Purpose: Gliomas are known to induce local and systemic immunosuppression, inhibiting T-cell–mediated cytotoxic responses to tumor growth. Tumor-associated macrophages are a significant component of the immune infiltrate in gliomas and may express immunosuppressive surface ligands, such as B7-H1.
Experimental Design: Tumor and peripheral blood samples from patients with glioblastoma (GBM) were analyzed by flow cytometry to evaluate the expression of B7-H1 in circulating and tumor-infiltrating macrophages. Human monocytes from healthy patients were stimulated with conditioned media from glioma cells to evaluate B7-H1 expression. Production of interleukin (IL)-10 by stimulated monocytes was measured by ELISA, and stimulation with IL-10 alone was evaluated for the ability to induce B7-H1 expression. The effect of inhibiting IL-10 and its receptor on glioma-induced B7-H1 expression in monocytes was evaluated.
Results: Circulating monocytes in patients with GBM had significantly increased expression of B7-H1 compared with healthy control patients. Tumor-associated macrophages from matched GBM tissue had even greater B7-H1 expression. Treatment of normal monocytes with glioma-conditioned media could significantly increase B7-H1 expression. Stimulation of monocytes with conditioned media resulted in substantial production of IL-10 and upregulation of the IL-10 receptor. Stimulation of monocytes with IL-10 alone could significantly increase B7-H1 expression, sufficient to induce T-cell apoptosis when cocultured with stimulated monocytes. Inhibition of IL-10 and the IL-10 receptor could knock down the effect of glioma media on B7-H1 by more than 50%.
Conclusions: Gliomas can upregulate B7-H1 expression in circulating monocytes and tumor-infiltrative macrophages through modulation of autocrine/paracrine IL-10 signaling, resulting in an immunosuppressive phenotype. Clin Cancer Res; 19(12); 3165–75. ©2013 AACR.
Patients with malignant gliomas are known to be systemically immunosuppressed and their tumors show multiple mechanisms of local immunoresistance. Innate immune monitoring and adaptive immunotherapy are rendered ineffective by these immune-suppressing factors, promoting tumor growth. Expression of B7-H1 by glioma cells has been shown to be a significant mechanism of local immunoresistance, resulting in quiescence and apoptosis of tumor-infiltrating cytotoxic T cells. In this study, we show that circulating monocytes and tumor-infiltrating macrophages in patients with glioma are induced to express B7-H1 as well, establishing a local and systemic immunosuppressive environment. Monocytes/macrophages are stimulated to express B7-H1 in response to a soluble factor produced by glioma cells that can amplify interleukin-10 autocrine/paracrine signaling in monocytes, resulting in upregulated B7-H1 expression. Inhibition of this pathway may represent a novel target to overcome glioma-mediated immune suppression and may be a necessary component of immunotherapy in the future.
Malignant gliomas are highly aggressive tumors with a uniformly poor prognosis (1, 2). Despite advances in medical and surgical management over the past decade, the median survival for the most aggressive form of glioma, glioblastoma (GBM), remains approximately 12 to 15 months (3, 4). One significant challenge to the development of new therapies for gliomas is their ability to induce local and systemic immunosuppression, limiting the innate defense to tumor growth and the efficacy of adaptive immunotherapy (5, 6). In the local tumor microenvironment, gliomas have been shown to suppress immune responses through upregulation of anti-inflammatory proteins (7–10), downregulation of antigen presentation (11, 12), and expansion of immunosuppressive effector cells such as regulatory T cells (13, 14). Gliomas can also express immunosuppressive ligands at the cell surface, including the costimulatory molecule B7-homologue 1 (B7-H1), also known as the programmed death ligand 1 (PD-L1; 15). Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) results in upregulated phosphoinositide-3 kinase (PI3K) activity in glioma cells and increased surface expression of B7-H1 (16). This ligand can bind to and stimulate the programmed death-1 (PD-1) receptor on activated T cells resulting in T cell quiescence and apoptosis (17, 18). Expression of B7-H1 by tumor cells is a common mechanism of immunoresistance in cancer, and has been shown in numerous malignancies including renal, lung, breast, and prostate cancer (19–22).
Despite adaptations to evade immune surveillance, gliomas do stimulate a significant immune response and are known to be infiltrated by immune effector cells (8). In addition to the expected lymphocytic response, high-grade gliomas have substantial macrophage and microglial infiltration (23, 24). Tumor-associated macrophages account for more than 10% of infiltrative cells in peritumoral GBM tissue, in comparison to less than 1% of cells in tumor-free brain (23). A common finding in multiple malignancies, tumor-associated macrophages can be paradoxically both immune-activating and immunosuppressive (25–28). Macrophages respond to signals from tumor cells by polarizing into proinflammatory (M1) or anti-inflammatory (M2) phenotypes, defined by their cascade of cytokine production and secondary effects on other immune cells (29, 30). Recent studies of the phenotype of glioma-associated macrophages suggest that they are largely immunosuppressive (26, 29, 31). In gliomas, the degree of macrophage infiltration has been positively correlated with tumor grade (32) and within high-grade lesions, higher levels of macrophage infiltration are associated with decreased survival (33).
While the standard definition of an anti-inflammatory (M2) tumor-associated macrophage is based on expression of immunosuppressive cytokines such as interleukin (IL)-10 and TGF-β, there is evidence that these macrophages can also express B7-H1 (31, 34). Studies from patients with hepatocellular carcinoma (HCC) show elevated levels of B7-H1 expression by tumor-infiltrating macrophages in human tumor samples, which correlate with poor outcomes (35). While peripheral blood monocytes are known to express low levels of B7-H1, exposure of monocytes to conditioned media from HCC cells is sufficient to significantly upregulate B7-H1 expression in culture (35). To date, substantial expression of B7-H1 in circulating monocytes and tumor-infiltrating macrophages in patients with glioma has not been documented. On the basis of in vitro studies and results from patients with other tumor types, we hypothesized that glioma-infiltrating macrophages can be stimulated by a glioma-derived soluble factor, inducing B7-H1 expression and rendering these macrophages capable of suppressing T cell activity. Through this mechanism, gliomas may be capable of inducing immunosuppression locally and in circulation, independent of PTEN status and B7-H1 expression on the glioma cell itself. Here, we present evidence from patients with GBM supporting this hypothesis.
Materials and Methods
Cell lines and specimens
Glioma cell lines (U251, U87) were obtained through the UCSF Brain Tumor Research Center, and normal human astrocytes (NHA) were obtained from Sciencell Laboratories. Fresh tumor tissue was obtained at surgery from patients undergoing initial operation for newly diagnosed GBM. Peripheral blood samples were also obtained at surgery from patients with GBM or from healthy donors. All patient specimens were obtained with written, informed consent under approval of the UCSF Committee on Human Research.
Peripheral blood leukocytes (PBL) were isolated from whole blood using Ficoll-Paque Plus (GE Healthcare) centrifugation. Tumor-infiltrating leukocytes (TIL) were isolated from resected tissue using a three-step density gradient, as previously described (36). Monocytes were then extracted from the PBL of healthy donors by CD14+ selection using magnetic nanoparticles (EasySep, Stem Cell Technologies) according to the manufacturer's instructions. Once separated, monocytes were suspended in RPMI-1640 25 mmol/L Hepes, 2.0 g/L NaHCO3 supplemented with 1% penicillin–streptomycin, 1 mmol/L sodium pyruvate, 10 mmol/L nonessential amino acids, and 2.5% FBS (UCSF Cell Culture Facility).
Glioma cells were plated at 5 × 104 cells per well in 24-well plates in RPMI-1640 media with 10% FBS and allowed to adhere 24 hours before co-culture. Isolated CD14+ monocytes were plated with glioma cells at 2 × 105 cells per well in contact or above a 0.2 μm pore Transwell permeable insert (Costar) and incubated for 24 hours at 37°C. Monocytes were then harvested from coculture suspension, spun at 300 × g for 5 minutes, and resuspended in RPMI before staining for flow cytometry.
Conditioned media culture
Supernatant from NHA and glioma cells cultured in T75 flasks was harvested at 90% confluence. Conditioned media was spun at 300 × g for 5 minutes to pellet any cellular material and the supernatant was transferred to 10 kDa Amicon ultrafiltration tubes (Milipore) and spun at 35, 000 rpm for 30 minutes to concentrate the media 20-fold. Isolated CD14+ monocytes were plated at 1 × 105 cells per well in 96-well plates and treated with the concentrated conditioned media added at 1:10 with normal media. In addition, monocytes were stimulated with varying doses of human recombinant cytokines including MCP-1 (BD Biosciences), MCP-3 (BD Biosciences), M-CSF (eBioscience), IL-6 (eBioscience), or IL-10 (eBioscience). Cells were incubated with conditioned media and/or cytokines for 24 hours at 37°C before staining for flow cytometry.
Cells were harvested and stained extracellularly with CD45 FITC (clone HI30, eBioscience), CD11b PeCy (clone ICRF44, eBioscience), HLA-DR APC (clone LN3, eBioscience), and B7-H1 PE (clone MIH1, eBioscience) or isotype control (eBioscience) in PBS with 2% bovine serum albumin on ice for 30 minutes. After washing, cells were fixed with 2% paraformaldehyde (Sigma) and read using a BD FACScaliber flow cytometer with CellQuest Software (Beckton Dickinson). Data was analyzed using FlowJo software (Treestar). For measurement of IL-10 receptor expression, IL-10R (CDw210) PE (clone 3F9, BD Bioscience) was used as an extracellular stain.
T cell apoptosis
T cells were enriched from donor PBL using a negative magnetic nanoparticle selection kit (StemCell Technologies). These cells were then activated by culture in 96-well plates with plate-bound human anti-CD3 (clone OKT3, 2 μg/mL) and soluble anti-CD28 (clone CD28.2, 4 μg/mL) for 48 hours (eBioscience). Activation was confirmed by blast formation. Concurrently, monocytes were extracted from the same donor PBL by CD14+ selection and stimulated as described. At 48 hours, activated monocytes and T cells were combined (1:1) in 96-well plates at a concentration of 1 × 105 cells per well in the presence of 10 U/mL recombinant human IL-2 (BD Bioscience). The coculture was allowed to incubate for 12 hours. Cells were harvested, washed, and resuspended in PBS with 2% bovine serum albumin. Cells were surface stained on ice for 30 minutes with CD3 PE (clone SK7, BD Bioscience) and CD11b FITC (clone ICRF44, eBioscience). After washing with Annexin buffer, cells were stained with Annexin V APC (eBioscience) for 10 minutes at room temperature and fixed in 2% paraformaldehyde before analysis by flow cytometry. The percentage of Annexin-positive cells was determined as a fraction of the total number of CD3+ T cells.
IL-10 concentration in glioma-conditioned media and in the media of treated monocytes was determined using a human IL-10 ELISA kit (BD Bioscience) according to manufacturer's instructions. Human cytomegalovirus (HCMV)-derived IL-10 was also measured in the glioma-conditioned media by ELISA. hcmvIL-10–specific IgG (100 ng/well; affinity purified, polyclonal; R&D systems, cat# AF117) was plated as a capture antibody and biotinylated hcmvIL-10–specific polyclonal IgG (10 ng/well; R&D systems, cat# BAF117) was used as a detection antibody. Standard curves were constructed using recombinant hcmvIL-10 (R&D systems, cat# 117-VL-025), as previously published (37).
Reverse transcribed-quantitative PCR
B7-H1 and IL-10 mRNA was extracted from treated monocytes using the RNeasy Mini Kit (Qiagen) and cDNA was generated using the Superscript III Kit (Invitrogen). Transcript levels were detected using the SYBR Green (Applied Biosystems) and the CFX96 Real-Time System (Bio-Rad Laboratories). Values of transcript levels were standardized using 18S ribosomal RNA. Sequences of primers used were: B7-H1 (5′-GCTGTT-GAAGGA-CCAGCT-CT/TGCTTG-TCCAGA-TGACTT-CG-3′), IL-10 (5′-GTGGAG-CAGGTG-AAGAAT-GC/ATAGAG-TCGCCA-CCCTGA-TG-3′), and 18S rRNA (5′-GTAACC-CGTTGA-ACCCCA-TT/CCATCC-AATCGG-TAGTAG-CG 3′).
Paraffin-embedded specimens were deparaffinized and rehydrated, followed by antigen retrieval for 30 minutes at 95°C using 10 mmol/L citrate buffer. Endogenous peroxidases were quenched with hydrogen peroxide. Samples were blocked in tyramide buffer (Invitrogen) with 1% Triton-X for 1 hour at room temperature. Incubation with a single primary antibody was conducted overnight at 4°C. Slides were washed in PBS with 0.1% Tween-20 and then incubated with a biotinylated secondary antibody (Vector Labs) at 1:500 for 30 minutes. Visualization was conducted using tyramide signal amplification (TSA) kit #21 (Invitrogen) as per manufacturer's protocol. Samples were washed then incubated with ABC-Elite (Vector Labs) for 30 minutes, then final labeling conducted using TSA kits #22 (Alexa Fluor 488), #25 (Alexa Fluor 594), or #26 (Alex Fluor 647). Samples were then blocked for 1 hour in tyramide buffer with avidin (Vector labs) and incubated overnight with a second primary antibody diluted in tyramide blocking buffer with biotin (Vector labs) at 4°C. The procedure above was repeated for the second and third primary antibodies. Dilution of primary antibodies was as follows: B7-H1 (Abcam ab55810, 1:500), CD163 (Abcam ab74604, prediluted), IL-10 (Thermo Scientific MA1-82664, 1:100). After completion of triple staining, nuclear visualization was conducted with DAPI (Invitrogen) and then mounted using VectaMount AQ (Vecor labs). Confocal images were generated on a Zeiss LSM 510 META laser-scanning microscope.
Monocytes extracted from PBL by CD14+ selection were plated at 1 × 105 cells per well in 96-well plates and pretreated with a soluble IL-10–neutralizing antibody (clone JES3-9D7, eBioscience) at 5 μg/mL or an IL-10 receptor anatagonist (clone 37607.11, Thermo Pierce Antibody) at 5 μg/mL for 2 hours. Cells were then stimulated with glioma-conditioned media and incubated for 24 hours at 37°C before staining for flow cytometry. The soluble IL-10–neutralizing antibody was redosed (5 μg/mL) at 12 hours to maintain an inhibitory effect.
Data for each experiment was collected from at least 3 independent specimens with testing of each patient specimen repeated in triplicate. The mean value from the multiple repetitions for each specimen was used for statistical analysis. Differences between treated specimens and controls were compared using a paired t test. Differences in the degree of change from control between different treatment conditions was evaluated by comparing the magnitude of change (Δ) using an independent t test for 2 test conditions or ANOVA from multiple groups. Correlations between protein expression and magnitude of functional change were evaluated using a Pearson's correlation coefficient. Statistical significance was accepted for P < 0.05. Comparisons were conducted using SPSS version 19 (IBM). Data is reported and shown graphically as mean ± SEM.
Identification of peritumoral macrophage infiltrates
Total leukocytes were isolated from peripheral blood and tumor of patients undergoing resection of GBM without prior therapy (n = 16) and analyzed by flow cytometry. The infiltrating macrophage fraction in the tumor was determined by gating on CD45bright/CD11b+ cells, distinguished from CD45dim/CD11b+ resident microglia (Fig. 1A). The cut-off intensity used to define CD45bright cells was selected from the lower boundary of CD45 positivity in the matched peripheral blood monocytes. A clear distinction between CD45bright and CD45dim groups was not always visible in the scatter plots, and therefore the macrophage fraction may contain some activated microglia as well.
Tumor-infiltrating macrophages have increased expression of B7-H1
Expression of B7-H1 on circulating monocytes from the peripheral blood of nonglioma control patients was found to be low (<15% B7-H1–positive cells). In comparison, peripheral blood and tumor-infiltrating monocytes/macrophages from patients with GBM had significantly increased expression of B7-H1 (Fig. 1B and C). Although there was significant variability in absolute expression between patients, in 15 of 16 (94%) patients B7-H1 expression was higher in the tumor-infiltrative macrophages compared with peripheral circulating monocytes, with nearly double the number of B7-H1–positive cells in tumor (63 ± 5.2% vs. 36.6 ± 5.3%, P < 0.001).
B7-H1 expression confers T cell immunoresistance
Peripheral blood monocytes from a nonglioma control patient and 3 patients with GBM were isolated from PBL by CD14+ selection. These selected patient monocytes had variable B7-H1 expression ranging from 8% to 78%-positive cells (Fig. 2A). Peripheral T cells were also isolated from the same patients and cocultured with their autologous monocytes after T cell activation. The percentage of T cells undergoing apoptosis was evaluated after 12 hours of culture among solitary T cells and T cells cocultured with monocytes (Fig. 2B). For all patients, the degree of T cell apoptosis was greater for cells cocultured with monocytes than in solitary culture (P < 0.01). The magnitude of increase in apoptosis (Δ% apoptosis) correlated linearly with degree of B7-H1 expression (R = 0.96, P < 0.001).
Gliomas can induce B7-H1 expression in peripheral monocytes
Having shown that monocytes/macrophages in patients with GBM have increased expression of B7-H1 that can functionally induce T cell apoptosis, we investigated the role of the glioma cell in stimulating expression of B7-H1 in macrophages. Peripheral monocytes from nonglioma control patients with low B7-H1 levels were cocultured with U251 glioma cells in contact or through a 0.2 μm filter. After 24 hours, the number of B7-H1–expressing cells had increased by more than 2-fold in monocytes cocultured through a filter (48.0 ± 5.2% vs. 13.0 ± 3.9%, P < 0.005), and more than 4-fold in monocytes cocultured in direct contact (83.9 ± 6.2% vs. 13.0 ± 3.9%, P < 0.001; Supplementary Fig. S1). The results of the filtered coculture suggest that gliomas can induce B7-H1 expression in monocytes through a secreted soluble factor. However, the increase in B7-H1 expression from the contact coculture was significantly greater than the filtered coculture (P < 0.01).
Glioma-derived soluble factors can induce B7-H1 expression in peripheral monocytes
To further investigate the role of a soluble glioma-derived factor that could upregulate macrophage B7-H1, peripheral monocytes were stimulated with conditioned media from glioma cells after removal of all cellular material. Conditioned media (CM) was obtained from normal human astrocytes (NHA), established glioma cell lines (U87, U251), and a primary GBM cell culture (SF1U). The conditioned media was concentrated 20-fold and added to peripheral monocytes from nonglioma control patients at a concentration of 1:10 with normal media. B7-H1 surface expression was assessed by flow cytometry after 24 hours of culture. There was no significant change in B7-H1 expression with the addition of NHA CM; however, the addition of CM from all 3 glioma cell lines resulted in significant increases in B7-H1 protein expression (Fig. 3A and B). In addition, using quantitative RT-PCR, B7-H1 (CD274) mRNA was shown to increase nearly 15-fold with the addition of glioma-conditioned media as compared with unstimulated monocytes, suggesting activation at the transcriptional level (Fig. 3C).
We then evaluated the physical properties of the soluble glioma-derived factor to identify the active component of the glioma CM responsible for induction of B7-H1 expression in macrophages. First, the glioma-conditioned media was spun through a 10 kDa filter and both filtered components were tested for the ability to upregulate B7-H1 expression in monocytes. Only the fraction with a molecular weight more than 10 kDa showed efficacy (Fig. 4A). The active fraction was then subjected to heat denaturation at 100°C for 10 minutes, which resulted in loss of activity (Fig. 4B). The glioma-conditioned media was also added to monocytes at various concentrations showing increases in B7-H1 in a dose–response relationship (data not shown). These characteristics suggested that the inducing factor was likely a protein with a molecular weight more than 10 kDa.
We subsequently used a candidate approach to screen a series of cytokines produced by gliomas that have been shown to stimulate monocytes/macrophages in previous studies. Monocyte chemotactic protein 1 and 3 (MCP1 and MCP3) as well as monocyte colony stimulating factor (M-CSF) are produced by a variety of tumors, including gliomas, and activate the PI3K/Akt pathway, which regulates B7-H1 expression in glioma cells (38, 39). In addition, IL-6 and IL-10 are produced by gliomas and activate the STAT1/3 pathways, which have been shown to drive anti-inflammatory phenotypes in monocytes/macrophages (40, 41). Therefore, these cytokines were screened for their ability to induce B7-H1 expression in monocytes at a range of concentrations compared with glioma-conditioned media. Even at supraphysiologic levels of individual cytokines, B7-H1 levels were not significantly increased in monocytes treated with MCP1, MCP3, MCSF, or IL-6 (Fig. 4C). However, monocytes treated with IL-10 did significantly upregulate B7-H1 expression (P < 0.01), albeit less than the induction observed with glioma-conditioned media treatment (P < 0.05).
Glioma-derived factors enhance IL-10 autocrine signaling in monocytes
Having shown that IL-10 can stimulate B7-H1 expression in monocytes, the levels of IL-10 produced by glioma cells were investigated. Using an ELISA, conditioned media from NHA, U251, U87, and SF1U cell cultures were analyzed and found to have no appreciable human IL-10. The CM was then added to unstimulated monocytes in culture for 24 hours and IL-10 levels were again measured from the culture media. Following stimulation with U251 CM and SF1U CM, monocyte production of IL-10 was significantly increased (Fig. 5A). Previous studies have shown that glioma stem cells from patients with prior exposure to cytomegalovirus (CMV) produce viral IL-10, which can phenotypically alter peripheral monocytes similar to human IL-10 (34). Therefore, the CM from glioma cells was retested before and after stimulation of monocytes using an ELISA to specifically detect hcmvIL-10. No hcmvIL-10 was detected in the CM before or after stimulation of monocytes.
In addition to IL-10, expression of the IL-10 receptor on monocytes was analyzed before and after stimulation with glioma-conditioned media. Although modest, there was a statistically significant increase in IL-10R expression after glioma-conditioned media stimulation (Fig. 5B). Together, these results suggest that a glioma-derived soluble factor enhances monocyte IL-10 production and IL-10R expression, possibly leading to a synergistic increase in IL-10 autocrine/paracrine signaling.
IL-10 expression precedes B7-H1 expression in stimulated monocytes
To determine the relative timing of IL-10 and B7-H1 expression following stimulation, normal monocytes were stimulated with glioma-conditioned media and both IL-10 and B7-H1 mRNA levels were evaluated by quantitative RT-PCR at 1, 6, 12, and 24 hours following stimulation (Fig. 5C). IL-10 transcript levels rose first, reaching near-peak expression by 6 hours, whereas B7-H1 expression lagged behind, rising steadily over 24 hours.
IL-10 and B7-H1 are coexpressed in immunosuppressive tumor-associated macrophages
To determine the source of IL-10 and B7-H1 in vivo, histopathologic specimens from patients with GBM were immunostained for CD163, B7-H1, and IL-10 (Fig. 5D). Immunosuppressive M2 macrophages, stained by CD163, were all found to be copositive for B7-H1 and IL-10.
IL-10 signaling is a necessary component of glioma-induced upregulation of B7-H1
To assess the functional consequences of upregulated B7-H1 expression in stimulated monocytes/macrophages, monocytes were cocultured with activated T cells and T cell apoptosis was measured by Annexin staining. Significant increases in T cell apoptosis were seen when cocultured with monocytes stimulated with IL-10 or glioma-conditioned media (Fig. 6A).
To determine whether IL-10 was a necessary component of the response to glioma-conditioned media, monocytes were stimulated with glioma CM in the presence of a soluble IL-10–neutralizing antibody or an IL-10 receptor inhibitor. With inhibition of IL-10 and the IL-10 receptor, monocyte expression of B7-H1 in response to stimulation was significantly reduced (Fig. 6B).
T cell–mediated cancer immunotherapy is dependent on initiation of a specific CD8+ T cell response that can effectively target and destroy the tumor cells (42). Immunoresistance in the local tumor microenvironment is a significant impediment to successful immunotherapy, allowing the tumor to evade a cytotoxic response. Gliomas have been shown to resist immune responses through several mechanisms including downregulation of immunostimulating antigens and expression of anti-inflammatory proteins (12, 13, 40, 41). We have previously linked expression of the immunosuppressive protein B7-H1 in glioma cells with activation of the PI3K/mTOR pathway, leading to quiescence and apoptosis of infiltrating T cells (16). This mechanism of local immunoresistance is well-recognized in multiple cancers including breast, lung, prostate, and renal cell (19, 20, 22). However, in addition to local immune resistance driven by the tumor, patients with glioma are known to have suppressed immune cell function, exhibiting a lack of response to normal immune activators (5). Therefore, gliomas must modulate the function of immune effectors in a global fashion. There have been several reports in the literature of mechanisms by which gliomas affect immunity, including expansion of circulating regulatory T cells and phenotypic modulation of circulating monocytes to myeloid-derived suppressor cells (14, 31, 43). In addition, studies have shown that monocytes exposed to glioma cells in vitro can be induced to express B7-H1 (31, 34). However, despite showing an association of anti-inflammatory macrophages with gliomas, the mechanism by which gliomas modulate macrophage function and overall immunity remains largely unknown. Expression of B7-H1 by circulating monocytes or infiltrating macrophages has not previously been shown in patients with glioma. We hypothesized that gliomas could induce expression of B7-H1 in macrophages/monocytes locally and systemically through production of a soluble cytokine.
The results presented in this study support our initial hypothesis. Circulating monocytes and tumor-infiltrating macrophages from patients with GBM have significantly increased expression of B7-H1 compared with monocytes from healthy control patients. An increase in B7-H1 can be induced in normal monocytes through stimulation with a soluble factor(s) produced by malignant glioma cells, and this factor showss the physical properties of a cytokine. As B7-H1 expression has been shown to be dependent on PI3K/Akt and STAT3 activity, we investigated activators of these signaling pathways as candidates for the glioma-derived stimulatory cytokine. Despite evidence that they are produced by glioma cells, MCP-1, MCP-3, M-CSF, TGF-β, and IL-6 all failed to modulate B7-H1 expression in normal monocytes. Only stimulation with IL-10 resulted in a significant increase in B7-H1 expression in monocytes with the functional consequence of increasing T-cell apoptosis when stimulated monocytes were co-cultured with activated T cells. These results suggested that IL-10 has a role regulating B7-H1 expression in glioma-associated macrophages.
We initially assumed that the glioma cells were the source of IL-10; however, no IL-10 was detectable in the CM from several glioma cell lines and low-passage primary cultures. IL-10 was produced by monocytes after stimulation with glioma-conditioned media. Previously published studies of IL-10 expression in human glioma tissue have shown that the primary source of IL-10 in tumors is tumor-infiltrative macrophages/microglia (44). Therefore, it is not surprising to find that gliomas can stimulate normal monocytes to produce IL-10, which has been described as part of the anti-inflammatory phenotype of M2 macrophages in gliomas. Using immunofluorescence, we have confirmed that CD163+ M2 macrophages in patient tumors do, in fact, express IL-10 and B7-H1.
The finding that IL-10 can stimulate B7-H1 expression in monocytes is also not entirely surprising, as this has been shown in macrophages associated with hepatocellular carcinoma (35). The novel finding in this study is that a glioma-derived factor can increase B7-H1 expression in macrophages indirectly by enhancing autocrine/paracrine IL-10 signaling, thereby promoting an immunosuppressive effect in the local microenvironment and systemic circulation. Furthermore, we have shown that by antagonizing IL-10 signaling, we can block upregulation of B7-H1 expression.
Supporting our findings, recent publications in the immunology literature have shown that stimulation of dendritic cells with agonists of the innate immune system (toll-like receptor ligands) such as lippopolysacharides results in production of IL-10 (45). These IL-10–expressing dendritic cells can then induce a phenotypic shift in naïve dendritic cells to B7-H1 expressing tolerogenic cells in the absence of a toll-like receptor agonist. This feedback system to suppress the response to pathogenic antigens likely exists to prevent overstimulation of the immune system and protect against autoimmunity. We now show that tumors can capitalize on this system to evade immune monitoring and promote tumor growth.
One limitation in our findings is that the degree of B7-H1 expression induced by stimulation with IL-10 alone, while enough to affect T cell apoptosis, is less than the expression achieved with glioma-conditioned media stimulation. Although this may be explained by the concurrent upregulation in the IL-10 receptor associated with glioma-conditioned media stimulation, we cannot rule out the possibility of a second glioma-derived stimulatory factor. As we have not yet identified the glioma-dervied factor that induces IL-10 production by monocytes, we cannot investigate the direct role that binding of this ligand has on B7-H1 expression. Having unsuccessfully used a candidate approach to identify this factor, we are now undertaking a protein purification approach to determine the active factor in glioma-conditioned media. On the basis of the known signaling pathways for IL-10, we hypothesize that B7-H1 expression is, at least in part, dependent on STAT3 activation. However, without having identified the IL-10–stimulating factor, we cannot predict what other pathways may be activated upstream and may modulate STAT3 activity. In addition, the degree of B7-H1 upregulation in monocytes cocultured with gliomas in contact is greater than the effect of culture with a soluble factor alone, suggesting that there may be further modulation by surface-bound cofactor on glioma cells. This has been shown in previous studies as well (31). However, inhibition of IL-10 signaling unquestionably resulted in a more than 50% reduction in B7-H1 expression, suggesting that targeting IL-10 may be a useful strategy to combat glioma-induced local and systemic immunosuppression. Further studies are necessary to clearly define all factors in this complex signaling pathway, and there is still much work to be done before developing a clinical strategy to decrease B7-H1 expression and increase the efficacy of adaptive immunity.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The funding organizations had no role in study design, data collection and analysis, or preparation of the article.
Conception and design: O. Bloch, A.T. Parsa
Development of methodology: O. Bloch, C.A. Crane, A.T. Parsa
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): O. Bloch, C.A. Crane, R. Kaur, M. Safaee, M.J. Rutkowski
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): O. Bloch, R. Kaur, M. Safaee, M.J. Rutkowski, A.T. Parsa
Writing, review, and/or revision of the manuscript: O. Bloch, C.A. Crane, R. Kaur, M.J. Rutkowski, A.T. Parsa
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.A. Crane, R. Kaur, M.J. Rutkowski, A.T. Parsa
Study supervision: A.T. Parsa
This work was supported by the NI H grants 1F32 CA153839-01, 1K99 NS078055-01 (to O. Bloch), 1K99 CA151412-02 (C.A. Crane), and the Reza and Georgianna Khatib Endowed Chair in Skull Base Surgery (A.T. Parsa.).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.