Type 1 Immune Mechanisms Driven by the Response to Infection with ...

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Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors Emily K. Bongiorno, Samantha A. Garcia, Sami Sauma and D. Craig Hooper

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http://www.jimmunol.org/content/suppl/2017/04/29/jimmunol.160144 4.DCSupplemental

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J Immunol published online 1 May 2017 http://www.jimmunol.org/content/early/2017/04/29/jimmun ol.1601444

Published May 1, 2017, doi:10.4049/jimmunol.1601444 The Journal of Immunology

Type 1 Immune Mechanisms Driven by the Response to Infection with Attenuated Rabies Virus Result in Changes in the Immune Bias of the Tumor Microenvironment and Necrosis of Mouse GL261 Brain Tumors Emily K. Bongiorno,* Samantha A. Garcia,* Sami Sauma,† and D. Craig Hooper*,†

S

tandard-of-care treatments for malignant glioma offer poor prognosis, contributing to an interest in immunotherapeutic strategies. Although certain early-phase trials of various cell-based vaccines and checkpoint inhibitors have shown some promise, most have failed to improve long-term survival of patients with highly malignant glioblastoma multiforme, and none have been approved as standard treatment (1). These studies have reaffirmed that the immunomodulatory nature of the glioma tumor microenvironment (TME) is a key hurdle that must be overcome for successful immunotherapy. Infiltrating tumor-associated macrophages (TAMs) are undoubtedly a major contributor to the immunomodulatory nature of the malignant glioma TME (2). These TAMs closely resemble M2 macrophages in phenotype, factor expression, and function and are *Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107; and †Department of Neurological Surgery, Thomas Jefferson University, Philadelphia, PA 19107 ORCIDs: 0000-0001-7278-341X (S.S.); 0000-0002-8578-5104 (D.C.H.). Received for publication August 18, 2016. Accepted for publication April 3, 2017. This work was supported by National Institutes of Health Grant AI093369 (to D.C.H.). Grant NCI 5 P30 CA056036 from the National Cancer Institute to the Sidney Kimmel Cancer Center provided support for the Laboratory Animal Facility Shared Resource used in the study. Address correspondence and reprint requests to Dr. D. Craig Hooper, Department of Cancer Biology, Thomas Jefferson University, 1020 Locust Street, JAH Room 452, Philadelphia, PA 19107-6731. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: f.f.u., focus-forming unit; i.c., intracranial; i.n., intranasal(ly); iNOS, inducible NO synthase; LCX, left cortex; PCA, principal component analysis; qPCR, quantitative real-time PCR; RABV, rabies virus; RCX, right cortex; TAM, tumor-associated macrophage; TME, tumor microenvironment; WT, wild-type. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601444

likely to arise from monocytes polarized in the periphery in response to M-CSF and other factors prior to their infiltration into tumor tissue (3–5). TAMs elaborate numerous products that can contribute to tumor promotion, including growth factors, angiogenic factors (e.g., VEGF), and anti-inflammatory cytokines (e.g., IL-10 and TGF-b) (6–8). In addition, TAMs express elevated levels of PD-L1 and decreased levels of costimulatory markers and MHC class II, which together result in inhibited antitumor T cell function (9, 10). Evidence that glioma malignancy is driven by the infiltration and intratumoral activity of cells either resembling M2 monocytes or with similar functional properties comes from studies in animal models and glioma patients. In the murine orthotopic GL261 glioma model, increasing the ratio of CD11b+ spleen cells implanted with tumor cells accelerates tumor growth (11). In humans, astrocytoma malignancy is closely associated with the levels of M2 TAMs; high levels of intratumoral and systemic M2 cells correlate with poor prognosis and resistance to therapy (12–15), and inhibition of M2 polarization inhibits glioma progression (16, 17). Although the CNS is considered somewhat immunologically privileged, immunity can be readily generated to brain tumor Ags in animal models. Mice immunized in the flank with GL261 tumor cells are protected against a subsequent intracranial (i.c.) challenge though GL261 cell–specific IgG1 isotype Abs generated in response to the immunization, revealing a type 2 immune bias (18). Similarly, sera from glioblastoma multiforme patients obtained prior to the onset of therapy generally contain tumor-reactive IgG2/IgG4 Abs, suggesting that there has been type 2/Th2 immune recognition of tumor Ags (19, 20). This is consistent with the polarization of monocytes to M2 and tumor progression, rather than therapeutic antitumor immunity, which is considered to require a type 1/Th1 response. Type 1 immunity is associated with the activation of Th1 CD4+ and CD8+ T cells, the production of cytokines, such as


Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor-associated macrophages (TAMs). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity toward type 2, away from the type 1 mechanisms with antitumor properties. To drive type 1 immunity in CNS tissues, we infected GL261 tumor–bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans-axonally, where they induce a strong type 1 immune response that involves Th1, CD8, and B cell entry across the blood–brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation, and high levels of the proinflammatory factors IFN-g, TNF-a, and inducible NO synthase in the TME merely 4 d postinfection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. Although the majority of infiltrating CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased M1 TAMs. Mice deficient in the Th1 transcription factor T-bet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype and highlighting the importance of type 1 mechanisms in this process. The Journal of Immunology, 2017, 198: 000–000.

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IMMUNOMODULATION OF THE BRAIN TME BY RABV INFECTION in 4˚C PBS. For i.c. implantations, mice were anesthetized with isoflurane (Vedco, St. Joseph, MO), and 105 GL261 cells in 2 ml PBS were stereotactically injected into the right cerebral cortex, 1 mm anterior to the bregma and 1 mm to the right of the midline at a depth of 3 mm. Normal control and RABV-infected control mice received surgeries during which PBS alone was injected.

Quantitative real-time PCR At 12 and 22 d after tumor implantation, CNS tissues from GL261-implanted animals were dissected and homogenized in TRI Reagent (MRC, Cincinnati, OH) by passaging through a 20-gauge needle, and total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA), according to the manufacturer’s protocol. cDNA was synthesized using oligo dT primers, dNTP, and Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI). Quantitative real-time PCR (qPCR) was performed using iQ Supermix or iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) for specific mRNAs with a Bio-Rad iCycler (Bio-Rad). The primer/probe sets (IDT, Coralville, IA) used for L13, SPBN-GAS, CD4, CD8, k-L chain, IFN-b, and IFN-g were described previously (31), with additional sets listed in Table I. All probes are dual labeled 59-6-FAM and 39-BHQ-1. Sample mRNA copy numbers were normalized to the housekeeping gene L13.

Viral infection and in vitro viral inhibition curves SPBN-GAS, a variant of the SAD B19 virus with two mutations in the glycoprotein that attenuate the virus and prevent reversion to a pathogenic strain, was propagated in NA cells, as described elsewhere (30). Mice were anesthetized with isoflurane and infected with 105 focus-forming units (f.f.u.) intranasally (i.n.) or with 104 f.f.u. in the left cortex (LCX) or right cortex (RCX). Virus titration in NA and GL261 cells was performed in 96-well plates when cells were ∼80% confluent. Virus was added to media in 10-fold dilutions. To assess viral spread inhibition, GL261 cells were grown in supplemented RPMI 1640, which was removed at various time points. Cellular debris was removed by centrifugation, and conditioned media were applied in 2-fold serial dilutions to NA cells that were then infected with virus at 104 f.f.u./ml. Alternatively, conditioned media were applied directly to NA cells, and virus was serially titrated. For both virus assays, plates were incubated for 48 h at 34˚C and then fixed in 80% acetone at 4˚C. FITC-conjugated anti-RABV RNP (Fujirebio Diagnostics, Malvern, PA) was applied with Evans Blue counterstain (J.T. Baker, Phillipsburg, NJ). Virus f.f.u. were counted using a fluorescent microscope.

ELISA and cell-based ELISA RABV-specific Ab isotypes were assessed by ELISA. Serum samples were collected 0, 8, and 12 d postinfection and incubated on UV-inactivated RABV-coated plates. Ab titers were determined using secondary Abs specific for IgG, as well as IgG1 and IgG2a, as described previously (31). A cell-based ELISA was used to detect the level and isotype of GL261specific Abs, as described previously (18). Ab isotypes were detected with alkaline phosphatase–conjugated anti-mouse IgG (1:1000), IgG1, IgG2a, or IgG2b (1:2000) (MP Biomedicals, Santa Ana, CA).

Histochemistry and immunofluorescence staining

Eight- to ten-week-old male wild-type (WT) C57BL/6 mice and Tbet2/2 mice on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) or were raised at Thomas Jefferson University from Jackson Laboratory founder animals. All procedures were conducted in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals under protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University (Animal Welfare Assurance Number A3085-01).

Whole brains were snap-frozen in Tissue-Tek O.C.T. compound at 12 and 22 d after implantation (Sakura Finetek, Torrance, CA), fixed in 95% ethanol, rinsed in water, stained with Mayer’s hematoxylin solution and Eosin Y solution (Sigma-Aldrich), dehydrated, and mounted with RichardAllen Scientific Mounting Medium (Thermo Fisher). Immunofluorescent staining was performed on sections fixed in cold methanol for 10 min at 220˚C, rinsed in PBS, and incubated with primary Abs diluted in PBS containing 2% BSA, 5% goat serum, and 0.25% Triton X-100, overnight at 4˚C. Abs for RABV nucleoprotein, NeuN, and CD4 have been described previously (31); additional reagents are listed in Table II. Slides were incubated with fluorescence-conjugated secondary Abs and mounted with VECTASHIELD HardSet Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA). Bright-field and fluorescent images were acquired with a Leica DM6000 microscope with the Leica Application Suite v4 program (Leica Microsystems, Heerbrugg, Switzerland). Image brightness and contrast were adjusted using Photoshop CS5 software.

GL261 maintenance and implantation

In vitro inducible NO synthase induction

The GL261 cell line was acquired from the National Cancer Institute. Cells were grown in RPMI 1640 supplemented with 10% FBS (DFBS; Corning, Corning, NY), 4 mM L-glutamine, 50 mg/ml gentamicin (both from Thermo Fisher, Waltham, MA), and 0.05 mM 2-ME (Sigma-Aldrich, St. Louis, MO) at 37˚C in 5% CO2. Prior to implantation, GL261 cells were harvested with 0.25% Trypsin (Corning) and then washed and suspended

GL261 cells were cultured in four-well Nunc Lab-Tek chamber slides (Thermo Fisher, Rochester, NY) until confluent and were treated with 0, 10, 100, or 1000 U of recombinant mouse IFN-g (BD Biosciences, San Jose, CA) for 24 h. Supernatant was removed from chamber wells, and cells were washed once with PBS before fixation with ice-cold methanol for 10 min and staining as described above.

Materials and Methods Mice


IFN-g, TNF-a, and IL-12, enhanced M1 polarization of monocytes/ macrophages, and a reduction in regulatory T cell activity, which, in the case of tumor Ags, can result in cell infiltration into tumor tissues and tumor cell destruction through a variety of mechanisms (21–25). The use of a dendritic cell–based vaccine to provoke Th1 antitumor immunity in glioma patients has shown therapeutic promise (26). In a mouse model of glioma, promoting a Th1 antitumor response via adenovirus-mediated intratumoral expression of IL-12 has been shown to enhance T cell infiltration and tumor cytotoxicity, survival, and long-lasting protection (27). Therapies that shift the myeloid and T cell responses toward M1 and Th1, respectively, can further enhance these antitumor effects (17). However, such therapies are dependent upon delivering the immune effectors into tumor tissues, which may be problematic, particularly at early treatable stages in glioma formation in which the blood–brain barrier may still be relatively intact. The difficulty in delivering type 1 immune effectors into CNS tissues can be overcome by the use of virus infection. In addition to the IL-12 adenoviral construct described above, the use for glioma therapy of several oncolytic viruses that typically induce type 1 immune responses has been assessed. However, the focus of these studies has been on the oncolytic properties of the virus, and the effects of the associated type 1 immune mechanisms have not been thoroughly examined (28, 29). To determine whether type 1 immune mechanisms induced by virus infection may impact glioma growth in the absence of tumor cell lysis, we have used the attenuated, neurotropic rabies virus (RABV) SPBN-GAS. This RABV strain contains multiple attenuating mutations in its glycoprotein that prohibit its reversion to pathogenicity, ensuring its safety even in immunocompromised animals (30, 31). Such viruses spread to the brain via axons, bypassing the blood–brain barrier, and then replicate in neurons and astrocytes causing minimal cell death (32). Unlike pathogenic strains, attenuated RABV triggers changes in the neurovasculature that allow immune effector entry into CNS tissues (33). In normal mice this results in the production of high levels of type 1 cytokines and virus-neutralizing Ab in the CNS tissues and, ultimately, clearance of the virus without histological or clinical evidence of pathology. Type 1 immune mechanisms are central to virus clearance from the CNS because mice lacking T-bet, the transcription factor associated with the development of Th1 cells, have a severe deficit in this process, despite developing a strong RABV-specific Th2 response (31, 34–36). To determine whether the induction of a type 1 response in CNS tissues would alter glioma growth, we infected congenic C57BL/6 and Tbet2/2 mice bearing i.c. GL261 tumors with SPBN-GAS and monitored the immune responses to tumor and viral Ags, cytokine production in the TME, and tumor growth.

The Journal of Immunology

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Luminex Serum and tumors were collected from mice 12 d after GL261 implantation. Tumors were homogenized with a 20-gauge needle, passed through a 70-mm strainer, and cultured overnight in GL261 media and culture conditions. The level of relevant factors in serum and tumor supernatant was measured using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panels (Millipore, Darmstadt, Germany), according to the manufacturer’s protocol. Duplicate samples were analyzed using a FLEXMAP 3D (Luminex, Austin, TX).

Statistical analysis

Results Infection with attenuated RABV extends the survival of normal, but not Th1-deficient, mice bearing i.c. GL261 tumors To determine whether attenuated RABV infection, which is known to generate a strong type 1 immune response in CNS tissue, would prolong survival of mice with glioma, mice were implanted in the cerebral cortex with 105 GL261 cells, a dose that causes close to 100% tumor growth, and were infected i.n. with 105 f.f.u. SPBNGAS 8 d later. Tumor-bearing C57BL/6 mice lived significantly longer when infected with RABV (Fig. 1A). This improved longevity evidently required the Th1 arm of the immune response, because there was no benefit from infection in glioma-bearing Tbet2/2 mice (Fig. 1B). Neither WT nor Tbet2/2 mice exhibited clinical symptoms of RABV infection, and all mice had substantial tumor burdens at sacrifice. RABV infection causes the early onset of tumor necrosis To gain insight into why RABV infection prolongs survival of C57BL/6 mice, but not Tbet2/2 mice, implanted with GL261 cells, we assessed brain and tumor tissues by histopathology at 12 and 22 d after tumor cell implantation. Differences in tumor tissues between the groups of mice were evident by gross examination, with only tumors from infected WT mice exhibiting extensive hemorrhage (data not shown). Consistent with their increased survival, tumor tissues from C57BL/6 mice that received SPBNGAS were considerably more necrotic by 4 d postinfection (12 d after GL261 cell implantation) than were those from uninfected C57BL/6 mice or infected and uninfected Tbet2/2 mice (Fig. 2A). Despite increasing necrotic areas in tumors from uninfected WT mice, infection continued to be associated with greater tumor necrosis in these animals over the next 10 d (Fig. 2C). A slight elevation in tumor necrosis was detected in Tbet2/2 mice as a consequence of infection at the early time point, but no difference between infected and uninfected Tbet2/2 mice was detected later (Fig. 2B, 2C). Notably, tumor necrosis remained significantly less in these animals than in C57BL/6 mice at 22 d after tumor implantation, regardless of treatment. In support of the concept that tumors in RABV-infected C57BL/6 mice are less viable, Ki67 staining revealed less tumor cell proliferation in these animals in comparison with uninfected counterparts and the Tbet2/2 cohort (Fig. 2D, 2E).

FIGURE 1. RABV infection prolongs survival of tumor-bearing mice via a Th1-dependent mechanism. Mice were stereotactically injected i.c. with 105 GL261 cells, given PBS (dashed line) or 105 FFU SPBN-GAS (solid line) i.n. on day 8, and euthanized when moribund. (A) C57BL/6 mouse survival presented over time, n = 10 per group. Data are representative of two experiments. (B) Tbet2/2 mouse survival data are presented as percentage survival over time. Tumor alone (n = 8), tumor + RABV (n = 9), difference NS. **p , 0.01, tumor-bearing mice versus infected tumor-bearing mice, Mantel–Cox test.

RABV does not spread to tumor-bearing cortex or tumor, likely due to secretion of antiviral factors by GL261 cells Tumor necrosis is evident in infected C57BL/6 mice as soon as 4 d postinfection (12 d posttumor cell implantation), which is considerably before SPBN-GAS given i.n. spreads through the cortex tissues of normal mice (31); this suggests the possibility that the presence of the glioma enhances virus spread. However, the pattern of staining for virus that emerged in tumor-bearing mice indicates otherwise. When virus becomes detectable in brain tissues by immunofluorescent staining for nucleoprotein at 7 d postinfection, it is observed predominantly in the LCX as opposed to the right tumor-bearing hemisphere (RCX) or the tumor itself (Fig. 3A). Analysis of tissues for RABV nucleoprotein mRNA confirms this observation (see Table I for the qPCR primer and probe sequences and Tables II for Abs used for staining). RABVinfected mice that had undergone implantation surgery but received an i.c. injection of PBS without cells had equal levels of viral mRNA in the LCX and RCX at all time points. When necrosis is observed in tumor tissues from mice infected 4 d previously, the level of viral mRNA in the CNS tissue is very low and undetectable in the tumors of C57BL/6 and Tbet2/2 mice. By 14 d postinfection, when virus is replicating at higher levels in the CNS, viral message was higher in the LCX compared with the RCX, and the levels in tumor tissues are significantly lower than in the RCX and, in turn, significantly lower than those measured in the LCX (Fig. 3B). These results suggested that the tumor may, in fact, be inhibiting virus spread to the area. To address this possibility, tumor-bearing and control mice were infected with 104 f.f.u.


Experimental data were plotted and statistical analyses were performed using Prism 5.01 (GraphPad, San Diego, CA). Survival curves were assessed for significance with the Mantel–Cox test. ELISA, qPCR, and in vitro experiments were analyzed using the Mann–Whitney U test. Statistically significant differences between groups are generally denoted as follows: *p # 0.05, **p # 0.01, and ***p # 0.001. Principal component analysis (PCA) and heat maps were generated to determine patterns in cytokine expression across samples in MeV (37). Luminex mean fluorescent intensity values were median centered across each cytokine. Three-component three-dimensional PCA plots were calculated for tumor samples and sera samples separately. Statistically significant differences in individual cytokines were determined using the Student t test, with p values based on permutation (critical p value = 0.05).

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IMMUNOMODULATION OF THE BRAIN TME BY RABV INFECTION

SPBN-GAS in the LCX or RCX, 12 d after tumor implantation in the RCX (Fig. 3C). Infection in either cortex resulted in a similar trend to i.n. infection: virus replicated preferentially in the nontumorbearing LCX and only minimally in tumor tissues.

To test the hypothesis derived from the in vivo studies, that products of GL261 cells may inhibit RABV replication and spread, we collected GL261 cell supernatants from cultures at various time points, measured secreted factors using a Luminex assay, and added

FIGURE 3. RABV does not infect tumor or tumor-bearing cortex, despite the route of infection. (A) Pattern of RABV spread assessed by immunofluorescence staining in infected tumor-bearing mice 15 d posttumor implantation (d.p.t.). Stains are NeuN (red), RABV N protein (green), and DAPI (blue). Images are representative of two mice per condition and six sections per mouse. Dotted white line and “T” indicate tumor border, Scale bars, 50 mm. (B) Mice with and without tumor were infected i.n. 8 d.p.t., and CNS tissue was collected 12 and 22 d.p.t. for virus qPCR detection (n = 5). (C) Tumorbearing C57BL/6 mice were infected 12 d.p.t. i.n with 105 f.f.u. RABV (n = 4) or i.c. in LCX (n = 5) or RCX (n = 5) with 104 FFU RABV; CNS tissue was collected 22 d.p.t. Viral mRNA expressed as mean (6 SEM) copies of mRNA per 1000 L13 in the LCX, RCX, or tumor. *p , 0.05, ***p , 0.001, Wilcoxon matched-pairs signed rank test. #p , 0.05 versus matching section in respective treatment groups, Mann–Whitney U test.


FIGURE 2. GL261 tumors become necrotic early after RABV infection. H&E staining was performed on C57BL/6 (A) and Tbet2/2 (B) mouse brains at 12 d posttumor implantation (d.p.t.) for each cohort of infection controls, tumor alone, and tumor + RABV infection. Scale bars, 100 mm. (C) Percentage necrosis was calculated in ImageJ as the total necrotic area per tumor area (pixels) from two to six sections per brain, n = 2 for each condition in C57BL/6 mice and Tbet2/2 mice at 12 and 22 d.p.t. Ki67 (red) staining of C57BL/6 (D) and Tbet2/2 (E) mouse brain tumors at 12 d.p.t. Fluorescent images are representative of six sections per mouse and two mice per condition. Scale bars, 50 mm. *p , 0.05, **p , 0.01, ***p , 0.001 infected versus uninfected mice and WT versus Tbet2/2 mice, Mann– Whitney U test. #p , 0.05, ##p , 0.01, ###p , 0.001 22 d.p.t. versus 12 d.p.t., Mann– Whitney U test.


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Table I. Primer and probe sequences for qPCR Gene

CXCL10 GZMB ICAM-1 iNOS TGF-b2 TNF-a

Forward Primer (59-39)

TAC TCG CTG TGG GTG AGG

TGT ACC CAG CTA GCT TTC

AAG CTA ACG CCA TCA TCT

Reverse Primer (59-39)

CTA TGT GGA GGT GCG CAT GGC CTT AC GAA GGC AGA TGG T CAT TGA AGA AGC TG CAA CAA AGA CA TCA AGG GAC AAG

AAC TTG GAG TCT TCG GCA

TTA CTG CTA GGC CTT GAG

GAA GGT AAG TCT TTA AGG

Probe (59-39)

CTG ACG AGC CTG AGC CTT CTC CTG TT GCA TGG CAC ACG TA TGA GCT GGA AGA AA TTC GGG ATG AT AGG TTG ACT TTC

the conditioned media to infected mouse neuroblastoma (NA) cells. Supernatants from NA cells, typically used to grow RABV, were used as controls. Compared with NA cells, GL261 cells secrete high levels of cytokines with antiviral properties including RANTES, CXCL10, and Lif (Supplemental Fig. 1A). Moreover, GL261-conditioned media were found to inhibit virus infection and spread in NA cells (Supplemental Fig. 1B). Finally, we compared the infectivity of SPBNGAS for NA and GL261 cells and found that the latter are highly resistant to infection with the virus (Supplemental Fig. 1C, 1D).

Attenuated RABV infection is known to promote the infiltration of immune cells specific for nonviral Ags into CNS tissues (34). Tumor tissues from infected C57BL/6 mice and, to a lesser extent, Tbet2/2 mice, show elevated levels of CD4+ T cell accumulation by immunofluorescence compared with noninfected controls at 4 d postinfection (Fig. 4A–C). CD4+ T cells were not seen in brain tissues from infected animals without tumors at this time point, and there were no CD4+ T cells present in the cortex surrounding the GL261 tumor in infected WT or Tbet2/2 mice. However, when tumor tissues were assessed for CD4 mRNA levels, no difference was seen between infected and noninfected animals (Fig. 4D). Consistent with this finding, Ki67 staining revealed that CD4+ cells in tumor tissue are not actively proliferating in C57BL/6 or Tbet2/2 tumor-bearing mice, regardless of whether they had been given RABV (Fig. 4E, 4F). RABV infection results in the enhanced expression of ICAM on neurovascular endothelial cells, which would be expected to support immune cell infiltration into CNS tissues (38). Infection increased ICAM mRNA expression in the tumors of C57BL/6 mice but not Tbet2/2 mice (Supplemental Fig. 2A). However, no increase in CD8 or GZMB expression was detected in tumors of C57BL/6 or Tbet2/2 mice 4 d following RABV infection (Supplemental Fig. 2B, 2C). Similarly, expression of the NK cell marker NKp46 was not significantly altered in tumors of infected WT and Tbet2/2 animals (data not shown). Comparison of sections of tumor tissue from the different groups of mice stained for NKp46 and CD8 also failed to reveal any increase in the numbers of

ACC CAC GCT CCA

ATG TTT GCC CCA

TGC CAT GCC CAG GCT GGG GGC CCA CA CAT CGG GGT GGT GAA AGC TGA ACT TGA GCG A

CAC ACC GTC AGC CGA TTT GCT ATC

NK or CD8 T cells as a consequence of RABV infection (data not shown). RABV infection of glioma-bearing mice has no impact on the tumor cell–specific humoral response GL261-specific Abs, which evidently contribute to immune protection against tumor growth in the mouse GL261 model (18), provide insight into the class and magnitude of the tumor-specific immune response. Therefore, we assessed GL261- and RABVspecific Ab production in the tumor-bearing mice following infection. Significantly higher levels of mRNA specific for the Ab k-L chain were detected in tumor tissues from infected C57BL/6 mice in comparison with uninfected tumor-bearing animals 4 d after virus infection (12 d posttumor implantation), as well as infected and uninfected tumor-bearing Tbet2/2 mice (Fig. 5A). Levels of mRNA for the B cell marker CD19 were also increased in the tumor tissues of only RABV-infected C57BL/6 mice, and staining of tumor sections revealed a slight increase in the numbers of CD19+ cells in these animals (data not shown). However, the development and isotypes of serum GL261-specific Abs in GL261 tumor-bearing C57BL/6 mice, analyzed using a cell-based ELISA, were unaltered by infection with RABV, becoming significant 22 d after cell implantation and remaining predominantly IgG1 (Fig. 5B, 5C). In contrast, the presence of tumor significantly reduced the levels of IgG2A RABV-specific Abs elicited by virus infection without altering the relatively low levels of virus-specific IgG1 detectable at 14 d postinfection (Fig. 5D, 5E). RABV infection of tumor-bearing mice results in a Th1-dependent proinflammatory shift in the TME The elevated numbers of infiltrating CD4+ T cells and increase in mRNA specific for Ab k-L chain in tumor tissues suggest that there may be an impact on the immune status of the TME 4 d after RABV infection, despite low levels of virus replication in CNS tissues at this time. To test this hypothesis, we first assessed tumor tissues from control and RABV-infected C57BL/6 and Tbet2/2 mice for levels of immunologically relevant mRNAs known to be induced in the CNS tissues of normal mice at the onset of rabies infection. At 12 d after tumor cell implantation, we observed

Table II. Primary and secondary Abs for immunohistochemistry Target

Primary Abs CD11b CD11c CD31 CD206 F4/80 iNOS Ki67 Secondary Abs Anti-rabbit Anti-rat

Tag

Clone

Host

Isotype

Dilution

Supplier and Reference

PE Allophycocyanin

M1/70 HL3 ER-MP12 MR5D3 Cl-A3-1 N20 16A8

Rat Hamster Rat Rat Rat Rabbit Rat

IgG2b IgG1 IgG2a IgG2a IgG2b IgG IgG2a

1/200 1/200 1/1000 1/100 1/100 1/100 1/100

BD Biosciences #553311 BD Biosciences #550261 AbD Serotec #MCA2388GA AbD Serotec #MCA2235A647 AbD Serotec #MCA497 Santa Cruz Biotechnology #sc-651 BioLegend #652405

1/200 1/1000

Life Technologies #A11008 Life Technologies #A21434

Alexa Fluor 647 Allophycocyanin Alexa Fluor 488 Alexa Fluor 555

Goat Goat


RABV infection promotes CD4, but not cytolytic CD8, T cell infiltration into glioma tissues

TTC TGT CCT TGG

6


significantly higher levels of IFN-g mRNA expression in the tumor tissues of C57BL/6 mice that received RABV 4 d previously but not in tumor tissues from uninfected mice, Tbet2/2 mice (Fig. 6A), or in the LCX or right cortical tissues surrounding the tumors (data not shown). IFN-b mRNA levels were increased in tumor tissues from the same cohort of animals (Fig. 6B), as were levels of mRNA encoding CXCL10 (Fig. 6C) and TNF-a (Fig. 6D). Of note, the levels of these mRNAs in the tumor tissues from infected C57BL/6 mice are higher than those seen in similarly infected mice without tumors at this early time point in the infection (data not shown). In contrast, levels of mRNA specific for the immunomodulatory cytokine TGF-b2 in tumor tissues were lowered by infection (Fig. 6E). Attenuated RABV infection is known to induce the expression of inducible NO synthase (iNOS), an enzyme responsible for the production of NO and associated radicals, primarily by cells closely associated with the neurovascular unit as opposed to infiltrating M2 macrophages (33). iNOS mRNA levels appeared to be selectively elevated in the tumor tissues of RABV-infected C57BL/6 mice but not Tbet2/2 mice (Fig. 6F). To provide further insight into the nature of iNOS+ cells, sections from the cortex and tumor tissues of C57BL/6 and Tbet2/2 mice, uninfected or infected 4 d previously, were stained for iNOS and CD31, a marker that is normally found on endothelial cells but can be expressed by intratumoral macrophages (39). In the tumor-bearing mice, CD31+ cells can be seen throughout the sections from both mouse strains, but substantial iNOS staining is only seen in tissues from RABV-infected C57BL/6 mice, colocalizing with CD31+ vasculature in the cortex (data not shown) and appearing in the cytoplasm and nucleus of CD312 cells in the tumor tissue (Fig. 6G, 6H). To determine whether macrophages are responsible for iNOS expression in the tumors of RABV-infected C57BL/6 mice, tumor tissues from

these animals were costained for iNOS and the macrophage markers F4/80 (Fig. 6I) and CD11b (Fig. 6J). Although there was evidence of macrophage infiltration into tumor tissues at this early time point, iNOS expression localized to a different cell type, which we speculate is primarily the GL261 tumor cell, based on morphology and the absence of macrophage markers for CD11b and F4/80. To investigate the possibility that the production of IFN-g in the TME of RABV-infected C57BL/6 mice may promote iNOS expression in GL261 cells, we incubated the cells with increasing concentrations of mouse rIFN-g in vitro and stained them for iNOS. In culture, untreated GL261 cells exhibited low, but detectable, nuclear and cytoplasmic iNOS staining that increased in intensity with the addition of increasing concentrations of IFN-g (Supplemental Fig. 3). To provide further insight into the changes in the tumor tissues caused by cell infiltration and any functional changes in the GL261 tumor–resident cells, supernatants from overnight cultures of dispersed cells from tumor tissues, excised from 4-d infected and uninfected C57BL/6 mice at 12 d post–GL261 cell implantation, were analyzed for mouse cytokine/chemokine production by Luminex. A heat map representation and PCA of the data reveal distinct differences in the factors secreted by cells from the tumor tissues of infected versus uninfected mice, with those from the former producing significantly higher levels of a number of primarily type 1 cytokines, including G-CSF, IFN-g, IL-6, IL-17, CXCL10, MIP-2, and TNF-a (Fig. 7). Statistically significant differences in cytokine/chemokine levels between sera obtained at the same time from infected and uninfected tumor-bearing mice were not detected (data not shown). RABV infection causes a shift in TAMs from M2 to M1 At 15 d post–GL261 cell implantation, similar numbers of CD206 + macrophages had accumulated in the tumor tissues,


FIGURE 4. Enhanced CD4+ T cell accumulation occurs in glioma tissue of RABV-infected mice. Immunofluorescence staining performed 12 d posttumor implantation (d.p.t.) in WT (A) and Tbet2/2 (B) mice in infected controls and in tumor-bearing mice, with and without infection. Immunolabeling of CD4+ cells (red) and DAPI (blue). Scale bars, 100 mm. (C) Quantification of CD4+ cells was performed with ImageJ; data are presented as total CD4+ cells per square millimeter of tumor area. Immunofluorescence images and quantification data are representative of two mice per condition and six sections per mouse. (D) qPCR analysis of tumor tissue from C57BL/6 and Tbet2/2 tumor-bearing mice, with and without infection performed 12 d.p.t., and expressed as CD4 mRNA copies per 100 L13. n = 5 for each condition in C57BL/6 and Tbet2/2 mice, differences NS. Immunofluorescence staining of Ki67 (green) and CD4 (red) staining of C57BL/6 (E) and Tbet2/2 (F) mouse brain tumors at 12 d.p.t. Fluorescent images are representative of six sections per mouse and two mice per condition. Scale bars, 50 mm.


7 Arg1 was not statistically significant. In contrast, levels of expression of Ym1, which is also a product of M2 cells, were increased in tumors of infected mice. In addition to CD11c, mRNA encoding CD38, a marker expressed by a variety of activated immune cells, including M1 macrophages, was found at significantly higher levels in tumors of RABV-infected mice (Supplemental Fig. 4).

Discussion

regardless of whether the mice had been infected (data not shown). However, when sections of tumors obtained from similar mice at 22 d postimplantation were stained for the macrophage markers CD11b and F4/80, the M2 marker CD206, and CD11c, which tends to be expressed at low levels on M2 macrophages and at high levels on M1 macrophages, different patterns emerged for infected and uninfected mice. Although the majority of the CD11b+ cells apparent in the tumor tissues of uninfected C57BL/6 mice coexpressed CD206, the majority of the CD11b+ cells in tumors from infected C57BL/6 mice did not (Fig. 8A). CD11b and CD206 were expressed by different cell populations in tumor tissues from Tbet2/2 mice, but these were unchanged by infection (Fig. 8B). When F4/80 was used to identify macrophages and CD11c was used to examine the cell subsets, TAMs in uninfected C57BL/6 mice were negative for CD11c, but they were positive for CD11c in tumor tissues from infected mice (Fig. 8C). In contrast, tumor tissues from uninfected Tbet2/2 mice contained large numbers of F4/80+CD11c+ populations, with infection resulting in lower numbers of these cells and cohorts of cells that were either F4/80+ with low levels of CD11c or individually positive for each marker (Fig. 8D). Analysis of mRNA from tumor tissue at this late time point (22 d postimplantation) also showed significant reductions in the expression of genes associated with M2 cells, including the phenotypic markers CD163 and CD206. The expression of genes encoding the M2 products restin like a (FIZZ1) and Arg1 was also reduced, although the reduction in


FIGURE 5. RABV infection of GL261-bearing mice does not enhance tumor-specific Ab production. (A) qPCR analysis of CNS tissue was performed 12 d posttumor implantation (d.p.t.) in C57BL/6 and Tbet2/2 mice; data are expressed as mean (6 SEM) copies k-L chain per 100,000 L13. Statistical differences between cohorts were not detected. GL261 cell– reactive IgG1 (B) and IgG2A (C) Ab measured via cell-based ELISA and presented as mean absorbance (6 SEM) in naive mice, at the infection time point and 22 d.p.t. No statistically significant differences were observed. RABV-specific IgG1 (D) and IgG2A (E) Ab isotyping performed via ELISA. n = 5 for mock tumor groups, n = 10 for infected and uninfected tumor-bearing cohorts. Background absorbance is indicated by a dotted line. *p , 0.05, **p , 0.01, ***p , 0.001, naive versus infected cohorts at 22 d.p.t., Mann–Whitney t test.

Infection of normal mice with attenuated neurotropic RABV drives the production of proinflammatory type 1 cytokines, the expression of iNOS-dependent radicals, and the accumulation of CD4 Th1 cells in brain tissue, which are initially detectable 6–8 d after i.n. instillation of the virus (34). In contrast, malignant gliomas, including those resulting from GL261 cell implantation, are characterized by the induction of type 2 immunity and the recruitment of anti-inflammatory M2 monocytes into the TME (3–5). In this study, we show that infection of GL261 glioma–bearing C57BL/6 mice with attenuated rabies causes a profound change in the immune bias of the TME from type 2 to type 1, which is associated with extensive tumor necrosis and results in prolonged survival. Because RABV does not spread to tumor tissues, TME alterations that occur as a consequence of RABV infection likely result from immunological signals originating in infected tissues distant to the tumor that trigger the production of proinflammatory cytokines in the tumor, ultimately leading to tumor cell death. We observe increased IFN-g, TNF-a, and iNOS within the tumor tissue as soon as 4 d after RABV infection in C57BL/6 mice but not in Th1-deficient Tbet2/2 mice. The outcome is consistent with prior reports that TNF-a and IFN-g provide a therapeutic benefit in glioma; their expression promotes upregulation of iNOS, which activates cell death cascades through the activity of its product, NO (40, 41). Accordingly, tumor-bearing uninfected C57BL/6 mice and uninfected and infected Tbet2/2 mice, in which there is minimal expression of proinflammatory cytokines, have lower iNOS expression and show little tumor necrosis. Although GL261 cells are positive for GFAP only at the tumor margin (data not shown) (42), iNOS+ cells were scattered throughout the tumor parenchyma in infected mice, generating a Th1 response. Astrocytes can be triggered to express iNOS by IFN-g and TNF-a, and iNOS+ astrocytes have been observed in inflammatory lesions in mice with experimental allergic encephalomyelitis (43). Moreover, GL261 can be induced to express iNOS in vitro by IFN-g treatment. Therefore, our data suggest that GL261 cells, transformed astrocytes, can also express iNOS in response to IFN-g, as well as that these are the predominant iNOS-expressing cell in tumors undergoing necrosis as a consequence of the response to attenuated RABV. Corresponding with the early onset of tumor necrosis at 4 d postinfection 12 d following GL261 cell implantation, increased CD4+ T cells are observed in the tumor tissues of mice capable of generating a type 1 antiviral response and likely contribute to the antitumor effect and altered TME. The presence of these CD4+ cells in tumor, but not in surrounding tissue, along with their appearance as early as 4 d postinfection suggest that they are not RABV specific. In nontumor-bearing mice infected i.n. with attenuated RABV, this is the point when virus first appears in CNS tissues, and it takes several additional days for Ag-specific cells to appear in the CNS. Moreover, virus spread to tumor tissues and the surrounding parenchyma is limited. Therefore, we consider that the CD4+ T cells are entering tumor tissues nonspecifically as a consequence of the local production of proinflammatory cytokines, such as TNF-a, and the effects of free radical activity in the tumor, processes that have been implicated in the activation of vasculature and effector entry into CNS tissue (34, 44, 45). Although

8


the early onset of CD4 cell entry, proinflammatory factor upregulation, and necrosis occur concomitantly and are likely related, there is a disparity between the high numbers of CD4+ T cells observed in the tumors and the relatively low levels of CD4 mRNA in tumor tissues. A similar phenomenon has been observed in Tbet2/2 mice clearing attenuated RABV; there is substantial recruitment of Th2 CD4 cells to the CNS, but the cells express low levels of activation markers and CD4 mRNA, unlike the Th1 CD4 T cells that enter the CNS tissues of C57BL/6 mice in response to the virus (31). This phenomenon has not been reported for other neuroimmune processes that are generally associated with Th1 or Th17 cell infiltration. Consequently, we speculate that the current results may reflect a CD4 infiltrate that is predominantly Th2, as is the natural response to

GL261 Ags (18), and a common mechanism whereby Th2 cells largely lose transcriptional activity in CNS tissues. Although this concept is supported by the lack of Ki67 expression by the tumorinfiltrating CD4 cells, it remains to be validated in other models. Although the activity of Th1 CD4 cells that enter tumor tissues is expected to be inhibited by the TME (46, 47), we expect that the antiinflammatory mechanisms responsible are counteracted by the response to attenuated RABV. The fact that the therapeutic effect of attenuated RABV infection is only seen in mice that can mediate a Th1 response suggests that elements of the type 1 response are responsible, possibly including the activity of a limited subset of tumor Ag-specific Th1 cells in tumor tissues underlying more extensive Th2 accumulation. Alternatively, NK or CD8 T cells in the tumors of


FIGURE 6. The TMEs of C57BL/6 RABV-infected mice exhibit an early proinflammatory shift. (A) IFN-g mRNA expression in C57BL/6 and Tbet2/2 mice at 12 d posttumor implantation (d.p.t.) presented as mean (6 SEM) mRNA copies per 100 L13. (B) IFN-b expression fold change shown as mean 6 SEM normalized to L13. (C) CXCL10 mRNA expression shown as mean (6 SEM) copies per 100 L13. (D) TNF-a expression shown as mean (6 SEM) mRNA copies per 100,000 L13. (E) TGF-b2 expression in tumors of infected and uninfected C57BL/6 and Tbet2/2 mice 12 d.p.t., represented as fold change. (F) iNOS mRNA expression presented as mean (6 SEM) copies mRNA per 100,000 L13. Fluorescent staining in tumors of C57BL/6 (G) and Tbet2/2 (H) mice 12 d.p.t., with and without RABV infection, for iNOS (green), CD31 (red), and DAPI (blue). Fluorescent staining of iNOS (green) and F4/80 (red) (I) and iNOS (green) and CD11b (red) (J) in tumors of C57BL/6 mice infected with RABV 12 d.p.t. Fluorescent images are representative of six sections per mouse and two mice per condition. Scale bars, 50 mm. *p , 0.05, **p , 0.01, Mann–Whitney U test.


9

infected C57BL/6 mice, although not increased in number, may have acquired enhanced activity. Further experimentation is necessary to characterize the precise cell subsets involved. A later effect of the high expression levels of type 1 factors in the TME of C57BL/6 mice infected with attenuated RABV, subsequent to the onset of necrosis, is a change in the TAM population. Approximately 10 d after the onset of tumor necrosis and the proinflammatory shift in the TME, there is a profound reduction in the expression of the M2 marker CD206 by TAMs and an increase in the expression of CD11c, which is generally considered an M1 marker (48, 49). Together with the data from our studies of macrophage subset gene expression in tumor tissues, this suggests that RABV infection induces a shift in TAMs away from a more typical M2 subset toward a less-differentiated or M1 phenotype. Although the latter bear the M1 phenotype marker CD11c, we were unable to determine whether they express the functional M1 marker iNOS because of the extensive expression of this enzyme in the infected tissues. Tbet 2/2 mice, which have large populations of CD206+ M2 and CD11c+ M1-like cells within their GL261 tumors, do not display any alterations in the polarization of these cells as a consequence of RABV infection, reaffirming that this is a type 1–dependent process. Polarization of macrophages away from the M2 phenotype can block glioma progression. Specifically, administration of a CSF-1R inhibitor resulted in the loss of M2 cells; although this was associated with an increase in macrophages with phagocytic functions, M1-related gene expression was not otherwise increased (16). M1 macrophages are known to produce certain of the type 1 factors that we observe in the infected animals, as well as iNOS, which can directly contribute to tumor cell lysis through the production of NO and associated cytotoxic radicals (50). Although iNOS is expressed in the tumor tissues 4 d after RABV infection of mice that had GL261 cells implanted 8 d previously, it does not appear to be expressed by the

macrophages at this time. Consequently, TAMs are unlikely to be contributing to iNOS-dependent tumor necrosis at its onset. TAMs are a heterogeneous continuum of highly plastic macrophage subsets that carry out diverse functions and respond to changes within the tumor (5, 51). Although we cannot rule out the possibility that M2 TAMs selectively undergo cell death and are replaced by infiltrating M1 macrophages, our data are consistent with the concept that macrophage populations in the glioma TME can be re-educated by the local cytokine milieu, as described by other investigators (16, 52, 53). The precise mechanisms responsible for the TAM repolarization observed in this study are unknown; however, they are undoubtedly dependent on type 1 immune processes acting in the TME, because TAM polarization occurs in the absence of any differences in serum cytokine levels between infected and uninfected tumor-bearing mice. Interestingly, Th1-dependent TME modulation, iNOS expression, tumor necrosis, TAM polarization, and the increased survival of RABV-infected tumor-bearing C57BL/6 mice occur without direct infection of the tumor, a requirement for oncolytic virus therapy. RABV replicates predominantly in the nontumor-bearing hemisphere and is largely excluded from the tumor, presumably due to antiviral factors secreted by GL261 cells. Also distinct from other immunotherapeutic strategies, the Th1-dependent changes in the TME are not accompanied by an enhanced Th1-biased gliomaspecific peripheral response, as measured by serum Ab. Instead, Ab isotype analysis indicates a shift in the RABV-specific response toward type 2 in the presence of a glioma. This bias has little impact on the pathogenicity of the virus, because it is safe and readily cleared in mice that lack a range of immune components, including Tbet (31). The failure of RABV infection to promote systemic Th1 antiglioma humoral immunity may be due to the transient nature of the antiviral response. This may contribute to the fact that the mice


FIGURE 7. Differential cytokine levels are secreted by tumor cells of infected and uninfected mice. (A) Heat map representation of cytokine levels from ex vivo tumor supernatant excised 12 d.p.t. from C57BL/6 mice, with and without RABV infection, as measured by Luminex. (B) PCA projection of Luminex data clustered by sample groups for tumor supernatant of infected (blue) and uninfected (green) tumor bearing mice. n = 5 mice per condition, replicates averaged for heat map expression and biological replicates reported in PCAs. **p , 0.01, ***p , 0.001, permutation test.

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eventually succumb to their tumor. However, we do not know whether tumor growth, which was severely curtailed, or other factors relevant to the antitumor immune response are responsible for the animals’ demise. At death, the extent of necrotic tissue in the brains of the animals was substantial. Conceivably, an adjustment in the timing of infection or concomitant vaccination with a type 1–biased glioma vaccine may improve the long-term outcome. In summary, this study reports that infection of GL261 tumor– bearing mice with attenuated neurotropic RABV prolongs survival and increases tumor necrosis via a Th1-dependent proinflammatory shift in the TME. Striking antitumor effects are achieved, despite the limited ability of RABV to infect GL261 tumor cells. The change in the TME associated with the onset of tumor necrosis is accompanied by the expression of IFN-g, TNF-a, and iNOS and is followed by the replacement of tumor-supportive M2 with potentially destructive M1 cells. Mice lacking the T-bet transcription factor responsible for generating type 1 immunity did not exhibit increased survival, tumor necrosis, a proinflammatory TME modulation, or a beneficial change in the TAM population, indicating that a Th1-dependent mechanism is responsible for these antitumor effects. A better understanding of how infection with attenuated neurotropic RABV leads to modulation of the TME has therapeutic implications for brain tumor immunotherapy.

Acknowledgments We thank Carla Portocarrero, Dr. Larry Harshyne, and Dr. Aurore Lebrun for technical assistance; Rhonda Kean for review of the manuscript; and Dr. David Andrews and Dr. Bernhard Dietzschold for constructive suggestions.

Disclosures The authors have no financial conflicts of interest.

References 1. Reardon, D. A., K. W. Wucherpfennig, G. Freeman, C. J. Wu, E. A. Chiocca, P. Y. Wen, W. T. Curry, Jr., D. A. Mitchell, P. E. Fecci, J. H. Sampson, and G. Dranoff. 2013. An update on vaccine therapy and other immunotherapeutic approaches for glioblastoma. Expert Rev. Vaccines 12: 597–615. 2. Parney, I. F., J. S. Waldron, and A. T. Parsa. 2009. Flow cytometry and in vitro analysis of human glioma-associated macrophages. Laboratory investigation. J. Neurosurg. 110: 572–582. 3. Sica, A., T. Schioppa, A. Mantovani, and P. Allavena. 2006. Tumourassociated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur. J. Cancer 42: 717–727. 4. Lawrence, T., and G. Natoli. 2011. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat. Rev. Immunol. 11: 750–761. 5. Sica, A., P. Larghi, A. Mancino, L. Rubino, C. Porta, M. G. Totaro, M. Rimoldi, S. K. Biswas, P. Allavena, and A. Mantovani. 2008. Macrophage polarization in tumour progression. Semin. Cancer Biol. 18: 349–355. 6. Lin, E. Y., J. F. Li, L. Gnatovskiy, Y. Deng, L. Zhu, D. A. Grzesik, H. Qian, X. N. Xue, and J. W. Pollard. 2006. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res. 66: 11238–11246.


FIGURE 8. RABV infection alters TAM polarization in GL261 tumors. Immunofluorescence staining 22 d posttumor implantation (d.p.t.) for CD206 (green), CD11b (red), and DAPI (blue) in tumors of infected and uninfected C57BL/6 (A) and Tbet2/2 (B) mice. Tumors stained for CD11c (green), F4/80 (red), and DAPI (blue) in WT (C) and Tbet2/2 (D) infected and uninfected mice at 22 d.p.t. Images are representative of six sections per mouse and two mice per condition. Scale bars, 50 mm.


32. Hooper, D. C., K. Morimoto, M. Bette, E. Weihe, H. Koprowski, and B. Dietzschold. 1998. Collaboration of antibody and inflammation in clearance of rabies virus from the central nervous system. J. Virol. 72: 3711–3719. 33. Fabis, M. J., T. W. Phares, R. B. Kean, H. Koprowski, and D. C. Hooper. 2008. Blood-brain barrier changes and cell invasion differ between therapeutic immune clearance of neurotrophic virus and CNS autoimmunity. Proc. Natl. Acad. Sci. USA 105: 15511–15516. 34. Phares, T. W., M. J. Fabis, C. M. Brimer, R. B. Kean, and D. C. Hooper. 2007. A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-a is neither necessary nor sufficient. J. Immunol. 178: 7334–7343. 35. Barkhouse, D. A., S. A. Garcia, E. K. Bongiorno, A. Lebrun, M. Faber, and D. C. Hooper. 2015. Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon. J. Virol. 89: 312–322. 36. Hooper, D. C., A. Roy, D. A. Barkhouse, J. Li, and R. B. Kean. 2011. Rabies virus clearance from the central nervous system. In Advances in Virus Research. A. C. Jackson, ed. Academic Press, San Diego, CA, p. 55–71. 37. Saeed, A. I., V. Sharov, J. White, J. Li, W. Liang, N. Bhagabati, J. Braisted, M. Klapa, T. Currier, M. Thiagarajan, et al. 2003. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34: 374– 378. 38. Phares, T. W., R. B. Kean, T. Mikheeva, and D. C. Hooper. 2006. Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J. Immunol. 176: 7666–7675. 39. McKenney, J. K., S. W. Weiss, and A. L. Folpe. 2001. CD31 expression in intratumoral macrophages: a potential diagnostic pitfall. Am. J. Surg. Pathol. 25: 1167–1173. 40. Ehtesham, M., K. Samoto, P. Kabos, F. L. Acosta, M. A. Gutierrez, K. L. Black, and J. S. Yu. 2002. Treatment of intracranial glioma with in situ interferongamma and tumor necrosis factor-alpha gene transfer. Cancer Gene Ther. 9: 925–934. 41. Kwak, J. Y., M. K. Han, K. S. Choi, I. H. Park, S. Y. Park, M. H. Sohn, U. H. Kim, J. R. McGregor, W. E. Samlowski, and C. Y. Yim. 2000. Cytokines secreted by lymphokine-activated killer cells induce endogenous nitric oxide synthesis and apoptosis in DLD-1 colon cancer cells. Cell. Immunol. 203: 84–94. 42. Wainwright, D. A., S. Sengupta, Y. Han, I. V. Ulasov, and M. S. Lesniak. 2010. The presence of IL-17A and T helper 17 cells in experimental mouse brain tumors and human glioma. PLoS One 5: e15390. 43. Tran, E. H., H. Hardin-Pouzet, G. Verge, and T. Owens. 1997. Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis. J. Neuroimmunol. 74: 121–129. 44. Klug, F., H. Prakash, P. E. Huber, T. Seibel, N. Bender, N. Halama, C. Pfirschke, R. H. Voss, C. Timke, L. Umansky, et al. 2013. Low-dose irradiation programs macrophage differentiation to an iNOS+/M1 phenotype that orchestrates effective T cell immunotherapy. Cancer Cell 24: 589–602. 45. Abadier, M., N. Haghayegh Jahromi, L. Cardoso Alves, R. Boscacci, D. Vestweber, S. Barnum, U. Deutsch, B. Engelhardt, and R. Lyck. 2015. Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier. Eur. J. Immunol. 45: 1043–1058. 46. Dong, H., S. E. Strome, D. R. Salomao, H. Tamura, F. Hirano, D. B. Flies, P. C. Roche, J. Lu, G. Zhu, K. Tamada, et al. 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 8: 793–800. 47. Hadrup, S., M. Donia, and P. Thor Straten. 2013. Effector CD4 and CD8 T cells and their role in the tumor microenvironment. Cancer Microenviron. 6: 123–133. 48. Movahedi, K., D. Laoui, C. Gysemans, M. Baeten, G. Stange´, J. Van den Bossche, M. Mack, D. Pipeleers, P. In’t Veld, P. De Baetselier, and J. A. Van Ginderachter. 2010. Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res. 70: 5728–5739. 49. Peterson, T. E., N. D. Kirkpatrick, Y. Huang, C. T. Farrar, K. A. Marijt, J. Kloepper, M. Datta, Z. Amoozgar, G. Seano, K. Jung, et al. 2016. Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages. Proc. Natl. Acad. Sci. USA 113: 4470–4475. 50. Lizotte, P. H., J. R. Baird, C. A. Stevens, P. Lauer, W. R. Green, D. G. Brockstedt, and S. N. Fiering. 2014. Attenuated Listeria monocytogenes reprograms M2polarized tumor-associated macrophages in ovarian cancer leading to iNOSmediated tumor cell lysis. OncoImmunology 3: e28926. 51. Murray, P. J., J. E. Allen, S. K. Biswas, E. A. Fisher, D. W. Gilroy, S. Goerdt, S. Gordon, J. A. Hamilton, L. B. Ivashkiv, T. Lawrence, et al. 2014. Macrophage activation and polarization: nomenclature and experimental guidelines. [Published erratum appears in 2014 Immunity 41: 339–340.] Immunity 41: 14–20. 52. Fujiwara, Y., Y. Komohara, T. Ikeda, and M. Takeya. 2011. Corosolic acid inhibits glioblastoma cell proliferation by suppressing the activation of signal transducer and activator of transcription-3 and nuclear factor-kappa B in tumor cells and tumor-associated macrophages. Cancer Sci. 102: 206–211. 53. Komohara, Y., Y. Fujiwara, K. Ohnishi, and M. Takeya. 2016. Tumor-associated macrophages: potential therapeutic targets for anti-cancer therapy. Adv. Drug Deliv. Rev. 99(Pt B): 180–185.


7. Ahn, G. O., and J. M. Brown. 2008. Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: role of bone marrow-derived myelomonocytic cells. Cancer Cell 13: 193–205. 8. Ostuni, R., F. Kratochvill, P. J. Murray, and G. Natoli. 2015. Macrophages and cancer: from mechanisms to therapeutic implications. Trends Immunol. 36: 229– 239. 9. Kennedy, B. C., R. C. Showers, D. E. Anderson, L. Anderson, P. Canoll, J. N. Bruce, and R. C. Anderson. 2013. Tumor-associated macrophages in glioma: friend or foe? J. Oncol. 2013: 486912. 10. Bloch, O., C. A. Crane, R. Kaur, M. Safaee, M. J. Rutkowski, and A. T. Parsa. 2013. Gliomas promote immunosuppression through induction of B7-H1 expression in tumor-associated macrophages. Clin. Cancer Res. 19: 3165–3175. 11. Chae, M., T. E. Peterson, A. Balgeman, S. Chen, L. Zhang, D. N. Renner, A. J. Johnson, and I. F. Parney. 2015. Increasing glioma-associated monocytes leads to increased intratumoral and systemic myeloid-derived suppressor cells in a murine model. Neuro Oncol. 17: 978–991. 12. Zhai, H., F. L. Heppner, and S. E. Tsirka. 2011. Microglia/macrophages promote glioma progression. Glia 59: 472–485. 13. Prosniak, M., L. A. Harshyne, D. W. Andrews, L. C. Kenyon, K. Bedelbaeva, T. V. Apanasovich, E. Heber-Katz, M. T. Curtis, P. Cotzia, and D. C. Hooper. 2013. Glioma grade is associated with the accumulation and activity of cells bearing M2 monocyte markers. Clin. Cancer Res. 19: 3776–3786. 14. Lu-Emerson, C., M. Snuderl, N. D. Kirkpatrick, J. Goveia, C. Davidson, Y. Huang, L. Riedemann, J. Taylor, P. Ivy, D. G. Duda, et al. 2013. Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma. Neuro Oncol. 15: 1079–1087. 15. Ye, X. Z., S. L. Xu, Y. H. Xin, S. C. Yu, Y. F. Ping, L. Chen, H. L. Xiao, B. Wang, L. Yi, Q. L. Wang, et al. 2012. Tumor-associated microglia/ macrophages enhance the invasion of glioma stem-like cells via TGF-b1 signaling pathway. J. Immunol. 189: 444–453. 16. Pyonteck, S. M., L. Akkari, A. J. Schuhmacher, R. L. Bowman, L. Sevenich, D. F. Quail, O. C. Olson, M. L. Quick, J. T. Huse, V. Teijeiro, et al. 2013. CSF1R inhibition alters macrophage polarization and blocks glioma progression. Nat. Med. 19: 1264–1272. 17. Kosaka, A., T. Ohkuri, and H. Okada. 2014. Combination of an agonistic antiCD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces antiglioma effects by promotion of type-1 immunity in myeloid cells and T-cells. Cancer Immunol. Immunother. 63: 847–857. 18. Morin-Brureau, M., K. M. Hooper, M. Prosniak, S. Sauma, L. A. Harshyne, D. W. Andrews, and D. C. Hooper. 2015. Enhancement of glioma-specific immunity in mice by “NOBEL”, an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide. Cancer Immunol. Immunother. 64: 447–457. 19. Harshyne, L. A., B. J. Nasca, L. C. Kenyon, D. W. Andrews, and D. C. Hooper. 2016. Serum exosomes and cytokines promote a T-helper cell type 2 environment in the peripheral blood of glioblastoma patients. Neuro Oncol. 18: 206–215. 20. Kumar, R., D. Kamdar, L. Madden, C. Hills, D. Crooks, D. O’Brien, and J. Greenman. 2006. Th1/Th2 cytokine imbalance in meningioma, anaplastic astrocytoma and glioblastoma multiforme patients. Oncol. Rep. 15: 1513–1516. 21. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655–669. 22. Deligne, C., A. Metidji, W. H. Fridman, and J. L. Teillaud. 2015. Anti-CD20 therapy induces a memory Th1 response through the IFN-g/IL-12 axis and prevents protumor regulatory T-cell expansion in mice. Leukemia 29: 947–957. 23. Nishimura, T., M. Nakui, M. Sato, K. Iwakabe, H. Kitamura, M. Sekimoto, A. Ohta, T. Koda, and S. Nishimura. 2000. The critical role of Th1-dominant immunity in tumor immunology. Cancer Chemother. Pharmacol. 46(Suppl.): S52–S61. 24. Nagarkatti, M., S. R. Clary, and P. S. Nagarkatti. 1990. Characterization of tumor-infiltrating CD4+ T cells as Th1 cells based on lymphokine secretion and functional properties. J. Immunol. 144: 4898–4905. 25. Haabeth, O. A., B. Bogen, and A. Corthay. 2012. A model for cancer-suppressive inflammation. OncoImmunology 1: 1146–1155. 26. Akasaki, Y., G. Liu, N. H. C. Chung, M. Ehtesham, K. L. Black, and J. S. Yu. 2004. Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2overexpressing glioma. J. Immunol. 173: 4352–4359. 27. Liu, Y., M. Ehtesham, K. Samoto, C. J. Wheeler, R. C. Thompson, L. P. Villarreal, K. L. Black, and J. S. Yu. 2002. In situ adenoviral interleukin 12 gene transfer confers potent and long-lasting cytotoxic immunity in glioma. Cancer Gene Ther. 9: 9–15. 28. Wollmann, G., K. Ozduman, and A. N. van den Pol. 2012. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 18: 69–81. 29. Parker, J. N., G. Y. Gillespie, C. E. Love, S. Randall, R. J. Whitley, and J. M. Markert. 2000. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc. Natl. Acad. Sci. USA 97: 2208–2213. 30. Faber, M., M. L. Faber, A. Papaneri, M. Bette, E. Weihe, B. Dietzschold, and M. J. Schnell. 2005. A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity. J. Virol. 79: 14141–14148. 31. Lebrun, A., C. Portocarrero, R. B. Kean, D. A. Barkhouse, M. Faber, and D. C. Hooper. 2015. T-bet is required for the rapid clearance of attenuated rabies virus from central nervous system tissue. J. Immunol. 195: 4358–4368.

11

Rantes

5000

(pg/ml)

4000

CXCL10

30000

300

20000

200

B

Lif

400

TNTC

2000

0

NA GL261

100

10000

NA (24h) GL261 (24h)

250

3000

1000

200

GL261 (72h)

150 100 50

6

24

48

NA

0

6

24

Hours

48

6

24

0

48

GL261

D

106

105

104

103

Virus titration

102

10

100

% Cells Infected

C

40000

FFU

A

NA GL261

80 60 40 20 0

.1

.01

.001

.0001 .00001

Virus dilution

Supplemental Figure 1. GL261 cells produce factors which inhibit viral infection. (A) Cytokines present in GL261 (red) or NA cell-conditioned media (blue) after 6-48 hour cultures plotted in pg/ml. (B) Infectivity of NA cells measured in FFU. NA (blue) or GL261 (red)24 or 72 hr cell-conditioned media applied to NA cells inhibit ability of virus to infect and spread in NA cells. (C) SPBN-GAS titration in NA and GL261 cells 48 hours post-infection, stained for RABV N protein (green), counterstained with Evans Blue (red), 1:10 dilution shown for NA and GL261 cells at 10x magnification. (D) Percent of virally-infected NA cells (blue) and GL261 cells (red) after virus titration performed at 48hr.

C57Bl/6

A ICAM mRNA

500 400 300

CD8 mRNA GZMb mRNA

*

RCX Tumor

400 300 200

100

100 Normal

RABV

Tumor

Tumor + RABV

0 Normal

25

25

20

20

15

15

10

10

5

5

0

C

500

LCX

200 0

B

Tbet-/-

Normal

RABV

Tumor

Tumor + RABV

80

60

60

40

40

20

20 Normal

RABV

Tumor

Tumor + RABV

Tumor

Tumor + RABV

0

80

0

RABV

0

Normal

RABV

Tumor

Tumor + RABV

Normal

RABV

Tumor

Tumor + RABV

Supplemental Figure 2. Tumor infiltrating effectors do not have increased cytolytic activity in tumors of RABV-infected mice. qPCR analysis of CNS tissue was performed 12d.p.t. in C57BL/6 and Tbet-/mice and expressed as mean ± SEM copies (A) ICAM, (B) CD8, (C) GZMb mRNA copies/ 100000 L13. Statistical differences between cortical and tumor tissues of different treatment cohorts measured by Mann Whitney test and represented by: *p