Abstract
Purpose: The objective of this study was to evaluate clinical significance and immunosuppressive mechanisms of B7-H4 (B7x/B7S1), a B7 family member, in glioma.
Experimental Design: B7-H4 levels in glioma tissue/cerebral spinal fluid (CSF) were compared between different grades of glioma patients. Survival data were analyzed with Kaplan–Meier to determine the prognostic value of B7-H4. Cytokines from CD133+ cells to stimulate the expression of B7-H4 on human macrophages (Mϕs) were investigated by FACS, neutralizing antibodies, and Transwell chemotaxis assay. shRNA, reporter vector, and chromatin immunoprecipitation were used to determine the binding of STAT3 to the B7-H4 promoter. The function of B7-H4+ Mϕs in vitro was evaluated through phagocytosis, T-cell proliferation/apoptosis, and cytokine production as well as in the xenografted model for in vivo analysis.
Results: We found that B7-H4 expression in tumors was associated with prognosis of human glioblastoma and correlated directly with malignant grades. Mechanistically, glioma initiating CD133+ cells and Mϕs/microglia cointeraction activated expression of B7-H4 via IL6 and IL10 in both tumor cells and microenvironment supporting cells. IL6-activated STAT3 bound to the promoter of B7-H4 gene and enhanced B7-H4 expression. Furthermore, CD133+ cells mediated immunosuppression through B7-H4 expression on Mϕs/microglia by silencing of B7-H4 expression on these cells, which led to increased microenvironment T-cell function and tumor regression in the xenograft glioma mouse model.
Conclusions: We have identified B7-H4 activation on Mϕs/microglia in the microenvironment of gliomas as an important immunosuppressive event blocking effective T-cell immune responses. Clin Cancer Res; 22(11); 2778–90. ©2016 AACR.
Glioblastoma (GBM) is the most malignant and lethal primary brain tumor. To understand the mechanisms underlying GBM progression is critical for clinical practice. This study revealed the complex immune interactions in the GBM tumor microenvironment involving immune checkpoint B7-H4 and IL6 regulatory mechanisms during tumor progression. Specifically, the results identified that B7-H4+ glioma–infiltrated macrophages (Mϕs)/microglia showed immunosuppressive activity, which could be autoregulated by IL6 production. IL6-activated STAT3 bound to the promoter of B7-H4 gene and enhanced B7-H4 expression on Mϕs. Our studies demonstrated a direct relationship between the grade of gliomas and the levels of B7-H4 expression, suggesting it as a potential immune-associated marker for progression of gliomas. The findings were further demonstrated using the in vivo xenografts model. Overall our study contributes to the understanding of GBM progression, and suggests that targeting the B7-H4 pathway holds therapeutic promise for advanced glioma patients.
Introduction
Glioblastoma (GBM; WHO Grade IV) is the most aggressive and frequent de novo intracranial neoplasm in adults, with less than half of patients surviving longer than a year after initial diagnosis. As views changed, more emphasis was placed on the tumor-induced immunosuppression as an important factor of the formation and development of the tumor. Immunosuppressive factors secreted by both tumor cells and microenvironment T-cell infiltrates are proposed to obstruct antitumor immunity (1, 2). Our hypothesis is that the tumor microenvironment cellular interactions between glioma-infiltrating macrophages/microglia (GIM) and glioma cells play a central role in synergistically promoting glioma malignancy and immunosuppression. It has been suggested that tumor-infiltrating macrophages/microglia (TIM) may contribute to the suppression of T-cell–mediated immunity (2, 3). Although some secreted factors (1, 4, 5) and coinhibitory immune molecules (4, 5) have been reported to contribute to the immune regulation in GBM, however, the precise molecular mechanisms underlying these pathways and cellular interaction within the GBM microenvironment are poorly understood.
B7-H4 (also called B7x or B7S1) is a member of the T-cell costimulatory and coinhibitory B7 family (6–8). Functionally, B7-H4 transmits negative signals to T cells to effectively inhibit activation, proliferation, and clonal expansion of CD4+ and CD8+ T cells (6–8). Elevated expression of B7-H4 is detected in human cancer tissues of multiple cancers (9, 10) and is often associated with poor prognosis. We have recently determined the crystal structure of human B7x IgV functional domain and further developed a new cancer immunotherapy with mAbs targeting the B7x IgV (11). Previously, we reported that B7-H4 can be expressed by malignant gliomas (12), but its clinical significance and immunologic role remain elusive. In addition, soluble B7-H4 (sB7-H4) is detected in blood from patients with ovarian, renal cell cancer, hepatocellular carcinoma, osteosarcoma, bladder urothelial carcinoma, and gastric cancer (13–18). However, the relationship between sB7-H4 and malignant grades is still unclear. We have suspected that B7-H4 is related to a subset of tumor-initiating cells in gliomas (12), but details underpinning these observations remain unknown. The evolving understanding of glioma-initiating cells and their importance in tumor pathophysiology (19–25) encourages us to consider that the interplay between glioma-initiating cells and other cell types (e.g., TIMs) may be important for tumor initiation and progression in GBM.
We demonstrated Mϕs-modulating cytokine production via the JAK–STAT3 pathway. B7-H4+ GIMs showed immunosuppressive activity and autoregulation by IL6 production. Also, it was observed that adoptive immune therapy of tumor-associated antigen (TAA)–specific T cells in conjunction with TIMs depleted of B7-H4 expression was able to induce tumor regression and prolonged survival of mice in xenograft human gliomas. These results revealed that circumventing the tumor-induced immunosuppression of B7-H4 can induce glioma regression. Overall, our finding indicated B7-H4 as a potential immunity-associated marker of GBM, suggesting that new cancer immunotherapy targeting the B7-H4 pathway holds promise for glioma patients.
Materials and Methods
Preparation of CD133+ glioma cells
To isolate human CD133+ glioma cells, fresh primary GBM surgical specimens were dissociated mechanically and digested with a type IV collagenase (Sigma) for 1 hour at 37°C. A single-cell suspension was then collected on a 30%/70% Percoll gradient and further purified by magnetic separation using anti-CD133 microbeads per the manufacturer's instructions (Miltenyi Biotec), CD133− cells were also obtained at the same time and cultured at conditions appropriate for growth (continued in Supplementary Methods).
mAb sandwich ELISA detection for sB7-H4, IL6, and IL10
Purified, unlabeled B7-H4–specific mAb (Clone MIH43, 2 μg/mL; AbDserotec) was used for coating and capture using a standard sandwich ELISA method. Thoroughly homogenized tumor tissues were analyzed by IL6 and IL10 ELISA kits (eBioscience). Purified samples were then spotted in duplicate to appropriately coated plates before horseradish peroxidase–conjugated detection antibodies were added. Tetramethylbenzidine was used as a substrate for color development. The optical density was analyzed at 450 nm with a microplate reader (Spectra Max 190; Molecular Devices) and concentrations were quantified using SoftMax Pro software (Molecular Devices).
FlowCytomix detection
Cytokine concentrations in serum, cerebral spinal fluid (CSF) and supernatant of CD133+ and CD133− cells were measured using FlowCytomix multiple kits according to the manufacturer's directions (eBioscience). Serum was obtained from fresh blood after centrifugation at 3,000 rpm. The supernatant was collected after culture for 72 hours and stored at −20°C. For FlowCytomix detection, the supernatant was added in duplicate to coated 96-well plates. After fluorescent polystyrol beads were coupled with antibodies specific to the cytokines, biotins, and streptavidin-PE were successively added. After being activated (690 nm), samples were analyzed by flow cytometry and quantified with FlowCytomix Pro 2.4 software. Detected cytokines included: MIP-1α, MCP-1, MIP-1β, IL8, RANTES, MIG, IL6, IL10, GM-CSF, and IL4.
Flow cytometry
To detect surface B7-H4, CD11b, or CD133 expression, cells were incubated on ice for 30 minutes with appropriate antibodies or isotype controls (PE-B7-H4, PE-mouse IgG1, APC-CD11b, APC-CD133, APC-mouse IgG1, all from eBioscience, 1:5), washed twice with FACS buffer (PBS containing 0.1% NaN3 and 5% FBS), and resuspended in 0.5 mL 1% formalin/PBS. To analyze intracellular B7-H4, cells were pre-treated with Fix and Perm cell permeabilization reagents (Caltag Laboratories) according to the manufacturer's instructions. Assays of immune function, cell proliferation, cell cycle, apoptosis, cytokines, and cytotoxicity were done under standard methods as previously described. All analyses were performed on a FACS calibur system (Becton Dickinson Immunocytometry Systems).
qRT-PCR
Total RNA from cells was isolated using an RNA purification kit (Qiagen) and treated with RNase-free DNase I to remove genomic DNA (Roche). cDNA libraries were generated using Superscript RNase H-Reverse Transcriptase (Invitrogen) and random hexamers (Sigma-Aldrich). qRT-PCR was performed in an ABI Prism7500 sequence detection system (Applied Biosystems) with SYBR Green Master Mix (Eurogentec) and primers (Sangon Biotech Co. Ltd.) at optimized concentrations. Primer sequences for B7-H4 were 5′-TCTGGGCATCCCAAGTTGAC-3′ (forward primer) and 5′-TCCGCCTTTTGATCTCCGATT-3′ (reverse primer), housekeeping GAPDH primers have been described previously. Standard curves were obtained for each gene and the amplification was 90% to 100% efficient. All values were normalized to GAPDH and relative RNA expression was determined and compared to control groups.
Regulation of B7-H4 expression
To regulate B7-H4 expression, Mϕs or microglia were cultured for 72 hours with CD133+ cells or CD133− cells at the ratio of 10:1, CD133+ supernatant or CD133− supernatant or different concentrations of recombinant IL10, IL6, IL4, and GM-CSF (Peprotech). Neutralizing monoclonal antibodies against human IL6 (anti-IL6, clone 6708, 500 ng/mL) and IL10 (anti-IL10, clone 23738, 50 ng/mL; Peprotech) were used as indicated. For B7-H4 blocking, fresh macrophages were transfected with lentiviral two pairs of short-hairpin (sh)-B7-H4 and sh-mock as directed (Shanghai gene chemical technology limited company, China).RT-PCR and flow cytometry were performed to quantify B7-H4 mRNA and surface protein expression.
In vitro TAA-specific T-cell immunosuppression
Dendritic cells (DC) extracted from peripheral blood mononuclear cells (PBMC) or mouse bone marrow by CD11c microbeads (Miltenyi Biotec) were incubated with irradiated apoptotic U87MG or GL261 cells at a ratio of 1:5 for 24 hours. These tumor-loaded DCs (2 × 104 cells/well) were then used to activate CD3+ T cells from blood or CD8+ T cells from spleen (2 × 105 cells/well) in the presence of 10 U/mlIL2 and 10 ng/mlIL7 for 1 week. To evaluate proliferation, cell-cycle stage, apoptosis, or cytokine secretion, TAA-specific T cells were cocultured with different macrophages at a 1:1 ratio, stained with CFSE, PI, annexin V/PI, or IL2/IFNγ, and sorted by FACS. To measure cytotoxicity, CD8+ T cells isolated from blood was stimulated by macrophages at various ratios and cocultured with CFSE-labeled U87MG cells. FACS was performed with annexin V/PI staining and cytotoxicity was quantified as the percentage of dead or apoptotic cells. For LDH release assay, Mϕs/microglias and activated CD8+ T cells (Mϕ/microglia:T = 40:1, 20:1, 10:1, 1:1, 1:10, 1:20) were added with or without 1 × 105 GL261 cells per well. After a 12 hours coculture, the supernatant from each well was collected and analyzed with the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega Corporation) and recorded at an absorbance of 490 nm.
Statistical analysis
The two-tailed Student t test was performed to assess the significance between experimental groups. The Pearson correlation or linear regression analysis was used for correlations between parameters. Cumulative survival time was calculated by the Kaplan–Meier method and comparison between the groups was tested by the log-rank test. GraphPad software (GraphPad Software, Inc.; version 5.02) was used for all statistical analyses. Statistically significant P values are indicated in the figures with asterisks: ***, P < 0.001; **, P < 0.01; *, P < 0.05.
Results
Elevated B7-H4 expression associated with human glioma progression
We examined the relationship between glioma B7-H4 expression and disease progression in the patients of different stages. B7-H4 mRNA levels in glioma tissues with WHO grade 1 to 4 and normal control brain tissue from epilepsy surgery (non-tumor controls) were compared. B7-H4 mRNA expression increased with higher grade tumor stage (Fig. 1A). Furthermore, to verify these findings, the expression of B7-H4 was examined by IHC in 138 glioma and six non-tumor control tissues (tissue microarray, TMA; Supplementary Table S1). We observed immunoreactivity of B7-H4 protein in both the cytoplasm and membrane (Fig. 1B), and a very low level of B7-H4 expression was observed in control compared with glioma tissues. Western blot (WB) analysis showed high expression of B7-H4 protein in high-grade gliomas (HGG), minimal expression in low-grade gliomas (LGG), and negligible detection in non-tumor controls (Fig. 1C). Significant differences for B7-H4 staining pattern were observed between controls and LGG (P < 0.001) and between LGG (n = 33) and HGG (n = 105; P < 0.001, Fig. 1D). We searched the Cancer Genome Atlas (TCGA) data GBM gene-expression profile for expression of B7-H4. Compared with normal brain tissue (n = 10), B7-H4 overexpressed in GBM tissue (n = 483). We futher analyzed the gene expression correlated with GBM phenotype, and found that B7-H4 gene upregulation mainly exists in proneural type (Supplementary Fig. S1).
Flow cytometry was used to assess glioma tissues, B7-H4+CD11b+ Mϕs/microglia in LGG and HGG were 24.5% and 69.9% (Fig. 1E, P < 0.01), respectively. We also assessed B7-H4 in peripheral blood cells (n = 8/healthy group, 13/LGG group, 10/HGG group) and glioma specimens (n = 8/LGG group, 8/HGG group) was characterized. In blood, B7-H4+CD11b+ monocytes in LGG and HGG were 8.5% and 14.4%, (Fig. 1E, P > 0.05), respectively.
Prior studies demonstrated a correlative relationship between sB7-H4 and severity of ovarian cancer and renal cancer (13, 14); however, the role of sB7-H4 in human glioma is unclear. We used ELISA to detect sB7-H4 in CSF from 52 patients (clinical characteristics of patients on Supplementary Table S1). We found that increasing concentrations of sB7-H4 in CSF was associated with higher malignancy stages (Fig. 1F). This analysis demonstrated sB7-H4 is released into the CSF of glioma patients and associated with advanced stages in the disease.
Retrospective analysis of B7-H4 expression in GBM to correlate with survival
A large cohort of GBM patients tumors (n = 70, Supplementary Table S2) was retrospectively analyzed for B7-H4 expression by IHC to determine the prognostic value. We identified an inverse correlation of tumor cell B7-H4 expression levels (negative vs. moderate vs. strong) and survival in terms of progression-free survival (PFS) and overall survival (OS; Fig. 1G. PFS: P < 0.001, OS: P < 0.001). This finding suggested a role for B7-H4 as a potential predictor of poor disease outcome.
B7-H4 distribution in both GIMs and tumor cells in glioma tissues
A murine glioma model in C57BL/6 mice was developed to further examine B7-H4 in tumors and GIMs (CD11b+/CD45+Mϕs). Higher levels of B7-H4 expression were found in GL261-derived intracranial tumors (Fig. 2A–B). B7-H4+ GIMs (Iba1+ Mϕs) were identified primarily at the glioma tumor boundary (Fig. 2C, 88.3% for boundary vs. 38.8% for intratumoral; ***, P < 0.001). We isolated GIM by CD11b magnetic microbeads from the mouse glioma tumor tissues. GIM were further identified in their role in GBM surveillance (26, 27) and found that the isolated cell population of GIMs expressed higher levels of surface B7-H4:74% ± 1% for GIMs versus 15% ± 7% for Mϕs/microglia from controls (Fig. 2D and E, P < 0.001). We compared levels of B7-H4 expression in GL261 cells cultured in vitro with GL261 cells implanted into mice and revealed a lower capacity for induction of B7-H4 expression in in vitro cultured GL261 cells, 2% ± 1.6% versus 6% ± 2%, P < 0.05 (Fig. 2F). To confirm this, GL261 cells from model mouse were isolated and analyzed by WB from in vitro cultured GL261 cells as controls. Isolated GIMs from xenografts expressed higher levels of B7-H4 from non-tumor controls (Supplementary Fig. S2).
Clinical tumors were assessed to determine whether the murine tumor observation also show a similar response. MRI and magnetic resonance spectroscopy (MRS) were used to characterize patient intracranial gliomas. For these studies, peritumoral (stromal) and tumor-nest regions were identified (Fig. 2G). Enhanced MRI images-guided brain tissue biopsies taken from these locations were sectioned to include both regions of interest and then employed IHC with anti-B7-H4, cell-specific, anti-Iba1, and anti-CD8 Abs for GIMs (Mϕs/microglia), and CTLs, respectively. The IHC analysis revealed higher amounts of B7-H4 on GIMs in the peritumoral regions (Fig. 2G). Immunofluorescence (IF) for GBM tissues proved that not only GIMs but also CD133+ tumor cells expressed B7-H4 as previously shown (Fig. 2H; ref. 12).
Together our results confirmed the presence of both cellular and soluble forms of B7-H4 and established GIMs and glioma cells (including CD133+ cells) as candidates for the sources of this protein. Considering their important role, we further explored the cross-talk between GBM CD133+ cells and GIMs in the glioma microenvironment.
CD133+ cells produce IL6 and IL10 to activate B7-H4 expression on Mϕs
We next examined whether GBM CD133+ cells could regulate B7-H4 expression in Mϕs. Fresh normal Mϕs were incubated in specific culture conditions: control medium only, medium and CD133+ GBM cells, medium and CD133− GBM cells, and supernatant of CD133+ or CD133− cells. After 72 hours of incubation, B7-H4 expression on Mϕs was assessed by flow cytometry. Mϕs cocultured with CD133+ cells expressed higher B7-H4 than those cultured devoid of CD133+ cells (56.1% for CD133+ cell coculture vs. 28.8% for CD133− cell coculture, P < 0.05, Fig. 3A). Mϕs exposed to CD133+ cell supernatant also had higher B7-H4 expression than those exposed to CD133− cell supernatant (49.3% for CD133+ supernatant vs. 16.6% for CD133− supernatant, P < 0.01, Fig. 3A). These findings were supported by RT-qPCR studies of B7-H4 expression of Mϕs in CD133+ and CD133− supernatant (Fig. 3B) in which marked significant elevation of B7-H4 expression was most notable in Mϕs cultured in CD133+ supernatant.
We hypothesized that factors secreted by CD133+ cells had a major role in upregulating B7-H4 expression. To test this, FlowCytomix was used to establish cytokine profiles for CD133+ and CD133− supernatant. Two significant chemokines, MIP-1α and MCP-1, were found to be at substantially higher concentrations in CD133+ supernatant relative to CD133− supernatant (Fig. 3C, P < 0.001). Interestingly, levels of IL6 and IL10 were also elevated in CD133+ media relative to CD133− supernatant (P < 0.001, Fig. 3C). To determine their roles, IL6 and IL10 were added to control cultures of Mϕs. We observed a dose-dependent upregulation of B7-H4 expression in Mϕs stimulated by these cytokines (Fig. 3D) and verified these findings by suppressing IL6 and IL10 activity with specific Ab, respectively (Fig. 3E).
Because these results suggested a primary role of interleukins IL6 and IL10 in regulating B7-H4 expression that occurs in the microenvironment, we further investigated systemic levels of these cytokines. FlowCytomix was performed with serum and CSF from a series of glioma patients (clinical characteristics in Supplementary Table S1). Only trace amounts of these cytokines were detected in serum and CSF. A relationship between the serologic interleukin levels and tumor grade was not readily delineated; ELISA analysis from tissue homogenates identified elevation of these two cytokines and showed a higher level of IL6 in HGG patients (P < 0.01) compared with control patients (Supplementary Fig. S3). This finding supports a role for these two cytokines in GBM and induction of immunosuppression, perhaps more confined to the tumor microenvironment than system-wide. Finally, we used a cell migration assays to examine whether CD133+ GBM cells could initiate recruitment of Mϕs/monocytes into the tumor environment. We found enhanced migration of Mϕs/monocytes with CD133+ supernatant as a chemoattractant (Fig. 3F, medium-only, CD133+ and CD133− supernatant: 1 ± 0.00, 5.17 ± 0.78, and 3.12 ± 0.22, respectively).
These findings support a novel mechanism by which CD133+ cells are able to induce Mϕs migration into the tumor microenvironment and induce them to express B7-H4 by cytokines IL6 and IL10.
IL6 induces B7-H4 expression via the JAK–STAT3 pathway
Considering that enhanced IL6 was found in glioma tissue homogenates (Supplementary Fig. S3), we then explored the molecular pathways through which B7-H4 expression was regulated. IL6 signaling has been extensively investigated through the JAK–STAT pathway (28), so we hypothesized that the expression of B7-H4 was regulated through this pathway. To confirm this, mouse BV2 microglia cells were stimulated with IL6 and analyzed the phosphorylation of STAT3 by WB. As shown in Fig. 4A, IL6 treatment upregulated phosphorylation of Stat3 and B7-H4 levels in BV2 cells in a dose- and gradient-dependent manner, and 25 ng/mL and 24 hours were suitable dosage and time point. Similar findings were reproduced in the mouse RAW Mϕs cell line (Fig. 4B) and Mϕs sorted from healthy human peripheral blood (Fig. 4C).
To further determine whether the expression of B7-H4 was regulated by the STAT3 pathway, four pairs of STAT3 shRNAs were individually transduced into BV2 cells to knockdown endogenous STAT3. STAT3 mRNA in BV2 cells transduced with lentiviral stocks expressing shRNA1, shRNA2, shRNA3, or shRNA4 were reduced by 54.2, 65.0, 85.3, and 78.6%, respectively (Fig. 4D, left). Because STAT3 protein was remarkably decreased in the BV2 cells that were transduced with shRNA3, we chose to use shRNA3 for the subsequent experiments. We found that silencing STAT3 with shRNA3 reduced B7-H4 expression with or without the stimulation of IL6 (Fig. 4E). WB analysis of BV2 microglia stimulated with CD133+ supernatant was also performed, which showed similar tendency as IL6 did (Supplementary Fig. S4).
We next determined whether STAT3 could be a transcriptional regulator of the B7-H4 gene. We transfected BV2 microglia cells with a reporter vector encoding Luciferase under control of the B7-H4 promoter (PGL3-B7H4.WT). Phosphorylation of STAT3 upregulated B7-H4 promoter activity, which was abrogated by knockdown of STAT3 with shRNA (Fig. 4F). These studies suggested that STAT3 regulated B7-H4 transcription via enhancing the B7-H4 promoter. We then carried out chromatin immunoprecipitation (ChIP) assays to assess whether STAT3 directly binds to the B7-H4 promoter. As shown in Fig. 4G, STAT3 protein bound to the B7-H4 promoter was significantly increased in BV2 microglia cells treated with IL6. The epistatic relationship between the expression of B7-H4 and IL6–STAT3 pathway (Supplementary Fig. S5) may provide a new line of evidence for the development of STAT3 inhibitor as anticancer chemotherapy drugs.
CD133+ cells mediate immunosuppression through B7-H4+ Mϕs in vitro and in vivo
To explore the role of B7-H4 in CD133+ cell-mediated immunosuppression, CD133+ cells were sorted from human GBM cell suspensions, and the supernatant of CD133+ cell culture was then used to treat normal Mϕs to induce expression of B7-H4. We found that CD133+ cell supernatant-treated Mϕs showed markedly reduced uptake of red fluorescent tag–labeled polystyrene latex beads compared with CD133− cell supernatant-treated Mϕs (Fig. 5A), suggesting that the supernatant of CD133+ cells inhibits the phagocytotic activity of Mϕs. We then examined the function of B7-H4 in Mϕs induced by the supernatant of CD133+ cells. B7-H4 expression in Mϕs was silenced by shRNA (Fig. 5B) and Mϕs were cocultured with T cells activated by U87MG-loaded CD11c+ DCs. We found that the B7-H4 silencing in Mϕs increased T-cell proliferation (Fig. 5C) and decreased T-cell apoptosis (Fig. 5D). Correspondingly, the B7-H4 silencing also reduced T cells that did not produce IL2 or IFNγ (Fig. 5E). Furthermore, B7-H4+ Mϕs cultured in CD133+ supernatant inhibited CTL-cytotoxic activity against U87MG glioma cells. However, the inhibitory effects were mitigated after B7-H4 expression was blocked (Fig. 5F).
To investigate the function of B7-H4 in Mϕs in vivo, we used the GL261-induced glioma mouse model. GL261 tumor cells derived from mouse glioma contained CD133+ cells and were able to inhibit both CD8+ and CD4+ T-cell function in vitro (Supplementary Fig. S6). Interestingly, the suppressive capacities were partially reversed by exposing Mϕs to anti–B7-H4 Ab (Supplementary Fig. S6). These results suggested that CD133+ cells can induce B7-H4 expression on Mϕs, which leads to T-cell inhibition.
Suppression of B7-H4 leads to T-cell activation and tumor regression in the xenograft glioma model
NOD/SCID xenograft gliomas derived from U87MG cells were further investigated. TAA-specific CD8+ T cells (CTL) were injected i.v. together with: (i) normal Mϕs (M), (ii) CD133+ glioma-initiating cells conditioned Mϕs (GCM), (iii) conditioned Mϕs transduced with sh1-mock, or (iv) conditioned Mϕs transduced with sh1-B7-H4 to silence B7-H4 expression. We observed tumor growth inhibition in mice injected together with CTL+M and CTL+ GCM+ sh1-B7-H4 Mϕs, whereas the other two groups showed rapid progression of nearly 200 mm3 per day (Fig. 5G and H). These results suggested that CD133+ cells played a primary role in driving B7-H4+ Mϕs to reduce TAA-specific T-cell immunity in vivo.
To determine whether B7-H4 could serve as a predictor of prognosis, we infused CTLs with CD133+ glioma-initiating cells conditioned sh1-mock or sh-B7-H4 Mϕs via a caudal vein injection of mice with U87MG-derived intracranial tumor. Mice with sh1-B7-H4–treated Mϕs survived significantly longer than the sh1-mock group (Fig. 5I). In contrast, mice infused with PBS and sh1-mock–treated Mϕs had U87MG cell invasion, which was also characterized by early lesions detected by MRI (Fig. 5J). Supporting the role of Mϕs in regulating T-cell function, human CD11b+ Mϕs/microglia and human CD8+ T cells appeared to comigrate into intracranial human tumors (Fig. 5K).
GIMs induce B7-H4 expression in CD133+ cells and autoregulation through IL6
GIMs were isolated from murine GL261 tumors to investigate their effects on CD133+ cells. Using a Transwell chemotaxis assay in a coculture system, migration abilities between GIMs and normal Mϕs/microglia (NM) were compared. GIMs exhibited higher time-dependent migration abilities (Fig. 6A).
Next, GL261-derived enriched CD133+ cells were separated from CD133− cells, and both cell populations were cultured in GIM-conditioned media. GIMs upregulated B7-H4 expression in CD133+ cells and CD133− cells according to flow cytometry, IF, and WB analysis (Fig. 6B–D). Considering IL6 and IL10 were involved in B7-H4 induction, we measured the level of these cytokines in the sera of: (i) normal mice, (ii) CD133− cells initiated tumor mice, (iii) CD133+ cells initiated the tumor group, and supernatants groups from NM and GIM cultures. There was no observable difference in serum IL6 levels across the three groups. However, we observed that GIM-secreted IL6 exceeded NM-secreted IL6 by >15-folds (Fig. 6E). No difference was observed for IL10 concentrations in these groups (data not shown). These results support a role for IL6-promoting B7-H4 expression in the tumor microenvironement. Finally, we also find that IL6 can upregulate B7-H4 expression in mice NMs in vitro as human Mϕs did (data not shown). The results showed that Mϕs/microglia in the tumor environment had markedly enhanced B7-H4 expression in the presence of CD133+ glioma-initiating cells.
Discussion
Immunosuppressive processes occurring both systemically and locally in the tumor microenvironment have limited the normal function of the immune system in primary brain malignancies. Our studies characterizing the immunologic roles of GIMs, tumor-derived cytokines, and tissue-derived glioma-initiating cells have revealed evidence for an immunosuppressive role of B7-H4 in human malignant glioma.
We found that increasing B7-H4 expression associated with progression of human glioma. Notably, the distribution of B7-H4–expressing cells was much higher at the peritumoral edges (i.e., stroma) when compared with the tumor nest. What was important, our studies demonstrated that PFS and OS are negatively correlated with expression of B7-H4 in human gliomas. Expression of B7-H4 was detected on the surface of CD11b+/Iba+ monocytes, microglia, and macrophages. CD8+ T cells have been shown to orchestrate autoimmunity in immune privileged locations (29) and antitumor immunity in malignancy (30, 31). Interestingly, we found that CD8+ T cells were also mainly present at the peritumoral edges. Colocalization of B7-H4, Mϕs/microglia, and CD8+ T cells at peritumoral edges suggests a functional connection in the tumor microenvironment on the periphery, but not internally. We found significantly higher levels of IL6 in the HGG group compared with LGG or LGG group compared with the normal, but not in serum, suggesting that the immunosuppressive effects of the cytokines are limited to the tumor environment.
B7-H4 in the tumor microenvironment, surrounding stroma and invasive edge, may contribute to the escape from immune surveillance during tumor cell invasion into adjacent brain tissue. We believe that recruitment of monocytes/Mϕs to the tumor at the glioma site occurs in the malignant state (32, 33). The development of these TIMs is stimulated by “polarization” induced by the glioma and other tumor cells, resulting in TIMs with a tumor-promoting phenotype (M2). A chemokine-mediated process that spawns tumor development in the microenvironment is demonstrated (27, 34, 35). It is also demonstrated that microglia derived from non-glioma human subjects could curb glioma cell growth by the secretion of chemokine like IL8 and MCP-1, but microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of tumor cells (36). Our studies showed that glioma-initiating cells mediated expression of B7-H4 on Mϕs. Moreover, the detection of Mϕ-recruiting chemokines, MIP-1α and MCP-1, which are secreted by CD133+ glioma-initiating cells, presents evidence to support a process whereby Mϕs are signaled to invade the tumor microenvironment.
Our experimental findings demonstrated that normal monocytes/Mϕs were induced to express B7-H4 via IL6 and IL10 by human CD133+ cells (Fig. 3) or U251 glioma-initiating cells (our published data; ref. 37). In turn, a suppressive potential conferred by Mϕs-expressing B7-H4 contributed to immunopathology based on several lines of evidence. First, forcing B7-H4 expression on glioma-initiating cells impaired phagocytosis in normal Mϕs, inhibited T-cell proliferation and cytokine production, and led to reduced cytotoxicity of T cells. Second, blocking B7-H4 expression significantly impaired the Mϕs' suppressive capacity. Third, despite the presence of potent TAA-specific effector T cells in vivo, CD133+ cell-conditioned Mϕs promoted intracranial and subcutaneous tumor growth in SCID/NOD mice bearing human gliomas. Colocalization of human CTL and Mϕs/microglia in vivo in our mouse model presents new evidence for the role of B7-H4+ GIMs in immunomodulatory function.
Antitumor effects appear to be obstructed by a dichotomous mechanism that eliminates M1 macrophage-mediated innate immune responses and impairs T-cell activation. We observed that CD133+ cells stimulated by GIMs, which secreted high levels of IL6, expressed B7-H4 but did not secrete notable amounts of IL6 or IL10 into sera. Functionally, IL6 resulted in B7-H4 expression in Mϕs. Other studies also demonstrated that IFNγ could upregulate B7-H4 expression on mouse embryo fibroblasts, but detailed pathway was unknown (38). It seems plausible that glioma-initiating cells native to the tumor microenvironment trigger immunosuppression in part by “recruiting and talking” to Mϕs. Following an initial exchange with the GSGs, the Mϕs became polarized to the M2 type and integrated into an external role in the developmental stage of this immunomodulatory process and conducted persistent “B7-H4 education” through autoregulation. This intimate process is further promoted by Glioma initiating cells and fosters the immunosuppressive conditions mounted in the tumor stroma. The immunosuppressive association between glioma-initiating cells and GIMs may thus be defined in the context of time (early vs. late) and space (niche-inhabiting vs. recruitment and marginalization) to explain the proposed role of B7-H4 as a potential immunity-associated marker for malignancy progression and prognosis (Supplementary Fig. S7).
In this study, we showed that CSF sB7-H4 concentration tended to correlate with malignancy grades of glioma. The blood–brain barrier (BBB) normally allows the permeability of small molecules (400–500 Da) and some small lipid-soluble proteins (39), B7-H4 (50–80 kDa) may be too large to pass across this natural barrier. Considering CSF comes in direct contact with the extracellular compartment of the central nervous system and the existence of BBB, CSF sB7-H4 may be a better potential immunity-associated marker for supratentorial tumors like gliomas when compared with serum sB7-H4. Enrolling more research subjects with intracranial immunologic diseases to reflect the enhanced utility of sB7-H4 in CSF over sB7-H4 from serum may be needed.
In summary, our studies demonstrated a direct relationship between WHO grading of gliomas and the levels of B7-H4, suggesting its utility as a potential immunity-associated marker for progression of the disease. We established crucial roles of B7-H4 in the cellular interplay of glioma-mediated immunosuppression between CD133+ glioma-initiating cells and TIMs/GIMs, and uncovered that such a “cross-talking” contributed to glioma pathology. This study revealed a previously unrecognized immune evasion route and provided an anti–B7-H4 therapeutic target in patients with malignant gliomas.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: Y. Yao, C.C. Chen, K. Jin, X. Zang, Y. Mao
Development of methodology: H. Ye, D.S.B. Hoon, Y. Wang, L. Zhou
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Yao, H. Ye, L. Mo, Q. Yue, A. Baral, L. Zhou
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Ye, L. Mo, Q. Yue, C.C. Chen, W. Hua, J. Zhang, K. Jin, Y. Mao
Writing, review, and/or revision of the manuscript: Y. Yao, Z. Qi, D.S.B. Hoon, J.C. Vera, J.D. Heiss, C.C. Chen, X. Zang, Y. Mao
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Yao, Z. Qi, Y. Wang, L. Zhou
Study supervision: Y. Mao
Acknowledgments
The authors thank Jian Hu (MD Anderson Cancer Center), Daniel A. Tennant (University of Birmingham) for helpful advice, Xin Zhang (Fudan University) for assistance with patients' follow-up, and Ran Tao (Shanghai Jiaotong University) for helpful discussions.
Grant Support
This work was supported by the National Science Foundation for Distinguished Young Scholars of China (grant no. 81025013; to Y. Mao), the National Natural Science Foundation of China (grant no. 81372708 and 81272797; to Y. Yao), the Project for National 985 Engineering of China (No.985-YFX0102; to Y. Mao), the “Dawn Tracking” Program of Shanghai Education Commission, and China grant 10GG01 (to Y. Mao),the Project for Science and Technology Commission of Shanghai Municipality grant 13JC1408000 (to L. Zhou), innovation Program of Shanghai Municipal Education Commission (grant no. 13ZZ010; to Y. Yao).
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