Murine Dendritic Cells Research Articles

Synthesis of sulfoCy5‐ and sulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

α‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer–antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7–αGalCer 1 and sulfoCy5–αGalCer 2. The fluorescent probes were obtained from a linker–functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5–αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer–antigen conjugates as shown here for DC2.4 cells.

Extracellular Silica Nanomatrices Promote In Vitro Maturation of Anti-tumor Dendritic Cells via Activation of Focal Adhesion Kinase.

The efficacy of dendritic cell (DC)-based cancer vaccines is critically determined by the functionalities of in vitro maturated DCs. The maturation of DCs typically relies on chemicals that are cytotoxic or hinder the ability of DCs to efficiently activate the antigen-specific cytotoxic T-lymphocytes (CTLs) against tumors. Herein, the maturation chemicals are replaced with extracellular silica nanomatrices, fabricated by glancing angle deposition, to promote in vitro maturation of murine bone marrow-derived DCs (mBMDCs). The extracellular nanomatrices composed of silica nanozigzags (NZs) enable the generation of mature mBMDCs with upregulated levels of co-stimulatory molecules, C-C chemokine receptor type-7, X-C motif chemokine recetpor-1, DC-specific ICAM-3 grabbing nonintegrin, and enhanced endocytic capacity. The in vitro maturation is partially governed by focal adhesion kinase (FAK) that is mechanically activated in the curved cell adhesions formed at the DC-NZ interfaces. The NZ-maturated mBMDCs can prime the antigen-specific CTLs into programmed cell death protein-1 (PD-1)lowCD44high memory phenotypes in vitro and suppress the growth of tumors in vivo. Meanwhile, the NZ-mediated beneficial effects are also observed in human monocyte-derived DCs. This work demonstrates that the silica NZs promote the anti-tumor capacity of in vitro maturated DCs via the mechanoactivation of FAK, supporting the potential of silica NZs being a promising biomaterial for cancer immunotherapy.

Dual A2A/ A2B adenosine receptor antagonist M1069 counteracts immuno-suppressive mechanisms of adenosine and reduces tumor growth in vivo.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.

Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma

BackgroundMARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. MethodsImmunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). ResultsElevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. ConclusionThese findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.

A pseudotyped adenovirus serotype 5 vector with serotype 49 fiber knob is an effective vector for vaccine and gene therapy applications

Adenoviruses (Ad) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector “ChAdOx1” in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the Coxsackie and Adenovirus Receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of a RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumour-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.

Preparation and characterization of Arctium lappa L. polysaccharide-Zn(II) complex and evaluation of its immune activity

A novel Arctium lappa L. polysaccharide zinc (ALP-Zn) complex was synthesized with success. The structural characteristics, stability, and in vitro digestive properties of ALP-Zn were studied. Moreover, the immunoreactivity of ALP-Zn on murine bone marrow-derived dendritic cells (BMDCs) was investigated by examining surface molecule expression, cytokine production and endocytosis. The results showed that the weight-average molecular weight (Mw) of ALP-Zn was 60,268 Da. ALP-Zn mainly consisted of fructose, arabinose, galactose, and glucose in a mass ratio of 50.74:27.94:12.49:7.26. Zn-O characteristic peak was identified by fourier transform infrared spectroscopy at 433.42 cm−1, indicating that zinc was successfully complexed with ALP. The stability and in vitro digestive properties suggested that ALP-Zn had good stability and biological acceptability. Furthermore, ALP and ALP-Zn demonstrated excellent immunological effects on dendritic cells (DCs). After being treated with ALP and ALP-Zn, CD80 and CD86 expression on the surface of DCs were increased and DCs maturation was induced. Moreover, ALP and ALP-Zn also significantly reduced DC endocytosis and increased cytokine (IFN-γ, TNF-α, IL-1β and IL-6). As a consequence, ALP-Zn complex could be as a potential zinc supplement with immune properties.

Macropinocytosis Is the Principal Uptake Mechanism of Antigen-Presenting Cells for Allergen-Specific Virus-like Nanoparticles.

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

Mycobacterium tuberculosis Utilizes Serine/Threonine Kinase PknF to Evade NLRP3 Inflammasome-driven Caspase-1 and RIPK3/Caspase-8 Activation in Murine Dendritic Cells.

Dendritic cells (DCs) are crucial for initiating the acquired immune response to infectious diseases such as tuberculosis. Mycobacterium tuberculosis has evolved strategies to inhibit activation of the NLRP3 inflammasome in macrophages via its serine/threonine protein kinase, protein kinase F (PknF). It is not known whether this pathway is conserved in DCs. In this study, we show that the pknF deletion mutant of M. tuberculosis (MtbΔpknF) compared with wild-type M. tuberculosis-infected cells induces increased production of IL-1β and increased pyroptosis in murine bone marrow-derived DCs (BMDCs). As shown for murine macrophages, the enhanced production of IL-1β postinfection of BMDCs with MtbΔpknF is dependent on NLRP3, ASC, and caspase-1/11. In contrast to macrophages, we show that MtbΔpknF mediates RIPK3/caspase-8-dependent IL-1β production in BMDCs. Consistently, infection with MtbΔpknF results in increased activation of caspase-1 and caspase-8 in BMDCs. When compared with M. tuberculosis-infected cells, the IL-6 production by MtbΔpknF-infected cells was unchanged, indicating that the mutant does not affect the priming phase of inflammasome activation. In contrast, the activation phase was impacted because the MtbΔpknF-induced inflammasome activation in BMDCs depended on potassium efflux, chloride efflux, reactive oxygen species generation, and calcium influx. In conclusion, PknF is important for M. tuberculosis to evade NLRP3 inflammasome-mediated activation of caspase-1 and RIPK3/caspase-8 pathways in BMDCs.

Realizing time-staggered expression of nucleic acid-encoded proteins by co-delivery of messenger RNA and plasmid DNA on a single nanocarrier.

Co-delivery of different protein-encoding polynucleotide species with varying expression kinetics of their therapeutic product will become a prominent requirement in the realm of combined nucleic acid(NA)-based therapies in the upcoming years. The current study explores the capacity for time-staggered expression of encoded proteins by simultaneous delivery of plasmid DNA (pDNA) in the core and mRNA on the shell of the same nanocarrier. The core is based on a Gelatin Type A-pDNA coacervate, thermally stabilized to form an irreversible nanogel stable enough for the deposition of cationic coats namely, protamine sulfate or LNP-related lipid mixtures. Only the protamine-coated nanocarriers remained colloidally stable following mRNA loading and could successfully co-transfect murine dendritic cell line DC2.4 with fluorescent reporter mRNA(mCherry) and pDNA (pAmCyan1).Further investigation of the protamine-coated nanosystem only, the transfection efficiency (percentage of transfected cells) and level of protein expression (mean fluorescence intensity, MFI) of mRNA and pDNA, simultaneously delivered by the same nanocarrier, were compared and kinetically assessed over 48h in DC2.4 using flow cytometry. The onset of transfection for both nucleotides was initially delayed, with levels < 5% at 6h. Thereafter, mRNA transfection reached 90% after 24h and continued to slightly increase until 48h. In contrast, pDNA transfection was clearly slower, reaching approximately 40% after 24h, but continuing to increase to reach 94% at 48h. The time course of protein expression (represented by MFI) for both NAs essentially followed that of transfection. Model-independent as well as model-dependent kinetic parameters applied to the data further confirmed such time-staggered expression of the two NA's where mRNA's rate of transfection and protein expression initially exceeded those of pDNA in the first 24h of the experiment whereas the opposite was true during the second 24h of the experiment where pDNA displayed the higher response rates. We expect that innovative nanocarriers capable of time-staggered co-delivery of different nucleotides could open new perspectives for multi-dosing, pulsatile or sustained expression of nucleic acid-based therapeutics in protein replacement, vaccination, and CRISPR-mediated gene editing scenarios.

Synthesis and Characterization of Thiolated Nanoparticles Based on Poly (Acrylic Acid) and Algal Cell Wall Biopolymers for the Delivery of the Receptor Binding Domain from SARS-CoV-2.

The COVID-19 pandemic required great efforts to develop efficient vaccines in a short period of time. However, innovative vaccines against SARS-CoV-2 virus are needed to achieve broad immune protection against variants of concern. Polymeric-based particles can lead to innovative vaccines, serving as stable, safe and immunostimulatory antigen delivery systems. In this work, polymeric-based particles called thiolated PAA/Schizo were developed. Poly (acrylic acid) (PAA) was thiolated with cysteine ethyl ester and crosslinked with a Schizochytrium sp. cell wall fraction under an inverse emulsion approach. Particles showed a hydrodynamic diameter of 313 ± 38 nm and negative Zeta potential. FT-IR spectra indicated the presence of coconut oil in thiolated PAA/Schizo particles, which, along with the microalgae, could contribute to their biocompatibility and bioactive properties. TGA analysis suggested strong interactions between the thiolated PAA/Schizo components. In vitro assessment revealed that thiolated particles have a higher mucoadhesiveness when compared with non-thiolated particles. Cell-based assays revealed that thiolated particles are not cytotoxic and, importantly, increase TNF-α secretion in murine dendritic cells. Moreover, immunization assays revealed that thiolated PAA/Schizo particles induced a humoral response with a more balanced IgG2a/IgG1 ratio. Therefore, thiolated PAA/Schizo particles are deemed a promising delivery system whose evaluation in vaccine prototypes is guaranteed.

Carboxylesterase 1 mediates a distinctive metabolic profile of dendritic cells to attain an inflammatory phenotype.

The metabolic profile of dendritic cells (DCs) shapes their phenotype and functions. Carboxylestrase 1 (CES1) enzyme is highly expressed in mononuclear myeloid cells however its exact role in DCs is elusive. We used a CES1 inhibitor (WWL113) and genetic overexpression to explore the role of CES1 in DCs differentiation in inflammatory models. CES1 expression was analyzed during CD14+ monocytes differentiation to DCs (MoDCs) using quantitative PCR. CES1 Inhibitor (WWL113) was applied during MoDCs differentiation. Surface markers, secreted cytokines, lactic acid production, phagocytic and T cell polarization capacity were analyzed. Transcriptomic and metabolic profile were assessed with RNA-sequencing and mass spectrometry. Cellular respiration was assessed with seahorse respirometry. Transgenic mice were used to assess CES1 overexpression in DCs in inflammatory models. CES1 expression peaks early during MoDCs differentiation. Pharmacological inhibition of CES1 led to higher expression of CD209, CD86 and MHCII. WWL113 treated MoDCs secreted higher quantities of IL6, IL8, TNF and IL10 and demonstrated stronger phagocytic ability and higher capacity to polarize Th17 differentiation in autologous DCs-T cells co-culture model. Transcriptomic profiling revealed enrichment of multiple inflammatory and metabolic pathways. Functional metabolic analysis shows impaired maximal mitochondrial respiration capacity, increased lactate production and decreased intracellular amino acids and TCA intermediates. Transgenic human CES1 overexpression in murine DCs generated less inflammatory phenotype and increased resistance to T cell mediated colitis. In conclusion, CES1 inhibition directs DCs differentiation towards more inflammatory phenotype, that shows stronger phagocytic capacity and supports Th17 skewing. This is associated with disrupted mitochondrial respiration and amino acids depletion.

TLR9-Dependent Activation by Inactivated Parapoxvirus Ovis in Murine Bone Marrow-Derived Dendritic Cells Is Associated with Specific Strain-Dependent Dendritic Cell Subsets

Introduction: Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is known about the signaling pathways that are involved in iPPVO-induced immune responses. Methods: In this study, we systematically analyzed how different types of dendritic cells (DCs) react to iPPVO (Zylexis, strain D1701) in both BALB/c and C57BL/6 mice by flow cytometry and ELISAs, and investigated which signaling pathway is related to DC activation by Western blotting and protein profiling. Results: We demonstrated that bone marrow-derived conventional DCs (BM-cDCs) and bone marrow-derived plasmacytoid DCs (BM-pDCs) matured and secreted type I interferons in response to Zylexis stimulation in both mouse strains. Similarly, Zylexis promoted the secretion of IL-12/23p40 and TNF by pDCs. However, IL-12/23p40 and TNF secretion by cDCs were induced in BALB/c mice but not in C57BL/6 mice. Analyzing the underlying signaling pathways revealed that iPPVO-induced maturation of cDCs was Toll-like receptor 9 (TLR9) independent, while the maturation of pDCs partially depended on the TLR9 pathway. Moreover, the production of proinflammatory cytokines by cDCs and the secretion of IFN-α/β by pDCs partially depended on the TLR9 pathway in both mouse strains. Therefore, other signaling pathways seem to participate in the response of DCs to iPPVO, supported by protein profiling. Conclusion: Our data provide useful insights into the diversity of iPPVO sensors and their varying effects across different strains and species.

Virion-surface display of a chimeric immunoglobulin Fc domain facilitating uptake by antigen-presenting cells

Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake.

Modulation of Mouse Dendritic Cells In Vitro by Lactobacillus gasseri Postbiotic Proteins.

Different lactobacilli are probiotics for their beneficial effects that confer to the host. Recently, some of these effects were associated with released metabolic products/constituents (postbiotics). In the present study, the potential immunomodulatory capacity of the probiotic Lactobacillus gasseri OLL2809 cell-free supernatant (sup) was investigated in murine bone marrow-derived dendritic cells (DCs). Bacteria induced significantly higher expression of all examined cytokines than those induced by the stimulatory lipopolysaccharide (LPS) itself. On the contrary, sup only induced the anti-inflammatory IL-10 similarly to LPS, whereas IL-12 and IL-6 secretions were stimulated at a lower level. Moreover, sup reduced the surface expression of the analyzed co-stimulatory markers CD40, CD80, and CD86. Treatments of sup with different digestive enzymes indicated the proteinaceous nature of these immunomodulatory metabolites. Western blot and immunoadsorption analyzes revealed cross-reactivity of sup with the surface-layer proteins (SLPs) isolated from OLL2809. Therefore, we directly tested the ability of OLL2809 SLPs to stimulate specifically cytokine expression in iDCs. Interestingly, we found that all tested cytokines were induced by SLPs and in a dose-dependent manner. In conclusion, our results highlighted distinct immune properties between L. gasseri OLL2809 and its metabolites, supporting the concept that bacterial viability is not an essential prerequisite to exert immunomodulatory effects.

Multiplex, high-throughput method to study cancer and immune cell mechanotransduction

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm−2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Saponin Esculeoside A and Aglycon Esculeogenin A from Ripe Tomatoes Inhibit Dendritic Cell Function by Attenuation of Toll-like Receptor 4 Signaling.

Dendritic cells (DCs) can initiate immune response through the presenting antigens to naïve T lymphocytes. Esculeoside A (EsA), a spirosolane glycoside, is reported as a major component in the ripe fruit of tomato. Little is known about the effect of tomato saponin on mice bone marrow-derived DCs. This study revealed that EsA and its aglycon, esculeogenin A (Esg-A), attenuated the phenotypic and functional maturation of murine DCs stimulated by lipopolysaccharide (LPS). We found that EsA/Esg-A down-regulated the expression of major histocompatibility complex type II molecules and costimulatory molecule CD86 after LPS stimulation. It was also determined that EsA-/Esg-A-treated DCs were poor stimulators of allogeneic T-cell proliferation and exhibited impaired interleukin-12 and TNF-α production. Additionally, EsA/Esg-A was able to inhibit TLR4-related and p-NFκB signaling pathways. This study shows new insights into the immunopharmacology of EsA/Esg-A, and represents a novel approach to controlling DCs for therapeutic application.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB

BackgroundA water-soluble ingredient of mature leaves of the tropical mahogany ‘Neem’ (Azadirachta indica), was identified as glycoprotein, thus being named as ‘Neem Leaf Glycoprotein’ (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells.MethodsSix glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of β-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs.ResultsConsidering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand’s peripheral β-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1.ConclusionNLGP’s binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.