PGE2-EP4 signaling steers cDC2 maturation toward the induction of suppressive T-cell responses.

Dendritic cells (DCs) shape adaptive immunity in response to environmental cues such as cytokines or lipid mediators, including prostaglandin E2 (PGE2). In cancer, tumors are known to establish an enriched PGE2 microenvironment. Tumor-derived PGE2 primes regulatory features across immune cells, including DCs, facilitating tumor progression. PGE2 shapes DC function by providing signaling via its two so-called E-prostanoid receptors (EPs) EP2 and EP4. Although studies with monocyte-derived DCs have shown the importance of PGE2 signaling, the role of PGE2-EP2/EP4 on conventional DCs type 2 (cDC2s), is still poorly defined. In this study, we investigated the function of EP2 and EP4 using specific EP antagonists on human cDC2s. Our results show that EP2 and EP4 exhibit different functions in cDC2s, with EP4 modulating the upregulation of activation markers (CD80, CD86, CD83, MHC class II) and the production of IL-10 and IL-23. Furthermore, PGE2-EP4 boosts CCR type 7-based migration as well as a higher T-cell expansion capacity, characterized by the enrichment of suppressive rather than pro-inflammatory T-cell populations. Our findings are relevant to further understanding the role of EP receptors in cDC2s, underscoring the benefit of targeting the PGE2-EP2/4 axis for therapeutic purposes in diseases such as cancer.

cDC2 plasticity and acquisition of a DC3-like phenotype mediated by IL-6 and PGE2 in a patient-derived colorectal cancer organoids model.

Metastatic colorectal cancer (CRC) is highly resistant to therapy and prone to recur. The tumor-induced local and systemic immunosuppression allows cancer cells to evade immunosurveillance, facilitating their proliferation and dissemination. Dendritic cells (DCs) are required for the detection, processing, and presentation of tumor antigens, and subsequently for the activation of antigen-specific T cells to orchestrate an effective antitumor response. Notably, successful tumors have evolved mechanisms to disrupt and impair DC functions, underlining the key role of tumor-induced DC dysfunction in promoting tumor growth, metastasis initiation, and treatment resistance. Conventional DC type 2 (cDC2) are highly prevalent in tumors and have been shown to present high phenotypic and functional plasticity in response to tumor-released environmental cues. This plasticity reverberates on both the development of antitumor responses and on the efficacy of immunotherapies in cancer patients. Uncovering the processes, mechanisms, and mediators by which CRC shapes and disrupts cDC2 functions is crucial to restoring their full antitumor potential. In this study, we use our recently developed 3D DC-tumor co-culture system to investigate how patient-derived primary and metastatic CRC organoids modulate cDC2 phenotype and function. We first demonstrate that our collagen-based system displays extensive interaction between cDC2 and tumor organoids. Interestingly, we show that tumor-corrupted cDC2 shift toward a CD14+ population with defective expression of maturation markers, an intermediate phenotype positioned between cDC2 and monocytes, and impaired T-cell activating abilities. This phenotype aligns with the newly defined DC3 (CD14+ CD1c+ CD163+) subset. Remarkably, a comparable population was found to be present in tumor lesions and enriched in the peripheral blood of metastatic CRC patients. Moreover, using EP2 and EP4 receptor antagonists and an anti-IL-6 neutralizing antibody, we determined that the observed phenotype shift is partially mediated by PGE2 and IL-6. Importantly, our system holds promise as a platform for testing therapies aimed at preventing or mitigating tumor-induced DC dysfunction. Overall, our study offers novel and relevant insights into cDC2 (dys)function in CRC that hold relevance for the design of therapeutic approaches.

Dendritic cell force-migration coupling on aligned fiber networks.

Dendritic cells (DCs) are antigen-presenting cells that reside in peripheral tissues and are responsible for initiating adaptive immune responses. As gatekeepers of the immune system, DCs need to continuously explore their surroundings, for which they can rapidly move through various types of connective tissue and basement membranes. DC motility has been extensively studied on flat 2D surfaces, yet the influences of a contextual 3D fibrous environment still need to be described. Using ECM-mimicking suspended fiber networks, we show how immature DCs (iDCs) engage in migratory cycles that allow them to transition from persistent migration to slow migratory states. For a subset of iDCs with high migratory potential, we report the organization of protrusions at the front of the cell body, which reverses upon treatment with inflammation agent PGE2. We identify an unusual migratory response to aligned fiber networks, whereby iDCs use filamentous protrusions to attach laterally and exert forces on fibers to migrate independent of fiber alignment. Increasing the fiber diameter from 200 to 500 nm does not significantly affect the migratory response; however, iDCs respond by forming denser actin bundles around larger diameters. Overall, the correlation between force-coupling and random migration of iDCs in aligned fibrous topography offers new insights into how iDCs might move in fibrous environments in vivo.

Quantifying superimposed protein flow dynamics in live cells using spatial filtering and spatiotemporal image correlation spectroscopy.

Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.

Clinical and biochemical footprints of inherited metabolic diseases. XII. Immunological defects.

Immunological problems are increasingly acknowledged manifestations in many inherited metabolic diseases (IMDs), ranging from exaggerated inflammation, autoimmunity and abnormal cell counts to recurrent microbial infections. A subgroup of IMDs, the congenital disorders of glycosylation (CDG), includes CDG types that are even classified as primary immunodeficiencies. Here, we reviewed the list of metabolic disorders reported to be associated with various immunological defects and identified 171 IMDs accompanied by immunological manifestations. Most IMDs are accompanied by immune dysfunctions of which immunodeficiency and infections, innate immune defects, and autoimmunity are the most common abnormalities reported in 144/171 (84%), 44/171 (26%) and 33/171 (19%) of IMDs with immune system involvement, respectively, followed by autoinflammation 17/171 (10%). This article belongs to a series aiming at creating and maintaining a comprehensive list of clinical and metabolic differential diagnoses according to organ system involvement.

Synthetic Semiflexible and Bioactive Brushes.

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide-alkyne cycloaddition reaction and the (photoactivated) tetrazole-ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

Certainty-based marking in a formative assessment improves student course appreciation but not summative examination scores.

BACKGROUND: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation. METHODS: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs. RESULTS: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs. CONCLUSIONS: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.

The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes.

Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses.

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4(+) and CD8(+) T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin-experienced CD4(+) T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1-specific CD8(+) T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients.

Enhanced receptor-clathrin interactions induced by N-glycan-mediated membrane micropatterning.

Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN-clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.

Nanoclustering as a dominant feature of plasma membrane organization.

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.

Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6.

At the immunological synapse, the activated leukocyte cell adhesion molecule (ALCAM) on a dendritic cell (DC) and CD6 molecules on a T cell contribute to sustained DC-T-cell contacts. However, little is known about how ALCAM-CD6 bonds resist and adapt to mechanical stress. Here, we combine single-cell force spectroscopy (SCFS) with total-internal reflection fluorescence microscopy to examine ALCAM-CD6-mediated cell adhesion. The combination of cells expressing ALCAM constructs with certain cytoplasmic tail mutations and improved SCFS analysis processes reveal that the affinity of ALCAM-CD6 bonds is not influenced by the linking of the intracellular domains of ALCAM to the actin cortex. By contrast, the recruitment of ALCAM to adhesion sites and the propensity of ALCAM to anchor plasma membrane tethers depend on actin cytoskeletal interactions. Furthermore, linking ALCAM to the actin cortex through adaptor proteins stiffens the cortex and strengthens cell adhesion. We propose a framework for how ALCAMs contribute to DC-T-cell adhesion, stabilize DC-T-cell contacts and form a mechanical link between CD6 and the actin cortex to strengthen cell adhesion at the immunological synapse.

Podosomes of dendritic cells facilitate antigen sampling.

Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here, we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for the podosomes of dendritic cells.

Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton.

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC-SIGN.

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C-type lectin dendritic cell-specific intracellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN), because a detailed characterization at the structural level is lacking. DC-SIGN recognizes specific Candida-associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan-branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope-based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC-SIGN. We demonstrate that slight differences in the N-mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC-SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 kB T. The single-bond affinity of tetrameric DC-SIGN for wild-type C. albicans is ~10.7 kB T and a dissociation constant kD of 23 µM, which is relatively strong compared with other carbohydrate-protein interactions described in the literature. In conclusion, this study shows that DC-SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC-SIGN and its pathogenic ligands will lead to a better understanding of how fungal-associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti-fungal drugs.

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion.

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Mechanobiology of myeloid cells.

Tissue-resident myeloid cells sense and transduce mechanical signals such as stiffness, stretch and compression. In the past two years, our understanding of the mechanosensitive signalling pathways in myeloid cells has significantly expanded. Moreover, it is increasingly clear which mechanical signals induce myeloid cells towards a pro- or anti-inflammatory phenotype. This is especially relevant in the context of altered matrix mechanics in immune-related pathologies or in the response to implanted biomaterials. A detailed understanding of myeloid cell mechanosensing may eventually lead to more effective cell-based immunotherapies for cancer, the development of mechanically inspired therapies to target fibrosis, and the engineering of safer implants. This review covers these recent advances in the emerging field of mechanoimmunology of myeloid cells.

EP2 and EP4 blockade prevents tumor-induced suppressive features in human monocytic myeloid-derived suppressor cells.

Tumors educate their environment to prime the occurrence of suppressive cell subsets, which enable tumor evasion and favors tumor progression. Among these, there are the myeloid-derived suppressor cells (MDSCs), their presence being associated with the poor clinical outcome of cancer patients. Tumor-derived prostaglandin E2 (PGE2) is known to mediate MDSC differentiation and the acquisition of pro-tumor features. In myeloid cells, PGE2 signaling is mediated via E-prostanoid receptor type 2 (EP2) and EP4. Although the suppressive role of PGE2 is well established in MDSCs, the role of EP2/4 on human MDSCs or whether EP2/4 modulation can prevent MDSCs suppressive features upon exposure to tumor-derived PGE2 is poorly defined. In this study, using an in vitro model of human monocytic-MDSCs (M-MDSCs) we demonstrate that EP2 and EP4 signaling contribute to the induction of a pro-tumor phenotype and function on M-MDSCs. PGE2 signaling via EP2 and EP4 boosted M-MDSC ability to suppress T and NK cell responses. Combined EP2/4 blockade on M-MDSCs during PGE2 exposure prevented the occurrence of these suppressive features. Additionally, EP2/4 blockade attenuated the suppressive phenotype of M-MDSCs in a 3D coculture with colorectal cancer patient-derived organoids. Together, these results identify the role of tumor-derived PGE2 signaling via EP2 and EP4 in this human M-MDSC model, supporting the therapeutic value of targeting PGE2-EP2/4 axis in M-MDSCs to alleviate immunosuppression and facilitate the development of anti-tumor immunity.

EP2/EP4 targeting prevents tumor-derived PGE2-mediated immunosuppression in cDC2s.

Tumor-derived prostaglandin E2 (PGE2) impairs anti-tumor immunity by priming suppressive functions on various immune cell types, including dendritic cells (DCs). In this way, tumors mediate DC dysfunction and hamper their anti-tumoral activity. PGE2 is known to modulate DC function via signaling through the E-prostanoid receptor type (EP) 2 and EP4. Preclinical studies have demonstrated the therapeutic value of targeting EP2/4 receptor signaling in DCs. Ongoing phase I clinical trials with EP antagonists have shown immunomodulation in cancer patients. However, the systemic drug administration leads to off-target events and subsequent side-effects. To limit the off-target effects of EP targeting, EP2 and EP4 antagonists were encapsulated in polymeric nanoparticles (NPs). In this study we evaluated the efficacy of EP2/4 specific antagonists encapsulated in NPs to protect cDC2s from suppressive effects of tumor-derived PGE2 in different tumor models. We show that tumor-derived PGE2 signals via EP2/4 to mediate the acquisition of a suppressive phenotype of cDC2s. EP2/4 antagonists encapsulated NPs impaired the conversion of cDC2s towards a suppressive state and inhibited the occurrence of suppressive features such as IL-10 production or the ability to expand Tregs. Importantly, the NPs abolished the transition towards this suppressive state in different tumor models: Melanoma-conditioned media, ascites fluid derived from ovarian cancer patients (2D), and upon coculture with colorectal cancer patient-derived organoids (3D). We propose that targeting the PGE2-EP2/4 axis using NPs can achieve immunomodulation in the immune system of cancer patients, alleviate tumor-derived suppression, and thus facilitate the development of potent anti-tumor immunity in cancer patients.

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

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.