CD3 aptamers promote expansion and persistence of tumor-reactive T cells for adoptive T cell therapy in cancer.

The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T cell activation are therefore of great interest for adoptive T cell therapy protocols. To identify CD3-specific T cell agonists, several RNA aptamers were selected against CD3 using Systematic Evolution of Ligands by EXponential enrichment combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through surface plasma resonance, P32 double-filter assays, and flow cytometry. Aptamer-mediated modulation of the threshold of T cell activation was observed in vitro and in preclinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T cell therapy by low-affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future.

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

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 is 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 observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects 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.

A DNA Origami-Based Biointerface to Interrogate the Spatial Requirements for Sensitized T-Cell Antigen Recognition.

When T cells scan the surface of antigen-presenting cells (APCs), they can detect the presence of just a few antigenic peptide/MHC complexes (pMHCs), in some cases even a single agonist pMHC. These are typically vastly outnumbered by structurally similar yet non-stimulatory endogenous pMHCs. How T cells achieve this enormous sensitivity and selectivity is still not clear, in particular in view of the rather moderate (1-100 µM) affinity that T-cell receptors (TCRs) typically exert for antigenic pMHCs. Experimental approaches that enable the control and quantification of physical input parameters within the context of the immunological synapse to precisely interrogate the molecular consequences of TCR-engagement, appear highly advantageous when searching for better answers.We here describe the implementation of a biointerface that allows to experimentally define molecular distances between T-cell ligands as a means to correlate them with molecular dynamics of antigen engagement, downstream signaling, and the overall T-cell response. The basis of this biointerface is DNA origami nanostructures, which are (i) rigid and highly versatile platforms that can (ii) be embedded as laterally mobile entities within supported lipid bilayers and functionalized (iii) in a site-specific and orthogonal manner with (iv) one or more proteins of choice.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

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 the 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 three 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 peptide nucleic acid (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.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

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 agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be 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. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's 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 reveal 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 allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein.

A complete understanding of signaling processes at the plasma membrane depends on a quantitative characterization of the interactions of the involved proteins. Fluorescence recovery after photobleaching (FRAP) is a widely used and convenient technique to obtain kinetic parameters on protein interactions in living cells. FRAP experiments to determine unbinding time constants for proteins at the plasma membrane, however, are often hampered by non-specific contributions to the fluorescence recovery signal. On the example of the interaction between the T cell receptor (TCR) and the Syk kinase ZAP70, we present here an approach based on protein micropatterning that allows the elimination of such non-specific contributions and considerably simplifies analysis of FRAP data. Specifically, detection and reference areas are created within single cells, each being either enriched or depleted in TCR, which permits the isolation of ZAP70-TCR binding in a straight-forward manner. We demonstrate the applicability of our method by comparing it to a conventional FRAP approach.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

Determination of the Membrane Environment of CD59 in Living Cells.

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known. Here, we refine a previously developed micropatterning approach combined with single molecule tracking to quantify the influence of the glycosylphosphatidylinositol-anchored (GPI-anchored) protein CD59 on its molecular environment directly in the live cell plasma membrane. We find that locally enriched and immobilized CD59 presents obstacles to the diffusion of fluorescently labeled lipids with a different phase-partitioning behavior independent of cell cholesterol levels and type of lipid. Our results give no evidence for either specific binding of the lipids to CD59 or the existence of nanoscopic ordered membrane regions associated with CD59.

Monomeric TCRs drive T cell antigen recognition.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns.

Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the feature size of patterns below 1 µm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment. Our approach is based on nanocontact printing and allows for the fabrication of protein patterns with feature sizes of 80 nm and periodicities down to 140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane) (X-PDMS) in a two-layer stamp layout for protein printing. In a proof of principle, different proteins at various scales were printed and the pattern quality was evaluated by atomic force microscopy (AFM) and super-resolution fluorescence microscopy.

Protein Micropatterning Assay: Quantitative Analysis of Protein-Protein Interactions.

Characterization, especially quantification, of protein interactions in live cells is usually not an easy endeavor. Here, we describe a straightforward method to identify and quantify the interaction of a membrane protein ("bait") and a fluorescently labeled interaction partner ("prey") (membrane-bound or cytosolic) in live cells using Total Internal Reflection Fluorescence microscopy. The bait protein is immobilized within patterns in the plasma membrane (e.g., via an antibody); the bait-prey interaction strength can be quantified by determining the prey bulk fluorescence intensity with respect to the bait patterns. This method is particularly suitable also for the analysis of weak, transient interactions that are not easily accessible with other methods.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.

With or without rafts? Alternative views on cell membranes.

The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity.

Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles.

Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, two-dimensional dissociation constants can be determined from the tracer diffusion or surface density.

GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.