Biology subjects

Huppa, J. B.

Publications and source records attributed to Huppa, J. B..

12 recordsLinked to original sources

bioRxiv2024

CD4+ T-cells create a stable mechanical environment for force-sensitive TCR:pMHC interactions

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition and ligand discrimination, yet their magnitude, frequency, and impact remain unclear. We quantitatively assess forces across various TCR:pMHC pairs with different bond lifetimes at single-molecule resolution, both before and during T-cell activation, on platforms that either include or exclude tangential force registration. Our results imply that CD4+ T-cell TCRs experience significantly lower forces than previously estimated, with only a small fraction of ligand-engaged TCRs being subjected to these forces during antigen scanning. These rare and minute mechanical forces do not impact the global lifetime distribution of the TCR:ligand bond. We propose that the immunological synapse is created as biophysically stable environment to prevent pulling forces from disturbing antigen recognition.

immunology↗

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

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↗

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

Strategies for the site-specific decoration of DNA origami nanostructures with functionally intact proteins

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to 3 proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral PNA in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

biophysics↗

bioRxiv2020

SARS-CoV-2 escapes CD8 T cell surveillance via mutations in MHC-I restricted epitopes

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control, though direct evidence has been lacking so far. Here, we identified non-synonymous mutations in MHC-I restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV- 2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding, which was associated with a loss of recognition and functional responses by CD8+ T cells isolated from HLA-matched COVID-19 patients. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through escape mutations in MHCI-restricted viral epitopes. This provides evolutionary evidence for CD8+ T cell immunity controlling SARS-CoV-2 with consequences for COVID-19 vaccine design.

immunology↗

bioRxiv2020

The SPPL3-defined glycosphingolipid repertoire regulates immune responses by improving HLA class I access

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

immunology↗

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

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↗