The ␤2-integrin LFA-1 facilitates extravasation of monocytes (MOs) into the underlying tissues, where MOs can differentiate into dendritic cells (DCs). Although DCs express LFA-1, unlike MOs, they cannot bind to ICAM-1. We hypothesized that an altered integrin organization on the DC plasma membrane might cause this effect and investigated the relationship between membrane organization and function of LFA-1 on MOs and DCs. High-resolution mapping of LFA-1 surface distribution revealed that on MOs LFA-1 function is associated with a distribution in well-defined nanoclusters (100 -150-nm diameter). Interestingly, a fraction of these nanoclusters contains primed LFA-1 molecules expressing the specific activation-dependent L16-epitope. Live imaging of MO-T-cell conjugates showed that only these primed nanoclusters are dynamically recruited to the cellular interface forming micrometer-sized assemblies engaged in ligand binding and linked to talin. We conclude that besides affinity regulation, LFA-1 function is controlled by at least three different avidity patterns: random distributed inactive molecules, well-defined ligand-independent proactive nanoclusters, and ligand-triggered micrometer-sized macroclusters.

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 nonintegrin (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.Key words: C-type lectin . DC . Endocytosis . Targeting . Virus Supporting Information available online IntroductionDendritic cells (DC) are professional Ag-presenting cells that operate at the interface of innate and adaptive immunity [1].Since DC are located in the mucosae and the lymphoid tissues, they are likely to be the first cells to encounter invading HIV-1 particles [2]. DC express several PRR that specifically recognize distinct pathogen-associated molecular patterns displayed on microbial surfaces including C-type lectin receptors (CLR) [3] and TLR [4].DC-specific ICAM-3-grabbing non-integrin (DC-SIGN; CD209) is a CLR initially described as an ICAM-3 binding protein [5] as well as a major HIV-1 receptor on DC [6]. In addition, DC-SIGN binds several other viruses as well as fungi, bacteria, and parasites [3]. Although DC-SIGN has been shown to bind to several viruses, its capacity to directly mediate viral entry has often been debated [7,8]. Recently, we demonstrated that on the plasma membrane of DC, DC-SIGN is organized in nanoclusters, some of which colocalize with lipid rafts, that specifically confer to the receptor its capacity of binding and internalizing viruses as well as Ag conjugated to fluorescent nanoparticles [9,10]. These findings were substantiated by a biophysical study by Neumann et al., who showed that these nanoclusters are enriched near the leading edge of living DC, but are preferentially endocytosed at lamellar sites posterior to the leading edge, suggesting a directed Binding of HIV-1 to DC-SIGN leads to non-fusogenic uptake of virions by DC, which is required for enhancement of T-cell infection [12] and is dependent on the cytoplasmic domain of the DC-SIGN molecule [13]. SHORT COMMUNICATIONAg internalized by DC-SIGN can be presented via both MHC class I and class II [14,15]. In addition, DC-SIGN has been shown to promote both MHC class I-a...

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The cover image depicts the co‐staining of DC‐SIGN and EEA‐1 in dendritic cells and shows the co‐localization of the endocytosed ligand with markers of the early endosomal compartments. The image was taken from the article by Cambi et al. (pp. 1923–1929), in which the authors analyze the role of the C‐type lectin DC‐SIGN in viral entry. The authors show that DC‐SIGN is involved in clathrin‐dependent HIV‐1 internalization by dendritic cells.