Dendritic Cells Maturation Markers Research Articles

Decoupling immunomodulatory properties from lipid binding in the α-pore-forming toxin Sticholysin II

Sticholysin II (StII), a pore-forming toxin from the marine anemone Stichodactyla helianthus, enhances an antigen-specific cytotoxic T lymphocyte (CTL) response when co-encapsulated in liposomes with a model antigen. This capacity does not depend exclusively on its pore-forming activity and is partially supported by its ability to activate Toll-like receptor 4 (TLR4) in dendritic cells, presumably by interacting with this receptor or by triggering signaling cascades upon binding to lipid membrane. In order to investigate whether the lipid binding capacity of StII is required for immunomodulation, we designed a mutant in which the aromatic amino acids from the interfacial binding site Trp110, Tyr111 and Trp114 were substituted by Ala. In the present work, we demonstrated that StII3A keeps the secondary structure composition and global folding of StII, while it loses its lipid binding and permeabilization abilities. Despite this, StII3A upregulates dendritic cells maturation markers, enhances an antigen-specific effector CD8+ T cells response and confers antitumor protection in a preventive scenario in C57BL/6 mice. Our results indicate that a mechanism independent of its lipid binding ability is involved in the immunomodulatory capacity of StII, pointing to StII3A as a promising candidate to improve the reliability of the Sts-based vaccine platform.

Lysosome-Associated Membrane Protein-3 (LAMP3) Expression in Oral Squamous Cell Carcinoma and Its Relationship With Clinicopathological Parameters: A Cross-Sectional Study.

Background Oral squamous cell carcinoma (OSCC) accounts for the majority of oral cancers globally. It is characterized by metastasis, poor prognosis, high recurrence rate, and poor five-year survival rate due to late detection or diagnosis at an advanced stage. Novel biomarkers that can predict the prognosis of patients with OSCC are needed to improve survival. Lysosome-associated membrane protein-3 (LAMP3) glycoprotein, a member of the LAMP protein family, is a molecular marker for mature dendritic cells. LAMP3 expression has been correlated with unfavorable prognosis in patients with various cancers. Few studies have examined the relationship between LAMP3 and clinicopathological parameters in OSCC. This study aims to analyze the immunohistochemical expression of LAMP3 in OSCC and its relationship with clinicopathological characteristics. Methodology In this study, 75 formalin-fixed, paraffin-embedded samples of cases diagnosed with primary OSCC were obtained and immunostained with LAMP3 antibody. Its expression was compared with clinicopathological parameters such as age, sex, tobacco and alcohol consumption habits, differentiation, TNM staging, tumor location, lymph node metastasis, lymphovascular invasion, perineural invasion, and pattern of invasion. Results Higher LAMP3 expression was highly significantly associated with the TNM stage (p = 0.001). High expression of LAMP3 was significantly associated with T stage (p = 0.002) and lymph node metastasis (p = 0.002). All poorly differentiated OSCC cases (n = 2, 100%) showed a high expression of LAMP3. Conclusions High LAMP3 expression and its significant association with TNM stage, T stage, and lymph node metastasis suggest a potential role for LAMP3 in OSCC carcinogenesis. High LAMP3 expression in poorly differentiated OSCC might indicate that it plays a pivotal role in oncogenic cell transformation. Our results indicate that LAMP3 may be a predictive marker for poor prognosis in OSCC.

Lipoproteins are key immunostimulatory components of Bacillus species for dendritic cell maturation and activation

Dendritic cells (DCs) play an important role in immunity by sensing and responding to invasive microbes. Bacillus species are rod-shaped sporulating bacteria that include the pathogenic Bacillus cereus and commensal Bacillus subtilis. Although the interaction between DC and these two Bacillus species has been studied, their key structural component that prompts DC activation is poorly understood. Here, we investigated the two Bacillus species in DC activation by whole cells and their representative microbe-associated molecular patterns (MAMPs). MAMPs including lipoteichoic acid (LTA), lipoprotein (LPP), and peptidoglycan (PGN) were purified from the two Bacillus species. Among the MAMPs, LPP from both species most potently induced the maturation and activation of DCs while PGN, but not LTA, moderately stimulated DCs. LPPs from both Bacillus species enhanced the expression of DC maturation markers including CCR7, CD40, CD80, CD83, CD86, CD205, MHC-I, and MHC-II. Among the MAMPs from B. cereus, PGN most considerably lowered the endocytic capacity of DCs implying DC maturation whereas PGN from B. subtilis lowered it to a similar degree to its LPP. Furthermore, DCs sensitized with LPPs from both Bacillus species and PGN from B. subtilis moderately induced TNF-α and IL-6 production. Notably, a combination of MAMPs did not show any synergistic effect on DC activation. Taken together, our results demonstrate that LPP is the key structural component in B. cereus and B. subtilis that leads to DC activation.

Immunomodulation by extracellular vesicle-like nanoparticles from marine macroalgae Sargassum fusiforme: Enhancing Type 1 T helper and cytotoxic T lymphocyte-mediated immune responses

This study investigated the immunological features and therapeutic potential of extracellular vesicle-like nanoparticles isolated from the marine macroalgae Sargassum fusiforme (SF-NPs). SF-NPs were approximately 266.4 nm in size, and comprehensive metabolomic analysis revealed 38 metabolites. SF-NPs can upregulate dendritic cell (DC) maturation markers, increase pro-inflammatory cytokine production, and enhance antigen presentation capacity. DC maturation is regulated by the mitogen-activated protein kinase and nuclear factor-kappa B pathways. Notably, SF-NPs-primed DCs orchestrated robust CD4+ and CD8+ T-cell proliferation, fostering potent Type 1 T-helper(Th1) responses and cytotoxic T lymphocyte (CTL) activity. In a cyclophosphamide-induced immunosuppression mouse model, SF-NPs restored and activated immune cell populations, including DCs, natural killer, CD4+ T, and CD8+ T cells. SF-NPs reversed cyclophosphamide-induced Type 2 T-helperand regulatory T cell-biased responses, favoring Th1 and CTL responses, including multifunctional T cell responses. This study highlighted the potential of SF-NPs as therapeutic candidates for diseases requiring Th1 and CTL responses.

A critical time window for leukapheresis product transportation to manufacture clinical-grade dendritic cells with optimal anti-tumor activities

Background aimsDendritic cell (DC)-based immunotherapy is a promising approach to treat cancer. However, key aspects governing the reproducible manufacturing of high-quality DC remain incompletely defined. Here, we show that the time window between leukapheresis and DC manufacturing is critical. MethodsTranscriptomic profiling by RNA-seq was used to unbiasedly characterize cellular states during each step of DC manufacturing process, and functional assays were used to determine the anti-tumor activities of DC. ResultsDuring preclinical development of a DC-based cytotherapy platform, CUD-002 (NCT05270720), we found that DC quality varied among different batches, even though commonly used DC maturation markers CD80, CD83 and CD86 were indistinguishable. Multivariate analysis indicated that DC quality was negatively associated with the shipping time from the leukapheresis site to the manufacturing center. To investigate the potential effect of shipping time, we stored leukapheresis materials from three donors for 0, 1, 2 or 3 days before DC manufacturing. For each step, we carried out RNA-seq analysis to unbiasedly characterize cellular states. Integrated bioinformatic analyses indicated that longer storage time reduced the expression of several transcription factors to attenuate interferon pathways. ConclusionsConsistently, we found that 3-day storage of leukapheresis materials significantly lowered the efficiency to generate DC but also impaired DC responses to inflammatory signals, resulting in inferior antigen-presentation and cytotoxic T-cell activities. Thus, we recommend using leukapheresis materials within 48 h to manufacture therapeutic DCs.

Biomaterial-assisted local oxygenation safeguards the prostimulatory phenotype and functions of human dendritic cells in hypoxia.

Dendritic cells (DCs), professional antigen-presenting cells, function as sentinels of the immune system. DCs initiate and fine-tune adaptive immune responses by presenting antigenic peptides to B and T lymphocytes to mount an effective immune response against cancer and pathogens. However, hypoxia, a condition characterized by low oxygen (O2) tension in different tissues, significantly impacts DC functions, including antigen uptake, activation and maturation, migration, as well as T-cell priming and proliferation. In this study, we employed O2-releasing biomaterials (O2-cryogels) to study the effect of localized O2 supply on human DC phenotype and functions. Our results indicate that O2-cryogels effectively mitigate DC exposure to hypoxia under hypoxic conditions. Additionally, O2-cryogels counteract hypoxia-induced inhibition of antigen uptake and migratory activity in DCs through O2 release and hyaluronic acid (HA) mediated mechanisms. Furthermore, O2-cryogels preserve and restore DC maturation and co-stimulation markers, including HLA-DR, CD86, and CD40, along with the secretion of proinflammatory cytokines in hypoxic conditions. Finally, our findings demonstrate that the supplemental O2 released from the cryogels preserves DC-mediated T-cell priming, ultimately leading to the activation and proliferation of allogeneic CD3+ T cells. This work emphasizes the potential of local oxygenation as a powerful immunomodulatory agent to improve DC activation and functions in hypoxia, offering new approaches for cancer and infectious disease treatments.

Deciphering immunological mechanisms underlying the efficacy of nanoparticle-based vaccines against opioid use disorder (OUD)

Abstract The highly complex OUD and overdose epidemic poses a huge public health and economic burden, urging the need for safe and more effective therapeutic options. Vaccines offer a promising strategy for OUD treatment and prevention of overdose. Vaccinating against opioids generates drug-specific polyclonal antibodies (Ab) that selectively bind targeted opioids, blocking their distribution to the brain and thus blunting opioid induced rewards, pharmacological and side effects. Previous clinical trials of addiction vaccines showed proof of efficacy only in subjects with the highest Ab titers, highlighting the need to design more effective vaccines. The generation of a robust anti-drug polyclonal Abs response relies on CD4+ T cell-dependent B cell activation, hence dendritic cells (DC) are crucial for initiating CD4+ T cells priming and B cell responses. To improve vaccine formulation, we are exploiting a lipid-polymer hybrid nanoparticle (LPNP) platform and toll-like receptor (TLR) agonists to potentiate antigen recognition by antigen presenting cells (APCs) and subsequently the efficacy of anti-opioid conjugate vaccines. Using a flow cytometry-based in vitro activation assay, we demonstrated that LPNP-based vaccines are more effective in inducing macrophages activation marker (iNOS) and DCs co-stimulatory molecules and maturation markers (CD86, CD40, and MHC II) compared to conventional vaccines. Adding selected TLR agonists further improved nanovaccine efficacy in inducing the maturation and activation of APCs. These findings were confirmed with RT-qPCR quantifying mRNA expression of above-mentioned markers. These studies will identify key APCs subsets contributing to vaccine efficacy against OUD and other targets. Supported by grants from NIH (UG3/UH3 DA048775)

Interferon-beta mediated STAT4up-regulation are distinct in the ex vivohuman CD4 +T-cells priming dendritic cells

Abstract STAT4 (Signal Transducer Activator of Transcription factor 4) polymorphisms are risk factors contributed to autoimmune disease. While STAT4 has a dominant role in promoting interferon-gamma (IFN-g) production in T and NK cells, its existence and function exert in human dendritic cells (DCs) are still unknown. To characterize the STAT4 expression in human peripheral DCs, we isolated DCs from buffy coat of health blood donors (n = 4). The ex vivo DCs were enriched and sorted into 3 subtypes including plasmacytoid DCs (pDCs, CD123 +), CD8 +T-cells priming DC (cDC1, CLEC9A +), and CD4 +T-cells priming DC (cDC2/3, CD1c +) through negative selection bead and flow cytometry. The presence of STAT4 as well as its potential target genes (interleukin-12, IL-12 and interleukin-23, IL-23) expression were examined in IFN-beta induced DCs maturation compared to immature DCs, using Real-Time PCR. We found the proportion of DCs subsets in peripheral mononuclear cells which are 0.05%, 0.02% and 0.3% for pDC, cDC1 and cDC2/3, respectively. The IFN-beta enhanced DC maturation markers (HLA-DR +CD86 +), especially in cDC2/3. Interestingly, up-regulated STAT4 expression (2.5-fold) were significantly prominent in mature cDC2/3 compared to immature (p < 0.01), while IFN-beta showed no influence on STAT4 expression in the cDC1 and pDC (p > 0.05). Interestingly, elevated IL-12 and IL-23 expression were significantly observed in activated cDC2/3 (p < 0.05). Further study to investigate the role of STAT4 in human cDC2/3 are needed. Our result firstly discover the existing of STAT4 expression in the ex vivo peripheral human DC. Supported by Chulalongkorn University (Fundamental Fund, CU_FRB65_hea (84)_179_37_09).

A novel defined TLR3 agonist as an effective vaccine adjuvant.

Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-β production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ+CD4+ and IFN-γ+CD8+ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines.

Eimeria tenella 14-kDa phosphohistidine phosphatase stimulates maturation of chicken dendritic cells and mediates DC-induced T cell priming in a Th1 cytokine interface

Given the central role of dendritic cells (DCs) in directing cell-mediated immunity, this study investigated the capability of Eimeria tenella 14-kDa phosphohistidine phosphatase (EtPHP14) to mature chicken DCs and initiate DC-induced T cell immunity. With the aim of identifying novel protective Eimeria antigen, EtPHP14 gene was successfully cloned and EtPHP14 recombinant protein (rEtPHP14) was expressed in Escherichia coli expression system. rEtPHP14 binding was identified on the surface of chicken DCs by Immunofluorescence assay. DC phenotypes were evaluated by flow cytometry and results indicated that MHCII, CD80, CD86, CD1.1 and CD11c were up-modulated in DCs following rEtPHP14 treatment. RT-qPCR showed increased transcript levels of DC maturation markers CCL5, CCR7 and CD83 in rEtPHP14-treated DCs. Moreover, transcript profile of genes associated with intracellular signaling pathways that characterize the immunogenic (TLR signaling) or tolerogenic (Wnt signaling) state of DCs revealed that TLR signaling was stimulated and Wnt signaling was inhibited in rEtPHP14-treated DCs. Furthermore, proliferation of T cells and differentiation of CD4+ cells were promoted when rEtPHP14-treated DCs were co-cultured with autologous T cells. DCs incubated with rEtPHP14 alone expressed increased IL-12 and IFN-γ levels while IL-10 and TGF-β levels remained unaffected. Likewise, similar trend of IFN-γ expression was noted in rEtPHP14 treated DC-T cell coculture, whereas IL-4 expression remained unchanged. These findings indicate that EtPHP14 is an important molecule that can upregulate host immune response, particularly Th1, during host-parasite interaction, suggesting its importance as a novel candidate for coccidiosis vaccine.

Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells.

Damage-associated molecular patterns (DAMPs) play a critical role in dendritic cells (DCs) ability to trigger a specific and efficient adaptive immune response for different physiological and pathological scenarios. We have previously identified constitutive DAMPs (HMGB1 and Calreticulin) as well as new putative inducible DAMPs such as Haptoglobin (HP), from a therapeutically used heat shock-conditioned melanoma cell lysate (called TRIMEL). Remarkably, HP was shown to be the most abundant protein in the proteomic profile of heat shock-conditioned TRIMEL samples. However, its relative contribution to the observed DCs phenotype has not been fully elucidated. Human DCs were generated from monocytes isolated from PBMC of melanoma patients and healthy donors. DC lineage was induced with rhIL-4 and rhGM-CSF. After additional stimulation with HP, the proteome of these HP-stimulated cells was characterized. In addition, DCs were phenotypically characterized by flow cytometry for canonical maturation markers and cytokine production. Finally, in vitro transmigration capacity was assessed using Transwell plates. Our results showed that the stimulation with HP was associated with the presence of exclusive and higher relative abundance of specific immune-; energy production-; lipid biosynthesis-; and DAMPs-related proteins. Importantly, HP stimulation enhanced the expression of specific DC maturation markers and pro-inflammatory and Th1-associated cytokines, and an in vitro transmigration of primary human DCs. Taken together, these data suggest that HP can be considered as a new inducible DAMP with an important role in in vitro DC activation for cancer immunotherapy.

Host humoral immune suppression by Clostridioides difficile Toxin A and Toxin B

Abstract Gram-positive Clostridioides difficile is the greatest cause of nosocomial enteric disease in the US and is associated with a high rate of morbidity and mortality. During C. difficile infection (CDI), two secreted toxins (TcdA and TcdB) drive pathogenesis. Although T-dependent humoral immunity, specifically high affinity TcdA- and TcdB-neutralizing IgG, is the best correlation for protection against CDI recurrence, this response is commonly lacking following CDI in both mice and patients, leading to a high chance of recurrence. We previously showed that in murine CDI, there was a lack of T follicular helper (Tfh) cell differentiation, reduced IgG class-switching, poor toxin neutralization, and lack of resistance to reinfection. Preliminary data also suggested that TcdB inhibited IgG responses following vaccination against C. difficile. We therefore hypothesize that TcdA and/or TcdB suppress C. difficile-specific T-dependent humoral immunity. Following either CDI or intraperitoneal injection with TcdA or TcdB, immune cells from mesenteric lymph nodes were analyzed for changes in gene expression. Changes in dendritic cell maturation markers were observed in the presence of TcdB but not TcdA. Multi-targeting mRNA profiling showed altered expression of antigen presentation and CD4+ T cell activation genes. Only one of the known TcdB cell entry receptors was expressed by immune cells, specifically by CD4+ T cells and B cells. Efforts are underway to measure the impact of TcdA and TcdB treatment on CD4+ T cell differentiation, Tfh proliferation, and T-B cell interactions. Our data indicate that toxins may hamper neutralizing IgG production by affecting multiple aspects of T-dependent humoral immunity. This work was supported by NIH grants AI134719 (M.L.L.) and AI119048 (J.D.B.).

Genetic Engineering of Dendritic Cells Using Partially Zwitterionic Dendrimer-Entrapped Gold Nanoparticles Boosts Efficient Tumor Immunotherapy.

Effective processing and cross-priming of tumor neoantigen by dendritic cells (DCs) to T cells for spontaneous immune response generation to effectively kill cancer cells remain challenging in cancer immunotherapy. Here, we report a general approach to genetically engineer DCs through silencing their YTHDF1 protein (an important reader protein responsible for RNA m6A methylation) expression via a dendrimeric non-viral vector to boost effective tumor immunotherapy. Poly(amidoamine) dendrimers of generation 5 were partially decorated with mannose and 1,3-propanesultone and then entrapped with gold (Au) nanoparticles. The created dendrimer nanoplatform has an Au core size of 1.8 nm; possesses desired stability, good cytocompatibility, and excellent YTHDF1 siRNA compression ability; and enables targeted gene silencing of DCs overexpressing mannose receptors to upregulate the expression of CD80 and CD86, markers of DCs maturation, potentially leading to tumor antigen cross-presentation. With these properties owned, the combination of YTHDF1 silencing of DCs with programmed cell death-ligand 1 antibody can boost the best immunotherapy of a xenografted melanoma tumor model through the created antitumor immune responses. Findings in this study demonstrate a general approach of genetic engineering of DCs via a dendrimeric non-viral vector to effectively boost antitumor immunotherapy.

Combined effects of oncolytic vaccinia virus and dendritic cells on the progression of melanoma B16-F10 in mice

Aim: We aimed to test the hypothesis that loading of dendritic cells (DCs) with both viral and tumor-specific antigens would enhance the efficacy antitumor DC-based therapy applied simultaneously with oncolytic virus. Methods: Vaccinia virus LIVP/GFP and melanoma B16-F10 were used in this study. DCs were pulsed with various combinations of viral and tumor-associated antigens. The maturation status of DCs was verified by expression of the markers CD80, CD86, and CCR7 and assessment of IL-6, TNF-α, and IL-12 secretion. The most efficient combination of antigens for DC loading was selected based on the analysis of the cytotoxic activity of T lymphocytes. Combination therapy using vaccinia virus LIVP/GFP and DCs pulsed with viral and tumor-specific antigens was administered to the B16-F10 melanoma/mouse C57Bl tumor model. Results: We found that loading of DCs with viral antigens, or with a combination of viral and tumor antigens, resulted in similar levels of expression of DC maturation markers. The maximal in vitro cytotoxicity against virus-infected and non-infected B16 melanoma cells exhibited T lymphocytes activated by DCs loaded with the heat inactivated lysate of vaccinia virus LIVP/GFP infected tumor cell. The results show that the combination of vaccinia virus LIVP/GFP and DCs loaded with both tumor and viral antigens inhibit tumor growth of B16-F10 murine melanoma by more than two-fold. Conclusions: Combination therapy with oncolytic vaccinia virus LIVP/GFP and tumor/virus antigen-loaded DCs limited the growth of established melanoma B16-F10, but no synergistic antitumor effects were observed. We propose that optimization of the therapy regimen could enhance the efficiency of combination therapy.

Investigation of Fascin1, a Marker of Mature Dendritic Cells, Reveals a New Role for IL-6 Signaling in CCR7-Mediated Chemotaxis.

Migration of mature dendritic cells (DCs) to lymph nodes is critical for the initiation of adaptive immunity. CCR7, a G-protein-coupled receptor for CCL19/21 chemokines, is known to be essential for chemotaxis of mature DCs, but the molecular mechanism linking inflammation to chemotaxis remains unclear. We previously demonstrated that fascin1, an actin-bundling protein, increases chemotaxis of mature mouse DCs. In this article, we demonstrated that fascin1 enhanced IL-6 secretion and signaling of mature mouse DCs. Furthermore, we demonstrated that IL-6 signaling is required for chemotaxis. Blockage of IL-6 signaling in wild-type DCs with an anti-IL-6 receptor α (IL-6Rα) Ab inhibited chemotaxis toward CCL19. Likewise, knockout of IL-6Rα inhibited chemotaxis of bone marrow-derived DCs. The addition of soluble IL-6Rα and IL-6 rescued chemotaxis of IL-6Rα knockout bone marrow-derived DCs, underscoring the role of IL-6 signaling in chemotaxis. We found that IL-6 signaling is required for internalization of CCR7, the initial step of CCR7 recycling. CCR7 recycling is essential for CCR7-mediated chemotaxis, explaining why IL-6 signaling is required for chemotaxis of mature DCs. Our results have identified IL-6 signaling as a new regulatory pathway for CCR7/CCL19-mediated chemotaxis and suggest that rapid migration of mature DCs to lymph nodes depends on inflammation-associated IL-6 signaling.

Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells.

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS–) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS– strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS– strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.

Potent priming by inactivated whole influenza virus particle vaccines is linked to viral RNA uptake into antigen presenting cells

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.

Methods of Studying Human Dendritic Cells Applicable to Assessing Vaccine Efficacy

Introduction: Vaccines are one of the most effective means of preventing infectious diseases. Their effectiveness and safety are guaranteed by studies of vaccine properties, during their development and during the mandatory preclinical and clinical trials of each new vaccine. Additional information on the mechanisms of vaccine action on human immune system cells can be obtained using in vitro immune response models. The objective of the study was to determine applicability of certain methods of studying human dendritic cells in vitro to assessing the effect of vaccines. Dendritic cells are the most active antigen presenting cells, which play a key role in triggering a primary immune response to an infection or vaccine. Materials and methods: We studied the effect of vaccines on the maturation of dendritic cells, their phagocytic activity and the ability to stimulate T-lymphocytes in vitro. Results: To test the methods, we used vaccines with a known pattern of action on the immune system. All the vaccines induced the expression of dendritic cell maturation markers. At the same time, different vaccines induced a different set of markers and the degree of expression of these molecules. Quantitative methods for assessing phagocytosis and stimulating activity of dendritic cells are described. Conclusion: Methods for evaluation of phagocytosis, phenotypic maturation and functional properties of dendritic cells have been shown to be useful for evaluation of vaccine action. In our opinion, these methods, as a complement to traditional methods for evaluating the immune response, can be used to investigate the action of prototype vaccines at the stage of their development and preclinical trials.

Immunosuppressive Regulation of Dendritic Cells and T Cells in Allergic Rhinitis by Semaphorin 3A.

Semaphrin3A (Sema3A) was found to play a major role in immune regulation in autoimmune diseases and to be of importance in allergic disease. However, the effect of Sema3A on allergic rhinitis (AR) is not fully clear. We sought to elucidate the effects of Sema3A on the regulation of dendritic cells (DCs) and naive CD4+ T cells in AR. The expression of Sema3A in nasal mucosa was measured by immunohistochemical staining and western blotting. Human peripheral blood mononuclear cells were separated by the Ficoll-Hypaque method. DCs and naive CD4+ T cells were purified by magnetic selection. A human Sema3A Fc chimera was added to DCs and naive CD4+ T cells in vitro to evaluate the effect of Sema3A on the function of DCs and T cells. Labeling T cells with CFSE was used to determine cell proliferation. Flow cytometry was used to detect the DC maturation markers (CD40 and CD83) and T helper 17 (Th17) and regulatory T cell (Treg) percentages. ELISA was used to detect the IL10, IL17, IL4, and IFNγ cytokine levels. The expression of Sema3A in AR inferior turbinate tissue was lower than that in healthy control tissue. Compared with healthy control DCs, AR DCs showed decreased levels of the DC maturation markers CD40 and CD83 after Sema3A treatment. Furthermore, Sema3A decreased naive CD4+ T cell proliferation in AR. In addition, Sema3A increased the percentage of Tregs but had no obvious effect on Th17 cells. Moreover, Sema3A significantly increased levels of IL10 and IFNγ, and decreased level of IL4, but had no obvious effect on level of IL17. AR presented with low expression of Sema3A in nasal mucosa, and Sema3A could decrease DC maturation, T cell proliferation, and Treg polarization.

Combined Treatment With H1 and H4 Receptor Antagonists Improves Th2 Inflammatory Responses in the Nasal Mucosa of Allergic Rhinitis Rats.

Histamine H1 receptor (H1R) antagonists are the first-line drugs for the treatment of allergic rhinitis (AR) at present. Emerging evidence supports an important role of histamine H4 receptor (H4R) in allergic diseases. However, information regarding the effects of combined treatment with H1 and H4 receptor antagonists in AR is limited. We aimed to assess the effects of combined treatment with H1R and H4R antagonists on Th2 inflammatory responses in the nasal mucosa of AR rats. Sprague Dawley rats were sensitized with ovalbumin and treated with H1R antagonist desloratadine or/and H4R antagonist JNJ7777120. Western blotting was used to assay the phenotypic markers of mature dendritic cells in the nasal mucosa, including major histocompatibility complex class II (MHC-II) and co-stimulatory molecules CD80, CD86 and OX40 ligand (OX40L). Th2 inflammatory cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were determined by using enzyme-linked immunoassay. The treatment with desloratadine alone down-regulated the CD86 expression, and decreased the production of Th2 cytokines, but had no impact on the expression of MHC-II, CD80 and OX40L. The administration of NJ7777120 alone reduced the levels of CD86, OX40L and Th2 cytokines, whereas MHC-II and CD80 expression was unaffected. The combination of desloratadine and JNJ7777120 showed more significant synergistic therapeutic effects than monotherapy. H4R antagonist acted synergistically with H1R antagonist to reduce Th2 inflammatory responses by down-regulating CD86 and OX40L expression in the nasal mucosa of AR rats. The combination with H1R and H4R antagonists might be a new strategy for AR treatment.