The indoleamine 2,3-dioxygenase pathway controls complement-dependent enhancement of chemo-radiation therapy against murine glioblastoma
- Minghui Li1, 2, 3,
- Aaron R Bolduc1, 2, 4,
- Md Nasrul Hoda5, 9,
- Denise N Gamble1, 2,
- Sarah-Bianca Dolisca3,
- Anna K Bolduc1, 2,
- Kelly Hoang3,
- Claire Ashley3,
- David McCall1, 2,
- Amyn M Rojiani1, 6,
- Bernard L Maria1, 3, 5, 7,
- Olivier Rixe1, 8,
- Tobey J MacDonald10,
- Peter S Heeger11, 12,
- Andrew L Mellor1, 2, 8,
- David H Munn1, 2, 3 and
- Theodore S Johnson1, 2, 3Email author
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 18 April 2014
Accepted: 2 July 2014
Published: 7 July 2014
Indoleamine 2,3-dioxygenase (IDO) is an enzyme with immune-suppressive properties that is commonly exploited by tumors to evade immune destruction. Anti-tumor T cell responses can be initiated in solid tumors, but are immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade. We hypothesized that IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction.
To test this hypothesis, we utilized a syngeneic orthotopic glioblastoma model in which GL261 glioblastoma tumor cells were stereotactically implanted into the right frontal lobes of syngeneic mice. These mice were treated with IDO-blocking drugs in combination with chemotherapy and radiation therapy.
Pharmacologic inhibition of IDO synergized with chemo-radiation therapy to prolong survival in mice bearing intracranial glioblastoma tumors. We now show that pharmacologic or genetic inhibition of IDO allowed chemo-radiation to trigger widespread complement deposition at sites of tumor growth. Chemotherapy treatment alone resulted in collections of perivascular leukocytes within tumors, but no complement deposition. Adding IDO-blockade led to upregulation of VCAM-1 on vascular endothelium within the tumor microenvironment, and further adding radiation in the presence of IDO-blockade led to widespread deposition of complement. Mice genetically deficient in complement component C3 lost all of the synergistic effects of IDO-blockade on chemo-radiation-induced survival.
Together these findings identify a novel mechanistic link between IDO and complement, and implicate complement as a major downstream effector mechanism for the beneficial effect of IDO-blockade after chemo-radiation therapy. We speculate that this represents a fundamental pathway by which the tumor regulates intratumoral vascular activation and protects itself from immune-mediated tumor destruction.
KeywordsIDO Indoleamine Complement Tumor Immunotherapy Chemotherapy Radiation therapy Indoximod Glioblastoma NLG919
Immune responses to established tumors, and particularly brain tumors, are commonly ineffective in controlling tumor progression [1, 2]. Nonetheless, vaccines against glioblastoma-specific antigens have resulted in improved survival in clinical trials, and chemotherapy appears to enhance this beneficial effect [3, 4]. Emerging evidence indicates that brain tumors are not irreversibly “immune-privileged,” but rather employ a number of regulatory mechanisms to prevent immune-mediated tumor destruction [2, 5]. Identifying and therapeutically targeting these poorly understood immune regulatory mechanisms could enable induction of protective, endogenous anti-tumor immunity.
Indoleamine 2,3-dioxygenase (IDO), an enzyme with immunosuppressive properties, has been detected in a wide variety of human tumors, including glioblastoma, and in tumor draining lymph nodes [6–8]. High tumor expression of IDO is an independent risk factor for poor outcome in a variety of human cancers, including glioblastoma, and orthotopic syngeneic GL261 tumors grown in C57BL/6 mice were shown in 2012 to exhibit an IDO-rich tumor microenvironment that protects tumors from T cell attack .
Mechanistically, IDO has been shown to suppress T cell responses and to promote activation of regulatory T cells (Treg cells) in settings as diverse as mucosal tolerance, pregnancy, chronic infection and organ transplantation [reviewed in ]. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade . IDO-dependent generation of the bioactive tryptophan metabolite kynurenine is a potent vasodilator  that appears to be critically important in preventing tissue rejection in one allogeneic heart graft model [12, 13]. Whether and how these mechanisms impact tumor growth are unclear, but the strong association between IDO expression and progressive tumor growth has led to ongoing early-phase trials to test whether targeting IDO has clinical efficacy.
In preclinical models, IDO-inhibitors were shown to have synergistic effects when administered with multiple classes of chemotherapy agents and radiation [14, 15]. Early-phase clinical trials combining standard chemotherapy with IDO-inhibitor drugs, such as 1-methyl-D-tryptophan (D-1MT, indoximod) and NLG919, for treatment of refractory solid tumors are in progress. Such approaches have not been previously attempted for brain tumors, and specific mechanisms through which IDO regulates tumor progression under these conditions are not known. Based on previously documented effects of radiation on inflammation and vascular activation [1, 16] and evidence that IDO-blockade has some effect on tumor therapy [14, 15], we tested the hypothesis that the IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction.
IDO-blockade synergizes with standard chemo-radiation therapy to enhance survival in mice with glioblastoma
Clinically, glioblastoma tumors are treated with surgical resection, followed by temozolomide (TMZ)-based chemotherapy plus local radiation. IDO-blockade has been shown to enhance the effects of both chemotherapy and radiation . We tested the hypothesis that blocking IDO synergizes with TMZ plus radiation therapy (RT) to enhance survival in a murine model of glioblastoma. We employed the mouse glioblastoma cell line GL261 which becomes a highly aggressive tumor with short survival time, so treatment was focused on a single cycle of therapy, and a synergistic effect was defined as prolongation of survival relative to controls.
Additional experiments in which we employed shielding to target the radiation dose to the cranium of the mice yielded results essentially identical to total-body irradiation (Additional file 1: Figure S2). In other control experiments, we observed that IDO-blockade had no survival-enhancing effect when combined with either TMZ alone (no RT; Additional file 1: Figure S3A), or RT alone (no chemotherapy; Additional file 1: Figure S3B). Together, these data indicate synergistic interactions among the three therapies when used together (IDO-blockade, TMZ, and RT), and the protective effect for the host was not influenced by the breadth of the radiation field.
IDO-blockade synergizes with subtherapeutic cyclophosphamide plus radiation therapy to enhance survival
We also tested the ability of IDO-inhibitors to synergize with a cyclophosphamide (CPM)-based chemo-radiation regimen using the same glioblastoma model. CPM has been used to treat glioblastoma in the clinic  and has been widely studied in the context of immunotherapy [14, 21]. We used a dose (100 mg/kg) that exhibited no therapeutic benefit as a single agent (Additional file 1: Figure S3C) and did not prolong survival when added to RT alone (Additional file 1: Figure S3B). However, when we added DL-1MT to CPM + RT we observed significantly prolonged survival compared to animals treated with CPM + RT alone (Figure 1C). Substituting the D-isomer of 1MT for the racemic mixture yielded the same results (Figure 1D). As with TMZ, IDO-blockade with CPM alone had no effect on survival (Additional file 1: Figure S3C). Thus, D-1MT and DL-1MT equivalently enhanced survival of mice with GL261 tumors treated with CPM + RT. In the subsequent experiments in this report, the D- and DL- preparations of 1MT were both used, and results were consistently equivalent.
IDO target protein is expressed by GL261 tumors in vivo
GL261 tumors have been shown by others to express IDO protein in vivo, even in mice genetically deficient in the IDO-1 isoform [7, 22]. We performed immunohistochemical staining on frozen sections of GL261 tumors to detect the presence of IDO protein in our system. We observed distinct patterns of IDO expression in tumor cells, in perivascular areas within and adjacent to tumors, and in peritumoral cells with astrocytic morphology (Additional file 1: Figure S4A). GL261 tumors were also found to express the IDO-2 isoform after 18 days of growth in either wild-type (WT) or IDO1-deficient host mice (Additional file 1: Figure S5). Because survival was improved when IDO-blockade was added to chemo-radiation therapy, we next analyzed GL261 tumors from treated host mice. We found that treatment of host mice with TMZ, TMZ + RT, or CPM + RT did not affect IDO target protein expression (Additional file 1: Figure S4B).
Chemotherapy alone promotes perivascular accumulation of bone marrow-derived leukocyte cell populations within tumors
Unexpectedly, instead of parenchymal infiltration of immune cells after chemotherapy treatment, we observed perivascular collections of CD45-positive leukocytes, as early as 3 days after chemotherapy treatment, which progressively increased over time (Figure 2A and C). In contrast, no perivascular leukocyte cuffs were observed in the contralateral (uninvolved) brain hemispheres. This vascular focus of leukocyte aggregation was due to chemotherapy, not the tumor itself, because only rare small collections were found in control, untreated, tumor-bearing mice (Figure 2B). Frequency analysis of these tissues for perivascular cuffs (defining a cuff as at least three layers of CD45-positive cells circumferentially around a CD31-positive vessel) demonstrated a significant increase in these large aggregates by day 7 after TMZ (Figure 2C). Finally, although TMZ can cause peripheral leukopenia, perivascular leukocyte cuffs were observed in tumors from mice treated with standard doses of either TMZ (100 mg/kg) or CPM (100 mg/kg) (Additional file 1: Figure S6).
The large perivascular cuffs from tumors harvested 7 days after TMZ (Additional file 1: Figure S7A) were predominantly comprised of CD68+ macrophages (CD45+) and microglia (CD45-lo), as well as substantial numbers of CD4+ T cells (Additional file 1: Figure S7B). A large proportion of the CD4 T cells co-stained for nuclear Foxp3, indicating a Treg cell phenotype (Additional file 1: Figure S7C). CD8 T cells were notably sparse in these leukocyte collections (Additional file 1: Figure S7B). Treating tumor-bearing mice with high dose chemotherapy plus RT caused the perivascular leukocyte cuffs and vessels to become disorganized with downregulation of endothelial CD31 expression (data not shown). Thus, while a single modest dose of TMZ alone had only minimal effect on survival, the intervention initiated a process of macrophage, microglia, and Treg cell aggregation at vascular sites within the tumor.
Blocking IDO during chemotherapy enhances vascular activation
IDO regulates complement deposition within the tumor
We found that GL261 tumors from untreated mice did not stain for C3, and tumors from mice treated with TMZ + RT showed only scattered interstitial C3 staining (Figure 4A). However, when we combined TMZ + RT with IDO-blockade using either 1MT or NLG919, we observed extensive and confluent C3 deposition (Figure 4B). To test whether IDO was required for complement deposition, we treated IDO-deficient mice with TMZ + RT. We observed the same deposition of C3 in tumors from IDO-deficient host mice treated with TMZ + RT as we found in tumors from WT mice treated with IDO-blockade plus TMZ + RT (Figure 4B). We also found that C3 accumulation in tumors required the full combination of IDO-blockade with chemo-radiation therapy, as complement deposition was not seen in mice treated with IDO-blockade alone, IDO-blockade with TMZ (no RT), or IDO-blockade with RT (no TMZ) (Additional file 1: Figure S8). We also found that C3 deposition was strictly confined to the tumor microenvironment, and adjacent brain tissue was completely spared (Figure 4B). Thus, host IDO activity regulated complement deposition in glioblastoma tumors after chemo-radiation therapy, and complement deposition was highly selective and confined to the tumor itself.Image analysis software was used to quantitate photomicrographs of tumors stained for complement C3. Fluorescence-intensity histograms were generated for quantitative analysis of mean fluorescence intensity (MFI) and percent of downfield-gated (positive) pixels (Figure 4C). Tumors from mice with inactive IDO, either due to IDO-blockade or genetic IDO deficiency, treated with TMZ + RT had significantly increased complement C3 MFI (Figure 4D) and Percent Downfield Pixels (Figure 4E) compared to controls. Taken together, these data indicate that pharmacological blockade or genetic ablation of IDO allows TMZ + RT to trigger widespread C3 deposition in tumors consistent with local activation of the complement cascade, which is not observed when WT hosts are treated with TMZ + RT alone.
Complement is mechanistically required for the beneficial effects of blocking IDO during chemo-radiation therapy
As before, when we added 1MT to the treatment regimen, we observed the expected additional enhancement in survival for WT host mice. In contrast, we found that C3-/- host mice completely lost the beneficial survival effect of blocking IDO, and their survival curves became essentially identical with or without 1MT. Thus, the survival benefit provided by adding IDO-blockade to chemo-radiation therapy was strictly dependent on the ability of this combination to activate the complement system, and was abrogated in C3-/- host mice.
Tryptophan catabolism resulting in production of immuno-active kynurenine compounds and local tryptophan depletion can be mediated by either IDO or tryptophan 2,3-dioxygenase (TDO). We cannot rule out a contribution by TDO in these GL261 tumors, and there is no single-cell assay for production of tryptophan breakdown products. We have therefore been careful not to assume that inability to measure bioactive kynurenines equates to their absence in the relevant microenvironments during IDO-blocking treatment. However, there is a clear biological effect of blocking IDO with 1MT, which does not inhibit TDO , and of genetic IDO-1 deficiency (Figure 4B-E). In the short-term, IDO activity is important in suppressing complement deposition, and TDO does not appear to compensate for this when IDO is blocked during chemo-radiation therapy. Although TDO may be a problem for long-term host survival, which has clinical and therapeutic implications, the mechanistic question has been answered by IDO-deficient host mice which do develop complement deposition in tumors after chemo-radiation treatment. Nonetheless, it would be desirable to develop dual-specificity inhibitors for clinical application.
We used this model to study the short-term effects of adding IDO-blocking drugs to a single cycle of chemo-radiation therapy. The goal of any single cycle of chemo-radiation therapy is for the tumor to be smaller before the next cycle begins, and any human cancer patient would be treated with multiple cycles to compound the damage to the tumor. We speculate that, in our short-term model, adding IDO-blockade damages the tumor acutely via intensified inflammation and vasculitis, effectively amplifying the chemotherapy/radiation dose.
An emerging theme in cancer immunotherapy is the importance of vascular activation and targeting in mediating certain forms of tumor regression. Previous work in the B16 melanoma model has shown that despite evidence of peripheral anti-tumor T cell responses, tumor-specific vaccines are ineffective in causing tumor rejection, even when combined with CTLA-4-blockade and depletion of host Treg cells, until radiation is used to drive vascular activation . Additional data from this model suggest that anti-tumor T cell responses are initiated in solid tumors, but immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO . Adoptive transfer of melanoma-specific CD8 T cells engineered to express IL-12 was found to cause tumor involution via collapse of tumor stroma and vasculature . IL-12 is known to promote IFNγ secretion, which in turn can induce IDO expression in myeloid cell subsets [reviewed in ]. Preliminary experiments in our model show that the adaptive immune system is necessary for the formation of perivascular leukocyte collections after chemotherapy (unpublished data). It is also known from previous work that T cell interactions with antigen-presenting cells and/or endothelial cells initiate alternative pathway-dependent complement activation yielding local production of C3a and C5a [26–28]. These anaphylatoxins signal through their receptors on T cells to promote effector T cell activation and expansion [26, 28] and inhibit the generation, function, and stability of Treg cells . We propose that IDO-blockade during inflammatory chemo-radiation therapy drives a positive feedback loop between complement activation/deposition and pre-primed tumor-specific effector T cells, which activates and amplifies both processes.
Based on the prominent connection between IDO and complement in settings as diverse as pregnancy and brain tumors, we propose that locally inhibiting the pro-inflammatory complement pathway is a fundamental mechanism by which IDO helps create immune tolerance. From a translational perspective, our data suggest that there is substantially more anti-tumor efficacy available from conventional chemo-radiation treatments if they are given in a setting in which IDO is blocked, thus allowing them to trigger the beneficial contribution of the complement pathway. Additional studies will be required to determine which components of the complement cascade are key mediators of the downstream biological effects of blocking IDO, which complement regulatory pathways are induced or maintained by IDO expression, and whether IDO activity directly affects complement production or activation.
Mice, glioma cell line, and reagents
C57BL/6, B6.129-Ido1tm1Alm/J (IDO1-deficient), and B6;129S4-C3tm1Crr/J (complement C3-deficient) mice were purchased from the Jackson Laboratory. Animal studies were approved by the Institutional Animal Care and Use Committee of Georgia Regents University. GL-261 cells were purchased from the National Institutes of Health Tumor Repository (Frederick, MD) and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Corning Cellgro). IDO-inhibitor drugs 1-methyl-D-tryptophan (D-1MT, catalog no. 452483) and 1-methyl-L-tryptophan (L-1MT, catalog no. 447439) were purchased from Sigma-Aldrich, and NLG919 was a generous gift from Mario Mautino (NewLink Genetics). Primary antibodies for immunohistochemical studies were purchased from: Abcam [rabbit anti-mouse CD31 polyclonal antibody (catalog no. ab 28364)], eBioscience [rat anti-mouse CD45 (clone 30-F11)], AbD Serotec [rat anti-mouse VCAM-1 (clone MVCAM A(429))], and Cedarlane [rat anti-mouse complement C3 (clone RmC11H9, specific for native mouse C3 and breakdown products C3b, iC3b, and C3dg)]. Secondary immunohistochemistry reagents labeled with Alexa Fluor 488 or Cy3 were purchased from Jackson ImmunoResearch Laboratories, Inc.
Tumors and animal treatments
Mice were anesthetized with 4% isoflurane, and the surgical plane of anesthesia was maintained with 2% isoflurane in oxygen. Mice were immobilized in a stereotactic frame (Stoelting Company) for tumor implantation. Briefly, the skull was shaved and exposed with a 0.5 cm skin incision. With antiseptic technique, 105 GL261 cells (suspended in 3 μL RPMI-1640) were injected at the following coordinates with respect to the bregma on the right side (antero-posterior, -2 mm; medio-lateral, 2 mm; dorso-ventral, 3 mm). This placement reproducibly yielded tumor growth in a paracortical area of the posterolateral right frontal lobe. Tumor-bearing mice were treated with combinations of oral DL-1MT (2 mg/mL D-1MT mixed with 2 mg/mL L-1MT) in drinking water, D-1MT (4 mg/mL) in drinking water, NLG919 (6 mg/mL) in drinking water, intraperitoneal cyclophosphamide (Baxter), intraperitoneal temozolomide (Merck), and/or total-body radiation (500 cGy from a 137Cs source), as detailed in figure legends. Mice were observed daily, and sacrificed when they became ill or moribund, which defined the ethical surrogate endpoint as approved by our animal-use committee.
Tumor tissue was harvested, snap-frozen, and stored at -80°C until 5 μm sections were cut for immunohistochemical staining. Tissue sections were fixed with ethanol and acetone (1:1). Nonspecific binding to tissue was then blocked with normal mouse serum and serum matched to the secondary reagent host species. Sections were incubated with primary antibody followed by fluorescence-labeled secondary reagents. Nuclear counterstain was performed using Hoechst (catalog no. 14530, Sigma-Aldrich). Fluorescent microscopy (Olympus AX70 upright compound microscope) with a SPOT digital camera and software (SPOT Imaging Solutions) was used to generate photomicrographs.
Quantitation of complement C3 staining
Photomicrographs of tumors stained for complement C3 (red) were obtained in a grid pattern at magnification ×400. Abobe Photoshop CS6 (Adobe Systems, Inc.) image analysis software was used to transform photomicrographs of complement C3-labeled tumors into fluorescence-intensity histograms for quantitative analysis of MFI and percent of downfield-gated (positive) pixels, defined as occurring above an arbitrarily-chosen negative threshold (above channel thirty-two). Histograms for reproduction in Figure 4 were generated by FastStone Image Viewer (FastStone Soft, 2013).
Statistical analysis was performed using NCSS 2007 statistical software (NCSS, LLC.). For survival data, Kaplan-Meier curves were analyzed by the log-rank test. For frequency analysis of perivascular cuff and quantitative analysis of complement C3 (MFI and Percent Downfield Pixels), ANOVA with Kruskal-Wallis test was used. Significance was defined as a P value less than 0.05. Time to ethical surrogate endpoint was treated as survival time for statistical analysis.
Mean fluorescence intensity
- Treg cell:
Regulatory T cell
Vascular cell adhesion molecule-1
The authors thank Joyce Wilson (GRU Cancer Center) for expert technical assistance in developing immunohistochemical techniques, Kimberly Smith (GRU Cancer Center) for assistance with tissue processing and histology, and Mario Mautino and Rick Metz (NewLink Genetics, Ames, Iowa) for providing NLG919 drug and novel IDO-specific antibody reagents. We also acknowledge Dr. Jian-Yue Jin (Georgia Regents University, Medical College of Georgia Department of Radiology) for assistance with radiation shielding experiments, and Dr. Ian Heger (Georgia Regents University, Medical College of Georgia Department of Neurosurgery) for valuable discussions.
This study was supported by the Alex’s Lemonade Stand Foundation (to TSJ), Hyundai Hope on Wheels Foundation (to TSJ), the Beez Foundation (to TSJ), the Elizabeth and Lovick Corn Foundation (to DHM), the Virendra Mahesh Research Award (to DHM), the National Cancer Institute, NIH (R01 CA112431 to DHM), and the National Institute of Allergy and Infectious Diseases, NIH (R01 AI071185 to PSH, R01 AI103447 to ALM, and U01 AI083005 to ALM).
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