Dendritic Cell-specific Intercellular Adhesion Molecule-3-grabbing Non-integrin Research Articles

Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation via TGF-β

Background & AimsMonocyte and macrophage (MΦ) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MΦs and monocytes recruited as precursors of MΦs into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MΦs in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MΦs.MethodsHealthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MΦ markers and cytokine levels. A similar analysis was performed on circulating monocytes and liver MΦs from HCV-infected patients and controls.ResultsHuh7.5/JFH-1 cells induced monocytes to differentiate into MΦs with increased expression of CD14 and CD68. HCV-MΦs showed M2 surface markers (CD206, CD163, and Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)) and produced both proinflammatory and anti-inflammatory cytokines. HCV-induced early interleukin 1β production promoted transforming growth factor (TGF)β production and MΦ polarization to an M2 phenotype. TGF-β secreted by M2-MΦ led to hepatic stellate cell activation indicated by increased expression of collagen, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. In vivo, we observed a significant increase in M2 marker (CD206) expression on circulating monocytes and in the liver of chronic HCV-infected patients. Furthermore, we observed the presence of a unique collagen-expressing CD14+CD206+ monocyte population in HCV patients that correlated with liver fibrosis.ConclusionsWe show an important role for HCV in induction of monocyte differentiation into MΦs with a mixed M1/M2 cytokine profile and M2 surface phenotype that promote stellate cell activation via TGF-β. We also identified circulating monocytes expressing M2 marker and collagen in chronic HCV infection that can be explored as a biomarker.

Vitamin C Attenuates Hemorrhagic Hypotension Induced Epithelial-Dendritic Cell Transformation in Rat Intestines by Maintaining GSK-3β Activity and E-Cadherin Expression.

To investigate the roles of epithelial-dendritic cell transformation (EDT) characterized by the expression of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) in the occurrence of tissue inflammation induced by hemorrhagic hypotension (HH), the protective effect of vitamin C (VitC), and the potential mechanisms. We conducted an in vitro study using the rat intestinal epithelial cells (IEC-6). After hypoxic culture with or without VitC for 2, 6, 24, and 48 h (n = 3 per group), the expression levels of DC-SIGN, E-cadherin, and Glycogen synthase kinase-3β-S9 (GSK-3β-S9) in IEC-6 cells, IL-1β, and IL-6 concentrations in the cell culture medium were measured. To investigate the potential mechanism, we inhibited E-cadherin expression by siRNA and GSK-3β activity by TDZD-8, respectively. The in vivo study was conducted by establishing SD rat HH model. We observed the expression levels and location of DC-SIGN in the intestines. We also showed histological damage, TNF-α and IL-6 concentrations, and organ injury scores at 2, 6, and 24 h after HH (n = 6 per group), with or without VitC pretreatment. Hypoxia-induced DC-SIGN expression in IEC-6 cells in a time-dependent manner and the inflammatory factors were also increased. VitC inhibited all these phenomena. Hypoxia inhibited GSK-3β activity and E-cadherin expression. VitC could ease these inhibitions. The inhibitory effect of VitC on DC-SIGN was diminished when E-cadherin expression was inhibited in advance. TDZD-8 diminished the protective effect of VitC on E-cadherin and abolished inhibitory effect of VitC on DC-SIGN expression. HH-induced DC-SIGN expression in rat intestine epithelial cells and the histological damage scores and pro-inflammatory cytokine levels were also increased. HH induces EDT in rat intestine epithelial cells. VitC maintains GSK-3β activity, attenuates the suppression of E-cadherin caused by hypoxia, and ultimately decreases DC-SIGN expression.

DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.

Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC-SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC-SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC-SIGN was co-expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC-SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti-DC-SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC-SIGN, major histocompatibility complex (MHC) class II and CD80 was up-regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)-γ/interleukin (IL)-4 ratio increased. However, anti-DC-SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC-like function through their expression of DC-SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC-SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.

Lectin binding to surface Ig variable regions provides a universal persistent activating signal for follicular lymphoma cells

AbstractThe vast majority of cases of follicular lymphoma (FL), but not normal B cells, acquire N-glycosylation sites in the immunoglobulin variable regions during somatic hypermutation. Glycans added to sites are unusual in terminating at high mannoses. We showed previously that the C-type lectins, dendritic cell–specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor, bound to FL surface immunoglobulin (sIg), generating an intracellular Ca2+ flux. We have now mapped further intracellular pathways activated by DC-SIGN in a range of primary FL cells with detection of phosphorylated ERK1/2, AKT, and PLCγ2. The SYK inhibitor (tamatinib) or the BTK inhibitor (ibrutinib) each blocked phosphorylation. Activation by DC-SIGN occurred in both IgM+ and IgG+ cases and led to upregulation of MYC expression, with detection in vivo observed in lymph nodes. Unlike cells of chronic lymphocytic leukemia, FL cells expressed relatively high levels of sIg, unchanged by long-term incubation in vitro, indicating no antigen-mediated downregulation in vivo. In contrast, expression of CXCR4 increased in vitro. Engagement of sIg in FL cells or normal B cells by anti-Ig led to endocytosis in vitro as expected, but DC-SIGN, even when cross-linked, did not lead to significant endocytosis of sIg. These findings indicate that lectin binding generates signals via sIg but does not mediate endocytosis, potentially maintaining a supportive antigen-independent signal in vivo. Location of DC-SIGN in FL tissue revealed high levels in sinusoidlike structures and in some colocalized mononuclear cells, suggesting a role for lectin-expressing cells at this site.

DC-SIGN–expressing macrophages trigger activation of mannosylated IgM B-cell receptor in follicular lymphoma

AbstractFollicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM+ FL B cells activated a stronger BCR signaling network than IgG+ FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM+ FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN–dependent adhesion of highly mannosylated IgM+ FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN–expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets.

In situ Delivery of Antigen to DC-SIGN+CD14+ Dermal Dendritic Cells Results in Enhanced CD8+ T-Cell Responses

CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN.

Dengue virus binding and replication by platelets

AbstractDengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500 000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti–DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

Role of Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin on Dendritic Cells in the Recognition of Hepatitis B Virus.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an essential process for virus infection, such as HIV and hepatitis C, and plays a role in immune escape. However, the role of DC-SIGN in hepatitis B virus (HBV) infection is still unknown. The aim of this study was to investigate the role of DC-SIGN in mediating the maturation and activation of dendritic cells (DCs) when infected by HBV. Highly mannosylated HBV particles were obtained by treating HBV-producing HepG2.2.15 cells with the a-mannosidase I-inhibitor kifunensine. Highly mannosylated HBV or wild type HBV was added to infect the DCs of the DC-SIGN gene-silencing group and normal group, respectively. Then, the expression of CDla, CD80, CD83, CD86 and HLA-DR on DCs was detected by flow cytometry, the capacity of stimulating lymphocyte proliferation was tested by MTT assay, the level of IL-12p70 that was released by DCs was measured by enzyme-linked immunosorbent assay, and the expression of the proteins NF-κBp65 and p38 was detected by western blot. Both wild type and highly mannosylated HBV could promote DCs maturation and activation. However, the highly mannosylated HBV could promote DCs immune activation more strongly. The difference in the effect on DCs between the two types of HBV could be eliminated by DC-SIGN gene silencing. DC-SIGN can promote the maturation and activation of DCs when recognized HBV, but wild type HBV can escape recognition by DC-SIGN to a certain extent with the help of demannosylated modification, leading to defective DCs function and chronic HBV infection.

Comparative evaluation of several docking tools for docking small molecule ligands to DC-SIGN.

Five docking tools, namely AutoDock, FRED, CDOCKER, FlexX and GOLD, have been critically examined, with the aim of selecting those most appropriate for use as docking tools for docking molecules to the lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This lectin has been selected for its rather non-druggable binding site, which enables complex interactions that guide the binding of the core monosaccharide. Since optimal orientation is crucial for forming coordination bonds, it was important to assess whether the selected docking tools could reproduce the optimal binding conformation for several oligosaccharides that are known to bind DC-SIGN. Our results show that even widely used docking programs have certain limitations when faced with a rather shallow and featureless binding site, as is the case of DC-SIGN. The FRED docking software (OpenEye Scientific Software, Inc.) was found to score as the best tool for docking ligands to DC-SIGN. The performance of FRED was further assessed on another lectin, Langerin. We have demonstrated that this validated docking protocol could be used for docking to other lectins similar to DC-SIGN.

Association Between Individual and Combined SNPs in Genes Related to Innate Immunity and Incidence of CMV Infection in Seropositive Kidney Transplant Recipients

In this study, we assessed the association between single-nucleotide polymorphisms (SNPs) in seven candidate genes involved in orchestrating the immune response against cytomegalovirus (CMV) and the 12-month incidence of CMV infection in 315 CMV-seropositive kidney transplant (KT) recipients. Patients were managed either by antiviral prophylaxis or preemptive therapy. CMV infection occurred in 140 patients (44.4%), including 13 episodes of disease. After adjusting for various clinical covariates, patients harboring T-allele genotypes of interleukin-28B (IL28B) (rs12979860) SNP had lower incidence of CMV infection (adjusted hazard ratio [aHR]: 0.66; 95% confidence interval [CI]: 0.46-0.96; p-value = 0.029). In the analysis restricted to patients not receiving prophylaxis, carriers of the TT genotype of toll-like receptor 9 (TLR9) (rs5743836) SNP had lower incidence of infection (aHR: 0.61; 95% CI: 0.38-0.96; p-value = 0.035), whereas the GG genotype of dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN) (rs735240) SNP exerted the opposite effect (aHR: 1.86; 95% CI: 1.18-2.94; p-value = 0.008). An independent association was found between the number of unfavorable SNP genotypes carried by the patient and the incidence of CMV infection. In conclusion, specific SNPs in IL28B, TLR9 and DC-SIGN genes may play a role in modulating the susceptibility to CMV infection in CMV-seropositive KT recipients.

Host Langerin (CD207) is a receptor for Yersinia pestis phagocytosis and promotes dissemination.

Yersinia pestis is a Gram‐negative bacterium that causes plague. After Y. pestis overcomes the skin barrier, it encounters antigen‐presenting cells (APCs), such as Langerhans and dendritic cells. They transport the bacteria from the skin to the lymph nodes. However, the molecular mechanisms involved in bacterial transmission are unclear. Langerhans cells (LCs) express Langerin (CD207), a calcium‐dependent (C‐type) lectin. Furthermore, Y. pestis possesses exposed core oligosaccharides. In this study, we show that Y. pestis invades LCs and Langerin‐expressing transfectants. However, when the bacterial core oligosaccharides are shielded or truncated, Y. pestis propensity to invade Langerhans and Langerin‐expressing cells decreases. Moreover, the interaction of Y. pestis with Langerin‐expressing transfectants is inhibited by purified Langerin, a DC‐SIGN (DC‐specific intercellular adhesion molecule 3 grabbing nonintegrin)‐like molecule, an anti‐CD207 antibody, purified core oligosaccharides and several oligosaccharides. Furthermore, covering core oligosaccharides reduces the mortality associated with murine infection by adversely affecting the transmission of Y. pestis to lymph nodes. These results demonstrate that direct interaction of core oligosaccharides with Langerin facilitates the invasion of LCs by Y. pestis. Therefore, Langerin‐mediated binding of Y. pestis to APCs may promote its dissemination and infection.

Human Milk Blocks DC-SIGN-Pathogen Interaction via MUC1.

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.

Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Enterocyte dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin expression in inflammatory bowel disease.

To investigate dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expression in intestinal epithelial cells (IECs) in inflammatory bowel disease (IBD). The expression of DC-SIGN in IECs was examined by immunohistochemistry of intestinal mucosal biopsies from 32 patients with IBD and 10 controls. Disease activity indices and histopathology scores were used to assess the tissue lesions and pathologic damage. Animal studies utilized BALB/c mice with dextran sodium sulfate (DSS)-induced colitis treated with anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). Controls, untreated and treated mice were sacrificed after 7 d, followed by isolation of colon tissue and IECs. Colonic expression of DC-SIGN, CD80, CD86 and MHC II was examined by immunohistochemistry or flow cytometry. The capacity of mouse enterocytes or dendritic cells to activate T cells was determined by co-culture with naïve CD4(+) T cells. Culture supernatant and intracellular levels of interleukin (IL)-4 and interferon (IFN)-γ were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively. The ability of IECs to promote T cell proliferation was detected by flow cytometry staining with carboxyfluorescein diacetate succinimidyl ester. Compared with controls, DC-SIGN expression was significantly increased in IECs from patients with Crohn's disease (P < 0.01) or ulcerative colitis (P < 0.05). DC-SIGN expression was strongly correlated with disease severity in IBD (r = 0.48; P < 0.05). Similarly, in the DSS-induced colitis mouse model, IECs showed upregulated expression of DC-SIGN, CD80, CD86 and MHC, and DC-SIGN expression was positively correlated with disease activity (r = 0.62: P < 0.01). IECs from mouse colitis stimulated naïve T cells to generate IL-4 (P < 0.05). Otherwise, dendritic cells promoted a T-helper-1-skewing phenotype by stimulating IFN-γ secretion. However, DC-SIGN expression and T cell differentiation were suppressed following treatment of mice with DSS-induced colitis with PsL-EGFmAb. The proliferation cycles of CD4(+) T cells from mice with DSS-induced colitis appeared as five cycles, which was more than in the control and treated groups. These results suggest that IECs can promote T cell proliferation. IECs regulate tissue-associated immune compartments under the control of DC-SIGN in IBD.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Development and characterization of a monoclonal antibody specific for bovine CD209

Dendritic cells (DC) play a central role in tailoring the immune response to pathogens. Effector activity is mediated through pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPS). C-type lectin receptors (CLR) comprise a group of PRRs that recognize a broad range of pathogens. CD209 (DC-specific ICAM3-grabbing non-integrin, DC-SIGN) is a CLR expressed on DC that plays a critical role on DC function and pathogen recognition. It facilitates DC migration to peripheral tissues and local lymph nodes and mediates T cell activation by binding ICAM-2 (CD102) and ICAM-3 (CD50). The absence of monoclonal antibody (mAb) to bovine CD209 has limited the ability to characterize the phenotype and function of DC in cattle. To address this issue we developed and used a mAb to CD209 to characterize the phenotype of CD209 expressing cells in bovine blood using flow cytometry. Initial analysis has revealed the CD209 positive population in blood is comprised of multiple phenotypically defined subsets.

Dendritic cells respond to nasopharygeal carcinoma cells through annexin A2-recognizing DC-SIGN.

Dendritic cells (DCs) play an essential role in immunity and are used in cancer immunotherapy. However, these cells can be tuned by tumors with immunosuppressive responses. DC-specific intercellular adhesion molecule 3-Grabbing Nonintegrin (DC-SIGN), a C-type lectin expressed on DCs, recognizes certain carbohydrate structures which can be found on cancer cells. Nasopharyngeal carcinoma (NPC) is an epithelial cell-derived malignant tumor, in which immune response remains unclear. This research is to reveal the molecular link on NPC cells that induces the immunosuppressive responses in DCs. In this article, we report identification of annexin A2 (ANXA2) on NPC cells as a ligand for DC-SIGN on DCs. N-linked mannose-rich glycan on ANXA2 may mediate the interaction. ANXA2 was abundantly expressed in NPC, and knockdown of ANXA2 suppressed NPC xenograft in mice, suggesting a crucial role of ANXA2 in NPC growth. Interaction with NPC cells caused DC-SIGN activation in DCs. Consequently DC maturation and the proinflammatory interleukin (IL)-12 production were inhibited, and the immunosuppressive IL-10 production was promoted. Blockage of either DC-SIGN or ANXA2 eliminated the production of IL-10 from DCs. This report suggests that suppression of ANXA2 at its expression or glycosylation on NPC may improve DC-mediated immunotherapy for the tumor.

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.

Isolation and propagation of Dengue virus in Vero and BHK-21 cells expressing human DC-SIGN stably

The “standard” methods of isolating dengue virus (DENV) utilize the mosquito cell line C6/36, monkey kidney LLC-MK2 cells, Vero cells, or baby hamster kidney (BHK-21) cells. However, these cells lines lack a particular DENV receptor, known as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is expressed on immature dendritic cells and monocytes/macrophages. This may result in less efficient virus isolation and propagation. The present study used a lentivirus vector to establish Vero and BHK-21 cell lines (Vero-DC and BHK-DC) that express human DC-SIGN stably. Five DENV strains, each passaged several times in C6/36 cells, replicated more efficiently in Vero-DC and BHK-DC than in the parental Vero or BHK-21 cells. Vero/Vero-DC and BHK-21/BHK-DC were used to isolate virus from buffy coats and plasma samples derived from 13 patients infected with DENV. Most of the viruses showed increased production in cell lines expressing DC-SIGN. However, the isolation rate was lower (15.4–46.2%) than that from C6/36 cells (84.6%). Interestingly, when the viruses were isolated in C6/36 cells prior to infecting Vero/Vero-DC and BHK-21/BHK-DC, the rate of virus production increased markedly, reaching levels higher than those initially achieved in C6/36 cells. These data suggest that Vero-DC and BHK-DC could be useful tools for virus propagation, and that human specimens may contain a factor that interferes with virus growth in mammalian cells.