OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy
- Ichiyo Shibahara1,
- Ryuta Saito1Email author,
- Rong Zhang1,
- Masashi Chonan1,
- Takuhiro Shoji1,
- Masayuki Kanamori1,
- Yukihiko Sonoda1,
- Toshihiro Kumabe2,
- Masahiko Kanehira3,
- Toshiaki Kikuchi3,
- Takanori So4,
- Takashi Watanabe5,
- Hiroaki Takahashi6,
- Erina Iwabuchi6,
- Yuetsu Tanaka7,
- Yukiko Shibahara6,
- Hironobu Sasano6,
- Naoto Ishii4 and
- Teiji Tominaga1
© Shibahara et al.; licensee BioMed Central. 2015
Received: 21 June 2014
Accepted: 28 January 2015
Published: 15 February 2015
Glioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy.
Tumor specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2).
OX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10.
Glioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.
Glioblastoma, classified as grade IV glioma by the World Health Organization, is associated with a dismal prognosis. Despite the best interventions achieved by surgery, radiotherapy, and chemotherapy, the median overall survival time is still only 14.6 months . Many innovative strategies, including immunotherapy, are under investigation in the hope of making a breakthrough in glioblastoma treatment. Some immunotherapy studies have demonstrated efficacy in establishing tumor-specific immunity against mouse glioma but there has been only limited success in clinical settings . In spite of these dilemmas, it is certain that there are long-term survivors of glioblastoma. We have hypothesized that, in some cases, adaptive immunity of the host may ameliorate glioblastoma progression. Activating this specific immunity may lead to potential immunotherapy against glioblastoma.
To test this hypothesis, we focused on OX40 and OX40 ligand (L), both of which are known to evoke a key signal for long-lasting immunity . OX40 is a member of the tumor necrosis factor receptor superfamily and interactions between OX40 and OX40L act as costimulatory signals [4,5]. We [4,5] and others have extensively studied the functions of OX40 and OX40L in the immune system, including the generation of memory T cells [3,6,7] and the stimulation of CD4 T cells, CD8 T cells, and natural killer T cells [5,8,9]. Triggering OX40 signaling has been demonstrated in various mouse tumor models [10-14] as a potential antitumor therapy and its translation to clinical trials is under investigation [8,15]. However, the mechanism by which OX40 signaling in glioblastoma regulates the antitumor adaptive immunity of the host remains unknown.
OX40L expression is believed to be restricted to cells related to the immune system and its surroundings [4,16-19]. However, we have discovered that OX40L is expressed in human glioblastoma cells. We therefore addressed the possible role of OX40L expressed in glioblastoma cells and immunotherapy targeting OX40 signaling.
Patients and glioblastoma specimens
Informed consent was obtained from all subjects. The ethics committee of Tohoku University School of Medicine approved the study. Tissue specimens were obtained from patients with primary glioblastoma (n = 110) and grade III glioma (n = 34). Among the 110 patients with glioblastoma, 79 patients underwent gross total resection. Human T cells were obtained from healthy human donors.
RNA was extracted from human glioblastoma tissue and from five human glioblastoma cell lines, using the RNeasy Lipid Tissue Mini Kit (Qiagen Science, Germantown, MD). Reverse transcription was performed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Carlsbad, CA). The expressions of OX40L mRNA and the internal control β-actin mRNA was analyzed using the TaqMan Gene Expression Assays (tumor necrosis factor ligand, superfamily, member 4, Assay ID: Hs00182411_m1, and β-actin Control Reagents, respectively, Applied Biosystems). A mixture (20 μl) of cDNA, the TaqMan Fast Advanced Master Mix (Applied Biosystems) and each probe was subjected to amplification with StepOnePlus Real-Time PCR Systems (Applied Biosystems), according to the manufacturer’s instructions.
Immunostaining was performed by the streptavidin-biotin method using a Histofine kit (Nichirei co Ltd. Tokyo, Japan). Tag34, a mouse monoclonal antibody against human OX40L  was used as the primary antibody (1:100). Paraffin-embedded sections of human glioblastoma specimens (n = 23) were deparaffinized with xylene and absolute ethanol. Antigen retrieval was performed by heating the slides with a microwave in 10 mM EDTA (pH8) for 10 minutes. A172 human glioblastoma cells were cultured on Millicell EZ slide (Millipore, Billerica, MA) and fixed by 4% paraformaldehyde at room temperature for 15 minutes. Both staining was followed by previously reported protocol . As for the positive control, we used tissues of human skin psoriasis  and for the negative control, normal mouse IgG was used. The degree of OX40L expression was scored by I.S. and two pathologists (Y.S. and H.S.).
The Mann–Whitney U test was used for comparison of continuous variables. To analyze whether OX40L mRNA expression was associated with progression-free survival (PFS), we used the univariate Cox proportional hazard model, with relative OX40L expression being the only covariate. OX40L expression data were distributed exponentially and were logarithmically transformed. When OX40L expression was zero, i.e., undetectable, we imputed 0.001 as the minimum value before transformation. The starting point for PFS and overall survival (OS) was the day of surgery. Tumor progression at the last follow-up examination or death was the end point for PFS and OS. Statistical analyses were performed with R version 3.0.2, and P values of <0.05 were considered statistically significant.
Functional analysis of OX40L expressed in human glioblastoma
Five human glioblastoma cell lines, U87, U251, U373, T98 and A172, were used in this study. Ethylendiamine tetraacetic acid (EDTA) solution was used to detach cells without altering the structure of OX40L protein. For detecting OX40L expression, antibodies specific for biotinylated Tag34 were used, followed by PE-streptavidin. Analysis was performed using FACS CantoII cytometer and FACS Diva software (BD Bioscience, Franklin Lakes, NJ). In the series of experiments, analyzing the effect of hypoxia on OX40L expression, A172 cells were cultured for 72 h under hypoxic (1.5% O2) or normoxic (21% O2) conditions. A172 cells were analyzed for OX40L mRNA and protein expression. A172 cells cultured on chamber slides were used for immunohistochemical analysis of OX40L expression, as described above. Cell culture conditions are described in the Additional file 1.
Human CD4 cells (1 × 105) obtained from healthy human donors were cocultured with irradiated A172 cells (3 × 104) in 100 μl of medium per well and either the Tag34 or IgG antibody (20 μg/ml each), under hypoxia or normoxia, in anti-CD3-antibody-coated 96-well plates (BioLegend, San Diego, CA). Irradiated A172 cells were prepared by irradiating 1 × 107 cells seeded in 1 ml PBS, in a 6-cm dish. Anti-CD3-antibody-coated plates were used to stimulate naïve T cells to express OX40 . After 72 h of incubation under normoxia, the supernatant was used for ELISA to measure interferon (IFN)-γ. Human CD4-positive cells (1 × 105) were pretreated with carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) and were detected in the fluorescein isothiocyanate fraction. The proliferation of activated CD4 cells was followed with flow cytometry. Details are in the Additional file 1. For cell sorting, MicroBeads and the AutoMACS system (Miltenyi Biotec, Gladbach, Germany) were used to isolate human CD4 cells from healthy human blood.
Mouse cell lines
The mouse cell lines used were GL261 glioma cell line , generously provided by Dr. Masaki Toda, Keio University and NSCL61 glioma-initiating cell-like cell line , generously provided by RIKEN (Kobe, Japan). NSCL61 cells were established by introducing an oncogenic HRas L61 in p53-deficient neural stem cells and were confirmed to retain features of glioma-initiating cell-like cells . Mouse OX40L cDNA or a parent vector (empty vector) was transduced in GL261 cells, using retroviruses to create GL261 cells expressing OX40L (GL261-mOX40L) or empty vector (GL261-mock). The expression of mouse OX40L was analyzed by the flow-cytometry with PE-conjugated anti-mouse OX40L antibody (eBioscience, San Diego, CA). Cell culture and transfection are described in the Additional file 1.
Mouse models of glioma
All animal experiments were reviewed and approved by the Animal Care and Use Committee, Tohoku University School of Medicine and performed in accordance with institutional ethical guidelines. Six to eight-week-old, female, C57BL/6 mice and BALB/c nude mice were purchased from SLC Japan, Inc. (Shizuoka, Japan). The generation of OX40-knockout (OX40KO) mice was described previously [24,25]. After anesthesia, each mouse was injected either with GL261 cells (1 × 105 or 2 × 105) or NSCL61 cells (1 × 104) into the right striatum. A small number of NSCL61 cells were inoculated because of their aggressive growth potential. Details are described in the Additional file 1.
Mouse brain frozen sections were stained with either the anti-CD4 or the anti-CD8 antibody (Abcam, Cambridge, MA), followed by anti-rat IgG Zenon® Alexa 568 (Invitrogen, Carlsbad, CA). Details are in the Additional file 1.
OX40 triggering on T-cell activation
Lymphocytes (1 × 105) harvested from the spleens of mice vaccinated with OX86 and irradiated GL261 cells were cultured with irradiated GL261 cells (3 × 104) for 3 days, to assess GL261 cell-specific T-cell activation. Supernatants were used for ELISA to detect mouse IFN-γ (BD OptEIA; BD Bioscience). MicroBeads and the AutoMACS system (Miltenyi Biotec, Gladbach, Germany) were used to isolate lymphocytes from mouse spleens. For detecting the effector T cells, CD4-Pacific Blue, CD44-APC and CD62L-FITC (eBioscience) were used. FACS analysis was performed, as described above. Treg cells (1 × 105) collected from spleens of untreated wild-type mice were cocultured with either OX86 or IgG antibodies, under hypoxia or normoxia, in anti-CD3 antibody-coated 96-well plates. Supernatants were used to measure IL-10 with ELISA (R&D Systems, Inc.).
Vaccination therapy in mice bearing transplanted glioma
For vaccination, mice received subcutaneous injections of the mixture that contained tumor cell lysates and 250 μg of purified rat IgG reagent (Sigma–Aldrich) or OX86, a mouse OX40 agonistic antibody, into the left flank. To prepare the tumor cell lysates, cells (1 × 107) were seeded in 1 ml of PBS in a 6-cm dish and irradiated (5,000–7,000 rad). Irradiated GL261 cells (5 × 105) or NSCL61 cells (5 × 104) were used as cell lysates. The OX86 antibody was obtained from the supernatant of OX86 hybridoma (European Collection of Cell Cultures). For comparison, mice were vaccinated with intraperitoneal injection of OX86 or IgG antibody. Vaccination was performed twice at a 5-day interval. In the study of tumor therapy, mice bearing the transplanted GL261 or NSCL61 cells were treated with respective tumor lysates in combination with OX86 or IgG control antibody. Mice were monitored daily for survival and general health. To assess the effect of memory T cells, mice with intracranial GL261 cells, which survived after OX86 vaccination, were reinjected with GL261 cells (2 × 105) into the contralateral hemisphere.
All statistical analyses, excluding human survival analyses, were performed with the Prism software (GraphPad Software, San Diego, CA). All P values were two tailed and P values of <0.05 were considered statistically significant.
Expression of OX40L in human glioblastoma
The immunohistochemical analysis showed that OX40L protein was expressed in cells with atypical nuclei (Figure 1b, left), indicating that OX40L protein was expressed in glioblastoma cells. However, there were cases in which OX40L protein expression was undetectable (right panel). In fact, among the 23 glioblastoma specimens analyzed, only six samples were strongly positive for OX40L expression (26%).
Because OX40L is a potent immunoinducer, we next explored its effect on the prognosis of glioblastoma patients. The univariate Cox proportional hazard model showed that higher OX40L expression levels were associated with longer PFS in patients with glioblastoma (n = 110), although the difference was not statistically significant (Hazard Ratio per log-relative OX40L expression, 0.898; 95% confidence interval, 0.775–1.041; P = 0.155). Importantly, among the glioblastoma patients who underwent gross total resection (n = 79), higher OX40L expression was associated with longer PFS (Hazard Ratio per log-relative OX40L expression, 0.776; 95% confidence interval, 0.611–0.985; P = 0.037). In contrast, OX40L expression was not associated with PFS in the 31 patients with glioblastoma, who did not undergo gross total resection. Moreover, there was no statistical significance between OX40L expression level and overall survival (OS), irrespective of the gross total resection (among gross totally resected patients, hazard ratio per log-relative OX40L expression, 0.936; 95% confidence interval, 0.762–1.15; P = 0.531). These results suggest that gross total resection may resolve a certain immunosuppressive condition in glioblastoma to enhance the growth inhibitory effect of OX40 signaling.
Functional implication of OX40L expressed in glioblastoma
OX40 signaling restricts glioma progression in mouse models
To confirm the role of OX40 signaling in mouse glioma, we next used GL261-mOX40L cells that were forced to express exogenous OX40L. The GL261-mOX40L cells or GL261-mock cells (Figure 3d) were transplanted into the wild-type mouse brain to establish the model that simulates human glioblastoma expressing OX40L. The mice transplanted with GL261-mOX40L cells did not die for at least 60 days after the cell transplantation (Figure 3e), whereas all mice transplanted with GL261-mock cells died by 50 days. GL261-mOX40L cells were also injected subcutaneously into the wild-type mice but none of them formed a tumor (data not shown). These results suggest that OX40L expression may restrict the growth of transplanted cells, thereby conferring survival advantage on the host. Moreover, the BALB/c nude mice bearing intracranial GL261-mOX40L cells died within 50 days (Figure 3e). The overall survival was indistinguishable between the nude mice bearing GL261-mOX40L cells and the wild-type mice bearing GL261-mock cells. Thus, GL261-mOX40L cells were effectively eliminated probably by OX40L-induced immunoreactive cells before forming a tumor mass.
OX40 triggering induces T-cell activation
OX40 triggering as immunotherapy in mouse glioma models
To further confirm this therapeutic effect, we conducted survival analysis in another glioma model. Glioma-initiating cell-like cells have been considered to regulate tumor initiation and formation  and are known to be resistant to treatment . Therefore, we assessed the antitumor effect of the subcutaneous OX86 vaccination in mice with the intracranial transplantation of NSCL61 cells (Figure 5d and e) . NSCL61 cells do not express OX40L, as judged by flow cytometry (Additional file 2: Figure S1b). Hematoxylin and eosin staining of intracranial NSCL61 cells showed aggressive features with many atypical cells (Figure 5d). In fact, all mice bearing intracranial NSCL61 cells died by 20 days after transplantation, despite the subcutaneous OX86 vaccination (Figure 5e and f); namely, their survival time was much shorter than that of the mice bearing intracranial GL261 cells (see Figure 5c). These results suggest that NSCL61 cells were more aggressive in proliferation and resistant to the subcutaneous OX86 vaccination than GL261 cells. However, the subcutaneous OX86 vaccination conferred a significant survival advantage on the mice bearing intracranial NSCL61 cells, compared with control IgG vaccination (P < 0.0001, Figure 5f). Thus, the subcutaneous OX86 vaccination had a strong antitumor effect in two mice glioma models.
Paradoxical effects of OX40L on glioblastoma growth
Consequently, we focused on Treg cells that express OX40  and are also abundant in the glioblastoma microenvironment [18,29,30]. Mouse Treg cells were cultured with OX86 or the control IgG antibody, under hypoxia or normoxia, in anti-CD3-antibody-coated plates. The level of IL-10, an immunosuppressive cytokine, was significantly increased in the conditioned medium of Treg cells with OX86 treatment under hypoxia, compared with that under hypoxia with IgG treatment or under normoxia with OX86 treatment (Figure 7d). Thus, hypoxia significantly increased IL-10 production from Treg cells, the degree of which was further enhanced by OX86. Taken together, these results suggest that glioblastoma may cooperate with Treg cells via OX40L, thereby enhancing IL-10 production. IL-10 in turn generates the immunosuppressive microenvironment. Such pro-tumor effects caused by Treg cells may be enhanced under hypoxic conditions.
Here, we show for the first time that glioblastoma cells express OX40L, which activates OX40 signaling and strengthens antitumor adaptive immunity. Furthermore, we show that higher OX40L expression is significantly associated with prolonged progression-free survival. An in vivo study, using OX40KO mice and GL261-mOX40L cells, showed the importance of OX40 signaling in antitumor immunity. Collectively, adaptive immunity evoked by OX40 signaling has the potential to suppress the proliferation of glioblastoma cells. OX40 triggering has been reported as an effective anti-cancer therapy, in which systemic i.p. administration was mainly used [12,13,31]. We found that s.c. injection of OX86 and tumor lysate induced stronger antitumor immunity than systemic i.p. injection, thereby providing persistent long-term antitumor effects. In terms of T-cell activation, subcutaneous OX86 vaccination, a mixture of both OX86 and tumor antigen (irradiated GL261 cells), may be an efficient method for generating tumor-specific T-cell activation.
The expression of OX40L in glioblastoma suggests a hitherto unrecognized role of OX40 signaling. It was initially surprising to us that glioblastoma expresses endogenous OX40L, which may result in self-attacking. To resolve this paradox, we focused on oxygen tension and thus discovered the potential pro-tumor effects of OX40 signaling. Under hypoxia, which simulates the tumor microenvironment, OX40 triggering induced IL-10 production from Treg cells, which may provide a suitable environment for tumor progression. Treg cells consistently express OX40  and are known to accumulate in the microenvironment of glioblastoma [29,30]. Thus, expressing OX40L might be a strategy of glioblastoma to create the immunosuppressive environment with the help of Treg cells. In fact, OX40L expression was upregulated in A172 cells under hypoxia. Our data also showed that OX40L-expressing glioma cells could not form a tumor mass in wild-type mouse brain. Kaneko et al. presented anti-lymphoma immunity by introducing OX40L into lymphoma cells, which was consistent with our data . Glioblastoma cells probably express OX40L only after tumor bulk reaches a critical size, which could generate the hypoxic environment suitable for tumor progression. In this context, we observed the survival benefit of OX40L only in patients that were able to undergo gross total removal. Thus, this immunostimulative aspect may be achieved via resolving the hypoxic microenvironment of glioblastoma.
Around 26% of the analyzed glioblastoma tissue presented high OX40L, indicating this phenomenon is not applicable to all glioblastoma. We showed that OX40 signaling achieved opposite functions, immunoinduction and immunosuppression, depending on oxygen tension. In fact, Ruby et al. reported that OX40 triggering had two opposite effects on Treg cells, depending on the local cytokine milieu . Moreover, ovarian cancer produced CC-chemokine ligand 28 under hypoxic conditions, resulting in Treg cell recruitment and the induction of tumor angiogenesis through VEGFA secretion . Thus, difference in the oxygen tension may account for the dual functions of OX40L.
There are a few limitations in the present study. First, for the mouse survival analysis, depletion analysis was not conducted, although we showed the effective induction of tumor specific antitumor immunity by numerous apoptotic cells. Second, concerning T-cell activation, pathway analysis was not performed. Studies on the downstream pathway of OX40 signals, such as NF-κB , need to be conducted. Lastly, T cells used in this study were mainly obtained from the mouse spleen because of the difficulty in the isolation of T cells from glioblastoma tissues or the cervical lymph node. Considering this limitation, we have presented the OX40-mediated immunoinduction using human T cells.
Here, we demonstrated OX40L expression in human glioblastoma cells. We also showed that the activation of OX40/OX40L signaling, by subcutaneous vaccination, induces strong and long-lasting immunity, with therapeutic antitumor effects using mouse glioma models. This result is in agreement with the clinical survival data on glioblastoma patients. On the other hand, we also provide evidence of crosstalk between glioblastoma cells and Treg cells under hypoxic conditions, which provides glioblastoma cells with immunosuppressive benefits. Thus, immunostimulation and immunosuppression are closely connected within glioblastoma, and targeting of these two aspects may unravel a clue for therapeutic approach against glioblastoma.
We thank M. Fue for technical assistance, M. Toda (Keio University School of Medicine, Tokyo, Japan) for providing the GL261 cells, T. Kondo (Ehime University School of Medicine, Ehime; and Riken, Kobe, Hyogo, Japan) for providing the NSCL61 cells, and Enago (www.enago.jp) for the English language review.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan to R.S. (#25670613).
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