Peripheral Blood Monocyte-derived Dendritic Cells Research Articles

Abstract LB120: Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors

Abstract Despite the remarkable success of chimeric antigen receptor T cell (CAR-T) therapies in treating hematological malignancies, their use in solid tumor indications has been met with distinct challenges. The immunosuppressive tumor microenvironment (TME) and tumor antigen heterogeneity are two significant factors that may adversely impact the efficacy of these ground-breaking therapies. Engineered natural killer T (NKT) cells harbor biological features distinct from CAR-T cell or CAR-NK cell therapies and thus represent a novel therapeutic approach to deploy “off the shelf” to patients. Appia Bio’s ACUA platform is capable of generating large quantities of highly pure CAR-NKT cells from cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) for clinical use. We have demonstrated that multiple distinct mechanisms of anti-tumor activity are operative in CAR-NKT cells, including CAR-mediated killing, NK receptor-mediated killing, adjuvant effects on antigen-specific T cells and immature dendritic cells, as well as the ability to deplete of pro-tumorigenic macrophages.When transduced with a CAR, ACUA-derived CAR-NKT cells show potent CAR-mediated tumor cell killing, similar to CAR-T cells. In contrast to conventional T cells, CAR-NKT cells also demonstrate potent CAR antigen-independent tumor cell killing, mediated partially through NKG2D and DNAM-1 activating NK receptors. CAR-NKT cells, but not conventional CAR-T cells, enhanced the function of NY-ESO specific T cells in controlling multiple rounds of tumor rechallenge, through the secretion of soluble components. In regard to other adjuvant effects which may provide additional benefit in vivo, ACUA-derived NKT cells enhanced the activation of peripheral blood monocyte-derived dendritic cells, which can regulate beneficial Th1 type immune responses. To explore how ACUA-derived NKT cells could reshape the TME, we generated immunosuppressive M2-polarized macrophages and observed that ACUA-derived NKT cells can either kill or reprogram these cells to a pro-inflammatory phenotype following co-culture. Notably, these immune cell adjuvant mechanisms are specific to ACUA-derived NKT cells and are not observed with conventional CAR-T cells. In summary, these mechanistic studies underscore the advantages of ACUA-derived NKT cells over conventional CAR-T cells and highlight their potential as a promising differentiated allogeneic therapy for the treatment of solid tumors. Citation Format: Xi Wang, Andrew J. Hou, Michael A. Christopher, Yannick Bulliard, Jason S. Damiano. Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB120.

Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1β and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

Recombinant Domain of Flagellin Promotes In Vitro a Chemotactic Inflammatory Profile in Human Immune Cells Independently of a Dendritic Cell Phenotype

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1β, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).

The role of the 47-kDa membrane lipoprotein of Treponema pallidum in promoting maturation of peripheral blood monocyte-derived dendritic cells without enhancing C-C chemokine receptor type 7-mediated dendritic cell migration.

The 47-kDa membrane lipoprotein (Tp47) is the most representative membrane protein of Treponema pallidum (T. pallidum). Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that connect innate and acquired immunity. The regulatory role of Tp47 on DCs remains unclear. To evaluate the effects of Tp47 on DC maturation and migration, and research the changes of the main chemokine C-C chemokine receptor type 7 (CCR7) involved in DC migration. A transwell assay was applied to assess the migration of DCs. Cytokines (interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α)) in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA), and the expression of cell surface markers (CD80, CD86, CD40, and human leukocyte antigen (HLA)-DR) and CCR7 was assessed using flow cytometry. The expression of CCR7 in DCs was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The Tp47 promoted DC phenotypic maturation, such as increased CD40, CD80, CD86, and HLA-DR expression, as well as DC functional maturation, thus stimulating DCs to secrete inflammatory cytokines, including IL-6, IL-10, IL-12, and TNF-α. At the same time, Tp47 did not enhance DC migration and did not increase the expression of CCR7. The Tp47 promoted the maturation of DCs while not enhancing CCR7-mediated DC migration ability. This may be one of the mechanisms by which T. pallidum escapes host immune clearance.

Evaluation of different combination of pam2CSK4, poly (I:C) and imiquimod enhance immune responses to H9N2 avian influenza antigen in dendritic cells and duck.

Current commercial H9 avian influenza viruses (AIVs) vaccines cannot provide satisfactory antibody titers and protective immunity against AIVs in duck. Toll like receptors (TLR) ligand as AIVs adjuvants can activate dendritic cells to improve immune responses in multiple animals, while the studies were absent in duck. Therefore, we investigated TLR ligands pam2CSK4, poly (I:C) and/or imiquimod enhance immune responses to inactivated H9N2 avian influenza antigen (H9N2 IAIV) in peripheral blood monocyte-derived dendritic cells (MoDCs) and duck. In vitro, we observed that transcription factor NF-κB, Th1/Th2 type cytokines (IFN-γ, IL-2 and IL-6) and the ability of catching H9N2 IAIV antigen were significantly up-regulated when H9N2 IAIV along with TLR ligands (pam2CSK4, poly (I:C) and imiquimod, alone or combination) in duck MoDCs. Also, the best enhancement effects were showed in combination of pam2CSK4, poly (I:C) and imiquimod group, whereas IFN-α showed no significant enhancement in all experimental groups. In vivo, the results demonstrated that the percentages of CD4+/ CD8+ T lymphocytes, the levels of Th1/Th2 type cytokines and H9N2 HI titers were significant enhanced in combination of pam2CSK4, poly (I:C) and imiquimod group. However, pam2CSK4 alone or combining with imiquimod showed no enhancement or additive effects on Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-6) and HI titers in Muscovy duck, respectively. Taken together, our results concluded that not all TLR ligands showed enhancement of immune responses to H9N2 IAIV in duck. The combination of poly (I:C), imiquimod and pam2CSK4 that can be an effectively adjuvant candidate for H9N2 AIVs inactivated vaccine in duck, which provide novel insights in explore waterfowl vaccine.

Phosphonate-Modified Cellulose Nanocrystals Potentiate the Th1 Polarising Capacity of Monocyte-Derived Dendritic Cells via GABA-B Receptor.

PurposePhosphonates, like 3-AminoPropylphosphonic Acid (ApA), possess a great potential for the therapy of bone tumours, and their delivery via cellulose nanocrystals (CNCs) seems a promising approach for their increased efficacy in target tissues. However, the immunological effects of CNC-phosphonates have not been investigated thoroughly. The main aim was to examine how the modification of CNCs with phosphonate affects their immunomodulatory properties in human cells.MethodsWood-based native (n) CNCs were modified via oxidation (ox-CNCs) and subsequent conjugation with ApA (ApA-CNCs). CNCs were characterised by atomic force microscopy (AFM) and nanoindentation. Cytotoxicity and immunomodulatory potential of CNCs were investigated in cultures of human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs)/T cells co-cultures by monitoring phenotype, cytokines production, allostimulatory and Th/Treg polarisation capacity.ResultsAFM showed an increase in CNCs' thickens, elasticity modulus and hardness during the modification with ApA. When applied at non-toxic doses, nCNCs showed a tolerogenic potential upon internalisation by MoDCs, as judged by their increased capacity to up-regulate tolerogenic markers and induce regulatory T cells (Treg), especially when present during the differentiation of MoDCs. In contrast, ox- and ApA-CNCs induced oxidative stress and autophagy in MoDCs, which correlated with their stimulatory effect on the maturation of MoDCs, but also inhibition of MoDCs differentiation. ApA-CNC-treated MoDCs displayed the highest allostimulatory and Th1/CTL polarising activity in co-cultures with T cells. These effects of ApA-CNCs were mediated via GABA-B receptor-induced lowering of cAMP levels in MoDCs, and they could be blocked by GABA-B receptor inhibitor. Moreover, the Th1 polarising and allostimulatory capacity of MoDCs differentiated with ApA-CNC were largely preserved upon the maturation of MoDCs, whereas nCNC- and ox-CNC-differentiated MoDCs displayed an increased tolerogenic potential.ConclusionThe delivery of ApA via CNCs induces potent DC-mediated Th1 polarisation, which could be beneficial in their potential application in tumour therapy.

Human Conventional and Plasmacytoid Dendritic Cells Differ in Their Ability to Respond to Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a commensal yeast colonizer of mucosal surfaces and an emerging opportunistic pathogen in the mucosa and bloodstream. The role of S. cerevisiae has been largely characterized in peripheral blood mononuclear cells and monocyte-derived dendritic cells, where yeast cells induce the production of inflammatory cytokines through the interaction with mannose receptors, chitin receptors, DC SIGN, and dectin1. However, the response of blood-circulating dendritic cells (DCs) to S. cerevisiae has never been investigated. Among blood DCs, conventional DCs (cDCs) are producers of inflammatory cytokines, while plasmacytoid DCs (pDCs) are a specialized population producing a large amount of interferon (IFN)-α, which is involved in the antiviral immune response. Here we report that both human DC subsets are able to sense S. cerevisiae. In particular, cDCs produce interleukin (IL)-6, express activation markers, and promotes T helper 17 cell polarization in response to yeasts, behaving similarly to monocyte-derived DCs as previously described. Interestingly, pDCs, not cDCs, sense fungal nucleic acids, leading to the generation of P1-pDCs (PD-L1+CD80–), a pDC subset characterized by the production of IFN-α and the induction of a Th profile producing IL-10. These results highlight a novel role of pDCs in response to S. cerevisiae that could be important for the regulation of the host microbiota–immune system balance and of anti-fungal immune response.

CX3CL1/fractalkine regulates the differentiation of human peripheral blood monocytes and monocyte-derived dendritic cells into osteoclasts

Osteoclast differentiation is promoted under inflammatory conditions and osteoclasts play a major role in bone destruction in rheumatoid arthritis (RA). Chemokine (C-X3-C motif) ligand 1 (CX3CL1), also known as fractalkine, functions as a chemoattractant and adhesion molecule, and is involved in the pathogenesis of RA. The blockade of CX3CL1 inhibits the migration of macrophages and osteoclast precursor cells into the inflamed synovium. In the present study, we investigated the direct stimulatory effects of CX3CL1 on osteoclast differentiation from human peripheral blood monocytes and monocyte-derived dendritic cells. A stimulation with CX3CL1 significantly promoted osteoclast differentiation from CD16- monocytes and also monocyte-derived dendritic cells induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). On the other hand, CD16+ monocytes treated with M-CSF and RANKL did not differentiate into osteoclasts, even with CX3CL1. Calcium resorption was significantly increased by monocyte-derived osteoclasts, but not by dendritic cell-derived osteoclasts, following the addition of CX3CL1. The present results suggest that CX3CL1 directly regulates osteoclast differentiation. CX3CL1 may play important roles in the pathogenesis of RA, not only through the accumulation of inflammatory cells, but also through osteoclastogenesis.

Isatis root polysaccharide promotes maturation and secretory function of monocyte-derived dendritic cells

BackgroundPseudorabies virus (PRV) is an animal virus that is globally responsible for the high economic losses in the swine industry. Isatis root is a traditional Chinese medicinal herb that possesses immune-enhancing and antiviral properties. However, the molecular mechanisms underlying the effects of the active component of the isatis root polysaccharide (IRPS) extract on immature dendritic cells remain elusive.MethodsIn this study, we investigated the molecular changes in primary porcine peripheral blood monocyte-derived dendritic cells (MoDCs) during PRV infection, using enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription-polymerase chain reaction. Additionally, we studied the effect of IRPS on PRV-infected DCs.ResultsThe results showed that IRPS stimulated the maturation of MoDCs, induced IL-12 secretion, and downregulated IL-6 expression.ConclusionsCollectively, these results suggest that IRPS is a promising candidate for promoting maturation of DCs and enhancing their secretory potential after PRV infection.

Diminished toll-like receptor response in febrile infection-related epilepsy syndrome (FIRES)

BackgroundDefective human TLR3 signaling causes recurrent and refractory herpes simplex encephalitis/encephalopathy. Children with febrile infection-related epilepsy syndrome with refractory seizures may have defective TLR responses. MethodsChildren with febrile infection-related epilepsy syndrome were enrolled in this study to evaluate TLR1-9 responses (IL-6, IL-8, IL-12p40, INF-α, INF-γ, and TNF-α) in their peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MDDCs), compared to those with febrile seizures and non-refractory epilepsy with/without underlying encephalitis/encephalopathy. ResultsAdenovirus and enterovirus were found in throat cultures of enrolled patients (2–13 years) as well as serologic IgM elevation of mycoplasma pneumonia and herpes simplex virus, although neither detectable pathogens nor anti-neural autoantibodies in the CSF could be noted. Their PBMCs and MDDCs trended to have impaired TLR responses and significantly lower in cytokine profiles of TLR3, TLR4, TLR7/8, and TLR9 responses but not other TLRs despite normal TLR expressions and normal candidate genes for defective TLR3 signaling. They also had decreased naïve T and T regulatory cells, and weakened phagocytosis. ConclusionChildren with febrile infection-related epilepsy syndrome (FIRES) could have impaired TLR3, TLR4, TLR7/8, and TLR9 responses possibly relating to their weakened phagocytosis and decreased T regulatory cells.

Immunotropic Effects and Proposed Mechanism of Action for 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase Inhibitors (Statins)

Inhibitors of HMG-CoA reductase (statins) are the major group of lipid-lowering drugs. Along with hypocholesterolemic activity, statins exhibit anti-inflammatory and immunomodulatory properties that expand their clinical use, particularly, in the treatment of chronic inflammatory and autoimmune disorders. In this review, we critically analyze the data of statin effects on immune cells (e.g., monocytes and T cells) involved in the development of atherosclerosis and other chronic inflammatory diseases. We (i) discuss the properties of statins and routes of cell entry, as well as their major intracellular targets; (ii) evaluate the data on the effects of statins on the subset composition of circulatory monocytes, ability of monocytes to migrate to the site of inflammation (cell motility and expression of adhesion molecules and chemokine receptors), production of cytokines, matrix metalloproteinases, and reactive oxygen species by monocytes/macrophages, and antigen-presenting activity in peripheral blood monocyte-derived dendritic cells; and (iii) summarize the data on the regulation of proliferation and differentiation of various CD4+ T cell subsets (type 1/2/17 helper T cells and regulatory T cells) by statins.

Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides.

The methanogenic archaeon Methanomassiliicoccus luminyensis strain B10T was isolated from human feces just a few years ago. Due to its remarkable metabolic properties, particularly the degradation of trimethylamines, this strain was supposed to be used as “Archaebiotic” during metabolic disorders of the human intestine. However, there is still no data published regarding adaptations to the natural habitat of M. luminyensis as it has been shown for the other two reported mucosa-associated methanoarchaea. This study aimed at unraveling susceptibility of M. luminyensis to antimicrobial peptides as well as its immunogenicity. By using the established microtiter plate assay adapted to the anaerobic growth requirements of methanogenic archaea, we demonstrated that M. luminyensis is highly sensitive against LL32, a derivative of human cathelicidin (MIC = 2 μM). However, the strain was highly resistant against the porcine lysin NK-2 (MIC = 10 μM) and the synthetic antilipopolysaccharide peptide (Lpep) (MIC>10 μM) and overall differed from the two other methanoarchaea, Methanobrevibacter smithii and Methanosphaera stadtmanae in respect to AMP sensitivity. Moreover, only weak immunogenic potential of M. luminyensis was demonstrated using peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (moDCs) by determining release of pro-inflammatory cytokines. Overall, our findings clearly demonstrate that the archaeal gut inhabitant M. luminyensis is susceptible to the release of human-derived antimicrobial peptides and exhibits low immunogenicity towards human immune cells in vitro–revealing characteristics of a typical commensal gut microbe.

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte-derived dendritic cells.

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro-inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte-derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF-α, -β and -γ were significantly upregulated in the H5N1 group. Pro-inflammatory cytokines (IL-1β, TNF-α and IL-18) were highly upregulated in early mid (IL-1), and late (IL-6) phases of H5N1 virus infection. IL-8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus-induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV-infected cells.

Single cell variability in pro-inflammatory and antiviral gene responses in dendritic cells

Abstract Type I interferons (IFNs) can have both protective and deleterious effects in chronic infection and autoimmune disease. Cellular responses to type I IFNs are mediated by IFN-stimulated genes (ISGs), are cell type-dependent, and likely cell specific. Varying responses of individual cells to immune signals, such as type I IFNs, may be critical in orchestrating context and microenvironment appropriate immune responses. Simulations of an agent-based mathematical model of viral infection suggested that within each cell, the levels of pro-inflammatory ISGs induced by STAT dimers and the antiviral ISGs induced by the ISGF-3 heterotrimer can show low correlation. We used RNAseq to study global single-cell correlation of ISG groups in human peripheral blood CD1c+ dendritic cells infected with an influenza A H1N1 virus. Consonant with the simulations, distinct groups of ISGs were identified that showed high levels of single cell expression correlation within, but low to negative correlations across ISG subgroups. Using multiple probe FISH we find low single cell correlation of the pro-inflammatory ISG IL6 and the antiviral ISG RIG-I induction in cells stimulated with type I IFN. The single cell correlations in the induction of the two genes were 0.56, 0.55 and 0.19 in human peripheral blood plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells respectively. The generation of uncorrelated pro-inflammatory and antiviral ISG single cell responses within a dendritic cell population can increase the breadth of the immune response to noxious stimuli.

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells.

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12μM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50μM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50μM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24h of exposure to 50μM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins.

Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway.

PRRS is one of the most important diseases in swine industry. Current PRRS inactivated vaccine provides only a limited protection and cannot induce sufficient cell-mediated immune responses. In this study, we first found that the mRNA and protein levels of Th1-type cytokines (IFN-γ, IL-12) and Th2-type cytokines (IL-6, IL-10) were significantly increased through TRIF/MyD88-NF-κB signaling pathway when porcine peripheral blood monocyte-derived dendritic cells (MoDCs) were treated with poly (I: C) of TLR3 ligand and imiquimod of TLR7 ligand, along with inactivated PRRSV antigen. Meanwhile, the ability of catching PRRSV antigen was also significantly enhanced. In mice experiment, it was found that the PRRSV-specific T lymphocyte proliferation, the percentages of CD4+, CD8+ T lymphocytes and PRRSV-specific CD3+ T cells producing IFN-γ and IL-4, the levels of Th1- and Th2-type cytokines and the titers of neutralization antibody were significantly enhanced in poly (I: C), imiquimod along with inactivated PRRSV group. Taken together, results of our experiments described for the first time that synergy of TLR3 and 7 ligands could significantly enhance the function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway and be used as adjuvant candidate for the development of novel PRRS inactivated vaccine.

Generation of regulatory dendritic cells after treatment with paeoniflorin.

Regulatory dendritic cells are a potential therapeutic tool for assessing a variety of immune overreaction diseases. Paeoniflorin, a bioactive glucoside extracted from the Chinese herb white paeony root, has been shown to be effective at inhibiting the maturation and immunostimulatory function of murine bone marrow-derived dendritic cells. However, whether paeoniflorin can program conventional dendritic cells toward regulatory dendritic cells and the underlying mechanism remain unknown. Here, our study demonstrates that paeoniflorin can induce the production of regulatory dendritic cells from human peripheral blood monocyte-derived immature dendritic cells in the absence or presence of lipopolysaccharide (LPS) but not from mature dendritic cells, thereby demonstrating the potential of paeoniflorin as a specific immunosuppressive drug with fewer complications and side effects. These regulatory dendritic cells treated with paeoniflorin exhibited high CD11b/c and low CD80, CD86 and CD40 expression levels as well as enhanced abilities to capture antigen and promote the proliferation of CD4(+)CD25(+) T cells and reduced abilities to migrate and promote the proliferation of CD4(+) T cells, which is associated with the upregulation of endogenous transforming growth factor (TGF)-β-mediated indoleamine 2,3-dioxygenase (IDO) expression. Collectively, paeoniflorin could program immature dendritic cells (imDCs) and imDCs stimulated with LPS toward a regulatory DC fate by upregulating the endogenous TGF-β-mediated IDO expression level, thereby demonstrating its potential as a specific immunosuppressive drug.

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

The presence of a galactosamine substituent on the arabinogalactan of Mycobacterium tuberculosis abrogates full maturation of human peripheral blood monocyte-derived dendritic cells and increases secretion of IL-10

Slow-growing and pathogenic Mycobacterium spp. are characterized by the presence of galactosamine (GalN) that modifies the interior branched arabinosyl residues of the arabinogalactan (AG) that is a major heteropolysaccharide cell wall component. The availability of null mutants of the polyprenyl-phospho-N-acetylgalactosaminyl synthase (Rv3631, PpgS) and the (N-acetyl-) galactosaminyl transferase (Rv3779) of Mycobacterium tuberculosis (Mtb) has provided a means to elucidate the role of the GalN substituent of AG in terms of host-pathogen interactions. Comparisons of treating human peripheral blood monocyte-derived dendritic cells (hPMC-DCs) with wild-type, Rv3631 and Rv3779 mutant strains of Mtb revealed increased expression of DC maturation markers, decreased affinity for a soluble DC-SIGN probe, reduced IL-10 secretion and increased TLR-2-mediated NF-κB activation among GalN-deficient Mtb strains compared to GalN-producing strains. Analysis of surface expression of a panel of defined or putative DC-SIGN ligands on both WT strains or either Rv3631 or Rv3779 mutant did not show significant differences suggesting that the role of the GalN substituent of AG may be to modulate access of the bacilli to immunologically-relevant receptor domains on DCs or contribute to higher ordered pathogen associated molecular pattern (PAMP)/pattern recognition receptor (PRR) interactions rather than the GalN-AG components having a direct immunological effect per se.

Toll-like receptor 1 N248S polymorphism affects T helper 1 cytokine production and is associated with serum immunoglobulin E levels in Taiwanese allergic patients.

This study was carried out to investigate whether toll-like receptor-1 (TLR1) rs4833095 (N248S) variant, common in the Taiwanese population, contributes to pathogenesis of allergy. TLR2/1 ligand Pam3CSK4-stimulated cytokine production in peripheral blood mononuclear cells and monocyte-derived dendritic cells of different genotypes were measured via enzyme-linked immunosorbent assay. Ninety-three Taiwanese allergic patients (with asthma, allergic rhinitis, or atopic dermatitis) and 76 controls were recruited for genotyping. Serum immunoglobulin E (IgE) levels were evaluated in 60 allergic patients. The homozygous TLR1 C variant allele carrier had increased Pam3CSK4-induced tumor necrosis factor-α and interleukin-12 responses in peripheral blood mononuclear cells and monocyte-derived dendritic cells, respectively. Furthermore, although the C/C genotype was not associated with susceptibility to atopic diseases, it was correlated with lower total IgE levels in sera of allergic patients. Our data suggest that the TLR1 N248S polymorphism might play a role in Th1/Th2 differentiation, and the determination of serum IgE levels. However, interactions with other genetic and environmental factors might be required to contribute to risk of allergic diseases in our population.