Subcutaneous biodegradable scaffolds for restimulating the antitumour activity of pre-administered CAR-T cells.

The efficacy of adoptive T-cell therapies based on chimaeric antigen receptors (CARs) is limited by the poor proliferation and persistence of the engineered T cells. Here we show that a subcutaneously injected biodegradable scaffold that facilitates the infiltration and egress of specific T-cell subpopulations, which forms a microenvironment mimicking features of physiological T-cell activation, enhances the antitumour activity of pre-administered CAR-T cells. CAR-T-cell expansion, differentiation and cytotoxicity were driven by the scaffold's incorporation of co-stimulatory bound ligands and soluble molecules, and depended on the types of co-stimulatory molecules and the context in which they were presented. In mice with aggressive lymphoma, a single, local injection of the scaffold following non-curative CAR-T-cell dosing led to more persistent memory-like T cells and extended animal survival. Injectable biomaterials with optimized ligand presentation may boost the therapeutic performance of CAR-T-cell therapies.

Mechanical forces amplify TCR mechanotransduction in T cell activation and function.

Adoptive T cell immunotherapies, including engineered T cell receptor (eTCR) and chimeric antigen receptor (CAR) T cell immunotherapies, have shown efficacy in treating a subset of hematologic malignancies, exhibit promise in solid tumors, and have many other potential applications, such as in fibrosis, autoimmunity, and regenerative medicine. While immunoengineering has focused on designing biomaterials to present biochemical cues to manipulate T cells ex vivo and in vivo, mechanical cues that regulate their biology have been largely underappreciated. This review highlights the contributions of mechanical force to several receptor-ligand interactions critical to T cell function, with central focus on the TCR-peptide-loaded major histocompatibility complex (pMHC). We then emphasize the role of mechanical forces in (i) allosteric strengthening of the TCR-pMHC interaction in amplifying ligand discrimination during T cell antigen recognition prior to activation and (ii) T cell interactions with the extracellular matrix. We then describe approaches to design eTCRs, CARs, and biomaterials to exploit TCR mechanosensitivity in order to potentiate T cell manufacturing and function in adoptive T cell immunotherapy.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors.

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Generation of functionally distinct T-cell populations by altering the viscoelasticity of their extracellular matrix.

The efficacy of adoptive T-cell therapies largely depends on the generation of T-cell populations that provide rapid effector function and long-term protective immunity. Yet it is becoming clearer that the phenotypes and functions of T cells are inherently linked to their localization in tissues. Here we show that functionally distinct T-cell populations can be generated from T cells that received the same stimulation by altering the viscoelasticity of their surrounding extracellular matrix (ECM). By using a model ECM based on a norbornene-modified collagen type I whose viscoelasticity can be adjusted independently from its bulk stiffness by varying the degree of covalent crosslinking via a bioorthogonal click reaction with tetrazine moieties, we show that ECM viscoelasticity regulates T-cell phenotype and function via the activator-protein-1 signalling pathway, a critical regulator of T-cell activation and fate. Our observations are consistent with the tissue-dependent gene-expression profiles of T cells isolated from mechanically distinct tissues from patients with cancer or fibrosis, and suggest that matrix viscoelasticity could be leveraged when generating T-cell products for therapeutic applications.

Enhancing CAR-T cell functionality in a patient-specific manner.

Patient responses to autologous CD19 chimeric antigen receptor (CAR) T-cell therapies are limited by insufficient and inconsistent cellular functionality. Here, we show that controlling the precise level of stimulation during T-cell activation to accommodate individual differences in the donor cells will dictate the functional attributes of CAR-T cell products. The functionality of CAR-T cell products, consisting of a diverse set of blood samples derived from healthy donors, acute lymphoblastic leukemia (ALL), and chronic lymphocytic lymphoma (CLL) patient samples, representing a range of patient health status, is tested upon culturing on artificial antigen-presenting cell scaffolds to deliver T-cell stimulatory ligands (anti-CD3/anti-CD28) at highly defined densities. A clear relationship is observed between the dose of stimulation, the phenotype of the T-cell blood sample prior to T-cell activation, and the functionality of the resulting CAR-T cell products. We present a model, based on this dataset, that predicts the precise stimulation needed to manufacture a desired CAR-T cell product, given the input T-cell attributes in the initial blood sample. These findings demonstrate a simple approach to enhance CAR-T functionality by personalizing the level of stimulation during T-cell activation to enable flexible manufacturing of more consistent and potent CAR-T cells.

Cytokine conjugation to enhance T cell therapy.

Adoptive T cell transfer (ACT) therapies suffer from a number of limitations (e.g., poor control of solid tumors), and while combining ACT with cytokine therapy can enhance effectiveness, this also results in significant side effects. Here, we describe a nanotechnology approach to improve the efficacy of ACT therapies by metabolically labeling T cells with unnatural sugar nanoparticles, allowing direct conjugation of antitumor cytokines onto the T cell surface during the manufacturing process. This allows local, concentrated activity of otherwise toxic cytokines. This approach increases T cell infiltration into solid tumors, activates the host immune system toward a Type 1 response, encourages antigen spreading, and improves control of aggressive solid tumors and achieves complete blood cancer regression with otherwise noncurative doses of CAR-T cells. Overall, this method provides an effective and easily integrated approach to the current ACT manufacturing process to increase efficacy in various settings.

The magnitude of LFA-1/ICAM-1 forces fine-tune TCR-triggered T cell activation.

T cells defend against cancer and viral infections by rapidly scanning the surface of target cells seeking specific peptide antigens. This key process in adaptive immunity is sparked upon T cell receptor (TCR) binding of antigens within cell-cell junctions stabilized by integrin (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) complexes. A long-standing question in this area is whether the forces transmitted through the LFA-1/ICAM-1 complex tune T cell signaling. Here, we use spectrally encoded DNA tension probes to reveal the first maps of LFA-1 and TCR forces generated by the T cell cytoskeleton upon antigen recognition. DNA probes that control the magnitude of LFA-1 force show that F>12 pN potentiates antigen-dependent T cell activation by enhancing T cell-substrate engagement. LFA-1/ICAM-1 mechanical events with F>12 pN also enhance the discriminatory power of the TCR when presented with near cognate antigens. Overall, our results show that T cells integrate multiple channels of mechanical information through different ligand-receptor pairs to tune function.

Massively Parallelized Molecular Force Manipulation with On-Demand Thermal and Optical Control.

In single-molecule force spectroscopy (SMFS), a tethered molecule is stretched using a specialized instrument to study how macromolecules extend under force. One problem in SMFS is the serial and slow nature of the measurements, performed one molecule at a time. To address this long-standing challenge, we report on the origami polymer force clamp (OPFC) which enables parallelized manipulation of the mechanical forces experienced by molecules without the need for dedicated SMFS instruments or surface tethering. The OPFC positions target molecules between a rigid nanoscale DNA origami beam and a responsive polymer particle that shrinks on demand. As a proof-of-concept, we record the steady state and time-resolved mechanical unfolding dynamics of DNA hairpins using the fluorescence signal from ensembles of molecules and confirm our conclusion using modeling.

Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

Variable incidence angle linear dichroism (VALiD): a technique for unique 3D orientation measurement of fluorescent ensembles.

A fundamental challenge with fluorophore orientation measurement is degeneracy, which is the inability to distinguish between multiple unique fluorophore orientations. Techniques exist for the non-degenerate measurement of the orientations of single, static fluorophores. However, such techniques are unsuitable for densely labeled and/or dynamic samples common to biological research. Accordingly, a rapid, widefield microscopy technique that can measure orientation parameters for ensembles of fluorophores in a non-degenerate manner is desirable. We propose that exciting samples with polarized light and multiple incidence angles could enable such a technique. We use Monte Carlo simulations to validate this approach for specific axially symmetric ensembles of fluorophores and obtain optimal experimental parameters for its future implementation.

Tunable DNA Origami Motors Translocate Ballistically Over µm Distances at nm/s Speeds.

Inspired by biological motor proteins, that efficiently convert chemical fuel to unidirectional motion, there has been considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, DNA motors that undertake discrete steps on RNA tracks have shown the greatest promise. Nonetheless, DNA nanomotors lack intrinsic directionality, are low speed and take a limited number of steps prior to stalling or dissociation. Herein, we report the first example of a highly tunable DNA origami motor that moves linearly over micron distances at an average speed of 40 nm/min. Importantly, nanomotors move unidirectionally without intervention through an external force field or a patterned track. Because DNA origami enables precise testing of nanoscale structure-function relationships, we were able to experimentally study the role of motor shape, chassis flexibility, leg distribution, and total number of legs in tuning performance. An anisotropic rigid chassis coupled with a high density of legs maximizes nanomotor speed and endurance.

DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates.

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.

DNA probes that store mechanical information reveal transient piconewton forces applied by T cells.

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

Mechanical Proofreading: A General Mechanism to Enhance the Fidelity of Information Transfer Between Cells.

The cells and receptors of the immune system are mechanically active. Single molecule force spectroscopy, traction force microscopy, and molecular tension probe measurements all point to the importance of piconewton (pN) molecular forces in immune function. For example, forces enhance the ability of a T cell to discriminate between nearly identical antigens. The role of molecular forces at these critical immune recognition junctions is puzzling because mechanical forces generally facilitate bond dissociation, potentially increasing the difficulty for a receptor to recognize its cognate antigen. The advantage molecular forces confer in the process of immune recognition is not clear. Why would cells expend energy to exert force on the critical, but tenuous bonds that mediate immune surveillance? Do molecular forces provide some advantage to the immune system? The premise of this review is that molecular forces provide a specificity advantage to immune cells. Inspired by the recent discovery that receptor forces regulate immune signaling in T cells and B cells, we dub this notion "mechanical proofreading," akin to more classic kinetic proofreading models. During the process of mechanical proofreading, cells exert pN receptor forces on receptor-ligand interactions, deliberately increasing the energy cost of the immune recognition process in exchange for increased specificity of signaling. Here, we review the role of molecular forces in the immune system and suggest how these forces may facilitate mechanical proofreading to increase the specificity of the immune response.

Mapping the 3D orientation of piconewton integrin traction forces.

Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations.