Biology subjects

Stockinger, H.

Publications and source records attributed to Stockinger, H..

15 recordsLinked to original sources

bioRxiv2024

Deconstructing CTL-mediated autoimmunity through weak TCR-cross-reactivity towards highly abundant self-antigen

T-cell antigen receptors (TCRs) exhibit inherent cross-reactivity which broadens the spectrum of epitopes that are recognizable by a finite TCR-repertoire but also carries the risk of autoimmunity. However, TCRs support also a high level of antigen specificity as they allow T-cells to discriminate single antigenic peptide/MHC complexes (pMHCs) against millions of structurally related self-pMHCs, in some cases based on the absence or presence of a single methyl-group. How TCRs manage to convey such seemingly contrary properties and why some T-cells become over time autoreactive despite negative thymic selection, has remained elusive. Here, we devised a non-invasive molecular live cell imaging platform to investigate the biophysical parameters governing stimulatory TCR:pMHC interactions in settings of autoreactivity and anti-viral responses - two extremes in T-cell antigen recognition. We show that CMV-specific CD8+ RA14-T-cells respond effectively to even a single HLA-A2/CMV (A2/CMV) antigen, with synaptic TCR:pMHC lifetimes lasting seconds. In contrast, cross-reactivity of type 1 diabetes (T1D)-associated CD8+ 1E6 T-cells towards HLA-A2/preproinsulin (A2/PPI) self-epitopes involved ten-fold less stable synaptic TCR interactions resulting in severely attenuated ZAP70 recruitment and downstream signaling. Compared to A2/CMV-engaged RA14 T-cells, 1E6-T-cells required for activation 4000 or more A2/PPI and at least 100-times as many simultaneously pMHC-engaged TCRs. In support of antigen discrimination, CD8 co-engagement of MHC class I (MHCI) strengthened both settings of TCR:pMHC interactions equally but was essential only for sensitized virus detection but not autorecognition (1000-versus 5-fold enhancement). We conclude that the binding dynamics of TCRs and CD8 with pMHC shape the boundaries of central tolerance in the physiological context of the phenomenal yet also differential T-cell antigen detection capacity, TCR-cross-reactivity and self-antigen abundance. Gained insights are integral to a molecular and quantitative understanding of CD8+ T-cell mediated autoimmunity and protective immunity against infections and cancer. ONE SENTENCE SUMMARYWith the use of newly devised molecular live-cell imaging modalities we measured with unprecedented precision T-cell antigen recognition dynamics in human T-cells in settings of anti-viral immunity and autoimmunity-causing cross-reactivity. These two extremes within the spectrum of T-cell antigen detection differed substantially with regard to synaptic TCR: antigen-engagement, the level of sensitization through the CD8-coreceptor and the overall efficiency of ensuing downstream signaling. Our results demarcate limits of central tolerance and protective immunity and set quantitative boundaries on the occurrence of autoimmunity with direct implications for T-cell-based designs of immunotherapies.

immunology↗

bioRxiv2023

Sodium chloride in the tumor microenvironment enhances T-cell metabolic fitness and cytotoxicity

Adoptive T-cell therapy has become a powerful weapon for cancer treatment. The efficacy of antitumor immunity is associated with the metabolic state of cytotoxic T cells, which is highly sensitive to the tumor microenvironment. It is therefore of considerable interest to bypass immunosuppressive signals in the tumor microenvironment and to identify factors that augment cytotoxic effector functions and ultimately tumor killing. Whether ionic signals serve as aberrant immune signals and influence the adaptive human antitumor immune response is still largely unexplored. We therefore investigated the effect of sodium on the phenotype, function and metabolic regulation of human CD8+ T cells using transcriptomic, metabolomic, high-dimensional flow cytometric and functional assays. We demonstrate a significant enrichment of sodium in solid tumors from patients with breast cancer, which leaves a transcriptomic imprint on intratumoral immune cells. Sodium chloride (NaCl) enhanced the activation state and effector functions of human CD8+ memory T cells. These functional alterations were associated with enhanced metabolic fitness, particularly increases in glycolysis, oxidative phosphorylation and overall nutrient uptake. These NaCl-induced effects translated into increased tumor cell killing in vitro and in a tumor mouse model in vivo. We therefore propose NaCl as a positive regulator of acute antitumor immunity that could be harnessed for ex vivo conditioning of adoptively transferred T cells, such as CAR T-cells.

immunology↗

bioRxiv2023

TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)-modified T-cells and call for alternative antigen receptor designs for effective T-cell-based cancer immunotherapy. Here we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousand-fold. TCC-based antigen recognition occurred without adverse non-specific signaling, which is typically observed in CAR-T-cells, and did not depend - unlike sensitized peptide/MHC detection by conventional T-cells - on CD4- or CD8- coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anti-cancer response.

immunology↗

bioRxiv2023

Monomeric agonist peptide/MHCII complexes activate T-cells in an autonomous fashion

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs has been postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observed pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR-engagement of highly abundant endogenous pMHCIIs was low or non-existent and affected neither TCR-engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches. SHORT SUMMARYPlatzer et al. revealed via highly quantitative and single molecule live cell microscopy the nature of peptide-loaded MHC class II molecules (pMHCII) as monomeric, densely populating, randomly distributed and predominantly rapidly diffusing entities on the surface of B-cells and dendritic cells. Low abundant stimulatory agonist pMHCII acted as autonomous units with the highest chance of T-cell detection when equally spread on APCs. The presence of bystander-pMHCII previously termed "co-agonist pMHC" affected neither synaptic agonist -TCR-binding nor efficiencies of T-cell recognition. "Co-agonist"-TCR-binding resembled random molecular collisions. Findings inform the design of T-cell-based immunotherapies.

immunology↗

bioRxiv2022

24-Nor-ursodeoxycholic acid counteracts TH17/Treg imbalance and ameliorates intestinal inflammation by restricting glutaminolysis in differentiating TH17 cells

Objective24-Nor-ursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid for treating primary sclerosing cholangitis (PSC), an immune-mediated cholestatic liver disease. Since PSC strongly associates with inflammatory bowel diseases (IBD) driven by TH17/Treg imbalance, we aimed to explore NorUDCAs immunomodulatory potential on intestinal TH17/Treg balance. DesignNorUDCAs impact on TH17/Treg tissue distribution was first assessed in Mdr2-/- mouse model of PSC. We specifically investigated NorUDCAs effect on modulating TH17/Treg balance in a CD4+ T cell driven colitis model induced by adoptive transfer of CD25-CD44lowCD45RBhighCD4+ TNaive cells into Rag2-/- mice, mimicking human IBD. Mechanistic studies were performed using molecular approaches, flow cytometry and metabolic assays in murine TH17 cells in vitro. NorUDCAs signaling effects observed in murine system were further validated in circulating CD4+ T cells from PSC patients with co-existing IBD. ResultsNorUDCA promoted Treg generation in both liver and intestine in the Mdr2-/- model. In the experimental IBD model, NorUDCA attenuated intestinal immunopathology. Mechanistically, NorUDCA demonstrated strong immunomodulatory efficacy in counteracting TH17/Treg imbalance by restricting glutaminolysis in differentiating TH17 cells, thus suppressed -Ketoglutarate-dependent mTORC1 activation, glycolysis and enhanced FOXP3 expression. NorUDCAs impact on mTORC1 signaling was further confirmed in circulating CD4+ T-cells from PSC patients with IBD. ConclusionNorUDCA possesses direct immunometabolic modulatory potency to counteract TH17/Treg imbalance and ameliorate excessive TH17 cell driven intestinal immunopathology. These findings extend future clinical applications of NorUDCA for treatment of TH17 cell-mediated disorders along the gut-liver axis and beyond. Significance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIPSC is an immune-mediated cholestatic liver disease highly associated with IBD where TH17/Treg imbalance drives immunopathogenesis; seeking effective therapeutics covering both liver and intestinal disease in PSC is of high clinical relevance. C_LIO_LIIndependent of anti-cholestatic effects, NorUDCA has recently been shown to possess direct immunomodulatory properties on CD8+ T cell metabolism, lymphoblastogenesis and clonal expansion through targeting mTORC1 signaling. C_LIO_LISince mTORC1 serves as critical metabolic checkpoint orchestrating TH17/Treg axis, inhibiting mTORC1 activity represents a potential treatment avenue counteracting TH17/Treg imbalance under intestinal inflammatory conditions. C_LI What are the new findings?O_LINorUDCA enriches FOXP3+ Treg population in both liver and intestinal tissue in the cholestatic Mdr2-/- mouse model of PSC. C_LIO_LINorUDCA exhibits direct immunomodulatory efficacies in suppressing excess TH17 cell-mediated intestinal immunopathology and promotes FOXP3+ Treg generation in an experimental IBD model. C_LIO_LIMechanistically, NorUDCA counteracts TH17/Treg imbalance by restricting glutaminolysis in differentiating TH17 cells, thus suppresses -Ketoglutarate-dependent mTORC1 activation, glycolysis and enhances FOXP3 expression. C_LIO_LINorUDCAs impact on mTORC1 signaling was further confirmed in circulating CD4+ T cells from patients with PSC and IBD. C_LI How might it impact on clinical practice in the foreseeable future?These findings advance our current understanding of therapeutic potentials of NorUDCA, which might represent a novel therapeutic strategy in the treatment of PSC and concomitant IBD and other TH17-mediated intestinal diseases.

molecular biology↗

bioRxiv2021

Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by the N-terminal peptide of lactoferrin

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.

immunology↗

bioRxiv2021

RAxML Grove: An empirical Phylogenetic Tree Database

The assessment of novel phylogenetic models and inference methods is routinely being conducted via experiments on simulated as well as empirical data. When generating synthetic data it is often unclear how to set simulation parameters for the models and generate trees that appropriately reflect empirical model parameter distributions and tree shapes. As a solution, we present and make available a new database called RAxML Grove currently comprising more than 60,000 inferred trees and respective model parameter estimates from fully anonymized empirical data sets that were analyzed using RAxML (1) and RAxML-NG (2) on two web servers. We also describe and make available two simple applications of RAxML Grove to exemplify its usage and highlight its utility for designing realistic simulation studies and analyzing empirical model parameter and tree shape distributions. RAxML Grove is freely available at https://github.com/angtft/RAxMLGrove.

bioinformatics↗

bioRxiv2021

ACE2 is the critical in vivo receptor for SARS-CoV-2 in a novel COVID-19 mouse model with TNF- and IFNγ-driven immunopathology

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modelling revealed how Spike mutations of maVie16 enhanced interaction with murine ACE2. MaVie16 induced profound pathology in BALB/c and C57BL/6 mice and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFN{gamma} and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. Key pointsO_LIThe mouse-adapted SARS-CoV-2 strain maVie16 causes fatal disease in BALB/c mice and substantial inflammation, pneumonia and immunity in C57BL/6 mice C_LIO_LITNF/IFN{gamma} blockade ameliorates maVie16-induced immunopathology C_LIO_LIMaVie16 infection depends on ACE2 and soluble ACE2 inhalation can prevent disease C_LI

immunology↗

bioRxiv2021

24-Nor-Ursodeoxycholic acid reshapes immunometabolism in CD8+ T cells and alleviates hepatic inflammation

Background & Aims24-NorUrsodeoxycholic acid (NorUDCA) is novel therapy for immune-mediated liver diseases such as primary sclerosing cholangitis (PSC) where dysregulated T cells including CD8+ T cells cause liver immunopathology. We hypothesized that NorUDCA may directly modulate CD8+ T cell effector function thus contributing to its therapeutic efficacy independent of anti-cholestatic effects. MethodsNorUDCA effects on CD8+ T cell function in vivo were investigated in a hepatic injury model system induced by excessive CD8+ T cell immune response upon non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Mechanistic studies included molecular and biochemical approaches, flow cytometry and metabolic assays in mouse CD8+ T cells in vitro. Mass spectrometry (MS) was used to identify potential targets modulated by NorUDCA in CD8+ T cells. NorUDCA signaling effects observed in murine systems were validated in peripheral T cells from healthy volunteers and PSC patients. ResultsIn vivo NorUDCA ameliorated hepatic injury and systemic inflammation upon LCMV infection. Mechanistically, NorUDCA demonstrated a strong immunomodulatory efficacy in CD8+ T cells affecting lymphoblastogenesis, mTORC1 signaling and glycolysis of CD8+ T cells. With MS, we identified that NorUDCA regulates CD8+ T cells via targeting mTORC1. NorUDCAs impact on mTORC1 signaling was further confirmed in circulating human CD8+ T cells. ConclusionsNorUDCA possesses a yet-unrecognized direct modulatory potency on CD8+ T cells and attenuates excessive CD8+ T cell hepatic immunopathology. These findings may be relevant for treatment of immune-mediated liver diseases such as PSC and beyond.

molecular biology↗

bioRxiv2020

DNA origami demonstrate the unique stimulatory power of single pMHCs as T-cell antigens

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How they convert a biochemical interaction into a signaling response is poorly understood, yet indirect evidence pointed to the spatial antigen arrangement on the APC surface as a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. We found that the minimum signaling unit required for efficient T-cell activation consisted of two ligated TCRs within a distance of 20 nanometers, if TCRs were stably engaged by monovalent antibody fragments. In contrast, antigenic pMHCs stimulated T-cells robustly as well-isolated entities. These results identify the minimal requirements for effective TCR-triggering and validate the exceptional stimulatory potency of transiently engaging pMHCs.

biophysics↗

bioRxiv2020

Temporal Analysis of T-Cell Receptor-Imposed Forces via Quantitative Single Molecule FRET Measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informs most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings do not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

biophysics↗

bioRxiv2019

Inefficient ZAP70-Signaling Blunts Antigen Detection by CAR-T-Cells

Rational design of chimeric antigen receptors (CARs) with optimized anti-cancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen-engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative single molecule live-cell imaging and found the sensitivity of CAR-T-cells towards antigen approximately 1000-times reduced when compared to T-cell antigen receptor (TCR)-mediated recognition of nominal peptide/MHC complexes. While CARs outperformed TCRs with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-kinase ZAP70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR-designs, which limit at present the efficacy of CAR-T-cell therapies to target tumors with diminished antigen expression.

immunology↗

bioRxiv2019

Enabling Semantic Queries Across Federated Bioinformatics Databases

MotivationData integration promises to be one of the main catalysts in enabling new insights to be drawn from the wealth of biological data available publicly. However, the heterogeneity of the different data sources, both at the syntactic and the semantic level, still poses significant challenges for achieving interoperability among biological databases.\n\nResultsWe introduce an ontology-based federated approach for data integration. We applied this approach to three heterogeneous data stores that span different areas of biological knowledge: 1) Bgee, a gene expression relational database; 2) OMA, a Hierarchical Data Format 5 (HDF5) orthology data store, and 3) UniProtKB, a Resource Description Framework (RDF) store containing protein sequence and functional information. To enable federated queries across these sources, we first defined a new semantic model for gene expression called GenEx. We then show how the relational data in Bgee can be expressed as a virtual RDF graph, instantiating GenEx, through dedicated relational-to-RDF mappings. By applying these mappings, Bgee data are now accessible through a public SPARQL endpoint. Similarly, the materialised RDF data of OMA, expressed in terms of the Orthology ontology, is made available in a public SPARQL endpoint. We identified and formally described intersection points (i.e. virtual links) among the three data sources. These allow performing joint queries across the data stores. Finally, we lay the groundwork to enable nontechnical users to benefit from the integrated data, by providing a natural language template-based search interface.\n\nProject URLhttp://biosoda.expasy.org, https://github.com/biosoda/bioquery

bioinformatics↗

bioRxiv2019

The ELIXIR Core Data Resources: fundamental infrastructure for the life sciences

MotivationLife science research in academia, industry, agriculture, and the health sector depends critically on free and open data resources. ELIXIR (www.elixir-europe.org), the European Research Infrastructure for life sciences data, has identified a set of Core Data Resources within Europe that are of most fundamental importance for the long-term preservation of biological data. We explore characteristics of their usage, impact and assured funding horizon to assess their value and importance as an infrastructure, to understand sustainability of the infrastructure, and to demonstrate a model for assessing Core Data Resources worldwide. ResultsThe nineteen resources currently designated ELIXIR Core Data Resources form a data infrastructure in Europe which is a subset of the worldwide open life science data infrastructure. We show that, from 2014 to 2018, data managed by the Core Data Resources more than tripled while staff numbers increased by less than a tenth. Additionally, support for the Core Data Resources is precarious: together they have assured funding for less than a third of current staff after four years. Our findings demonstrate the importance of the ELIXIR Core Data Resources as repositories for research data and knowledge, while also demonstrating the uncertain nature of the funding environment for this infrastructure. ELIXIR is working towards longer-term support for the Core Data Resources and, through the Global Biodata Coalition, aims to ensure support for the worldwide life science data resource infrastructure of which the ELIXIR Core Data Resources are a subset. Contactrachel.drysdale@elixir-europe.org Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2019

Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via PALM

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophores blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform we revealed complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allowed for robust evaluation of molecular clustering based on individually recorded single molecule localization maps. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=45 SRC="FIGDIR/small/545152v5_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b0930org.highwire.dtl.DTLVardef@883897org.highwire.dtl.DTLVardef@13b7304org.highwire.dtl.DTLVardef@1fc136c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗