Modulation of Immune Tolerance via Siglec-Sialic Acid Interactions

Lubbers, Joyce, Rodriguez, Ernesto, van Kooyk, Yvette


One of the key features of the immune system is its extraordinary capacity to discriminate between self and non-self and to respond accordingly. Several molecular interactions allow the induction of acquired immune responses when a foreign antigen is recognized, while others regulate the resolution of inflammation, or the induction of tolerance to self-antigens. Post-translational signatures, such as glycans that are part of proteins
(glycoproteins) and lipids (glycolipids) of host cells or pathogens, are increasingly appreciated as key molecules in regulating immunity vs. tolerance. Glycans are sensed by glycan binding receptors expressed on immune cells, such as C-type lectin receptors (CLRs) and Sialic acid binding immunoglobulin type lectins (Siglecs), that respond to specific glycan signatures by triggering tolerogenic or immunogenic signaling pathways.
Glycan signatures present on healthy tissue, inflamed and malignant tissue or pathogens provide signals for “self” or “non-self” recognition. In this review we will focus on sialic acids that serve as “self” molecular pattern ligands for Siglecs. We will emphasize on the function of Siglec-expressing mononuclear phagocytes as sensors for sialic acids in tissue homeostasis and describe how the sialic acid-Siglec axis is exploited by tumors and pathogens for the induction of immune tolerance. Furthermore, we highlight how the sialic acid-Siglec axis can be utilized for clinical applications to induce or inhibit immune tolerance.…

C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining type I interferon signaling in dendritic cells

Troegeler, A; Mercier, I; Cougoule, C; Pietretti, D; Colom, A; Duval, C; Manh, TPV; Capilla, F; Poincloux, R; Pingris, K; Nigou, J; Rademann, J; Dalod, M; Verreck, FAW; Al Saati, T; Lugo-Villarino, G; Lepenies, B; Hudrisier, D; Neyrolles, O

Proc Natl Acad Sci U S A. 2017 Jan 24; 114(4): E540–E549

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary
lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen’s control through sustaining type I IFN
signaling in DCs.…

CD169(+) Macrophages Capture and Dendritic Cells Instruct: The Interplay of the Gatekeeper and the General of the Immune System

Grabowska, Joanna; Lopez-Venegas, Miguel A.; Affandi, Alsya J.; den Haan, Joke M. M.

FRONTIERS IN IMMUNOLOGY, 26 October 2018, Volume 9, Article 2472

Since the seminal discovery of dendritic cells (DCs) by Steinman and Cohn in 1973, there has been an ongoing debate to what extent macrophages and DCs are related and perform different functions. The current view is that macrophages and DCs originate from different lineages and that only DCs have the capacity to initiate adaptive immunity. Nevertheless, as we will discuss in this review, lymphoid tissue resident CD169+ macrophages have been shown to act in concert with DCs to promote or suppress adaptive immune responses for pathogens and self-antigens, respectively. Accordingly, we propose a functional alliance between CD169+ macrophages and DCs in which a division of tasks is established. CD169+ macrophages are responsible for the capture of pathogens and are frequently the first cell type infected and thereby provide a confined source of antigen. Subsequently, cross-presenting DCs interact with these antigen-containing CD169+ macrophages, pick up antigens and activate T cells. The
cross-priming of T cells by DCs is enhanced by the localized production of type I interferons (IFN-I) derived from CD169+ macrophages and plasmacytoid DCs (pDCs) that induces DC maturation. The interaction between CD169+ macrophages and DCs appears not only to be essential for immune responses against pathogens, but also plays a role in the induction of self-tolerance and immune responses against cancer. In this review we will discuss the studies that demonstrate the collaboration between CD169+
macrophages and DCs in adaptive immunity.…

On-chip screening of a glycomimetic library with C-type lectins reveals structural features responsible for preferential binding of dectin-2 over DC-SIGN/R and langerin

Medve, L; Achilli, S; Serna, S; Zuccotto, F; Varga, N; Thépaut, M; Civera, M; Vivès, C; Fieschi, F; Reichardt, N; Bernardi, A.

Chem. Eur. J. 2018, 24, 14448 – 14460

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the
first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.…

Automated glycan assembly as an enabling technology

Pardo-Vargas, A. ; Delbianco, M. ; Seeberger, P.

Current Opinion in Chemical Biology 2018, 46:48–55

Access to complex carbohydrates remains a limiting factor for the development of the glycosciences. Automated glycan assembly (AGA) has accelerated and simplified the synthetic process and, with the first commercially available instrument and building blocks, glycan synthesis can now be practiced by any chemist. All classes of glycans, including sulfated or sialylated carbohydrates and polysaccharides as long as 50mers are now accessible owing to optimized reaction conditions and new methodologies. These synthetic glycans have helped to understand many biological functions and to advance diagnostic and vaccine development. Establishing detailed structure–function relationships will eventually enable the production of unnatural materials with tuned properties.…

Fucosylated Antigens in Cancer: An Alliance toward Tumor Progression, Metastasis, and Resistance to Chemotherapy

Athanasios Blanas, Neha M. Sahasrabudhe, Ernesto Rodríguez, Yvette van Kooyk, Sandra J. van Vliet

Frontiers in Oncology 8:39 2018

Aberrant glycosylation of tumor cells is recognized as a universal hallmark of cancer pathogenesis. Overexpression of fucosylated epitopes, such as type I (H1, Lewisa, Lewisb, and sialyl Lewisa) and type II (H2, Lewisx, Lewisy, and sialyl Lewisx) Lewis antigens, frequently occurs on the cancer cell surface and is mainly attributed to upregulated expression of pertinent fucosyltransferases (FUTs). Nevertheless, the impact of fucose-containing
moieties on tumor cell biology is not fully elucidated yet. Here, we review the relevance of tumor-overexpressed FUTs and their respective synthesized Lewis determinants in critical aspects associated with cancer progression, such as increased cell survival and proliferation, tissue invasion and metastasis, epithelial to mesenchymal transition,
endothelial and immune cell interaction, angiogenesis, multidrug resistance, and cancer stemness. Furthermore, we discuss the potential use of enhanced levels of fucosylation as glycan biomarkers for early prognosis, diagnosis, and disease monitoring in cancer patients.…

ABO Antigens Active Tri- and Disaccharides Microarray to Evaluate C-type Lectin Receptor Binding Preferences

Chethan D. Shanthamurthy, Prashant Jain, Sharon Yehuda, João T. Monteiro, Shani Leviatan Ben-Arye, Balamurugan Subramani, Bernd Lepenies, Vered Padler-Karavani, Raghavendra Kikkeri

Sci Rep. 2018, Apr 26;8(1):6603

Understanding blood group antigen binding preferences for C-type lectin receptors holds promise for modulating immune responses, since several Gram-negative bacteria express blood group antigens as molecular mimicry to evade immune responses. Herein, we report the synthesis of ABO blood group antigen active tri and disaccharides to investigate the binding specificity with various C-type lectin receptors using glycan microarray. The results of binding preferences show that distinct glycosylation on the galactose and fucose motifs are key for C-type lectin receptor binding and that these interactions occur in a Ca2+-dependent fashion.…

Chemoenzymatic Synthesis of N-glycan Positional Isomers and Evidence for Branch Selective Binding by Monoclonal Antibodies and Human C-type Lectin Receptors

Begoña Echeverria, Sonia Serna, Silvia Achilli, Corinne Vivès, Julie Pham, Michel Thépaut, Cornelis H. Hokke, Franck Fieschi, Niels-Christian Reichardt

ACS Chem Biol, 2018, 13 (8), 2269-2279

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of Nglycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4-[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeX monoclonal IgM antibodies raised against S. mansoni glycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular

C-type Lectin Receptor (CLR)–Fc Fusion Proteins As tools to screen for Novel CLR/Bacteria Interactions: An exemplary study on Preselected Campylobacter jejuni Isolates

Sabine Mayer, Rebecca Moeller, Joao T. Monteiro, Kerstin Ellrott, Christine Josenhans and Bernd Lepenies

Frontiers in Immunology 9 (2018) Art. 213


C-type lectin receptors (CLRs) are carbohydrate-binding receptors that recognize their ligands often in a Ca2+-dependent manner. Upon ligand binding, myeloid CLRs in innate immunity trigger or inhibit a variety of signaling pathways, thus initiating or modulating effector functions such as cytokine production, phagocytosis, and antigen presentation. CLRs bind to various pathogens, including viruses, fungi, parasites, and bacteria. The bacterium Campylobacter jejuni (C. jejuni) is a very frequent Gram-negative zoonotic pathogen of humans, causing severe intestinal symptoms. Interestingly, C. jejuni expresses several glycosylated surface structures, for example, the capsular polysaccharide (CPS), lipooligosaccharide (LOS), and envelope proteins. This “Methods” paper describes applications of CLR–Fc fusion proteins to screen for yet unknown CLR/bacteria interactions using C. jejuni as an example. ELISA-based detection of CLR/bacteria interactions allows a first prescreening that is further confirmed by flow cytometry-based binding analysis and visualized using confocal microscopy. By applying these methods, we identified Dectin-1 as a novel CLR recognizing two selected C. jejuni isolates with different LOS and CPS genotypes. In conclusion, the heredescribed applications of CLR–Fc fusion proteins represent useful methods to screen for and identify novel CLR/bacteria interactions.

Facile access to pseudo-thio-1,2-dimannoside, a new glycomimetic DC-SIGN antagonist

Alice Tamburrini, Silvia Achilli, Francesca Vasile, Sara Sattin, Corinne Vivès, Cinzia Colombo,
Franck Fieschi, Anna Bernardi

Bioorganic & Medicinal Chemistry 25 (2017) 5142–5147


The synthesis and conformational analysis of pseudo-thio-1,2-dimannoside are described. This molecule
mimics mannobioside (Mana(1,2)Man) and is an analog of pseudo-1,2-dimannoside, with expected
increased stability to enzymatic hydrolysis. A short and efficient synthesis was developed based on an
epoxide ring-opening reaction by a mannosyl thiolate, generated in situ from the corresponding thioacetate.
NMR-NOESY studies supported by MM3⁄ calculations showed that the pseudo-thio-1,2-dimannoside
shares the conformational behavior of the pseudo-1,2-dimannoside and is a structural mimic of
the natural disaccharide. Its affinity for DC-SIGN was measured by SPR and found to be comparable to
the corresponding O-linked analog, offering good opportunities for further developments.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH–p Stacking Interactions by Different Fluorination Patterns

Luca Unione, María Alcalá, Begoña Echeverria, Sonia Serna, Ana Ardá, Antonio Franconetti, F. Javier Cañada, Tammo Diercks, Niels-Christian Reichardt, Jesús Jiménez-Barbero

Chem. Eur. J. 2017, 23, 3957 – 3965


We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar–protein interactions by simple 1H and/or 19F NMR spectroscopy methods. In this process, we have chosen the binding of N,N’-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2FCONHgroup and the natural molecule (with the CH3CONH- fragment). In contrast, the molecule with the CF3CONH- isoster displayed the weakest intermolecular interaction (one order
of magnitude weaker). Because sugar–aromatic CH–p interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

Myeloid C-Type Lectin Receptors in Viral Recognition and Antiviral Immunity

João T. Monteiro, Bernd Lepenies

Viruses 2017, 9, 59; doi:10.3390/v9030059


Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into hosT cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.

NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features

Ana Gimeno, Niels-Christian Reichardt, F. Javier Cañada, Lukas Perkams, Carlo Unverzagt, Jesús Jiménez-Barbero, Ana Ardá

ACS Chemical Biology 2017 12 / 4 (1104-1112)


Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate–protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.

Specific anti-glycan antibodies are sustained during and after parasite clearance in Schistosoma japonicum-infected rhesus macaques

Y. Y. Michelle Yang, Xiao Hong Li, Katarzyna Brzezicka, Niels-Christian Reichardt, R. Alan Wilson, Angela van Diepen, Cornelis H. Hokke

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005339


Schistosomes express many glycan antigens to which antibodies are raised by the infected host. These glycans may therefore form potential vaccine targets. Unlike humans where the disease persists chronically if not treated, schistosome-infected rhesus macaques are able to elicit a self-cure process naturally. To find out if anti-glycan responses could contribute to the natural clearance process, we followed the dynamics of anti-glycan serum antibodies in Schistosoma-infected macaques in a longitudinal study starting from the onset of infection until 22 weeks post-infection, when the macaques had eliminated most of the parasites. We found that sera of macaques taken after 22 weeks of infection contained high IgG titres towards specific schistosome glycan epitopes highly abundant on schistosome larvae. Moreover, infected macaque serum at week 22 was able to kill schistosomula in vitro. Our results suggest that anti-glycan antibodies play an important role in the self-cure process and the acquired resistance to re-infection in Schistosoma infected macaques.

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