Inflammatory cytokine-mediated evasion of virus-induced tumors from NK cell control

Abstract

Infections with DNA tumor viruses, including members of the polyomavirus family, often result in tumor formation in immune-deficient hosts. The complex control involved in antiviral and antitumor immune responses during these infections can be studied in murine polyomavirus (PyV)-infected mice as a model. We found that NK cells efficiently kill cells derived from PyV-induced salivary gland tumors in vitro in an NKG2D (effector cell) -RAE-1 (target cell) - dependent manner, but in T cell-deficient mice NK cells only delay but do not prevent the development of PyV-induced tumors. Here we show that the PyV-induced tumors have infiltrating functional NK cells. The freshly removed tumors, however, lack surface RAE-1 expression, and the tumor tissues produce soluble factors that down-regulate RAE-1. These factors include the pro-inflammatory cytokines IL-1α, IL-1β, IL-33, and TNF. Each of these cytokines down-regulate RAE-1 expression and susceptibility to NK cell mediated cytotoxicity. CD11b+F4/80+ macrophages infiltrating the PyV-induced tumors produce high amounts of IL-1β and TNF. Thus, our data suggest a new mechanism whereby inflammatory cytokines generated in the tumor environment lead to evasion of NK cell-mediated control of virus-induced tumors.

Introduction

Virus-associated cancers constitute more than 20% of human cancers and often occur in immune compromised individuals [1]. Recently, a polyomavirus (Merkel cell polyomavirus) has been shown to be associated with Merkel cell carcinomas, adding this virus to the list of cancercausing human viruses [2]. Oncogenic viruses, including mouse PyV, which is phylogenetically close to the Merkel cell polyomavirus, efficiently cause cellular transformation in vitro and often induce tumor development in infected hosts under immune compromised conditions, highlighting the importance of a functional immune system in the control of cancers [3,4]. PyV induces a variety of tumors after neonatal infection in some “tumor susceptible” mouse strains, such as C3H/BiDa, DBA/2, BALB/c, but not in other “tumor resistant strains”, such as C57/BL6 (B6) mice [5]. Adult immune-competent mice are generally resistant to tumor formation regardless of their genetic background. However, PyV infection of adult mice will lead to tumor development under severely immune suppressive conditions, like whole body irradiation, neonatal thymectomy or in congenitally thymus-deprived nude mice [6,7]. The resistance to PyV-induced tumor development has been primarily attributed to CD8 T cell responses against viral antigens expressed by PyV-infected cells and PyV-induced tumors [3]. Some findings are consistent with this view. For example, endogenous super-antigens encoded by mouse mammary tumor provirus Mtv-7 increase the susceptibility of neonatally infected H2^k mice to tumor formation by eliminating Vβ6+ cytotoxic T lymphocytes from the CD8+ T cell repertoire [8]. As most of the CD8 T cells in H2^k mice that are specific for the immunodominant PyV middle T antigen epitope expressed both in cells productively infected or transformed by PyV are Vβ6+, this finding suggests that these CD8 T cells are critically important for the control of PyV-induced tumors [9]. Moreover, adult mice lacking β2 microglobulin (on the B6 genetic background) are highly susceptible to PyV-induced tumor development, further suggesting an important role for class I MHC-restricted αβ CD8+ cells. On the other hand, some H2^k mouse strains are susceptible to tumors induced by PyV despite possessing Vβ6+ CTLs, and CD8-deficient mice can resist tumor formation [10], suggesting the importance of other factors in addition to CD8 T cell repertoire in tumor resistance. Our recent studies comparing PyV-induced tumor formation in TCRβ KO, TCRβxδ KO and E26 (NK and T cell-deficient) mice after infection as adults showed that γδ T cells and NK cells play a protective role in the control of PyV-induced tumors, as most TCRβxδ KO mice developed virus-induced tumors, whereas TCRβ KO mice did not, and E26 mice developed tumors faster in comparison to TCRβxδ KO mice [11] .

NK cells, in addition to playing a major role in the control of some virus infections, such as MCMV, are also thought to protect against tumor growth both in humans and in mouse models. For example, NK cells are found among the tumor infiltrating lymphocytes in many human carcinomas, such as pulmonary adenocarcinomas, colorectal cancers and gastric cancers, and their presence in the tumor environment in general shows a positive correlation with better prognosis [12-14]. In mice, implanted syngeneic tumors, including those induced by tumor viruses, grow more aggressively if no functional NK cells are present [15]. The role of NK cells in the control of naturally developing virus-induced tumors, however, has not been thoroughly investigated.

NK cell activation is determined by the cytokine milieu and by the balance of signals transduced from activating and inhibitory receptors that have been engaged by their ligands [16]. NKG2D is one of the activating receptors, and it is present on NK cells, on activated CD8+αβT cells, and on some γδ T cells [17]. NKG2D binds to a family of ligands which are expressed on “stressed” cells, including cells infected by pathogens, undergoing cellular transformation, or exposed to other stress conditions such as heat shock or DNA damage [18]. So far in humans eight (MICA/MICB, ULBP 1-4, RAET1G and RAET1L) and in mice nine (RAE-1α, β, γ, δ, ε, H60 a-c and MULT-1) NKG2D ligands have been identified [19]. The expression of these ligands is tightly regulated. Many primary human cancers, mouse tumors and established tumor cell lines express NKG2D ligands, and the recognition of these ligands by NKG2D can tip the balance and lead to the activation of NK cells and NK cell-mediated cytolysis of the cells expressing the stress ligands [20]. Expression of NKG2D ligands on tumors transplanted into mice made them susceptible to rejection by NK cells in vivo [21,22]. Tumors appear earlier in mice genetically engineered to develop transgenic adenocarcinomas (by expressing SV40 T antigens in their prostate epithelium) or myc-driven B cell lymphomas, if these transgenic mice lack NKG2D. These tumors also express higher levels of NKG2D ligands in NKG2D KO mice compared to NKG2D-sufficient ones. These data taken together strongly suggest the importance of NKG2D receptor in tumor surveillance [23].

The expression of NKG2D ligands is often modulated in tumor bearing or virus-infected hosts, and this modulation serves as a mechanism exploited by the tumor cells or infected cells to evade NK cell-mediated cytotoxicity. HCMV and MCMV both express several proteins that bind to NKG2D ligands in the infected cells and prevent their expression on the cell surface [24-30]. In addition, both cell- and virus-encoded miRNAs, including those encoded by the human polyomaviruses JC and BK, were reported to down-regulate the expression of NKG2D ligands [31-33]. Cytokines can also alter NKG2D ligand expression under certain conditions. TGF-β was shown to suppress the expression of MICA and ULBPs in human gliomas and in murine models of head and neck carcinoma [34,35], and IFNγ was found to decrease H60 expression in methylcholantrene (MCA)- induced sarcomas in mice [36] .

We have previously reported that cultured cell lines established from PyV- induced salivary gland tumors express the NKG2D ligand RAE-1 at high levels. Moreover, these tumor cells activated NK cells to produce IFNγ and granzyme B and were killed in an NKG2D-dependent manner in vitro by NK cells [11]. We show here that PyV-induced tumors in vivo contain a population of infiltrating functional NK cells which express NKG2D and produce IFNγ and granzyme B. However, the ex vivo freshly removed primary tumor cells do not express RAE-1 protein on their surfaces, and soluble factors produced by tumor infiltrating leukocytes down-regulate RAE-1 expression on established tumor cell lines. These factors include the pro-inflammatory cytokines IL-1α, IL-1β, IL-33, and TNF, each of which may contribute to the in vivo down-modulation of RAE-1. The decrease in RAE-1 expression correlates with diminished sensitivity to NK cell-mediated cytotoxicity. Thus we propose that the inflammatory environment in virus-induced tumors can lead to immune evasion by decreasing tumor cell recognition by NK cells without significantly affecting NK cell function.

Materials and Methods

Mice and infections

Mice used in the studies were all on the C57BL/6 (B6) genetic background. TCRβ KO, TCRβ×δ KO and SCID mice were originally purchased from the Jackson Laboratory (Bar Harbor, Maine), and colonies of these mice were maintained in the Department of Animal Medicine of the University of Massachusetts Medical School under specific pathogen-free conditions. NKG2D KO mice [50] were given by Dr Wayne M. Yokoyama (Washington U). To obtain TCRβ x NKG2D double KO mice, NKG2D KO mice were crossbred with TCRβ KO mice. Mice were used between 8 and 12 weeks of age except for tumor-bearing mice, which were older at the time of sacrifice. Virus infections were done intraperitoneally with 2×10⁶ PFU of PyV strain A2 per mouse. All the procedures using animals were done according to the protocols “Immunology of virus infections” approved by the University of Massachusetts Medical School Animal Care and Use Committee.

Transwell experiments

Several polyomavirus-induced salivary gland tumor cell lines (e.g. PyVTu1, PyVTu2 and PyVTu3) were independently derived from TCRβ×δ KO mice bearing PyV-induced salivary gland tumors. For transwell experiments the PyVTu cell lines were plated in the lower compartment either in 6 well plates with 24 mm inserts or 100mm plates with 75mm inserts with 0.4μm pore size (Transwell permeable support Corning NY 14831) overnight. Tumors were aseptically excised from euthanized PyV-infected TCRβ×δ KO mice. The tumor tissue was rinsed with sterile DMEM containing antibiotics, cut into small pieces and digested with type I collagenase (100U/ml) in 10 ml of DMEM containing 10% FCS for 1 hr. The cells were then harvested and plated in the upper compartment of a transwell plate at a ratio of 8:1 to the cells plated in the lower chamber. For experiments with spent media, primary tumor infiltrating cells were harvested and plated in DMEM containing 10% FCS. Subsequently the culture media was collected, centrifuged to remove debris and added to the cell cultures.

In vitro cytotoxicity assays

Standard 4 h ⁵¹Cr release microcytotoxicity assays were used to determine NK cell activity [38]. Activated peritoneal exudate cells (PECs) from TCRβ KO, TCRβ×NKG2D double KO or spleen cells from naive SCID mice were used as effector cells. The PECs were activated in vivo by injection of PyVTu cells (4 to 5 × 10⁶) i.p. 2 to 3 days prior to their harvest. The injection of PyVTu cells was previously shown to increase the numbers of NK cells in the peritoneum and to increase their ability to produce IFNγ and Granzyme-B [11]. ⁵¹Cr-labelled PyVTu cells were used as targets, and 10⁴ target cells were plated into wells of microtiter plates with varying numbers of effectors to achieve the planned effector to target (E:T) ratios. After 4 hours of incubation ⁵¹Cr release into the supernatants was measured. The percentage of specific ⁵¹Cr release was calculated as described before [39]. For blocking RAE-1, polyclonal goat anti-mouse RAE-1γ antibodies (clone AF-1136; R&D systems) were used at 2μg/ml concentration.

Reverse transcriptase (RT) PCR and qRT PCR to detect RAE-1

RNA samples from various organs, tumors or cell lines were isolated using the RNeasy mini kit (Qiagen) following the manufacturer’s protocol. Two μg of total RNA from each sample was used to synthesize 1st strand cDNA using 0.5μg of oligo dT (Invitrogen) and superscript II RT (Invitrogen) following the manufacturer’s protocol. The PCR amplification was carried out in a total volume of 50μl containing 0.2mM of each dNTP, 0.5 U of Taq polymerase (Invitrogen) in 1x PCR buffer supplied by the manufacturer and 20 pmol each of forward and reverse primer (Invitrogen). The amplified PCR mix were run on a 1% agarose gel and stained with ethidium bromide for visualization. For qRT PCR SYBR green master mix (Applied Biosystem) or QuantiFast SYBR Green PCR Kit was used. PCR amplification with the β-actin primers started with one cycle at 95°C for 10 minutes, then 37 cycles of 95°C for 30 sec, 62°C for 25 sec, and 72°C for 25 sec. Negative controls included samples with no reverse transcriptase. The RAE-1 primers were designed to amplify all RAE-1 isoforms. The PCR cycles started with 95°C for 10 minutes, then 32 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. For determining relative expression, the target gene copy numbers were normalized for β-actin by using mathematical formula (ΔΔCt) methods. The following primers were used: β-actin forward primer 5′ CGA GGC CCA GAG CAA GAG AG and β-actin reverse primer 5′ CGG TTG GCC TTA GGG TTC AG; RAE-1 forward primer 5′TGA GCT GGA GAT CAG CTA ATG A and RAE-1 reverse primer 5′ GAA GCG GGG AAG TTG ATG TA.

Cytokine treatment, surface and intracellular staining

Purified recombinant mouse IL-1β, IL-10, IL-12, TNF and TGF-β were obtained from PeproTech, U.S.A, IL-1α from Biolegend, U.S.A. and IL-33 from eBiosciences, U.S.A. All these cytokines were re-suspended in PBS containing BSA and stored at −20°C or −80°C in smaller aliquots for further use. For most experiments cells were treated with 2ng/ml of purified recombinant cytokines except for TNF, which was given at the dose of 5ng/ml in all experiments unless otherwise stated. Control cells were treated with PBS containing BSA.

For RAE-1, MHC I, CD155, IL-1R1 and IL-33 receptor staining 2 to 4×10⁵ PyVTu cells were treated with anti-CD16/32 (Fc block; clone 2.4G2; BD Pharmingen) and then stained with the following antibodies: PE-anti-mouse RAE-1 (pan-specific, clone 186107 R&D Systems), PE-anti-mouse MHC class I H-2K^b (clone AF6-88.5; BD Bioscience), PE-anti-mouse CD155 (clone TX56; Biolegend), PE anti-mouse CD121a type I/80 (clone JAMA-147; Biolegend), PE-anti-mouse ST2/IL-1R4 (R&D Systems) and Rat IgG_2A isotype control-PE (clone 54447; R&D Systems), Armenian Hamster IgG isotype control-PE (clone 299Arm; eBiosciences), Mouse IgG2_A isotype control-PE (BD Pharmingen) were used.

For surface and intracellular staining freshly isolated tumor cells of salivary gland tumors from TCR KO mice were homogenized to make single cell suspensions as described above. Also single cell suspensions were prepared from spleen and PECs. For IFNγ, Granzyme B, IL-1β,TNF staining 2X10⁶ spleen, or tumor-infiltrating leukocytes were cultured in vitro for four to five hours. For the final 3 hours of the incubation time 0.2μl of golgi plug (BD Bioscience) and 0.13 μl of golgi stop (BD Bioscience) was added to allow accumulation of intracellular proteins. The cells were then treated with anti-CD16/32 (Fc block; clone 2.4G2; BD Pharmingen) and surface stained with the required surface antibodies (anti-mouse NK.1.1 (clone- PK136; BD Pharmingen), anti-mouse CD11b (clone-M1/70; BD Pharmingen), anti-mouse F4/80 (clone BM8; eBiosciences), anti mouse CD3e (clone 145-2C11; BD Pharmingen), anti-mouse CD11c (clone HL3; BD Pharmingen) anti-mouse CD226/DNAM-1 (clone TX42.1 Biolegend), or anti-mouse CD134/NKG2D (clone CX5 eBiosciences), for 25 minutes at 4°C. For live/ dead staining Live/Dead Fixable Aqua Dead cells staining kit (Invitrogen) was used along with surface stains. Cells were then washed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), and stained for the required intracellular antibodies (anti-mouse-IFNγ (clone XMG1.2; BD Pharmingen), anti-human and mouse granzyme-B (clone GB11; Invitrogen), anti-mouse IL1-β (clone NJTEN3; eBiosciences), or anti-mouse TNF (clone MP6-XT22; Biolegend) for 20-25 minutes at 4°C. For CD107a/b staining FITC-anti-mouse-CD107a (clone-1D4B; BD Pharmingen) and FITC-anti-mouse-CD107b (clone-ABL-93; BD Pharmingen) antibodies were used along with golgi plug and golgi stop for four hours and then the cells were treated with anti-CD16/32 (Fc block; clone 2.4G2; BD Pharmingen) and stained with surface markers.

Immunocytochemistry, western blotting and Elisa

Formalin-fixed tumor tissue samples were embedded in paraffin, sectioned and stained with polyclonal anti-RAE-1γ antibodies (clone AF-1136; R&D systems) and developed with the peroxidase- anti-peroxidase procedure (DAKO Co. Carpinteria, CA). RAE-1 expressing tumor cells were mixed with agarose, fixed in formalin and embedded in paraffin, and then were used as positive controls. For western blots and Elisa, proteins were isolated from PyVTu cell lines, tumors or organs after homogenizing them with Tissue Extraction Reagent I (Invitrogen) following the manufacturer’s protocols. The protein concentrations were measured using BCA Protein Assay Kit (Thermo Scientific) and BSA standards. Western blots for RAE-1 were performed with polyclonal anti-RAE-1γ antibodies (clone AF-1136; R&D systems, which also detects RAE-1δ, RAE-1α, RAE-β and RAE-1ε) at 0.2μg/ml concentration and anti-goat HRP secondary antibody (Santa Cruz biotech). The blots were developed using Super Signal West Pico Chemiluminescent Substrate (Thermo scientific). Mouse IL-1β ELISA Ready-SET-Go and Mouse IL-33 Ready-SET-Go ELISA kits (eBiosciences) were used to do determine IL-1β and IL-33 concentrations in tumor and organ lysates, following the manufacturer’s protocols

Results

PyV- induced tumors contain infiltrating functional NK cells

We found previously that cell lines established from salivary gland tumors of PyV-infected adult TCRβxδ KO mice express RAE-1, a ligand for the activating receptor NKG2D, but not other NKG2D ligands, such as MULT1 or H60 [11]. NK cells lysed the cell lines in vitro in an NKG2D-RAE-1-dependent manner, as anti-RAE-1 polyclonal antibody treatment substantially decreased NK cell-mediated killing of these tumor cell lines (Fig.1A). Similarly, peritoneal NK cells that were pre-activated by intraperitoneal injection of PyVTu cells for 2-3 days mediated reduced killing of the RAE-1 expressing PyVTu cell targets in vitro when they were taken from NKG2D KO or TCRβxNKG2D double KO mice compared to effector cells taken from the NKG2D-sufficient counterparts (Fig. 1B and C). Thus, specific interaction of NKG2D and RAE-1 played a major role in killing of the PyV- induced tumor cell lines.

Figure 1. NK cells kill PyVTu cells in a NKG2D-dependent manner.

In vitro cytotoxicity assays were performed with (A) PyVTu1 and PyVTu2 targets and spleen cell effectors from naïve SCID mice, with and without blocking polyclonal anti-RAE-1 antibodies; one of three similar experiments is shown; (B) PyVTu1 targets and pre-activated PEC effectors from B6 and NKG2D KO mice; (C) PyVTu1 target cells and pre-activated PEC effectors from TCRβ KO and TCRβxNKG2D KO mice. A representative of three experiments with similar results is shown.

Despite the ability of NK cells to kill PyV- induced tumor cell lines in vitro, NK cells seemed only to delay but not prevent PyV-induced tumor formation in vivo. This conclusion is based on the observation that, although PyV-infected TCRβxδ KO mice have tumors with longer latency time than mice that lack both T cells and NK cells (the E26 strain), most T cell-deficient mice which have NK cells eventually succumb to PyV-induced tumors [11].

The salivary gland is a major site for tumor development in adult PyV-infected TCRβxδ KO mice on the C57BL/6 background, although tumors at others sites were also seen. To understand the in vivo role of NK cells in tumor resistance, we first tested for the presence of NK cells in tumors developing in TCRβxδ KO mice by analyzing the cell populations infiltrating the salivary gland tissue at different stages post PyV infection and in PyV-induced salivary gland tumors. We found that the tumors had a high percentage of infiltrating CD3-NK1.1+ NK cells (~18% of infiltrating leukocytes, average of 11 tumors). Acute (8 days post PyV infection) and persistently infected (60 days post PyV infection) salivary gland tissues also had a higher percentage of CD3-NK1.1+ NK cell infiltrates than salivary glands from uninfected control mice (Fig. 2A and B). There were no significant differences in the percentages of NKG2D+ NK cells and in the levels of NKG2D expression on them (judged by the mean fluorescent intensity (MFI)) whether they were isolated from PyV-induced tumors or spleens of tumor bearing mice. NK cells from uninfected, acutely or chronically PyV- infected salivary glands also expressed similar levels of NKG2D (Fig. 2C).

Figure 2. Ex vivo tumors have functional NK cell infiltrates.

(A.) Percentage of NK cells in the spleen and in salivary gland tumor of tumor bearing mice (~4-8 months post PyV infection) (top) and in salivary gland tissues (pools of 3 mice) of naïve, day 7 (acute) and day 60 (chronic) PyV-infected mice (bottom). (B) Percentage of infiltrating NK1.1+CD3- NK cells in PyV-induced salivary gland tumors from TCRβxδ KO mice. Each dot represents a single tumor. (C) NKG2D expression on CD3- NK1.1+ NK cells in the spleen and tumor tissue of tumor bearing mice (top) and in salivary gland tissues (pools of 3 mice) of naïve, day 7 (acute) and day 60 (chronic) PyV-infected mice (bottom). (D) IFNγ (top), granzyme B (middle) production, and CD107a/b expression (bottom) by NK cells from spleens of naïve and tumor bearing mice and from tumor tissue shown by intracellular staining. The FACs plots were gated on CD3- NK1.1+ NK cells. A representative of three similar experiments is shown.

To assess whether the tumor infiltrating NK cells were functional we tested their ability to produce IFNγ and granzyme B. NK cells isolated from the tumors produced spontaneously more IFNγ compared to splenic NK cells. Summarizing the results of four experiments, we found that the percentage of IFNγ producing NK cells was on average 4.5-fold (+/−2.9) higher in the tumor infiltrating NK cells than in NK cells of the spleens of PyV-infected tumor bearing or uninfected mice (p=0.05). Granzyme B production by the tumor infiltrating NK cells was also significantly higher (48.3 %+/−12 of NK cells were granzyme B+) than by spleen NK cells from the same tumor bearing mice (24.5%+/− 5.5, p=0.01) or from naïve TCRβxδ KO mice (13.02%+/− 3.9, p=0.01) (Fig. 2D). CD107a and CD107b (LAMP-1 and LAMP-2) are markers of degranulation and often used as a measure of the cytotoxic capacity of NK and CD8 T cells. We found that tumor infiltrating NK cells expressed high levels of CD107a/b (16.3% +/−1.9 of the NK cells were CD107a/b+, summarizing the data of 3 experiments), similar to the NK cells from the spleens of the same tumor bearing mice (13.5%+/−7.3 CD107a/b +) (Figure 2D). Therefore we concluded that the tumor infiltrating NK cells were functional.

Freshly isolated PyV-induced tumor cells express little RAE-1 protein

Virus-infected cells and cancer cells are thought to evade NK cell recognition by modulating NKG2D ligand expression on their surface. Therefore we tested for the expression of NKG2D ligand RAE-1 on PyV-induced advanced tumors ex vivo. In contrast to the established PyVTu cell lines, primary ex vivo tumor cells did not express RAE-1 protein, as determined by either cell surface or intracellular antibody staining and flow cytometry or by immunohistochemistry (Fig. 3A and B). Nevertheless, RAE-1 RNA transcripts were still observed in these tumor cells (Fig. 3C), suggesting that either the RAE-1 protein expression was down regulated or that the NK cells selectively killed and eliminated the RAE-1 protein-expressing cells in-vivo at the advanced tumor stage.

Figure 3. Ex vivo PyV-induced tumors lack RAE-1 expression.

(A) RAE-1 (left) and MHC-I (right) expression on the cell surface (top) or inside the cells (bottom) of the PyVTu1 cell line and freshly removed PyV-induced salivary gland tumors (grey shaded histograms are isotype controls and open black histogram represents for RAE-1 and MHC-I antibody staining). Representatives of 3 tumor cell lines and 6 tumors are shown. (B) Immunohistochemistry of paraffin embedded section of a primary tumor and an established tumor cell line PyVTu1 stained with polyclonal RAE-1 antibody. (C) RAE-1 and β-actin transcripts in freshly removed PyV-induced salivary gland tumors (Tumor-1, Tumor-2, Tumor-3), determined by RT-PCR. The control lanes include the PyVTu1 cell line and control plasmid with RAE-1 insert.

PyV-induced tumors produce soluble factors that down-modulate RAE-1 expression

To test whether ex vivo PyV-induced tumors affect the expression of RAE-1 on PyVTu cells by making soluble factors we co-cultured the PyVTu cell lines with cells freshly isolated from primary tumor tissue in trans-well plates. This allowed soluble material produced by the ex vivo tumors to reach the PyTu cells without physical contact between the two kinds of cells. Three and a half days of co-culture with freshly isolated tumors decreased RAE-1 surface expression on the established tumor cell lines; the MFI of RAE-1 was significantly lower on these cells compared to untreated controls (Fig. 4A). Summarizing data obtained with three different ex vivo tumors, the RAE-1 MFA on the PyVTu1 cells decreased by 54.8% (+/−2.6) following a 3.5 day long co-culture, compared to untreated PyVTu1 cells. This effect was also observed after adding culture medium of freshly isolated ex-vivo tumor cells to the cultures of the PyVTu cell lines (Fig. 4B), and the magnitude of decrease in RAE-1 MFI was similar to the one observed in the transwell experiments, 43.6% (+/−18). Thus, soluble factors produced by the freshly removed tumor tissue were responsible for down-modulating RAE-1 expression on the tumor cell lines. In contrast, the expression of MHC class I or CD155 (ligands for other NK receptors) on the PyVTu cells did not change upon co-culture (Fig. 4C and D). The RAE-1 down-regulating effect of the soluble factors produced by freshly removed tumors increased with time of co-culture (Fig 4E). Western blots of proteins extracted from PyV tumor cell lines with or without co-culture with ex-vivo tumor infiltrating cells also showed a substantial decrease in RAE-1 protein (Fig.4F). The RAE-1 down-modulating effect appeared to be post-transcriptional, as there were no consistent and statistically significant changes in the amounts of RAE-1 transcripts measured by RT q-PCR after co-culture with freshly isolated tumor cells or after the addition of tumor culture fluid (Fig. 4G).

Figure 4. Down-modulation of RAE-1 by soluble factors derived from ex vivo PyV-induced tumors.

(A) Expression of RAE-1 on PyVTu cells with (open dark) and without (filled grey histogram) co-culture with ex vivo tumor tissues in transwell plates for three and a half days. The open grey histograms show unstained samples, the dashed line histograms show cells stained with irrelevant isotype control antibodies. The two plots show experiments with two different ex vivo tumors, the MFI values of RAE-1 with/without treatment are indicated on the top of the plots. (B) RAE-1expression on PyVTu cells with (open dark) and without (filled grey histogram) treatment with supernatants of ex vivo tumor tissues for three days. MFI is indicated as in (A). (C) MHC class I expression on PyVTu cells shown on (A) (top) and (B) (bottom). (D) DNAM-1 ligand CD-155 on PyVTu cells with (open dark) and without (filled grey histogram) co-culture with ex vivo tumor tissues in transwell plates (E) RAE-1 expression on PyVTu cells co-cultured with freshly removed PyV-induced tumor tissue for 1, 3 and 5 days (open histograms). Grey filled histograms are untreated controls. (F) Western blot of PyVTu1 and PyVTu2 cell lysates with or without co-culture with ex vivo tumors in transwell plates. Top shows RAE-1 protein detected by polyclonal antibodies to RAE-1, bottom is Hsp70 control. (G) RAE-1 specific mRNA expression in PyVTu cells untreated, co-cultured with ex vivo tumors (left) or treated with tumor supernatants (right) for 3.5 days. Results of qRT-PCR are expressed as fold changes compared to the untreated samples, using the ΔΔCt method. (H) In vitro cytotoxicity assays using as targets PyVTu1 (left ) and PyVTu2 (right) cells with or without co-culturing with ex vivo tumors for 3.5 days, and spleen cells of naïve SCID mice as effectors.

The decrease in RAE-1 expression on PyVTu cells resulting from exposure to the factors produced by ex-vivo tumors was associated with reduced sensitivity of these cells to NK cell-mediated killing when used as targets in vitro in cytotoxicity assays (Fig 4H). The decreased killing by NK cells demonstrated that the reduction in RAE-1 expression on the cell surface after exposure to the factors, although moderate in magnitude, is biologically relevant.

IL1-β, IL-1α, IL-33 and TNF administration decreases RAE-1 expression on PyVTu cells

Because we found that soluble factors from the tumors affected the susceptibility of PyVTu to NK cells, we initiated studies to examine cytokine production within the tumors. Multiarraycytokine Elisa analysis of fresh tumor lysates and intracellular staining of cells from the tumors indicated the presence of many cytokines, including IL-1β, IL-33, IL-6, TGF-β, TNF, MCP-1, MIP-1α, and MIP-1β. By ELISA the IL-1β concentrations in the fresh tumor lysates were 15-50 pg/mg and IL-33 concentrations were 17-37 pg/mg of total protein for three individual tumors that have been tested (Fig 5A). Of note is that gene expression studies on freshly isolated PyVTu cells revealed high expression of both the IL-1 receptor (IL1R1) and the IL-33 receptor (ST2), and expression of these receptors on the cell surface was confirmed by flow cytometry (Fig 5B).

Figure 5. Pro-inflammatory cytokines regulate RAE-1 expression on PyVTu cells.

(A) IL-33 and IL-1β produced by ex vivo tumors, shown by ELISA assays done with total lysates of the ex vivo tumor tissue. One experiment of two with 3 tumors each is shown. (B) Surface expression of IL-33 receptor ST2 and IL-1 receptor on PyVTu1 and PyVTu2 cell lines (grey histogram represents isotype control, the open histogram shows staining with ST2 or IL-1R-specific antibodies). (C) RAE-1 expression on PyVTu cells with (open dark) and without (filled grey histogram) co-culture with IL-α, IL-1β, IL-33 (at 2ng/ml each) or TNF (5ng/ml) for 3 days (top) or 6 days (middle). The open grey histograms show unstained samples, the dashed dark histograms show cells stained with irrelevant isotype control antibodies. Bottom Table: MFI of RAE-1 on the untreated/ treated cells. A representative of 4 experiments is shown. (D) Western blot of PyVTu1 cell lysates treated with 2ng/ ml IL-1α, IL-1β, IL-33 or 5ng/ml TNF for 6 days or left untreated (the same experiment as shown on C). (E) RAE-1 transcript after treatment with IL-1α, IL-1β, IL-33 or TNF as in (C) for 3 (left) or 6 days (right). Mean values and sd of 3 samples/ group are shown. (F) In vitro cytotoxicity assays using naïve SCID mouse spleen cells as effectors and PyVTu cell targets that were treated previously with the indicated cytokines for the indicated time. A representative of three similar experiments is shown here.

The cytokines found in the tumor environment were tested for their ability to modulate RAE-1 expression on PyVTu cells. Administration of exogenous recombinant mouse IL-1 β, IL-33, IL- α or TNF for three days or six days significantly decreased RAE-1 surface expression on PyVTu cells, as measured by flow cytometry (Fig. 5C & Table-1). Summarizing data of three independent experiments, three days treatment with IL-1α decreased RAE-1 MFI on PyVTu cells by 45.5% (+/−6.8), with IL-1β by 43% (+/−3.1), with IL-33 by 41.9% (+/−2.6), and with TNF by 39.5% (+/−3.7). Six days treatment with IL-1α results in decrease of RAE-1 MFI on PyVTu cells by 66.5% (+/−4.2), with IL-1β by 66.5% (+/−6), with IL-33 by 59.3% (+/−4.8), and with TNF by 51.6% (+/−10). In contrast, many other cytokines, including IL-10, IL-12, and TGF-β (Supplemental Fig. 1) did not change RAE-1 surface expression. IL-6 was found to be produced by both established PyVTu cell lines and primary tumors. Addition of IL-6 blocking antibody, however, did not change RAE-1 surface expression (data not shown) on PyVTu cells.

Treatment of PyVTu cell lines with IL-1α, IL-1β, IL-33 and TNF for six days (two passages) also resulted in substantial decrease in total RAE-1 protein of PyVTu cells as seen on Western blots performed with protein lysates (Fig 5D) using polyclonal antibody to RAE-1. The effects of the cytokines on RAE-1 expression were mainly post-transcriptional, similarly to the effects of cell culture supernatants of ex vivo primary tumors, as we didn’t observe consistent changes in the levels of RAE-1 transcripts in cells treated with different cytokines after 3 or 6 days post cytokine treatment (Fig. 5E). Treatment with the cytokines didn’t decrease the expression of MHC I or ligands of other activating NK receptors, such as the DNAM-1 ligand CD155 (Supplemental Fig. 1B & C). The only change noted in addition to RAE-1 down regulation was an up-regulation of MHC class I by TNF treatment; others have reported that TNF can up-regulate MHC-I expression [40]. PyVTu cell lines treated with these cytokines also became resistant to NK cell- mediated lysis (Fig. 5F).

Tumor-infiltrating activated macrophages secrete IL1 β and TNF

In PyV-infected TCRβxδ KO mice the tumor infiltrates contained monocytes (CD11b+ F4/80-), activated macrophages (CD11b+ F4/80+), dendritic cells (CD11c+CD11b+), NK cells and B cells. The tumor-infiltrating CD11b+ F4/80+ macrophages produced high amounts of IL-1β and TNF (Fig 6). These cells also expressed high levels of CD11c on their surface. Tumor infiltrating NK cells were found to produce low levels of TNF, but no IL-1β (data not shown). Based on these data we conclude that tumor infiltrating CD11b+F/80+ macrophages are the primary producers of the inflammatory cytokines IL-1β and TNF in the PyV-induced salivary gland tumor tissue of TCRβxδ KO mice.

Figure 6. TNF and IL-β are mostly produced by tumor infiltrating macrophages.

Intracellular IL-1β and TNF staining of cells prepared from ex vivo tumors. IL-1β+ or TNF + cells were mostly F4/80+ CD11b+ macrophages.

Decrease in RAE-1 expression by proinflammatory cytokines is dependent on NFκB

The finding that multiple cytokines have a negative regulatory role in RAE-1 expression of PyV-induced tumor cells raised the question whether these factors act additively, synergistically, or redundantly, by activating the same pathways Addition of IL-1β and IL-33 together in comparison to treatment with IL-1β or IL-33 alone (Fig. 7A) did not result in enhanced RAE-1 suppression. Thus, these two cytokines had neither additive nor synergistic effects, suggesting that common pathways downstream of the receptors for these two cytokines were involved in the down-regulation of RAE-1. The inflammatory cytokines, including IL-1, IL-33 and TNF, that decrease RAE-1 expression on PyVTu cells are all known to be potent activators of NFκB. Inhibition of the NFκB pathway by using the IKK2 inhibitor SC-514 at 50μM concentration partially abolished the RAE-1 down-regulating effect of these cytokines (Fig 7B). Western blots performed with PyVTu cells treated with SC-514 and IL-1β confirmed these findings (data not shown). Similar effects to SC-514 were also observed with the IKK2 inhibitor type VII (a cell-permeable benzamido-pyrimidine compound that acts as a potent, selective inhibitor of IKK) (data not shown). These data taken together suggest that the effects of the proinflammatory cytokines on RAE-1 are, at least in part, NFκB pathway-dependent.

Figure 7. Effect of multiple cytokines on RAE-1 expression is mediated by NFkB pathways.

(A) RAE-1 expression on PyVTu cells treated with IL-1β(5ng/ml), IL-33 (5ng/ml) or TNF (10ng/ml) individually or with a combination of these cytokines for 3 days. The MFI of RAE1 without/with treatments is indicated. (B) RAE-1 expression on PyVTu cells treated with the IKK2 inhibitor SC-514, IL-1α, IL-1β and a combination of each cytokine (2ng/ml) with SC-514 for 2 days. The MFI of RAE-1 without/with treatments is indicated.

Discussion

In PyV-infected αβ T cell-deficient mice used as a mouse model of virus-induced tumor formation, NK cells and γδ T cells together prevent tumor development. Moreover, although tumors develop in mice devoid of all T cells (αβ T and γδ T), there is an increased latency time if the mice have functional NK cells (11). The studies described here were initiated to question why NK cells can only delay but not prevent PyV-induced tumor formation in TCRβxδ KO mice. We found that, in contrast to RAE-1-expressing cell lines established from PyV-induced salivary gland tumors, large salivary gland tumors freshly removed from the host lack RAE-1 expression. We then demonstrated that cells in the tumor environment, mostly tumor-infiltrating macrophages, produce proinflammatory cytokines including IL-1α, IL-1β, IL-33 and TNF, which can each down-regulate RAE-1 on the tumor cells, resulting in the resistance of tumor cells to NK cell-mediated killing. These findings taken together suggest a novel immune evasion mechanism mediated by tumor infiltrating inflammatory cells.

There are several ways NK cells may delay the formation and outgrowth of virus-induced tumors. They may facilitate virus clearance by killing virus-infected cells or by secreting antiviral cytokines, and the consequential reduction of persistent virus load, in turn, may lead to reduced tumor outgrowth. NK cells can also have immunoregulatory roles, affecting the function of other cell types that participate in antiviral immune responses. Alternatively, NK cells may directly kill tumor cells. Our data suggest that NK cells do not regulate PyV load, as SCID mice with or without NK cell depletion had no difference in viral titers after PyV infection [39]. Moreover, comparison of the virus load of PyV-infected TCRβ KO mice and NK cell-depleted TCRβ KO mice did not show significant differences in virus load, as determined by qPCR measuring viral genome copies (data not shown). Therefore the NK cell responses in T cell-deficient mice infected with PyV seem likely to be primarily directed against the tumor cells.

Our studies suggest that NKG2D-NKG2D ligand (RAE-1) interactions are essential for the killing of PyVTu cells by NK cells, as blocking antibodies specific for NKG2D or using NKG2D KO NK cells prevent PyVTu killing in vitro in Cr release assays. In the absence of NKG2D-NKG2D ligand interactions, however, NK cells can be still activated by the PyVTu cells to produce IFNγ and granzymes, suggesting that other surface molecules in addition to NKG2D ligands may also contribute to NK cell activation by PyVTu cells. Indeed PyVTu cells expressed CD155, which is a ligand for the activating receptor DNAM-1 (data not shown).

The failure of NK cells in vivo to protect against tumor formation in the absence of T cells, despite delaying the appearance of tumors, could be due to several factors. NK cells may not get to the tumor site, they may become dysfunctional within the tumor, or the tumor cells may change their phenotype to avoid recognition. Our study demonstrated that the latter scenario is likely, because, in mice bearing advanced PyV-induced tumors, functional NK cells encounter tumor cells that lack RAE-1 on their surface. When the freshly removed salivary gland tumor cells that lack RAE-1 surface proteins are cultured in vitro, after a few passages the cells become RAE-1+ by surface staining. This finding suggests that either rare RAE-1+ tumor cells selectively proliferate under the in vitro culture conditions or that removal from the in vivo tumor tissue releases them from the effect of factors that inhibit RAE-1 expression. Our data are consistent with the latter scenario, as the cultured PyVTu cell population gradually shifts with time as a single peak with higher and higher MFI for RAE-1 on the plots obtained by FACS analysis.

NKG2D is an activating receptor controlling the function of NK cells, γδ T cells and some αβ T cells. Therefore, it is not surprising that the expression of ligands that engage NKG2D is tightly regulated on cells. These ligands, including RAE-1, H60 and MULT-1 in mice and MICA, MICB and ULBPs in humans, are not expressed on normal cells, but their expression is induced by various pathological conditions representing “cellular stress”, including infections and oncogenic transformation. DNA damage is one manifestation of cellular stress that was shown to lead to NKG2D ligand expression, but the regulation of NKG2D ligand expression is not well understood. PyV infection in vivo does not induce detectable RAE-1 expression on spleen cells or salivary gland tissues when taken at the acute phase of infection in SCID mice with high virus load. This finding includes cell types which do not support PyV replication, such as lymphocytes, and cells, for example macrophages and DCs, which do. Mouse embryonic fibroblasts (MEF), however, express a basal level of RAE-1, and this level of expression is increased after PyV-infection (data not shown). We don’t know if tumors at early stages of their development express significant levels of RAE-1 protein, and it may be very difficult to find early stage incipient tumors and test their RAE-1 expression, as it is likely that RAE-1+ tumor cells are rapidly eliminated by infiltrating NK cells.

It has been previously observed that tumors (human and murine) can evade NK cell-mediated killing via down-regulation of NKG2D ligands on their surface. This can occur by shedding soluble NKG2D ligands, and human tumors were shown to shed MICA or ULBP2 [41-44]. Sequestration of MICA inside the cell in the ER was seen in human melanomas or in HCMV-infected cells, in this latter case due to complex formation with the viral protein UL16 [24,25,44,45]. PyV-induced tumors did not shed RAE-1 detectable by ELISA into the culture medium (data not shown) and the freshly removed tumors lacked RAE-1 both on their surface and intracellularly (Fig. 3A).

In this study soluble factors secreted by the tumor tissue were found to have a RAE-1 down-regulating effect, suggesting that in vivo these secreted factors may suppress RAE-1 expression, thereby aiding tumor escape from NK cell-mediated surveillance. By analyzing the factors produced by the ex vivo tumor tissues a group of cytokines was identified, including IL-1, IL-33 and TNF, each with RAE-1 down-regulating activity. Administration of IL-1α or IL-1β at as low as 500 pg/ml concentration was effective in reducing RAE-1 expression of PyVTu cells in culture. As far as we know, these cytokines were not previously known to modulate NKG2D ligand levels. IFNγ was reported to decrease H60 (but not RAE-1) expression on murine sarcomas or on human melanomas and glioma [36] , but IFNγ was not among the cytokines abundant in the tumor environment in our model.

IL-1α, IL-1β, IL-33 and TNF seemed to act post-transcriptionally, as no consistent and significant decrease was seen in RAE-1 mRNA levels after treatment with these cytokines. Human NKG2D ligands MICB and ULBP3 were reported to be regulated by viral microRNAs post-transcriptionally in EBV-, KSHV-, HCMV, JC and BK virus-infected cells [19]. PyV is known to encode a miRNA with unknown function [46]. Moreover, several cellular miRNAs are differentially expressed in PyVTu cells with or without exposure to ex vivo tumor tissueproduced soluble factors (data not shown). Future studies will test the involvement of these microRNAs in the modulation of RAE-1 levels in the PyV-induced tumor model.

The cytokines do not act by enhancing RAE-1 shedding into the medium. PyVTu cells cultured with or without the cytokines did not have soluble RAE-1 detectable by ELISA in their culture media (data not shown). Moreover, we have performed surface RAE-1 staining with limiting amounts of RAE-1-specific antibodies on PyVTu cells incubated with culture supernatants of IL-1 or IL-33-treated or –untreated tumor cells to test whether the binding of the anti-RAE-1 antibody to RAE-1 is outcompeted with soluble RAE-1 in the culture supernatants. We have also done Western blots of homogenates of freshly removed PyV-induced tumor tissue, which would have detected RAE-1 that is shed in the tumor environment. None of these experiments indicated the presence of appreciable amounts of soluble RAE-1, thus making RAE-1 shedding highly unlikely.

In our studies the effect of IL-1α, IL-1β, IL-33 and TNF was not additive, as adding combinations of these cytokines together did not result in greater reduction of RAE-1 expression than adding just one of these cytokines. These data suggest that the downstream pathways mediating the specific RAE-1 down-regulating effect of the cytokines may be shared. Activation of the PI3 kinase pathways was reported to cause RAE-1 up-regulation in some models [47]. IL-1 can activate the PI3 kinase pathways, but has a RAE-1 down-regulating effect on PyVTu. Addition of the PI3 kinase inhibitor LY294002 along with IL-1β or IL-33 caused a more pronounced down regulation of RAE-1 than LY294002 or IL-33 treatment alone (data not shown). These data suggest that the PI3K pathways don’t play a major role in mediating the effects of IL-1 and IL-33 in the studies reported here, although PI3K activation can reduce RAE-1 on PyVTu cells, contrary to its effects in other models [47]. Our studies with NFkB inhibitors, on the other hand, suggest a major role for the NFkB pathways in down-regulating RAE-1 expression, and this observation is consistent with known signaling pathways activated by IL-1α, IL-1β, IL-33 and TNF.

The observations that the pro-inflammatory cytokines regulate RAE-1 expression on the tumor cells by an NF-κB-mediated mechanism, and that RAE-1 expressions seems to be down-regulated post-transcriptionally are not contradicting each other. NF-κB is known to regulate the expression of several microRNAs (e.g. miR18a, miR21, miR34a, miR146, and miR155 [48]). Some of these microRNAs, in turn, inhibit NF-κB expression, acting as components of a negative feedback loop. Most microRNAs known to have multiple targets, therefore we speculate some of the NF-κB-induced microRNAs may prevent the transcription of RAE-1 messages in the PyV-induced tumor cells.

The fact that four distinct proinflammatory cytokines produced in the tumor environment act in a similar fashion by reducing RAE-1 expression on the surface of tumor cells suggest that this may be a biologically important, therefore redundantly regulated step, and may represent a major mechanism of immunoevasion. Although the decrease in the expression of RAE-1 in response of the proinflammatory cytokines is gradual and modest (judged by RAE-1 MFI on the treated tumor cells), this change seem to be important biologically, as it leads to a decrease target sensitivity to NK cells.

The predominant sources of IL-1α, IL-1β, IL-33 and TNF in the tumor tissue were tumor-infiltrating macrophages. Macrophages associated with advanced tumors exhibit a wide diversity. Some, called M1 macrophages, are classically activated and produce mediators typical of acute inflammation. Others, the M2 macrophages, have immunosuppressive roles and thereby promote tumor growth [49]. In our model the pro-inflammatory macrophages may contribute to tumor progression by producing IL-1 and TNF, which can promote immune-evasion of the tumors by making them to display less RAE-1 and escape NK cell-mediated killing.

As an inflammatory environment is common for many tumors, especially the virus-induced ones, the capacity of the inflammatory tumor environment to reduce the susceptibility of tumor cells to NK cell-mediated killing may be a general phenomenon and may provide a target for therapeutic interventions.