Murine Bone Marrow-derived Dendritic Cells Research Articles

Birch Pollen Induces Toll-Like Receptor 4-Dependent Dendritic Cell Activation Favoring T Cell Responses.

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation.

β-(1→4)-Mannobiose Acts as an Immunostimulatory Molecule in Murine Dendritic Cells by Binding the TLR4/MD-2 Complex.

Some β-mannans, including those in coffee bean and soy, contain a mannose backbone with β-(1→4) bonds. Such mannooligosaccharides could have immunological functions involving direct interaction with immune cells, in addition to acting as prebiotics. This study aimed at assessing the immunological function of mannooligosaccharides with β-(1→4) bond, and elucidating their mechanism of action using bone marrow-derived murine dendritic cells (BMDCs). When BMDCs were stimulated with the mannooligosaccharides, only β-Man-(1→4)-Man significantly induced production of cytokines that included IL-6, IL-10, TNF-α, and IFN-β, and enhanced CD4+ T-cell stimulatory capacity. Use of putative receptor inhibitors revealed the binding of β-Man-(1→4)-Man to TLR4/MD2 complex and involvement with the complement C3a receptor (C3aR) for BMDC activation. Interestingly, β-Man-(1→4)-Man prolonged the production of pro-inflammatory cytokines (IL-6 and TNF-α), but not of the IL-10 anti-inflammatory cytokine during extended culture of BMDCs, associated with high glucose consumption. The results suggest that β-Man-(1→4)-Man is an immunostimulatory molecule, and that the promotion of glycolysis could be involved in the production of pro-inflammatory cytokine in β-Man-(1→4)-Man-stimulated BMDCs. This study could contribute to development of immune-boosting functional foods and a novel vaccine adjuvant.

Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death.

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically via host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4+ T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1β, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis.

Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Priming of the cGAS-STING-TBK1 Pathway Enhances LPS-Induced Release of Type I Interferons.

Cytoplasmic nucleic acids sensing through cGAS-STING-TBK1 pathway is crucial for the production of antiviral interferons (IFNs). IFN production can also be induced by lipopolysaccharide (LPS) stimulation through Toll-like receptor 4 (TLR4) in appropriate conditions. Of note, both IFN production and dysregulated LPS-response could play a role in the pathogenesis of Systemic Lupus Erythematosus (SLE). Indeed, LPS can trigger SLE in lupus-prone mice and bacterial infections can induce disease flares in human SLE. However, the interactions between cGAS and TLR4 pathways to IFNs have been poorly investigated. To address this issue, we studied LPS-stimulation in cellular models with a primed cGAS-STING-TBK1 pathway. cGAS-stimulation was naturally sustained by undigested self-nucleic acids in fibroblasts from DNase2-deficiency interferonopathy, whilst it was pharmacologically obtained by cGAMP-stimulation in THP1 cells and murine bone marrow-derived dendritic cells. We showed that cells with a primed cGAS-STING-TBK1 pathway displayed enhanced IFNs production after TLR4-challenge. STING-inhibition did not affect IFN production after LPS alone, but prevented the amplified IFN production in cGAMP-primed cells, suggesting that functional STING is required for priming-dependent enhancement. Furthermore, we speculated that an increased PIK3AP1 expression in DNase2-deficient fibroblasts may link cGAMP-priming with increased LPS-induced IFN production. We showed that both the hyper-expression of PIK3API and the enhanced LPS-induced IFN production can be contrasted by STING inhibitors. Our results may explain how bacterial LPS can synergize with cGAS-pathway in promoting the development of SLE-like autoimmunity.

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

TLR2 and the NLRP3 inflammasome mediate IL-1β production in Prevotella nigrescens-infected dendritic cells.

Prevotella nigrescens is an oral pathogen that is frequently observed in the subgingival plaque of periodontitis patients. Interleukin-1β (IL-1β) is known to be involved in the immunopathology of periodontal diseases and has been implicated in the destruction of bone. In this study, we investigated the mechanism of IL-1β production by P. nigrescens in murine bone marrow-derived dendritic cells (BMDCs). Our results showed that a host receptor, Toll-like receptor 2 (TLR2), but not TLR4 is required for pro-IL-1β induction and nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) priming in BMDCs in response to P. nigrescens and activation of the NLRP3 inflammasome is necessary for processing of pro-IL-1β into mature IL-1β. In addition, an inhibitor assay revealed that production of reactive oxygen species, P2X7R activity, and release of cathepsin B are involved in IL-1β production in BMDCs in response to P. nigrescens.

Differential infection of murine and human dendritic cell subsets by oncolytic vesicular stomatitis virus variants

ABSTRACT Oncolytic viruses (OVs) can eradicate tumor cells and elicit antitumor immunity. VSV-GP, a chimeric vesicular stomatitis virus (VSV) with the glycoprotein (GP) of the lymphocytic choriomeningitis virus, is a promising new OV candidate. However, the interaction of VSV-GP with host immune cells is not fully understood. Dendritic cells (DCs) are essential for inducing efficient antitumor immunity. Thus, we aimed to investigate the interaction of VSV-GP with different murine and human DCs subsets in direct comparison to the less cytopathic variant VSV-dM51-GP and wild type VSV. Immature murine bone marrow-derived DCs (BMDCs) were equally infected and killed by VSV and VSV-GP. Human monocyte-derived DCs (moDCs) were more permissive to VSV. Interestingly, VSV-dM51-GP induced maturation instead of killing in both BMDCs and moDCs as well as a pronounced release of pro-inflammatory cytokines. Importantly, matured BMDCs and moDCs were no longer susceptible to VSV-GP infection. Mouse splenic conventional DC type 1 (cDC1) could be infected ex vivo by VSV and VSV-GP to a higher extent than cDC2. Systemic infection of mice with VSV-GP and VSV-dM51-GP resulted in strong activation of cDCs despite low infection rates in spleen and tumor tissue. Human blood cDC1 were equally infected by VSV and VSV-GP, whereas cDC2 showed preferential infection with VSV. Our study demonstrated differential DC infection, activation, and cytokine production after the treatment with VSV and VSV-GP variants among species and subsets, which should be taken into account when investigating immunological mechanisms of oncolytic virotherapy in mouse models and human clinical trials.

Functionalized multifunctional nanovaccine for targeting dendritic cells and modulation of immune response.

Multifunctional gelatin nanoparticles modified by NIR-emitting gold/silver alloy nanoclusters and loaded with ovalbumin (OVA) as a model antigen were developed. Two different designs of nanoparticles were introduced; positively charged NPs with OVA displayed over the surface (S-NPs) versus OVA encapsulated in the NPs' matrix and the surface is functionalized by dextran (Dex-NPs) for dendritic cell targeting. The nanoparticles showed average particle sizes of 210 and 305nm and zeta potentials of +25.6 and -23.9mV, for S-NPs and Dex-NPs, respectively. Both types of NPs succeeded to induce maturation of murine bone marrow-derived dendritic cells (BMDCs) as indicated by the upregulated surface expression of MHC-II and co-stimulatory molecules CD86, CD80 and CD40. Dex-NPs induced no cytotoxicity in BMDCs, in contrast to S-NPs. Functionalization of NPs with dextran increased their uptake by BMDCs, enhanced secretion of immune stimulatory chemokines, and boosted their T cell stimulation capacity. Co-culture of NP loaded BMDCs with OVA-specific CD4 or CD8 T cells, induced enhanced T cell proliferation and release of IL-2 from both CD8 and CD4 cells and IFN-γ from CD8 T cells. This highlights the potential of the developed NPs as vaccines for inducing T helper 1 type CD4 as well as CD8 responses, such as vaccines for cancer or viral infections.

Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and Tcell activation against a vesicularstomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) invitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I Tcells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 Tcell response invivo without altering the anti-viral CD8 Tcell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Irreversible electroporation can alter immune cell composition and enhance antigen presentation potential in pancreatic cancer

Abstract Pancreatic cancer has a devastating survival rate of only 9%. A major treatment limitation is the immunosuppressive nature of pancreatic tumors created by high amounts of tumor-associate stromal cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). These form a barrier that inhibits anti-tumor immunity and has yet to be overcome by current immunotherapy options. Irreversible electroporation (IRE), an ablative technology that utilizes short electrical pulses to disrupt tumor cell membranes, shows evidence of inducing proinflammatory cell death in pancreatic tumors, but its effect on innate and adaptive immune responses, vital for long-term survival, has not been investigated. We hypothesize that IRE treatment for pancreatic cancer can reduce pro-tumor immune cells, recruit anti-tumor immune cells such as cytotoxic T-cells, and increase tumor-specific antigen presentation potential. We utilized in vivo immunocompetent Pan02 murine pancreatic cancer models to assess progression-free survival and tumor immune cell populations via flow cytometry, as well as an ex vivo antigen presentation model with murine bone marrow-derived dendritic cells and splenic T-cells to determine the ability of IRE to generate viable antigens. Our findings show a temporal increase in anti-tumor immune cell populations, reduction in tumor-associated immune cell populations, and strong antigen presentation potential. However, we also observed an increase in Pdl-1, normally down-regulated in pancreatic cancer, in response to increased immune signaling. This may make IRE a candidate for cotherapy options such as anti-Pdl-1 antibodies or other immunotherapies that could increase patient survival.

MicroRNA-155 Suppresses the Translation of p38 and Impairs the Functioning of Dendritic Cells in Endometrial Cancer Mice.

BackgroundDendritic cells (DCs) are reported to play an important role in activating the anti-tumor immune responses. p38 MAPK14 signaling plays an important role in controlling their activity. Here, we identified that miR-155 suppressed the translation of p38 and impaired the functioning of dendritic cells in endometrial cancer.MethodsHEC1A endometrial cancer cell lines were used for the study which was transfected in the C57BL/6 mice. Murine bone marrow-derived dendritic cells (BMDCs) were isolated from the mice. Target prediction was done by TargetScan which was confirmed by RT-PCR analysis. The protein expression was carried by Western blot analysis. Levels of IL-12 were evaluated by ELISA. Mice injected with HEC1A cells were subjected to tumor challenge study.ResultsOn screening the binding sites of p38 MAPK14 gene, miR-155 was found to bind the 3ʹUTR directly and blocked its translation. The levels of miR-155 were upregulated in dendritic cells and RAW264.7 cells, miR-155 showed inhibitory effect on expression levels of p38. In dendritic cells, miR-155 was found to regulate the expression of IL-12, also miR-155 inhibitor stimulated the differentiation of Th1 cells in mice induced with endometrial cancer. In dendritic cells, miR-155 inhibited the expression of p38 gene and decreased their ability to interfere in tumor growth.ConclusionThe study concludes suppressive role of miR-155 in the process of dendritic cells mediated anti-tumor immunity, also inhibiting miR-155 provides a novel strategy for countering endometrial cancer.

Abstract B21: Dual immunotherapy targeting macrophages and PD-1 immune checkpoint for treatment of melanoma

Abstract Blockading antibodies against PD-1/PD-L1 immune checkpoints are approved for melanoma treatment; however, only a minority of patients experience durable remission. Our previous work suggested that pegylated liposomal alendronate (PLA) targets tumor-associated macrophages, another mediator of immunosuppression, and polarized them towards an antitumor phenotype. We hypothesized that dual immunotherapy with PLA and PD-1 inhibitor would result in enhanced efficacy by targeting nonoverlapping immune escape mechanisms. In vitro cytotoxicity studies were performed in B16-OVA melanoma and murine bone marrow-derived dendritic cells. In vivo efficacy/toxicity studies were conducted in seven-week old C57BL/6 mice with 100,000 B16-OVA cells implanted on the rear flank. Once tumors were palpable, mice (n=4-5/group) were randomized to receive PLA (alendronate 4 mg/kg) + α-PD-1 mAb (10 mg/kg), vehicle + IgG-isotype, PLA + IgG-isotype, or vehicle + α-PD-1. Tumor growth was monitored, and at endpoint, tissues (tumors, lymph nodes, livers, kidneys, spleen, intestines, and lungs) were harvested for analysis by flow cytometry and histopathology. Neither PLA nor α-PD-1 were cytotoxic to melanoma or dendritic cells in vitro when compared to doxorubicin (cytotoxic chemotherapy). Mice treated with PLA + α-PD-1 had significantly smaller tumors compared to placebo controls (p=0.0015) and α-PD-1 monotherapy (p=0.0168).There were no significant pathologic changes in tissues. Assessment of immune responses in tumors and sentinel lymph nodes is ongoing. Dual immunotherapy targeting tumor-associated macrophages and the PD-1 checkpoint is safe and more efficacious than monotherapies solely targeting the PD-1 checkpoint in the treatment of murine melanoma. This is rapidly translatable to clinical trials, as the separate components are already approved for use in humans. Citation Format: Claire E. Shudde, Jalpa Patel, Robin Rajan, Hilary Shmeeda, Dung Nguyen, Alberto A. Gabizon, Ninh M. La-Beck. Dual immunotherapy targeting macrophages and PD-1 immune checkpoint for treatment of melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B21.

Proteomic analyses reveal that Orf virus induces the activation and maturation of mouse bone marrow-derived dendritic cells

Orf virus (ORFV) is known for its immunostimulatory capacities and has been utilized as an efficient viral vector vaccine in non-permissive host species. Murine bone marrow-derived dendritic cells (BMDCs) are able to react with ORFV. In this study, we aimed to identify pivotal differentially expressed proteins involved in the process of DCs' differentiation in response to ORFV. Our findings showed that ORFV activates the maturation and differentiation of DCs. We further identified and validated seven differentially expressed proteins following ORFV stimulation. With functions in biological processes such as stimulus response, DCs maturation, antigen presentation and Th1 cell activation. Western blot analyses validated the respective changes in protein expression. The huge number of differentially expressed proteins identified in this study will be valuable for elucidating the mechanisms underlying ORFV-induced immunomodulation of murine BMDCs.

Upregulation of Cytokines and Differentiation of Th17 and Treg by Dendritic Cells: Central Role of Prostaglandin E2 Induced by Mycobacterium bovis

Mycobacterium bovis (M. bovis) is a zoonotic pathogen that causes bovine and human tuberculosis. Dendritic cells play a critical role in initiating and regulating immune responses by promoting antigen-specific T-cell activation. Prostaglandin E2 (PGE2)-COX signaling is an important mediator of inflammation and immunity and might be involved in the pathogenesis of M. bovis infection. Therefore, this study aimed to reveal the character of PGE2 in the differentiation of naïve CD4+ T cells induced by infected dendritic cells (DCs). Murine bone marrow-derived DCs were pre-infected with M. bovis and its attenuated strain M. bovis bacillus Calmette-Guérin (BCG). Then, the infected DCs were co-cultured with naïve CD4+ T cells with or without the cyclooxygenase (COX) inhibitor indomethacin. Quantitative RT-PCR analysis and protein detection showed that PGE2/COX-2 signaling was activated, shown by the upregulation of PGE2 production as well as COX-2 and microsomal PGE2 synthase (mPGES1) transcription in DCs specifically induced by M. bovis and BCG infection. The further co-culture of infected DCs with naïve CD4+ T cells enhanced the generation of inflammatory cytokines IL-17 and IL-23, while indomethacin suppressed their production. Following this, the differentiation of regulatory T cells (Treg) and Th17 cell subsets was significantly induced by the infected DCs rather than uninfected DCs. Meanwhile, M. bovis infection stimulated significantly higher levels of IL-17 and IL-23 and the differentiation of Treg and Th17 cell subsets, while BCG infection led to higher levels of TNF-α and IL-12, but lower proportions of Treg and Th17 cells. In mice, M. bovis infection generated more bacterial load and severe abnormalities in spleens and lungs, as well as higher levels of COX-2, mPGE2 expression, Treg and Th17 cell subsets than BCG infection. In conclusion, PGE2/COX-2 signaling was activated in DCs by M. bovis infection and regulated differentiation of Treg and Th17 cell subsets through the crosstalk between DCs and naive T cells under the cytokine atmosphere of IL-17 and IL-23, which might contribute to M. bovis pathogenesis in mice.

The immunostimulatory activity of polysaccharides from Glycyrrhiza uralensis.

BackgroundThe enhancement of immunity is very important for immunocompromised patients such as cancer patients with radiotherapy or chemotherapy. Glycyrrhiza uralensis has been used as food and medicine for a long history. G. uralensis polysaccharides (GUPS) were prepared and its immunostimulatory effects were investigated.MethodsHuman monocyte-derived dendritic cells (DCs) and murine bone marrow-derived DCs were treated with different concentrations of GUPS. The DCs maturation and cytokine production were analyzed by flow cytometry and ELISA, respectively. Inhibitors and Western blot were used to study the mechanism of GUPS. The immunostimulatory effects of GUPS were further evaluated by naïve mouse model and immunosuppressive mouse model induced by cyclophosphamide. ResultsGUPS significantly promoted the maturation and cytokine secretion of human monocyte-derived DCs and murine bone marrow-derived DCs through TLR4 and down-stream p38, JNK and NF-κB signaling pathways. Interestingly, the migration of GUPS treated-DCs to lymph node was increased. In the mouse model, GUPS increased IL-12 production in sera but not for TNF-α. Moreover, GUPS ameliorated the side effect of cyclophosphamide and improved the immunity of immunosuppressive mice induced by cyclophosphamide. These results suggested that GUPS might be used for cancer therapy to ameliorate the side effect of chemotherapy and enhance the immunity.

Quantification of Mushroom-Derived Soluble β-1,6-Glucan Using the Function-Modified Recombinant β-1,6-Glucanase.

Mushroom is one of the major sources of β-glucan used in medical applications and traditional therapies. Thus, structure analysis and quantification of β-glucan content is crucial to evaluate medicinal mushrooms. Most studies concerning mushroom-derived β-glucan have been focused on β-1,3-glucans. However, recent investigations suggest that β-1,6 glucans have important roles for immunomodulating activity. Therefore, to elucidate the fine structure of various mushroom-derived β-glucans, we recently developed a novel β-1,6 glucan detection system using the function-modified recombinant β-1,6-glucanase. In this study, we performed an ELISA-like assay using modified β-1,6-glucanase and soluble dectin-1-Fc as the probes for β-1,6-glucan and β-1,3-glucan, respectively. Reactivity of ELISA to crude hot water extracts of edible mushrooms (Grifola frondosa, Agaricus bisporus, Pleurotus tuoliensis, P. eryngii, P. ostreatus, Hypsizygus marmoreus, and Lentinus edodes) was compared and L. edodes showed the strongest reactivity among them. An additional 19 different products of fresh L. edodes (shiitake mushroom) commercially available in Japan were also analyzed. This revealed limited differences in amounts of β-1,6-glucan and β-1,3-glucan in each shiitake mushroom. Furthermore, structural analysis of some purified β-glucans derived from medicinal mushrooms was performed, and their action for inducing tumor necrosis factor-α production from the murine bone marrow-derived dendritic cells was investigated. We found relation between reactivity to modified β-1,6-glucanase and its cytokine inducing activity. This assay could be useful for evaluating the strains of edible or medicinal mushrooms, which may be used as alternative medicines.