Primary Human Dendritic Cells Research Articles

Nanolipoprotein delivery enhances immunostimulatory properties of innate immune agonists and provides protection against lethal influenza challenge (P4218)

Abstract Therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms. As such, we have employed a nanolipoprotein (NLP) platform to enhance the efficacy of synthetic innate immune agonists. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. We demonstrate that the synthetic Toll-like receptor (TLR) ligands monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. Further, conjugation of both MPLA and CpG ODNs to NLPs significantly enhances their immunostimulatory profiles in vivo compared to administration of agonists alone. Specifically, conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, enhances and prolongs stimulation of innate immune cells, significantly increases cytokine production, and enhances upregulation of myriad immunoregulatory genes in vivo. Importantly, enhancement of cytokine production by agonists conjugated to NLPs was also observed in primary human dendritic cells. Strikingly, BALB/c mice pretreated with NLP-CpG constructs prior to a lethal influenza challenge were protected from weight-loss induced death, whereas pretreatment with CpG alone had no effect on survival. Taken together, these data suggest that NLP:agonist conjugates potentially represent a novel therapeutic measure against emerging pathogens.

333. High-Efficiency Transduction of Primary Human Monocyte-Derived Dendritic Cells by Recombinant AAV6 Vectors Containing Mutations in Surface-Exposed Serine and Threonine Residues

333. High-Efficiency Transduction of Primary Human Monocyte-Derived Dendritic Cells by Recombinant AAV6 Vectors Containing Mutations in Surface-Exposed Serine and Threonine Residues

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: A comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical’s sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential.

Ragweed pollen extract intensifies lipopolysaccharide‐induced priming of NLRP3 inflammasome in human macrophages

Ragweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1β (IL-1β), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1β production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1β production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1β production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1β production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1β and key components of the inflammasome via a ROS-dependent mechanism.

Farm‐derived Gram‐positive bacterium Staphylococcus sciuri W620 prevents asthma phenotype in HDM‐ and OVA‐exposed mice

Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation. We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co-culture with autologous T cells. The allergo-protective properties of S. sciuri W620 were proven in a T(H)2-driven OVA model as well as in a mixed T(H)1/T(H)2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell T(H)1/T(H)2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in moDC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported T(H)1 cytokine release by co-cultured T cells. Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.

Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format

Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5–7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

Inflammatory cytokine and microRNA responses of primary human dendritic cells cultured with Helicobacter pylori strains

Primary human dendritic cells (DC) were used to explore the inflammatory effectors, including cytokines and microRNAs, regulated by Helicobacter pylori. In a 48 h ex-vivo co-culture system, both H. pylori B38 and B45 strains activated human DCs and promoted a strong inflammatory response characterized by the early production of pro-inflammatory TNFα and IL-6 cytokines, followed by IL-10, IL-1β, and IL-23 secretion. IL-23 was the only cytokine dependent on the cag pathogenicity island status of the bacterial strains. DC activation and cytokine production were accompanied by an early miR-146a upregulation followed by a strong miR-155 induction, which mainly controlled TNFα production. These results pave the way for further investigations into the nature of H. pylori antigens and the subsequently activated signaling pathways involved in the inflammatory response to H. pylori infection, the deregulation of which may likely contribute to gastric lymphomagenesis.

The Need for Thorough in Vitro Testing of Biomaterial Scaffolds: Two Case Studies

Tissue engineered scaffolds are an integral part of future regenerative efforts, whether as carriers for stem cells and growth factors, or as acellular supports on their own. These scaffolds will be inserted at the site of injury, aim to provide temporary mechanical support and promote appropriate cell attachment and growth. Millions of dollars are spent on the development of biomaterial scaffolds, with many put into animal studies and clinical use without proper evaluation, often leading to unfavourable outcomes and associated patient morbidity.We exposed two novel scaffolds designed for use in tendon regeneration to a series of in vitro tests, the first a decellularised extracellular matrix (ECM), the second a silk-like material. SEM images were taken to evaluate scaffold nanostructure. Primary human dendritic cells were exposed to scaffolds, with FACS analysis of cell-surface activation markers determining scaffold immunogenicity. Growth of primary tenocytes was analysed using live-dead staining and alamarBlue® fluorescence, for cytocompatability. While, phenotypic retention was assessed through real-time PCR analysis of tenocytic genes.The decellularised ECM demonstrated low activation in the dendritic cell assay, suggesting low immunogenicity. The cytocompatability assays and real-time PCR analyses suggested that the material was indeed suitable for cell growth and differentiation. However, during the third biological repeat, no cells grew on the material, nor did they on the fourth and fifth repeat. In retrospect it appears that following the second repeat a new batch was tested, with very different properties to the first, which accounted for the reduced cytocompatability.The silk-like material produced some of the most repeatable and promising results with the cytocompatability and real-time PCR analyses, while the SEM demonstrated intertwined fibres suitable for cell alignment. However, the immunogenicity assay demonstrated that this material invoked high activation of the primary human dendritic cells, suggesting the material would be unsuitable for in vivo implantation.These studies have highlighted that without stringent testing in vitro, great time and expense would have been spent taking these materials forward for animal studies and potentially clinical studies, when clearly they are not suitable for these in their current incarnation.

The influence of the co-monomer ratio of poly[acrylonitrile-co-(N-vinylpyrrolidone)]s on primary human monocyte-derived dendritic cells

ABSTRACTA major goal in the field of regenerative medicine is to improve our understanding of how biomaterial properties affect cells of the immune system. Systematic variation of defined chemical properties could help to understand which factors determine and modulate cellular responses. A series of copolymers poly[acrylonitrile-co-(N-vinylpyrrolidone)]s (P(AN-co-NVP)) served as model system, in which increasing hydrophilicity was adjusted by increasing the content related to the NVP based repeating units (nNVP) (0, 4.6, 11.8, 22.3, and 29.4 mol%). The influence of increasing nNVPcontents on cellular response of human primary monocyte derived dendritic cells (DC), which play a key role in the initiation of immune responses, was investigated. It was shown using the LAL-Test as well as a macrophage-based assay, that the materials were free of endotoxins and other microbial contaminations, which could otherwise bias the readout of the DC experiments. The increasing nNVPcontent led to a slightly increased cell death of DC, whereas the activation status of DC was not systematically altered by the different P(AN-co-NVP)s as demonstrated by the expression of co-stimulatory molecule and cytokine secretion. Similarly, under inflammatory conditions mimicked by the addition of lipopolysaccharides (LPS), neither the expression of co-stimulatory molecules nor the release of cytokines was influenced by the different copolymers. Conclusively, our data showed that this class of copolymers does not substantially influence the viability and the activation status of DC.

Contribution of Double-Stranded RNA and CPSF30 Binding Domains of Influenza Virus NS1 to the Inhibition of Type I Interferon Production and Activation of Human Dendritic Cells

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.

PS2 - 10. Enterovirus-Infected Human Pancreatic Islets Produce Pro-inflammatory Cytokines and Chemokines and Activate Primary Human Myeloid Dendritic Cells

Enteroviruses (e.g. Coxsackie B viruses, CVBs) have been implicated as environmental factors in type 1 diabetes, but the underlying mechanism(s) remain elusive.

Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Pre-existing humoral immunity in military smallpox vaccinees temporally effects MVA-vectored transgene expression in dendritic cells

Background Modified Vaccinia Ankara (MVA), a vector-based vaccine that targets dendritic cells (DC) and induces cell-mediated immunity, is a promising HIV vaccine candidate. As MVA vaccination strategies continue to be explored, concerns arise regarding transferability to individuals with pre-existing immunity to vaccina, particularly military personnel. Prior reports suggest long-lasting vaccinia immunity after childhood vaccination. This study, conducted in a unique cohort of adult primary vaccinees, explores how pre-exisiting humoral immunity to vaccinia affects entry and transgene expression of MVA-vectored vaccines in a primary human DC infection model. Methods

Bifunctional CD4–DC-SIGN Fusion Proteins Demonstrate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination

Early stages of mucosal infection are potential targets for HIV-1 prevention. CD4 is the primary receptor in HIV-1 infection whereas DC-SIGN likely plays an important role in HIV-1 dissemination, particularly during sexual transmission. To test the hypothesis that an inhibitor simultaneously targeting both CD4 and DC-SIGN binding sites on gp120 may provide a potent anti-HIV strategy, we designed constructs by fusing the extracellular CD4 and DC-SIGN domains together with varied arrangements of the lengths of CD4, DC-SIGN and the linker. We expressed, purified and characterized a series of soluble CD4-linker-DC-SIGN (CLD) fusion proteins. Several CLDs, composed of a longer linker and an extra neck domain of DC-SIGN, had enhanced affinity for gp120 as evidenced by molecular-interaction analysis. Furthermore, such CLDs exhibited significantly enhanced neutralization activity against both laboratory-adapted and primary HIV-1 isolates. Moreover, CLDs efficiently inhibited HIV-1 infection in trans via a DC-SIGN-expressing cell line and primary human dendritic cells. This was further strengthened by the results from the human cervical explant model, showing that CLDs potently prevented both localized and disseminated infections. This is the first time that soluble DC-SIGN-based bifunctional proteins have demonstrated anti-HIV potency. Our study provides proof of the concept that targeting both CD4 and DC-SIGN binding sites on gp120 represents a novel antiviral strategy. Given that DC-SIGN binding to gp120 increases exposure of the CD4 binding site and that the soluble forms of CD4 and DC-SIGN occur in vivo, further improvement of CLDs may render them potentially useful in prophylaxis or therapeutics.

Multiple-checkpoint inhibition of thymic stromal lymphopoietin–induced TH2 response by TH17-related cytokines

The interplay between allergy and autoimmunity has been a matter of long debate. Epidemiologic studies point to a decreased frequency of allergy in patients with autoimmune diseases. However, recent studies suggest that IL-17 and related cytokines, which play a central role in autoimmunity, might also promote allergy. To address this controversy, we systematically studied the interactions between T(H)17-related cytokines and the thymic stromal lymphopoietin (TSLP)-mediated proallergic pathway. We used human primary dendritic cells (DCs), T cells, and skin explants. A novel geometric representation and multivariate ANOVA were used to analyze the T(H) cytokine profile. We show that IL-17A specifically inhibits TSLP production but increases proinflammatory IL-8 production in human skin explants exposed to TNF-α and IL-4. This inhibitory activity was confirmed in cultured skin explants of atopic dermatitis lesions. At the T-cell level, T(H)17-polarizing cytokines (IL-1β, IL-6, TGF-β, and IL-23) inhibited T(H)2 differentiation induced by TSLP-activated DCs. This led to a global dominance of a T(H)17-polarizing environment over TSLP-activated DCs, as revealed by clustering and computational analysis. Our data indicate that T(H)17-related cytokines are negative regulators of the TSLP immune pathway. This might explain the decreased frequency of allergy in patients with autoimmunity and suggests new means of manipulating proallergic responses.

368. High-Efficiency Transduction of Primary Human Monocyte-Derived Dendritic Cells by Recombinant Adeno-Associated Virus 2 Vectors Containing Mutations in Surface-Exposed Serine and Threonine Residues

368. High-Efficiency Transduction of Primary Human Monocyte-Derived Dendritic Cells by Recombinant Adeno-Associated Virus 2 Vectors Containing Mutations in Surface-Exposed Serine and Threonine Residues

Influenza A virus infection of human primary dendritic cells impairs their ability to cross-present antigen to CD8 T cells.

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.

A large‐scale 19F MRI‐based cell migration assay to optimize cell therapy

Adoptive transfer of cells for therapeutic purposes requires efficient and precise delivery to the target organ whilst preserving cell function. Therefore, therapeutically applied cells need to migrate and integrate within their target tissues after delivery, e.g. dendritic cells (DCs) need to migrate to lymph nodes to elicit an antigen-specific immune response. Previous studies have shown that inappropriate cell delivery can hinder DC migration and result in insufficient immune induction. As migration can be extremely difficult to study quantitatively in vivo, we propose an in vitro assay that reproduces key in vivo conditions to optimize cell delivery and migration in vivo. Using DC migration along a chemokine gradient, we describe here a novel (19)F MR-based, large-scale, quantitative assay to measure cell migration in a three-dimensional collagen scaffold. Unlike conventional migration assays, this set-up is amenable to both large and small cell numbers, as well as opaque tissue samples and the inclusion of chemokines or other factors. We labeled primary human DCs with a (19)F label suitable for clinical use; (0.5-15) × 10(6) cells in the scaffolds were imaged sequentially, and migration was assessed using two independent methods. We found no migration with larger numbers of cells, but up to 3% with less than one million cells. Hence, we show that the cell density in cell bolus injections has a decisive impact on migration, and this may explain the limited migration observed using large cell numbers in the clinic.

Respiratory Syncytial Virus Glycoprotein G Interacts with DC-SIGN and L-SIGN To Activate ERK1 and ERK2

Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.