Ben Joosten

The C‐type lectin DC‐SIGN (CD209) is an antigen‐uptake receptor for Candida albicans on dendritic cells

Dendritic cells (DC) that express the type II C‐type lectin DC‐SIGN (CD209) are located in the submucosa of tissues, where they mediate HIV‐1 entry. Interestingly, the pathogen Candida albicans,the major cause of hospital‐acquired fungal infections, penetrates at similar submucosal sites. Here we demonstrate that DC‐SIGN is able to bind C. albicans both in DC‐SIGN‐transfected cell lines and in human monocyte‐derived DC. The binding was shown to be time‐ as well as concentration‐dependent, and live as well as heat‐inactivated C. albicans were bound to the same extent. Moreover, in immature DC, DC‐SIGN was able to internalize C. albicans in specific DC‐SIGN‐enriched vesicles, distinct from those containing the mannose receptor, the other known C. albicans receptor expressed by DC. Together, these results demonstrate that DC‐SIGN is an exquisite pathogen‐uptake receptor that captures not only viruses but also fungi.

Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro.

Vaccine efficacy is strongly enhanced by antibody-mediated targeting of vaccine components to dendritic cells (DCs), which are professional antigen presenting cells. However, the options to link antigens or immune modulators to a single antibody are limited. Here, we engineered versatile nano- and micrometer-sized slow-release vaccine delivery vehicles that specifically target human DCs to overcome this limitation. The nano- (NPs) and microparticles (MPs), with diameters of approximately 200nm and 2microm, consist of a PLGA core coated with a polyethylene glycol-lipid layer carrying the humanized targeting antibody hD1, which does not interact with complement or Fc receptors and recognizes the human C-type lectin receptor DC-SIGN on DCs. We studied how these particles interact with human DCs and blood cells, as well as the kinetics of PLGA-encapsulated antigen degradation within DCs. Encapsulation of antigen resulted in almost 38% degradation for both NPs and MPs 6days after particle ingestion by DCs, compared to 94% when nonencapsulated, soluble antigen was used. In contrast to the MPs, which were taken up rather nonspecifically, the NPs effectively targeted human DCs. Consequently, targeted delivery only improved antigen presentation of NPs and induced antigen-dependent T cell responses at 10-100 fold lower concentrations than nontargeted NPs.

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Hotspots of GPI-anchored proteins and integrin nanoclusters function as nucleation sites for cell adhesion

Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their cellular function. In particular, rafts have been implicated in regulation of integrin-mediated cell adhesion, although the underlying mechanism remains elusive. We used single-molecule near-field optical microscopy (NSOM) with localization accuracy of approximately 3 nm, to capture the spatio-functional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Dual color nanoscale imaging revealed the existence of a nanodomain GPI-AP subpopulation that further concentrated in regions smaller than 250 nm, suggesting a hierarchical prearrangement of GPI-APs on resting monocytes. We previously demonstrated that in quiescent monocytes, LFA-1 preorganizes in nanoclusters. We now show that integrin nanoclusters are spatially different but reside proximal to GPI-AP nanodomains, forming hotspots on the cell surface. Ligand-mediated integrin activation resulted in an interconversion from monomers to nanodomains of GPI-APs and the generation of nascent adhesion sites where integrin and GPI-APs colocalized at the nanoscale. Cholesterol depletion significantly affected the reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and LFA-1 adhesion to its ligand. As such, our data demonstrate the existence of nanoplatforms as essential intermediates in nascent cell adhesion. Since raft association with a variety of membrane proteins other than LFA-1 has been documented, we propose that hotspots regions enriched with raft components and functional receptors may constitute a prototype of nanoscale inter-receptor assembly and correspond to a generic mechanism to offer cells with privileged areas for rapid cellular function and responses to the outside world.

DCIR is endocytosed into human dendritic cells and inhibits TLR8-mediated cytokine production

C‐type lectin receptors (CLRs) expressed on APCs play a pivotal role in the immune system as pattern‐recognition and antigen‐uptake receptors. In addition, they may signal directly, leading to cytokine production and immune modulation. To this end, some CLRs, like dectin‐1 and dendritic cell immunoreceptor (DCIR), contain intracellular ITIMs or ITAMs. In this study, we explored expression and function of the ITIM‐containing CLR DCIR on professional APCs. DCIR is expressed on immature and mature monocyte‐derived DCs (moDC) but also on monocytes, macrophages, B cells, and freshly isolated myeloid and plasmacytoid DCs. We show that endogenous DCIR is internalized efficiently into human moDC after triggering with DCIR‐specific mAb. DCIR internalization is clathrin‐dependent and leads to its localization in the endo‐/lysosomal compartment, including lysosome‐associated membrane protein‐1+ lysosomes. DCIR triggering affected neither TLR4‐ nor TLR8‐mediated CD80 and CD86 up‐regulation. Interestingly, it did inhibit TLR8‐mediated IL‐12 and TNF‐α production significantly, and TLR2‐, TLR3‐, or TLR4‐induced cytokine production was not affected. Collectively, the data presented characterize DCIR as an APC receptor that is endocytosed efficiently in a clathrin‐dependent manner and negatively affects TLR8‐mediated cytokine production. These data provide further support to the concept of CLR/TLR cross‐talk in modulating immune responses.

Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells

Clustering of cell surface receptors into micro‐domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near‐field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C‐type lectin DC‐SIGN on the membrane of dendritic cells, both in air and in liquid.

Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation.

Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8(+) T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies.

PGE2-mediated podosome loss in dendritic cells is dependent on actomyosin contraction downstream of the RhoA–Rho-kinase axis

Podosomes are dynamic adhesion structures found in dendritic cells (DCs) and other cells of the myeloid lineage. We previously showed that prostaglandin E2 (PGE2), an important proinflammatory mediator produced during DC maturation, induces podosome disassembly within minutes after stimulation. Here, we demonstrate that this response is mediated by cAMP elevation, occurs downstream of Rho kinase and is dependent on myosin II. Whereas PGE2 stimulation leads to activation of the small GTPase RhoA, decreased levels of Rac1-GTP and Cdc42-GTP are observed. These results show that PGE2 stimulation leads to activation of the RhoA–Rho-kinase axis to promote actomyosin-based contraction and subsequent podosome dissolution. Because podosome disassembly is accompanied by de novo formation of focal adhesions, we propose that the disassembly/formation of these two different adhesion structures is oppositely regulated by actomyosin contractility and relative activities of RhoA, Rac1 and Cdc42.

The C-type lectin DC-SIGN internalizes soluble antigens and HIV-1 virions via a clathrin-dependent mechanism.

Dendritic cells (DC), professional Ag‐presenting cells located in mucosae and lymphoid organs, operate at the interface of innate and adaptive immunity and are likely the first cells to encounter invading HIV‐1. Although the C‐type lectin DC‐Specific ICAM‐3‐grabbing non‐integrin (DC‐SIGN) binds to several viruses, including HIV‐1, its direct involvement in viral entry remains controversial. Despite its central role in DC function, little is known about the underlying molecular mechanism(s) of DC‐SIGN‐mediated Ag uptake. Here, we analyzed the early stages of DC‐SIGN‐mediated endocytosis and demonstrate that both membrane cholesterol and dynamin are required. Confocal microscopy and clathrin RNAi showed that DC‐SIGN‐mediated internalization occurs via clathrin‐coated pits. Electron microscopy of ultrathin sections showed the involvement of DC‐SIGN in clathrin‐dependent HIV‐1 internalization by DC. Currently, DC‐specific C‐type lectins are considered potential target in anti‐tumor clinical trials. Detailed information about how different Ag are internalized via these receptors will facilitate the rational design of targeted therapeutic strategies.

The neck region of the C-type lectin DC-SIGN regulates its surface spatiotemporal organization and virus-binding capacity on antigen presenting cells

Background: Nanoclusters of the C-type lectin DC-SIGN on dendritic cells act as docking sites for viral binding. Results: The extracellular neck region is responsible for nanocluster formation and necessary for virus binding. Conclusion: Heterogeneous nanocluster density and spatial distribution confer broad binding capabilities to DC-SIGN. Significance: Insights into how virus receptors preorganize and assemble into docking platforms contribute to clarifying mechanisms of virus entry. The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection.