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Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+but not for Langerin+cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN’s carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.

Fragment-based drug discovery is a powerful complement to conventional high-throughput screening, especially for difficult targets. Screening low-molecular-weight fragments usually requires highly sensitive biophysical methods, because of the generally low affinity of the identified ligands. Here, we developed a cell-based fragment screening assay (cellFy) that allows sensitive identification of fragment hits in a physiologically more relevant environment, in contrast to isolated target screenings in solution. For this, a fluorescently labeled multivalent reporter was employed, enabling direct measurement of displacement by low-molecular-weight fragments without requiring enzymatic reactions or receptor activation. We applied this technique to identify hits against two challenging targets of the C-type lectin receptor (CLR) family: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) and Langerin. Both receptors are involved in pathogen recognition and initiation of an immune response, which renders them attractive targets for immune modulation. Because of their shallow and hydrophilic primary binding site, hit identification for CLRs is challenging and druglike ligands for CLRs are sparse. Screening of a fragment library followed by hit validation identified several promising candidates for further fragment evolution for DC-SIGN. In addition, a multiplexed assay format was developed for simultaneous screening against multiple CLRs, allowing a selectivity counterscreening. Overall, this sensitive cell-based fragment screening assay provides a powerful tool for rapid identification of bioactive fragments, even for difficult targets.

Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay.

Severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including infectivity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of the SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analyzed using data-independent acquisition–mass spectrometry. This resulted in a comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across four time points. Most notably, the activation of interferon type-I response was observed, which is surprisingly absent in several proteome studies. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced abundance of the viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyze the innate immunity.

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-β1, and the Notch ligand DLL4 differentiate within 3 days into CD1a+Langerin+cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-α, and stimulate proliferation and cytokine production in allogeneic CD4+and CD8+T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro‒generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

We evaluated the effects of a single application of granular carbaryl made against nymphal Ixodes scapularis Say on the diversity and abundance of forest arthropods taken in pitfall traps in oak and mixed oak–pine forest sites for 12 wk after treatment in central New Jersey. Significant short-term changes in arthropod assemblages were detected immediately posttreatment. Effects were not distributed equally across taxa. Seasonal changes in numbers and diversity of forest arthropods in the study areas may have affected the impact of the acaricide in the treatment area. Comparison with control areas indicated that reductions in abundance of some arthropod taxa in the treatment area were detectable 12 wk after treatment.