Biology subjects

Dustin, M.

Publications and source records attributed to Dustin, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Immune receptor LILRB1 mediates cis-signalling which is targeted by RIFINs of the malaria parasite

The malaria parasite, Plasmodium falciparum, replicates within human erythrocytes, where it is susceptible to clearance by immune cells. It uses erythrocyte surface proteins, the RIFINs, to signal through immune receptors and to suppress immune cell function. Previous studies identified two groups of RIFINs which bind different sites on inhibitory immune receptor LILRB1. While some RIFINs bind an elongated LILRB1 conformation, triggering inhibitory immune signalling in trans, we show that other RIFINs stabilise a c-shaped LILRB1 conformation. This buckled LILRB1 binds MHC class I found on the same immune cell, which triggers inhibitory cis signalling. Therefore, LILRB1 exists in dynamic equilibrium, with an elongated conformation able to bind to ligands in trans on a target cell, while a buckled conformation signals through MHC class I in cis, setting the signalling threshold. Different clades of RIFINs exist to mediate inhibitory signalling through each of these LILRB1 conformations to prevent parasite destruction.

microbiology↗

bioRxiv2026

PD-1 blockade enhances T cell activation by reorchestrating CD28 and CTLA4 ligand interactions

The network of interactions comprising CD28, CTLA4 and PD-1 and their ligands CD80, CD86, and PD-L1 have profound implications for T cell activation. Despite their critical role, factors that determine their receptor occupancy when all three ligands are present remains incompletely understood. Using a supported lipid bilayer to replicate the antigen-presenting cell membrane facilitated a quantitative analysis of CD80, CD86, and PD-L1 interactions. Our observations reveal that PD-1 blockade operates through a previously unrecognized competitive mechanism beyond conventional checkpoint inhibition: liberated PD-L1 molecules redistribute to form CD80-PD-L1 heterodimers that strengthen CD28-CD80 interactions and outcompete CD86 for CD28 binding. This preferential binding enhances T cell activation through superior CD80 co-stimulation. Furthermore, the redistribution by PD-1 blockade reduces CD80 homodimer availability, limiting CTLA4 binding to CD80 and enhancing CD28 signaling. These findings provide vital insights into the competitive principles that allow B7-CD28 family receptors to regulate T cell activation through network-level effects.

immunology↗

bioRxiv2025

CD4⁺ T cells confer transplantable rejuvenation via Rivers of telomeres

The role of the immune system in regulating organismal lifespan remains poorly understood. Here, we show that CD4 T cells release "telomere Rivers" into circulation after acquiring telomeres from antigen-presenting cells (APCs). River formation requires fatty acid oxidation at the T cell-APC synapse, which selectively excludes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the telomere vesicles. The resulting Rivers are depleted of glycolytic enzymes but enriched in T cell-derived stemness factors, enabling targeted rejuvenation of senescent tissues across multiple organs. In aged mice, adoptive transfer of young or metabolically reprogrammed CD4 T cells triggered River production in vivo, and Rivers isolated from these animals could be transplanted into other aged mice to propagate the rejuvenation phenotype independently of T cells. River therapy extended median lifespan by [~]17 months, with several mice surviving to nearly five years. This immune-driven telomere transfer pathway is conserved across kingdoms, including plants, defining the first systemic, transplantable programme of youth.

cell biology↗

bioRxiv2025

Studying macromolecular composition in cell-cell interfaces using 3D membrane reconstitution systems

During direct communication between two cells, the plasma membranes of each cell serve as a platform for ligand-receptor interaction initiating downstream signalling cascades. In immune cell signalling, this cell-cell interface - the immune synapse - is highly spatiotemporally organized. Multiple stimulatory and co-stimulatory signals need to be integrated over time to ensure proper immune cell function. This process is still not fully understood given the vast complexity of interactions between proteins, lipids, glycocalyx and associated cortical actin cytoskeleton. To examine the impact of a single component, the use of model membrane systems has increased. Here, we developed a fully artificial system to study the interface between two vesicles and a semi-artificial one between a live cell and a vesicle to reconstitute 3D contacts. We investigated the distribution and reorganization of immune cell proteins at artificial and semi-artificial contacts. Using our vesicle-vesicle system, we show the enrichment and depletion of different proteins in the synapse. Using the cell-vesicle system we showed how different peptides with varying affinity presented by the same MHC class I affect T cell activation. We further explored the distribution of glycocalyx elements at the cell-cell contact and showed differential partitioning of different sugar moieties in the interface. While we focused on the T cell interface here, our model systems are powerful tools to study distribution and reorganization of lipids, proteins and glycocalyx components at any cell-cell contact.

biophysics↗

bioRxiv2025

SpatioEv: Spatial evolution of protein and morphological features reveals development dynamics of cells and spatial neighbourhoods

Understanding cellular function in tissues demands sophisticated tools to decode complex microenvironmental interactions. Current spatial analysis methods often lack the comprehensive framework needed to systematically analyse cell morphology, dynamics, interactions, and extracellular matrix (ECM) architecture. We introduce SpatioEv, a unified computational framework for highly multiplexed tissue imaging that addresses these critical gaps. SpatioEv integrates automated quality control, cell phenotyping, neighbourhood identification, multi-scale spatial characterization, niche boundary analysis, ECM fiber-cell interaction mapping, and spatiotemporal trajectory inference. This pipeline enables reproducible cell annotation, reveals novel ECM-cell interactions, characterizes tissue neighbourhood boundaries, and infers developmental progressions directly from spatial data. Using this, we can identify disease-specific spatial signatures distinguishing rheumatoid arthritis from osteoarthritis, characterize diverse tumour boundary phenotypes in cancer metastases in liver, and map evolutionary trajectories in pancreatic ductal adenocarcinoma (PDAC) at single-niche resolution. Our findings highlight the significance of spatial context in shaping cell behaviour and underscore its potential to uncover emergent tissue architecture and cellular dynamics. By addressing major analytical challenges, SpatioEv provides a scalable, adaptable platform for advancing spatial biology and translational research.

systems biology↗

bioRxiv2025

PTPN22 Regulates T Cell Synapse Formation through PSTPIP1-Dependent Actin Remodeling

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a critical regulator of T cell signaling, working in concert with C-terminal SRC kinase (Csk) to dephosphorylate key signaling proteins and suppress lymphocyte activation. The R620W variant of PTPN22, one of the most prevalent mutations associated with autoimmune diseases, has been implicated in altered T cell responses, although its broader effects on T cell activation are not fully understood. Recent studies have uncovered a novel interaction between PTPN22 and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP-1), a cytoskeletal adaptor protein involved in F-actin remodeling. PSTPIP-1 recruits Wiskott-Aldrich Syndrome Protein (WASp) to facilitate actin foci formation, a process integral to stabilizing TCR microclusters and amplifying downstream signaling. Given that mutations in PSTPIP-1 impair actin remodeling and localize within the PTPN22 binding domain, we hypothesized that PTPN22 modulates actin dynamics through its interaction with PSTPIP-1. Using live and fixed multi-color super-resolution fluorescence imaging, we demonstrate that PTPN22 deficiency or inhibition of its phosphatase activity leads to aberrant Arp2/3-dependent actin remodeling and exaggerated calcium signaling, particularly under low- affinity TCR stimulation. Single-protein resolution imaging via DNA-PAINT further revealed disrupted nanoscale clustering of PSTPIP-1 and TCR in PTPN22-deficient T cells, uncovering a previously unrecognized PSTPIP-1-TCR interaction in the absence of PTPN22. These findings highlight a novel PTPN22-PSTPIP-1 signaling axis, offering new insights into the molecular mechanisms that may contribute to autoimmune disease susceptibility. One Sentence SummaryPTPN22 and PSTPIP-1 control actin remodeling upon T cell synapse formation, modulating TCR clustering and activation.

immunology↗

bioRxiv2024

Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8+ T cells

Interleukin 2 (IL2) promotes T cell proliferation and differentiation, making it a central target in immunotherapies. T cells fine-tune their sensitivity to and consumption of IL2 by regulating surface expression and composition of the IL2 receptor. Following antigen recognition, IL2 receptor signaling is shared through polarized interactions in T cell aggregates. However, how IL2 function is integrated during earlier antigen-dependent T cell synapses is unknown. Here, we demonstrate a synergistic effect between the T cell receptor (TCR) and IL2 receptor signaling at the immunological synapse of CD8+ T cells with supported lipid bilayers. TCR and IL2 signaling overlapped in space and time, potentiating each other when simultaneously triggered. Immuno-STATs, a safe and effective new class of immunotherapeutics, which fuse IL2 and peptide-major histocompatibility complex (pMHC) in a single molecule to expand antigen-specific CD8+ T cells, enhanced both TCR and IL2 signaling and promoted antigen specific T-T immunological synapses.

immunology↗

bioRxiv2024

Sympathetic neuron-derived NPY protects from obesity by sustaining the mural progenitors of thermogenic adipocytes.

Neuropeptide Y (NPY) is secreted by sympathetic nerves1,2, but its direct impact on thermogenic adipocytes is unknown. Here we uncover the mechanism by which peripheral NPY protects from obesity. Our imaging of cleared murine brown and white adipose tissue (BAT and WAT) established that NPY+ sympathetic axons are only a minority that mostly maps to the peri-vasculature; our analysis of single-cell RNA-sequencing datasets identifies mural cells as the main NPY-responsive cells in adipose tissues. We show that NPY sustains mural cells, which are known to be a source of beige cells in both BAT and WAT3-5 and that NPY facilitates the differentiation to thermogenic adipocytes. We found that diet-induced-obesity leads to neuropathy of NPY+ axons and concomitant depletion of the mural cell pool of beige fat progenitors. This defect is replicated in conditional knockout (cKO) mice with NPY specifically abrogated from sympathetic neurons. These cKO mice have whitened BAT with reduced thermogenic ability and lower energy expenditure even before the onset of obesity; they develop adult-onset obesity on a regular chow diet and are more susceptible to diet induced obesity without increasing food consumption. Our results indicate that, relative to central NPY, peripheral NPY produced by the sympathetic nerves has the opposite effect on body weight homeostasis by sustaining the proliferation of the mural cell progenitors of thermogenic adipocytes.

physiology↗

bioRxiv2024

A general algorithm for consensus 3D cell segmentation from 2D segmented stacks

Cell segmentation is the foundation of a wide range of microscopy-based biological studies. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation, and computation. However, 3D cell segmentation, requiring dense annotation of 2D slices still poses significant challenges. Manual labeling of 3D cells to train broadly applicable segmentation models is prohibitive. Even in high- contrast images annotation is ambiguous and time-consuming. Here we develop a theory and toolbox, u- Segment3D, for 2D-to-3D segmentation, compatible with any 2D method generating pixel-based instance cell masks. u-Segment3D translates and enhances 2D instance segmentations to a 3D consensus instance segmentation without training data, as demonstrated on 11 real-life datasets, >70,000 cells, spanning single cells, cell aggregates, and tissue. Moreover, u-Segment3D is competitive with native 3D segmentation, even exceeding when cells are crowded and have complex morphologies.

bioinformatics↗

bioRxiv2024

Regulation of temporal cytokine production by co-stimulation receptors in TCR-T cells is lost in CAR-T cells

CD8+ T cells contribute to immune responses by producing cytokines when their T cell receptors (TCRs) recognise peptide antigens on major-histocompability-complex (pMHC) class I. However, excessive cytokine production can be harmful. For example, cytokine release syndrome (CRS) is a common toxicity observed in treatments that activate T cells, including chimeric antigen receptor (CAR)-T cell therapy. While engagement of costimulatory receptors is well known to enhance cytokine production, we have limited knowledge of their ability to regulate the kinetics of cytokine production by CAR-T cells. Here we compare early (0-12 hours) and late (12-20 hours) production of IFN-{gamma}, IL-2, and TNF- production by T cells stimulated via TCR or CARs in the presence or absence ligands for CD2, LFA-1, CD28, CD27, and 4-1BB. For T cells expressing TCRs and 1st-generation CARs, activation by antigen alone was sufficient to stimulate early cytokine production, while co-stimulation by CD2 and 4-1BB was required to maintain late cytokine production. In contrast, T cells expressing 2nd-generation CARs, which have intrinsic costimulatory signalling motifs, produce high levels of cytokines in both early and late periods in the absence of costimulatory receptor ligands. Losing the requirement for costimulation for sustained cytokine production may contribute to the effectiveness and/or toxicity of 2nd-generation CAR-T cell therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/581341v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1d59d68org.highwire.dtl.DTLVardef@1512832org.highwire.dtl.DTLVardef@126134corg.highwire.dtl.DTLVardef@bd104d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

LAT1 enables T cell activation under inflammatory conditions: a new therapeutic target for rheumatoid arthritis

ObjectiveTo assess the L-type amino acid transporter-1 (LAT1) as a possible therapeutic target for rheumatoid arthritis (RA). MethodsSynovial LAT1 expression was monitored by immunohistochemistry and transcriptomic datasets. The contribution of LAT1 to gene expression and immune synapse formation was assessed by RNA-sequencing and total internal reflection fluorescent (TIRF) microscopy, respectively. Mouse models of RA were used to assess the impact of therapeutic targeting of LAT1. ResultsLAT1 was strongly expressed by CD4+ T cells in the synovial membrane of patients with active RA and the level of expression correlated with levels of ESR and CRP as well as DAS-28 scores. Deletion of LAT1 in murine CD4+ T cells inhibited the development of experimental arthritis and prevented the differentiation of CD4+ T cells expressing IFN-{gamma} and TNF-, without affecting regulatory T cells. LAT1 deficient CD4+ T cells demonstrated reduced transcription of genes associated with TCR/CD28 signalling, including Akt1, Akt2, Nfatc2, Nfkb1 and Nfkb2. Functional studies using TIRF microscopy revealed a significant impairment of immune synapse formation with reduced recruitment of CD3{zeta} and phospho-tyrosine signalling molecules in LAT1 deficient CD4+ T cells from the inflamed joints but not the draining lymph nodes of arthritic mice. Finally, it was shown that a small molecule LAT1 inhibitor, currently undergoing clinical trials in man, was highly effective in treating experimental arthritis in mice. ConclusionsIt was concluded that LAT1 plays a critical role in activation of pathogenic T cell subsets under inflammatory conditions and represents a promising new therapeutic target for RA. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LILAT1 is an amino acid transporter that has previously been shown to play a role in T cell activation. C_LI What does this study add?O_LILAT1 is expressed by synovial T cells in human rheumatoid arthritis and the level of expression correlates with disease severity. C_LIO_LILAT1 expression by T cells is necessary for development of severe arthritis in animal models. C_LIO_LILAT1 is required for immune synapse formation and activation of pathogenic CD4+ T cell subsets in the inflamed joint, but not the lymph nodes. C_LIO_LIA small molecular weight LAT1 inhibitor, currently in clinical trials for cancer, is highly effective in animal models of rheumatoid arthritis. C_LI How might this impact on clinical practice of future developments?O_LIThe context-specific nature of LAT1 involvement in T cell activation positions it as an ideal therapeutic target to distinguish between pathogenic and protective T cell responses and this study provides the scientific rationale for clinical evaluation of LAT1 inhibitors in the treatment of rheumatoid arthritis. C_LI

immunology↗

bioRxiv2022

Alternative paths to immune activation: the role of costimulatory risk genes for polygenic inflammatory disease in T helper cells

T cell activation pathways have been repeatedly implicated by genetic studies as being enriched for risk genes for immune and inflammatory diseases. Many of these risk genes code for costimulatory receptors or ligands. Costimulatory receptors are cell surface proteins on T cells, which are engaged by costimulatory ligands on antigen-presenting cells. Both costimulation and antigen binding are required to trigger T cell activation. In order to study the different pathways activated by these costimulatory risk molecules, and the role they may play in inflammatory disease genetics, we carried out gene expression (RNA-seq) and chromatin accessibility (ATAC-seq) profiling of naive and memory CD4+ T cells (N=5 donors) activated via four different costimulatory receptors: CD28 (the standard molecule used for in vitro activation studies), along with alternative costimulatory molecules ICOS, CD6, and CD27. Most, but not all, activation genes and regions are shared by different costimulation conditions. Alternative costimulation induced lower proliferation and cytokine production, but higher lysosome production, altered metabolic processing, and indications of "signal seeking" behaviour (homing and expression of costimulatory and cytokine receptors). We validated a number of these functions at the surface protein level using orthogonal experimental techniques. We found the strongest enrichment of heritability for inflammatory bowel disease in shared regions upregulated by all costimulatory molecules. However, some risk variants and genes were only induced by alternative costimulation, and the impact of these variants on expression were less often successfully mapped in studies of T cells activated by traditional CD28 costimulation. This suggests that future genetics studies of gene expression in activated T cells may benefit from including alternative costimulation conditions.

genomics↗

bioRxiv2022

Dynamic mitochondrial transcription and translation in B cells control germinal centre entry and lymphomagenesis

Germinal centre (GC) B cells undergo proliferation at very high rates in a hypoxic microenvironment, but the cellular processes driving this are incompletely understood. Here we show that the mitochondria of GC B cells are highly dynamic, with significantly upregulated transcription and translation rates associated with the activity of transcription factor mitochondrial A (TFAM). TFAM, whilst also necessary for normal B cell development, is required for entry of activated GC-precursor B cells into the germinal centre reaction, and deletion of Tfam significantly impairs GC formation, function, and output. Loss of TFAM in B cells compromises the actin cytoskeleton and impairs cellular motility of GC B cells in response to chemokine signalling, leading to their spatial disorganisation. We show that B cell lymphoma substantially increases mitochondrial translation, and deletion of Tfam in B cells is protective against the development of lymphoma in a c-Myc transgenic model. Finally, we show that pharmacologic inhibition of mitochondrial transcription and translation inhibits growth of GC-derived human lymphoma cells, and induces similar defects in the actin cytoskeleton.

immunology↗

bioRxiv2022

Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse through distinct adaptors

Ligation of the T cell receptor (TCR) to peptide-MHC complexes initiates signaling leading to T cell activation. Regulation of T cell responses also requires mechanisms to stop this signaling and to downregulate surface expression of the receptor. T cells achieve this both by TCR internalization and by releasing TCR loaded vesicles directly from the plasma membrane. How these distinct fates are coordinated is unknown. Here we show that clathrin is recruited to TCR microclusters by HRS and STAM2 and that this process is essential for TCR release. Subsequently, EPN1 recruits clathrin to the remaining antigen conjugated TCRs to enable a late wave of TCR endocytosis. With these results we demonstrate two clathrin-dependent mechanisms and show how the clathrin machinery participates in membrane evagination and invagination depending upon the adaptor recruiting it. These sequential mechanisms mediate bi-directional membrane exchange at the immunological synapse, providing a scaffold for critical communication.

cell biology↗

bioRxiv2021

3D single molecule localization microscopy reveals the topography of the immunological synapse at isotropic precision below 15 nm

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. A variety of protein-protein interactions in trans-configuration defines the topography of the synapse and orchestrates the antigen-recognition process. In turn, the synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. For better understanding it is hence critical to map the 3D topography of the immunological synapse at high precision. Current methods, however, provide only rather coarse images of the protein distribution within the synapse, which do not reach the dimension of the protein ectodomains. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows 3-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

biophysics↗

bioRxiv2021

Coordination of KIF3A and KIF13A regulates leading edge localization of MT1-MMP in cancer cells

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF9 competes with KIF3A/KIF13A to bring MT1-MMP vesicles to different locations in the plasma membrane. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

cell biology↗

bioRxiv2021

A Simple Ultrafast Multicolor Immunolabelling and Clearing Approach for Whole-Organ and Large Tissue 3D Imaging

High-resolution whole-organ imaging of cleared tissues captures cellular and molecular insights within the intact tissue and tumour microenvironments. However, current immunolabelling and clearing methods are complicated and time-consuming; extending to several weeks. Here, we developed Simple Ultrafast Multicolor Immunolabelling and Clearing or SUMIC, a method that enables multicolor immunolabelling and clearing of whole murine organs and human tissues within 2 to 2.5 days. Moreover, SUMIC is simple, robust, non-hazardous and versatile comprising antigen retrieval, permeabilization, collagenase-based digestion, immunolabelling, dehydration, and clearing. SUMIC permits quantitative and singlecell resolution analysis and detection of rare cells in whole organs, for example, round SMA positive cells in the thymus. Upon volumetric imaging, SUMIC-processed samples retain normal tissue architecture and can be used for paraffin-embedding and histology. We employed the SUMIC method for whole-organ mapping of lymphatic vessels across different ages and organs. This analysis revealed the expansion of lymphatic vessels in endocrine tissues but not in any other organs with aging. Hence, SUMIC will accelerate discoveries compared to other whole organ imaging pipelines.

physiology↗

bioRxiv2020

Allosteric activation of T cell antigen receptor signalling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCR{beta} or CD3{zeta} that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCR{beta} and CD3{zeta}. We found that, similar to the activating mutations, pMHC binding reduced TCR{beta} cohesion with CD3{zeta}. This event occurred prior to CD3{zeta} phosphorylation and at 0{degrees}C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCR{beta} cohesion with CD3{zeta} in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCR{beta} to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3{zeta} phosphorylation and initiate signal propagation.

immunology↗