CD8⁺ T Cell-Independent Immune-Mediated Mechanisms of Anti-Tumor Activity.

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Cancer immunotherapy seeks to recruit an effective immune response to eliminate tumor cells. To date, cancer vaccines have shown only limited effectiveness because of our incomplete understanding of the necessary effector cells and mechanisms that yield efficient tumor clearance. CD8+ T cell cytotoxic activity has long been proposed as the primary effector function necessary for tumor regression. However, there is increasing evidence that indicates that components of the immune system other than CD8+ T cells play important roles in tumor eradication and control. The following review should provide an understanding of the mechanisms involved in an effective antitumor response to guide future therapeutic designs. The information provided suggests an alternate means of effective tumor clearance in malignant glioma to the canonical CD8+ cytotoxic T cell mechanism.

Victory and defeat in the induction of a therapeutic response through vaccine therapy for human and canine brain tumors: a review of the state of the art.

Anti-tumor immunotherapy using tumor lysate-based vaccines has made great advances over recent decades. Cancer vaccines aim to elicit adaptive immune responses through various pathways by providing tumor and tumor-associated antigens with an immune stimulant or adjuvant. These anti-tumor vaccines are therefore developed as personalized treatments. Utilizing tumors as a source of vaccine antigens in immunotherapy has demonstrated promising results with minimal toxicity. However, to date, researchers have failed to overcome the overpowering immune suppressive effects within the tumor microenvironment. Immune suppression occurs naturally via multiple mechanisms. These mechanisms serve an important homeostatic role restoring a normal tissue microenvironment following an inflammatory response. Due to these suppressive mechanisms and the inherent heterogeneity of tumors, it is imperative to then elicit and maintain a specific tumoricidal response if vaccine therapy or some other combination of reagents is chosen. In this review, we focus on the historical use of tumors as a source of antigens to elicit a tumoricidal response and the limitations encountered that prevent greater success in immunotherapy. We describe the advantages and disadvantages of various vaccines and their ineffectiveness due to tumor-induced immune suppression.

Vaccine injection site matters: qualitative and quantitative defects in CD8 T cells primed as a function of proximity to the tumor in a murine glioma model.

Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in multiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice. Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is being overlooked.

Transcriptomic observations of intra and extracellular immunotherapy targets for pediatric brain tumors.

OBJECTIVES: Despite surgical resection, chemoradiation, and targeted therapy, brain tumors remain a leading cause of cancer-related death in children. Immunotherapy has shown some promise and is actively being investigated for treating childhood brain tumors. However, a critical step in advancing immunotherapy for these patients is to uncover targets that can be effectively translated into therapeutic interventions. METHODS: In this study, our team performed a transcriptomic analysis across pediatric brain tumor types to identify potential targets for immunotherapy. Additionally, we assessed components that may impact patient response to immunotherapy, including the expression of genes essential for antigen processing and presentation, inhibitory ligands and receptors, interferon signature, and overall predicted T cell infiltration. RESULTS: We observed distinct expression patterns across tumor types. These included elevated expression of antigen genes and antigen processing machinery in some tumor types while other tumors had elevated inhibitory checkpoint receptors, known to be associated with response to checkpoint inhibitor immunotherapy. CONCLUSION: These findings suggest that pediatric brain tumors exhibit distinct potential for specific immunotherapies. We believe our findings can guide investigators in their assessment of appropriate immunotherapy classes and targets in pediatric brain tumors.

Establishment and characterization of two novel patient-derived lines from canine high-grade glioma.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

Randomized trial of neoadjuvant vaccination with tumor-cell lysate induces T cell response in low-grade gliomas.

BACKGROUNDLong-term prognosis of WHO grade II low-grade gliomas (LGGs) is poor, with a high risk of recurrence and malignant transformation into high-grade gliomas. Given the relatively intact immune system of patients with LGGs and the slow tumor growth rate, vaccines are an attractive treatment strategy.METHODSWe conducted a pilot study to evaluate the safety and immunological effects of vaccination with GBM6-AD, lysate of an allogeneic glioblastoma stem cell line, with poly-ICLC in patients with LGGs. Patients were randomized to receive the vaccines before surgery (arm 1) or not (arm 2) and all patients received adjuvant vaccines. Coprimary outcomes were to evaluate safety and immune response in the tumor.RESULTSA total of 17 eligible patients were enrolled - 9 in arm 1 and 8 in arm 2. This regimen was well tolerated with no regimen-limiting toxicity. Neoadjuvant vaccination induced upregulation of type-1 cytokines and chemokines and increased activated CD8+ T cells in peripheral blood. Single-cell RNA/T cell receptor sequencing detected CD8+ T cell clones that expanded with effector phenotype and migrated into the tumor microenvironment (TME) in response to neoadjuvant vaccination. Mass cytometric analyses detected increased tissue resident-like CD8+ T cells with effector memory phenotype in the TME after the neoadjuvant vaccination.CONCLUSIONThe regimen induced effector CD8+ T cell response in peripheral blood and enabled vaccine-reactive CD8+ T cells to migrate into the TME. Further refinements of the regimen may have to be integrated into future strategies.TRIAL REGISTRATIONClinicalTrials.gov NCT02549833.FUNDINGNIH (1R35NS105068, 1R21CA233856), Dabbiere Foundation, Parker Institute for Cancer Immunotherapy, and Daiichi Sankyo Foundation of Life Science.

CD200 Immune-Checkpoint Peptide Elicits an Anti-glioma Response Through the DAP10 Signaling Pathway.

Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8+ T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immune-stimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.

Targeting Neuroinflammation in Brain Cancer: Uncovering Mechanisms, Pharmacological Targets, and Neuropharmaceutical Developments.

Gliomas are one of the most lethal types of cancers accounting for ∼80% of all central nervous system (CNS) primary malignancies. Among gliomas, glioblastomas (GBM) are the most aggressive, characterized by a median patient survival of fewer than 15 months. Recent molecular characterization studies uncovered the genetic signatures and methylation status of gliomas and correlate these with clinical prognosis. The most relevant molecular characteristics for the new glioma classification are IDH mutation, chromosome 1p/19q deletion, histone mutations, and other genetic parameters such as ATRX loss, TP53, and TERT mutations, as well as DNA methylation levels. Similar to other solid tumors, glioma progression is impacted by the complex interactions between the tumor cells and immune cells within the tumor microenvironment. The immune system's response to cancer can impact the glioma's survival, proliferation, and invasiveness. Salient characteristics of gliomas include enhanced vascularization, stimulation of a hypoxic tumor microenvironment, increased oxidative stress, and an immune suppressive milieu. These processes promote the neuro-inflammatory tumor microenvironment which can lead to the loss of blood-brain barrier (BBB) integrity. The consequences of a compromised BBB are deleteriously exposing the brain to potentially harmful concentrations of substances from the peripheral circulation, adversely affecting neuronal signaling, and abnormal immune cell infiltration; all of which can lead to disruption of brain homeostasis. In this review, we first describe the unique features of inflammation in CNS tumors. We then discuss the mechanisms of tumor-initiating neuro-inflammatory microenvironment and its impact on tumor invasion and progression. Finally, we also discuss potential pharmacological interventions that can be used to target neuro-inflammation in gliomas.

CD200 Checkpoint Reversal: A Novel Approach to Immunotherapy.

PURPOSE: Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. EXPERIMENTAL DESIGN: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. RESULTS: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. CONCLUSIONS: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.

Treatment Combining CD200 Immune Checkpoint Inhibitor and Tumor-Lysate Vaccination after Surgery for Pet Dogs with High-Grade Glioma.

Recent advances in immunotherapy have included inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. We combined these approaches in pet dogs with high-grade glioma. Administration of a synthetic peptide targeting the immune checkpoint protein, CD200, enhanced the capacity of antigen-presenting cells to prime T-cells to mediate an anti-glioma response. We found that in canine spontaneous gliomas, local injection of a canine-specific, CD200-directed peptide before subcutaneous delivery of an autologous tumor lysate vaccine prolonged survival relative to a historical control treated with autologous tumor lysate alone (median survivals of 12.7 months and 6.36 months, respectively). Antigen-presenting cells and T-lymphocytes primed with this peptide suppressed their expression of the inhibitory CD200 receptor, thereby enhancing their ability to initiate immune reactions in a glioblastoma microenvironment replete with the immunosuppressive CD200 protein. These results support consideration of a CD200 ligand as a novel glioblastoma immunotherapeutic agent.

In vitro and in vivo apoptosis detection using membrane permeant fluorescent-labeled inhibitors of caspases.

Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer's disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research.

Tumor-derived exosomes, microRNAs, and cancer immune suppression.

Originally considered to be part of a cellular waste pathway, expansive research into exosomes has shown that these vesicles possess a vast array of functional utilities. As vital transporters of materials for communications between cells, particular interest has been generated in the ability of cancer cells to use exosomes to induce immune suppression, and to establish a thriving microenvironment, ideal for disease progression. Exosomes carry and transfer many types of cargo, including microRNAs (miRNAs; miRs), which are important modulators of messenger RNA (mRNA) expression. These miRNAs have been shown to be noteworthy components of the mechanisms used by tumor-derived exosomes to carry out their functions. Alternatively, research has been expanding into using exosomes and miRNAs as both biomarkers for detecting cancer and disease progression, and as potential treatment tools. Here, we discuss some of the progress that researchers have made related to cancer exosomes, their suppression of the immune system and the importance of the miRNAs they shuttle, along with some of the shortcomings, obstacles, and challenges that lie ahead.

Tumor-derived vaccines containing CD200 inhibit immune activation: implications for immunotherapy.

There are over 400 ongoing clinical trials using tumor-derived vaccines. This approach is especially attractive for many types of brain tumors, including glioblastoma, yet so far the clinical response is highly variable. One contributor to poor response is CD200, which acts as a checkpoint blockade, inducing immune tolerance. We demonstrate that, in response to vaccination, glioma-derived CD200 suppresses the anti-tumor immune response. In contrast, a CD200 peptide inhibitor that activates antigen-presenting cells overcomes immune tolerance. The addition of the CD200 inhibitor significantly increased leukocyte infiltration into the vaccine site, cytokine and chemokine production, and cytolytic activity. Our data therefore suggest that CD200 suppresses the immune system's response to vaccines, and that blocking CD200 could improve the efficacy of cancer immunotherapy.

Monomeric annexin A2 is an oxygen-regulated toll-like receptor 2 ligand and adjuvant.

BACKGROUND: Annexin A2 (ANXA2) is a pleiotropic, calcium-dependent, phospholipid-binding protein with a broad tissue distribution. It can be intracellular, membrane-bound, or secreted, and it exists as a monomer or heterotetramer. The secreted ANXA2 heterotetramer signals human and murine macrophages to produce IL-1, IL-6, and TNF-α through TLR4/MyD88- and TRIF-dependent pathways. METHODS: GL261 glioma cells were cultured in 5 % or 20 % O2. Monomeric ANXA2 (ANXA2m) was identified as a TLR2-binding protein enriched in 5 % O2 by mass spectrometry. Purified ANXA2m and ANXA2-derived peptides were added to TLR2-expressing reporter cells and immature dendritic cells (DCs) cells in vitro. ANXA2m was then mixed with chicken ovalbumin (OVA) for vaccination of TLR2 (+/+) and TLR2 (-/-) mice for subsequent quantification of antigen-specific CD8(+) T cell responses. The TLR2-binding region of ANXA2m was determined using various peptides derived from the ANXA2 amino terminus on TLR2 reporter cells and in vaccinated mice. RESULTS: ANXA2m is overexpressed by murine glioblastoma GL261 cells grown under 5 % O2, but not atmospheric 20 % O2, and acts as an adjuvant by inducing murine and human DC maturation through TLR2. ANXA2m upregulates CD80 and CD86 expression, enhances antigen cross-presentation, and induces the secretion of IL-12p70, TNF-α, and IFN-γ. The amino-terminal 15 amino acids of ANXA2m are necessary and sufficient for TLR2 binding and DC activation. CONCLUSION: This novel finding adds to the known functions of ANXA2 and suggests ways to exploit it as a vaccine adjuvant. ANXA2-antigen fusion peptides could be developed for patients as "off-the-shelf" agents containing common tumor antigens. Alternatively, they could be "personalized" and synthesized after tumor sequencing to identify immunogenic tumor-specific neo-antigens. As the amino terminal 15 amino acids of ANXA2 are required to stimulate TLR2 activity, a fusion peptide could be as short as 30 amino acids if one or two CD8 T cell epitopes are fused to the ANXA2 amino terminal portion. Future work will address the efficacy of ANXA2 peptide fusions alone and in combination with established TLR agonists to induce synergy in preclinical models of glioma as observed in other vaccines.

Gammadelta T-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry.

Gamma Delta (gammadelta) T lymphocytes contain the unique capability of responding to pathogens in both an innate and acquired immune response. Previously, gammadelta lymphocytes have been reported to respond to Mycobacteria tuberculosis determined by proliferation and IFN-gamma production. Unlike alpha beta (alphabeta) lymphocytes, gammadelta lymphocytes constitutively express a natural killer receptor providing gammadelta lymphocytes the capability for innate cytolytic functions. A new cytolytic assay by flow cytometry was reported capable of determining natural killer activity using K562 cells as targets without the need for radioactive materials. The objectives of this study were to first apply the flow cytometer-based assay to assess gammadelta lymphocytes natural killer activity following animal vaccination with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). Secondly, to optimize the flow cytometer assay in order to detect antigen specific cytolytic activity to mycobacterium and to compare the cytolytic activity of gammadelta lymphocytes to CD-8 lymphocytes. gammadelta lymphocytes increased in NK activity (P=0.012) following animal vaccination with M. bovis BCG. Both innate (P=0.02) and acquired antigen-specific cytolytic activity (P=0.04) increased following incubation with M. bovis-infected monocytes. In conclusion, flow cytometric-based assay is a sensitive and reliable tool to determine cytolytic activity of gammadelta T-lymphocytes against mycobacterium.

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.

Vaccination with dendritic cells loaded with allogeneic brain tumor cells for recurrent malignant brain tumors induces a CD4(+)IL17(+) response.

BACKGROUND: We tested the hypothesis that a novel vaccine developed from autologous dendritic cells (DC) loaded with cells from a unique allogeneic brain tumor cell line (GBM6-AD) would be well-tolerated and would generate an immune response. METHOD: Patients with recurrent primary brain tumors underwent vaccination with GBM6-AD/DC vaccine. Subjects were treated at escalating DC cell doses: 5 × 10(6) (one patient), 10 × 10(6) (one patient) and 15 × 10(6) (6 patients). Subcutaneous injections were planned for days 0, 14, 28, 42, 56, and monthly thereafter. The primary endpoint was the safety of the GBM6-AD/DC vaccination. The secondary endpoints were immune response, measured by flow cytometry, and the clinical outcome of tumor response defined by time to progression and overall survival. RESULTS: Eight patients were treated. The first three patients were treated in the dose escalation phase of the trial; the remaining five patients received the maximum dose of 15 × 10(6) DC. No dose limiting toxicity was observed. The best response per modified McDonald criteria was partial response in one patient. Flow cytometric immune profiling revealed significant differences in CD4(+)IL17(+) lymphocytes and myeloid derived suppressor cell populations between patients characterized as having stable vs. non-stable disease. CONCLUSION: This first-in-human study shows that the GBM6-AD/DC vaccine was well tolerated and was associated with an immune response in a subset of patients. No MTD was achieved in this trial. This small-scale pilot provides information for larger scale investigations into the use of this allogeneic vaccine source.

CD200 in CNS tumor-induced immunosuppression: the role for CD200 pathway blockade in targeted immunotherapy.

BACKGROUND: Immunological quiescence in the central nervous system (CNS) is a potential barrier to immune mediated anti-tumor response. One suppressive mechanism results from the interaction of parenchyma-derived CD200 and its receptor on myeloid cells. We suggest that CD200/CD200R interactions on myeloid cells expand the myeloid-derived suppressor cell (MDSC) population and that blocking tumor-derived CD200 will enhance the efficacy of immunotherapy. METHODS: CD200 mRNA expression levels in human brain tumor tissue samples were measured by microarray. The amount of circulating CD200 protein in the sera of patients with brain tumors was determined by ELISA and, when corresponding peripheral blood samples were available, was correlated quantitatively with MDSCs. CD200-derived peptides were used as competitive inhibitors in a mouse model of glioblastoma immunotherapy. RESULTS: CD200 mRNA levels were measured in human brain tumors, with different expression levels being noted among the sub groups of glioblastoma, medulloblastoma and ependymoma. Serum CD200 concentrations were highest in patients with glioblastoma and correlated significantly with MDSC expansion. Similarly, in vitro studies determined that GL261 cells significantly expanded a MDSC population. Interestingly, a CD200R antagonist inhibited the expansion of murine MDSCs in vitro and in vivo. Moreover, inclusion of CD200R antagonist peptide in glioma tumor lysate-derived vaccines slowed tumor growth and significantly enhanced survival. CONCLUSION: These data suggest that CNS-derived tumors can evade immune surveillance by engaging CD200. Because of the homology between mouse and human CD200, our data also suggest that blockade of CD200 binding to its receptor will enhance the efficacy of immune mediated anti-tumor strategies for brain tumors.