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Hemophagocytic lymphohistiocytosis (HLH) is a severe complication after allogeneic hematopoietic cell transplantation (HCT) and can cause graft failure or multi-organ failure. Here, we report two children with refractory HCT-associated HLH treated with ruxolitinib. In the first patient, ruxolitinib resolved fever, cytopenia and hyperferritinemia. In another patient, although severe hepatic failure, which developed and worsened before the administration of ruxolitinib, was irreversible, rapid improvement in fever, leukopenia and hyperferritinemia was observed. Of note, multiplex cytokine profiling showed amelioration of cytokine storm in both patients. Ruxolitinib may be an encouraging option for HCT-associated HLH.

Although programmed death (PD)-1 immune checkpoint therapies target the immune system, the relationship between inflammatory factors and the clinical outcome of anti-PD-1 therapy for nonsmall cell lung cancer (NSCLC) is not fully understood. Here we examined the association between soluble immune mediators and the outcome of treatment with PD-1 inhibitors in patients with advanced/recurrent NSCLC. In two independent cohorts, we assessed the levels of 88 different soluble immune mediators in peripheral blood before and after anti-PD-1 treatment, and evaluated their associations with clinical outcomes. In the training cohort, the plasma levels of chitinase 3-like-1 and GM-CSF before treatment (p = 0.006 and p = 0.005, respectively) and changes in the plasma levels of CXCL2, VEGF, IFNα2, and MMP2 after treatment (p < 0.001, p = 0.019, p = 0.019, and p = 0.012, respectively) were significantly correlated with PFS. The change in the plasma CXCL2 level was also significantly associated with treatment-related AEs (p = 0.017). In the validation cohort, however, only the changes in the plasma levels of CXCL2 and MMP2 after treatment were associated with PFS (p = 0.003 and p = 0.006, respectively), and these changes were maintained during the course of anti-PD-1 therapy in patients who showed better clinical outcomes, even in those with tumor pseudoprogression. Since CXCL2 and MMP2 can be easily measured by minimally invasive blood sampling, they could be useful for monitoring of clinical outcomes in NSCLC patients receiving PD-1 inhibitor therapy.

Introduction: Accumulating evidence for the feasibility and efficacy of cancer immunotherapy is ushering in a paradigm shift for cancer treatment. Notably, immune checkpoint inhibitors, suc...

Overcoming the immunosuppressive state in tumor microenvironments is a critical issue to improve the efficacy of cancer immunotherapy. Interleukin (IL)-6, a pleiotropic cytokine, is highly produced in the tumor-bearing host. Previous studies have indicated that IL-6 suppresses the antigen presentation ability of dendritic cells (DCs) through activation of signal transducer and activator of transcription 3 (STAT3). Thus, we focused on the precise effect of the IL-6/STAT3 signaling cascade on human DCs and the subsequent induction of antitumor T cell immune responses. Tumor-infiltrating CD11b+CD11c+ cells isolated from colorectal cancer tissues showed strong induction of the IL-6 gene, downregulated surface expression of human leukocyte antigen (HLA)-DR, and an attenuated T cell-stimulating ability compared with those from peripheral blood mononuclear cells, suggesting that the tumor microenvironment suppresses antitumor effector cells. In vitro experiments revealed that IL-6-mediated STAT3 activation reduced surface expression of HLA-DR on CD14+ monocyte-derived DCs. Moreover, we confirmed that cyclooxygenase 2, lysosome protease, and arginase activities were involved in the IL-6-mediated downregulation of the surface expression levels of HLA class II on human DCs. These findings suggest that IL-6-mediated STAT3 activation in the tumor microenvironment inhibits functional maturation of DCs to activate effector T cells, blocking introduction of antitumor immunity in cancers. Therefore, we propose in this review that blockade of the IL-6/STAT3 signaling pathway and target molecules in DCs may be a promising strategy to improve the efficacy of immunotherapies for cancer patients. This article is protected by copyright. All rights reserved.

Neoantigens derived from tumor-specific genetic mutations might be suitable targets for cancer immunotherapy because of their high immunogenicity. In the current study, we evaluated the immunogenicity of 10 driver mutations that are frequently expressed in various cancers using peripheral blood mononuclear cells from healthy donors (n = 25). Of the 10 synthetic peptides (27-mer) derived from these mutations, the six peptides from KRAS-G12D, KRAS-G12R, KRAS-G13D, NRAS-Q61R, PIK3CA-H1047R, and C-Kit-D816V induced T cell responses, suggesting that frequent driver mutations are not always less immunogenic. In particular, immune responses to PIK3CA-H1047R, C-Kit-D816V, KRAS-G13D, and NRAS-Q61R were observed in more than 10% of the donors. All six peptides induced human leukocyte antigen (HLA) class II-restricted CD4+ T cell responses, notably, PIK3CA-H1047R contained at least two different CD4+ T cell epitopes restricted to different HLA class II alleles. In addition, PIK3CA-H1047R and C-Kit-D816V induced antigen-specific CD8+ T cells as well, indicating that they might contain both HLA class I- and class II-restricted epitopes. Since the identified neoantigens might be shared by patients with various types of cancers and are not easily lost due to immune escape, they have the potential to be promising off-the-shelf cancer immunotherapy targets in patients with the corresponding mutations.

The fields of cancer immunology and immunotherapy have made dramatic progress in the past 20 years. Various approaches to cancer immunotherapies, including cancer vaccines using peptides, proteins, DNA, and dendritic cells, have been developed to activate antigen-specific immune cells and their efficacy has been clinically examined. Herein, we summarize previously performed clinical trials of various immunotherapies against colorectal cancer (CRC), including our own novel immunotherapeutic approach, the “personalized peptide vaccine”, in which human leukocyte antigen (HLA)-matched vaccine peptides are selected based on pre-existing host immunity before vaccination. In the near future, neoepitopes identified through massive DNA sequencing of the genetic alterations in tumor cells, combined with robust T cell epitope predictions in each patient might be targeted as a personalized immunotherapy for CRC.

Colorectal cancer(CRC)is one of the increasing cancers, and development of novel treatments is imperative for advanced CRC. In recent years, immune checkpoint inhibitors have demonstrated impressive clinical efficacy in various types of cancers, but only limited clinical responses were reported in CRC. It has been reported that such poor therapeutic effects might be possibly due to immune suppressive mechanisms other than immune checkpoints in the CRC. Therefore, comprehensive grasp of the immune environment is considered to be of significance for CRC patients. Our study aims to comprehensively assess the phenotypes and functions of various immune cells isolated from various tissues, including primary tumors and related lymph nodes, in primary CRC patients. Our preliminary results by flow cytometry analysis suggest that the immune suppression, particularly in regional lymph nodes, may be related to tumor progression. Further studies remain to be performed to more thoroughly elucidate immune suppressive mechanisms, and thereby to develop a novel immunotherapeutic strategy in the CRC.

Although programmed death (PD)-1 immune checkpoint therapies target the immune system, the relationship between inflammatory factors and the clinical outcome of anti-PD-1 therapy for nonsmall cell lung cancer (NSCLC) is not fully understood. Here we examined the association between soluble immune mediators and the outcome of treatment with PD-1 inhibitors in patients with advanced/recurrent NSCLC. In two independent cohorts, we assessed the levels of 88 different soluble immune mediators in peripheral blood before and after anti-PD-1 treatment, and evaluated their associations with clinical outcomes. In the training cohort, the plasma levels of chitinase 3-like-1 and GM-CSF before treatment (p = 0.006 and p = 0.005, respectively) and changes in the plasma levels of CXCL2, VEGF, IFNα2, and MMP2 after treatment (p0.001, p = 0.019, p = 0.019, and p = 0.012, respectively) were significantly correlated with PFS. The change in the plasma CXCL2 level was also significantly associated with treatment-related AEs (p = 0.017). In the validation cohort, however, only the changes in the plasma levels of CXCL2 and MMP2 after treatment were associated with PFS (p = 0.003 and p = 0.006, respectively), and these changes were maintained during the course of anti-PD-1 therapy in patients who showed better clinical outcomes, even in those with tumor pseudoprogression. Since CXCL2 and MMP2 can be easily measured by minimally invasive blood sampling, they could be useful for monitoring of clinical outcomes in NSCLC patients receiving PD-1 inhibitor therapy.

Immunosuppression in tumor microenvironments critically affects the success of cancer immunotherapy. Here, we focused on the role of interleukin (IL)-6/signal transducer and activator of transcription (STAT3) signaling cascade in immune regulation by human dendritic cells (DCs). IL-6-conditioned monocyte-derived DCs (MoDCs) impaired the presenting ability of cancer-related antigens. Interferon (IFN)-γ production attenuated by CD4(+) T cells co-cultured with IL-6-conditioned MoDCs corresponded with decreased DC IL-12p70 production. Human leukocyte antigen (HLA)-DR and CD86 expression was significantly reduced in CD11b(+)CD11c(+) cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors by IL-6 treatment and was STAT3 dependent. Arginase-1 (ARG1), lysosomal protease, cathepsin L (CTSL), and cyclooxygenase-2 (COX2) were involved in the reduction of surface HLA-DR expression. Gene expressions of ARG1, CTSL, COX2, and IL6 were higher in tumor-infiltrating CD11b(+)CD11c(+) cells compared with PBMCs isolated from colorectal cancer patients. Expression of surface HLA-DR and CD86 on CD11b(+)CD11c(+) cells was down-regulated, and T cell-stimulating ability was attenuated compared with PBMCs, suggesting that an immunosuppressive phenotype might be induced by IL-6, ARG1, CTSL, and COX2 in tumor sites of colorectal cancer patients. There was a relationship between HLA-DR expression levels in tumor tissues and the size of CD4(+) T and CD8(+) T cell compartments. Our findings indicate that IL-6 causes a dysfunction in human DCs that activates cancer antigen-specific Th cells, suggesting that blocking the IL-6/STAT3 signaling pathway might be a promising strategy to improve cancer immunotherapy.

Background: Neurotransmitters, including substance P (SP) and neurokinin A (NKA), are widely distributed in both the central and peripheral nervous system and their receptors, neurokinin-1 receptor (NK1R) and neurokinin-2 receptor (NK2R), are expressed on immune cells. However, the role of the NKA-NK2R axis in immune responses relative to the SP-NK1R signaling cascade has not been elucidated. Objective: We sought to examine the effect of neuropeptide signaling through NK1Rand NK2R on antigen presentation by dendritic cells (DCs) and the subsequent activation of effector Th cells. Methods: Expression levels of NK1R, NK2R, HLA-class II and costimulatory molecules of human MoDCs and cytokine production by birch pollen antigen-specific CD4+ T cells cocultured with MoDCs in the presence of NK1R and NK2R antagonists were evaluated by quantitative RT-PCR, flow cytometry or ELISA. NK1R and NK2R expression in the lung of patients with asthma and hypersensitivity pneumonitis was evaluated by immunohistochemistry. Results: Human MoDCs significantly upregulated NK2R and NK1R expression in response to poly I:C stimulation in a STAT1-dependent manner. Both NK2R and NK1R were expressed on alveolar macrophages and lung DCs from patients with asthma and pneumonitis hypersensitivity. Surface expression levels of HLA-class II and costimulatory molecules on DCs were modulated by NK1R or NK2R antagonists. Activation of birch pollen-derived antigen-specific CD4+ T cells and their production of cytokines including IL-4 and IFN-γ as well as IL-12 production by MoDCs, were suppressed by blocking NK1R or NK2R after in vitro antigen stimulation. Conclusions: NK1R- and NK2R-mediated neuropeptide signaling promotes both innate and acquired immune responses through activation of human DCs.

Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen. Indeed, the MEIOB gene is expressed only in the testis and not in other normal tissues. The mRNA expression of MEIOB was greatly enhanced in several lung cancer cell lines after the treatment with DAC. Furthermore, we identified a variety of helper epitopes of the MEIOB antigen, which were recognized by MEIOB antigen-specific T cells in a HLA-restriction manner. Finally, we demonstrated that IFN-γ production of MEIOB peptide-specific helper T cells in response to HLA-matched cancer cells was greatly augmented by treatment with DAC and IFN-γ. Taken together, these findings show DAC to be a promising tool for finding novel CT antigens and for developing a future novel combination cancer vaccine chemotherapy.

Cancer immunotherapy has attracted attention worldwide owing to the recent development of immune checkpoint inhibitors. However, these therapies have shown limited efficacy, and further advancements are needed before these modalities can progress to widespread use. Immune checkpoint inhibitors are a type of nonspecific cancer immunotherapy, and antitumor effects are only observed when cancer-specific T cells are found within the nonspecifically activated T-cell group. In order to facilitate the development of potent immunotherapies, selective enhancement of cancer-specific T cells is essential. In this report, we discuss current and future perspectives, including the latest clinical trials of cancer-specific immunotherapies, particularly cancer peptide vaccines.

Epidermal growth factor receptor (EGFR) is a prototypic cell-surface receptor belonging to the ErbB/HER onocogene family. Overexpression or somatic mutations of EGFR have been reported to play an important role in tumorigenesis in various types of epithelial cancers. Therefore, targeting of EGFR with specific blocking antibodies or inhibitors have been developing for treatment for EGFR-associated tumors. Immune responses to HER2, another molecule of the ErbB/HER onocogene family, have been well studied, but only limited information on the immune responses to EGFR in cancer has been currently available. In this review, we have summarized the available data and discussed potential clinical importance of the anti-EGFR immune responses and EGFR-mediated immune regulation in cancer. Several lines of evidence suggest that cellular and humoral immune responses to EGFR might be useful as a marker and/or target for cancer therapy against EGFR-associated tumors. In addition, recent studies suggest the critical roles of EGFR-mediated signaling in regulation of expression of an immune checkpoint molecule, programmed death-ligand 1 (PD-L1) in tumor cells. Further studies are warranted to clarify the impact of the anti-EGFR immune responses and EGFR-mediated immunomodulation for clinical application for cancer treatment.

Colorectal cancer (CRC) is one of the most increasing cancers worldwide, and novel treatment modalities remain to be developed for advanced CRC patients. In recent years, immune checkpoint inhibitors have demonstrated impressive clinical efficacy in various types of cancers, but limited clinical responses were reported in CRC, except for MSI-high or MMR-deficient tumors. It has thus been suggested that immune suppressive mechanisms other than immune checkpoints might contribute to tumor progression in CRC. In the current study, various types of immune cells were isolated from peripheral blood or resected tumor/normal tissues and regional lymph nodes in 25 primary CRC patients, and their phenotypes and functions were comprehensively assessed. Flow cytometry analysis demonstrated a significant increase of CD4+FoxP3+ T cells, which might represent regulatory T cells, in proximal lymph nodes (N1 region), compared to distal (N2 or N3 region) or unrelated lymph nodes. In addition, the functional analysis of T cells assessed by cytokine production after non-specific stimulation (PMA and A23187) showed a significant decrease in the frequencies of IFN γ-producing CD8+ T cells in N1 region. Notably, reduction of IFN γ-producing CD8+ T cells seemed to be associated with advanced stages of disease. These results indicate that the immunosuppressive microenvironment, particularly in regional lymph nodes, may be related to tumor progression in CRC. The current study suggests that monitoring of immunosuppressive microenvironment would be useful as a predictive biomarker for cancer progression and patient prognosis in CRC. Further studies remain to be performed to more thoroughly elucidate immune suppressive mechanisms, and thereby to develop a novel immunotherapeutic strategy in CRC. Citation Format: Tetsuro Sasada, Junya Ohtake, Tetsuta Satoyoshi, Keisuke Kazama, Satoshi Wada, Erika Yada, Manabu Shiozawa. Elucidation of immunosuppressive microenvironment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4739.

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.

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.

Neoantigens derived from tumor-specific genetic mutations can be recognized as foreign by the host immune system, and might be suitable as target for cancer immunotherapy possibly due to their higher immunogenicity. In this study, to really know the immunogenicity of tumor-specific neoantigens, we comprehensively investigated T cell responses against neoantigens derived from genetic mutations in gastric cancer. Using next-generation sequencing, 156 missense mutations were identified in tumor cells from two gastric cancer patients. From them, we selected 30 potentially immunogenic amino acid sequences, which were derived from the mutations and predicted to potentially bind to HLA-class I (A*0201, A*0206, or A*2402) by an epitope prediction server, IEBD. We synthesized 30 kinds of 27mer long peptides, in which the mutated sequences were located in the center, and then cultured peripheral blood mononuclear cells (PBMC) from healthy donors in the presence of the synthetic peptides to evaluate whether they could really induce antigen-specific T cell responses. In the analysis with PBMC from 18 healthy donors, 27/30 (90%) synthetic peptides showed an ability to induce antigen-specific T cell responses in at least one donor, assessed by cytokine production assay. Among them, 15 peptides were immunogenic in more than one donor. The antigen-specific T cell responses were detected more frequently in CD4+ T cells (70%) than in CD8+ T cells (43%). The specificity of T cell responses to mutated sequences, but not to the corresponding wild type sequences, were confirmed in 5 of 8 (63%) peptides examined. In addition, antigen-specific T cell responses induced by mutated peptides were shown to be much higher than those induced by the corresponding wild type peptides. These findings clearly demonstrated high immunogenicity and specificity of neoantigens derived from tumor-specific genetic mutations. Further studies would be recommended to develop a novel immunotherapeutic approach, “personalized cancer vaccination”, targeting mutation-derived neoantigens. Citation Format: Tetsuro Sasada, Junya Ohtake, Satoshi Wada, Erika Yada, Shintaro Yoshida. Comprehensive analysis of T cells responsive to neoantigens derived from tumor-specific genetic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2017-636

Wilms’ tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A*24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epit opes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A*24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A*24:02-restricted WT1-specific CTLs. Here we induced sever al WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vβ usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer pa tients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.

590 Background: Immunosuppression in tumor microenvironments is a critical issue for cancer immunotherapy. Recently, the effectiveness of immuno-check point therapy has been reported on various types of solid tumors. But, the effectiveness in colorectal cancer was poor compared to other cancers, such as melanoma and renal cell carcinoma. Correct regulation of dendritic cell (DC) function in tumors is important for inducing anti-tumor immunity. We have been demonstrated that IL-6 inhibits antigen presentation by DCs through STAT3 activation in tumor-bearing mice. In this study, we focused on the role of the IL-6/STAT3 signaling cascade in human DCs. Methods: Both IL-6 and pSTAT3 expressions in tumor sites of colorectal cancers was verified by immunohistostaining. CD11b+CD11c+DCs in cancer tissues and PBMCs were isolated by fluorescence-activated cell sorting system and investigated about the surface molecules such as HLA-DR, T cell stimulating ability, and effector gene expression levels. Moreover, we investigated influence of IL-6/STAT3 signaling in human DCs in vitro. Results: The results of IHC revealed that IL-6 was preferentially produced by cancer-associated fibroblasts and immune cells in the tissues of colorectal cancers. In addition, it was confirmed that STAT3 was activated in tumor-infiltrating immune cells. Tumor infiltrating CD11b+CD11c+ DCs highly induced IL-6 gene, down-regulated surface expression of HLA-DR, and attenuated T cell stimulating ability. In vitro experiments showed that IL-6-mediated STAT3 activation reduced surface expression of HLA-DR. COX2, cathepsin L (CTSL), and arginase activity were are involved in the IL-6-mediated down-regulation of surface expression levels of HLA-DR expression levels on DCs. Gene expressions of CTSL, ARG1 as well as IL6 in tumor infiltrating CD11b+CD11c+DCs were much higher than those of PBMCs. Conclusions: IL-6-mediated STAT3 activation inhibits functional maturation of DCs, causing suppression of anti-tumor immunity in colorectal cancer. Therefore, inhibition of the IL-6/STAT3 signaling pathway could be a promising strategy for improving immunotherapies for colorectal cancer patients.

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

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

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

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

Neurotransmitters such as Substance P (SP) and Neurokinin A (NKA) are widely distributed in both central and peripheral nervous system. Recently, we demonstrated that IFN-γ remarkably induced neurokinin-2 receptor (NK2R) expression in human dendritic cells (DCs). These findings strongly suggested that neuropeptide-signaling might regulate function of human DCs DC-mediated immune responses. In this study, we examined the effect of neuropeptide signaling on DC function. Human DCs generated from peripheral blood mononuclear cells (PBMCs) were treated with various type I cytokines or toll-like receptor ligands. Both neurokinin-1 receptor (NK1R) and NK2R mRNA expressions in DCs were significantly enhanced by poly I:C stimulation. The upregulation of NK1R and NK2R mRNA expressions was blocked in the presence of STAT-1 inhibitor. Surface expression levels of HLA-DR and costimulatory molecules on DCs, augmented by poly I:C, were modulated by NK1R or NK2R inhibitor. In addition, we found that blockade of NK1R- and NK2R-mediated signaling significantly suppressed various cytokine productions by antigen-specific CD4+ T cells stimulated with DCs. These findings indicate that NK1R- and NK2R-dependent neuropeptide signaling regulate Type-1 immunity through the activation of DCs, suggesting that such neuro-immune cross-talk through NK1R and NK2R signaling might be involved in various diseases with chronic inflammation caused by excessive Type-1-dominant immunity.

Immunosuppression in tumor microenvironments is one of the critical issues for cancer immunotherapy. We have been demonstrated that IL-6, a pleiotropic cytokine that regulate the growth, differentiation and survival of a variety of cells, significantly inhibited maturation of murine dendritic cells (DCs) via STAT3 activation both in vitro and in vivo. In this study, we focused on IL-6/STAT3-signaling cascade in human dendritic cells (DCs), and investigated the effects of IL-6 on antigen-presenting ability of DCs. Surface HLA class I, HLA class II, and costimulatory molecules of human DCs were evaluated after the stimulation with IL-6 in vitro. As a result, HLA-DR and CD86 expressions were significantly reduced by IL-6 treatment of human DCs. The reduction was remarkably blocked in the presence of STAT3 inhibitor. In addition, IFN-γ production by T cells after TCR-stimulation was significantly suppressed in the presence of IL-6-treated DCs compared with control DCs. Moreover, we found that activation of antigen-specific CD4+ T cells by DCs was remarkably reduced by the IL-6-treatment. In this study, we demonstrated that IL-6/STAT3-signaling cascade was one of the regulating factors for antigen presentation of DCs. From these findings suggest that IL-6/STAT3 signaling cascade regulates immunosuppressive function of human DCs, which would be a promising target for improving the effects of cancer immunotherapy.

We have proposed that IL-17 produced by γδ T cells promoted angiogenesis and subsequent tumor progression in tumor microenvironment. However, the precise effect of IL-17 on immune system was remained unclear. Since dendritic cells (DCs) play a crucial role in immune regulation, we investigated the impact of IL-17 on DC function. Bone marrow-derived DCs were treated with IL-17 for 16 h and then stimulated with various toll-like receptor (TLR) ligands. IL-17 treatment significantly reduced IL-12, TNF-α, or IL-6 production by the DCs in a dose dependent manner. Interestingly, we found that IL-12 production was most clearly decreased compared to the other cytokines. In addition, IL-17 markedly suppressed the upregulation of MHC class II, CD40, and CD86 expressions on DCs after the stimulation. To evaluate effects of IL-17 on T cell-stimulating activity, IL-17-treated DCs were co-cultured with OVA antigen-specific T cells in the presence of OVA peptides or OVA proteins. We confirmed that IFN-γ production and T cell proliferation were suppressed by the IL-17-treatment. To determine effects of IL-17 on in vivo DC function, we established a novel tumor-bearing model using IL-17-producing cancer cells. As a result, expression levels of MHC class II and costimulatoly molecules on DCs were reduced in this model. These findings clearly indicated that IL-17 would inhibit activation of DCs including cytokine production and T cell activation in tumor microenvironment.