Potent Immunostimulatory Properties Research Articles

Enhancing farmed striped catfish (Pangasianodon hypophthalmus) robustness through dietary β-glucan.

β-glucan is a well-documented feed additive for its potent immunostimulatory properties in many farmed fish species. This study examined how it can also be a promising growth promoter, modulate antioxidant enzyme activities, and act as an anti-stress agent in striped catfish (Pangasianodon hypophthalmus). A 12-week feeding experiment was untaken to determine the effects of dietary β-glucan supplementation at graded levels (0, 0.5, 1.0, and 1.5 g kg-1). Measured indicators suggest that a dietary inclusion level of 1.5 g kg-1 β-glucan gave the highest positive responses: weight gain (120.10 g fish-1), survival (98.30%), and lower FCR (1.70) (P<0.05). Whole body proximate analysis had only revealed that crude protein was significantly affected by the dietary inclusion of β-glucan (P<0.05), with the highest protein content (19.70%) being in fish that were fed with 1.5 g kg-1 β-glucan. Although other inclusion levels (i.e., 0.5 and 1 g kg-1) of β-glucan did not enhance body protein content (P>0.05). The assessment of fatty acid composition in muscle, liver, and adipose tissues showed modifications with the inclusion of β-glucan. Antioxidative-related enzyme activities (inc. catalase, glutathione peroxidase, and superoxide dismutase) that were measured in the liver had higher levels when fed with β-glucan inclusion diets (P<0.05). Following the feed trial, fish were subjected to crowding stress treatment. It was subsequently found that catfish fed with β-glucan-based diet groups had lower levels of blood stress-related indicators compared to the control group with no dietary β-glucan. The use of 1.5 g kg-1 of dietary β-glucan resulted in the lowest measured levels of cortisol (43.13 ng mL-1) and glucose (50.16 mg dL-1). This study has demonstrated that the dietary inclusion of β-glucan can have functional benefits beyond the immunological enhancements in striped catfish. Furthermore, its use can increase production levels and mitigate the stress associated with intensive farming practices.

Recombinant lipidated FLIPr effectively enhances mucosal and systemic immune responses for various vaccine types

Formyl peptide receptor-like 1 inhibitor protein (FLIPr) is an immune evasion protein produced by Staphylococcus aureus, and FLIPr is a potential vaccine candidate for reducing Staphylococcus aureus virulence and biofilm formation. We produced recombinant lipidated FLIPr (rLF) to increase the immunogenicity of FLIPr and showed that rLF alone elicited potent anti-FLIPr antibody responses to overcome the FLIPr-mediated inhibition of phagocytosis. In addition, rLF has potent immunostimulatory properties. We demonstrated that rLF is an effective adjuvant. When an antigen is formulated with rLF, it can induce long-lasting antigen-specific immune responses and enhance mucosal and systemic antibody responses as well as broad-spectrum T-cell responses in mice. These findings support further exploration of rLF in the clinic as an adjuvant for various vaccine types with extra benefits to abolish FLIPr-mediated immunosuppressive effects.

Neem leaf glycoprotein mediated epigenetic modification in oral squamous cell carcinoma

BackgroundRecent studies have found out that the initiation and progression of cancer, traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic alterations. Studies also emphasize development of drugs targeting histone deacetylases (HDACs) and DNA methyltransferase inhibitors as an emerging anticancer strategy. Oral squamous cell carcinoma (OSCC) is a deadly disease that comprises 60% of all head and neck squamous cell cancers. The leaves of the Neem tree (Azadirachtaindica) have been used in traditional Ayurvedic medicine for centuries to treat numerous oral maladies and are known to have significant anti-inflammatory properties. NLGP (a glycoprotein isolated from neem leaf) is famous for its potent anti-inflammatory, immunostimulatory and anti-cancer properties. Thus, in the current research proposal we investigated the NLGP mediated various epigenetic modifications in Oral squamous cell carcinoma (OSCC). PurposeThe aim of this work was to clarify if the epigenetic changes in oral squamous cell carcinoma could be mediated by Neem Leaf Glycoprotein (NLGP), which has already been demonstrated as a potent anti-cancer drug. The study's objectives were further expanded to include investigating the impact of NLGP-mediated epigenetic alterations on the Wnt and PI3K pathways. Study DesignIn this research work, we would like to show the epigenetic modification mediated by NLGP on OSCC cell line, SSC-9. We would also like to investigate the involvement of NLGP in certain signal transduction pathway(s) which may play a crucial role in epigenetic modification of oral squamous cell carcinoma. MethodologyChIP assays were performed with MNase-digested chromatin isolated from the NLGP treated and non-treated SCC-9 cells after fixing protein-DNA interactions with 1% formaldehyde. ChIP-grade antibodies and their isotype-Ig antibodies were used to pull-down DNA:Protein complexes. ChIPed samples were used for quantitative polymerase chain reaction (qPCR) analysis and the derived Ct values converted to absolute copy numbers using cloned-DNA plasmid standard dilution curve. Nonspecific signals obtained with IgG-ChIP were subtracted from test samples. Whole cell extracts were prepared from NLGP-treated and non-treated SSC9 (Oral Cancer Squamous) cells with the help of Cell Lysis Buffer (Sigma). Using differentially treated whole cell extracts coimmunoprecipitations were performed with the Universal Magnetic Co-immunoprecipitation kit (Active Motif), as per the manufacturer's specifications. Single blot was sequentially reprobed with multiple antibodies. For each co-immunoprecipitation experiment, isotype IgG antibody served as a negative control and immunoblotting the corresponding whole cell extracts with the primary antibody served as a positive control (input), and most images presented are from the same experiment and same gel exposure. ResultTreatment with NLGP increased percentage enrichment of Histone H3 trimethylation (H3K4me3) and acetylation (H3K14Ac) markers and decreased percentage enrichment of Histone H3 demethylation (H3K9me3, JMJD3) and deacetylation (HDAC1) markers respectively on DNA repair gene loci (ATM, ATR, DDB2), tumour suppressor gene (TP53), Signal transducer and activator of transcription 1 (STAT1) and DNA-dependent RNA polymerase (RNApol2). On further investigation of the molecular pathways that may be involved, we found that NLGP treatment blocks the activation of the DNA PKCs, a component of the Akt pathway and its subsequent phosphorylation of H2AFX and it also inhibits ß-catenin, an integral molecule of the Wnt pathway via increased percentage enrichment of the tri-methylation (H3K4Me3) and acetylation (H3K14Ac) markers thus inhibiting tumorigenesis and cancer progression in OSCC. ConclusionNeem Leaf Glycoprotein has been successfully involved in immuno-regulating the various aspects of the immune system in order to mount an effective anti-cancer action and thus has come forward as an emerging cancer-therapeutic strategy based on natural products. This study also contributes to a better understanding of NLGP's epigenetic modulatory function and paves the path for epigenetic-based therapy for OSCC.

Abstract 5591: Anti-FolRα ADC STRO-002 induces immunogenic cell death (ICD) to enhance anti-tumor activity

Abstract STRO-002 is an antibody-drug conjugate (ADC) composed of an anti-folate receptor alpha (FolRα) antibody conjugated to a tubulin-targeting hemiasterlin (SC209) warhead via a cleavable linker currently in clinical studies for ovarian and endometrial cancer. FolRα exhibits minimal expression in normal tissues but is overexpressed in several cancers such as ovarian, endometrial, and NSCLC. We previously showed that STRO-002 induces in vitro hallmarks of immunogenic cell death (ICD), such as increased calreticulin exposure and HMGB1 and ATP release in a FolRα dependent manner. Follow-up vaccination studies with murine MC38 tumor cells engineered to express human FolRα (MC38-hFolRa) were performed to further explore the significance of STRO-002-induced ICD and protective immunity in vivo. A single vaccination with STRO-002 or SC209 in vitro-treated MC38-hFolRa cells in immunocompetent mice resulted in 60% of animals rejecting a subsequent rechallenge with living MC38-hFolRa cells, while relapse was observed in most control mice. Including an additional booster vaccination further increased the proportion ( 90%) of animals remaining tumor free after rechallenge. These results demonstrate that tumor cells pre-treated with STRO-002 or SC209 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo. The ability of STRO-002 to induce ICD that elicits an effective and robust anti-tumor immune response may translate into several advantages in the clinic. Induction of ICD is likely to offer a complementary anti-tumor mechanism in combination therapy regimens such as with check point inhibitors or VEGF blockade. We have demonstrated that STRO-002 in combination with avelumab in MC38-hFolRα-bearing mice significantly enhanced efficacy and durable anti-tumor immunity. More recent rechallenge studies reveal that this combination treatment suppresses growth of not only MC38-hFolRα but also MC38 wild-type tumor cells, indicating formation of immunological memory with signs of epitope spreading. Additionally, co-administration of STRO-002 with anti-VEGF therapy demonstrates improved TGI compared to monotherapy (p &amp;lt; 0.0001) in human ovarian OV-90 tumors. The potent cytotoxic and immunostimulatory properties of STRO-002 may also contribute to enhancing efficacy in indications with low FolRα expression. Such tumor types include endometrial and NSCLC, in which STRO-002 has displayed potent monotherapy activity in preclinical studies. In a panel of endometrial PDX models, STRO-002 was significantly efficacious in 9/17 (53%) FolRα-positive tumors, including models with moderate and weak FolRα levels. In a NSCLC PDX model, a single dose of STRO-002 induced tumor regression and suppressed tumor growth for up to three months post-dose. In conclusion, the induction of ICD by STRO-002 has potential to increase anti-tumor activity in cancers with low FolRα expression. Citation Format: Cristina L. Abrahams, Andrew McGeehan, Jennifer Smith, Robert Yuan, Kshama A. Doshi, Sihong Zhou, Xiaofan Li, Cuong Tran, Gang Yin, Arturo Molina, Kristin Bedard, Trevor Hallam. Anti-FolRα ADC STRO-002 induces immunogenic cell death (ICD) to enhance anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5591.

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid.

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Abstract P030: Making cold tumors hot: Multi-faceted immune ignition by USP6

Abstract Ewing sarcoma (ES) is the second most common bone cancer, affecting predominantly children and young adults. While patients with localized disease have a five-year survival rate of ~75%, this plummets to 20% for metastatic and recurrent patients. Immunotherapy has had limited success in ES, in large part because it is considered immunologically cold, with few tumor-infiltrating T lymphocytes (TILs). However, TILs actually vary significantly, and high levels portend greatly improved survival. In ES, TIL abundance correlates with the interferon (IFN)g-inducible chemokines CXCL9/CXCL10/CCL5. These immune features (i.e. IFNg response, CXCL9/10 production, and high CD8+ TIL recruitment) define a hot TME, and predict both improved overall survival and response to immune checkpoint inhibitors (ICIs) across many malignancies. However, the tumor-intrinsic factors that confer this favorable immune phenotype, in ES or any other cancer, are largely unknown, and their identification represents a window of opportunity for therapeutic intervention to improve patient outcomes. We have discovered that high USP6 expression in ES patients is associated with dramatically improved overall survival. Our work has revealed that USP6 has potent and multi-faceted immunostimulatory properties: not only is it capable of triggering all hallmark features of a hot TME, but its expression in ES cells directly activates the cytolytic function of NK cells, and also induces secretion of a complex array of immune-activating, anti-tumorigenic chemokines that affect both innate and adaptive immune lineages. In vivo, USP6 inhibits growth of human ES cell xenografts in nude mice, and enhances intratumoral infiltration/activation of natural killer (NK) cells and myeloid lineages. We hypothesize that USP6 improves ES patient outcome due to these pleiotropic immunostimulatory functions, which engender a hot TME with resultant activation of multiple cytolytic immune lineages that effect tumor cell elimination. We have sought to harness the multi-faceted immune-igniting properties of USP6 into a novel immunotherapeutic by delivering USP6 mRNA using lipid nanoparticles (LNPs) customized for in vivo delivery into ES cells. Preliminary data validate the efficacy of intratumorally administered USP6 LNPs in stimulating immune cell recruitment and activation, and suppressing xenograft growth, with a fraction of tumors undergoing complete regression. Furthermore, we have observed that intratumoral expression of USP6 can trigger systemic activation of the immune system, including increased levels and activation NK cells. Notably, increased circulating levels of NK cells have been associated with improved patient prognosis. Despite the success of CAR-T cells and ICIs, there are significant hurdles limiting their efficacy, and it is essential to pursue therapeutics that engender an immunologically hospitable TME, as well as activate other cytolytic immune lineages, such as NK cells, both of which are encompassed by USP6 LNP therapy. Citation Format: Ian C. Henrich, Kanika Jain, Laura Quick, Robert Young, Margaret M. Chou. Making cold tumors hot: Multi-faceted immune ignition by USP6 [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P030.

Enzymatic bioconversion of ginseng powder increases the content of minor ginsenosides and potentiates immunostimulatory activity

BackgroundGinsenosides are biologically active components of ginseng and have various functions. In this study, we investigated the immunomodulatory activity of a ginseng product generated from ginseng powder (GP) via enzymatic bioconversion. This product, General Bio compound K-10 mg solution (GBCK10S), exhibited increased levels of minor ginsenosides, including ginsenoside-F1, compound K, and compound Y. MethodsThe immunomodulatory properties of GBCK10S were confirmed using mice and a human natural killer (NK) cell line. We monitored the expression of molecules involved in immune responses via enzyme-linked immunosorbent assay, flow cytometry, NK cell-targeted cell destruction, quantitative reverse-transcription real-time polymerase chain reaction, and Western blot analyses. ResultsOral administration of GBCK10S significantly increased serum immunoglobulin M levels and primed splenocytes to express pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ. Oral administration of GBCK10S also activated NK cells in mice. Furthermore, GBCK10S treatment stimulated a human NK cell line in vitro, thereby increasing granzyme B gene expression and activating STAT5. ConclusionGBCK10S may have potent immunostimulatory properties and can activate immune responses mediated by B cells, Th1-type T cells, and NK cells.

The two-faces of NK cells in oncolytic virotherapy

Oncolytic viruses (OVs) are immunotherapeutics capable of directly killing cancer cells and with potent immunostimulatory properties. OVs exert their antitumor effect, at least partially, by activating the antitumor immune response, of which NK cells are an important component. However, if on the one hand increasing evidence revealed that NK cells are important mediators of oncolytic virotherapy, on the other hand, NK cells have evolved to fight viral infections, and therefore they can have a detrimental effect for the efficacy of OVs. In this review, we will discuss the dichotomy between the antitumor and antiviral functions of NK cells related to oncolytic virotherapy. We will also review NK cell-based and OV-based therapies, engineered OVs aimed at enhancing immune stimulation, and combination therapies involving OVs and NK cells currently used in cancer immunotherapy.

IL-15 superagonist RLI has potent immunostimulatory properties on NK cells: implications for antimetastatic treatment

BackgroundAs the immune system is compromised in patients with cancer, therapeutic strategies to stimulate immunity appear promising, to avoid relapse and increase long-term overall survival. Interleukin-15 (IL-15) has similar properties...

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential “shock and kill” therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

Bifidobacterium animalis subspecies lactis engineered to produce mycosporin-like amino acids in colorectal cancer prevention.

Colorectal cancer is the third most common cancer and the third leading cause of cancer-related death. The pathogensesis of colorectal cancer involves a multi-step and multi-factorial process. Disruption of the gut microbiota has been associated with gastrointestinal diseases such as colorectal cancer. The genus Bifidobacterium is considered an important component of the commensal microbiota and plays important roles in several homeostatic functions: immune, neurohormonal, and metabolic. Bifidobacterium animalis subsp. lactis is a well-documented probiotic within the species Bifidobacterium. Mycosporin-like amino acids are low molecular weight amino acids demonstrated to exert prebiotic effects and to modulate host immunity by regulating the proliferation and differentiation of intestinal epithelial cells, macrophages and lymphocytes, as well as cytokine production.Their modulation of the metabolism of the immune system and transcription factors could exert a beneficial effect on colorectal cancer. B. animalis does not produce mycosporin-like amino acids. If one could create a B. animalis–producing mycosporin-like amino acids via genetic open reading frame engineering it should exert more potent immuno-stimulatory properties and, thereby, become a potent strain-specific microbial based therapy in colorectal cancer prevention.

Monitoring MHC Ubiquitination by MARCH Ubiquitin Ligases.

Ubiquitination is a reversible process that controls the intracellular transport of many transmembrane molecules. Ubiquitination of MHC I, MHC II, and CD1a by different members of the MARCH family of E3 ubiquitin ligases is a key event in the regulation of the potent immunostimulatory properties of activated dendritic cells. We describe here methods to monitor and quantify the ubiquitination levels of these different antigen presentation molecules and its impact on their cell surface accumulation.

Acetalated Dextran Microparticles for Codelivery of STING and TLR7/8 Agonists.

Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.

An Immunologically Modified Nanosystem Based on Noncovalent Binding Between Single-Walled Carbon Nanotubes and Glycated Chitosan.

Functionalized single-walled carbon nanotubes are currently being explored as novel delivery vehicles for proteins and therapeutic agents to treat various diseases. In order to maximize treatment efficacy, a strong binding between single-walled carbon nanotubes and their functionalized molecules is necessary. Glycated chitosan, a polymer with potent immunostimulatory properties for cancer treatment, has been used as a surfactant of single-walled carbon nanotubes to form an immunologically modified nanosystem for biomedical applications. In this study, we investigated the binding characteristics of single-walled carbon nanotube and glycated chitosan using molecular dynamics simulations. The mean square displacement, radius of gyration, interaction energy, and radial distribution function of the single-walled carbon nanotube-glycated chitosan system were analyzed. The results from the simulations demonstrated that glycated chitosan was bound to single-walled carbon nanotubes by a strong, noncovalent interaction. The stability of glycated chitosan on the single-walled carbon nanotubes surface was enhanced by the length of glycated chitosan, and the binding energy of the 2 molecules was closely related to the diameter and chirality of single-walled carbon nanotubes, with the most stable single-walled carbon nanotube-glycated chitosan system being formed by the combination of long polymer, large single-walled carbon nanotube, and armchair single-walled carbon nanotube. The understanding of the interactions between single-walled carbon nanotube and glycated chitosan and the structure of single-walled carbon nanotube-glycated chitosan allows the modifications of the novel nanosystem for disease diagnostics and therapeutics.

The influence of radiation in the context of developing combination immunotherapies in cancer.

In addition to tumouricidal activity, radiotherapy is now recognized to display potent immunostimulatory properties that can contribute to the generation of anti-cancer immune responses. Treatment with radiation can induce a variety of pro-immunogenic and phenotypic changes in malignant cells, and recalibrate the immune contexture of the tumour microenvironment, leading to enhanced activation of the innate immune system, and priming of tumour-specific T-cell immunity. The immune-dependent effects of radiotherapy provide a sound rationale for the development of combination strategies, whereby the immunomodulatory properties of radiation can be exploited to augment the activity of immunotherapeutic agents. Encouraged by the recent success of breakthrough therapies such as immune checkpoint blockade, and a wealth of experimental data demonstrating the efficacy of radiotherapy and immunotherapy combinations, the clinical potential of this approach is now being explored in numerous trials. Successful translation will require careful consideration of the most suitable dose and fractionation of radiation, choice of immunotherapy and optimal sequencing and scheduling regimen. Immunological control of cancer is now becoming a clinical reality. There is considerable optimism that the development of effective radiotherapy and immunotherapy combinations with the capacity to induce durable, systemic immunity will further enhance patient outcome and transform the future management of cancer.

Targeting of C‐type lectin‐like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides

Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoproteinVI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods Invitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. Invivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.

Vaccine-induced but not tumor-derived Interleukin-10 dictates the efficacy of Interleukin-10 blockade in therapeutic vaccination

ABSTRACTBlocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immunostimulatory properties but still inducing IL-10 led to superior antitumor immunity and complete tumor rejection. These results suggest that for therapeutic antitumor vaccination, blockade of vaccine-induced IL-10 is more relevant than tumor-associated IL-10.

A novel TLR2 agonist from Bordetella pertussis is a potent adjuvant that promotes protective immunity with an acellular pertussis vaccine.

Bordetella pertussis causes whooping cough, a severe and often lethal respiratory infection in infants. A recent resurgence of pertussis has been linked with waning or suboptimal immunity induced with acellular pertussis vaccines (Pa) that were introduced to most developed countries in the 1990s because of safety concerns around the use of whole-cell pertussis vaccines (Pw). Pa are composed of individual B. pertussis antigens absorbed to alum and promote strong antibody, T helper type 2 (Th2) and Th17 responses, but are less effective at inducing cellular immunity mediated by Th1 cells. In contrast, Pw, which include endogenous Toll-like receptor (TLR) agonists, induce Th1 as well as Th17 responses. Here we report the identification and characterization of novel TLR2-activating lipoproteins from B. pertussis. These proteins contain a characteristic N-terminal signal peptide that is unique to Gram-negative bacteria and we demonstrate that one of these lipoproteins, BP1569, activates murine dendritic cells and macrophages and human mononuclear cells via TLR2. Furthermore, we demonstrated that a corresponding synthetic lipopeptide LP1569 has potent immunostimulatory and adjuvant properties, capable of enhancing Th1, Th17, and IgG2a antibody responses induced in mice with an experimental Pa that conferred superior protection against B. pertussis infection than an equivalent vaccine formulated with alum.

CpG oligonucleotides bind TLR9 and RRM-Containing proteins in Atlantic Salmon (Salmo salar)

BackgroundBacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates.ResultsIn the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes.ConclusionThe presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.

DEC-205 is a cell surface receptor for CpG oligonucleotides

Synthetic CpG oligonucleotides (ODN) have potent immunostimulatory properties exploited in clinical vaccine trials. How CpG ODN are captured and delivered to the intracellular receptor TLR9, however, has been elusive. Here we show that DEC-205, a multilectin receptor expressed by a variety of cells, is a receptor for CpG ODN. When CpG ODN are used as an adjuvant, mice deficient in DEC-205 have impaired dendritic cell (DC) and B-cell maturation, are unable to make some cytokines such as IL-12, and display suboptimal cytotoxic T-cell responses. We reveal that DEC-205 directly binds class B CpG ODN and enhances their uptake. The CpG-ODN binding function of DEC-205 is conserved between mouse and man, although human DEC-205 preferentially binds a specific class B CpG ODN that has been selected for human clinical trials. Our findings identify an important receptor for class B CpG ODN and reveal a unique function for DEC-205.