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Several parasitic helminthes, such as the human parasite Schistosoma mansoni, express glycoconjugates that contain terminal GalNAc beta1-4(Fuc alpha1-3)GlcNAc beta-R (LDNF) moieties. These LDNF glycans are dominant antigens of the parasite and are recognized by human dendritic cells via the C-type lectin DC-SIGN. To study the functional role of the LDNF antigen in interaction with the immune system, we have developed an easy chemoenzymatic method to synthesize multivalent neoglycoconjugates carrying defined amounts of LDNF antigens. An acceptor substrate providing a terminal N-acetylglucosamine was prepared by coupling a fluorescent hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N'-diacetylchitobiose. By the subsequent action of recombinant Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase and human alpha1,3-fucosyltransferase VI (FucT-VI), this substrate was converted to the LDNF antigen. We showed that human FucT-VI has a relatively high affinity for the unusual substrate GalNAc beta1-4GlcNAc (LDN), and this enzyme was used to produce micromolar amounts of LDNF-DAP. The synthesized LDNF-DAP was coupled to carrier protein via activation of the DAP moiety by diethyl squarate. By varying the molar glycan:protein ratio, neoglycoconjugates were constructed with defined amounts of LDNF, as was determined by MALDI-TOF analysis and ELISA using an anti-LDNF antibody.

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3(+) regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3(+) Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8(+) T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity.

Toll-like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro-inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced cross-presentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8(+) T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.

The C-type lectin macrophage galactose-type lectin (MGL) exerts an immunosuppressive role reflected by its interaction with terminal GalNAc moieties, such as the Tn antigen, on CD45 of effector T cells, thereby down-regulating T cell receptor signaling, cytokine responses, and induction of T cell death. Here, we provide evidence for the pathways that control the specific expression of GalNAc moieties on human CD4(+) T cells. GalNAc epitopes were readily detectable on the cell surface after T cell activation and required de novo protein synthesis. Expression of GalNAc-containing MGL ligands was completely dependent on PKC and did not involve NF-κB. Instead, activation of the downstream ERK MAPK pathway led to decreased mRNA levels and activity of the core 1 β3GalT enzyme and its chaperone Cosmc, favoring the expression of Tn antigen. In conclusion, expression of GalNAc moieties mirrors the T cell activation status, and thus only highly stimulated T cells are prone to the suppressive action of MGL.

It is becoming increasingly clear that glycosylation plays important role in intercellular communication within the immune system. Glycosylation-dependent interactions are crucial for the innate and adaptive immune system and regulate immune cell trafficking, synapse formation, activation, and survival. These functions take place by the cis or trans interaction of lectins with glycans. Classical immunological and biochemical methods have been used for the study of lectin function; however, the investigation of their counterparts, glycans, requires very specialized methodologies that have been extensively developed in the past decade within the Glycobiology scientific community. This mini-review intends to summarize the available technology for the study of glycan biosynthesis, its regulation and characterization for their application to the study of glycans in immunology.

Dendritic cells are the most efficient professional antigen-presenting cells in pathogen recognition and play a pivotal role in the control of the immune response. Pathogen recognition is ensured by the expression of a vast variety of pattern-recognition receptors. Amongst them are C-type lectins, a large family of receptors characterized by a domain that - in many cases - mediates calcium-dependent glycan binding. C-type lectins facilitate antigen uptake for efficient processing and presentation and, in some cases, also trigger signaling to modulate T cell responses. These properties make C-type lectin receptors ideal candidates for the targeting of antigens to dendritic cells for vaccination. DC-SIGN is a paradigmatic example of C-type lectin receptors on dendritic cells that facilitate vaccination strategies. DC-SIGN is highly expressed on immature conventional dendritic cells, particularly at the mucosa and the dermis, where DCs first encounter pathogens, but also can easily be accessed for vaccination. Upon ligand binding, DC-SIGN rapidly internalizes and directs its cargo into the endo-lysosomal pathway, which results in MHC-II presentation. But antigens targeted to DC-SIGN are also presented efficiently to CD8(+) T cells, suggesting there is an additional endocytic route that leads to cross-presentation. Simultaneous triggering of DC-SIGN and TLRs results in the modulation of cytokine responses and facilitates cross-presentation to enhance CD4(+) and CD8(+) T cell responses. Because the glycan specificity of DC-SIGN has been characterized in detail, glycans can be used for the targeting of antigens to DCs in a DC-SIGN-dependent manner. Glycans represent a great advantage over monoclonal antibodies, they diminish the risk of side effects, are very small, and their production can rely entirely in organic chemistry approaches. Here, we discuss the capacity of glycan-based vaccines to enhance antigen-specific CD4(+) and CD8(+) T cell responses in human skin and mouse model systems.

The structural characterization and quantification of the glycome of cells and glycoproteins is necessary for the understanding of glycan functions in Biology, the development of diagnostics tests, and the monitoring of glycoprotein pharmaceuticals. Classical N-glycan characterization methods involve enzymatic release followed by derivatization with a fluorochrome and separation by normal-phase HPLC. We have recently developed glycan nanoprofiling, a method for the simultaneous quantification and characterization of the N-glycans without the need of external standardization. Although glycan nanoprofiling allows the characterization of both neutral and sialylated glycans within the same chromatographic run, a significant drawback is the coelution of similar glycans when complex glycan mixtures are analyzed. To overcome this problem, we have developed enhanced glycan nanoprofiling. This new method introduces a weak anion-exchange HPLC separation step to fractionate glycans according to their sialic acid content followed by a mild acid desialylation. Glycans are then resolved by nano-LC-coupled ESI-MS with an intercalated nanofluorescence detector. Neutral glycans have a better analytical separation, better ionization profiles, and provide significantly higher MS signals allowing a detailed characterization of rare glycan species. Enhanced glycan nanoprofiling is a powerful approach that provides a fast and sensitive alternative to available N-glycan profiling methods.

Dendritic cells (DCs) are antigen-presenting cells efficient in capturing pathogens, and processing their antigenic determinants for presentation to antigen-specific T cells to induce robust immune responses. Their location at peripheral tissues and the expression of pattern-recognition receptors, among them DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), facilitates the capture of pathogens before spreading. However, some pathogens have developed strategies to escape the immune system. One of the most successful is HIV-1, which targets DC-SIGN for transport to the lymph node where the virus infects CD4(+) T cells. Contact of HIV-1 with DC-SIGN is thus the first event in the pathogenic cascade and, therefore, it is the primary target point for therapies aimed at HIV infection prevention. DC-SIGN recognizes specific glycans on HIV-1 and this interaction can be blocked by competitive inhibition through glycans. Although the affinity of glycans is relatively low, multivalency may increase avidity and the strength to compete with HIV-1 virions. We have designed multivalent dendrimeric compounds based on Lewis-type antigens that bind DC-SIGN with high selectivity and avidity and that effectively block gp120 binding to DC-SIGN and, consequently, HIV transmission to CD4(+) T cells. Binding to DC-SIGN and gp120 inhibition was higher on glycodendrimers with larger molecular diameter, indicating that the geometry of the compounds is an important factor determining their functionality. Our compounds elicited DC-SIGN internalization, a property of the receptor upon triggering, but did not affect the maturation status of DCs. Thus, Le(X) glycodendrimers could be incorporated into topic prophylactic approaches for the prevention of HIV-1 transmission.

We recently showed that MGL2 specifically binds tumour-associated glycan N-acetylgalactosamine (GalNAc). We here demonstrate that modification of an antigen with tumour-associated glycan GalNAc, targets antigen specifically to the MGL2 on bone marrow derived (BM)-DCs and splenic DCs. Glycan-modification of antigen with GalNAc that mimics tumour-associated glycosylation, promoted antigen internalisation in DCs and presentation to CD4 T cells, as well as differentiation of IFN-γ producing CD4 T cells. Furthermore, GalNAc modified antigen enhanced cross-presentation of both BM-DCs and primary splenic DCs resulting in enhanced antigen specific CD8 T cell responses. Using MyD88-TRIFF(-/-) BM-DCs we demonstrate that the enhanced cross-presentation of the GalNAc modified antigen is TLR independent. Our data strongly suggest that tumour-associated GalNAc modification of antigen targets MGL on DCs and greatly enhances both MHC class II and class I presentation in a TLR independent manner.

Dendritic cells (DCs) are key antigen presenting cells that have the unique ability to present antigens on MHC molecules, which can lead to either priming or suppression of T cell mediated immune responses. C-type lectin receptors expressed by DCs are involved in antigen uptake and presentation through recognition of carbohydrate structures on antigens. Here we have explored the feasibility of modification of liposomes with glycans for targeting purposes to boost immune responses. The potential of targeting glycoliposomal constructs to the C-type lectin DC-SIGN on DCs was studied using either PEGylated or non-PEGylated liposomes. Our data demonstrate that formulation of the glycoliposomes as PEGylated negatively affected their potential to target to DCs.

Mother-to-child transmission of human immunodeficiency virus-1 (HIV-1) occurs frequently via breast-feeding. HIV-1 targets DC-SIGN+ dendritic cells (DCs) in mucosal areas that allow efficient transmission of the virus to T cells. Here, we demonstrate that the epithelial mucin MUC1, abundant in milk, efficiently bound to DC-SIGN on DC. The O-linked glycans within the mucin domain contained Lewis X structures, that were specifically recognized by the receptor. Interestingly, MUC1 prevented DC-SIGN-mediated transmission of HIV-1 from DCs to CD4+ T cells. We hypothesize that repetitive units of Lewis X, within the mucin domain, play an important role in inhibiting transmission of HIV-1 from mother to child. (C) 2009 Elsevier Ltd. All rights reserved

Dendritic cells have gained much interest in the field of anti-cancer vaccine development because of their central function in immune regulation. One of the receptors that facilitate DC-specific targeting of antigens is the DC-specific C-type lectin DC-SIGN. Although DC-SIGN is specifically expressed on human DCs, its murine homologue is not present on any murine DC subsets, which makes in vivo evaluation of potential DC-SIGN targeting vaccines very difficult. Here we describe the use of DC-SIGN transgenic mice, as a good model system to evaluate DC-SIGN targeting vaccines. We demonstrate that glycan modification of OVA with DC-SIGN targeting glycans, targets antigen specifically to bone marrow (BM)** derived DCs and splenic DCs. Glycan modification of OVA with Lewis X or Lewis B oligosaccharides, that target DC-SIGN transgenic DCs, resulted in efficient 10-fold induction of OT-II compared to unmodified OVA. Interestingly, glycan modified OVA proteins were significantly cross-presented to OT-I T cells by wild type DC, 10-fold more than native OVA, and the expression of DC-SIGN further enhanced this cross-presentation. Targeting of glycosylated OVA was neither accompanied with any DC maturation, nor the production of inflammatory or anti-inflammatory cytokines. Thus, we conclude that glycan modification of antigens and targeting to DC-SIGN enhance both CD4 and CD8 T cell responses. Furthermore, our data demonstrate that DC-SIGN transgenic mice are valuable tool for optimisation and efficiency testing of DC vaccination strategies that are designed to target in particular the human DC-SIGN receptor.

Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large amounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are approximately 100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the d-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing.

The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans. We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans.

Cancer immunotherapy requires potent tumor-specific T cell responses, initiated by dendritic cells (DCs). Our aim is to develop a therapy that specifically targets DCs in vivo in human skin using glycosylated liposomes that contain tumor antigen and Toll-like receptor (TLR) ligand. The use of glycans specific for certain C-type-lectin receptors (CLRs) allows directing of the liposomes to specific DC subsets. Targeting the CLR DC-SIGN, which is a potent uptake receptor specifically expressed on human dermal DCs, by using Lewis (Le)X-modified liposomes, resulted in increased internalization of liposomes by human monocyte-derived (mo)DCs. Targeting moDCs with Melanoma antigen MART-1-encapsulated liposomes modified with LeX led to enhanced antigen presentation to CD8+ T cells. For good CD8+ T cell responses, maturation of DCs with LPS was necessary. Incorporation of TLR ligands MPLA, Poly I:C, R848, R837 or Pam3CSK4 into LeX-modified liposomes resulted in maturation of moDCs. Moreover, injection of TLR ligand-glycoliposomes in human skin led to their enhanced uptake by skin DCs and DC maturation. The enhanced uptake of glycoliposomes by skin DCs resulted in enhanced antigen presentation to CD8+ T cells. Our data demonstrate the potency of glycoliposomes as anti-cancer vaccine targeting DC-SIGN+ skin DCs in vivo and inducing tumor-specific T cell responses.

In vivo dendritic-cell targeting constitutes a promising strategy for anticancer vaccination. Here, we discuss the usage of multivalent DC-SIGN-targeting glycan platforms that allow for the efficient routing of antigens to the endo-lysosomal pathway as well as to a yet uncharacterized cross-presentation mechanism inducing CD4+ and CD8+ T-cell responses.