Biology subjects

Spencer, K.

Publications and source records attributed to Spencer, K..

8 recordsLinked to original sources

bioRxiv2026

The membrane distal domain of CD16a allosterically regulates NK cell ADCC

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (Fc{gamma}RIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3{zeta} phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of [~]9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor Fc{varepsilon}RI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

immunology↗

bioRxiv2026

Molecular and Structural Characterization Reveals Divergent Extracellular Vesicle Profiles Between Wild Type and Alzheimer's Disease Cerebrocortical Organoids

Alzheimers disease (AD) is a neurodegenerative disorder affecting millions of patients globally. Despite significant efforts from researchers in recent decades, there are still many unanswered questions about AD pathogenesis. AD patient brains manifest changes in extracellular vesicles (EVs) secreted from diseased neurons, and the effect of this phenomenon remains poorly understood. EVs contain a variety of biomolecules and play a critical role in cell-to-cell communication in all eukaryotic organisms. Here, we report a thorough characterization of small EVs purified from cultures of human cerebrocortical organoids. These organoids are differentiated from human patient-derived stem cells that bear a familial AD mutation in the presenilin 1 (PSEN1) gene, or from an isogenic wildtype (WT) control. The organoid conditioned media was aspirated from cultures and processed for EV enrichment using a non-invasive technique that requires no cellular disruption. EVs purified from AD organoid conditioned media have a wider size distribution and show differential expression of tetraspanins CD63, CD9, and CD81 when compared to WT organoid-derived EVs. AD organoid-derived EVs can have single, double, and even triple membranes and display luminal fibrillar material. A deep proteomic profiling of the EVs reveals several statistically significant differences, including evidence for modifications in secretory autophagy. EV isolates from both WT and AD organoids show strong binding to amyloid detecting dyes, both in bulk fluorescence and fluorescence microscopy assays. After a 1-week co-culture of AD organoids with WT organoids, there is evidence of endosomal membrane transfer between the isogenic cultures with an increase in amyloid-{beta} peptides in the WT organoids. These observations support the notion that non-cell-autonomous spread of amyloid-containing EVs in human AD brains can be modeled in a cerebral organoid system.

neuroscience↗

bioRxiv2025

Neutrophil and NET-driven pulmonary microvascular injury following myocardial injury: attenuation by S100A8/A9 inhibition

Myocardial infarction (MI) triggers not only local cardiac damage but also a systemic inflammatory response that extends to remote organs. The pulmonary microcirculation, by virtue of its dense capillary network and direct anatomical proximity to the heart, is particularly vulnerable. Neutrophils and their effector mechanisms, including neutrophil extracellular traps (NETs) and the alarmin S100A8/A9, have been implicated in adverse cardiovascular outcomes. However, their role in remote damage post-MI remains unclear. Using intravital in vivo imaging in murine MI models and analysis of human lung tissues, we show that MI induces rapid pulmonary neutrophil and platelet recruitment, formation of platelet-neutrophil aggregates within capillaries, and endothelial activation. These changes are accompanied by NET release, fibrin deposition, and microvascular obstruction, leading to impaired vascular perfusion and necrosis. These pulmonary disturbances closely parallel those in the infarcted myocardium and exceed responses observed in other organs such as the kidney and liver, highlighting the lung as a vulnerable target organ. Increased neutrophil recruitment was associated with marked upregulation of the neutrophil-derived, NET-associated alarmin S100A8/A9 in mouse and human lungs, where it co-localised with infiltrating neutrophils, NETs, and platelet aggregates. Additionally, we show that short-term pharmacological inhibition of S100A8/A9 with ABR-238901 significantly attenuated pulmonary neutrophil infiltration, reduced NETosis and fibrin deposition, and restored capillary perfusion while rebalancing the pulmonary immune landscape. Together, these findings identify the lung as a principal site of remote thrombo-inflammatory injury after MI and implicate S100A8/A9, a neutrophil-derived, NET-associated alarmin, as a mechanistic driver of pulmonary microvascular dysfunction. We propose that targeting this pathway could provide dual protection for both cardiac and pulmonary microcirculations in the acute phase of myocardial injury. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/675647v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@289725org.highwire.dtl.DTLVardef@db2011org.highwire.dtl.DTLVardef@16790dorg.highwire.dtl.DTLVardef@1655b17_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

The Quantification of Drug Accumulation within Gram-Negative Bacteria

Intrabacterial drug accumulation, mediated by the bacterial permeability barrier, efflux, and intrabacterial drug metabolism, is of general significance to the interaction between small molecules and bacteria. For example, the ability of a small molecule to accumulate within a bacterium influences its ability to serve as a chemical probe of an intracellular protein target and/or its efficacy as an antibacterial drug discovery entity. A general method to quantitatively interrogate both intrabacterial drug accumulation and metabolism (IBDM) is presented for Gram-negative bacteria and exemplified with Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa in both single-compound and high-throughput formats. The liquid chromatography-mass spectrometry based platform does not depend on drug labelling and its utility is highlighted through the demonstrated correlation of drug accumulation with drug minimum inhibitory concentration (MIC) in both wild type and efflux deficient strains of E. coli and a matched pair of K. pneumoniae clinical and laboratory strains of varying degrees of drug resistance. Furthermore, an investigation of drug synergy implicates the selective enhancement of the accumulation of one drug by its partner therapy. Finally, a high-throughput format is validated and deployed which provides a readily adaptable approach to screening assays. We anticipate the further applications of this platform to both the translational and the fundamental studies of the interactions of small molecules with bacteria.

microbiology↗

bioRxiv2024

Spatial localization of CD16a at the human NK cell ADCC lytic synapse

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc{gamma}RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

immunology↗

bioRxiv2022

RaMP-DB 2.0: a renovated knowledgebase for deriving biological and chemical insight from genes, proteins, and metabolites

RaMP-DB 2.0 is a web interface, API, relational database and R package designed for straightforward and comprehensive functional interpretation of metabolomic and multi-omic data. Since its first release in 2018, RaMP-DB 2.0 has been upgraded with an expanded breadth and depth of functional and chemical annotation. Content from the source databases (Reactome, HMDB, and Wikipathways) has been updated, and new data types related to metabolite annotations have been incorporated. Structural information incorporated in RaMP-DB 2.0 includes SMILES strings, InChIs, InChIKeys. Chemical classes have been sourced from ClassyFire and LIPID MAPS. Accordingly, the RaMP-DB 2.0 R package has been updated and supports queries on pathways, common reactions, ontologies, chemical classes, and chemical structures. Additionally, RaMP-DB 2.0 now supports enrichment analyses on pathways and chemical classes. Our process for integrating annotations across resources has also been upgraded to lessen the burden of harmonization, thereby supporting more frequent updates. The code used to build all components of RaMP-DB 2.0 is freely available on GitHub at https://github.com/ncats/ramp-db and https://github.com/ncats/RaMP-Backend.

bioinformatics↗

bioRxiv2021

Leukocyte proliferation mediates disease pathogenesis in the Ndufs4(KO) mouse model of Leigh syndrome

Symmetric, progressive, necrotizing lesions in the brainstem are a defining feature of Leigh syndrome (LS). A mechanistic understanding of the pathogenesis of these lesions has been elusive. Here, we report that leukocyte proliferation is causally involved in the pathogenesis of Leigh syndrome. Directly depleting leukocytes with a colony-stimulating factor 1 receptor (CSF1R) inhibitor dramatically attenuates disease, including complete prevention of CNS lesion formation and substantial extension of survival. Leukocyte depletion rescues a range of symptoms including hyperlactemia, seizures, respiratory function, and neurologic symptoms. These data provide a mechanistic explanation for the beneficial effects of mTOR inhibition. More importantly, these findings dramatically alter our understanding of the pathogenesis of LS, demonstrating that immune involvement directly drives disease. These findings have significant implication for the mechanisms of disease resulting from mitochondrial dysfunction, and may lead to novel therapeutic strategies. One-Sentence SummaryPharmacologic targeting of leukocytes prevents CNS lesions, neurological disease, and metabolic dysfunction in the Ndufs4(KO) mouse model of Leigh syndrome.

pathology↗

bioRxiv2021

Light-induced asymmetries in the embryonic retina are mediated by the vascular system and extracellular matrix

Left-right asymmetries in the nervous system (lateralisation) influence a broad range of behaviours, from social responses to navigation and language. The role and pathways of endogenous and environmental mechanisms in the ontogeny of lateralisation remains to be established. The domestic chick is a model of both endogenous and experience-induced lateralisation driven by light exposure. Following the endogenous rightward rotation of the embryo, the asymmetrical position in the egg results in a greater exposure of the right eye to environmental light. To identify the genetic pathways activated by asymmetric light stimulation, and their time course, we exposed embryos to different light regimes: darkness, 6 hours of light and 24 hours of light. We used RNA-seq to compare gene expression in the right and left retinas and telencephalon. As expected, no differential expression between left and right was present in darkness. We detected differential gene expression in right vs left retina after 6 hours of light exposure. This difference disappeared before 24 hours of light exposure, suggesting that light-induced activation is a self-terminating phenomenon. This transient effect of light exposure was associated with a downregulation of the sensitive-period mediator gene DIO2 (iodothyronine deiodinase 2) in the right retina. No differences between genes expressed in the right vs. left telencephalon were detected. Gene networks associated with lateralisation were connected to vascularisation, cell motility, and the extracellular matrix. Interestingly, we know that the extracellular matrix- including the differentially expressed PDGFRB (platelet-derived growth factor receptor {beta}) gene - is involved in both sensitive periods and in the endogenous chiral mechanism of primary cilia, that drives lateralisation. Our data show a similarity between endogenous and experience-driven lateralisation, identifying functional gene networks that affect lateralisation in a specific time window.

neuroscience↗