Mechanisms of Immune Evasion by Gliomas

53

CHAPTER 5

MECHANISMS OF IMMUNE EVASION BY GLIOMAS

Cleo E. Rolle,1 Sadhak Sengupta1 and Maciej S. Lesniak*,2

1Department of Surgery, Section of Neurosurgery, The University of Chicago Pritzker School of Medicine, Chicago, Illinois, USA; 2Section of Neurosurgery, The University of Chicago, Chicago, Illinois, USA *Corresponding Author: Maciej S. Lesniak—Email: [email protected]

Abstract: A major contributing factor to glioma development and progression is its ability to evade the immune system. This chapter will explore the mechanisms utilized by glioma to mediate immunosuppression and immune evasion. These include intrinsic mechanisms linked to its location within the brain and interactions between glioma cells and immune cells. Lack of recruitment of naïve effector immune cells perhaps accounts for most of the immune suppression mediated by these tumor cells. This is enhanced by increased recruitment of microglia which resemble immature antigen presenting cells that are unable to support T-cell mediated immunity. Furthermore, secreted factors like TGF-�, COX-2 and IL-10, altered costimulatory molecules and inhibition of STAT-3 all contribute to the recruitment and expansion of regulatory T cells, which further modulate the immunosuppressive environment of glioma. In light of these findings, multiple immunotherapeutic treatment modalities are currently being explored.

INTRODUCTION

The brain has classically been considered an immune privileged organ isolated from the immune system on the basis of several key observations. Seminal transplant immunology studies conducted by Medewar found that foreign tissues implanted in the brain were not immediately rejected.1 Later work suggested that this was due to the fact that the brain was physical isolated from the immune system by the blood-brain barrier (BBB). Further, it was suggested that the brain was not connected to the lymphatic system and that lymphocytes did not traffic into the brain. However, with improvements in techniques and assessment of immune responses the concept of immune privilege was

Glioma: Immunotherapeutic Approaches, edited by Ryuya Yamanaka. ©2012 Landes Bioscience and Springer Science+Business Media.

54 GLIOMA: IMMUNOTHERAPEUTIC APPROACHES

refined. Subsequent studies demonstrated the rejection of intracerebral xenografts and allografts in immunocompetent hosts.2

It is now well established that there is exchange between the peripheral immune compartment and the brain. Although naive T cells fail to traffic into the brain, activated T cells are capable of traversing the BBB. Additionally, Tsugawa and colleagues demonstrated that brain interstitial fluid drained into the cervical lymph nodes.3 Using an animal model, dendritic cells injected into brain tumors were later detected in cervical lymph nodes. Our laboratory and others have characterized T cells in experimental animal models of glioma and primary patient samples.4-7 Collectively, these data indicated that the brain was not isolated from the immune system as previously thought, rather immune responses are tightly regulated within the brain.

Many studies regarding glioma-mediated immunosuppression have been undertaken in attempts to develop more effective treatment strategies. Patients with glioma exhibit a suppression of cell-mediated immunity, which has been linked to the skewing of the immune response within the central nervous system (CNS) towards a humoral, rather than an inflammatory or cytotoxic immune response. This chapter will discuss the mechanisms proposed to mediate glioma immunosuppression, including intrinsic mechanisms directly related to its location within the brain and interactions between glioma cells and immune cells.

INTRINSIC MECHANISMS OF IMMUNOSUPPRESSION

Altered Human Leukocyte Antigen Expression

Human leukocyte antigens (HLA) are the MHC molecules in humans. The class I antigens are comprised of three major (classical) genes—HLA-A, -B and -C; and three minor (nonclassical) genes—HLA-E, -F and -G. The classical HLA class I molecules present intracellular antigenic peptides on the surface of altered cells, thus targeting the cells for lysis by cytotoxic CD8� T cells (CTL). They are typically expressed by most nucleated cells in the body. Analysis of HLA class I antigens expression in 47 glioblastoma multiforme (GBM) lesions revealed that expression was lost in approximately 50% of the samples.8 More detailed analysis found a selective HLA-A2 antigen loss. There was a significant positive correlation between HLA class I antigen loss and tumor grade (P 0.025). The selective loss of HLA class I antigens would suggest a defect in antigen presentation by glioma cells and a concomitant impairment of CTL lysis. Accordingly, Mehling et al found that antigen processing and accessory antigen presentation molecules were also downregulated and the extent of downregulation was correlated with tumor grade.9 Interestingly, HLA class II antigen expression was lost in only approximately 30% of the 44 lesions analyzed.8 These data would suggest that while antigen presentation to CD8� T cells might be impaired, glioma cells retain the ability to present antigen to CD4� T cells.

The major HLA class II antigens are HLA-DR, -DP and -DQ. The expression of class II molecules is restricted to antigen presenting cells. HLA-DR presents antigenic peptides to CD4� T cells. In vitro studies of HLA-DR expression by glioma cells found that its expression was modulated by transforming growth factor-� (TGF-�).10 Interestingly, the expression of other HLA-related molecules, including HLA class I molecules and beta 2-microglobulin were not modulated by TGF-�. Immunohistochemical studies

55MECHANISMS OF IMMUNE EVASION BY GLIOMAS

found that approximately 67.2-83% of glioma patient samples expressed HLA-DR.11,12 Unlike the expression of HLA class I which decreased with tumor grade, HLA-DR expression increased as tumor grade increased. The increased expression of HLA-DR suggests a skewing of the immune response towards helper T cells, rather than CTL. Further, this would explain that although CTL are found in glioma lesions they appear to be unable to lyse glioma cells.

Recently, HLA-G, a nonclassical HLA class I molecule, has been identified in malignant tumors and glioma cell lines and may be involved in tumor immune escape.13 The normal function of HLA-G is to maintain immune tolerance at the maternal-fetal interface by inhibiting maternal NK cell activity against fetal tissue. The major isoforms of HLA-G are HLA-G1 and HLA-G5, which exist as membrane bound surface molecules, as well as in a soluble form. Expression of soluble HLA-G is increased in the plasma of patients with malignant melanoma, glioma, breast and ovarian cancer.14,15 The expression of membrane-bound HLA-G1 and soluble HLA-G5 by tumor cells inhibited alloreactive and Ag-specific immune responses in vitro. Ectopic expression of HLA-G1 or HLA-G5 in HLA-G-negative glioma cells (U87MG) conferred resistance to lysis and prevented efficient priming of cytotoxic T cells. Interestingly, HLA-G expression by a few tumor cells within a population of predominantly HLA-G-negative tumor cells was sufficient to confer significant inhibitory effects on immune cells.16 Therefore, the aberrant expression of HLA-G by glioma cells is a mechanism by which they are able to evade NK cell-mediated lysis, as well as prevent T-cell activation.

Another inhibitory molecule is the nonclassical MHC class I molecule HLA-E. It is the only known ligand for CD94/NKG2A, an inhibitory receptor, expressed on NK cells and CD8� T cells. The over-expression of HLA-E by glioma cells would render them resistant to NK cell and CTL cytotoxicity. Analysis of human long-term glioma cell lines, primary ex vivo polyclonal glioblastoma cell cultures and surgical glioblastoma specimens, revealed high expression of HLA-E.17,18 There was a massive over-expression of HLA-E in Grade IV glioblastomas compared to normal CNS tissue. HLA-E expression levels and immune cell infiltration were positively correlated in WHO Grade IV glioblastomas. According to Wischhusen et al, siRNA-mediated silencing of HLA-E or antibody-mediated blocking of CD94/NKG2A enables NKG2D-mediated lysis of tumor cells by NK cells.17

The altered expression pattern of HLA molecules is one potential mechanism by which glioma cells are able to evade the immune system. The coordinated down-regulation of HLA-A and up-regulation of HLA-DR could explain the defective CTL responses and enhanced CD4� T-cell responses observed in glioma patients. In addition to the down-regulation of HLA-A, accessory antigen processing and presentation molecules are also down-regulated, suggesting a mechanism by which glioma cells prevent antigen presentation to CD8� T cells. Conceivably, HLA-E and HLA-G inhibit NK cell and T-cell lysis of glioma cells.

The Role of Microglia

Microglia are the brain’s resident macrophages. They differentiate from monocytes which migrate into the brain during embryogenesis.19 Microglia are distinguished from macrophages based on low expression of CD45 and high expression of CD11b (CD45dim CD11b�). Under normal physiological conditions, microglia roam the CNS, phagocytosing debris and maintaining homeostasis. Microglia were first described in brain tumors in the


early 1920s.20 This finding was later confirmed using animal models and primary patient samples.11,21,22 More recently, primary patient samples obtained from 9 newly diagnosed malignant gliomas quantified microglial infiltration.23 While these studies clearly identified microglia in gliomas, their exact role in immunosuppression was not described until much later. Studies have suggested that the extent of microglia infiltration correlates to tumor grade. Roggendorf et al proposed early on that tumors actively recruited migroglia.24 The expression of MCP-1, a monocyte chemoattractant, by glioma cells was later confirmed and shown to be functional.25,26

Although the presence of microglia in glioma has long been established, the actual role of tumor infiltrating microglia during glioma remains controversial. This is related to conflicting data regarding the immunostimulatory ability of microglia. On the basis of the expression of MHC and costimulatory molecules, microglia resemble immature antigen presenting cells. However, upon activation, microglia up-regulate the expression of MHC and B7, making them more likely to stimulate T cells. Therefore, some studies have found that microglia are able to prime T cells.27 Microglia derived from normal brains are inefficient at presenting glioma-derived antigens to naïve T cells and CTL in vitro.28 In contrast, microglia isolated from normal brain and expanded in vitro with growth factors, express MHC class II, efficiently process antigen and stimulate T cells.27 Primary microglia isolated from human glioma samples only weakly induced T-cell cytotoxicity against glioma cells.29These data indicate that with appropriate stimuli microglia are able to activate T cells, however in the tumor microenvironment they are unable to support T-cell-mediated cytotoxicity, and in some cases directly inhibit T-cell proliferation.

It is likely that microglia may play dual roles during glioma development through the expression of cytokines. Microglia have been postulated to support glioma cell proliferation through the production of IL-10 and IL-6.30,31 IL-10 is also an immunosuppressive cytokine which inhibits T-cell proliferation. Furthermore, microglia express a clearly Th2 skewed cytokine profile, which suppresses cytotoxic responses. Taken together, glioma infiltrating microglia support glioma tumorigenesis while promoting immune evasion.

The Regulation of Th1/Th2 Cytokine Responses

The cytokine milieu of the brain ensures that immune responses are primarily humoral responses in order to prevent damage due to inflammation. The normal humoral response is further skewed in glioblastoma patients. Analysis of gene expressions of Th1/Th2 cytokines in 62 specimens of human glioma tissues, 4 glioma cell lines and 5 specimens of normal adult brain tissue determined predominant expression of Th2 cytokines in glioma cell lines (P 0.01) and specimens of human glioma tissues (P 0.01).32 The total positive rates of Th1 and Th2 cytokines genes in human glioma cells were 14.77% and 75%, respectively.33 In addition to glioma cells, the cytokine profiles of glioma infiltrating lymphocytes were also Th2 skewed.34 Therefore, the immunosuppressive cytokine milieu of gliomas is not only influenced by glioma cells, but also the infiltrating lymphocytes.

IL-10 is a Th2 cytokine that is has been described in CNS responses. There was high expression of IL-10 reported at the mRNA level in 87.5% of Grade III and IV, but only 4% of Grade II tumors. Elevation of IL-10 serum levels was found in 11% of low grade and in 63.6% of high grade glioma patients.30 IL-10 was found to stimulate the proliferation of glioma cells. This glioma cell proliferation was blocked by the addition of blocking IL-10 antibody.35 Additonally, glioma-derived IL-10 greatly downregulated HLA-DR expression on monocytes. In vitro, IL-10 increased the proliferation and


migratory capacity in human glioma cell lines. Interestingly, blocking IL-10 antibody was not sufficient to inhibit IL-2 production.36 These data suggest that IL-10 in the cytokine milieu of glioma is sufficient to inhibit T-cell proliferation but does not induce T-cell anergy. During glioma tumorigenesis, it is plausible that IL-10 plays dual roles, acting as a growth factor for glioma cells and as an immunosuppresive factor.

Since this early work assessed IL-10 mRNA levels in patient tumor samples and not purified glioma cells, it was proposed that IL-10 was produced by glioma cells. However, in situ hybridization for IL-10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL-10.37 More recently, Samaras et al assessed the expression of IL-10 in paraffin-embedded neoplastic tissue of 12 glioma patients. In comparison to healthy controls, IL-10 secretion from peripheral mononuclear and tumor tissue from glioma patients was higher (P � 0.0002).38 As in the earlier study, IL-10 expression in glioblastomas was restricted to microglia/macrophage cells. IL-10 production by microglia supports the growth and proliferation of glioma cells and mediates immune evasion.

IMPAIRMENT OF GLIOMA AND IMMUNE CELL INTERACTIONS

Cell-to-cell contact is required for lymphocytes to lyse tumor cells. Specifically, the release of the cytotoxic molecules, perforin and granzymes, by natural killer (NK) cells and CTL is cell contact dependent. Glioma cells have been shown to alter the extracellular matrix and this may have an impact on cell-to-cell contact. Recently, tenascin-C expression has been described in glioma cells. Tenascin-C belongs to a specialized class of ECM proteins, matricellular proteins, which function as adaptors and modulators of cell-matrix interactions.39,40 The exact role of tenascin-C is unresolved as it has been implicated in both cell adhesion and migration.41-43 Tenascin-C has also been shown to inhibit T-cell proliferation and IFN� production.44,45 The infiltration of lymphocytes into tumors was inhibited by tenascin-C.46 Through the expression of tenascin-C, glioma cells could potentially enhance cell migration, while suppressing T-cell responses by limiting cell-to-cell contact.

MECHANISMS OF GLIOMA-MEDIATED IMMUNOSUPPRESSION

Secreted Immunosuppressive Factors

TGF-�

Glioma cells secrete multiple factors that have been shown to suppress antitumor immune responses, namely TGF-�. There are three isoforms of TGF-�: TGF-�1, -�2, -�3. TGF-�2 has been shown to enhance tumor growth and invasion, angiogenesis and immunosuppression. The expression of TGF�-1 and -2 in two glioblastoma cell lines and newly isolated patient samples was confirmed at the mRNA level.47 However, only TGF�-2 was detected in the supernatant of glioma cell lines and in the cerebral spinal fluid of patients with malignant glioma.48 Interestingly, TGF-�2 was originally isolated and cloned from glioblastoma cell lines and was named for its ability to suppress T-cell growth and IL-2 production.49,50 More recently, TGF-�1 and -�2 have been shown to induce FoxP3


expression in anti-CD3 activated CD4� T cells, resulting in inducible regulatory T cells (Tregs), whose role in glioma-mediated immunosuppression will be discussed further. Thus, the secretion of TGF-�� by glioma cells mediates immunosuppression not only by acting directly on T cells and NK cells, but also indirectly through the induction of Tregs.

Cyclooxygenase-2 and Prostaglandin E2

Cyclooxygenase-2 (COX-2) is an isoform of the cyclooxygenase enzyme or prostaglandin synthase responsible for the formation of important biological mediators called prostanoids (including prostaglandins, prostacyclin and thromboxane). COX-2 is an inducible enzyme, becoming abundant in activated macrophages and other cells at sites of inflammation. Inhibitors of COX-2 are used as nonsteroidal anti-inflammatory drugs (NSAID) for treating inflammation and pain. The over-expression of cyclooxygenase-2 (COX-2), the key enzyme for the synthesis of prostaglandin E2 (PGE2) from arachidonic acid, has also been well characterized in many types of cancers including colorectal, lung, urinary bladder and malignant gliomas.51-55

PGE2 suppresses cell-mediated immune responses while enhancing humoral immune responses.56,57 In macrophages the down-regulation of cell-mediated immune responses by PGE2 is characterized by a remarkable drop in LPS-mediated TNF-� and IL-12 production.58,59 PGE2 modulates a variety of physiological processes, including APC function and the production of inflammatory cytokines.60,61 PGE2 is also a potent inducer of IL-10,62 which is produced by a variety of cells including monocytes, and exerts suppressive effects on dendritic cell maturation and Th1 responses.63-64

PGE2, contributes to cellular immune suppression in cancer patients through an unknown mechanism. In 2004 Akasaki et al, reported that Tr1 Tregs were induced by CD11c� mature dendritic cells that phagocytosed allogeneic and autologous COX-2-overexpressing glioma.65 These gliomas did not express IL-10 receptors and recombinant IL-10 did not suppress their COX-2 expression. Exposure to COX-2-over-expressing glioma induced mature dendritic cells (DC) to over-express IL-10 and decreased IL-12p70 production. These dendritic cells (DC) induced a Tr1 response, which is characterized by secretion of IL-10 and TGF-ß with negligible IL-4 secretion by CD4� T cells and an inhibitory effect on lymphocytes. Peripheral CD4� T-cell populations isolated from a glioma patient also predominantly demonstrated a Tr1 response. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this Tr1 response and instead elicited Th1 activity. Stable transfection of COX-2 in glioma cell lines also induced a Tr1 response. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.

PGE2 levels were significantly higher in patient glioma samples compared to control brain samples.66 Furthermore, surgical removal of malignant brain tumors resulted in a decrease of PGE2 levels.67 PGE2 has been linked to tumor metastasis and immune evasion, such that the inhibition of PGE2 synthesis using COX inhibitors suppressed tumor growth.68-71 The increased biosynthesis of PGE2 in glioma cells is due to increased expression of mPGES-1, an enzyme which catalyses the isomerization of PGH2 into PGE2.72 PGE2 has been implicated in tumorigenesis, mediating tumor growth and angiogenesis, as well as immunosupression.

PGE2 has been shown to increase vascular permeability and edema and at physiological concentrations blocks IL-2 production.73-75 It also inhibits the induction of CTLs, NK


cells and lymphokine activated killer cells (LAK).76 Lauro et al found that serum-free supernatant from glioma cells inhibited the proliferation of PHA-stimulated T cells.77 The expression of PGE2 by glioma cells in vitro was sufficient to inhibit the induction of LAK cells from PBMCs and tumor infiltrating lymphocytes, even at high concentrations of IL-2.78

Not only do glioma cells themselves produce PGE2, but they can also stimulate tumor infiltrating microglia to produce PGE2.79 Coculture of glioma cells with microglia or microglia cultured in the presence of glioma conditioned media increased the expression of mPGES1 and enhanced the production PGE2.80 Therefore, the production of PGE2 by the glioma cells themselves and the enhancement of PGE2 production in microglia both contribute to immunosuppression.

Induction of T-Cell Anergy

Altered B7 Expression

While T cells infiltrate gliomas, in both animal models and primary patient samples, they often lack cytotoxic activity and fail to produce IL-2. This observed T-cell anergy has been linked to defective antigen presentation and poor costimulation by tumor cells. As we discussed previously, antigen presentation to CD8� T cells is decreased during glioma tumorigenesis due to the loss of HLA class I expression. Additionally, glioma cells express very low levels of B7 costimulatory molecules.81 Functional assays using heterogeneous ex vivo tumor preparations or pure populations of ex vivo tumor cells and microglia, demonstrated CD4+ T-cell activation only in the presence of exogenous B7 costimulation (provided by addition of soluble agonist anti-CD28 monoclonal antibody). Therefore, to overcome immunosuppression in glioma, strategies need to overcome the low levels of B7 costimulation.

STAT3

In recent years, signal transducer and activator of transcription-3 (STAT3) has been identified as a major molecular hub of several signaling pathways in several types of cancer including glioblastoma, breast, lung, ovarian, pancreatic, skin and prostate cancer.82,83 The binding of STAT3 to its gene targets affects proliferation, survival, differentiation and development. It is a member of the STAT family of cytoplasmic latent transcription factors. Receptor engagement by members of IL-6 cytokine family like IL-6, oncostatin M and Leukemia inhibitory factor, or growth factors like platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epithelial growth factor (EGF) activate STAT3 by tyrosine phosphorylation. Kinases that induce STAT3 activation are receptor kinases like Janus kinase (JAK) family memebers associated with, FGFR, EGFR, PDGFR or nonreceptor-associated kinases like Ret, Src or Bcl-Abl. Postactivation STAT3 works together with other transcription factors to regulate expression of bcl2, bclxL, mcl1 and cyclinD1 among others.83 STAT3 activity is attenuated by suppressors of cytokine signaling (SOCS) by down-regulating its upstream kinase activity, while protein inhibitors of activated STAT (PIAS) and protein tyrosine phosphatases target STAT3 directly.84-85

Other than promoting oncogenesis, active STAT3 also enables tumor growth by suppressing tumor recognition by the immune system.86 STAT3 promotes tumor immune evasion by inhibiting pro-inflammatory cytokine signaling and amplifying


Tregs. Inhibiting STAT3 activity by dominant-negative or antisense STAT3 expression increased expression of proinflammatory IFN-�, TNF-�, IL-6, RANTES and IP-10, while activating STAT3 hindered expression of IL-6 and RANTES in experimental systems.87 STAT3 is also involved in maintaining immature dendritic cells and promoting tumor immune tolerance. Mice lacking STAT3 had elevated levels of MHC class II, CD80 and CD86. Antitumor activity of T cells, NK cells and neutrophils was increased and dendritic cell maturation was enhanced by inhibition of STAT3.88,89 STAT3 activity was shown to be elevated in tumor-associated Treg cells that maintain tumor immune evasion.88,90 In these Tregs, elevated activation of STAT3 increased proliferation and promoted expression of FoxP3, TGF-� and IL-10, all of which inhibit CD8� T-cell differentiation, dendritic cell maturation and promote proliferation of Tregs.86,90

In gliomas, activation of STAT3 has been associated with the inhibition of T-cell response. Tregs have been shown in the blood and within the tumor microenvironment of glioblastoma patients and are thought to contribute to the lack of effective immune responsiveness against glioblastomas, although the STAT3 activation status of these cells was not examined in these studies.29,91 In a tissue microarray using tumor samples from 129 glioma patients, Abou-Ghazal et al92 showed correlation between prognosis of activated STAT3 residues and tumor-infiltrating Tregs. Low incidence of activated STAT3 was observed in normal brain or low-grade astrocytomas. While the incidence of activated STAT3 varied in different grades of gliomas, it correlated with the tumor-infiltrating immune-cells but not with that of Tregs. Glioma-infiltrating CD8� T cells were characterized as a marker of immunosuppressive environment as they were neither activated nor proliferating.29 Using a novel small molecule inhibitor of STAT3 in monocytes from glioma patients, the expression of costimulatory molecules such as CD80 and CD86 were up-regulated.93 This apparently enhanced T-cell activation which is critical in overcoming immune suppression.

PDL-1

Programmed Death Ligand-1 (PDL-1) or B7-H1 is a member of the B7 family and is a ligand for PD-1(Programmed Death-1), an extended member of CD28 family of T-cell regulators. B7-H1 is primarily inhibitory when expressed on tumors. It is significantly expressed on the surface of many human cancers, while undetectable in normal tissues.94 B7-H1 is found to be highly expressed in carcinomas of colon, breast, ovarian, lung and melanoma cancer, oral squamous cell carcinoma, head and neck cancers and glioma among others.95-98

B7-H1 induction of IFN-� and IL-10, granulocyte macrophage colony-stimulating factor production and proliferation of T cells in vitro and the late downstream effects of signaling initiated through the B7-H1:PD-1:unidentified receptor network result in negative regulation of immune function.99,100 The inhibitory effects of B7-H1:PD-1 interactions in tumor immunity were described by models using either PD-1 knockout mice or blocking monoclonal antibodies for B7-H1 or PD-1. Both increase cytokine production and antitumor CTL activity in experimental tumor models in mice.101-105 Another possible mechanism is by induction of apoptosis in effector CTL cells through PD-1 after exposure to B7-H1 on tumor cells.101 Several models in which both human and murine tumors are transfected with B7-H1 become resistant to CTL attack, resistant to lysis and more resistant to apoptosis induction, while actually inducing lysis of CTLs through PD-1.102,106

Expression and immune regulatory activity of B7-H1 has been recently identified in human glioma cells in vitro and in vivo. In 12 different glioma cell lines, Wintrelle


et al observed constitutive expression of B7-H1 mRNA, although these cells lacked B7.1/2 (CD80/86).98 Upon exposure to IFN-�, B7-H1 expression was strongly enhanced. High B7-H1 expression was observed in all 10 malignant glioma specimens upon immunohistochemical analysis, whereas no B7-H1 expression could be detected on normal brain tissues. Functional significance of glioma cell-related B7-H1 expression was elucidated by coculture experiments of glioma cells with alloreactive CD4� and CD8� T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4� as well as CD8� T-cell activation as assessed by increased production of IFN-�, IL-2 and IL-10 and expression of CD69 in the presence of a neutralizing antibody against B7-H1. These observations infer that B7-H1 may significantly influence the outcome of T-cell tumor interactions and correspond to a novel mechanism by which glioma cells evade immune recognition and destruction.

Inhibition of Natural Killer Cells

Natural killer (NK) cells are innate immune cells that lyse tumor cells or infected cells. Upon activation they also participate in the initiation of adaptive immune responses, primarily through the production of cytokines. Unlike T and B cells which express an antigen-specific receptor and only become activated following antigen presentation via MHC molecules, NK cells are not MHC-restricted. In fact, NK cells were named for their ability to lyse target cells lacking MHC class I molecules.107 Natural killer cells are cytotoxic lymphocytes, expressing perforin and granzymes, resulting in the induction of apotosis or necrosis in target cells. The activation of NK cells is tightly regulated and requires increased signaling via activating receptors coupled with decreased signaling via inhibitoty receptors. Interferons, IL-2, IL-12 and IL-8, also serve to activate NK cells. Natural killer cells express the following activating and inhibitory receptors: NKG2, Ly49, killer cell immunoglobulin-like receptors (KIR) and leucocyte inhibitory receptors (LIR). Engagement of NKG2D, an activating receptor, has been shown to be critical for NK cell-mediated tumor rejection.108,109 Despite an increased fraction of NK cells in the blood and tumor-infiltrating lymphocytes of glioma patients, MHC-unrestricted NK activity is impaired.110 As tumor grade increases, NK cell cytotoxicity becomes impaired.111

Lectin-Like Transcript 1

Although it is well established that malignant brain tumors have significantly altered HLA expression they are able to escape NK cell mediated rejection. This would suggest that activating signals are overridden by inhibitory signals.112 As we discussed earlier, the expression of TGF�, HLA-E and HLA-G by glioma cells, all contribute to inhibit NK cell activity against malignant brain tumors. More recently, lectin-like transcript 1 (LLT1) has been shown to inhibit NK cell activity via an interaction with CD161 on human NK cells. Lectin-like transcript 1 was identified as a ligand for CD161 based on its sequence homology to ligands for other CD161 receptor family members.113 Engagement of CD161 expressed on NK cells by LLT1 on target cells inhibited NK cell cytotoxicity and IFN� secretion. The expression of LLT1 was primarily restricted to monocytes and B cells in the peripheral blood and PBMCs, NK cells and T cells expressed it upon activation.

According to Roth et al, human glioma cells expressed LLT1 mRNA and protein in vitro and in vivo.114 Although LLT1 was expressed by normal brain cells, it was expressed at significantly lower levels compared to glioma cell lines and primary glioma samples


(p 0.0001). There was a positive correlation between WHO tumor grade and level of LLT1 expression. Interestingly, TGF� treatment enhanced the expression of LLT1 by glioma cells in vitro. In order to assess the role of LLT1 in glioma immunosuppression, siRNA was used to inhibit LLT1 expression in glioma cells. Natural killer cells were able to lyse glioma cells in which LLT1 levels were successfully knocked down. Therefore, the expression of LLT1 by glioma cells mediates immune evasion.

Regeneration and Tolerance Factor

According to a recent report by Roth and colleagues, RTF mRNA and protein were expressed in human glioma cells in vitro and in vivo. Originally identified in the placenta for its role in fetal allograft survival, RTF has been implicated in the inhibition of T-cell activation and the induction of T-cell apoptosis.115 In animal models of glioma, suppression of RTF using RNAi promoted the lysis of glioma cells by NK cells and T cells and by extension RTF-depleted glioma cells failed to grow tumors in nude mice.116 In vivo, RTF-mediated tumor rejection was reversed in NK cell depleted hosts. Collectively, these data suggests that RTF expression by glioma cells enhances immunosuppression by inhibiting NK cells.

The Induction of Apoptosis

Fas

The induction of apoptosis is a key immune evasion strategy for multiple malignancies, including glioma. Fas/APO-1 is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or anti-Fas antibody. The expression of Fas has been demonstrated on perinecrotic glioma cells, a histological hallmark of glioblastomas, suggesting a correlation between Fas expression and cell death. Since glioma cells are not always able to turn off Fas expression they must use other strategies to evade Fas-induced apoptosis. A potential endogenous antagonist of Fas is soluble Fas which lacks the transmembrane domain. The soluble form of the Fas mRNA was detected in one anaplastic astrocytoma and in two glioblastomas.117 Interestingly, some glioma cells co-expressed Fas and FasL suggesting that soluble Fas expression by the glioma cells prevented tumor cell-induced apoptosis.

The expression of FasL was observed in 10 glioblastoma cell lines and in 14 astrocytic brain tumors (three low-grade astrocytomas and 11 glioblastomas).118 The expression of FasL by glioma cells may act as a glioma immune evasion mechanism. Apoptosis was induced in T cells by FasL� tumor explants and tumor cell lines.119 According to Didenko et al, T cells that had undergone apoptosis expressed Fas and were colocalized with FasL-expressing tumor cells.120

Since glioma cells have the ability to express both Fas and FasL, they have evolved mechanisms to prevent tumor cell apoptosis, while simultaneously inducing apoptosis in T cells. In a recent report by Roth et al, the expression of decoy receptor 3 (DcR3), a soluble FasL molecule with anti-apoptotic properties, was detected in human glioma


cells and in GBM samples.121 The expression of DcR3 was correlated to tumor grade and was not detected in WHO Grade II tumors. The protective effect of DcR3 was specific to Fas induced cell death and did not prevent apoptosis induced by TRAIL ligands. Ectopic expression in the rat glioma cell line 9L inhibited T-cell and microglia infiltration. These data demonstrate a mechanism by which glioma are able to ensure tumor propagation by evading the immune system and prevent death in tumor cells.

CD70

CD70, TNF-related cell surface ligand, is normally expressed on mature dendritic cells, activated T and B cells.122 The binding of CD70 to its cognate receptor CD27, a TNF receptor family member, is thought to play an important role in T-cell, B-cell and NK cell activation. CD27 is constitutively expressed by T cells, B cells and NK cells.123 Signaling via CD27 by activated T cells is critical for survival and memory cell formation.124 In contrast, the ligation of CD27 has also been shown to induce apoptosis in activated T and B cells.125 CD70-CD27 induced cell death has been implicated in models of chronic immune activation as a means to maintain self tolerance.126 Moreover, CD27 costimulation has been shown to enhance Treg function.127

According to a study conducted by Wischhusen et al, 11 of 12 human glioma cell lines expressed CD70 mRNA and protein.16 CD70� glioma cells were able to induce apoptosis in PBMC. In vitro experiments using glioma cells, in which Fas and TNF� were blocked, revealed that CD70 was sufficient to induce apoptosis in T cells.128 The expression of CD70 is a novel mechanism utilized by glioma cells to induce apoptosis in T cells and consequently evade the immune response.

Gangliosides

Previous studies demonstrated the role of tumor-derived gangliosides as important mediators of T-cell apoptosis and hence, as one mechanism by which tumors evade immune destruction.129 Gangliosides are most highly expressed in cells of the CNS, where they comprise approximately 5-10% of the lipids.130 Using a human glioblastoma biopsy brain tumor nude rat xenograft model, glioma cells were shown to express multiple gangliosides: GD3, GM2 and 3�-isoLM1.131 Ganglioside GD3 was detected in the tumor mass while ganglioside 3�-isoLM1 was more abundantly expressed in the periphery of the tumor, associated with areas of tumor cell invasion. The ganglioside GM2 was only detected within the main tumor mass. According to a study conducted by Markowska-Woyciechowska et al, high and low grade gliomas had similar ganglioside profiles.132 The expression of gangliosides GD2 and GD3 was restricted to the cytoplasm of highly fibrillary (gemistocytic) astrocytes in all grades of gliomas.133 Moreover, the increase of GD3 was associated with a decrease of normal brain gangliosides and this was correlated with a higher grade of malignancy.134 This work supports the notion that gangliosides play a role in apoptosis and indicates that antibodies or ligands directed against GD3 and 3�-isoLM1 might be complementary when applied in the treatment of human glioblastomas.131


RECRUITMENT OF IMMUNOSUPPRESSIVE LYMPHOCYTES

Expression of Chemokines

Chemokines are a family of cytokines helping in the migration of responder cells by inducing directed chemotaxis. These proteins are very small in size (8-10 kDa) and have a characteristic two cysteine residues joined by disulfide bonds (cystine). The different members of chemokine family share gene sequence and amino acid homology. They are classified into several categories according to spacing of the cystine moieties that are key to forming their 3-dimensional shape and accordingly, different families of receptors for these molecules have been described.135 Their role has been implicated in various processes including angiogenesis and CNS development. Most importantly, chemokines play a central role within the immune system, as the secretion of these molecules leads to migration of leucocytes.136-138 Chemokines appear to play a significant role in various human diseases, including cancer.139 As chronic inflammation can predispose to cancer formation and progression, the expression of chemokines is suspected to contribute to this process.140 Alternatively, chemokines might elicit an intrinsic effect on tumor cells. For instance, multiple human cancers including leukemias, lymphomas, gliomas and various epithelial carcinomas express CXC receptor 4 (CXCR4) and respond to its ligand CXC ligand 12 (CXCL12). This ligand-receptor interaction promotes the migration and metastatic establishment of tumor cells.141,142

One of the initial reports of chemokines secretion by gliomas was made in 2001 by Choi and others, where the authors demonstrated that human glioma cell lines secrete MCP-1 (CCL2) and IL-8 on stimulation with anti-Fas antibody or soluble Fas ligand in a time and dose-dependent manner.143 However, at that time its function was not correlated to Treg migration and was instead attributed to proinflammatory properties.

With regards to the tumoral migration of Tregs, cancers express a series of chemokines that promote the infiltration by these regulatory lymphocytes. For instance, chemokine CCL22 promotes the migration of Tregs into prostate and ovarian carcinomas.144 Human glioma cell lines have been reported to express CCL22 in addition to CCL2, although only CCL2 was secreted by samples from GBM patients.91 This has been investigated in the human glioma cell lines D-54, U-87, U-251 and LN-229 as well as in tumor cells from eight patients with GBM. The authors further reported that Tregs from these brain tumor patients had significantly higher expression of the CCL2 receptor CCR4 than the Tregs from healthy controls. Migration experiments have suggested that Treg migration is mediated by CCL2 and CCL22, which was blocked by antibodies to the chemokine receptors CCR2 and CCR4.

Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) were originally named natural suppressors because they functioned independently of MHC interactions and inhibited the antigen-induced proliferation of T cells and antibody production by B cells.145-147 MDSC were isolated from the bone marrow of tumor patients and named for their ability to suppress T-cell proliferation.148 They lack expression of surface molecules characteristic of T, B, macrophage, or NK cells. Myeloid-derived suppressor cells are immature myeloid cells that have the potential to generate mature granulocytes, macrophages and dendritic cells.149-151 In mice, they are identified based on co-expression


of CD11b and Gr-1.152,153 Human MDSC equivalents are CD34� CD33�, CD15- and CD13�, with variable expression of CD11c and HLA-DR and were originally identified in patients with head and neck cancer.154 Further studies of patients with tumors showed an increased population of myeloid-derived suppressor cells in secondary lymphoid organs, blood and tumor site. In fact, tumor-derived factors promoted MDSC recruitment and maturation into immunosuppressive cells and inhibited the differentiation of dendritic cells. The effect of tumor-derived factors was abrogated with neutralizing antibodies to IL-6 and colony stimulating factor-1 (CSF-1).155,156 Interestingly, glioma cells are known to produce both IL-6 and CSF-1, which may aid in the recruitment and/or expansion of MDSC in patients.

Regulatory T Cells

An increased fraction of Regulatory T cells (Tregs; CD4�CD25�FOXP3�) has been reported to infiltrate the tumor contributing to the immunodeficient status associated with glioma.4-6,29,157 Tregs are a fraction of the T-cell population that suppress immune activation and thereby maintain immune system homeostasis and tolerance to self-antigens. Functional deletion of Treg cells induce autoimmunity, facilitate transplantation tolerance and also increases immunity to tumors.5,158,159 A lack of immune rejection of neoplastic cells is believed to be maintained by Tregs in many malignancies including colorectal, esophageal, pancreatic, breast, lung, ovarian and brain tumors.5,160-163 It is therefore very important to understand the biology and function of Tregs for its potential therapeutic benefits.

Sakaguchi et al, demonstrated that a unique subset of CD4� T cells expressing CD25 (IL-2 receptor �-chain) in normal rodents displayed potent immunoregulatory functions in vitro and in vivo.164 Treg cells consist of 1-10% of total CD4� T cells in thymus, peripheral blood and lymphoid tissues and could conceivably recognize a wide spectrum of self and nonself antigens.165 Treg development takes place directly in the thymus and leave thymus as mature with defined phenotype.166 This is unlike the development of other T-cell subsets, which are induced upon antigen exposure.

They are dependent on IL-2 stimulation for their development, peripheral expansion and suppression function.164 Experimental mice which are deficient for IL-2 and IL-2R have a reduced pool of Treg cells and die prematurely from a severe lymphoproliferative and autoimmune syndrome.167-169 In IL-2R� knock-out mice, introduction of IL-2R� transgene, which is expressed predominantly in the thymus, CD4�CD25� T-cell development is restored indicating that an intact IL-2/IL-2R pathway is also required for thymic generation of Treg.170 In experimental mouse models, adoptive transfer of wild-type (WT) Treg cells prevents autoimmunity in mice lacking a functional IL-2R, but not in those lacking IL-2. These donor Treg cells undergo rapid and extensive IL-2-dependent proliferation in lymph nodes and spleen.168 These studies imply that IL-2 is a critical growth and differentiation factor for Treg cells.171 Blocking the IL-2R on Treg cells leads to a loss of their regulatory activity, suggesting a possible role for IL-2 for suppressor function.172 These studies show that IL-2 may be required in the production of Treg and alterations in this pathway may block Treg cell development. Functionally, IL-2 promotes proliferation and survival in T cells either by activating the signal transducer and activator of transcription 5 (STAT5) transcription factor or by up-regulation of antiapoptotic molecules, Bcl-2.173,174 Blocking IL-2 signaling in Treg cells does not disrupt an essential survival pathway, whereas abrogation of STAT5 show autoimmune pathology similar to IL-2 and IL-2R knock-out


mice and a decrease in number of CD4� CD25� Treg cells. Mice transgenic for the active form of STAT5 possess a greater frequency of Treg cells.175-177

Tregs express forkhead box P3 (FoxP3), a transcription factor that plays a central role in defining their function. FoxP3 is essentially expressed in CD4�CD25�CD8� thymocytes but not in any other thymic cells.178-180 FoxP3 deficient mice suffer from autoimmune symptoms and die from inflammatory diseases. However, this condition can be abrogated by adoptive transfer of CD4�CD25�FOXP3� cells from immune-competent mice.170 In humans, Type I diabetes, thyroiditis and inflammatory bowel diseases is associated with mutations on the FoxP3 gene.

Existence of other populations of T regulatory lymphocytes has been reported in recent publications. Unlike classic CD4�CD25� FoxP3� Tregs, these cells are induced in the periphery via T-cell receptor (TCR)-MHC/peptide stimulation. Qiao et al, described the existence of a different population of regulatory T cells called iTregs or induced regulatory T cells.181 These were developed from CD4�CD25� cells when exposed to CD4�CD25�FoxP3� Tregs and suppressive conditions like TGF-� and suppressed responder T-cell activation by cell-to-cell contact and secreted factors. These cells did not express Foxp3.181 Another example of nonclassical regulatory T cells are Tr1 cells which are induced in the periphery in a process that is dependent on IL-10 and interactions with immature dendritic cells which lack the expression of costimulatory molecules.182 These cells are characterized by the expression of CD4� CD25int/high and mediate suppression by secreting large amounts of IL-10. In contrast to Tregs, FoxP3 is not constitutively expressed in Tr1 cells.183 Third of this kind are Th3 cells, induced in the periphery through a TGF-� dependent process and these cells require IL-10 for expansion. Th3 cells suppress via the secretion of TGF-�.184 Both Tr1 and Th3 cells are implicated in oral tolerance.185 They play a role in neoplasia as a member of the tumor infiltrating lymphocytes from gastric cancer patients.161,186

The precise means by which Tregs suppress effector T-cell-mediated immune responses have not been definitively characterized. Some studies suggest roles of cytokines in their regulation and others support the contribution of cell-to-cell contact with effector T cells on APC, where membrane bound TGF-� and cytotoxic T-lymphocyte protein (CTLA-4) plays an important role.187-189 Heme oxygenase-1 (HO-1), a rate limiting enzyme in heme metabolism also plays a role in Treg mediated immune suppression. HO-1 is constitutively expressed in human Tregs and is induced by FoxP3 expression.190,191 It is suggested that HO-1 suppresses effector T cell by carbon-monoxide production.192,193

In 2006, a groundbreaking observation made by our laboratory demonstrated tumor infiltration of Tregs in glioblastoma multiforme (GBM) patients.4 The expression of FoxP3� Tregs was significantly higher in patients with GBM than in controls. A mean of 24.7% of Tregs among the glioma-infiltrating lymphocytes were observed, whereas these cells were absent from control brain specimens (p �0.01). Higher levels of FoxP3 expression were observed in regulatory T cells isolated from the tumor tissue (55.1%) in comparison to autologous patient blood (33.4%) and blood from control individuals (15.6%) (p �0.01). In an in vitro suppression assay Tregs inhibited T-cell proliferation in a dose-dependent manner. Among various markers analyzed, the expression of CD62L and CTLA-4 was elevated in the glioma infiltrating Tregs in comparison to that of the controls. Following depletion of Tregs with anti-CD25 monoclonal antibody, in mice with experimental brain tumors, we showed that an increased survival.

At the same time, another study was reported from the Heimberger laboratory at the M.D. Anderson Cancer Center.29 GBM patient tumors were analyzed for glioma-infiltrating


microglia/macrophage (GIM) and their effect on antitumor immune responses. The authors revealed that GIMs failed to induce T-cell proliferation. Although GIMs expressed MHC class II expression, they lacked costimulatory molecules CD86, CD80 and CD40 critical for T-cell activation. They demonstrated a corresponding lack of effector/activated T cells and there was a prominent population of Tregs (CD4�CD25�FoxP3�) infiltrating the tumor.

Fecci et al found that absolute counts of both CD4� T cells and FoxP3�CD45RO� Tregs were greatly diminished in the peripheral pool of patients with malignant glioma, but the Treg fraction was increased in the remaining CD4 compartment in 5 out of the 8 patients evaluated.6 The proportion of Tregs in the peripheral blood of patients with GBM was 2.63 times higher than that found in the blood of normal volunteers (p � 0.004). Interestingly, their experiments suggested that despite the reduction in their total number, the increased Treg fraction (p � 0.0003 versus controls) was sufficient to elicit the characteristic impairment of T-cell responsiveness in vitro. The patients with an elevated Treg fraction showed significant CD4� T-cell lymphopenia (p �0.0001), whereas the patients without Treg elevation possessed normally proliferating CD4� T-cell levels. T cells from the patients bearing malignant gliomas regained their function after Treg depletion in vitro and proliferated to levels equivalent to those of healthy controls.

Curtin et al observed that efficacy of immunotherapy using anti-CD25 depleting antibody (PC61), although glioma progression in experimental animals was dependent upon the tumor load.194 Systemic depletion of Tregs 15 days after tumor implantation improved long-term survival, but Tregs depleted 24 days after tumor implantation showed no improvement in survival. Immunotherapy was supplemented with intratumoral delivery of oncolytic adenovirus and herpes simplex Type 1, to further enhance tumor regression and long-time survival. It is very important to note that this observation suggests that immunotherapy in combination with Treg depeletion is a more effective therapeutic strategy, provided that Treg depletion occurs prior to immunotherapy and when tumor burden is low.

Heme Oxygenase-1

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme to biliverdin, carbon monoxide (CO) and free iron. There are three isoforms of mammalian HO that have been identified: HO-1, HO-2 and HO-3.195 HO-1 is inducible by a variety of stimuli, particularly oxidative stress.196,197 Induction of HO-1 expression has been associated with neuroprotection during hyperthermia in glial cells and during hypoxia.198,199 HO-1 expression has also been observed in a wide range of experimental diseases of the rodent brain such as traumatic brain injury, ischemia and human Alzheimer’s disease.200-202 In brain tumors, elevated HO-1 expression has been observed and in at least one report, it would appear that HO-1 accumulates during oligodenodroglioma progression.203-205

The precise function of HO-1 expression in T cells is not fully understood. Nevertheless, growing evidence has shown that human Tregs constitutively express HO-1 and that HO-1 inhibits T-cell proliferation.190 Over-expression of HO-1 renders T cells resistant to Fas-mediated apoptosis.206 Most recently, FoxP3 expression, which encodes a forkhead/winged-helix transcription repressor specifically expressed in Treg, has been shown to induce HO-1 expression.191 These studies suggest that HO-1 may be an important effector of FoxP3-mediated immune suppression and an important target for further clinical development.

Our group investigated the correlation between glioma infiltrating Tregs and tumor grade, as well as the expression of HO-1. Patients with Grade IV tumors (11.54%)


showed the highest level of FoxP3 expression versus Grade III (6.74%) or Grade II (2.53%) (p �0.05).207 Moreover, in Grade IV tumors, the frequency of HO-1 expressing Treg cells was 11.8 ��2.45% vs 7.42 ��0.31% in Grade III and 2.33 ��0.12% in Grade II. HO-1 accumulated during glioma progression and apparently, plays a role in FoxP3 mediated immune suppression. Tumor infiltrating Tregs stained positively with anti-HO-1 antibody and the expression of HO-1 correlated with CD4�CD25�FoxP3� infiltration (r � 0.966). These results suggested that the induction of HO-1 expression is linked to the expression of FoxP3 in glioma infiltrating Tregs. Collectively, this data supports the notion that HO-1 is a key suppressive factor for regulatory T cells during the growth of malignant brain tumors.

CONCLUSION

The anatomical location of glioma within the CNS has many advantages for tumor progression. First, the cytokine milieu of the CNS is intrinsically Th2 skewed in order to protect vital CNS structures from damage due to inflammation. Gliomas exploit the primarily humoral response to suppress cytotoxic responses, not only through the production of Th2 cytokines by tumor cells, but also by enhancing Th2 cytokine production by TIL. Second, entry into the CNS and immune cell activation are tightly regulated. In general, gliomas are under less immune surveillance than tumors exposed to the peripheral immune compartment. Furthermore, the predominant effector cells in glioma appear to be suppressor cells, Tregs and myeloid-derived suppressor cells. These suppressor cells act to prevent cytotoxic responses mediated by NK cells and CTL. The efficient immune evasion and suppression mediated by glioma suggests that novel strategies to overcome these mechanisms may be beneficial for the treatment of this disease.

In light of the studies of CNS immunobiology and gliomas, more selective immunotherapeutic strategies have emerged and have resulted in moderate clinical successes. Gliomas not only recruit, but also aid in the expansion, of suppressor cells. In studies where these chemotaxic factors were blocked, either by chemotherapeutic drugs or blocking antibodies, immune cell recruitment was also inhibited.91 In addition, TGF� has been implicated in the expansion of Tregs and has been shown to be overexpressed in gliomas. Antisense TGF� ODN, (AP 12009) has been used successfully to inhibit TGF� expression in primary glioma samples, in vitro.208 Additionally, small molecule inhibitors of TGF� signaling effectively block signaling in glioma cells209 and immune cells93 and restore immune surveillance in animal models of glioma. There are dual therapeutic benefits of inhibiting chemokines and cytokines in gliomas, not only inhibiting glioma cell proliferation, but also reducing the recruitment of immunosuppressive cells into tumors, thus preventing immunosuppressive mechanisms.

Another novel strategy that has emerged is the depletion of Tregs in tumor patients. Treg depletion strategies have been successful in the treatment of ovarian cancer. Using ONTAK (IL-2 fused to diptheria toxin),210 Barnett and colleagues demonstrated the selective depletion of Tregs, with a concomitant increase of IFN�� effector cells.211 Our laboratory and others have shown that the depeletion of Tregs improves the surivival of glioma bearing mice.5,157 Recently, Grauer and colleagues illustrated that precisely timed Treg depletion may enhance adoptive immunotherapy for the treatment of glioma.212 Therefore, whether used alone or in combination with other immunotherapeutic strategies, Treg depletion may be critical for the treatment of glioma.

Despite the fact that effector cells have been detected in glioma tumor lesions, these cells are unable to eradicate the tumor. In light of this, vaccination strategies aimed at increasing the frequency of tumor antigen-specific T cells have been developed. One such


vaccine activates the T cells which recognize the tumor specific antigen, EGFRviii.213 In clinical trials, the EGFRviii vaccine increased the time to progression and improved the mean survival in glioma patients.214 Additionally, DC-based vaccines are currently being evaluated in glioma patients. Early results from the clinical trials showed improved patient survival over historical norms. Although there is great potential for vaccination strategies, more studies are necessary before concrete benefits are realized. An improved understanding of the myriad of immunosuppressive mechanisms in play during glioma progression will lead to the development of optimal treatment strategies and with hope, should improve the prognosis of glioma patients.

REFERENCES

1. Medawar PB. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29:58-69.

2. Freed WJ, Dymecki J, Poltorak M et al. Intraventricular brain allografts and xenografts: studies of survival and rejection with and without systemic sensitization. Prog Brain Res 1988; 78:233-241.

3. Tsugawa T, Kuwashima N, Sato H et al. Sequential delivery of interferon-alpha gene and DCs to intracranial gliomas promotes an effective antitumor response. Gene Ther 2004; 11:1551-1558.

4. El Andaloussi A, Lesniak MS. An increase in CD4�CD25�FOXP3� regulatory T-cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme. Neuro Oncol 2006; 8:234-243.

5. El Andaloussi A, Han Y, Lesniak MS. Prolongation of survival following depletion of CD4�CD25� regulatory T-cells in mice with experimental brain tumors. J Neurosurg 2006; 105:430-437.

6. Fecci PE, Mitchell DA, Whitesides JF et al. Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma. Cancer Res 2006; 66:3294-3302.

7. Heimberger AB, Abou-Ghazal M, Reina-Ortiz C et al. Incidence and prognostic impact of FoxP3� regulatory T-cells in human gliomas. Clin Cancer Res 2008; 14:5166-5172.

8. Facoetti A, Nano R, Zelini P et al. Human leukocyte antigen and antigen processing machinery component defects in astrocytic tumors. Clin Cancer Res 2005; 11:8304-8311.

9. Mehling M, Simon P, Mittelbronn M et al. WHO grade associated downregulation of MHC class I antigen-processing machinery components in human astrocytomas: does it reflect a potential immune escape mechanism? Acta Neuropathol 2007; 114:111-119.

10. Zuber P, Kuppner MC, De Tribolet N. Transforming growth factor-beta 2 down-regulates HLA-DR antigen expression on human malignant glioma cells. Eur J Immunol 1988; 18:1623-1626.

11. Rossi ML, Esiri MM, Jones NR et al. Characterization of the mononuclear cell infiltrate and HLA-Dr expression in 19 oligodendrogliomas. Surg Neurol 1991; 36:119-125.

12. Rossi ML, Jones NR, Karr GF et al. HLA-Dr expression by tumor cells compared with survival in high grade astrocytomas. Tumori 1991; 77:122-125.

13. Mouillot G, Marcou C, Rousseau P et al. HLA-G gene activation in tumor cells involves cis-acting epigenetic changes. Int J Cancer 2005; 113:928-936.

14. Rebmann V, Regel J, Stolke D et al. Secretion of sHLA-G molecules in malignancies. Semin Cancer Biol 2003; 13:371-377.

15. Pistoia V, Morandi F, Wang X et al. Soluble HLA-G: Are they clinically relevant? Semin Cancer Biol 2007; 17:469-479.

16. Wiendl H, Mitsdoerffer M, Hofmeister V et al. A functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape. J Immunol 2002; 168:4772-4780.

17. Wischhusen J, Friese MA, Mittelbronn M et al. HLA-E protects glioma cells from NKG2D-mediated immune responses in vitro: implications for immune escape in vivo. J Neuropathol Exp Neurol 2005; 64:523-528.

18. Mittelbronn M, Simon P, Loffler C et al. Elevated HLA-E levels in human glioblastomas but not in grade I to III astrocytomas correlate with infiltrating CD8� cells. J Neuroimmunol 2007; 189:50-58.

19. Guillemin GJ, Brew BJ. Microglia, macrophages, perivascular macrophages and pericytes: a review of function and identification. J Leukoc Biol 2004; 75:388-397.

20. Penfield W. Microglia and the process of phagocytosis in gliomas. Am J Path 1925; 1:77-97.21. Rossi ML, Hughes JT, Esiri MM et al. Immunohistological study of mononuclear cell infiltrate in malignant

gliomas. Acta Neuropathol 1987; 74:269-277.22. Rossi ML, Jones NR, Candy E et al. The mononuclear cell infiltrate compared with survival in high-grade

astrocytomas. Acta Neuropathol 1989; 78:189-193.


23. Parney IF, Waldron JS, Parsa AT. Flow cytometry and in vitro analysis of human glioma-associated macrophages. J Neurosurg. 2009 Mar;110(3):572-82.

24. Roggendorf W, Strupp S, Paulus W. Distribution and characterization of microglia/macrophages in human brain tumors. Acta Neuropathol 1996; 92:288-293.

25. Leung SY, Wong MP, Chung LP et al. Monocyte chemoattractant protein-1 expression and macrophage infiltration in gliomas. Acta Neuropathol 1997; 93:518-527.

26. Platten M, Kretz A, Naumann U et al. Monocyte chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas. Ann Neurol 2003; 54:388-392.

27. Aloisi F, Ria F, Penna G et al. Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 cell activation. J Immunol 1998; 160:4671-4680.

28. Flugel A, Labeur MS, Grasbon-Frodl EM et al. Microglia only weakly present glioma antigen to cytotoxic T-cells. Int J Dev Neurosci 1999; 17:547-556.

29. Hussain SF, Yang D, Suki D et al. The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses. Neuro Oncol 2006; 8:261-279.

30. Huettner C, Paulus W, Roggendorf W. Messenger RNA expression of the immunosuppressive cytokine IL-10 in human gliomas. Am J Pathol 1995; 146:317-322.

31. Goswami S, Gupta A, Sharma SK. Interleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MG. J Neurochem 1998; 71:1837-1845.

32. Hu YS, Zhang QL, Tian ZG et al. [Significance of the unbalanced expression of Th1/Th2 type cytokines in human glioma]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2001; 23:594-598.

33. Li G, Hu YS, Li XG et al. Expression and switching of TH1/TH2 type cytokines gene in human gliomas. Chin Med Sci J 2005; 20:268-272.

34. Roussel E, Gingras MC, Grimm EA et al. Predominance of a type 2 intratumoural immune response in fresh tumour-infiltrating lymphocytes from human gliomas. Clin Exp Immunol 1996; 105:344-352.

35. Huettner C, Czub S, Kerkau S et al. Interleukin 10 is expressed in human gliomas in vivo and increases glioma cell proliferation and motility in vitro. Anticancer Res 1997; 17:3217-3224.

36. Hishii M, Nitta T, Ishida H et al. Human glioma-derived interleukin-10 inhibits antitumor immune responses in vitro. Neurosurgery 1995; 37:1160-1166; discussion 1166-1167.

37. Wagner S, Czub S, Greif M et al. Microglial/macrophage expression of interleukin 10 in human glioblastomas. Int J Cancer1999; 82:12-16.

38. Samaras V, Piperi C, Korkolopoulou P et al. Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors. Mol Cell Biochem 2007; 304:343-351.

39. Murphy-Ullrich JE. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J Clin Invest 2001; 107:785-790.

40. Sage EH. Regulation of interactions between cells and extracellular matrix: a command performance on several stages. J Clin Invest 2001; 107:781-783.

41. Erickson HP. Gene knockouts of c-src, transforming growth factor beta 1 and tenascin suggest superfluous, nonfunctional expression of proteins. J Cell Biol 1993; 120:1079-1081.

42. Erickson HP. A tenascin knockout with a phenotype. Nat Genet 1997; 17:5-7.43. Jones PL, Jones FS. Tenascin-C in development and disease: gene regulation and cell function. Matrix

Biol 2000; 19:581-596.44. Hemesath TJ, Marton LS, Stefansson K. Inhibition of T-cell activation by the extracellular matrix protein

tenascin. J Immunol 1994; 152:5199-5207.45. Puente Navazo MD, Valmori D, Ruegg C. The alternatively spliced domain TnFnIII A1A2 of the

extracellular matrix protein tenascin-C suppresses activation-induced T-lymphocyte proliferation and cytokine production. J Immunol 2001; 167:6431-6440.

46. Parekh K, Ramachandran S, Cooper J et al. Tenascin-C, over expressed in lung cancer down regulates effector functions of tumor infiltrating lymphocytes. Lung Cancer 2005; 47:17-29.

47. Bodmer S, Strommer K, Frei K et al. Immunosuppression and transforming growth factor-beta in glioblastoma. Preferential production of transforming growth factor-beta 2. J Immunol 1989; 143:3222-3229.

48. Tada T, Yabu K, Kobayashi S. Detection of active form of transforming growth factor-beta in cerebrospinal fluid of patients with glioma. Jpn J Cancer Res 1993; 84:544-548.

49. de Martin R, Haendler B, Hofer-Warbinek R et al. Complementary DNA for human glioblastoma-derived T-cell suppressor factor, a novel member of the transforming growth factor-beta gene family. EMBO J 1987; 6:3673-3677.

50. Wrann M, Bodmer S, de Martin R et al. T-cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta. EMBO J 1987; 6:1633-1636.

51. Eberhart CE, Coffey RJ, Radhika A et al. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994; 107:1183-1188.


52. Wolff H, Saukkonen K, Anttila S et al. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res 1998; 58:4997-5001.

53. Mohammed SI, Knapp DW, Bostwick DG et al. Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res 1999; 59:5647-5650.

54. Shono T, Tofilon PJ, Bruner JM et al. Cyclooxygenase-2 expression in human gliomas: prognostic significance and molecular correlations. Cancer Res 2001; 61:4375-4381.

55. Joki T, Heese O, Nikas DC et al. Expression of cyclooxygenase 2 (COX-2) in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS-398. Cancer Res 2000; 60:4926-4931.

56. Phipps RP, Stein SH, Roper RL. A new view of prostaglandin E regulation of the immune response. Immunol Today 1991; 12:349-352.

57. Roper RL, Phipps RP. Prostaglandin E2 regulation of the immune response. Adv Prostaglandin Thromboxane Leukot Res 1994; 22:101-111.

58. Kunkel SL, Spengler M, May MA et al. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression. J Biol Chem 1988; 263:5380-5384.

59. van der Pouw Kraan TC, Boeije LC, Smeenk RJ et al. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 1995; 181:775-779.

60. Harizi H, Juzan M, Grosset C et al. Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells. Cell Immunol 2001; 209:19-28.

61. Williams JA, Shacter E. Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. J Biol Chem 1997; 272:25693-25699.

62. Strassmann G, Patil-Koota V, Finkelman F et al. Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2. J Exp Med 1994; 180:2365-2370.

63. Fiorentino DF, Zlotnik A, Vieira P et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J Immunol 1991; 146:3444-3451.

64. Harizi H, Juzan M, Pitard V et al. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production of endogenous IL-10, which down-regulates dendritic cell functions. J Immunol 2002; 168:2255-2263.

65. Akasaki Y, Liu G, Chung NH et al. Induction of a CD4� T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma. J Immunol 2004; 173:4352-4359.

66. Kokoglu E, Tuter Y, Sandikci KS et al. Prostaglandin E2 levels in human brain tumor tissues and arachidonic acid levels in the plasma membrane of human brain tumors. Cancer Lett 1998; 132:17-21.

67. Loh JK, Hwang SL, Lieu AS et al. The alteration of prostaglandin E2 levels in patients with brain tumors before and after tumor removal. J Neurooncol 2002; 57:147-150.

68. Rozic JG, Chakraborty C, Lala PK. Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis. Int J Cancer 2001; 93:497-506.

69. Attiga FA, Fernandez PM, Weeraratna AT et al. Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases. Cancer Res 2000; 60:4629-4637.

70. Kundu N, Fulton AM. Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease in a murine model of breast cancer. Cancer Res 2002; 62:2343-2346.

71. Connolly EM, Harmey JH, O’Grady T et al. Cyclo-oxygenase inhibition reduces tumour growth and metastasis in an orthotopic model of breast cancer. Br J Cancer 2002; 87:231-237.

72. Lalier L, Cartron PF, Pedelaborde F et al. Increase in PGE2 biosynthesis induces a Bax dependent apoptosis correlated to patients’ survival in glioblastoma multiforme. Oncogene 2007; 26:4999-5009.

73. Chouaib S, Bertoglio JH. Prostaglandins E as modulators of the immune response. Lymphokine Res Fall 1988; 7:237-245.

74. Goodwin JS, Ceuppens J. Regulation of the immune response by prostaglandins. J Clin Immunol 1983; 3:295-315.

75. Rappaport RS, Dodge GR. Prostaglandin E inhibits the production of human interleukin 2. J Exp Med 1982; 155:943-948.

76. Kuppner MC, Sawamura Y, Hamou MF et al. Influence of PGE2- and cAMP-modulating agents on human glioblastoma cell killing by interleukin-2-activated lymphocytes. J Neurosurg 1990; 72:619-625.

77. Lauro GM, Di Lorenzo N, Grossi M et al. Prostaglandin E2 as an immunomodulating factor released in vitro by human glioma cells. Acta Neuropathol 1986; 69:278-282.

78. Sawamura Y, Diserens AC, de Tribolet N. In vitro prostaglandin E2 production by glioblastoma cells and its effect on interleukin-2 activation of oncolytic lymphocytes. J Neurooncol 1990; 9:125-130.

79. Nakano Y, Kuroda E, Kito T et al. Induction of macrophagic prostaglandin E2 synthesis by glioma cells. J Neurosurg 2006; 104:574-582.

80. Nakano Y, Kuroda E, Kito T et al. Induction of prostaglandin E2 synthesis and microsomal prostaglandin E synthase-1 expression in murine microglia by glioma-derived soluble factors. Laboratory investigation. J Neurosurg 2008; 108:311-319.


81. Anderson RC, Anderson DE, Elder JB et al. Lack of B7 expression, not human leukocyte antigen expression, facilitates immune evasion by human malignant gliomas. Neurosurgery 2007; 60(6):1129-1136; discussion 1136.

82. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002; 109(9):1139-1142.83. Brantley EC, Benveniste EN. Signal transducer and activator of transcription-3: a molecular hub for signaling

pathways in gliomas. Mol Cancer Res 2008; 6(5):675-684.84. Chung CD, Liao J, Liu B et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science 1997;

278(5344):1803-1805.85. Pillemer BB, Xu H, Oriss TB et al. Deficient SOCS3 expression in CD4�CD25�FoxP3� regulatory T-cells

and SOCS3-mediated suppression of Treg function. Eur J Immunol 2007; 37(8):2082-2089.86. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour

microenvironment. Nat Rev Immunol 2007; 7(1):41-51.87. Wang T, Niu G, Kortylewski M et al. Regulation of the innate and adaptive immune responses by STAT3

signaling in tumor cells. Nat Med 2004; 10(1):48-54.88. Kortylewski M, Kujawski M, Wang T et al. Inhibiting Stat3 signaling in the hematopoietic system elicits

multicomponent antitumor immunity. Nat Med 2005; 11(12):1314-1321.89. Almand B, Resser JR, Lindman B et al. Clinical significance of defective dendritic cell differentiation in

cancer. Clin Cancer Res 2000; 6(5):1755-1766.90. Kasprzycka M, Marzec M, Liu X et al. Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK)

oncoprotein induces the T regulatory cell phenotype by activating STAT3. Proc Natl Acad Sci USA 2006; 103(26):9964-9969.

91. Jordan JT, Sun W, Hussain SF et al. Preferential migration of regulatory T-cells mediated by glioma-secreted chemokines can be blocked with chemotherapy. Cancer Immunol Immunother 2008; 57(1):123-131.

92. Abou-Ghazal M, Yang DS, Qiao W et al. The incidence, correlation with tumor-infiltrating inflammation and prognosis of phosphorylated STAT3 expression in human gliomas. Clin Cancer Res 2008; 14(24):8228-8235.

93. Hussain SF, Kong LY, Jordan J et al. A novel small molecule inhibitor of signal transducers and activators of transcription 3 reverses immune tolerance in malignant glioma patients. Cancer Res 2007; 67(20):9630-9636.

94. Ichikawa M, Chen L. Role of B7-H1 and B7-H4 molecules in down-regulating effector phase of T-cell immunity: novel cancer escaping mechanisms. Front Biosci 2005; 10:2856-2860.

95. Ghebeh H, Mohammed S, Al-Omair A et al. The B7-H1 (PD-L1) T-lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia 2006; 8(3):190-198.

96. Tsushima F, Tanaka K, Otsuki N et al. Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma. Oral Oncol 2006; 42(3):268-274.

97. Strome SE, Dong H, Tamura H et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma. Cancer Res 2003; 63(19):6501-6505.

98. Wintterle S, Schreiner B, Mitsdoerffer M et al. Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis. Cancer Res 2003; 63(21):7462-7467.

99. Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity. J Mol Med 2003; 81(5):281-287.

100. Tamura H, Dong H, Zhu G et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood 2001; 97(6):1809-1816.

101. Dong H, Strome SE, Salomao DR et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002; 8(8):793-800.

102. Hirano F, Kaneko K, Tamura H et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res 2005; 65(3):1089-1096.

103. Blank C, Brown I, Peterson AC et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T-cell receptor (TCR) transgenic CD8� T-cells. Cancer Res 2004; 64(3):1140-1145.

104. Blank C, Kuball J, Voelkl S et al. Blockade of PD-L1 (B7-H1) augments human tumor-specific T-cell responses in vitro. Int J Cancer 2006; 119(2):317-327.

105. Iwai Y, Ishida M, Tanaka Y et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci USA 2002; 99(19):12293-12297.

106. Blank C, Gajewski TF, Mackensen A. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T-cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol Immunother 2005; 54(4):307-314.

107. Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 1986; 319(6055):675-678.

108. Diefenbach A, Jensen ER, Jamieson AM et al. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 2001; 413(6852):165-171.


109. Cerwenka A, Baron JL, Lanier LL. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc Natl Acad Sci USA 2001; 98(20):11521-11526.

110. Dix AR, Brooks WH, Roszman TL et al. Immune defects observed in patients with primary malignant brain tumors. J Neuroimmunol 1999; 100(1-2):216-232.

111. Braun DP, Penn RD, Harris JE. Regulation of natural killer cell function by glass-adherent cells in patients with primary intracranial malignancies. Neurosurgery 1984; 15(1):29-33.

112. Proescholdt MA, Merrill MJ, Ikejiri B et al. Site-specific immune response to implanted gliomas. J Neurosurg 2001; 95(6):1012-1019.

113. Aldemir H, Prod’homme V, Dumaurier MJ et al. Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor. J Immunol 2005; 175(12):7791-7795.

114. Roth P, Mittelbronn M, Wick W et al. Malignant glioma cells counteract antitumor immune responses through expression of lectin-like transcript-1. Cancer Res 2007; 67(8):3540-3544.

115. Boomer JS, Derks RA, Lee GW et al. Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis. Hum Immunol 2001; 62(6):577-588.

116. Roth P, Aulwurm S, Gekel I et al. Regeneration and tolerance factor: a novel mediator of glioblastoma-associated immunosuppression. Cancer Res 2006; 66(7):3852-3858.

117. Tachibana O, Nakazawa H, Lampe J et al. Expression of Fas/APO-1 during the progression of astrocytomas. Cancer Res 1995; 55(23):5528-5530.

118. Gratas C, Tohma Y, Van Meir EG et al. Fas ligand expression in glioblastoma cell lines and primary astrocytic brain tumors. Brain Pathol 1997; 7(3):863-869.

119. Saas P, Walker PR, Hahne M et al. Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? J Clin Invest 1997; 99(6):1173-1178.

120. Didenko VV, Ngo HN, Minchew C et al. Apoptosis of T-lymphocytes invading glioblastomas multiforme: a possible tumor defense mechanism. J Neurosurg 2002; 96(3):580-584.

121. Roth W, Isenmann S, Nakamura M et al. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 2001; 61(6):2759-2765.

122. Hintzen RQ, Lens SM, Koopman G et al. CD70 represents the human ligand for CD27. Int Immunol 1994; 6(3):477-480.

123. Sugita K, Hirose T, Rothstein DM et al. CD27, a member of the nerve growth factor receptor family, is preferentially expressed on CD45RA� CD4 T-cell clones and involved in distinct immunoregulatory functions. J Immunol 1992; 149(10):3208-3216.

124. Hendriks J, Gravestein LA, Tesselaar K et al. CD27 is required for generation and long-term maintenance of T-cell immunity. Nat Immunol 2000; 1(5):433-440.

125. La Rosa FG, Adams FS, Krause GE et al. Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations. Cancer Lett 1997; 113(1-2):55-60.

126. Tesselaar K, Arens R, van Schijndel GM et al. Lethal T-cell immunodeficiency induced by chronic costimulation via CD27-CD70 interactions. Nat Immunol 2003; 4(1):49-54.

127. Koenen HJ, Fasse E, Joosten I. CD27/CFSE-based ex vivo selection of highly suppressive alloantigen-specific human regulatory T-cells. J Immunol 2005; 174(12):7573-7583.

128. Chahlavi A, Rayman P, Richmond AL et al. Glioblastomas induce T-lymphocyte death by two distinct pathways involving gangliosides and CD70. Cancer Res 2005; 65(12):5428-5438.

129. Kudo D, Rayman P, Horton C et al. Gangliosides expressed by the renal cell carcinoma cell line SK-RC-45 are involved in tumor-induced apoptosis of T-cells. Cancer Res 2003; 63(7):1676-1683.

130. Ledeen RW, Wu G. Nuclear lipids: key signaling effectors in the nervous system and other tissues. J Lipid Res 2004; 45(1):1-8.

131. Hedberg KM, Mahesparan R, Read TA et al. The glioma-associated gangliosides 3�-isoLM1, GD3 and GM2 show selective area expression in human glioblastoma xenografts in nude rat brains. Neuropathol Appl Neurobiol 2001; 27(6):451-464.

132. Markowska-Woyciechowska A, Bronowicz A, Ugorski M et al. Study on ganglioside composition in brain tumours supra- and infratentorial. Neurol Neurochir Pol 2000; 34(6 Suppl):124-130.

133. Mennel HD, Bosslet K, Geissel H et al. Immunohistochemically visualized localisation of gangliosides Glac2 (GD3) and Gtri2 (GD2) in cells of human intracranial tumors. Exp Toxicol Pathol 2000; 52(4):277-285.

134. Wagener R, Rohn G, Schillinger G et al. Ganglioside profiles in human gliomas: quantification by microbore high performance liquid chromatography and correlation to histomorphology and grading. Acta Neurochir (Wien) 1999; 141(12):1339-1345.

135. Ward SG, Westwick J. Chemokines: understanding their role in T-lymphocyte biology. Biochem J 1998; 333(Pt3):457-470.


136. Mantovani A. Chemokines. Introduction and overview. Chem Immunol 1999; 72:1-6.137. Mantovani A, Allavena P, Sozzani S et al. Chemokines in the recruitment and shaping of the leukocyte

infiltrate of tumors. Semin Cancer Biol 2004; 14:155-160.138. Rollins BJ. Chemokines. Blood 1997; 90:909-928.139. Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001; 2:108-115.140. Rollins BJ. Inflammatory chemokines in cancer growth and progression. Eur J Cancer 2006; 42:760-767.141. Zhou Y, Larsen PH, Hao C et al. CXCR4 is a major chemokine receptor on glioma cells and mediates

their survival. J Biol Chem 2002; 277:49481-49487.142. Li M, Ransohoff RM. Multiple roles of chemokine CXCL12 in the central nervous system: a migration

from immunology to neurobiology. Prog Neurobiol 2008; 84:116-131.143. Choi C, Xu X, Oh JW et al. Fas-induced expression of chemokines in human glioma cells: involvement

of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Cancer Res 2001; 61:3084-3091.

144. Curiel TJ, Coukos G, Zou L et al. Specific recruitment of regulatory T-cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004; 10:942-949.

145. Mazzoni A, Bronte V, Visintin A et al. Myeloid suppressor lines inhibit T-cell responses by an NO-dependent mechanism. J Immunol 2002; 168:689-695.

146. Duwe AK, Singhal SK. The immunoregulatory role of bone marrow. II. Characterization of a suppressor cell inhibiting the in vitro antibody response. Cell Immunol 1979; 43:372-381.

147. Duwe AK, Singhal SK. The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow. Cell Immunol 1979; 43:362-371.

148. Young MR, Newby M, Wepsic HT. Hematopoiesis and suppressor bone marrow cells in mice bearing large metastatic Lewis lung carcinoma tumors. Cancer Res 1987; 47:100-105.

149. Bronte V, Kasic T, Gri G et al. Boosting antitumor responses of T-lymphocytes infiltrating human prostate cancers. J Exp Med 2005; 201:1257-1268.

150. Kusmartsev S, Cheng F, Yu B et al. All-trans-retinoic acid eliminates immature myeloid cells from tumor-bearing mice and improves the effect of vaccination. Cancer Res 2003; 63:4441-4449.

151. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species. J Leukoc Biol 2003; 74:186-196.

152. Bronte V, Apolloni E, Cabrelle A et al. Identification of a CD11b(�)/Gr-1(�)/CD31(�) myeloid progenitor capable of activating or suppressing CD8(�) T-cells. Blood 2000; 96:3838-3846.

153. Gabrilovich DI, Bronte V, Chen SH et al. The terminology issue for myeloid-derived suppressor cells. Cancer Res 2007; 67:425; author reply 426.

154. Pak AS, Wright MA, Matthews JP et al. Mechanisms of immune suppression in patients with head and neck cancer: presence of CD34(�) cells which suppress immune functions within cancers that secrete granulocyte-macrophage colony-stimulating factor. Clin Cancer Res 1995; 1:95-103.

155. Lin EY, Gouon-Evans V, Nguyen AV et al. The macrophage growth factor CSF-1 in mammary gland development and tumor progression. J Mammary Gland Biol Neoplasia 2002; 7:147-162.

156. Menetrier-Caux C, Montmain G, Dieu MC et al. Inhibition of the differentiation of dendritic cells from CD34(�) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. Blood 1998; 92:4778-4791.

157. Grauer OM, Nierkens S, Bennink E et al. CD4�FoxP3� regulatory T-cells gradually accumulate in gliomas during tumor growth and efficiently suppress antiglioma immune responses in vivo. Int J Cancer 2007; 121:95-105.

158. Yong Z, Chang L, Mei YX et al. Role and mechanisms of CD4�CD25� regulatory T-cells in the induction and maintenance of transplantation tolerance. Transpl Immunol 2007; 17:120-129.

159. Brusko TM, Putnam AL, Bluestone JA. Human regulatory T-cells: role in autoimmune disease and therapeutic opportunities. Immunol Rev 2008; 223:371-390.

160. Ling KL, Pratap SE, Bates GJ et al. Increased frequency of regulatory T-cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun 2007; 7:7.

161. Ichihara F, Kono K, Takahashi A et al. Increased populations of regulatory T-cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res 2003; 9:4404-4408.

162. Liyanage UK, Moore TT, Joo HG et al. Prevalence of regulatory T-cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 2002; 169:2756-2761.

163. Okita R, Saeki T, Takashima S et al. CD4�CD25� regulatory T-cells in the peripheral blood of patients with breast cancer and nonsmall cell lung cancer. Oncol Rep 2005; 14:1269-1273.


164. Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T-cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155:1151-1164.

165. Toda A, Piccirillo CA. Development and function of naturally occurring CD4�CD25� regulatory T-cells. J Leukoc Biol 2006; 80:458-470.

166. Itoh M, Takahashi T, Sakaguchi N et al. Thymus and autoimmunity: production of CD25�CD4� naturally anergic and suppressive T-cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol 1999; 162:5317-5326.

167. Schimpl A, Berberich I, Kneitz B et al. IL-2 and autoimmune disease. Cytokine Growth Factor Rev 2002; 13:369-378.

168. Bayer AL, Yu A, Adeegbe D et al. Essential role for interleukin-2 for CD4(�)CD25(�) T regulatory cell development during the neonatal period. J Exp Med 2005; 201:769-777.

169. Bayer AL, Yu A, Malek TR. Function of the IL-2R for thymic and peripheral CD4�CD25� Foxp3� T regulatory cells. J Immunol 2007; 178:4062-4071.

170. Malek TR, Yu A, Vincek V et al. CD4 regulatory T-cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 2002; 17:167-178.

171. Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol 2003; 74:961-965.

172. de la Rosa M, Rutz S, Dorninger H et al. Interleukin-2 is essential for CD4�CD25� regulatory T-cell function. Eur J Immunol 2004; 34:2480-2488.

173. Moriggl R, Topham DJ, Teglund S et al. Stat5 is required for IL-2-induced cell cycle progression of peripheral T-cells. Immunity 1999; 10:249-259.

174. Van Parijs L, Refaeli Y, Lord JD et al. Uncoupling IL-2 signals that regulate T-cell proliferation, survival and Fas-mediated activation-induced cell death. Immunity 1999; 11:281-288.

175. Antov A, Yang L, Vig M et al. Essential role for STAT5 signaling in CD25�CD4� regulatory T-cell homeostasis and the maintenance of self-tolerance. J Immunol 2003; 171:3435-3441.

176. Snow JW, Abraham N, Ma MC et al. Loss of tolerance and autoimmunity affecting multiple organs in STAT5A/5B-deficient mice. J Immunol 2003; 171:5042-5050.

177. Burchill MA, Goetz CA, Prlic M et al. Distinct effects of STAT5 activation on CD4� and CD8� T-cell homeostasis: development of CD4�CD25� regulatory T-cells versus CD8� memory T-cells. J Immunol 2003; 171:5853-5864.

178. Hori S, Nomura T, Sakaguchi S. Control of regulatory T-cell development by the transcription factor Foxp3. Science 2003; 299:1057-1061.

179. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4�CD25� regulatory T-cells. Nat Immunol 2003; 4:330-336.

180. Yagi H, Nomura T, Nakamura K et al. Crucial role of FOXP3 in the development and function of human CD25�CD4� regulatory T-cells. Int Immunol 2004; 16(:1643-1656.

181. Qiao M, Thornton AM, Shevach EM. CD4� CD25� [corrected] regulatory T-cells render naive CD4� CD25� T-cells anergic and suppressive. Immunology 2007; 120:447-455.

182. Sundstedt A, O’Neill EJ, Nicolson KS et al. Role for IL-10 in suppression mediated by peptide-induced regulatory T-cells in vivo. J Immunol 2003; 170:1240-1248.

183. Vieira PL, Christensen JR, Minaee S et al. IL-10-secreting regulatory T-cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4�CD25� regulatory T-cells. J Immunol 2004; 172:5986-5993.

184. Miller A, Lider O, Roberts AB et al. Suppressor T-cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering. Proc Natl Acad Sci USA 1992; 89:421-425.

185. Zhang X, Izikson L, Liu L et al. Activation of CD25(�)CD4(�) regulatory T-cells by oral antigen administration. J Immunol 2001; 167:4245-4253.

186. Levings MK, Gregori S, Tresoldi E et al. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25�CD4� Tr cells. Blood 2005; 105:1162-1169.

187. Takahashi Y, Onda M, Tanaka N et al. Establishment and characterization of two new rectal neuroendocrine cell carcinoma cell lines. Digestion 2000; 62:262-270.

188. Manzotti CN, Tipping H, Perry LC et al. Inhibition of human T-cell proliferation by CTLA-4 utilizes CD80 and requires CD25� regulatory T-cells. Eur J Immunol 2002; 32:2888-2896.

189. Tang Q, Boden EK, Henriksen KJ et al. Distinct roles of CTLA-4 and TGF-beta in CD4�CD25� regulatory T-cell function. Eur J Immunol 2004; 34:2996-3005.

190. Pae HO, Oh GS, Choi BM et al. Differential expressions of heme oxygenase-1 gene in CD25- and CD25� subsets of human CD4� T-cells. Biochem Biophys Res Commun 2003; 306:701-705.


191. Choi BM, Pae HO, Jeong YR et al. Critical role of heme oxygenase-1 in Foxp3-mediated immune suppression. Biochem Biophys Res Commun 2005; 327:1066-1071.

192. Pae HO, Oh GS, Choi BM et al. Carbon monoxide produced by heme oxygenase-1 suppresses T-cell proliferation via inhibition of IL-2 production. J Immunol 2004; 172:4744-4751.

193. Song R, Zhou Z, Kim PK et al. Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells. J Biol Chem 2004; 279:44327-44334.

194. Curtin JF, Candolfi M, Fakhouri TM et al. Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials. PLoS ONE 2008; 3:e1983.

195. Maines MD, Polevoda B, Coban T et al. Neuronal overexpression of heme oxygenase-1 correlates with an attenuated exploratory behavior and causes an increase in neuronal NADPH diaphorase staining. J Neurochem 1998; 70:2057-2069.

196. Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci USA 1987; 84:5918-5922.

197. Stocker R, Yamamoto Y, McDonagh AF et al. Bilirubin is an antioxidant of possible physiological importance. Science 1987; 235:1043-1046.

198. Ewing JF, Haber SN, Maines MD. Normal and heat-induced patterns of expression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein. J Neurochem 1992; 58:1140-1149.

199. Panahian N, Yoshiura M, Maines MD. Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice. J Neurochem 1999; 72:1187-1203.

200. Fukuda K, Panter SS, Sharp FR et al. Induction of heme oxygenase-1 (HO-1) after traumatic brain injury in the rat. Neurosci Lett 1995; 199:127-130.

201. Nimura T, Weinstein PR, Massa SM et al. Heme oxygenase-1 (HO-1) protein induction in rat brain following focal ischemia. Brain Res Mol Brain Res 1996; 37:201-208.

202. Schipper HM, Cisse S, Stopa EG. Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann Neurol 1995; 37:758-768.

203. Hara E, Takahashi K, Tominaga T et al. Expression of heme oxygenase and inducible nitric oxide synthase mRNA in human brain tumors. Biochem Biophys Res Commun 1996; 224:153-158.

204. Nishie A, Ono M, Shono T et al. Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas. Clin Cancer Res 1999; 5:1107-1113.

205. Deininger MH, Meyermann R, Trautmann K et al. Heme oxygenase (HO)-1 expressing macrophages/microglial cells accumulate during oligodendroglioma progression. Brain Res 2000; 882:1-8.

206. Choi BM, Pae HO, Jeong YR et al. Overexpression of heme oxygenase (HO)-1 renders Jurkat T-cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1. Free Radic Biol Med 2004; 36:858-871.

207. El Andaloussi A, Lesniak MS. CD4� CD25� FoxP3� T-cell infiltration and heme oxygenase-1 expression correlate with tumor grade in human gliomas. J Neurooncol 2007; 83:145-152.

208. Schlingensiepen KH, Schlingensiepen R, Steinbrecher A et al. Targeted tumor therapy with the TGF-beta 2 antisense compound AP 12009. Cytokine Growth Factor Rev 2006; 17:129-139.

209. Iwamaru A, Szymanski S, Iwado E et al. A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo. Oncogene 2007; 26:2435-2444.

210. Foss FM. DAB(389)IL-2 (denileukin diftitox, ONTAK): a new fusion protein technology. Clin Lymphoma 2000; (1 Suppl 1):S27-31.

211. Barnett B, Kryczek I, Cheng P et al. Regulatory T-cells in ovarian cancer: biology and therapeutic potential. Am J Reprod Immunol 2005; 54:369-377.

212. Grauer OM, Sutmuller RP, van Maren W et al. Elimination of regulatory T-cells is essential for an effective vaccination with tumor lysate-pulsed dendritic cells in a murine glioma model. Int J Cancer 2008; 122:1794-1802.

213. Heimberger AB, Crotty LE, Archer GE et al. Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors. Clin Cancer Res 2003; 9:4247-4254.

214. Sampson JH, Archer GE, Mitchell DA et al. Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma. Semin Immunol 2008; 20:267-275.

Mechanisms of Immune Evasion by Gliomas

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