Kazutaka Masuko

Anti-IL-6 receptor mAb eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T-cell responses

CD11b+Gr‐1+ immature myeloid cells (ImCs), which are abnormally increased in tumor‐bearing mice, were classified into three different subsets according to their phenotypic and morphological characteristics: Gr‐1low F4/80+ macrophages (MΦ‐ImCs), Gr‐1mid stab neutrophils (Neutstab‐ImCs), and Gr‐1high segmented neutrophils (Neutseg‐ImCs). In the spleen, only MΦ‐ImCs but not Neutstab‐ImCs and Neutseg‐ImCs exhibited a significant immunosuppressive activity in MLR. In contrast, tumor‐infiltrating leukocytes (TILs) contained only two ImC subsets, MΦ‐ImCs and Neutseg‐ImC, both of which exhibited stronger inhibitory activity against T cells compared with spleen‐MΦ‐ImCs. Thus, we concluded that tumor‐infiltrating MΦ‐ImCs and Neutseg‐ImCs were fully differentiated myeloid‐derived suppressor cells (MDSCs) with stronger T‐cell inhibitory activity. Indeed, spleen MΦ‐ImCs were converted into stronger MΦ‐MDSCs by tumor‐derived factor (TDF). Moreover, both spleen Neutstab‐ImCs and Neutseg‐ImCs differentiated into Neutseg‐MDSCs with suppressive activity after culture with TDF. We first demonstrated that administration of anti‐IL‐6R mAb could downregulate the accumulation of MΦ‐MDSCs and Neutseg‐MDSCs in tumor‐bearing mice. The elimination of those MDSCs caused subsequent enhancement of antitumor T‐cell responses, including IFN‐γ‐production. The therapeutic effect of anti‐IL‐6R mAb was further enhanced by combination with gemcitabine (GEM). Thus, we propose that anti‐IL‐6R mAb could become a novel tool for the downmodulation of MDSCs to enhance antitumor T‐cell responses in tumor‐bearing hosts.

The Key Role of IL-6–Arginase Cascade for Inducing Dendritic Cell–Dependent CD4+ T Cell Dysfunction in Tumor-Bearing Mice

Evaluation of immune dysfunction during the tumor-bearing state is a critical issue in combating cancer. In this study, we initially found that IL-6, one of the cachectic factors, suppressed CD4+ T cell–mediated immunity through downregulation of MHC class II by enhanced arginase activity of dendritic cells (DC) in tumor-bearing mice. We demonstrated that administration of Ab against IL-6R (anti–IL-6R mAb) greatly enhanced T cell responses and inhibited the growth of tumor in vivo. We also found that IL-6 upregulated the expression of arginase-1 and arginase activity of DC in vitro. Tumor-infiltrating CD11c+ DC exhibited upregulated mRNA expression of arginase-1 but reduced expression of MHC class II in parallel with the increase in serum IL-6 levels at the late stage in tumor-bearing hosts. However, the administration of anti–IL-6R mAb into tumor-bearing mice inhibited both the downmodulation of MHC class II and the upregulation of arginase-1 mRNA levels in DC. Furthermore, we noted that Nω-hydroxy-L-arginine or L-arginine, an arginase-1 inhibitor, blocked the reduction in MHC class II levels on CD11c+ DC during the tumor-bearing state. Finally, we demonstrated that the administration of Nω-hydroxy-L-arginine at the peritumor site significantly enhanced CD4+ T cell responses and inhibited tumor growth. Thus, IL-6–mediated arginase activation and the subsequent reduction in MHC class II expression on DC appeared to be critical mechanisms for inducing dysfunction of the immune system in the tumor-bearing state. Blockade of the IL-6–arginase cascade is a promising tool to overcome the dysfunction of antitumor immunity in tumor-bearing hosts.

Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.

Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): preparation and immunological analysis of vaccine efficacy.

To elucidate the immunologic mechanisms of artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which indicated a great vaccine efficacy in human cancers, we prepared ovalbumin (OVA)-H/K-HELP by conjugating killer and helper epitopes of OVA-model tumor antigen via a glycine-linker. Vaccination of C57BL/6 mice with OVA-H/K-HELP (30 amino acids) but not with short peptides mixture of class I-binding peptide (8 amino-acids) and class II-binding peptide (17 amino-acids) combined with adjuvant CpG-ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotides), induced higher numbers of OVA-tetramer-positive CTL with concomitant activation of IFN-γ-producing CD4(+) Th1 cells. However, replacement of glycine-linker of OVA-H/K-HELP with other peptide-linker caused a significant decrease of vaccine efficacy of OVA-H/K-HELP. In combination with adjuvant CpG-ODN, OVA-H/KHELP exhibited greater vaccine efficacy compared with short peptides vaccine, in both preventive and therapeutic vaccine models against OVA-expressing EG-7 tumor. The elevated vaccine efficacy of OVAH/K-HELP might be derived from the following mechanisms: (i) selective presentation by only professional dendritic cells (DC) in vaccinated draining lymph node (dLN); (ii) a long-term sustained antigen presentation exerted by DC to stimulate both CTL and Th1 cells; (iii) formation of three cells interaction among DC, Th and CTL. In comparative study, H/K-HELP indicated stronger therapeutic vaccine efficacy compared with that of extended class I synthetic long peptide, indicating that both the length of peptide and the presence of Th epitope peptide were crucial aspects for preparing artificially synthesized H/K-HELP vaccine.

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

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