Anti-oxidative Amino Acid L-ergothioneine Modulates the Tumor Microenvironment to Facilitate Adjuvant Vaccine Immunotherapy.

Cancer vaccines consist of a tumor-associated antigen (TAA) and adjuvant. These vaccines induce and activate proliferation of TAA-specific cytotoxic T lymphocytes (CTLs), suppressing tumor growth. The therapeutic efficacy of TAA-specific CTLs depends on the properties of tumor microenvironment. The environments make immunosuppressive by function of regulatory T cells and tumor-associated myeloid cells; thus, regulation of these cells is important for successful cancer immunotherapy. We report here that L-ergothioneine (EGT) with the adjuvant Toll-like receptor 2 (TLR2) ligand modulated suppressive microenvironments to be immune-enhancing. EGT did not augment DC-mediated CTL priming or affect CTL activation in draining lymph node and spleen. However, EGT decreased the immuno-suppressive function of tumor-associated macrophages (TAMs). TLR2 stimulation accompanied with EGT administration downregulated expression of PD-L1, CSF-1R, arginase-1, FAS ligand, and TRAIL in TAMs, reflecting reduction of CTL suppression. An anti-oxidative thiol-thione residue of EGT was essential to dampening CTL suppression. The effect was specific to the thiol-thione residue of EGT because no effect was observed with another anti-oxidant N-acetyl-L-cysteine (NAC). A CTL-suppressive environment made by TLR2 is relieved to be improved by the addition of EGT, which may ameliorate the efficacy of vaccine immunotherapy.

Vaccine adjuvant ARNAX promotes mucosal IgA production in influenza HA vaccination.

Adjuvant stimulates pattern-recognition receptors (PRRs) expressed by dendritic cells, which causes immune-enhancing of T lymphocytes. Adjuvant also induces innate immune response in whole-body cells via PRRs to evoke cytokinemia. A cytokine-mediated immune response is important for the systemic protection of a host from microbial infections. Using an influenza subcomponent vaccine in a mouse model, we intranasally administered a TLR3-specific adjuvant ARNAX + HA split vaccine to mice. ARNAX efficiently induced mucosal IgA and systemic IgG production by nasal drop. Moreover, ARNAX + HA simultaneously induced CD8 and CD4 T cell activation. We have previously shown that ARNAX does not induce harmful systemic cytokine production. Thus, our findings indicate that the ARNAX + HA vaccine is a harmless prophylactic vaccine for flu that induces HA-specific T cell activation and IgA/IgG production. These results suggested that ARNAX + antigen enhanced the immune response without inducing inflammatory toxicity for vaccination against infectious diseases.

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models.

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll-like receptor (TLR)3-adjuvant and tumor antigen overcomes anti-programmed death ligand-1 (PD-L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)-expressing tumor cell lines were implanted into syngeneic mice and subjected to anti-tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3-specific agonist that does not activate the mitochondrial antiviral-signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD-L1-low cell line of EG7. Addition of anti-PD-L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD-L1-high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor-infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD-L1. The tumor-infiltrating immune cells in ARNAX-susceptible tumors contained fewer immunosuppressive myeloid cells with low PD-L1 expression. Combination with anti-PD-L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8+ T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti-PD-L1 therapy enabled permanent remission against some tumors that stably present antigens.

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy.

The immune system eliminates advanced cancer when treated with programmed cell death protein-1 (PD-1) or its ligand (PD-L1) blockade, but PD-1 therapy is effective in only ∼20% of patients with solid cancer. The PD-1 antibody mainly acts on the effector phase of cytotoxic T lymphocytes (CTLs) in tumors but induces no activation of the priming phase of antigen-presenting dendritic cells (DCs). It is reasonable that both DC-priming and PD-1/L1 blocking are mandatory for efficient CTL-mediated tumor cytolysis. For DC-priming, a therapeutic vaccine containing Toll-like receptor (TLR) agonists, namely a priming adjuvant, is a good candidate; however, a means for DC-targeting by TLR adjuvant therapy remains to be developed. TLR adjuvants usually harbor cytokine toxicity, which is a substantial barrier against drug approval. Here, we discuss the functional properties of current TLR adjuvants for cancer immunotherapy and introduce a TLR3-specific adjuvant (ARNAX) that barely induces cytokinemia in mouse models.

Toll-like receptor 3 signal augments radiation-induced tumor growth retardation in a murine model.

Radiotherapy induces anti-tumor immunity by induction of tumor antigens and damage-associated molecular patterns (DAMP). DNA, a representative DAMP in radiotherapy, activates the stimulator of interferon genes (STING) pathway which enhances the immune response. However, the immune response does not always parallel the inflammation associated with radiotherapy. This lack of correspondence may, in part, explain the radiation-resistance of tumors. Additive immunotherapy is expected to revive tumor-specific CTL facilitating radiation-resistant tumor shrinkage. Herein pre-administration of the double-stranded RNA, polyinosinic-polycytidylic acid (polyI:C), in conjunction with radiotherapy, was shown to foster tumor suppression in mice bearing radioresistant, ovalbumin-expressing Lewis lung carcinoma (LLC). Extrinsic injection of tumor antigen was not required for tumor suppression. No STING- and CTL-response was induced by radiation in the implant tumor. PolyI:C was more effective for induction of tumor growth retardation at 1 day before radiation than at post-treatment. PolyI:C targeted Toll-like receptor 3 with minimal effect on the mitochondrial antiviral-signaling protein pathway. Likewise, the STING pathway barely contributed to LLC tumor suppression. PolyI:C primed antigen-presenting dendritic cells in draining lymph nodes to induce proliferation of antigen-specific CTL. By combination therapy, CTL efficiently infiltrated into tumors with upregulation of relevant chemokine transcripts. Batf3-positive DC and CD8+ T cells were essential for therapeutic efficacy. Furthermore, polyI:C was shown to stimulate tumor-associated macrophages and release tumor necrosis factor alpha, which acted on tumor cells and increased sensitivity to radiation. Hence, polyI:C treatment prior to radiotherapy potentially induces tumor suppression by boosting CTL-dependent and macrophage-mediated anti-tumor responses. Eventually, polyI:C and radiotherapy in combination would be a promising therapeutic strategy for radiation-resistant tumors.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

BACKGROUND: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. METHODS: We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. RESULTS: We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. CONCLUSION: These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.

The Anti-Oxidant Ergothioneine Augments the Immunomodulatory Function of TLR Agonists by Direct Action on Macrophages.

L-Ergothioneine (EGT) is a naturally-occurring amino acid which is characterized by its antioxidant property; yet, the physiological role of EGT has yet to be established. We investigated the immune-enhancing properties of EGT, and found that it acts as a potentiator of toll-like receptor (TLR) signaling. When mouse bone marrow-derived macrophages (BMDMs) were pretreated with EGT, TLR signal-mediated cytokine production was augmented in BMDMs. The results were reproducible with TLR2, 3, 4 and 7 agonists. In particular, IL-6 and IL-12p40 were elevated further by pretreatment with EGT in BMDMs, suggesting the induction of M1 polarization. In co-culture assay with OT-II CD4+ T cells and splenic F4/80+ macrophages, EGT significantly induced Th17 skewing in CD4+ T cells. Thus, EGT is an immune modifier as well as a redox controller under TLR stimulation that induces M1 macrophages and a Th17 shift in inflammation.

Toll-like receptor 2 ligand and interferon-γ suppress anti-tumor T cell responses by enhancing the immunosuppressive activity of monocytic myeloid-derived suppressor cells.

CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) suppress activation/proliferation of cytotoxic T cells, thereby hindering cancer immunotherapy. MDSCs are increased after adjuvant therapy with toll-like receptor (TLR) 2 ligands, such as Pam2CSK4, in tumor-bearing mice. However, it remains unknown if the activation of TLR2 in MDSCs affects their function and the therapeutic efficacy of TLR2 ligand. Here, we show that TLR2 signaling in CD11b+Ly6G-Ly6Chigh monocytic MDSCs (M-MDSCs), but not CD11b+Ly6G+Ly6Clow granulocytic MDSCs (G-MDSCs), enhances their immunosuppressive activity, thereby limiting anti-tumor T cell responses induced by TLR2-activated dendritic cells (DCs). iNOS induction was critical for Pam2CSK4-enhanced T cell suppression by M-MDSCs. iNOS was expressed in M-MDSC-derived macrophages, but not undifferentiated M-MDSCs, in cocultures with CD8+ T cells, CD11c+ DCs, antigen peptide and Pam2CSK4. Pam2CSK4 increased the differentiation frequency of M-MDSCs to macrophages, and iNOS expression required interferon-γ (IFN-γ) production by CD8+ T cells that had been transiently stimulated by M-MDSC-derived macrophages in an antigen/TLR2-dependent manner. Although Pam2CSK4 triggered DC maturation and tumor regression via induction of tumor antigen-specific cytotoxic T lymphocyte (CTL) responses in tumor-bearing mice, Pam2CSK4 plus antigen increased the frequency of iNOS+ macrophages in the tumor. Treatment with iNOS inhibitor enhanced the therapeutic efficacy of Pam2CSK4. Hence, the results suggest that TLR2 ligand and T cell-derived IFN-γ enhance M-MDSC-mediated immunosuppression, which may negatively regulate anti-tumor CTL response.

First Successful Pre-Distribution of Stable Iodine Tablets Under Japan's New Policy After the Fukushima Daiichi Nuclear Accident.

Stable iodine tablets are effective in reducing internal exposure to radioactive iodine, which poses a risk for thyroid cancer and other conditions. After the Fukushima Daiichi nuclear power plant accident, the Japanese government shifted its policy on stable iodine tablet distribution from "after-the-fact" to "before-the-fact" and instructed local governments to pre-distribute stable iodine tablets to residents living within a 5-km radius of nuclear facilities. The nation's first pre-distribution of stable iodine tablets was carried out in June and July of 2014 in Kagoshima Prefecture. Health surveys were conducted so that the medication would not be handed out to people with the possibility of side effects. Of the 4715 inhabitants in the area, 132 were found to require a physician's judgment, mostly to exclude risks of side effects. This was considered important to prevent the misuse of the tablets in the event of a disaster. The importance of collective and individualized risk communication between physicians and inhabitants at the community health level was apparent through this study. Involvement of physicians through the regional Sendai City Medical Association was an important component of the pre-distribution. Physicians of the Sendai City Medical Association were successfully educated by using the Guidebook on Distributing and Administering Stable Iodine Tablets prepared by the Japan Medical Association and Japan Medical Association Research Institute with the collaboration of the National Institute of Radiological Sciences and the Japanese government. Thus, the physicians managed to make decisions on the dispensing of stable iodine tablets according to the health conditions of the inhabitants. All physicians nationwide should be provided continuing medical education on stable iodine tablets. (Disaster Med Public Health Preparedness. 2017;11:365-369).

The Communication of Information Such as Evacuation Orders at the Time of a Nuclear Power Station Accident: -Recommendations for responses by the national government and electric power utilities to the "Information Disaster".

This research was carried out from the perspective that the damage to the people of Fukushima and others from the Fukushima Daiichi Nuclear Power Station (NPS) accident was an "information disaster." It evaluated the critical problems raised by and actual condition analysis on the process of events in the Fukushima Daiichi NPS disaster and responses of the governments and others, notification of the occurrence of the accident and evacuation order by the national and local governments and the evacuation of residents, and guidance for distribution and intake of stable iodine tablets. The research aimed to provide a basis for the implementation of effective distribution and intake of stable iodine tablets and responses to the "information disaster" in the nuclear power disaster. On March 15 at the time that the most radioactive substances were dispersed, even when the average wind speed at the site area was 1.6 m/s, the radioactive substances had reached the outer boundary of Urgent Protective action planning Zone (UPZ, the region with a radius of 30 km) within about five hours. Because of this, every second counted in the provision of information about the accident and the issuance of evacuation orders. This study evaluated the actual condition of information provision by the national government and others from the perspective of this awareness of the importance of time. On the basis of the results of this kind of consideration, we come to the following recommendations: The Nuclear Emergency Response Guidelines and the system for communication of information to medical providers should be revised. The national government should make preparations for the effective advance distribution and intake of stable iodine tablets.