Biology subjects

Dustin, M. L.

Publications and source records attributed to Dustin, M. L..

34 records · Page 2Linked to original sources

bioRxiv2023

Single-cell immune multi-omics and repertoire analyses in pancreatic ductal adenocarcinoma reveal differential immunosuppressive mechanisms within different tumour microenvironments

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. Understanding the multiple mechanisms by which the tumour evades immune control, and how these mechanisms may be disrupted is critical to developing targeted immunotherapies. Previous studies have shown that higher lymphocyte infiltration is associated with better survival, and here we investigated what mediates these differences. We performed a comprehensive analysis of PDAC-associated immune cells using single cell multi-omics coupled with re-analysis of public PDAC scRNA-seq datasets. We introduce novel single-cell and repertoire analyses that have uncoupled diverse roles and contributions of various immune cell populations within different tumour microenvironments (TMEs). They revealed clear distinctions in the clonal characteristics among different patient groups, provided valuable insights into the mechanisms of immune cell migration and tissue adaptation underlying these disparities. These results point to differential CD4 polarisation of intra-tumoural T cells, differential B cell differentiation, GC reactions, antigen presentation pathways, and distinct cell-cell communication between the myeloid-enriched and adaptive-enriched groups. Overall, we identified two major distinct themes for future immune intervention within PDAC patients between those with higher adaptive versus myeloid immune cell infiltration.

immunology↗

bioRxiv2023

Using CombiCells, a platform enabling titration and combinatorial display of cell surface ligands, to investigate the sensitivity and costimulatory requirements of TCRs and CARs

Understanding how cellular decisions by receptor/ligand interactions at cell/cell interface has been challenging because it is difficult to independently vary the surface density of multiple ligands. Here, we exploit the SpyCatcher/SpyTag split-protein system for rapid combinatorial display of native ligands on cells (Combicells). We use this platform to assess T cell antigen sensitivity and the impact of T cell co-stimulation/co-inhibition receptors. The TCR displayed much greater sensitivity to pMHC than CARs and BiTES did to CD19. While TCR sensitivity was greatly enhanced by CD2 ligand, CAR sensitivity to CD19 was primarily but more modestly enhanced by LFA-1 ligand. Lastly, we show that the PD-1/ligand engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor/ligand interactions at cell/cell interfaces. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/545075v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@d1ec95org.highwire.dtl.DTLVardef@33c0forg.highwire.dtl.DTLVardef@5bfed7org.highwire.dtl.DTLVardef@1b80ee2_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryUsing CombiCells, a platform for the combinatorial display of cell surface ligands, to compare T cell antigen sensitivity mediated by TCRs, CARs, and BiTEs and its dependence on co-stimulation/co-inhibition receptor ligands

immunology↗

bioRxiv2022

The kinase occupancy of T-cell coreceptors reconsidered

The sensitivity of the {beta} T-cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8{beta}, which are expressed primarily by cells of the helper and cytotoxic T-cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor-kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4-Lck interaction was stoichiometric (~100%) and that the CD8{beta}-Lck interaction was also substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor-Lck co-diffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to show that stoichiometric Lck coupling required an amphipathic -helix present in CD4 but not CD8. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 out-competed CD8{beta} for Lck. In T cells, TCR signaling induced CD4-Lck oligomerization but did not affect the high levels of CD4-Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting. Significance statementCD4 and CD8{beta} are archetypal coreceptor proteins that potently enhance T-cell antigen sensitivity but how they function is still debated. A fundamental question that remains incompletely resolved is: what fractions of the coreceptors bind the signal-initiating kinase, Lck? Using in vitro assays and non-invasive fluorescence fluctuation spectroscopy in live cells, we show that most coreceptors are occupied by Lck at the surface of live cells. The structural basis for important differences in the kinase occupancy of CD4 and CD8{beta} is also identified. These results provide important context for refining current models of both TCR antigen recognition and cell fate decisions made during thymopoiesis.

immunology↗

bioRxiv2022

Segmental flexibility of bispecific T-cell engagers regulates the dynamics of immune synapse formation

Bispecific T-cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact ([≤]13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A-D) targeting CD3{varepsilon} and Her2, designed to create varying intermembrane distances (A B=C>D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A=B=C>D. Addition of close-contact requiring CD58 co-stimulation revealed phospholipase C-{gamma} activation matching cytotoxicity with A>B=C>D. Our findings suggest that, when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close-contact/high flexibility) and C (far-contact/low flexibility). Significance statementBispecific T-cell engagers (TcEs) are immunotherapeutic drugs that trigger the destruction of cancer cells by linking T cells to cancer cell through specific surface molecules (antigens). We designed a series of TcEs with varying distances between their binding sites and flexibilities of the TcE-antigen complexes. By combining structural and functional analyses, we confirmed close-contact formation between T cells and cancer cells as a critical determinant, mediated by co-activating receptors. Furthermore, we also identified molecular flexibility of the TcE-antigen complex as a further critical parameter for TcE potency. These findings provide novel insights into TcE function and highlight the importance of both parameters for future research and the design of improved immunotherapies.

immunology↗

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2020

Artificial Antigen Presenting Cells for Detection and Desensitisation of Auto-reactive T cells Associated with Type 1 Diabetes

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients and using mouse models we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

bioengineering↗

bioRxiv2020

The Bardet-Biedl Syndrome complex component BBS1 regulates proteasome-dependent F-actin clearance from the centrosome to enable its translocation to the T cell immune synapse

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are exploited by the non-ciliated T cell to orchestrate polarized endosome recycling to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We show that BBS1 allows for centrosome polarization towards the immune synapse by promoting its untethering from the nuclear envelope. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.

immunology↗

bioRxiv2020

In silico characterization of mechanisms positioning costimulatory and checkpoint complexes in immune synapses

Integrin and small immunoglobulin superfamily (sIGSF) adhesion complexes function physiologically in human immunological synapses (IS) wherein sIGSF complexes form a corolla of microdomains around an integrin ring and secretory core. The corolla recruits and retains the major costimulatory and checkpoint complexes that regulate the response to T cell receptor (TCR) engagement, making forces that govern corolla formation of particular interest. We developed a phenomenological agent-based model in order to test different hypotheses concerning the mechanisms underlying molecular reorganization during IS formation. The model showed that sIGSF complexes are passively excluded to the distal aspect of the IS as long as their interaction with the ramified F-actin transport network is absent or weaker than that of integrins. An attractive force between sIGSF adhesion and costimulatory/checkpoint complexes relocates the latter from the centre of the IS to the corolla. The simulations suggest that size based sorting interactions with large glycocalyx components as well as a short-range self-attraction between sIGSF complexes explain the corolla "petals". These molecular and mechanistic features establish a general model that can recapitulate complex pattern formation processes observed in cell-bilayer and cell-cell interfaces. One Sentence SummaryComputer simulations of immunological synapses reveal the localization mechanisms of immunoglobulin superfamily adhesion and costimulatory/checkpoint complexes.

immunology↗

bioRxiv2019

Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells

Semaphorin-3A (Sema3A) regulates tumor angiogenesis, but its role in modulating anti-tumor immunity is unclear. We demonstrate that Sema3A secreted within the tumor microenvironment (TME) suppresses tumor-specific CD8+ T cell function via Neuropilin-1 (NRP1), a receptor that is upregulated upon activation with T cells cognate antigen. Sema3A inhibits T cell migration, assembly of the immunological synapse, and tumor killing. It achieves these functional effects through hyper-activating the acto-myosin system in T cells leading to cellular paralysis. Finally, using a clear cell renal cell carcinoma patient cohort, we demonstrate that human tumor-specific CD8+ T cells express NRP1 and are trapped in Sema3A rich regions of tumors. Our study establishes Sema3A as a potent inhibitor of anti-tumor immunity.

immunology↗

bioRxiv2019

Single-cell glycolytic activity regulates membrane tension 1 and HIV-1 fusion

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 viruses were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane-order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells. As glycolytic activity sets membrane tension levels by altering cellular cholesterol surface levels, our results suggest additional previously unknown benefits of cholesterol-lowering medication in HIV-1 infection.

immunology↗

bioRxiv2019

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T-cells

Cell communication through extracellular vesicles is an emerging topic in biology, including communication between cells of the immune system. Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and intercellular adhesion molecule-1 (ICAM-1) induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but their ability to incorporate other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. Our results demonstrate selective enrichment of CD40 ligand (CD40L) and inducible T-cell costimulator (ICOS) in SE in response to addition of CD40 and ICOS ligand (ICOSL), respectively, to SLB presenting TCR ligands and ICAM-1. TCR triggering mobilized intracellular CD40L to the T cells surface at the IS, where it engaged CD40 to enable sorting into SE. SEs were enriched in tetraspanins and bone marrow stromal cell antigen 2 (BST-2) by immunofluorescence and TCR signalling and endosomal sorting complexes required for transport by proteomics. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2 microclusters. CD40L in SE retains the capacity to induce dendritic cell (DC) maturation and cytokine production. SE enabled helper T cells to release effectors physically linked to TCR. One Sentence SummaryTCR and CD40L microclusters can be linked in synaptic ectosomes (extracellular vesicles) that are released in the immunological synapse by helper T cells and induce dendritic cell maturation and cytokine production.

immunology↗

bioRxiv2019

CD2 expression acts as a quantitative checkpoint for immunological synapse structure and T-cell activation.

The CD2 receptor has been described as an adhesion and costimulatory receptor on T cells. Here, transcriptional profiling of colorectal cancers (CRC) revealed a negative correlation between CD2 expression and \"exhausted CD8+ T-cells\" gene signatures. Furthermore, we detected reduced surface CD2 levels in exhausted CD127lowPD-1hi CD3+CD8+ tumour infiltrating lymphocytes (TILs) in CRC. We describe a CD2 expression-level-dependent switch in CD2-CD58 localization between central and peripheral domains in the immunological synapse (IS). A peripheral \"CD2 corolla\" formed when CD2 surface expression was sufficiently high and its cytoplasmic domain intact. The corolla recruited other ligated receptors like CD28, boosted recruitment of activated Src-family kinases (pSrc), LAT and PLC-{gamma} in the IS and consequently T-cell activation in response to a tumour antigen. Corolla formation and pSrc in the IS increased linearly with CD2 expression, whereas pSrc signals were reduced by high, \"exhausted-like\" levels of PD-1, which invaded the corolla. These results suggest two levels of inhibition of Src-family kinases in CD3+CD8+ TILs: reduced CD2 expression and high PD-1 expression.

immunology↗

bioRxiv2019

A tissue-like platform for studying engineered quiescent human T-cells’ interactions with dendritic cells

Research in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situ activation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 help to CD8 T-cells through enhanced maturation of DCs and effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.

immunology↗

bioRxiv2018

Synapse propensity of human memory CD8 T cells confers competitive advantage over naïve counterparts

Memory T cells are endowed with multiple functional features that enable them to be more protective than naive T cells against infectious threats. It is not known if memory cells have a higher synapse propensity, i.e. increased probability to form immature immunological synapses that then provide an entry into different modes of durable interaction with antigen presenting cells. Here we show that only human memory CD8 T cells have remarkably high synapse propensity compared to naive counterparts. Such a dichotomy between naive and memory cells is not observed within the human CD4 or murine CD8 T cell population. Increased surface expression of LFA1 contributes to the higher synapse propensity in human memory CD8 T cells. Finally, we show that higher synapse propensity in human memory CD8 T cells allows them to compete out naive CD8 T cells from getting recruited to the response. This observation has implications for original antigenic sin and aging of the immune system in humans.

immunology↗

bioRxiv2018

Multifaceted influence of pre-mitotic cytotoxicity of primed CD8 T cells on immunity and infection

Granzyme B mRNA is expressed in primed CD8 T cells within 12 hours, but the consequences of this for the immune response are unknown. We observed that substantial portion of the naive CD8 T cell repertoire expressed granzyme B and became pre-mitotic cytotoxic cells (PMCs) immediately in response to Listeria monocytogenes or Lymphocytic choriomeningitis virus infections. The surprising breadth arose from sufficiency of low potency peptide-MHC to induce granzyme B expression in the context of infection. PMCs killed antigen bearing dendritic cells (DCs) in a granzyme B-dependent but largely perforinindependent fashion between 1-2 days post infection. This terminated antigen presentation at 3 days and resulted in reduced clonal expansion. As additional consequences, we highlight that PMCs reduced the burden of DC-borne infectious agents, but also opened a window of vulnerability for secondary infection. Thus, PMCs serve antigen-specific, regulatory and host defence functions, that are innate-like in scale, at the onset of the adaptive immune response.

immunology↗

bioRxiv2018

Reconstitution of immune cell interactions in free-standing membranes

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. It is important, therefore, to be able to elucidate molecular processes occurring at these interfaces. However, the detailed investigation of each components contribution to the formation and regulation of the contact is hampered by the complexity of cellular composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially for using advanced imaging technology. One approach to circumventing these problems is to establish in vitro systems that faithfully mimic immune cell interactions, incorporating complexity that can be dialled-in as needed. Here, we present an in vitro system making use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we begin to investigate the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and the intracellular rearrangements that accompany the initiation of signalling in T cells. The model system presented here is expected to be widely applicable.\n\nSummary StatementImmune cell-cell interactions are reconstituted in free-standing vesicles wherein spatiotemporal aspects of immune synapse formation can be investigated.

immunology↗