Systemic Inhibition of Transforming Growth Factor- in Glioma-Bearing Mice Improves the Therapeutic Efficacy of Glioma-Associated Antigen Peptide Vaccines

Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Clinical Cancer Research (Impact Factor: 8.72). 11/2009; 15(21):6551-9. DOI: 10.1158/1078-0432.CCR-09-1067
Source: PubMed


A variety of cancers, including malignant gliomas, overexpress transforming growth factor-beta (TGF-beta), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8(+) CTLs and enhancing the generation of regulatory T (T(reg)) cells. We hypothesized that inhibition of TGF-beta would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.
Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-beta-neutralizing monoclonal antibody, 1D11, with or without s.c. vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679), emulsified in incomplete Freund's adjuvant.
Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA vaccines alone, or mock treatments alone. TGF-beta neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain-infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/IFN-gamma-producing CD8(+) T cells, and reduced CD4(+)/FoxP3(+) T(reg) cells in the glioma microenvironment. Neutralization of TGF-beta also upregulated plasma levels of interleukin-12, macrophage inflammatory protein-1 alpha, and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma interleukin-15 levels and promoted the persistence of GAA-reactive CD8(+) T cells in glioma-bearing mice.
These data suggest that systemic inhibition of TGF-beta by 1D11 can reverse the suppressive immunologic environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA vaccines.

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Available from: Xinmei Zhu, Aug 22, 2014
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    • "Analysis of animal subjects from this study revealed significantly elevated CTL activity within the lymph nodes and spleens, as well as greater immune cell infiltration, with concurrent reduction of Tregs, in the brains of the mouse hosts. Overall, treatment was associated with promoting the Th1 phenotype [88]. "
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    ABSTRACT: Glioblastoma remains a lethal diagnosis with a 5-year survival rate of less than 10%. (JAMA 352:987-96, 2005) Although immunotherapy-based approaches are capable of inducing detectable immune responses against tumor-specific antigens, improvements in clinical outcomes are modest, in no small part due to tumor-induced immunosuppressive mechanisms that promote immune escape and immuno-resistance. Immunotherapeutic strategies aimed at bolstering the immune response while neutralizing immunosuppression will play a critical role in improving treatment outcomes for glioblastoma patients. In vivo murine models of glioma provide an invaluable resource to achieving that end, and their use is an essential part of the preclinical workup for novel therapeutics that need to be tested in animal models prior to testing experimental therapies in patients. In this article, we review five contemporary immunocompetent mouse models, GL261 (C57BL/6), GL26 (C57BL/6) CT-2A (C57BL/6), SMA-560 (VM/Dk), and 4C8 (B6D2F1), each of which offer a suitable platform for testing novel immunotherapeutic approaches.
    Journal of Translational Medicine 04/2014; 12(1):107. DOI:10.1186/1479-5876-12-107 · 3.93 Impact Factor
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    • "In particular, TGF-β1 has a critical role in immune-suppressive mechanisms such as reducing the number and function of circulating DCs [22], inactivation of CTLs [23], and generation of Tregs [24]. Thus, several strategies to target immune-suppressing signaling from tumor cells for whole tumor cell-based cancer vaccines have been developed using neutralizing antibodies [25], small molecular inhibitors [26], specific small interfering RNAs (siRNAs) [27], or expression of a soluble TGF-β receptor by tumor cells [28]. However, the complexity of all these methods may limit its use, especially in clinical settings. "
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    ABSTRACT: The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-β1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed "eat-me" signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90α and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90α and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-β1 and up-regulated the production of IL-12p70 and HSP90α. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-γ, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.
    PLoS ONE 05/2013; 8(5):e63498. DOI:10.1371/journal.pone.0063498 · 3.23 Impact Factor
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    • "Given the microenvironment of the brain, including the unique contribution of the BBB, unique mode of lymphatic drainage and highly immunosuppressive environment, even under normal conditions, the mechanisms regulating Treg accumulation in brain tumors may be independent of the TGF-β signaling pathway. However, it is important to note that TGF-β neutralization leads to a decrease in the level of brain tumor-infiltrating Tregs (Ueda et al., 2009) suggesting that this cytokine somehow plays a role in Treg recruitment and/or expansion. "
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    ABSTRACT: One of the hallmark features of glioblastoma multiforme (GBM), the most common adult primary brain tumor with a very dismal prognosis, is the accumulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Regulatory T cells (Tregs) segregate into two primary categories: thymus-derived natural Tregs (nTregs) that develop from the interaction between immature T cells and thymic epithelial stromal cells, and inducible Tregs (iTregs) that arise from the conversion of CD4(+)FoxP3(-) T cells into FoxP3 expressing cells. Normally, these Treg subsets complement one another's actions by maintaining tolerance of self-antigens, thereby suppressing autoimmunity, while also enabling effective immune responses toward non-self-antigens, thus promoting infectious protection. However, Tregs have also been shown to be associated with the promotion of pathological outcomes, including cancer. In the setting of GBM, nTregs appear to be primary players that contribute to immunotherapeutic failure, ultimately leading to tumor progression. Several attempts have been made to therapeutically target these cells with variable levels of success. The blood brain barrier-crossing chemotherapeutics, temozolomide, and cyclophosphamide (CTX), vaccination against the Treg transcriptional regulator, FoxP3, as well as mAbs against Treg-associated cell surface molecules CD25, CTLA-4, and GITR are all different therapeutic approaches under investigation. Contributing to the poor success of past approaches is the expression of indoleamine 2,3-dioxygenase 1 (IDO), a tryptophan catabolizing enzyme overexpressed in GBM, and critically involved in regulating tumor-infiltrating Treg levels. Herein, we review the current literature on Tregs in brain cancer, providing a detailed phenotype, causative mechanisms involved in their pathogenesis, and strategies that have been used to target this population, therapeutically.
    Frontiers in Immunology 05/2013; 4:116. DOI:10.3389/fimmu.2013.00116
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