Published papers

Purpose: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel vaccination with α-type 1 polarized dendritic cells (αDC1) loaded with synthetic peptides for glioma-associated antigen (GAA) epitopes and administration of polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in HLA-A2 + patients with recurrent malignant gliomas. GAAs for these peptides are EphA2, interleukin (IL)-13 receptor-α2, YKL-40, and gp100. Patients and Methods: Twenty-two patients (13 with glioblastoma multiforme [GBM], five with anaplastic astrocytoma [AA], three with anaplastic oligodendroglioma [AO], and one with anaplastic oligoastrocytoma [AOA]) received at least one vaccination, and 19 patients received at least four vaccinations at two αDC1 dose levels (1 × or 3 × 107/dose) at 2-week intervals intranodally. Patients also received twice weekly intramuscular injections of 20 μg/kg poly-ICLC. Patients who demonstrated positive radiologic response or stable disease without major adverse events were allowed to receive booster vaccines. T-lymphocyte responses against GAA epitopes were assessed by enzyme-linked immunosorbent spot and HLA-tetramer assays. Results: The regimen was well-tolerated. The first four vaccines induced positive immune responses against at least one of the vaccination-targeted GAAs in peripheral blood mononuclear cells in 58% of patients. Peripheral blood samples demonstrated significant upregulation of type 1 cytokines and chemokines, including interferon-α and CXCL10. Nine (four GBM, two AA, two AO, and one AOA) achieved progression-free status lasting at least 12 months. One patient with recurrent GBM demonstrated sustained complete response. IL-12 production levels by αDC1 positively correlated with time to progression. Conclusion: These data support safety, immunogenicity, and preliminary clinical activity of poly-ICLC-boosted αDC1-based vaccines. © 2010 by American Society of Clinical Oncology.

Malignant gliomas are lethal cancers in the brain and heavily infiltrated by myeloid cells. Interleukin-4 receptor-α (IL-4Rα) mediates the immunosuppressive functions of myeloid cells, and polymorphisms in the IL-4Rα gene are associated with altered glioma risk and prognosis. In this study, we sought to evaluate a hypothesized causal role for IL-4Rα and myeloid suppressor cells in glioma development. In both mouse de novo gliomas and human glioblastoma cases, IL-4Rα was upregulated on glioma-infiltrating myeloid cells but not in the periphery or in normal brain. Mice genetically deficient for IL-4Rα exhibited a slower growth of glioma associated with reduced production in the glioma microenvironment of arginase, a marker of myeloid suppressor cells, which is critical for their T-cell inhibitory function. Supporting this result, investigations using bone marrow-derived myeloid cells showed that IL-4Rα mediates IL-13-induced production of arginase. Furthermore, glioma-derived myeloid cells suppressed T-cell proliferation in an IL-4Rα-dependent manner, consistent with their identification as myeloid-derived suppressor cells (MDSC). Granulocyte macrophage colony-stimulating factor (GM-CSF) plays a central role for the induction of IL-4Rα expression on myeloid cells, and we found that GM-CSF is upregulated in both human and mouse glioma microenvironments compared with normal brain or peripheral blood samples. Together, our findings establish a GM-CSF-induced mechanism of immunosuppression in the glioma microenvironment via upregulation of IL-4Rα on MDSCs. © 2013 American Association for Cancer Research.

© Springer Science+Business Media, LLC 2012. All rights reserved. Recent clinical trials of immunotherapies indicate that tumor reactive autologous T cells are able to regress even advanced, large tumors in melanoma patients. For example, the adoptive transfer of CD8+ cytotoxic T lymphocytes (CTL) specifically targeted for identified tumor antigens following lymphodepletive treatment, such as fludarabine/cyclophosphamide administration and total body irradiation, led to objective tumor responses in more than 70% of patients with melanoma (Dudley et al. 2008). Immunological analyses on these tumor tissues demonstrated that administered T cells may eliminate tumor cells through direct killing and cytokine secretion. Therefore, CD8+ CTLs that recognize MHC class I positive cancer cells are important for in vivo tumor rejection.

To identify tumor antigens for glioma, a human testis cDNA library was screened by serological identification of antigens by recombinant expression cloning with sera from glioma patients. In this screening, the most frequently isolated antigen was SOX6, an Sry-related high-mobility group (HMG) box-containing gene. SOX6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. IgG antibodies against SOX6 were detected in sera from 12 of 36 glioma patients (33.3%), 0 of 14 patients with other brain disease (0%), and one of 54 other cancer patients (1.9%). In sera from 37 healthy individuals, no IgG responses against SOX6 were detected, except in an elderly female. Furthermore, Western blot and ELISA analyses with sera from glioma patients revealed that the DNA-binding domain, the HMG box of SOX6, might be a dominant epitope of IgGs against SOX6. RT-PCR and Northern blot analysis revealed that the SOX6 gene was more highly expressed in glioma tissues than in normal adult tissues, except testis. Western blot analysis with an anti-SOX6 antibody demonstrated that the SOX6 protein was expressed in glioma tissues, but not in normal adult brain tissue. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analysed expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in non-neoplastic tissues from the cerebral cortex. In summary, these results indicate that the developmentally regulated transcription factor SOX6 is aberrantly expressed in glioma and specifically recognized by IgGs from glioma patients' sera.

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma-bearing mice. In this study, we attempted to identify SOX6-derived peptides as specific targets for effective and safe T-cell-mediated immunotherapy targeting SOX6-positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)-A*2402 (A24)-restricted peptides, RFENLGPQL (SOX6504) and PYYEEQARL (SOX6628) or the HLA-A*0201 (A2)-restricted peptide, ALFGDQDTV (SOX6447) was capable of inducing SOX6 peptide-specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I-restricted and an antigen-dependent manner. Furthermore, peptide vaccines of SOX6628, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H-2d, induced CTLs specific for SOX6628 in H-2d mice. Normal autologous cells from mice, in which SOX6-specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA-A24 or -A2 positive glioma patients and should be considered as a promising strategy for safe and effective T-cell-based immunotherapy of patients with gliomas. © 2009 UICC.

Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly higher levels of the very late antigen (VLA)-4 and VLA-6 integrins when compared with T cells cultured under type 2 or nonpolarizing (type 0) conditions. This difference appears due to the action of interleukin (IL)-4, as loss of VLA-4/-6 expression on Th cells was prevented by inclusion of neutralizing anti-IL-4 mAb during the initial culture period. We also observed that CD4 T cells deficient in Stat6, a critical component of the IL-4R signaling cascade, retained high levels of VLA-4 and VLA-6 expression, regardless of IL-4 status in the culture conditions. When applied to committed Th1 cells, rIL-4 readily inhibited VLA-4 and VLA-6 expression to levels observed for Th2 cells, without altering the type 1 functional status of these cells. Conversely, low levels of VLA-4/VLA-6 expressed by committed Th2 cells could not be resurrected by culture in the presence of the Th1-kines IL-12p70 and interferon-γ. Predictably, among the Th populations evaluated, Th1 cells alone adhered efficiently to, and were costimulated by, plate-bound VCAM-1 and laminin in a VLA-4-dependent or VLA-6-dependent manner, respectively. Finally, adoptive-transferred Th1 (but not Th2) cells developed from OT-II mice were uniquely competent to traffick into OVA M05 melanoma lesions in vivo, thereby enhancing the therapeutic benefits associated with cotransferred OVA-specific type 1 CD8 (OT-I) cells. These data suggest that treatment strategies capable of sustaining/enhancing VLA-4/VLA-6 expression on Th1 effector cells may yield improved clinical efficacy in the cancer setting. Copyright © 2009 by Lippincott Williams & Wilkins.

We previously demonstrated that the developmentally regulated gene, SOX6, is strongly expressed in glioma cells and in the fetal brain, but only faintly in the normal adult brain. Recent studies have indicated that brain tumor cells may share antigens, signaling systems, and behavior with neural stem/progenitor cells. To test the validity of this proposition, we analyzed the expression of SOX6 in various human central nervous system (CNS) tumors. Immunohistochemical analysis revealed that astrocytic and oligodendroglial tumors expressed SOX6; neuronal-glial cell tumors (central neurocytoma) and embryonal tumors (medulloblastoma), which arise from multipotential stem cell precursors, also showed a high intensity of SOX6 staining. In contrast, ependymal tumors (ependymoma and subependymoma), meningioma, and schwannoma, which are all well differentiated tumors, showed either no staining or only faint staining for SOX6. These results suggest that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells. SOX6 may be a useful marker for the diagnosis of tumors arising from immature bipotential cells that may differentiate into neuronal and glial cells.

The development of effective immunotherapy strategies for glioma requires adequate understanding of the unique immunological microenvironment in the central nervous system (CNS) and CNS tumors. Although the CNS is often considered to be an immunologically privileged site and poses unique challenges for the delivery of effector cells and molecules, recent advances in technology and discoveries in CNS immunology suggest novel mechanisms that may significantly improve the efficacy of immunotherapy against gliomas. In this review, we first summarize recent advances in the CNS and CNS tumor immunology. We address factors that may promote immune escape of gliomas. We also review advances in passive and active immunotherapy strategies for glioma, with an emphasis on lessons learned from recent early-phase clinical trials. We also discuss novel immunotherapy strategies that have been recently tested in non-CNS tumors and show great potential for application to gliomas. Finally, we discuss how each of these promising strategies can be combined to achieve clinical benefit for patients with gliomas. © 2009 by Begell House, Inc.

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma. © 2008 Wiley-Liss, Inc.

A variety of cancers, including malignant gliomas, show aberrant activation of STAT3, which plays a pivotal role in negative regulation of antitumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T cell adoptive transfer therapy by reversal of STAT3-induced immunosuppression in a murine GL261 intracranial glioma model. In vitro treatment of GL261 cells with JSI-124, a STAT3 inhibitor, reversed highly phosphorylated status of STAT3. Systemic i.p. administration of JSI-124 in glioma-bearing immunocompetent mice, but not athymic mice, resulted in prolonged survival, suggesting a role of adaptive immunity in the antitumor effect. Furthermore, JSI-124 promoted maturation of tumor-infiltrating CD11c+ dendritic cells and activation of tumor-conditioned cytotoxic T cells, enhanced dendritic cells and GL261 production of CXCL-10, a critical chemokine for attraction of Tc1 cells. When i.p. JSI-124 administration was combined with i.v. transfer of Pmel-I mouse-derived type-1 CTLs (Tc1), glioma-bearing mice exhibited prolonged survival compared with i.p. JSI-124 or i.v. Tc1 therapy alone. Flow cytometric analyses of brain infiltrating lymphocytes revealed that JSI-124-treatment enhanced the tumor-homing of i.v. transferred Tc1 cells in a CXCL-10-dependent fashion. Systemic JSI-124 administration also up-regulated serum IL-15 levels, and promoted the persistence of transferred Tc1 in the host. These data suggest that systemic inhibition of STAT3 signaling can reverse the suppressive immunological environment of intracranial tumor bearing mice both systemically and locally, thereby promoting the efficacy of adoptive transfer therapy with Tc1. Copyright © 2008 by The American Association of Immunologists, Inc.

Three female patients aged 50-79 years (mean 61.0 years) presented with extremely rare intradural C-1 root schwannoma manifesting as foramen magnum syndrome. Magnetic resonance imaging revealed the intradural extra-axial tumors extending from the anterior aspect of the medullospinal junction, with a mean major diameter of 2.7 cm. Total resection of the tumors was performed via the lateral suboccipital transcondylar approach after a mean period of 11.7 months from the symptom onset. The final diagnosis of schwannoma of the C-1 ventral root was based on the intraoperative and histological findings. All three patients were successfully rehabilitated, with symptomatic improvement and no evidence of tumor recurrence. All three patients were successfully treated by surgical resection via the lateral suboccipital transcondylar approach. This approach is effective and safe provided individual anatomy, drilling of the occipital condyle, and patient position are considered carefully.

Jugular foramen meningioma is very rare. Papillary meningioma, the histological aspects and clinical behavior of which are highly malignant, is also very rare. Only 55 cases of jugular foramen meningioma have been reported in the literature. To our knowledge, this is the first report of a jugular foramen papillary meningioma. A 25-year-old woman presented with hoarseness and right hearing disturbance. Neurological examination showed deficits of the right hypoglossal and vagus nerves with palsy. Magnetic resonance imaging (MRI) revealed a mass on the right jugular foramen extending intracranially and extracranially, with weak contrast enhancement. The intracranial tumor was removed via the right lateral suboccipital approach. Histopathological examination confirmed the diagnosis of papillary meningioma. We report here the first case of jugular foramen papillary meningioma. No completely effective treatment for papillary meningiomas exists at present, with the exception of surgery.

A 36-year-old man presented with a tumor in the anterior skull base manifesting as headache and visual disturbance. Neurofibromatosis type 1 (NF-1) was identified in early childhood in the patient, and also in his father. Subtotal excision of the tumor was performed, leaving the portion extending outside of the cranium. The histological diagnosis was malignant peripheral nerve sheath tumor. Local radiotherapy was instituted postoperatively. Facial paralysis and dysphagia appeared 7 months after the first operation. Magnetic resonance imaging revealed new lesions in the lateral ventricle and around the brainstem. These tumors were also subtotally excised, but the patient died 10 months after the first operation. The tumor very likely originated from the meningeal branch of the trigeminal nerve. Treatment of such tumors developing inside the cranium should include the widest resection possible, followed by irradiation of the entire neuraxis including the spinal cord to inhibit dissemination through the cerebrospinal fluid. Treatment should be started as quickly as possible if the tumor is associated with NF-1, because of the poor prognosis associated with this condition.

Background: Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated.Methods: We performed miR microarray analyses on in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells.Results: Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4+ T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-γ production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD).Conclusion: The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy. © 2010 Sasaki et al; licensee BioMed Central Ltd.

An 18-year-old woman presented with a malignant fibrous histiocytoma (MFH) originating in the orbit and invading the frontal and temporal base of the skull manifesting as exophthalmos and double vision that had persisted for 2 months. Magnetic resonance imaging revealed a tumor in the left orbit that extended as far as the frontal and temporal base of the skull. The tumor was treated by radical resection with conservation of the eyeball and its contents, followed by orbit wall reconstruction. The histological diagnosis was MFH. Local radiotherapy was administered postoperatively. The preoperative symptoms improved, and there has been no evidence of local recurrence or metastasis in the year since the surgery. In this case, radical resection of the tumor was essential. Furthermore, the adjuvant therapy was apparently successful, probably because this histological type of tumor is highly sensitive to radiotherapy.

Stimulation of double-stranded (ds)RNA receptors can increase the effectiveness of cancer vaccines, but the underlying mechanisms are not completely elucidated. In this study, we sought to determine critical roles of host IFN-α and IFN-γ pathways in the enhanced therapeutic efficacy mediated by peptide vaccines and polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in the murine central nervous system (CNS) GL261 glioma. C57BL/6-background wild type (WT), IFN-α receptor-1 (IFN-αR1)-/- or IFN-γ -/- mice bearing syngeneic CNS GL261 glioma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes with or without intramuscular (i.m.) injections of poly-ICLC. The combinational treatment induced a robust transcription of CXCL10 in the glioma site. Blockade of CXCL10 with a specific monoclonal antibody (mAb) abrogated the efficient CNS homing of antigen-specific type-1 CTL (Tc1). Both IFN-αR -/- and IFN-γ -/- hosts failed to up-regulate the CXCL10 mRNA and recruit Tc1 cells to the tumor site, indicating non-redundant roles of type-1 and type-2 IFNs in the effects of poly-ICLC-assisted vaccines. The efficient trafficking of Tc1 also required Tc1-derived IFN-γ. Our data point to critical roles of the host-IFN-α and IFN-γ pathways in the modulation of CNS glioma microenvironment, and the therapeutic effectiveness of poly-ICLC-assisted glioma vaccines. © 2010 Springer-Verlag.

We previously reported to identify SOX5 as a glioma antigen by serological screening using a testis cDNA library. The present study was designed to analyze SOXS expression, its immunoreactivity, and the correlation between SOX5 IgG responses and clinical features in glioma patients to evaluate the possibility of its use as a diagnostic marker. Quantitative RT-PCR and Western blot analysis revealed that SOX5 was expressed in glioma tissues, but not in normal adult tissues, except in the testis. An immunohistochemical analysis showed that SOX5 was expressed in glioma cells, but only a few SOX5-positive cells were detected in non-neoplastic tissues from the cerebral cortex. IgG antibodies against SOX5 were detected in sera from 8 of the 27 glioma patients (27.6%), 0 of the 14 patients with other brain diseases (0%), 1 of the 54 other cancer patients (1.9%) and 1 of the 37 healthy individuals (2.7%). Patients with glioblastotna (GBM) who showed IgG responses against SOX5 exhibited significantly better survival periods than GBM patients without SOX5 antibodies. In summary, SOX5 is aberrantly expressed in glioma and can be recognized as a glioma antigen using IgGs from the sera of glioma patients. Furthermore, there is a statistically significant correlation between the presence of SOX5 IgGs and survival in GBM patients, suggesting that the glioma antigen SOX5 may be useful not only as a diagnostic marker, but also as a prognostic marker in glioma patients. © 2007 Wiley-Liss, Inc.

Background: In patients with high grade glioma, little is known regarding existence of naturally occurring adaptive T cell reactivity against glioma-associated antigens (GAAs). In this report, we characterized GAA-specific CD8+ T cells and innate immune cells in a patient who has survived with anaplastic astrocytoma (AA) for over 12 years without recurrence. Methods: Peripheral blood mononuclear cells (PBMCs) derived from the long term survivor with AA were evaluated for the frequency, cytotoxic T lymphocyte (CTL) activity and differentiation status of CD8+ cells recognizing GAA-derived epitopes as well as relative numbers of other immune cell subsets. This patient's AA tissue was evaluated for expression of two GAAs EphA2 and interleukin-13 receptor α2 subunit (IL-13Rα2) by immunohistochemistry. Results: The patient's tumor expressed both EphA2 and IL-13Rα2, and in vitro stimulated PBMC demonstrated superior EphA2883-891 and IL-13Rα2345-353-specific CTL reactivity compared to PBMC samples from two other patients with progressing malignant glioma. Unstimulated EphA2883-891- reactive CD8+ T cells contained high numbers ofCD45RA-/ CCR7- late effector and CD45RA-/CCR7+ central memory cells. Among other leukocyte subsets, elevated numbers of NK-T cells were found. Conclusion: To our knowledge, the current study is one of the first demonstrating the presence of antigen-experienced, GAA-reactive CD8+ T cells in a patient who has survived with AA for over 12 years without recurrence. Further studies are warranted to determine whether the status of GAA-reactive CD8+ T cells dictates survival of patients and/or response to therapeutic vaccines. © 2007 Ueda et al; licensee BioMed Central Ltd.

VLA-4 plays a critical role in T cell trafficking into inflammatory sites. Our recent studies have suggested that VLA-4 expression on CD8+ T cells is negatively controlled by IL-4 and serves as a functionally distinguishing variable for why Type-1, but not Type-2, CD8+ T cells are able to traffic into tumors. In this study, using in vitro culture of murine CD8+ T cells under Type-1 and Type-2 cytokine conditions, we show that IL-4-mediated down-regulation of VLA-4 expression is completely abrogated in Stat6-deficient CD8+ T cells. Conversely, CD8+ T cells expressing a constitutively active mutant form Stat6 (Stat6VT) failed to express VLA-4 even in the absence of IL-4-stimuIation. Notably, Type-2 CD8+ T cells developed from Stat6-/- but not wild-type mice were competent to migrate into tumor lesions in vivo. These results suggest that Stat6-signaling is necessary and sufficient to restrict CD8+ T cell expression of VLA-4 (by IL-4), thereby serving as a regulator for CD8 + T cell infiltration into tumors. Copyright © 2008 by The American Association of Immunologists, Inc.