Takuto Kishikawa

IL-11 induces differentiation of myeloid-derived suppressor cells through activation of STAT3 signalling pathway

Myeloid-derived suppressor cells (MDSCs) are immune negative regulators in the tumour microenvironment. Interleukin (IL)-11, a member of IL-6 family cytokines, functions through the unique receptor IL-11 receptor α coupled with the common signal transducer gp130. IL-11-gp130 signalling causes activation of the JAK/STAT3 pathway. IL-11 is highly upregulated in many types of cancers and one of the most important cytokines during tumourigenesis and metastasis. However, the precise effect of IL-11 on differentiation into MDSCs is still unknown. Here, we found that CD11b+CD14+ monocytic MDSCs were generated from peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence of IL-11. IL-11-conditioned PBMCs induced higher expression of immunosuppressive molecules such as arginase-1. A reduction of T-cell proliferation was observed when MDSCs generated in the presence of IL-11 were co-cultured with CD3/CD28-stimulated, autologous T cells of healthy donors. Culture of normal PBMCs with IL-11 led to STAT3 phosphorylation and differentiation into MDSCs via STAT3 activation. We confirmed expressions of both IL-11 and phosphorylated STAT3 in tumour tissues of colorectal cancer patients. These findings suggest that monocytic MDSCs may be induced by IL-11 in the tumour microenvironment. Thus, IL-11-mediated regulation in functional differentiation of MDSCs may serve as a possible target for cancer immunotherapy.

Neuropeptide signaling through neurokinin-1 and neurokinin-2 receptors augments antigen presentation by human dendritic cells.

Title Neuropeptide signaling through neurokinin-1 and neurokinin-2 receptors augments antigen presentation by human dendritic cells Author(s) Ohtake, Junya; Kaneumi, Shun; Tanino, Mishie; Kishikawa, Takuto; Terada, Satoshi; Sumida, Kentaro; Masuko, Kazutaka; Ohno, Yosuke; Kita, Toshiyuki; Iwabuchi, Sadahiro; Shinohara, Toshiya; Tanino, Yoshinori; Takemura, Tamiko; Tanaka, Shinya; Kobayashi, Hiroya; Kitamura, Hidemitsu Citation Journal of Allergy and Clinical Immunology, 136(6): 1690-1694

Abstract 3615: IL-6/STAT3-dependent immunosuppressive function of tumor-infiltrating dendritic cells in colorectal cancer

Introduction Immunosuppression in tumor microenvironments is one of the critical issues for cancer immunotherapy. To develop more effective treatment, it is essential to overcome the dysfunction of immunity in cancer patients. Recently, it has been demonstrated that myeloid derived suppressor cells (MDSCs) have crucial roles for such immunosuppression. In the current reports, it was indicated that activation of STAT3 was required for immunosuppressive function of human MDSCs. In this study, we focused on IL-6/STAT3-signaling pathway in human monocyte-derived dendritic cells (DCs), and investigated the effects of IL-6 on antigen-presenting ability of DCs. Materials and Methods Tumor-infiltrating myeloid cells and PBMCs were collected from specimen of patients with colorectal cancers. Surface molecules , such as HLA-DR, of the cells were investigated by flowcytometry. Then, the surface molecules of monocyte-derived DCs from healthy volunteers were evaluated after the stimulation with IL-6 in vitro. IL-6-treated DCs were co-cultured with autologous T cells stimulated by using anti-CD3 antibody and cytokine production levels were investigated by ELISA. MAGE-A4-specific CD4+ T cells were generated from PBMCs ofhealthy healthy volunteers in vitro and co-cultured with IL-6-pretreated DCs in the presence of MAGE-A4 antigen. Cytokine production by antigen specific T cells were measured by ELISA. Results In this study, we found that expression levels of HLA-DR and CD86 are down regulated in tumor infiltrating CD11c+CD14+ DCs compared with peripheral monocytes. In addition, we confirmed that HLA-DR and CD86 expressions were significantly reduced by IL-6 treatment of CD11c+CD14+ DCs in vitro. The reduction of HLA-DR and CD86 expression levels were remarkably blocked in the presence of STAT3 inhibitor. IFN-γ production by T cells after TCR-stimulation was suppressed in the presence of IL-6-treated DCs compared with control DCs. Moreover, activation of MAGE-A4 antigen-specific CD4+ T cells were remarkably reduced by IL-6-treatment of DCs. Discussion Previously, it was reported that CD14+HLA-DRlow/- cells increased in peripheral blood of cancer patients and that these cells were an important population for immunosuppression. In this study, we demonstrated that IL-6/STAT3-signaling cascade was one of the regulating factors for surface expression levels of HLA-DR on DCs. In addition, we confirmed that IL-6-treatment significantly suppressed the antigen presentation by DCs for cancer antigen-specific CD4+ T cells. Generally, MDSCs were known to induce several immunosuppressive and tumor promoting factors such as VEGF, ARG1, and COX2. We are now investigating target molecules of IL-6 for dysfunction of DCs in tumor microenvironments. Conclusion IL-6/STAT3 signaling pathway regulates immunosuppressive function of human DCs, which would be a promising target for improving the effects of cancer immunotherapy. Citation Format: Yosuke Ono, Jyunya Ohtake, Shun Kaneumi, Kazutaka Masuko, Kentaro Sumida, Takuto Kishikawa, Satoshi Terada, Toshiyuki Kita, Norihiko Takahashi, Akinobu Taketomi, Hidemitsu Kitamura. IL-6/STAT3-dependent immunosuppressive function of tumor-infiltrating dendritic cells in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3615. doi:10.1158/1538-7445.AM2014-3615

Regulation of Antigen Presentation by Dendritic Cells and Its Application to Cancer Immunotherapy

Dendritic cells (DCs) are one of the most powerful antigen-presenting cells and play a crucial role in bridging between innate and acquired immunity. Cancer antigens or the long peptides are engulfed by DCs, digested into helper and killer epitope peptides, transported to the cell surface, and presented to CD4+ and CD8+ T cells through major histocompatibility complex (MHC) class II and MHC class I to induce effector T helper cells and cytotoxic killer T cells, respectively. In addition, DCs produce type 1 cytokines such as interleukin (IL)-12 and interferon (IFN)-α/-β to facilitate differentiation of naive T cells into effector T cells and activation of memory T cells. Therefore, proper regulation of DC function is essential for induction and augmentation of anti-tumor immunity in cancer patients. Generally, DCs are immediately activated by various maturation signals including Toll-like-receptor (TLR) ligands, type 1 cytokines, and CD40/40L interaction. On the other hand, IL-6 produced in tumor microenvironments caused dysfunction of DCs through reduction of MHC class II expression and IL-12 production. It has recently been reported that zinc transporter-mediated intracellular zinc levels and neuropeptide signaling through the receptors are involved in the regulation of the antigen-presenting function of DCs in type 1 immune responses, including TLR-mediated inflammatory response. In this chapter, we report on regulation of the antigen-presenting function and the potential benefit of DC-mediated cancer immunotherapy.

Abstract 3661: Crucial roles of cytokine-signaling for alteration in functions of myeloid-derived suppressor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Various types of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) were generated from immature myeloid cells (ImCs) according to the state of tumor microenvironments. In human cancer patients, MDSCs are generally defined as monocytic (CD11b+, CD14+, and HLA-DR-/low) or granulocytic (CD11b+ and CD15+) myeloid cells with antitumor and immunosuppressive activities. In order to develop the effective cancer immunotherapy, these immune suppressor populations should be restrictlly controlled in tumor bearing hosts. In the present research, we evaluated the immunological functions of monocytic or granulocytic myeloid cells, which were collected from tumor tissues of colorectal cancer patients. We found here that myeloid populations in tumor tissues significantly induced immuno-modulating factors such as IL-6, IL-10, VEGF, iNOS, ARG1, IDO1, and IDO2. In addition, we confirmed that tumor-infiltrating myeloid-populations exhibited a significant immunosuppressive activity than those prepared from peripheral blood mononuclear cells (PBMCs). To investigate how tumor-infiltrating MDSCs acquired the immunosuppressive activity, we focused on the effect of tumor-derived factors (TDFs) on immunosuppressive activity. PBMCs from healthy volunteers were cultured in the presence of IL-6, TGF-β, and granulocyte macrophage colony-stimulating factor (GM-CSF) for 6 days, and then differentiated into Mo-MDSCs (CD11b+CD14+CD33+)-like phenotype cells. The generated Mo-MDSCs were further isolated by using cell sorter system and cultured with donor matched T cells and stimulated with anti-CD3/CD28 beads for 3 days. As a result, we confirmed that T cell proliferation was significantly inhibited by the addition of Mo-MDSCs generated with IL-6, TGF-β and GM-CSF. On the other hand, these populations showed immunostimulating ability in the presence of IFN-g in vitro, suggesting the Mo-MDSCs might have multi-differencing potentials. These results indicated that tumor-infiltrating MDSCs acquired stronger immunosuppressive activity by tumor-derived IL-6 and TGF-β, however the function might be altered in response to cytokine conditions. Thus, we concluded that IL-6 and TGF-β induced functional maturation of MDSCs in tumor microenvironments, suggesting promising targets to improve the immunological responses by immunotherapy for cancer patients. Citation Format: Kentaro Sumida, Yosuke Ono, Junya Ohtake, Kazutaka Masuko, Satoshi Terada, Shun Kaneumi, Takuto Kishikawa, Toshiyuki Kita, Hidemitsu Kitamura. Crucial roles of cytokine-signaling for alteration in functions of myeloid-derived suppressor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3661. doi:10.1158/1538-7445.AM2014-3661

Abstract 3620: Neuropeptide signaling activates Type-1 immunity through the NK1 and NK2 receptors on human dendritic cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Dendritic cells (DCs), representative antigen-presenting cells, effectively induce antigen-specific immune responses through activation of CD4+T and CD8+T cells. In tumor microenvironments, DCs uptake tumor-derived antigens, and generate helper T cells and cytotoxic T lymphocytes (CTLs), which recognize and kill the target tumor cells in response to the antigens. Thus, proper regulation of DC function is important for tumor immunology. Substance P (SP) and Neurokinin A (NKA), neurotransmitters, are widely distributed in both central and peripheral nervous system. Recently, we demonstrated that IFN-γ remarkably induced neurokinin-2 receptor (NK2R) expression human monocyte-derived dendritic cells. These findings strongly suggested that neuropeptide-signaling cascade might be closely related with regulation of DC-mediated immune responses. To elucidate the precise role of such neuro-immune crosstalk, we further investigated the effect of neuropeptide signaling on function of human DCs. At first, we found that both neurokinin-1 receptor (NK2R) and NK2R mRNA expressions were significantly enhanced by IFN-β, IFN-γ, LPS, or poly I:C stimulation with human DCs generated from peripheral blood mononuclear cells (PBMCs). In addition, we confirmed that the upregulation of NK1R and NK2R mRNA expressions was induced in a STAT-1-dependent manner. Surface expression levels of HLA-DR and costimulatory molecules on DCs, augmented by poly I:C, were modulated in the presence of specific inhibitors against NK1R or NK2R. On the other hand, we confirmed that blockade of NK1R- and NK2R-mediated signaling cascade significantly suppressed cytokine productions by antigen-specific CD4+ T cells after the antigen stimulation with DCs. Finally, we found that human DCs also enhanced expression level of TAC-1 gene, which encodes SP and NKA, after the IFN-γ stimulation. Thus, these findings indicate that NK1R- and NK2R-dependent neuropeptide signaling regulate Type-1 immunity through the activation of DC function, suggesting that such neuro-immune cross-talk through SP-NK1R and NKA-NK2R cascade might be involved in various diseases caused by excessive Type-1-dominant immunity including cancer and infection with chronic inflammation. Citation Format: Hidemitsu Kitamura, Junya Ohtake, Shun Kaneumi, Kazutaka Masuko, Kentaro Sumida, Satoshi Terada, Takuto Kishikawa, Yosuke Ono, Toshiyuki Kita, Hiroya Kobayashi. Neuropeptide signaling activates Type-1 immunity through the NK1 and NK2 receptors on human dendritic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3620. doi:10.1158/1538-7445.AM2014-3620

Abstract 4085: Crucial roles of helper and killer epitopes in tumor antigens for developing dendritic cell-mediated cancer immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Dendritic cells (DCs), powerful antigen presenting cells, play central role for induction of the antigen-specific immune responses through activation of CD4+T and CD8+T cells. Tumor antigen-pulsed DCs strongly generate helper T cells and cytotoxic T lymphocytes (CTLs), which recognize and kill the target tumor cells in response to the antigens. Thus, proper design of antigen is required for application of DC-mediated cancer immunotherapy. Many investigators have performed clinical trials of cancer immunotherapy. Numerous vaccinations with tumor antigen-derived peptides have been able to induce tumor specific immune responses to eradicate cancer with superior specificity and without severe adverse effects. However, the therapeutic efficacy of cancer vaccine therapy using MHC class I-binding peptides for CTLs have been limited to induce complete regression in cancer patients. To overcome limitation of antitumor effects, strong and persisting activation of tumor specific CTLs are required for eradication of tumor tissues to induce a complete cure in tumor-bearing hosts. Recently, it has been demonstrated that CD4+ helper T cells play a critical role for inducing fully activated antitumor CTLs. Moreover, long peptides composed of both MHC class I- and class II-binding epitopes exhibited superior vaccine efficacy compared with shorter peptides. Thus, the existence of helper epitopes and the length of peptides appeared to be key factors for designing a promising peptide for DC-mediated immunotherapy. In this work, we focused natural tumor antigens such as Birc5, a member of the inhibitor of apoptosis gene family, which was abundantly expressed in the majority of cancer cells. We first prepared a Birc5 long peptide containing helper and killer epitopes and the shorter peptides containing helper and/or killer epitope. The long peptide was injected alone or with bone marrow-derived DCs into several types of tumor-bearing mice. The tumor growth was remarkably decreased by in vivo injection of the long peptide compared with the short peptide. In addition, we confirmed that long peptides were superior to the short peptides for inducing antigen-specific immune responses in vivo. These data indicated that long peptides containing helper and killer epitopes might be critical for inducing effective antitumor immunity in cancer immunotherapy, suggesting that long peptide vaccination containing helper and killer epitope would become a promising therapeutic vaccine strategy for cancer patients. Citation Format: Kazutaka Masuko, Shun Kaneumi, Junya Ohtake, Kentaro Sumida, Satoshi Terada, Takuto Kishikawa, Yosuke Ohno, Toshiyuki Kita, Hidemitsu Kitamura. Crucial roles of helper and killer epitopes in tumor antigens for developing dendritic cell-mediated cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4085. doi:10.1158/1538-7445.AM2014-4085

Abstract 3621: Regulation of antigen-presentaion by dendritic cells by TLR ligands and its application to cancer vaccine immunotherapy

Dendritic cells (DCs), one of the most powerful antigen presenting cells (APCs), are crucial not only for the innate immunity but also for the central role of the antigen-specific immune responses through activation of CD4+T and CD8+T cells. Generally, professional APCs such as DCs have a characteristic to initiate the strong T cell-immune responses because of the signals from costimulatory molecules and cytokines in addition to antigen-loaded MHCs compared with other APCs. Tumor antigen-pulsed DCs strongly prime both CD4+T and CD8+T cells capable of recognizing and killing tumor cells in response to the antigens on the target cells. Therefore, the effective antigen presentation by DCs is required for application of DC-mediated cancer immunotherapy. Numerous clinical studies using the HLA class I binding short peptides have been performed to activate tumor antigen-specific cytotoxic T cells (CTLs) in cancer patients. However, remarkable results such as the complete disappearance of tumors is still limited. Therefore, we focused on the function of DCs as professional APCs. In this study, we first prepared long peptides of tumor antigens including MHC class I and class II epitopes to uptake by DCs and to activate effector CTLs and Th cells, effectively. Next, we examined the effect of adjuvants or cytokines on DC function to activate tumor-antigen specific CTLs and Th cells, because it was thought that the maturation of DCs was an important factor for the induction of antigen specific T cells. We used OK-432, poly I:C, LPS, IFN-α, IFN-β, IFN-γ, IL-4, IL-6, and TGF-β in addition to GM-CSF for induction or stimulation with DCs. As a result, we observed that upregulation of MHC class I and class II expressions on DCs after stimulation with TLR ligands and cytokines except IL-6 and TGF-β. In this study, we found that IL-6 suppressed the function of DCs, whereas TGF-β inhibited the differentiation of DCs. Furthermore, we confirmed that IL-12 production by DCs after stimulation with OK-432 and poly I:C, but not LPS or other cytokines. Then, we examined the antigen-uptake and activation of antigen-specific CD4+T and CD8+T cells by using OK-432- or poly I:C-treated DCs. As a result, it became clear that we could induce antigen-specific Th cells and CTLs more effectively by using long peptides than short peptides. In addition, we confirmed that antigen-specific cytokine production by T cells was attenuated by the blockade of endocytosis and proteasome of DCs, suggesting the dynamics of antigen processing of the long peptides in DCs. We are now investigating for the novel regulator of DCs to generate antigen-specific T cells in cancer patients, which would promote the effect of the cancer vaccines. Citation Format: Junya Ohtake, Takuto Kishikawa, Shun Kaneumi, Kazutaka Masuko, Yosuke Ono, Kentaro Sumida, Satoshi Terada, Toshiyuki Kita, Hidemitsu Kitamura. Regulation of antigen-presentaion by dendritic cells by TLR ligands and its application to cancer vaccine immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3621. doi:10.1158/1538-7445.AM2014-3621