Near-infrared photoimmunotherapy as a complementary modality to in situ vaccine in a preclinical pancreatic cancer model.

Pancreatic cancer is one of the most refractory malignancies. In situ vaccines (ISV), in which intratumorally injected immunostimulatory adjuvants activate innate immunity at the tumor site, utilize tumor-derived patient-specific antigens, thereby allowing for the development of vaccines in patients themselves. Near-infrared photoimmunotherapy (NIR-PIT) is a novel therapy that selectively kills cancer cells exclusively in the NIR-irradiated region. Extending our previous research showing that ISV using the unique nanoparticulate Toll-like receptor 9 (TLR9) ligand K3-SPG induced effective antitumor immunity, here we incorporated NIR-PIT into K3-SPG-ISV so that local tumor destruction by NIR-PIT augments the antitumor effect of ISV. In the mouse model of pancreatic cancer, the combination of K3-SPG-ISV and CD44-targeting NIR-PIT showed synergistic systemic antitumor effects and enhanced anti-programmed cell death-1 (PD-1) blockade. Mechanistically, strong intratumoral upregulation of interferon-related genes and dependency on CD8+ T cells were observed, suggesting the possible role of interferon and cytotoxic T cell responses in the induction of antitumor immunity. Importantly, this combination induced immunological memory in therapeutic and neoadjuvant settings. This study represents the first attempt to integrate NIR-PIT with ISV, offering a promising new direction for cancer immunotherapy, particularly for pancreatic cancer.

5,6-dimethylxanthenone-4-acetic acid (DMXAA), a partial STING agonist, competes for human STING activation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

Akkermansia muciniphila induces slow extramedullary hematopoiesis via cooperative IL-1R/TLR signals.

Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the spleen, a process termed extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria regulate not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbes on hematopoietic pathology remains unclear. Here, we find that systemic single injections of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium, rapidly activate BM myelopoiesis and slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we term delayed EMH. Mechanistically, delayed EMH triggered by A. m. is mediated entirely by the MYD88/TRIF innate immune signaling pathway, which persistently stimulates splenic myeloid cells to secrete interleukin (IL)-1α, and in turn, activates IL-1 receptor (IL-1R)-expressing splenic HSPCs. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) or IL-1α partially diminishes A. m.-induced delayed EMH, while inhibition of both pathways alleviates splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Deleting interleukin-10 from myeloid cells exacerbates atherosclerosis in Apoe-/- mice.

Atherosclerosis is initiated by subendothelial retention of lipoproteins and cholesterol, which triggers a non-resolving inflammatory process that over time leads to plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary drivers of the inflammatory response and plaque formation. Several immune cells including macrophages, T cells and B cells secrete the anti-inflammatory cytokine IL-10, known to be essential for the atherosclerosis protection. The cellular source of IL-10 in natural atherosclerosis progression is unknown. This study aimed to determine the main IL10-producing cell type in atherosclerosis. To do so, we crossed VertX mice, in which IRES-green fluorescent protein (eGFP) was placed downstream of exon 5 of the Il10 gene, with atherosclerosis-prone Apoe-/- mice. We found that myeloid cells express high levels of IL-10 in VertX Apoe-/- mice in both chow and western-diet fed mice. By single cell RNA sequencing and flow cytometry analysis, we identified resident and inflammatory macrophages in atherosclerotic plaques as the main IL-10 producers. To address whether IL-10 secreted by myeloid cells is essential for the protection, we utilized LyzMCre+Il10fl/fl mice crossed into the Apoe-/- background and confirmed that macrophages were unable to secrete IL-10. Chow and western diet-fed LyzMCre+Il10fl/fl Apoe-/- mice developed significantly larger atherosclerotic plaques as measured by en face morphometry than LyzMCre-Il10 fl/flApoe-/-. Flow cytometry and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated CCL2, and TNFα in blood plasma. We conclude that macrophage-derived IL-10 is critical for limiting atherosclerosis in mice.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

In situ vaccination using unique TLR9 ligand K3-SPG induces long-lasting systemic immune response and synergizes with systemic and local immunotherapy.

Although checkpoint inhibitors (CPIs) have changed the paradigm of cancer therapy, low response rates and serious systemic adverse events remain challenging. In situ vaccine (ISV), intratumoral injection of immunomodulators that stimulate innate immunity at the tumor site, allows for the development of vaccines in patients themselves. K3-SPG, a second-generation nanoparticulate Toll-like receptor 9 (TLR9) ligand consisting of K-type CpG oligodeoxynucleotide (ODN) wrapped with SPG (schizophyllan), integrates the best of conventional CpG ODNs, making it an ideal cancer immunotherapy adjuvant. Focusing on clinical feasibility for pancreaticobiliary and gastrointestinal cancers, we investigated the antitumor activity of K3-SPG-ISV in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). K3-SPG-ISV suppressed tumor growth more potently than K3-ISV or K3-SPG intravenous injections, prolonged survival, and enhanced the antitumor effect of CPIs. Notably, in PDAC model, K3-SPG-ISV alone induced systemic antitumor effect and immunological memory. ISV combination of K3-SPG and agonistic CD40 antibody further enhanced the antitumor effect. Our results imply that K3-SPG-based ISV can be applied as monotherapy or combined with CPIs to improve their response rate or, conversely, with CPI-free local immunotherapy to avoid CPI-related adverse events. In either strategy, the potency of K3-SPG-based ISV would provide the rationale for its clinical application to puncturable pancreaticobiliary and gastrointestinal malignancies.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

Cyclic GMP-AMP Triggers Asthma in an IL-33-Dependent Manner That Is Blocked by Amlexanox, a TBK1 Inhibitor.

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.

Oncolytic Reovirus Inhibits Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in a TLR3-Dependent Manner.

Oncolytic reovirus, which possesses 10 segments of dsRNA genome, mediates antitumor effects via not only virus replication in a tumor cell-specific manner, but also activation of antitumor immunity; however, the mechanism(s) of reovirus-induced activation of antitumor immunity have not been fully elucidated. Recent studies have demonstrated that overcoming an immunosuppressive environment in tumor-bearing hosts is important to achieve efficient activation of antitumor immunity. Among the various types of cells involved in immunosuppression, it has been revealed that myeloid-derived suppressor cells (MDSCs) are significantly increased in tumor-bearing hosts and play crucial roles in the immunosuppression in tumor-bearing hosts. In this study, we examined whether reovirus inhibits the immunosuppressive activity of MDSCs, resulting in efficient activation of immune cells after in vivo administration. The results showed that splenic MDSCs recovered from PBS-treated tumor-bearing mice significantly suppressed the Ag-specific proliferation of CD8+ T cells. In contrast, the suppressive activity of MDSCs on T cell proliferation was significantly reduced after reovirus administration. Reovirus also inhibited the immunosuppressive activity of MDSCs in IFN-ß promoter stimulator-1 knockout (KO) mice and in wild-type mice. In contrast, the immunosuppressive activity of MDSCs in TLR-3 KO mice was not significantly altered by reovirus treatment. The activation levels of CD4+ and CD8+ T cells were significantly lower in TLR3 KO mice than in wild-type mice after reovirus administration. These results indicate that reovirus inhibits the immunosuppressive activity of MDSCs in a TLR3, but not IFN-ß promoter stimulator-1, signaling-dependent manner.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. METHODS AND RESULTS: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. CONCLUSIONS: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8+ T Cells in Nonhuman Primates.

The priming, boosting, and restoration of memory cytotoxic CD8+ T lymphocytes by vaccination or immunotherapy in vivo is an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic acid-based compounds have been shown to act as anticancer monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, a humanized CpG DNA (K3) complexed with schizophyllan (SPG), K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously shown to act as a potent monoimmunotherapeutic agent against established tumors in mice in vivo. In this study we extend the monoimmunotherapeutic potential of K3-SPG to a nonhuman primate model. K3-SPG activated monkey plasmacytoid dendritic cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo. A single injection s.c. or i.v. with K3-SPG significantly increased the frequencies of activated memory CD8+ T cells in circulation, including Ag-specific memory CTLs, in cynomolgus macaques. This increase did not occur in macaques injected with free CpG K3 or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx were lower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, we show that K3-SPG has potent Ag-specific memory CTL response-boosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

Induction of humoural and cellular immunity by immunisation with HCV particle vaccine in a non-human primate model.

OBJECTIVE: Although HCV is a major cause of chronic liver disease worldwide, there is currently no prophylactic vaccine for this virus. Thus, the development of an HCV vaccine that can induce both humoural and cellular immunity is urgently needed. To create an effective HCV vaccine, we evaluated neutralising antibody induction and cellular immune responses following the immunisation of a non-human primate model with cell culture-generated HCV (HCVcc). DESIGN: To accomplish this, 10 common marmosets were immunised with purified, inactivated HCVcc in combination with two different adjuvants: the classically used aluminum hydroxide (Alum) and the recently established adjuvant: CpG oligodeoxynucleotide (ODN) wrapped by schizophyllan (K3-SPG). RESULTS: The coadministration of HCVcc with K3-SPG efficiently induced immune responses against HCV, as demonstrated by the production of antibodies with specific neutralising activity against chimaeric HCVcc with structural proteins from multiple HCV genotypes (1a, 1b, 2a and 3a). The induction of cellular immunity was also demonstrated by the production of interferon-γ mRNA in spleen cells following stimulation with the HCV core protein. These changes were not observed following immunisation with HCVcc/Alum preparation. No vaccination-related abnormalities were detected in any of the immunised animals. CONCLUSIONS: The current preclinical study demonstrated that a vaccine included both HCVcc and K3-SPG induced humoural and cellular immunity in marmosets. Vaccination with this combination resulted in the production of antibodies exhibiting cross-neutralising activity against multiple HCV genotypes. Based on these findings, the vaccine created in this study represents a promising, potent and safe prophylactic option against HCV.

Essential Role of CARD14 in Murine Experimental Psoriasis.

Caspase recruitment domain family member 14 (CARD14) was recently identified as a psoriasis-susceptibility gene, but its immunological role in the pathogenesis of psoriasis in vivo remains unclear. In this study, we examined the role of CARD14 in murine experimental models of psoriasis induced by either imiquimod (IMQ) cream or recombinant IL-23 injection. In all models tested, the psoriasiform skin inflammation was abrogated in Card14-/- mice. Comparison of the early gene signature of the skin between IMQ-cream-treated Card14-/- mice and Tlr7-/-Tlr9-/- mice revealed not only their similarity, but also distinct gene sets targeted by IL-23. Cell type-specific analysis of these mice identified skin Langerinhigh Langerhans cells as a potent producer of IL-23, which was dependent on both TLR7 and TLR9 but independent of CARD14, suggesting that CARD14 is acting downstream of IL-23, not TLR7 or TLR9. Instead, a bone marrow chimera study suggested that CARD14 in radio-sensitive hematopoietic cells was required for IMQ-induced psoriasiform skin inflammation, controlling the number of Vγ4+ T cells producing IL-17 or IL-22 infiltrating through the dermis to the inflamed epidermis. These data indicate that CARD14 is essential and a potential therapeutic target for psoriasis.

T Helper 17 Promotes Induction of Antigen-Specific Gut-Mucosal Cytotoxic T Lymphocytes following Adenovirus Vector Vaccination.

Few current vaccines can establish antigen (Ag)-specific immune responses in both mucosal and systemic compartments. Therefore, development of vaccines providing defense against diverse infectious agents in both compartments is of high priority in global health. Intramuscular vaccination of an adenovirus vector (Adv) has been shown to induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut-mucosal compartments. We previously found that type I interferon (IFN) signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that T helper 17 (Th17)-polarizing cytokine expression was down-regulated in the inguinal lymph nodes (iLNs) of Ifnar2-/- mice, resulting in the reduction of Ag-specific Th17 cells in the iLNs and gut mucosa of the mice. We also found that prior transfer of Th17 cells reversed the decrease in the number of Ag-specific gut-mucosal CTLs in Ifnar2-/- mice following Adv vaccination. Additionally, prior transfer of Th17 cells into wild-type mice enhanced the induction of Ag-specific CTLs in the gut mucosa, but not in systemic compartments, suggesting a gut mucosa-specific mechanism where Th17 cells regulate the magnitude of vaccine-elicited Ag-specific CTL responses. These data suggest that Th17 cells translate systemic type I IFN signaling into a gut-mucosal CTL response following vaccination, which could promote the development of promising Adv vaccines capable of establishing both systemic and gut-mucosal protective immunity.

CD63-Mediated Antigen Delivery into Extracellular Vesicles via DNA Vaccination Results in Robust CD8+ T Cell Responses.

DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8+ T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4+ and CD8+ T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8+ T cells. CD63 delivery into EVs led to better CD8+ T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8+ T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.

DNA-Containing Exosomes Derived from Cancer Cells Treated with Topotecan Activate a STING-Dependent Pathway and Reinforce Antitumor Immunity.

Danger-associated molecular patterns derived from damaged or dying cells elicit inflammation and potentiate antitumor immune responses. In this article, we show that treatment of breast cancer cells with the antitumor agent topotecan (TPT), an inhibitor of topoisomerase I, induces danger-associated molecular pattern secretion that triggers dendritic cell (DC) activation and cytokine production. TPT administration inhibits tumor growth in tumor-bearing mice, which is accompanied by infiltration of activated DCs and CD8+ T cells. These effects are abrogated in mice lacking STING, an essential molecule in cytosolic DNA-mediated innate immune responses. Furthermore, TPT-treated cancer cells release exosomes that contain DNA that activate DCs via STING signaling. These findings suggest that a STING-dependent pathway drives antitumor immunity by responding to tumor cell-derived DNA.

Advax, a Delta Inulin Microparticle, Potentiates In-built Adjuvant Property of Co-administered Vaccines.

Advax, a delta inulin-derived microparticle, has been developed as an adjuvant for several vaccines. However, its immunological characteristics and potential mechanism of action are yet to be elucidated. Here, we show that Advax behaves as a type-2 adjuvant when combined with influenza split vaccine, a T helper (Th)2-type antigen, but behaves as a type-1 adjuvant when combined with influenza inactivated whole virion (WV), a Th1-type antigen. In addition, an adjuvant effect was not observed when Advax-adjuvanted WV vaccine was used to immunize toll-like receptor (TLR) 7 knockout mice which are unable to respond to RNA contained in WV antigen. Similarly, no adjuvant effect was seen when Advax was combined with endotoxin-free ovalbumin, a neutral Th0-type antigen. An adjuvant effect was also not seen in tumor necrosis factor (TNF)-α knockout mice, and the adjuvant effect required the presences of dendritic cells (DCs) and phagocytic macrophages. Therefore, unlike other adjuvants, Advax potentiates the intrinsic or in-built adjuvant property of co-administered antigens. Hence, Advax is a unique class of adjuvant which can potentiate the intrinsic adjuvant feature of the vaccine antigens through a yet to be determined mechanism.

Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells.

Recent evidence suggest that a ß-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.