Membrane Fluctuation Model for Understanding the Effect of Receptor Nanoclustering on the Activation of Natural Killer Cells through Biomechanical Feedback.

We investigated the role of ligand clustering and density in the activation of natural killer (NK) cells. To that end, we designed reductionist arrays of nanopatterned ligands arranged with different cluster geometries and densities and probed their effects on NK cell activation. We used these arrays as an artificial microenvironment for the stimulation of NK cells and studied the effect of the array geometry on the NK cell immune response. We found that ligand density significantly regulated NK cell activation while ligand clustering had an impact only at a specific density threshold. We also rationalized these findings by introducing a theoretical membrane fluctuation model that considers biomechanical feedback between ligand-receptor bonds and the cell membrane. These findings provide important insight into NK cell mechanobiology, which is fundamentally important and essential for designing immunotherapeutic strategies targeting cancer.

A Novel Approach for Colloidal Lithography: From Dry Particle Assembly to High-Throughput Nanofabrication.

We introduce a novel approach for colloidal lithography based on the dry particle assembly into a dense monolayer on an elastomer, followed by mechanical transfer to a substrate of any material and curvature. This method can be implemented either manually or automatically and it produces large area patterns with the quality obtained by the state-of-the-art colloidal lithography at a very high throughput. We first demonstrated the fabrication of nanopatterns with a periodicity ranging between 200 nm and 2 µm. We then demonstrated two nanotechnological applications of this approach. The first one is antireflective structures, fabricated on silicon and sapphire, with different geometries including arrays of bumps and holes and adjusted for different spectral ranges. The second one is smart 3D nanostructures for mechanostimulation of T cells that are used for their effective proliferation, with potential application in cancer immunotherapy. This new approach unleashes the potential of bottom-up nanofabrication and paves the way for nanoscale devices and systems in numerous applications.

Multifunctional Nanoscale Platform for the Study of T Cell Receptor Segregation.

T cells respond not only to biochemical stimuli transmitted through their activating, costimulatory, and inhibitory receptors but also to biophysical aspects of their environment, including the receptors' spatial arrangement. While these receptors form nanoclusters that can either colocalize or segregate, the roles of these colocalization and segregation remain unclear. Deciphering these roles requires a nanoscale platform with independent and simultaneous spatial control of multiple types of receptors. Herein, using a straightforward and modular fabrication process, we engineered a tunable nanoscale chip used as a platform for T cell stimulation, allowing spatial control over the clustering and segregation of activating, costimulatory, and inhibitory receptors. Using this platform, we showed that, upon blocked inhibition, cells became sensitive to changes in the nanoscale ligand configuration. The nanofabrication methodology described here opens a pathway to numerous studies, which will produce an important insight into the molecular mechanism of T cell activation. This insight is essential for the fundamental understanding of our immune system as well as for the rational design of future immunotherapies.

Elastic Microstructures: Combining Biochemical, Mechanical, and Topographical Cues for the Effective Activation and Proliferation of Cytotoxic T Cells.

The ex vivo activation and proliferation of cytotoxic T cells are critical steps in adoptive immunotherapy. Today, T cells are activated by stimulation with antibody-coated magnetic beads, traditionally used for cell separation. Yet, efficient and controlled activation and proliferation of T cells require new antibody-bearing materials, which, in particular, deliver mechanical and topographic cues sensed by T cells. Here, we demonstrate a new approach for the activation and proliferation of human cytotoxic T cells using an elastic microbrush coated with activating and costimulatory antibodies. We found that the microbrush topography affects the protrusion of the cell membrane and the elastic response to the forces applied by cells and can be optimized to yield the strongest activation of T cells. In particular, T cells stimulated by a microbrush showed a three-fold increase in degranulation and release of cytokines over T cells stimulated with state-of-the-art magnetic beads. Furthermore, the microbrush induced a T-cell proliferation of T cells that was more prolonged and yielded much higher cell doubling than that done by the state-of-the-art methods. Our study provides an essential insight into the physical mechanism of T-cell activation and proliferation and opens the floodgates for the design of novel stimulatory materials for T-cell-based immunotherapy.

Fabrication of Nanoscale Arrays to Study the Effect of Ligand Arrangement on Inhibitory Signaling in NK Cells.

Molecular scale nanopatterns of bioactive molecules have been used to study the effect of transmembrane receptor arrangement on a variety of cell types, including immune cells and their immune response in particular. However, state-of-the-art fabrication approaches have thus far enabled the production of patterns with control over one receptor type only. Herein, we describe a protocol to fabricate arrays for the molecular scale control of the segregation between activating and inhibitory receptors in NK cells. We used this platform to study how ligand segregation regulates NK cell inhibitory signaling and function. The arrays are based on patterns of nanodots of two metals, selectively functionalized with activating and inhibitory ligands. Due to the versatility of our functionalization approach, this protocol can be applied to configurate virtually any combination of extracellular ligands into controlled multifunctional arrays.

Enhancement of electronic effects at a biomolecule-inorganic interface by multivalent interactions.

The binding of peptides and proteins through multiple weak interactions is ubiquitous in nature. Biopanning has been used to "hijack" this multivalent binding for the functionalization of surfaces. For practical applications it is important to understand how multivalency influences the binding interactions and the resulting behaviour of the surface. Considering the importance of optimization of the electronic properties of surfaces in diverse electronic and optoelectronic applications, we study here the relation between the multivalency effect and the resulting modulation of the surface work function. We use 12-mer peptides, which were found to strongly bind to oxide surfaces, to functionalize indium tin oxide (ITO) surfaces. We show that the affinity of the peptides for the ITO surface, and concurrently the effect on the ITO work function, are linearly affected by the number of basic residues in the sequence. The multivalent binding interactions lead to a peptide crowding effect, and a stronger modulation of the work function for adodecapeptide than for a single basic amino acid functionalization. The bioderived molecular platform presented herein can pave the way to a novel approach to improve the performance of optoelectronic devices in an eco-friendly manner.

Nanocomposite coatings for the prevention of surface contamination by coronavirus.

The current Covid-19 pandemic has a profound impact on all aspects of our lives. Aside from contagion by aerosols, the presence of the SARS-CoV-2 is ubiquitous on surfaces that millions of people handle daily. Therefore, controlling this pandemic involves the reduction of potential infections via contaminated surfaces. We developed antiviral surfaces by preparing suspensions of copper and cupric oxide nanoparticles in two different polymer matrices, poly(methyl methacrylate) and polyepoxide. For total copper contents as low as 5%, the composite material showed remarkable antiviral properties against the HCoV-OC43 human coronavirus and against a model lentivirus and proved well-resistant to accelerated aging conditions. Importantly, we showed that the Cu/CuO mixture showed optimal performances. This product can be implemented to produce a simple and inexpensive coating with long-term antiviral properties and will open the way to developing surface coatings against a broad spectrum of pathogens including SARS-CoV-2.

Dynamic Surface Layer Coiled Coil Proteins Processing Analog-to-Digital Information.

Surface layer proteins perform multiple functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. However, mimicking the complex and dynamic behavior of such two-dimensional biochemical systems is challenging, and hence research has so far focused mainly on the design and manipulation of the structure and functionality of protein assemblies in solution. Motivated by the new opportunities that dynamic surface layer proteins may offer for modern technology, we herein demonstrate that immobilization of coiled coil proteins onto an inorganic surface facilitates complex behavior, manifested by reversible chemical reactions that can be rapidly monitored as digital surface readouts. Using multiple chemical triggers as inputs and several surface characteristics as outputs, we can realize reversible switching and logic gate operations that are read in parallel. Moreover, using the same coiled coil protein monolayers for derivatization of nanopores drilled into silicon nitride membranes facilitates control over ion and mass transport through the pores, thereby expanding the applicability of the dynamic coiled coil system for contemporary stochastic biosensing applications.

Templated Assembly of Nanoparticles into Continuous Arrays.

The templated assembly of nanoparticles has been limited so far to yield only discontinuous nanoparticle clusters confined within lithographically patterned cavities. Here, we explored the templated assembly of nanoparticles into continuous 2D structures, using lithographically patterned templates with topographical features sized as the assembled nanoparticles. We found that these features act as nucleation centers, whose exact arrangement determines four possible assembly regimes (i) rotated, (ii) disordered, (iii) closely packed, and (iv) unpacked. These regimes produce structures strikingly different from their geometry, orientation, long-range and short-range orders, and packing density. Interestingly, for templates with relatively distant nucleation centers, these four regimes are replaced with three new ones, which produce large monocrystalline domains that are either (i) uniformly rotated, (ii) uniformly aligned, or (iii) nonuniformly rotated relative to the nucleation lattice. We rationalized our experimental data using a mathematical model, which examines all the alignment possibilities between the nucleation centers and the ideal hexagonal assembly. Our finding provides a new approach for the à la carte obtainment of various nanoscale structures unachievable by natural self-assembly and opens a route for the fabrication of numerous functional nanodevices and nanosystems that could not be realized so far by the standard bottom-up approach.

Molecular-scale spatio-chemical control of the activating-inhibitory signal integration in NK cells.

The role of juxtaposition of activating and inhibitory receptors in signal inhibition of cytotoxic lymphocytes remains strongly debated. The challenge lies in the lack of tools that allow simultaneous spatial manipulation of signaling molecules. To circumvent this, we produced a nanoengineered multifunctional platform with molecular-scale spatial control of ligands, which was applied to elucidate KIR2DL1-mediated inhibition of NKG2D signaling-receptors of natural killer cells. This platform was conceived by bimetallic nanodot patterning with molecular-scale registry, followed by a ternary functionalization with distinct moieties. We found that a 40-nm gap between activating and inhibitory ligands provided optimal inhibitory conditions. Supported by theoretical modeling, we interpret these findings as a consequence of the size mismatch and conformational flexibility of ligands in their spatial interaction. This highly versatile approach provides an important insight into the spatial mechanism of inhibitory immune checkpoints, which is essential for the rational design of future immunotherapies.

Antibody-Functionalized Nanowires: A Tuner for the Activation of T Cells.

T cells sense both chemical cues delivered by antigen molecules and physical cues delivered by the environmental elasticity and topography; yet, it is still largely unclear how these cues cumulatively regulate the immune activity of T cells. Here, we engineered a nanoscale platform for ex vivo stimulation of T cells based on antigen-functionalized nanowires. The nanowire topography and elasticity, as well as the immobilized antigens, deliver the physical and chemical cues, respectively, enabling the systematic study of the integrated effect of these cues on a T cell's immune response. We found that T cells sense both the topography and bending modulus of the nanowires and modulate their signaling, degranulation, and cytotoxicity with the variation in these physical features. Our study provides an important insight into the physical mechanism of T cell activation and paves the way to novel nanomaterials for the controlled ex vivo activation of T cells in immunotherapy.

Nanowire Based Guidance of the Morphology and Cytotoxic Activity of Natural Killer Cells.

The cytotoxic activity of natural killer (NK) cells is regulated by many chemical and physical cues, whose integration mechanism is still obscure. Here, a multifunctional platform is engineered for NK cell stimulation, to study the effect of the signal integration and spatial heterogeneity on NK cell function. The platform is based on nanowires, whose mechanical compliance and site-selective tip functionalization with antigens produce the physical and chemical stimuli, respectively. The nanowires are confined to micron-sized islands, which induce a splitting of the NK cells into two subpopulations with distinct morphologies and immune responses: NK cells atop the nanowire islands display symmetrical spreading and enhanced activation, whereas cells lying in the straits between the islands develop elongated profiles and show lower activation levels. The demonstrated tunability of NK cell cytotoxicity provides an important insight into the mechanism of their immune function and introduces a novel technological route for the ex vivo shaping of cytotoxic lymphocytes in immunotherapy.

Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells.

Mechanosensing has been recently explored for T cells and B cells and is believed to be a part of their activation mechanism. Here, we investigated the mechanosensing of the third type of lymphocyte - natural killer (NK) cells, by showing that they modulate their immune activity in response to changes in the stiffness of a stimulating surface. Interestingly, we found that this immune response is bell-shaped and peaks for a stiffness of a few hundreds of kPa. This bell-shaped behavior was observed only for surfaces functionalized with the activating ligand major histocompatibility complex class I polypeptide-related sequence A but not for control surfaces, lacking immunoactive functionalities. We found that stiffness does not affect uniformly all the cells but increases the size of a little group of extra-active cells, which in turn contributes to the overall activation effect of the entire cell population. We further imaged the clustering of costimulatory adapter protein DAP10 on the NK cell membrane and found the same bell-shaped dependence to surface stiffness. Our findings reveal what seems to be ″the tip of the iceberg″ of mechanosensation of NK cells and provide an important insight into the mechanism of their immune signaling.

Cell-Cell Adhesion-Driven Contact Guidance and Its Effect on Human Mesenchymal Stem Cell Differentiation.

Contact guidance has been extensively explored using patterned adhesion functionalities that predominantly mimic cell-matrix interactions. Whether contact guidance can also be driven by other types of interactions, such as cell-cell adhesion, still remains a question. Herein, this query is addressed by engineering a set of microstrip patterns of (i) cell-cell adhesion ligands and (ii) segregated cell-cell and cell-matrix ligands as a simple yet versatile set of platforms for the guidance of spreading, adhesion, and differentiation of mesenchymal stem cells. It was unprecedently found that micropatterns of cell-cell adhesion ligands can induce contact guidance. Surprisingly, it was found that patterns of alternating cell-matrix and cell-cell strips also induce contact guidance despite providing a spatial continuum for cell adhesion. This guidance is believed to be due to the difference between the potencies of the two adhesions. Furthermore, patterns that combine the two segregated adhesion functionalities were shown to induce more human mesenchymal stem cell osteogenic differentiation than monofunctional patterns. This work provides new insight into the functional crosstalk between cell-cell and cell-matrix adhesions and, overall, further highlights the ubiquitous impact of the biochemical anisotropy of the extracellular environment on cell function.

Remote imaging of single cell 3D morphology with ultrafast coherent phonons and their resonance harmonics.

Cell morphological analysis has long been used in cell biology and physiology for abnormality identification, early cancer detection, and dynamic change analysis under specific environmental stresses. This work reports on the remote mapping of cell 3D morphology with an in-plane resolution limited by optics and an out-of-plane accuracy down to a tenth of the optical wavelength. For this, GHz coherent acoustic phonons and their resonance harmonics were tracked by means of an ultrafast opto-acoustic technique. After illustrating the measurement accuracy with cell-mimetic polymer films we map the 3D morphology of an entire osteosarcoma cell. The resulting image complies with the image obtained by standard atomic force microscopy, and both reveal very close roughness mean values. In addition, while scanning macrophages and monocytes, we demonstrate an enhanced contrast of thickness mapping by taking advantage of the detection of high-frequency resonance harmonics. Illustrations are given with the remote quantitative imaging of the nucleus thickness gradient of migrating monocyte cells.

Advanced Materials and Devices for the Regulation and Study of NK Cells.

Natural Killer (NK) cells are innate lymphocytes that contribute to immune protection by cytosis, cytokine secretion, and regulation of adaptive responses of T cells. NK cells distinguish between healthy and ill cells, and generate a cytotoxic response, being cumulatively regulated by environmental signals delivered through their diverse receptors. Recent advances in biomaterials and device engineering paved the way to numerous artificial microenvironments for cells, which produce synthetic signals identical or similar to those provided by the physiological environment. In this paper, we review recent advances in materials and devices for artificial signaling, which have been applied to regulate NK cells, and systematically study the role of these signals in NK cell function.

Nanoscale Mechanosensing of Natural Killer Cells is Revealed by Antigen-Functionalized Nanowires.

Cells sense their environment by transducing mechanical stimuli into biochemical signals. Commonly used tools to study cell mechanosensing provide limited spatial and force resolution. Here, a novel nanowire-based platform for monitoring cell forces is reported. Nanowires are functionalized with ligands for cell immunoreceptors, and they are used to explore the mechanosensitivity of natural killer (NK) cells. In particular, it is found that NK cells apply centripetal forces to nanowires, and that the nanowires stimulate cell contraction. Based on the nanowire deformation, it is calculated that cells apply forces of down to 10 pN, which is the smallest value demonstrated so far by microstructured platforms for cell spreading. Furthermore, the roles of: i) nanowire topography and ii) activating ligands in the cell immune function are studied and it is found that only their combination produces enhanced population of activated NK cells. Thus, a mechanosensing mechanism of NK cells is proposed, by which they integrate biochemical and mechanical stimuli into a decision-making machinery analogous to the AND logic gate, whose output is the immune activation. This work reveals unprecedented mechanical aspects of NK cell immune function and introduces an innovative nanomaterial for studying cellular mechanics with unparalleled spatial and mechanical resolution.

Directed Assembly of Au-Tipped 1D Inorganic Nanostructures via Nanolithographic Docking.

Controlled assembly of nanostructures is a key challenge in nanotechnology. In this work, we introduce an approach for the controlled assembly of 1D nanodumbbells-Au-tipped semiconductor nanorods-into arbitrary 2D higher architectures, by their chemical docking to nanopatterned functionalities. We realized the docking functionalities via nanoimprinted metallic nanodots functionalized with an organic monolayer, whose terminal thiol groups chemically bind the nanodumbbell tips. We demonstrated that the functional nanopattern encodes the nanodumbbell assembly and can be designed to deterministically position nanodumbbells in two possible modes. In the single-docking mode, the nanodot arrays are designed with a spacing that exceeds the nanodumbbell length, restricting each nanodumbbell to dock with one edge and physically connect with its free edge to one of the neighboring nanodumbbells. Alternatively, in the double-docking mode, the nanodots are spaced to exactly fit the nanodumbbell length, allowing nanodumbbell docking with both edges. We found that the docking kinetics can be described by a random attachment model, and verified that for the used docking chemistry, nanodumbbells that are docked to the same dot do not interact with each other. Our work demonstrates the possibility for massively parallel positioning of sub-100 nm 1D semiconductor nanostructures, and can potentially enable their future integration into functional nanodevices and nanosystems.

Natural killer cells' immune response requires a minimal nanoscale distribution of activating antigens.

NK cells recognize cancer and viral cells by binding their activating receptors to antigens presenting on the membrane of target cells. Although the activation mechanism of NK cells is a subject of extensive research today, the role of the composition and spatial distribution of activating ligands in NK cell cytotoxicity is barely understood. In this work, we engineered a nanochip whose surface was patterned with matrices of antigens for NKG2D activating receptors. These matrices mimicked the spatial order of the surface of antigen presenting cells with molecular resolution. Using this chip, we elucidated the effect of the antigen spatial distribution on the NK cell spreading and immune activation. We found that the spatial distribution of the ligand within the 100 nm length-scale provides the minimal conditions for NKG2D regulated cell spreading. Furthermore, we found that the immune activation of NK cells requires the same minimal spatial distribution of activating ligands. Above this threshold, both spreading and activation plateaued, confirming that these two cell functions work hand in hand. Our study provides an important insight on the spatial mechanism of the cytotoxic activity of NK cells. This insight opens the way to rationally designed antitumor therapies that harness NK cytotoxicity.

Spatial and Chemical Surface Guidance of NK Cell Cytotoxic Activity.

Studying how different signaling pathways spatially integrate in cells requires selective manipulation and control of different transmembrane ligand-receptor pairs at the same time. This work explores a novel method for precisely arranging two arbitrarily chosen ligands on a micron-scale two-dimensional pattern. The approach is based on lithographic patterning of Au and TiO2 films, followed by their selective functionalization with Ni-nitrilotriacetic acid-histidine and biotin-avidin chemistries, respectively. The selectivity of chemical and biological functionalizations is demonstrated by X-ray photoelectron spectroscopy and immunofluorescence imaging, respectively. This approach is applied to produce the first type of bifunctional surfaces with controllably positioned ligands for activating the receptors of natural killer (NK) immune cells. NK cells were used as a model system to demonstrate the potency of the surface in guiding site-selective cell attachment and activation. Upon applying the suitable ligand or ligand combination, the surfaces guided the appropriate single- or bifunctional attachment and activation. These encouraging results demonstrate the effectiveness of the system as an experimental platform aimed at the comprehensive understanding of the immunological synapse. The great simplicity, modularity, and specificity of this approach make it applicable for a myriad of combinations of other biomolecules and applications, turning it into the "Swiss knife" of biointerfaces.