Eirikur Saeland

Sweet preferences of MGL: carbohydrate specificity and function.

C-type lectins play important roles in both innate and adaptive immune responses. In contrast to the mannose- or fucose-specific C-type lectins DC-SIGN and mannose receptor, the galactose-type lectins, of which only macrophage galactose-type lectin (MGL) is found within the immune system, are less well known. MGL is selectively expressed by immature dendritic cells and macrophages with elevated levels on tolerogenic or alternatively activated subsets. Human MGL has an exclusive specificity for rare terminal GalNAc structures, which are revealed on the tumor-associated mucin MUC1 and CD45 on effector T cells. These findings implicate MGL in the homeostatic control of adaptive immunity. We discuss here the functional similarities and differences between MGL orthologs and compare MGL to its closest homolog, the liver-specific asialoglycoprotein receptor (ASGP-R).

Targeting glycan modified OVA to murine DC-SIGN transgenic dendritic cells enhances MHC class I and II presentation

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.

The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma.

The epithelial mucin MUC1 is a high molecular weight membrane glycoprotein frequently overexpressed and aberrantly glycosylated in adenocarcinoma. Mucins normally contain high amounts of O-linked carbohydrate structures that may influence immune reactions to this antigen. During malignant transformation, certain glyco-epitopes of MUC1, such as Tn-antigen, TF-antigen and their sialylated forms become exposed. The role of these glycan structures in tumor biology is unknown, but their presence is known to correlate with poor prognosis in several adenocarcinomas. We analyzed the potency of MUC1 containing Tn-antigens (MUC1-Tn) to target C-type lectins that function as carbohydrate recognition and uptake molecules on dendritic cells (DC). We identified the macrophage galactose type C-type lectin (MGL), expressed by both DC and macrophages, as the receptor for recognition and binding of MUC1-Tn. To validate the occurrence of MGL–MUC1 interactions in situ, we studied the binding of MGL to MUC1 in primary colon carcinoma tissue. Isolation of MUC1 out of colon carcinoma tissue showed strong binding activity to MGL. Interestingly, MGL binding to MUC1 was highly correlated to binding by the lectin Helix pomatia agglutinin (HPA), which is associated with poor prognosis in colorectal cancer. The detection of MGL positive cells in situ at the tumor site together with the modified glycosylation status of MUC1 to target MGL on DC suggests that MGL positive antigen presenting cells may play a role in tumor progression.

MUC1 in human milk blocks transmission of human immunodeficiency virus from dendritic cells to T cells

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.

Galectin-9 induced by dietary synbiotics is involved in suppression of allergic symptoms in mice and humans

Prebiotic galacto‐ and fructo‐oligosaccharides (scGOS/lcFOS) resembling non‐digestible oligosaccharides in human milk reduce the development of atopic disorders. However, the underlying mechanisms are still unclear. Galectins are soluble‐type lectins recognizing β‐galactoside containing glycans. Galectin‐9 has been shown to regulate mast cell degranulation and T‐cell differentiation. In this study, the involvement of galectin‐9 as a mechanism by which scGOS/lcFOS in combination with Bifidobacterium breve M‐16V protects against acute allergic symptoms was investigated.

N-glycosylated proteins and distinct lipooligosaccharide glycoforms of Campylobacter jejuni target the human C-type lectin receptor MGL

An increasing number of bacterial pathogens produce an array of glycoproteins of unknown function. Here we report that Campylobacter jejuni proteins that are modified by the N‐linked glycosylation machinery encoded by the pgl locus bind the human Macrophage Galactose‐type lectin (MGL). MGL receptor binding was abrogated by EDTA and N‐acetylgalactosamine (GalNAc) and was successfully transferred to Escherichia coli by introducing the C. jejuni pgl locus together with a glycan acceptor protein. In addition to glycoproteins, C. jejuni lipooligosaccharide with a terminal GalNAc residue was recognized by MGL. Recombinant E. coli expressing the C. jejuni pgl locus in the absence of a suitable glycan acceptor protein produced altered lipopolysaccharide glycoforms that gained MGL reactivity. Infection assays demonstrated high levels of GalNAc‐dependent interaction of the recombinant E. coli with MGL‐transfected mammalian cells. In addition, interleukin‐6 production by human dendritic cells was enhanced by C. jejuni lacking N‐linked glycans compared with wild‐type bacteria. Collectively, our results provide evidence that both N‐linked glycoproteins and distinct lipooligosaccharide glycoforms of C. jejuni are ligands for the human C‐type lectin MGL and that the C. jejuni N‐glycosylation machinery can be exploited to target recombinant bacteria to MGL‐expressing eukaryotic cells.

Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C‐type lectins MGL and DC‐SIGN, and Galectin‐3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen‐Friedenreich antigen (TF‐antigen) (Core 1, Galβ1‐3GalNAc‐Ser/Thr). Only small amounts of Tn‐antigen (GalNAcα‐Ser/Thr) expression was observed, but the human C‐type lectin MGL showed increased binding to tumour‐associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour‐associated CEA (CEACAM5) contained high levels of the blood‐group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C‐type lectin DC‐SIGN to tumour‐associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N‐glycans were prominent, and this correlated well with human Galectin‐3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Characterization of murine MGL1 and MGL2 C-type lectins: Distinct glycan specificities and tumor binding properties

Antigen presenting cells (APC) express a variety of pattern recognition receptors, including the C-type lectin receptors (CLR) that specifically recognize carbohydrate structures expressed on self-tissue and pathogens. The CLR play an important role in antigen uptake and presentation and have been shown to mediate cellular interactions. The ligand specificity of the human macrophage galactose-type lectin (MGL) has been characterized extensively. Here, we set out to determine the glycan specificity of the murine homologues, MGL1 and MGL2, using a glycan array. Murine MGL1 was found to be highly specific for Lewis X and Lewis A structures, whereas mMGL2, more similar to the human MGL, recognized N-acetylgalactosamine (GalNAc) and galactose, including the O-linked Tn-antigen, TF-antigen and core 2. The generation of MGL1 and MGL2-Fc proteins allowed us to identify ligands in lymph nodes, and MGL1-Fc additionally recognized high endothelial venules. Strikingly, MGL2 interacted strongly to adenocarcinoma cells, suggesting a potential role in tumor immunity.

Design of neo-glycoconjugates that target the mannose receptor and enhance TLR-independent cross-presentation and Th1 polarization.

Cross‐presentation is an important mechanism by which DCs present exogenous antigens on MHC‐I molecules, and activate CD8+ T cells, cells that are crucial for the elimination of tumors. We investigated the feasibility of exploiting the capacity of the mannose receptor (MR) to improve both cross‐presentation of tumor antigens and Th polarization, processes that are pivotal for the anti‐tumor potency of cytotoxic T cells. To this end, we selected two glycan ligands of the MR, 3‐sulfo‐LewisA and tri‐GlcNAc (N‐acetylglucosamine), to conjugate to the model antigen OVA and assessed in vitro the effect on antigen presentation and Th differentiation. Our results demonstrate that conjugation of either 3‐sulfo‐LewisA or tri‐GlcNAc specifically directs antigen to the MR. Both neo‐glycoconjugates showed, even at low doses, improved uptake as compared with native OVA, resulting in enhanced cross‐presentation. Using MR−/− and MyD88‐TRIFF−/− bone marrow‐derived DCs (BMDCs), we show that the cross‐presentation of the neo‐glycoconjugates is dependent on MR and independent of TLR‐mediated signaling. Whereas proliferation of antigen‐specific CD4+ T cells was unchanged, stimulation with neo‐glycoconjugate‐loaded DCs enhanced the generation of IFN‐γ‐producing T cells. We conclude that modification of antigen with either 3‐sulfo‐LewisA or tri‐GlcNAc enhances cross‐presentation and permits Th1 skewing, through specific targeting of the MR, which may be beneficial for DC‐based vaccination strategies to treat cancer.

Tumour-associated glycan modifications of antigen enhance MGL2 dependent uptake and MHC class I restricted CD8 T cell responses

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.