Y. van Kooyk

The Actin Cytoskeleton Regulates LFA-1 Ligand Binding through Avidity Rather than Affinity Changes

To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (αLβ2), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete β2cytoplasmic tail and/or the conserved KVGFFKR sequence in the αL cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.

The LFA-1 integrin supports rolling adhesions on ICAM-1 under physiological shear flow in a permissive cellular environment.

The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A β2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium–host interaction

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC‐SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose‐dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC‐SIGN‐transfected Raji cells, but no differences in IL‐10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose‐capped LAM does not dominate the Mycobacterium–host interaction.

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells

Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells in order to achieve an appropriate uptake, processing, and presentation to Ag-specific T cells. C-type lectins have shown to be ideal receptors for the targeting of antigens to dendritic cells and allow the use of their natural ligands - glycans - instead of antibodies. Amongst them, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is an interesting candidate due to its biological properties and the availability of its natural carbohydrate ligands. Using Le(b)-conjugated poly(amido amine) (PAMAM) dendrimers we aimed to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16-32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases.

CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG

Human myelin oligodendrocyte glycoprotein is decorated with fucosylated N-glycans that are recognized by DC-SIGN+ DCs and microglia that control immune homeostasis.

Antibody-Opsonized Bacteria Evoke an Inflammatory Dendritic Cell Phenotype and Polyfunctional Th Cells by Cross-Talk between TLRs and FcRs

During secondary immune responses, Ab-opsonized bacteria are efficiently taken up via FcRs by dendritic cells. We now demonstrate that this process induces cross-talk between FcRs and TLRs, which results in synergistic release of several inflammatory cytokines, as well as altered lipid metabolite profiles. This altered inflammatory profile redirects Th1 polarization toward Th17 cell responses. Interestingly, GM-CSF–producing Th cells were synergistically evoked as well, which suggests the onset of polyfunctional Th17 cells. Synergistic cytokine release was dependent on activation via MyD88 and ITAM signaling pathways through TLRs and FcRs, respectively. Cytokine regulation occurred via transcription-dependent mechanisms for TNF-α and IL-23 and posttranscriptional mechanisms for caspase-1–dependent release of IL-1β. Furthermore, cross-talk between TLRs and FcRs was not restricted to dendritic cells. In conclusion, our results support that bacteria alone initiate fundamentally different immune responses compared with Ab-opsonized bacteria through the combined action of two classes of receptors and, ultimately, may refine new therapies for inflammatory diseases.

Using the glycan toolbox for pathogenic interventions and glycan immunotherapy

Glycans play a crucial role to discern between self and foreign entities by providing key recognition elements for C-type lectin receptors (CLRs) and Siglec receptors expressed on immune cells. The glycan recognition of CLRs has illustrated a potent immune modulatory role affecting not only innate pathogen binding and immune signalling, but also Thelper differentiation, cytokine production and antigen presentation. This broad range of influence has implicated glycans in the pathogenesis of infectious diseases but also revealed their extraordinary properties in cancer. Glycan binding by CLRs and Siglecs can be exploited for immunotherapy and the design of glycan-based therapeutics and their multivalent requirements will aspire new biotechnological approaches to effectively interfere in immunological processes in cancer and infectious diseases.

The human glycoprotein salivary agglutinin inhibits the interaction of dc-sign and langerin with oral micro-organisms

Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1 gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavity contains dendritic cells (DC) and Langerhans cells (LC), which express the C-type lectin receptors (CLR) DC-SIGN and Langerin, respectively. Both DC-SIGN and Langerin recognise mannose and fucose carbohydrate structures on pathogens and self-glycoproteins to regulate immunity and homeostasis. The purpose of this study was to investigate whether SAG interacts with these CLR and whether this interferes with the binding to oral pathogens. We show that whole parotid saliva and SAG, when coated to microplates, strongly interact with DC-SIGN and Langerin, probably via mannose and fucose structures. Also, primary human DC and LC bind parotid saliva and SAG via DC-SIGN and Langerin, respectively. Furthermore, SAG binding to DC-SIGN or Langerin prevented binding to the micro-organisms Candida albicans and Escherichia coli which express mannose and fucose-containing glycan structures. Thus, binding of saliva glycoprotein SAG to DC-SIGN and Langerin may inhibit pathogen-DC/LC interactions, and could prove to be a new immunomodulatory mechanism of SAG.

A1.45 Hyperglycosylation of ACPA-IGG variable domains modulates reactivity to citrullinated antigens

Background and Objectives Autoantibodies specific for citrullinated antigens are highly relevant diagnostic and prognostic biomarkers in rheumatoid arthritis and have been considered to be involved in disease pathogenesis. Previous studies have indicated that the ACPA-specific immune response differs from conventional B cell responses by generating polyclonal, cross-reactive antibodies of mostly low-avidity. In addition, ACPA were found to carry aberrant glycosylation patterns at the IgG-Fc tail. The present study was undertaken to further characterise the molecular make-up of ACPA and its potential functional consequences in the context of RA. Materials and Methods Serum components of RA patients were fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from RA patient serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA with variations in ACPA molecular structure were used to study binding affinity by surface plasmon resonance. Results We discovered that ACPA-IgG from RA patients have a higher apparent molecular weight as compared to other IgG molecules including antibodies against recall antigens and other autoantibodies. This higher molecular weight was explained by the overrepresentation of N-linked glycans in the variable domain (Fab region) of ACPA-IgG. Detailed structural analysis of these glycans demonstrated that ACPA-IgG linked Fab glycans are complex-type biantennary N-glycans that differ from the conventional Fc-linked N-glycans by a high degree of sialylation, galactosylation, and fucosylation together with the presence of bisecting N-acetylglucosamine. Using recombinant ACPA-IgG monoclonal antibodies with and without Fab-glycans, we found that Fab-glycans modulate binding affinity of ACPA-IgG for citrullinated antigens. Finally, lectin-immunoblotting showed that ACPA Fab-glycans can bind to sialic acid-binding immunoglobulin-type lectins. Conclusions This study describes an unusual and novel molecular feature of the citrulline-specific immune response in RA. ACPA-IgG, in contrast to non-citrulline-specific IgG, are highly glycosylated in the variable region, which modulates recognition of citrullinated antigens. Moreover, ACPA-IgG linked Fab glycans can be the target of specific lectins, suggesting additional functional features potentially involved in ACPA-mediated pathogenetic effects. This finding points to aberrations in the development of ACPA-specific B cells and further elucidates our understanding of basic disease mechanisms in RA.