Highlights
- •AP-BG augments the anti-tumor immune responses.
- •AP-BG changes the phenotype of TADCs from suppressive to promotive.
- •AP-BG enhances the production of cytolytic granules and cytokine in the TILs.
- •AP-BG improves DCs’ antigen-specific priming of T cells in vivo and in vitro.
Abstract
Dendritic cells (DCs) are recognized as the most potent antigen-presenting cells, capable of priming both naïve and memory T cells. Thus, tumor-resident DCs (tumor-associated DCs: TADCs) play a crucial role in the immune response against tumors. However, TADCs are also well known as a “double-edged sword” because an immunosuppressive environment, such as a tumor microenvironment, maintains the immature and tolerogenic properties of TADCs, resulting in the deterioration of the tumor. Therefore, it is essential to maintain and enhance the anti-tumoral activity of TADCs to aid tumor elimination. This study demonstrated the potential for tumor growth inhibition of Aureobasidium pullulan-derived β-glucan (AP-BG). Administration of AP-BG dramatically limited the development of different types of tumor cell lines transplanted into mice. Examination of the tumor-infiltrating leukocytes revealed that AP-BG caused high expression of co-stimulatory molecules on TADCs and enhanced the production of cytolytic granules as well as pro-inflammatory cytokines by the tumor-resident T cells. Furthermore, the syngeneic mixed lymphoid reaction assay and popliteal lymph node assay showed the significant ability of AP-BG to improve DCs’ antigen-specific priming of T cells in vitro and in vivo. Taken together, β-glucan might be an immune-potentiating adjuvant for cancer treatment. This highly widely-used reagent will initiate a new way to activate DC-targeted cancer immune therapy.
Introduction
Cancer is one of the leading causes of death worldwide. Many anticancer drugs cause undesirable toxic and other side effects, including damage to the immune system, hindering the host response during chemotherapeutic treatment. Therefore, it is important to investigate novel antitumor drugs that stimulate the immune response and lower toxicity profiles of other therapeutic agents [1]. Hence, the discovery and identification of new safe drugs that are active against tumors is an important goal. Immunotherapy represents an attractive option for the management of advanced malignancies that are resistant to conventional treatments.
Dendritic cells (DCs) play an important role in initiating innate and adaptive immune responses. They are the most powerful antigen-presenting cells and are therefore viewed as critical regulators of the adaptive immune responses. In the context of tumor immunity, DCs are potent antitumor initiators. However, DCs in the TME (tumor microenvironment) can be a “double-edged sword.” The tumor microenvironment (TME) is an immunosuppressive microenvironment that supports tumor growth and metastasis. The TME causes downregulation of MHC on DCs within itself, resulting in the acquisition of a profound immune suppressive effect on T cells by DCs. Through this process, tumor-specific CD8+ T cells in a growing tumor are often immunosuppressing with low IFN-γ secretion and reduced cytolytic ability [2]. In addition, immunosuppressive DCs in the TME can support neovascularization, furthering tumor growth. Finally, DCs in the TME can block antitumor immunity and stimulate cancer cell growth and spreading. Several studies have shown positive correlations between the presence of infiltrating immune cells, including DCs, in the TME and the prognosis of many cancers. However, the negative roles of the immunosuppressive DCs in the TME as tumor-associated dendritic cells (TADCs) are widely demonstrated. Because of these conflicting observations, the significance of TADCs has been subject to debate regarding their functions [3], [4], [5]. TADCs play an integral role in enhancing the immune response and are a subset of cells in the TME to activate antitumor T cells; however, they may alter their role from being immunostimulatory to immunosuppressive during cancer progression [6]. The imbalance of cytokines in the TME impairs DC differentiation and maturation. Elicit in T cell expansion by TADCs is an integral part of successful immunotherapy and can be augmented by transferring antigen-pulsed DCs to the host [7].
β-Glucan is an immuno-stimulating agent that has been used to treat cancer and infectious diseases for many years with varying and unpredictable efficacy. Recently, several reports showed that β-glucan has immunostimulant, anti-inflammatory, anti-microbial, anti-infective, anti-viral, anti-tumor, anti-oxidant, anti-coagulant, cholesterol-lowering, radioprotective, and wound healing effects [8], [9], [10], [11], [12], [13]. β-glucan has been recognized as a major fungal pathogen-associated molecular pattern, which can strongly influence natural and adaptive host immune responses, mostly through engagement of the C-type lectin receptor Dectin-1 [14]. β-glucans might promote the cytotoxic activities of leukocytes; therefore, they are recognized as potent immunological stimulators in humans. Currently, β-glucans are used for the clinical treatment of different types of cancers clinically in China [15].
In the present study, we investigated the anti-tumor effects of Aureobasidium pullulan-produced β-glucan (AP-BG). We excised tumor tissue from the tumor-bearing mouse models, and isolated tumor-infiltrating leukocytes (TILs), including cytotoxic T lymphocytes (CTLs: CD3+CD8+), helper T cells (Th: CD3+CD4+), and TADCs as CD11c+CD11b+ phenotype. After administration of AP-BG, tumor-infiltrating CTLs showed the potential to produce cytolytic granules and to secrete inflammatory cytokines. We also found that TADCs acquired immune stimulatory potency after administration of AP-BG. Furthermore, to explore the potential mechanism of AP-BG, we employed a syngeneic mixed lymphoid reaction (MLR) assay. We co-cultured OVA-loaded bone marrow-derived DCs (BMDCs) with OVA-specific T cells isolated from OT-I and OT-II mice. In the presence of AP-BG, the antigen-specific T cells showed not only a significant increase in proliferation but also a large enhancement of CTL functions. Our study explores the potential of AP-BG as an immune-potentiating adjuvant to activate DC-targeted immunotherapies.
Section snippets
Mice, cell line, and tumor models
Six to eight-week-old female C57BL/6NCrSlc (B6) and BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were housed with filtered water and food for one week before the experiment to maintain consistency. C57BL/6-Tg (OT-I) and C57BL/6-Tg (OT-II) transgenic mice were kindly supplied by Dr. N. Ishii (Graduate School of Medicine, Tohoku University), S. Nakae (The Institute of Medical Science, The University of Tokyo), respectively. Mice were cared for in accordance with the
AP-BG treatment significantly enhanced the anti-tumor response
In comparison to the no-treatment control group, AP-BG administration initiated potent growth inhibition of the tumor burden in murine melanoma cell line (MO4-Luc)-bearing mice (Fig. 1A, B). The anti-tumor efficacy of AP-BG was dose-dependent, and 200 mg/kg had greatest effect. AP-BG showed a similar level of anti-tumor efficacy even when the other type of cancer cell line was challenged. As shown in Fig. 1C, D, C26 (murine colon carcinoma cell line) transplanted into Balb/c mice were also
Discussion
DCs are potent antigen-presenting cells that possess the ability to present antigens to antigen-specific naïve T cells, as well as to maintain both innate and adaptive immune responses. However, specific microenvironmental signaling might prevent maturation of DCs or polarize their differentiation, resulting in the formation of DC subsets with tolerogenic and immunosuppressive potential accountable for antigen-specific unresponsiveness in the lymphoid organs and periphery [1]. Gabrilovich et al
CRediT authorship contribution statement
Yifang Shui: Conceptualization, Methodology, Data curation, Formal analysis, Writing-original draft, Writing-review & Editing. Xin Hu: Conceptualization, Methodology, Data curation, Formal analysis, Writing-original draft, Writing-review & Editing. Hiroshi Hirano: Conceptualization, Writing-original draft, Writing-review & Editing. Kisato Kusano: Conceptualization, Formal analysis, Resources. Hirotake Tsukamoto: Conceptualization, Resources. Mengquan Li: Conceptualization. Kenichiro Hasumi:
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [KK is an employee of Aureo Co., Ltd.].
Acknowledgement
The authors are grateful to thank Miss Zhidan Wang and Yalin Gao for their invaluable technical assistance. This study was supported by research grants from the Grants of Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid 16K11064, 24/17H04277, 18K08558); grants from the National Center for Child Health and Development (29-09); Science and Technology Innovation Talents in Henan Universities (No.19HASTIT003).
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