Signaling through GITR, using agonist anti-GITR antibodies or GITR ligands abrogates the suppressive effects of Tregs (7,10) and enhances T cell responses (6,8,9,11). Administration of agonist anti-GITR antibodies promotes the activation of CTLs, and interferon

We attempted to enhance the antitumor effects of tumor lysate-pulsed dendritic cells by eliminating regulatory T cells. The combinatorial effects of dendritic cells and agonist anti-glucocorticoid-induced tumor necrosis factor receptor (anti-GITR) antibodies were investigated with respect to enhancement of the systemic immune response, elimination of regulatory T cells, and inhibition of tumor growth. To determine whether the combination of dendritic cells and anti-GITR antibodies could enhance systemic immune responses and inhibit primary tumor growth in a murine osteosarcoma (LM8) model. We established the following 4 groups of C3H mice (20 mice in total): i), control IgG-treated mice; ii), tumor lysate-pulsed dendritic cell-treated mice; iii), agonist anti-GITR antibody-treated mice; and iv), agonist anti-GITR antibodyand tumor lysate-pulsed dendritic cell-treated mice. The mice that received the agonist anti-GITR antibodies and tumor lysate-pulsed dendritic cells displayed inhibited primary growth, prolonged life time, reduced numbers of regulatory T lymphocytes in the spleen, elevated serum interferon-γ levels, increased number of CD8+ T lymphocytes. The mice that received combined therapy had reduced level of immunosuppressive cytokines in tumor tissue and serum. Combining agonist anti-GITR antibodies with tumor lysate-pulsed dendritic cells enhanced the systemic immune response. These findings provide further support for the continued development of agonist anti-GITR antibodies as an immunotherapeutic strategy for osteosarcoma. We suggest that our proposed immunotherapy could be developed further to improve osteosarcoma treatment. Introduction Osteosarcoma is the most common primary malignant tumor of the bone. Remarkable advances in the treatment of osteosarcoma have been made in the past 2-3 decades. These include the introduction of adjuvant chemotherapy and appropriate surgical excision (1,2). However, there have also been advances in the field of immunotherapy for osteosarcoma that have received less attention (3,4). We developed a method using dendritic cells (DCs) to enhance tumor-specific immunoreactions based on the premise that DCs are the main antigen-presenting cells initiating cell-mediated immune responses in vivo (5). Our current strategy involves eliminating immunosuppressive factors such as regulatory T cells (Tregs) and enhancing cell-mediated immunity. The glucocorticoid-induced tumor necrosis factor receptor (GITR) family-related protein is constitutively expressed at high levels on Tregs and presented ubiquitously at lower levels on various immune subsets including cytotoxic T lymphocytes (CTLs) (6,7). GITR ligation provides a costimulatory signal that enhances CD4+ and CD8+ T cell proliferation and effector functions, particularly in the context of suboptimal T cell receptor stimulation (8,9). Signaling through GITR, using agonist anti-GITR antibodies or GITR ligands abrogates the suppressive effects of Tregs (7,10) and enhances T cell responses (6,8,9,11). Administration of agonist anti-GITR antibodies promotes the activation of CTLs, and interferon (IFN)-γ is reportedly required for the antitumor response induced by anti-GITR antibodies (12,13). Recently, several studies showed that in vivo GITR ligation by using anti-GITR antibodies can augment antitumor T cell responses and induce tumor rejection (14-16). However, the efficacy of the combination of tumor lysate-pulsed DCs and agonist anti-GITR antibodies in an osteosarcoma model has not been evaluated. Therefore, we hypothesized that an antitumor effect may be triggered if Tregs are controlled, resulting in the activation of CTLs and inhibition of tumor growth. We investigated how immunotherapies that target the inhibitory pathways of Tregs using anti-GITR-mAbs can potentially synergize the effects of cryotreated tumor lysate-pulsed DCs to generate systemic antitumor immunity. We verify that, in contrast to tumor lysate-pulsed DC or anti-GITR-Ab treatment alone, the combination therapy enhanced antitumor immunity and slowed the growth. Dendritic cells combined with anti-GITR antibody produce antitumor effects in osteosarcoma MASANORI KAWANO, KAzuHIRO TANAKA, ICHIRO ITONAGA, TATSuYA IWASAKI, MASASHI MIYAzAKI, SHINICHI IKEDA and HIROSHI TSuMuRA Department of Orthopaedic Surgery, Faculty of Medicine, Oita university, Oita 879-5593, Japan Received April 28, 2015; Accepted June 2, 2015 DOI: 10.3892/or.2015.4161 Correspondence to: Dr Masanori Kawano, Department of Orthopaedic Surgery, Faculty of Medicine, Oita university, Oita