There appeared to be an IL-6 concentration threshold. At concentrations of 10 to 20 ng/ml, IL-6 significantly restored LAK cell killing activity, regardless of whether TGF-β1 levels were low (0.5 ng/ml) or high (5 ng/ml). IL-6, at 5 ng/ml and below, had no effect. However, extremely high doses of IL-6 suppressed NK and LAK activity (34). The functions of IL-6 are multiple and different, depending on the sources and/or types of tumors. In some cases, IL-6 enhances tumor growth, whereas in others, it assists host immune activity against tumor cells (19, 35). IL-6 function may depend, in part, on the type of cell that produces it. Thus, IL-6 secreted by a tumor cell may protect the tumor from host immune attack (36), but IL-6 produced by TIL, as in this study, stimulates host immune responses.

In addition, we found that IL-6 alone did not significantly increase LAK killing activity, which implies that IL-6 does not act directly on LAK. Instead, IL-6 antagonized inhibition of LAK killing activity by TGF-β1, which enabled LAK to resume normal activities.

IL-6 also activates CTLs and induces T cell-mediated antitumor effects (34). IL-6 has been implicated in the induction of IL-2 receptor expression and in T cell proliferation following stimulation of T cell Ag receptors (37). Therefore, high concentrations of IL-6 secreted by TIL to block TGF-β1 inhibition of killing activity, also may enhance T cell cytotoxicity when MHC molecules are up-regulated on tumor cells. Interestingly, TIL secreted IL-6 during the growth phase, although in small amounts. The effect of low concentrations of IL-6 on host-tumor interactions merits further study.

In conclusion, CTVT secretes TGF-β1 to inhibit killing activity of TIL and escape host immunosurveillance, allowing the tumor to grow vigorously. However, some TIL were attracted to CTVT and produced low levels of IL-6. Over time, TIL were able to express higher concentrations of IL-6, antagonizing TGF-β1 inhibition of LAK activity. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-β1 anti-LAK activity, has potential applications in cancer immunotherapy.