Based on the evidence that IL-17 is a key cytokine involved in various inflammatory diseases, we explored the critical role of IL-17-producing γδ T cells for tumor development in tumor-bearing mouse model. IL-17−/− mice exhibited a significant reduction of tumor growth, concomitantly with the decrease of vascular density at lesion area, indicating a pro-tumor property of IL-17. Among tumor-infiltrating lymphocytes (TIL), γδ T cells were the major cellular source of IL-17. Analysis of TCR repertoires in TIL-γδ T cells showed that circulating γδ T cells, but not skin resident Vγ5+γδ T cells, produced IL-17. Neutralizing antibodies against IL-23, IL-6, and TGF-β, which were produced within the tumor microenvironment, inhibited the induction of IL-17-producing γδ T cells. IL-17 production by tumor-infiltrating γδ T cells was blocked by anti-γδTCR or anti-NKG2D antibodies, indicating that these ligands, expressed within the tumor microenvironment, are involved in γδ T-cell activation. The IL-17-producing TIL-γδ T cells exhibited reduced levels of perforin mRNA expression, but increased levels of COX-2 mRNA expression. Together, our findings support the novel concept that IL-17-producing γδ T cells, generated in response to tumor microenvironment, act as tumor-promoting cells by inducing angiogenesis.
In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop anti-tumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. The tumor microenvironment, which is mainly composed of tumor cells, stromal cells, and tumor-infiltrating immune cells, is entirely different from noncancerous tissues. This unique microenvironment potently inhibits immune responses against tumor cells via various soluble mediators and contact-dependent mechanisms 1, 2. Previously, it was suggested that T-cell responses within the local tumor tissue are completely inhibited. However, this concept was abandoned following the discovery of the regulatory T-cell and Th17 cell subsets, which are activated rather than suppressed in the tumor microenvironment via TGF-β and/or IL-6. Thus, the tumor microenvironment is conducive to IL-17 production. In fact, it has been shown that IL-17 is produced in human and murine tumor tissues 3–5. Tumor cells promote neo-vascularization into tumor tissues through hyper-production of angiogenic factors, which also support their own abnormal proliferation and survival 6. It has been reported that tumor cells over-expressing IL-17 significantly promote new vessel growth into the tumor tissues 5; however, physiological effects of IL-17 on tumor progression remain to be defined.
Th17 differentiation from naïve CD4+ T cells is regulated by TGF-β and IL-6. Proliferation, maintenance and full maturation of these cells are controlled by IL-23 7–9. Recently, it has been shown that IL-17 is produced by diverse T-cell subsets, including CD4+ T cells, CD8+ T cells, NKT cells and γδ T cells 9–12. Physiologically, IL-17 plays a pivotal role in protecting the host against certain infectious microorganisms through the recruitment of neutrophils, by increasing the expression of multiple chemokines 13, 14. Furthermore, IL-17 has been implicated in many inflammatory and autoimmune disorders such as colitis, rheumatoid arthritis, and asthma 8, 9, 14, 15.
Th17 cells, as a helper T-cell subset distinct from Th1, Th2, and Treg, have a critical role in regulating the immune balance in co-operation with other T-cell subsets. We have proposed that the induction of Th1-dominant immune responses is an essential step in inducing strong anti-tumor immunity in response to adjuvants or tumor-specific Th1 cells 16–20. However, within the tumor microenvironment, the balance between IL-12 and IL-23, which directly reflects Th1 and Th17 cell responses, is shifted toward IL-23 via activation of STAT3 21, 22. Much attention has been paid to the finding that tumor stromal cells and tumor-associated macrophages, in part by producing IL-23, mainly contribute to the induction of cancer by promoting inflammation, whereas T cells suppress tumor development as part of the immunosurveillance system 23–29. Recent research has provided evidence for the involvement of T cells in promoting tumor progression 28–35; however, the precise effects of T cells and the cytokines they produce on tumorigenesis remain controversial.
In this paper, we demonstrate that (i) IL-17 is a pro-tumor cytokine, which supports tumor growth by promoting angiogenesis; (ii) γδ T cells, but neither TCRαβCD4+ T cells nor TCRαβCD8+ T cells, are the major cellular source of IL-17 in tumor-infiltrating lymphocytes (TIL); (iii) blood circulating γδ T cells, but not skin-resident Vγ5+ γδ T cells, infiltrate into the tumor site; (iv) tumor-infiltrating γδ T cells differentiate into IL-17-producing cells but downmodulate their cytotoxic activity within the tumor microenvironment; (v) IL-17 produced by tumor-infiltrating γδ T cells promotes tumor progression by inducing angiogenesis. Thus, this paper proposes the novel concept that IL-17-producing γδ T cells play a crucial role as tumor-promoting T cells during tumor development. These findings provide new insights that can be employed for the development of novel cancer immunotherapies.
IL-17 promotes tumor development by enhancing angiogenesis
It has been demonstrated that IL-17 is expressed in tumor microenvironments 3–5. The precise role of IL-17 during tumor development, however, remains unclear. Here, we examined the physiological role of IL-17 in the growth of the transplantable methylcholanthrene (MCA)-induced tumor cell line CMS-G4. We found that the growth of CMS-G4 tumor cells was markedly decreased in IL-17−/− mice compared with WT mice (Fig. 1A). We further examined IL-17 contribution to the growth inhibition of transplantable tumor cell lines. Firstly, we considered the possibility that immune responses in IL-17−/− mice at tumor site were decreased as compared with WT mice. However, the ratio and absolute numbers of distinct T-cell subsets and prevalence of IFN-γ-producing cells among TIL, including CD4+, CD8+, and γδ T cells were similar between WT and IL-17−/− mice. These findings suggested that the reduction of tumor growth in IL-17−/− mice was not due to impaired immune responses (Supporting Information Fig. 1). Secondly, to investigate the possibility that IL-17−/− mice failed to produce certain tumor-promoting factors, we surveyed the vascular density in tumor tissues obtained from WT and IL-17−/− mice. Immunohistochemical analyses revealed that the mean numbers of blood vessels in the tumor tissues were markedly decreased in IL-17−/− mice (Fig. 1B and C). In addition, IL-17 markedly increased the transcripts of Ang-2 and vascular endothelial growth factor (VEGF), which are angiogenesis-related genes, in CMS-G4 fibrosarcoma cells in vitro (Fig. 1D). These findings indicate that IL-17 can promote tumor progression by the enhancement of angiogenesis.
IL-17 within tumors is predominantly produced by γδ T cells
To identify the cellular source of IL-17 production during tumor development, we prepared TIL from tumor-bearing mice 2 wk after CMS-G4 inoculation. IL-17 production by distinct subsets of lymphocytes was analyzed by intracellular cytokine staining assay after stimulation with immobilized anti-CD3 and anti-CD28 mAb in vitro. We found that IL-17 was predominantly produced by γδ T cells rather than CD4+ T cells or CD8+ T cells infiltrated into the tumor tissues (Fig. 2A and B). Similar results were obtained in the case of TIL of CMC-1 skin carcinoma or CT26 colon carcinoma (Supporting Information Fig. 2). The majority of IL-17-producing cell population was γδ T cells, but not CD4+ T cells or CD8+ T cells, even in the MCA-induced primary tumor tissues, though CD4+ T cells preferentially accumulate into the tissues (Fig. 2C and D). Furthermore, more than half of the γδ T cells infiltrating into the tumor tissues secreted IL-17, but these cells produced negligible levels of IFN-γ (Fig. 3A). These results demonstrate that tumor-infiltrating IL-17-producing γδ T cells, but not CD4+ T cells or CD8+ T cells, play a critical role in promoting tumor growth by influencing angiogenesis.
It has been reported that the Vγ5+ subset of γδ T cells resides in epithelial tissues, including skin, and represents a major T-cell subpopulation within this location. In our experimental model, transplantable tumor cells were inoculated into the skin of BALB/c mice. Therefore, we hypothesized that skin-resident Vγ5+ γδ T cells might be expanded within the tumor tissue and produce IL-17. Unexpectedly, however, FACS analysis showed that most γδ T cells infiltrating into transplanted tumor tissues were not Vγ5+ γδ T cells, whereas intraepithelial T cells within the skin were predominantly Vγ5+ γδ T cells (Supporting Information Fig. 3). We further examined the distribution of TCR-Vγ gene expression in normal skin, spleen, and tumor tissues by RT-PCR. Consistent with the results of the FACS analysis, TCR-Vγ5 mRNA was detected in normal skin, but not in tumor tissue or spleens of tumor-bearing mice (Fig. 3B). γδ T cell subsets with diverse TCR-Vγ gene expression were present within the tumor tissues, and composition of γδ T cells in tumor tissues was the same as found in spleen (Fig. 3B). These findings suggest that circulating γδ T cells but not skin resident γδ T cells migrated into the tumor tissues.
Recently, IL-17-producing γδ T cells have been defined as a CD27−CCR6+ γδTCR+ population 36–38. Accordingly, we analyzed the expression of these markers on IL-17-producing TIL and found that IL-17-producing γδ T cells did not express the CD27 molecule; however, both CCR6+ and CCR6− γδ T cells produced IL-17 in the tumor site (Fig. 3C). Although CCL20, a ligand of CCR6, is expressed by the tumor cells (data not shown), higher numbers of CCR6− γδ T cells migrated into tumor tissues in comparison with CCR6+ γδ T cells. Thus, these results indicate that a subset of the IL-17-producing γδ T cells in TIL was the pre-differentiated CD27−CCR6+ γδ T-cell population, however, most CCR6− γδ T cells might differentiate into IL-17-producing cells after exposure to the tumor microenvironment.
Generation of IL-17-producing γδ T cells requires the local tumor microenvironment
Next, we investigated the precise mechanisms involved in γδ T-cell accumulation and activation to produce IL-17. As shown in Fig. 4, although the ratio of IL-17-producing γδ T cells was markedly increased in tumor-growing skin tissue compared with normal skin, the ratio of IL-17-producing γδ T cells in draining lymph nodes and spleen of tumor-bearing mice was not different from that of normal mice. These results suggest that the local tumor site provides a microenvironment that is conducive to the induction of IL-17-producing γδ T cells.
Given that we have detected mRNA expression of IL-17-inducing cytokines such as IL-6, TGF-β, and IL-23 in the tumor tissue (Supporting Information Fig. 4), we next examined the effect of mAb against IL-6 receptor (IL-6R), TGF-β, or IL-23p19 on the induction of IL-17-producing γδ T cells in vivo. As shown in Fig. 5A, the mice treated with anti-IL-6R mAb, anti-TGF-β mAb, or anti-IL-23p19 mAb, partially but significantly reduced the ratio of IL-17-producing γδ T cells that migrated into the tumor tissue. In addition, we further showed that tumor-infiltrating γδ T cells, in the absence of TCR stimuli, markedly produced IL-17 in response to IL-23 in vitro, whereas IL-6 plus TGF-β did not induce IL-17 production (Fig. 5B). Therefore, these findings indicate that IL-6 and TGF-β were involved in the differentiation of tumor-infiltrating γδ T cells into IL-17-producing cells, and that IL-23 had a strong impact on IL-17 production by the differentiated γδ T cells.
γδTCR- and NKG2D-mediated signaling is critical for IL-17 production in the tumor microenvironment
T-cell activation typically requires stimulation via antigen-specific TCR and co-stimulatory molecules. To determine whether TCR-mediated recognition of specific antigen is essential for IL-17 production by γδ T cells, we first prepared a single cell suspension of CMS-G4 tumor tissues and cultured these cells in high-density cell culture conditions in the absence of exogenous stimuli. By using intracellular cytokine staining, we detected IL-17-producing cells only in γδ T cells (Fig. 5C). Thus, we measured IL-17-producing cells as IL-17-producing γδ T cells in this experimental condition. Since we detected NKG2D on tumor-infiltrating γδ T cells (Supporting Information Fig. 5), we added anti-γδ TCR or anti-NKG2D mAb in the cultures and evaluated the prevalence of IL-17-producing cells. As shown in Fig. 5D, IL-17 production was profoundly blocked by anti-γδ TCR mAb, and partially blocked by anti-NKG2D mAb. These findings suggest that γδ TCR engagement is essential for IL-17 production by tumor-infiltrating γδ T cells within the tumor microenvironment and NKG2D enhances these effects.
Tumor-infiltrating IL-17-producing γδ T cells play a crucial role in promoting tumor development
Our findings demonstrate that tumor-infiltrating γδ T cells exhibit an effector cell phenotype: NKG2D+, CD44high and CD62Llow (Supporting Information Fig. 5 and Fig. 6A). We isolated CD44high and CD62Llow γδ T cells from the spleen or tumor tissues and performed RT-PCR analyses to evaluate mRNA expression of effector molecules. As shown in Fig. 6B, γδ T cells isolated from tumor tissues expressed higher levels of Th17-related molecules, such as RORγt and IL-17 in comparison with γδ T cells from the spleen; however, mRNA levels of perforin, a key molecule for cytotoxic function, was reduced, whereas mRNA levels of COX-2, which is well known as a tumor-promoting factor, was increased in tumor-infiltrating γδ T cells compared with splenic γδ T cells. These findings indicate that IL-17-producing tumor-infiltrating γδ T cells had a role as tumor-promoting cells, i.e. lost their cytotoxic activity and instead acquired the capacity to express tumor-promoting molecules such as IL-17 and COX-2.
In the current study, we first demonstrated that γδ T cells, rather than αβ T cells, were the major cellular source of IL-17 and exhibited properties of tumor-promoting cells in both transplantable sarcoma model and MCA-induced primary tumor model. In the transplantable tumor model by i.d. inoculation of CMS-G4 fibrosarcoma cell line, we showed that tumor-infiltrating γδ T cells were differentiated into IL-17-producing cells by exposing with IL-6 and TGF-β. Furthermore, it was found that the differentiated γδ T cells produced substantial levels of IL-17 in response to IL-23. In addition to the soluble factors, ligands of γδTCR and NKG2D, expressed within the tumor microenvironment, critically activated tumor-infiltrating γδ T cells, and significantly induced IL-17 production by the cells. IL-17-deficient mice markedly reduced angiogenesis and subsequent tumor progression, suggesting the significant roles for tumor growth of IL-17-producing γδ T-cell-dependent inflammatory responses in the microenvironment.
Although prior studies have reported that CD4+ or CD8+ T cells were implicated in IL-17 production in tumor-bearing hosts 3, 34, 35, these reports have not examined γδ T cells. In our experiments, we showed that whereas small numbers of CD4+ T cells produced IL-17, there were much more IL-17-producing γδ T cells in the transplanted tumor tissues (Fig. 2A and B). Moreover, this finding was generalized by detecting the prevalence of IL-17-producing γδ T cells, even in the established primary tumor tissues, induced by MCA injection (Fig. 2C and D). As shown in Supporting Information Fig. 2, we also obtained the same results in the transplantable tumor model with skin and colon carcinoma cells. Therefore, these findings provide clear evidence that γδ T cells would represent a dominant IL-17-producing lymphocyte subset during physiological tumor development. Consistently, previous papers demonstrated that a substantial number of γδ T cells were observed in human breast, prostate, and renal cancer tissues 39. As we confirmed that human γδ T cells differentiated into IL-17-producing cells, at least in the presence of IL-6, TGF-β and IL-23 (data not shown), IL-17 might be produced by γδ T cells in these types of cancer. However, recent studies have shown that CD4+ T cells were the main cellular source of IL-17 in human ovarian cancer tissues 40, 41. Therefore, in human cases, it is possible that the IL-17-producing cell subsets may vary among organs depending on the type of cancer.
In the latest report, Wu et al.42 suggested that IL-17 was produced by γδ T cells in addition to CD4+ T cells in the infection-induced colonic carcinogenesis model. However, they evaluated IL-17 production only at the early inflammatory phase during carcinogenesis, but not in established cancer. In addition, because of the infection-induced carcinogenesis, there is the possibility that bacterial stimuli generated IL-17-producing γδ T cells in the colon. In this current study, we first demonstrated that carcinogen-induced established tumor microenvironment preferentially induced IL-17-producing γδ T cells without pathogenic stimuli. In addition, we have already detected the prevalence of IL-17-producing γδ T cells in MCA-induced inflammatory sites and demonstrated the reduction of tumor incidence in IL-17−/− mice by MCA-induced carcinogenesis model (unpublished data). These findings strongly suggest that γδ T cells would be the subset responsible for controlling both tumor initiation and development in our model. We have clearly demonstrated that the established tumor progression was significantly diminished in IL-17−/− mice, due to a decrease of angiogenesis. Consistent with this finding, prior studies have shown that substantial IL-17 expression levels in human cancer tissues were correlated with increased vascularity 4, 5. Moreover, our findings are consistent with other reports that IL-17 directly influenced on the proliferation and survival of tumor cells and that IL-6-STAT3 pathway promoted tumor growth in IL-17-dependent manner 34, 43. However, it is an unresolved controversial issue whether IL-17 acts as a pro- or anti-tumor agent. Indeed, it was reported that B16 melanoma cells formed increased numbers of lung metastatic foci in IL-17−/− mice compared with WT mice 44. Thus, in the lung metastasis model, IL-17-induced inflammatory responses appears to be involved in anti-tumor immunity rather than tumor formation; whereas in cutaneous tissues, chronic inflammation mediated by IL-17 may facilitate tumor development, which is supported by our findings. In addition, in our intradermal transplantable tumor models, the growth of CMS-G4 sarcoma, CMC-1 skin carcinoma, and CT26 colon carcinoma cells, but not B16 melanoma cells were inhibited in IL-17−/− mice compared with WT mice (data not shown). This observation suggests that, in the case of B16 melanoma cells, IL-17 appears not to be involved in the promoting tumor growth. Resolving the mechanisms underlying these discrepancies would shed light on the regulation of anti-tumor immunity.
It has been demonstrated that T cells, including αβTCR+CD4+, αβTCR+CD8+, and γδTCR+ T cells, play a pivotal role in tumor immunosurveillance to eliminate tumor cells 23–29. Recently, it has been shown that αβTCR+CD4+ and αβTCR+CD8+ T cells also act as tumor-promoting cells in various mouse models 28–35. However, the previous studies have not examined a pro-tumor function of γδ T cells. Here, we show that circulating Vγ5− γδ T cells preferentially accumulate in the tumor site, and then convert into IL-17-producing pro-tumor cells at the skin tumor local site, though it has been reported that the skin-resident Vγ5+ γδ T-cell subset plays an essential role in immune surveillance during skin carcinogenesis 27. We also show a decreased expression of perforin and an increased expression of COX-2 in the infiltrating γδ T cells, indicating a pro-tumor cell phenotype for tumor development. To our knowledge, this is the first report demonstrating tumor-promoting properties of γδ T cells, which is quite different from the generally reported tumor-inhibiting properties of skin-resident Vγ5+ γδ T cells.
Recent studies have shown that tumor antigen-specific Th17 or Tc17 cells, which were induced in vitro, were converted into IFN-γ-producing cells after transfer into tumor-bearing mice and subsequently eradicated tumor cells 45, 46. In contrast, IL-17-producing γδ T cells did not produce IFN-γ in the tumor microenvironment in parallel with the decrease of their cytotoxic function. Thus, our results indicate that IL-17-producing γδ T cells act as pro- but not anti-tumor effector cells in tumor-growing microenvironment. In our previous work 16–20, we have proposed that the introduction of Th1-dominant immunity overcomes strong immunosuppression in tumor-bearing host and induces complete cure of the tumor-bearing mice. Therefore, we now investigated whether Th1 cell therapy or introduction of Th1 immunity by IL-12 can induce a plasticity in IL-17-producing γδ T cells, namely, alter pro- to anti-tumor IL-17-producing γδ T cells. Recently, it has been reported that IL-17-producing cells exhibited anti-tumor immunity in vivo in an IFN-γ-dependent manner 46. Therefore it might be possible to control pro-tumor and anti-tumor properties of IL-17-producing γδ T cells by introducing Th1-dominant immunity at tumor-local site. Our findings provide novel insights into the role of IL-17-producing γδ T cells as tumor-promoting cells in cancer-related inflammatory environment, indicating that γδ T cells would be a potential target for developing novel cancer immunotherapies.
Materials and methods
WT BALB/c mice were purchased from Charles River Japan. BALB/c background IL-17−/− mice were generated as described previously 15. All mice were maintained under specific pathogen-free conditions and used at 6–8 wk of age.
Tumor-bearing mice model
The fibrosarcoma cell line CMS-G4 and skin carcinoma cell line CMC-1 were generated by injection of MCA into BALB/c mice. BALB/c background WT or IL-17−/− mice were inoculated intradermally with 2x106 CMS-G4 cells, and tumor growth was monitored at two day intervals. Tumor size was measured by micrometer calipers and the volume was calculated by the following formula: tumor volume=0.4×length (mm)×[width (mm)]2. In the antibody treatment experiments, CMS-G4 cells were inoculated i.d. into BALB/c mice (day 0), and anti-IL-6 receptor, anti-TGF-β or anti-IL-23p19 mAb was intravenously injected at day −1, 2, 5, 8, and 11. Anti-IL-6 receptor mAb (MR16-1) was a kind gift from Chugai Pharmaceutical, Shizuoka, Japan. Anti-TGF-β mAb (1D11) was purified from hybridoma cells, which was purchased from the ATCC. Anti-IL-23p19 mAb (G23–8) was purchased from e-Bioscience. Primary tumors were induced by i.d. injection of MCA (500 μg in corn oil) into BALB/c mice.
Preparation of tissue-infiltrating lymphocytes
CMS-G4, CMC-1, and CT26 cells were injected i.d. into BALB/c mice, and 2 wk after the injection, tumor tissues were dissected. In the primary tumor model, MCA was i.d. injected in to BALB/c mice, and 2–4 months after injection, primary tumor tissues were dissected. Tumor tissues were minced and digested with 1 mg/mL collagenase (Sigma). Single-cell suspensions were prepared, live lymphocytes were collected by density gradient centrifugation and used for the following experiments.
Flow cytometric analysis
For cell-surface molecules, cell samples were stained with fluorescent dye-conjugated mAb against selected markers on ice. For intracellular cytokine staining, lymphocytes were stimulated with plate-bound anti-CD3 and anti-CD28 mAb (BD Bioscience) in 96-well flat-bottomed plates for 6 h. For detection of cytoplasmic cytokines without exogenous stimuli, un-separated tumor-infiltrating cells including tumor cells (5×105 cells) were cultured in high cell density condition in 96-well V-bottomed palets for 12 h. Brefeldin A was added at 4 h. Then, cells were harvested and stained with 7-AAD, anti-CD4, -CD8, -CD45, CD27, CCR6, and/or −γδTCR mAb (BD Bioscience). In the blocking analysis in vitro, anti-γδ TCR (GL3, 10 μg/mL) or anti-NKG2D (C7, 10 μg/mL) antagonizing mAb were added in high cell density condition and cells were stained with 7-AAD alone. After fixation and permeabilization, cells were stained with anti-IFN-γ and anti-IL-17 mAb (e-Bioscience and BD Bioscience, respectively). Data were acquired on a FACSCalibur (BD Bioscience) and analyzed using CellQuest software (BD Bioscience).
Expression of cytokine genes
For analysis of gene expression, tumor tissues and local lesions of mice injected with MCA were collected 2 wk after injection. Then, CD44high CD62low γδ T cells were isolated from spleen or tumor tissue of CMS-G4-bearing mice by FACSAria (BD Biosecience). To examine the effect of IL-17 on tumor cells, CMS-G4 cells were treated with IL-17 (10 ng/mL) for 12 h in vitro. Total RNA was extracted by using ISOGEN RNA extraction kit (Nippongene) and RNeasy Mini kit (QIAGEN). First-strand cDNA synthesis was performed using Superscript II (Invitrogen), according to the manufacturer's instructions, and amplified with a thermal cycler system (Perkin Elmer) using gene-specific primer pairs as follows: mouse β-actin, 5′-GTGATGGTGGGAATGGGTCAG-3′ and 5′-TTTGATGTCACGCACGATTTCC-3′: IL-1β, 5′-TTGAAGAAGAGCCCATCCTCTG-3′ and 5′-GCTGATGTACCAGTTGGGGAAC-3′: IL-6, 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′: IL-17, 5′-GCTCCAGAAGGCCCTCAGA-3′ and 5′-CTTTCCCTCCGCATTGACA-3″: TGF-β, 5′-GTTGTACGGCAGTGGCTGAAC-3′ and 5′-CCGGGTTGTGTTGGTTGTAGA-3′: IL-12p19, 5′-CAGCAGCTCTCTCGGAATCT-3′ and 5′-TAGAACTCAGGCTGGGCATC-3′: RORγt, 5′-CGCGGAGCAGACACACTTAC-3′ and 5′-CTCCCGTGAAAAGAGGTTGG-3′: perforin, 5′-CCGGTCCTGAACTCCTGGCCAC-3′ and 5′-CCCCTGCACACATTACTGGAAG-3′. The primer pairs for analyzing TCR-Vγ expression were described previously 12. For real-time PCR analysis, the indicated cDNA was specifically amplified by thermal cycler (LightCycler, Roche) using the corresponding primer pairs and probes for mouse Ang-2, VEGF-A, and β-actin. The sequences used were as follows: Ang-2, (sense) 5′- CATCTGCAAGTGTTCCCAGA-3′, (antisense) 5′- TTCAAGTTGGAAGGACCACA-3′, (probe) Mouse Universal probe library ♯75 (Roche); VEGF-A, (sense) 5′-GCAGCTTGAGTTAAACGAACG-3′, (antisense) 5′-GGTTCCCGAAACCCTGAG-3′, (probe) Mouse Universal probe library ♯64 (Roche); β-Actin, (sense) 5′-AAGGCCAACCGTGAAAAGAT-3′, (antisense) 5′-GTGGTACGACCAGAGGCATAC-3′, (probe) Mouse Universal probe library ♯56 (Roche).
CMS-G4 cells were inoculated into BALB/c mice. After 9 days, tumor tissues were harvested from the mice, embedded in optimal cutting temperature compound, and cryosections (5 μm) were prepared. The sections were fixed for 10 min in cold acetone and stained with purified anti-pan-edothelial cell antigen mAb (BD Bioscience) for 60 min at room temperature. Subsequently, sections were incubated for 10 min at room temperature with peroxidase-conjugated secondary antibody (Histfine symplestain MAX PO; Nichirei). Peroxidase activity was detected with diaminobenzidine (DAB; sigma-Aldrich) and counter stained with hematoxiline. Blood vessels were counted under light microscopy at ×200. Any distinct area of positive staining was counted as a single positive region regardless of size. The results are shown as mean±SD per 6 high power fields per tumor section.
The data were evaluated by two-tailed Student's t test. p<0.05 was considered significant in the present experiments.
We thank Chugai Pharmaceutical Company Ltd. for their gift of anti-IL-6R antibody (MR16-1) and recombinant IL-6. We would like to thank Dr. Luc Van Kaer for his critical reading of this manuscript. This work was supported in part by a Grant-in-Aid for Ministry of Education, Culture, Sports, Science and Technology (H.K. and T.N.), by a National Project “Knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-S”), Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), and by a JSPS Research Fellowships for Young Scientists (D.W.).
Conflict of interest: The authors declare no financial or commercial conflict of interest.