Two Modes of Th1 Polarization Induced by Dendritic-Cell-Priming Adjuvant in Vaccination.

Viral infections are usually accompanied by systemic cytokinemia. Vaccines need not necessarily mimic infection by inducing cytokinemia, but must induce antiviral-acquired immunity. Virus-derived nucleic acids are potential immune-enhancers and particularly good candidates as adjuvants in vaccines in mouse models. The most important nucleic-acid-sensing process involves the dendritic cell (DC) Toll-like receptor (TLR), which participates in the pattern recognition of foreign DNA/RNA structures. Human CD141+ DCs preferentially express TLR3 in endosomes and recognize double-stranded RNA. Antigen cross-presentation occurs preferentially in this subset of DCs (cDCs) via the TLR3-TICAM-1-IRF3 axis. Another subset, plasmacytoid DCs (pDCs), specifically expresses TLR7/9 in endosomes. They then recruit the MyD88 adaptor, and potently induce type I interferon (IFN-I) and proinflammatory cytokines to eliminate the virus. Notably, this inflammation leads to the secondary activation of antigen-presenting cDCs. Hence, the activation of cDCs via nucleic acids involves two modes: (i) with bystander effect of inflammation and (ii) without inflammation. In either case, the acquired immune response finally occurs with Th1 polarity. The level of inflammation and adverse events depend on the TLR repertoire and the mode of response to their agonists in the relevant DC subsets, and could be predicted by assessing the levels of cytokines/chemokines and T cell proliferation in vaccinated subjects. The main differences in the mode of vaccine sought in infectious diseases and cancer are defined by whether it is prophylactic or therapeutic, whether it can deliver sufficient antigens to cDCs, and how it behaves in the microenvironment of the lesion. Adjuvant can be selected on a case-to-case basis.

Effects of elevated ozone and carbon dioxide on the dynamic photosynthesis of Fagus crenata seedlings under variable light conditions.

Ozone (O3) is an air pollutant that is toxic to trees. O3 reduces steady-state net photosynthetic rate (A), and the adverse effects of O3 are mitigated under elevated CO2 condition. However, the combined effects of O3 and elevated CO2 on dynamic photosynthesis under variable light conditions have not yet been clarified. In this study, we investigated the effects of O3 and elevated CO2 on dynamic photosynthesis in the leaves of Fagus crenata seedlings under variable light conditions. The seedlings were grown under four gas treatments comprising two levels of O3 concentration (lower and two times higher than the ambient O3 concentration) and two levels of CO2 concentration (ambient and 700 ppm). Although O3 significantly decreased steady-state A under ambient CO2 concentrations, no significant decrease was observed under elevated CO2 concentrations, indicating the mitigating effect of elevated CO2 on O3-induced adverse effects on steady-state A. During photosynthetic induction, the response of A to the change in photosynthetic photon flux density (PPFD) from 50 (low light) to 1000 µmol m-2 s-1 (high light) showed that the increase in A was slowed by O3 and accelerated by elevated CO2. Under fluctuating light condition of repeating low light for 4 min and high light for 1 min, A at end of each high light period gradually decreased in all treatments, and O3 and elevated CO2 accelerated the reduction of A. In contrast to steady-state A, no mitigating effect of elevated CO2 was observed for any parameters related to dynamic photosynthesis. We conclude that the combined effects of O3 and elevated CO2 on A of F. crenata are different under steady-state and variable light conditions, and the O3-induced decrease in leaf A may not be mitigated by elevated CO2 in the field under variable light conditions.

Toward Establishing an Ideal Adjuvant for Non-Inflammatory Immune Enhancement.

The vertebrate immune system functions to eliminate invading foreign nucleic acids and foreign proteins from infectious diseases and malignant tumors. Because pathogens and cancer cells have unique amino acid sequences and motifs (e.g., microbe-associated molecular patterns, MAMPs) that are recognized as "non-self" to the host, immune enhancement is one strategy to eliminate invading cells. MAMPs contain nucleic acids specific or characteristic of the microbe and are potential candidates for immunostimulants or adjuvants. Adjuvants are included in many vaccines and are a way to boost immunity by deliberately administering them along with antigens. Although adjuvants are an important component of vaccines, it is difficult to evaluate their efficacy ex vivo and in vivo on their own (without antigens). In addition, inflammation induced by currently candidate adjuvants may cause adverse events, which is a hurdle to their approval as drugs. In addition, the lack of guidelines for evaluating the safety and efficacy of adjuvants in drug discovery research also makes regulatory approval difficult. Viral double-stranded (ds) RNA mimics have been reported as potent adjuvants, but the safety barrier remains unresolved. Here we present ARNAX, a noninflammatory nucleic acid adjuvant that selectively targets Toll-like receptor 3 (TLR3) in antigen-presenting dendritic cells (APCs) to safely induce antigen cross-presentation and subsequently induce an acquired immune response independent of inflammation. This review discusses the challenges faced in the clinical development of novel adjuvants.

Prophylactic Vaccine Targeting TLR3 on Dendritic Cells Ameliorates Eosinophilic Pneumonia in a Mouse SARS-CoV Infection Model.

Putative subcomponent vaccines of severe acute respiratory syndrome coronavirus spike protein and ARNAX (TLR3-specific adjuvant for priming dendritic cells) were examined and compared with spike protein + Alum in a mouse BALB/c model. Survival, body weight, virus-neutralizing Ab titer in the blood, and viral titer in the lung were evaluated for prognosis markers. The infiltration degrees of eosinophils in the lung were histopathologically monitored at 10 d postinfection. The results were: (1) adjuvant was essential in vaccines to achieve a complete recovery from infection, (2) ARNAX displayed optimal body weight recovery compared with Alum, (3) ARNAX was optimal for the amelioration of eosinophilic pneumonia, and (4) the eosinophil infiltration score was not associated with the neutralizing Ab titer in the blood or viral titer in the lung. Although the pathological link between the TLR3 vaccine and lung eosinophil infiltration remains unclear, severe acute respiratory syndrome-mediated eosinophilic pneumonia can be blocked by the prior induction of dendritic cell priming by ARNAX.

Growth and photosynthetic responses to ozone of Siebold's beech seedlings grown under elevated CO2 and soil nitrogen supply.

Ozone (O3) is a phytotoxic air pollutant, the adverse effects of which on growth and photosynthesis are modified by other environmental factors. In this study, we examined the combined effects of O3, elevated CO2, and soil nitrogen supply on Siebold's beech seedlings. Seedlings were grown under combinations of two levels of O3 (low and two times ambient O3 concentration), two levels of CO2 (ambient and 700 ppm), and three levels of soil nitrogen supply (0, 50, and 100 kg N ha-1 year-1) during two growing seasons (2019 and 2020), with leaf photosynthetic traits being determined during the second season. We found that elevated CO2 ameliorated O3-induced reductions in photosynthetic activity, whereas the negative effects of O3 on photosynthetic traits were enhanced by soil nitrogen supply. We observed three-factor interactions in photosynthetic traits, with the ameliorative effects of elevated CO2 on O3-induced reductions in the maximum rate of carboxylation being more pronounced under high than under low soil nitrogen conditions in July. In contrast, elevated CO2-induced amelioration of the effects of O3 on stomatal function-related traits was more pronounced under low soil nitrogen conditions. Although we observed several two- or three-factor interactions of gas and soil treatments with respect to leaf photosynthetic traits, the shoot to root dry mass (S/R) ratio was the only parameter for which a significant interaction was detected among seedling growth parameters. O3 caused a significant increase in S/R under ambient CO2 conditions, whereas no similar effects were observed under elevated CO2 conditions. Collectively, our findings reveal the complex interactive effects of elevated CO2 and soil nitrogen supply on the detrimental effects of O3 on leaf photosynthetic traits, and highlight the importance of taking into consideration differences between the responses of CO2 uptake and growth to these three environmental factors.

Targeting Toll-like receptor 3 in dendritic cells for cancer immunotherapy.

INTRODUCTION: Activation of innate immune system is a key step to develop anti-tumor immunity. Antigen-presenting dendritic cells (DCs) cross-present tumor-associated antigens to cytotoxic CD8+ T cells (CTLs). Signaling from pattern-recognition receptors (PRRs) in DCs is required to induce tumor-specific CTLs. AREAS COVERED: This review summarizes the properties of PRRs expressed by antigen-presenting DCs, especially TLR3, and provides the recent knowledge of their function in anti-tumor immunity. We also summarize the characteristics of newly-developed TLR3-specific agonist, ARNAX, which efficiently primes DCs to induce anti-tumor immunity without systemic inflammation in mice. EXPERT OPINION: In cancer immunotherapy, the induction of tumor-specific CTLs is significant for tumor regression and to augment the efficacy of PD-1/PD-L1 blockade. Non-inflammatory TLR3 adjuvant ARNAX that can induce tumor-specific CTLs without inducing inflammation benefits cancer immunotherapy. Development of appropriate protocols for ARNAX vaccine therapy would be useful to overcome the PD-1/PD-L1 blockade resistance.

Phospholipase A2 from bee venom increases poly(I:C)-induced activation in human keratinocytes.

Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly(I:C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly(I:C)-mediated interleukin 8 production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly(I:C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly(I:C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.

A Toll-like receptor 3 (TLR3) agonist ARNAX for therapeutic immunotherapy.

Vaccine immunotherapy consisting of tumor antigens combined with an immune-enhancing adjuvant fosters cytotoxic T cell (CTL) proliferation. Clinically, polyI:C has been used as an adjuvant to enhance cancer vaccine protocols. However, according to its long history, polyI:C promotes inflammation that causes cytokine toxicity. Although checkpoint inhibitor immunotherapy has improved the prognoses of patients with progressive cancer, over 75% of patients continue to experience resistance to antibody (Ab) against anti-programmed cell death-protein 1 (PD-1) or its ligand, PD-L1 therapy. In most cases, patients suffer from adverse events resulting from inflammation during anti-PD-1/L1 Ab therapy, which is a serious obstacle to patients' quality of life. We have studied the functional properties of double-stranded (ds)RNA and polyI:C, and developed a nucleic acid adjuvant that barely induces a significant increase in the level of serum inflammatory cytokines in mouse models. This adjuvant, termed ARNAX, consists of DNA-capped dsRNA that specifies the endosomal target for Toll-like receptor 3 (TLR3) in dendritic cells (DCs). We expect that this adjuvant is safe for administration in elderly patients with cancer receiving immunotherapy. Here, we summarize the properties of ARNAX for immunotherapy in mice. We suggest that DC-priming is essential to induce anti-tumor immunity; neither exogenous inflammation nor the administration of tumor antigens is always a prerequisite for DC-mediated CTL proliferation. If our mouse data can be extrapolated to humans, ARNAX and the liberated endogenous tumor antigens may facilitate effect of current therapies on patients with therapy-resistant tumors.

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.

Extracellular RNA Sensing by Pattern Recognition Receptors.

RNA works as a genome and messenger in RNA viruses, and it sends messages in most of the creatures of the Earth, including viruses, bacteria, fungi, plants, and animals. The human innate immune system has evolved to detect single- and double-stranded RNA molecules from microbes by pattern recognition receptors and induce defense reactions against infections such as the production of type I interferons and inflammatory cytokines. To avoid cytokine toxicity causing chronic inflammation or autoimmunity by sensing self-RNA, the activation of RNA sensors is strictly regulated. All of the Toll-like receptors that recognize RNA are localized to endosomes/lysosomes, which require internalization of RNA for sensing through an endocytic pathway. RIG-I-like receptors sense RNA in cytosol. These receptors are expressed in a cell type-specific fashion, enabling sensing of RNA for a wide range of microbial invasions. At the same time, both endosomal and cytoplasmic receptors have strategies to respond only to RNA of pathogenic microorganisms or dying cells. RNA are potential vaccine adjuvants for immune enhancement against cancer and provide a benefit for vaccinations. Understanding the detailed molecular mechanisms of the RNA-sensing system will help us to broaden the clinical utility of RNA adjuvants for patients with incurable diseases.

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.

The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells.

The TLR2 agonist, dipalmitoyl lipopeptide (Pam2LP), has been used as an immune adjuvant without much success. Pam2LP is recognised by TLR2/6 receptors in humans and in mice. This study examined the proliferative activity of cytotoxic T lymphocytes (CTL) using mouse Ag-presenting dendritic cells (DCs) and OT-I assay system, where a library of synthetic Pam2LP was utilised from the Staphylococcus aureus database. Ag-specific CTL expansion and IFN-γ levels largely depended on the Pam2LP peptide sequence. The first Aa is cysteine (Cys), which has an active SH residue to bridge fatty acids, and the second and third Aa are hydrophilic or non-polar. The sequence structurally adapted to the residual constitution of the reported TLR2/6 pocket. The inactive sequence contained proline or leucine/isoleucine after the first Cys. Notably, no direct activation of OT-I cells was detected without DCs by stimulation with the active Pam2LP having the Cys-Ser sequence. MyD88, but not TICAM-1 or IFN pathways, in DCs participates in DC maturation characterised by upregulation of CD40, CD80 and CD86. Hence, the active Pam2LPs appear suitable for dimeric TLR2/6 on DCs, resulting in induction of DC maturation.

Mucosal Immune Response in Nasal-Associated Lymphoid Tissue upon Intranasal Administration by Adjuvants.

The nasal administration of vaccines directed against diseases caused by upper respiratory tract infections of pathogens, such as the influenza virus, mimics the natural infection of pathogens and induces immunoglobulin A (IgA) production in the nasal cavity to effectively protect viral entry. Therefore, the development of a nasally administered vaccine is a research objective. Because the antigenicity of influenza split vaccines is low, nasal inoculation with the vaccine alone does not induce strong IgA production in the nasal cavity. However, the addition of adjuvants activates the innate immune response, enhancing antigen-specific IgA production and the T-cell response. Although the development of suitable adjuvants for nasal vaccinations is in progress, the mechanism by which adjuvants promote the immune response is still unclear. In this review, we discuss the mucosal immune response, especially in the nasal-associated lymphoid tissue, induced in response to the intranasal inoculation of an influenza vaccine and adjuvants in animal models.

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.

TICAM-1 is dispensable in STING-mediated innate immune responses in myeloid immune cells.

Stimulator of interferon genes (STING) is an essential molecule for the production of type I interferon (IFN), and other inflammatory cytokines, in response to cytosolic DNA. STING contributes to host defense against infection and anti-tumor responses. Previous reports have demonstrated that STING signaling is required by the adaptor Toll-IL-1 receptor-containing adaptor molecule-1 (TICAM-1), which has been identified as a TLR3-adaptor molecule using mouse embryonic fibroblasts. Here, we demonstrate that TICAM-1 does not affect STING-mediated innate immune responses, as increases in the mRNA expression levels of IFN-ß, IL-6, and CCL5 were observed in bone marrow-derived or splenic myeloid cells. Moreover, STING ligand-enhanced co-stimulatory molecule expression, including CD80, CD86, and CD40, was detected on splenic CD11c + DCs, even in Ticam-1-deficient mice. Our results suggest that STING-mediated innate immune responses and dendritic cell maturation do not require TICAM-1 in myeloid lineage immune cells. TICAM-1 is ubiquitously expressed, even in cell types which do not express TLR3. Therefore, TICAM-1 may possess different functions depending on cell type and signaling purposes.

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.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

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.

DNAJB1/HSP40 Suppresses Melanoma Differentiation-Associated Gene 5-Mitochondrial Antiviral Signaling Protein Function in Conjunction with HSP70.

Melanoma differentiation-associated gene 5 (MDA5) is a pattern recognition receptor that recognizes cytoplasmic viral double-stranded RNA (dsRNA) and initiates rapid innate antiviral responses. MDA5 forms a filament-like multimer along the dsRNA leading to oligomerization, which in turn activates the adaptor protein mitochondrial antiviral signaling protein (MAVS) to provide a signal platform for the induction of type I interferon (IFN) and proinflammatory cytokines. The conformational switch of MDA5 causes antiviral defense, but excessive activation of the MDA5-MAVS pathway may result in autoimmune diseases. The regulatory mechanisms of MDA5 activation remain largely unknown. By yeast 2-hybrid, we identified DNAJB1, a member of the HSP40 (heat shock protein 40) family, as an MDA5-binding protein. HSP40s usually cowork with HSP70s. We found that dsRNA stimulation with physiological conditions upregulated the expression levels of DNAJB1 and HSP70; then the proteins were coupled and translocated into the stress granules, where MDA5 encounters dsRNA. DNAJB1 disrupted MDA5 multimer formation, resulting in the suppression of type I IFN induction. The disruption of endogenous DNAJB1 increased MDA5- and MAVS-mediated IFN promoter activation and rendered cells virus resistant. HSP70 inhibitor also enhanced the IFN-inducing function of MDA5 and MAVS. These results suggest that the DNAJB1-HSP70 complex functions for the natural maintenance of RNA sensing by interacting with MDA5/MAVS.

cGAMP Promotes Germinal Center Formation and Production of IgA in Nasal-Associated Lymphoid Tissue.

Induction of immunoglobulin (Ig) A in the mucosa of the upper respiratory tract and the nasal cavity protects against influenza virus infection. Cyclic dinucleotides (CDNs) are used as mucosal adjuvants to enhance the immunogenicity of intranasal influenza hemagglutinin (HA) vaccines. The adjuvant activity of 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) on Ig production was investigated in nasal-associated lymphoid tissue (NALT), serum of wild-type C57BL/6J, and stimulator of interferon genes (STING)-deficient mice, which do not recognize cGAMP. Mice were vaccinated intranasally with a HA vaccine with or without the cGAMP adjuvant. IgA and IgG production, T-cell responses, germinal center formation, and cytokine expression in NALT were assayed. cGAMP enhanced IgA and IgG production, and promoted T-cell responses. Intranasal administration of cGAMP activated both NALT and systemic immune cells, induced a favorable cytokine environment for IgA induction, and promoted germinal center formation. The cGAMP effect was STING-dependent. Taken together, cGAMP as an HA vaccine adjuvant promoted a STING-dependent NALT environment suitable for the enhancement of IgA production.