Advances in ligand-based surface engineering strategies for fine-tuning T cell mechanotransduction toward efficient immunotherapy

Abstract

T cell-based immunotherapy has recently emerged as a promising strategy to treat cancer, requiring the activation of antigen-directed cytotoxicity to eliminate cancer cells. Mechanical signaling, although often overshadowed by its biochemical counterpart, plays a crucial role in T cell anticancer responses, from activation to cytolytic killing. Rapid advancements in the fields of chemistry, biomaterials, and micro/nanoengineering offer an interdisciplinary approach to incorporating mechano- and immunomodulatory ligands, including but not limited to synthetic peptides, small molecules, cytokines, and artificial antigens, onto the biomaterial-based platforms to modulate mechanotransducive processes in T cells. The surface engineering of these immunomodulatory ligands with optimization of ligand density, geometrical arrangement, and mobility has been proven to better mimic the natural ligation between immunoreceptors and ligands to directly enhance or inhibit mechanotransduction pathways in T cells, through triggering upstream mechanosensitive channels, adhesion molecules, cytoskeletal components, or downstream mechanoimmunological regulators. Despite its tremendous potential, current research on this new biomaterial surface engineering approach for mechanomodulatory T cell activation and effector functions remains in a nascent stage. This review highlights the recent progress in this new direction, focusing on achievements in mechanomodulatory ligand-based surface engineering strategies and underlying principles, and outlooks the further research in the rapidly evolving field of T cell mechanotransduction engineering for efficient immunotherapy.

Significance

Recent advancements in surface engineering have led to the development of sophisticated mechanochemical stimulatory platforms that can effectively modulate T cell mechanotransducive pathways, leveraging the unique properties of bioactive immunomodulatory materials, including but not limited to peptides, synthetic peptides, small molecules, cytokines, and artificial antigens, on biomaterial-based platforms to modulate mechanotransducive processes in T cell. Surface decoration of specific ligands on biomaterial-based platforms, with proper design principles, can mimic and enhance the natural mechanical and chemical cues that T cells encounter during activation and effector functions in T cell-based cancer immunotherapy. This review highlights the cutting-edge ligand-based surface engineering strategies and the underlying biophysical mechanisms to modulate T cell mechanotransduction for efficient immunotherapy.

Introduction

T cells play a pivotal role in the adaptive immune system, capable of exerting antitumor functions by recognizing and eliminating cancerous cells at the tumor site. While the importance of immunobiological factors in T cell function is well established, the crucial roles of mechanobiological signals in T cell antitumor processes have emerged as a fascinating area of research (1). T cell mechanotransduction, the conversion of mechanical stimuli into biochemical signals, is fundamental to their function, mediating various stages of T cell antitumor processes, from their infiltration and activation in the tumor, formation of immunological synapses, to the execution of cytotoxic functions. For example, mechanical forces and mechanosensitive signaling play critical roles in T cell activation through T cell receptor (TCR) recognition of peptides engaged with the major histocompatibility complex (MHC) (2,3,4). A decline in T cell mechanical fitness, such as aberrant mechanosensing mechanisms, disrupted cytoskeletal machinery, and downstream signaling pathways, leads to irregular genetic and epigenetic transcription (5). These changes are often implicated in dysfunctional T cell phenotypes in aging (6) and cancers (7,8). For example, altered mechanotransduction in tumor-infiltrating T cells, due to a mechanically soft tumor microenvironment, results in reduced force generation and impaired tumor cell killing (9). In chimeric antigen receptor (CAR) T cell therapy, a paradigm-shifting approach in cancer treatment, dysfunctional mechanical signaling could contribute to poor quality of patient-derived CAR T cell products and suboptimal clinical responses (10). CAR T cells are genetically engineered to express a synthetic antibody-like CAR that recognizes a specific tumor-associated antigen (e.g., CD19 and BCMA on B cells (11,12), EpCAM on breast cancer cells (13), mesothelin on pancreatic cancer cells (14)) with enhanced potency, resulting in their distinguished synaptic force dynamics and mechanotransducive mechanisms from the conventional TCR activation (4,15). Therefore, strategies to manipulate and augment T cell mechanotransduction are critically needed to advance therapeutic outcomes of immunotherapy.

Surface engineering of mechano- and immunomodulators such as synthetic peptides, antibodies, cytokines, and small molecules on bioengineered platforms provides a unique and powerful approach to improve T cell anticancer functions (Fig. 1). Traditionally, T cell stimulation with immunomodulators, such as antibodies and cytokines, is often in soluble forms (16,17) or via conjugation with beads and microbubbles (18,19,20). The surface presentation of anti-CD3 and anti-CD28 antibodies on beads is particularly crucial for T cell activation, which is essential for both cell expansion and efficient transduction of CAR lentivirus during CAR T cell production ex vivo (21). While priming with soluble ligands can serve as a simple way for T cell modulation, surface engineering of immunomodulatory ligands with precise control of ligand density, geometrical arrangement, and mobility was proven to better mimic mechanical cues and natural ligation between immunoreceptor and ligand at extracellular and cellular interfaces, thus effectively modulating T cell mechanotransduction pathways to boost T cell activation and effector functions (22,23). Cutting-edge nanoengineering techniques such as nanolithography, nanoarrays, and DNA-based nanostructures have been leveraged to precisely control the spatial distribution of ligands, optimizing their configuration on substrates for efficient TCR activation signaling (24,25,26,27). Moreover, advanced chemical approaches for dynamic ligand presentation such as the supramolecular host-guest system, offer promising strategies to more accurately mimic natural receptor-ligand interactions (28). A diverse array of immunomodulatory ligands, including synthetic peptides, small molecules, and artificial antigens, have been incorporated into these engineered surfaces to modulate mechanosignaling in T cells. However, despite their immense potential, the integration of surface-engineered platforms with immunomodulatory ligands remains underexploited in current research efforts.

Figure 1.

Surface engineering of mechanomodulatory ligands for fine-tuning T cell mechanotransduction toward efficient immunotherapy. Peptides, antibodies, and artificial antigens are commonly used to engineer ligand-based surfaces that can mimic the interplay between surface ligands, cell membrane receptors, and intracellular components in transducing mechanical signals (left panel). In T cells, these engineered surfaces can be decorated with various ligands (right panel), including ICAM1, which interacts with LFA-1 on T cells, and MHC molecules, which interact with the TCR. Peptides and artificial antigens are also used for surface decoration to modulate mechanosensitive ion channels and adhesion proteins to fine-tune mechanosignaling pathways, such as Piezo1 and noncanonical Wnt signaling.

This review highlights the cutting-edge ligand-based surface engineering strategies and underlying principles to modulate T cell mechanotransduction and anticancer responses. Firstly, we summarize the fundamental mechanisms of T cell mechanotransduction involved in T cell activation and cytotoxicity process. Thereafter, we provide a deeper discussion on the recent nanoengineering approaches for fine-tuning ligand presentation, such as ligand density and spatial organization to optimize TCR activation, and novel surface decoration strategies developed for direct enhancement or inhibition of mechanotransduction pathways in T cells. These include modulations of mechanosensitive channels (e.g., Piezo1, TRPV4), adhesion molecules (e.g., integrins, CD44), the downstream cytoskeletal (e.g., RhoA/ROCK, Rac1), and transcriptional regulators (e.g., NFAT, YAP/TAZ). As research continues to uncover the critical roles of mechanical signaling in T cell antitumor functions, the development of sophisticated surface engineering strategies tailored to modulate mechanoresponse may lead to more effective and personalized immunotherapies for cancer and other immune-related disorders. At the end, we outline the extant challenges and the potential avenues for T cell mechanoimmunoengineering toward efficient immunotherapy, setting the stage for future research endeavors.

Principles of T cell mechanotransduction and mechanosignaling pathways

Ligand geometric arrangement, mobility, and force transmission are critical for TCR activation

Mechanical forces and signaling are involved in all cellular processes in T cell antitumor activities from activation to cytotoxicity (Fig. 2). T cell activation is initiated through the recognition of MHC-peptide molecules by the TCR, and costimulation via costimulatory receptors such as CD28 and CD3 interacting with their corresponding ligands on antigen-presenting tumor cells. This antigen recognition process is regulated by force-sensitive catch-slip bonds, allowing T cells to discriminate between different peptides based on the force transmitted to the ligand-receptor complex. In this context, slip bonds weaken under force and are associated with nonagonist peptides and minimal T cell activation, while catch bonds strengthen under moderate force and are associated with agonist peptides (29,30). When a TCR first binds to an MHC, the interaction begins as a slip bond, which then transitions into catch bonds under applied mechanical force, strengthening the interaction and prolonging bond lifetime, thereby amplifying T cell activation (29). Studies have shown that the application of tensile forces to the ligand-receptor bond can enhance T cell activation. Specifically Liu et al. found that T cells utilize cytoskeletal coupling to transmit 12–19 pN of tensile force to the TCR-peptide-MHC (pMHC) complexes for activation, and the activation force threshold of a single TCR is 12 pN (31). As CARs have much lower antigen sensitivity compared with TCRs (32), CAR T cells require a substantially greater binding force between the synthetic CAR and the antigen for effective activation (1,33). Conversely, nonagonist peptides maintain slip bonds that rapidly weaken under force, resulting in minimal activation. The efficacy of this mechanotransduction is influenced by various biophysical factors, including the geometric arrangement of ligands (such as spacing, hinge length, and stoichiometry), ligand mobility, and the magnitude of tensile force applied to the ligand-receptor bonds. The stiffness of the target cell surface also influences T cell activation and the formation and stability of the immunological synapse. Specifically, more rigid cell surfaces are able to provide higher activation forces required for TCR-pMHC complexes, promoting more effective mechanistic feedback and immune responses (34). If the applied force or substrate stiffness fails to achieve the activation force threshold or yield intracellular signaling, T cell activation may remain suboptimal. While they may initially appear to be primarily biochemical cues, both the geometric arrangement (ligand spacing, hinge length, stoichiometry, etc.) and mobility of ligands are critical biophysical factors in TCR activation, intimately connected to how forces are generated, distributed, and sensed at the T cell-target cell interface to efficiently activate downstream mechanosignaling in T cells (35).

Figure 2.

Mechanotransduction pathways in T cell. It mainly involves TCR activation, mechanosensitive channels (e.g., Piezo1, TRPV4), adhesion molecules (e.g., integrins, CD44), the downstream cytoskeletal (e.g., RhoA/ROCK, Rac1) and transcriptional regulators (e.g., Wnt, YAP/TAZ).

The spatial distribution of ligands on the target cell surface significantly influences force transmission during T cell activation, where clustered ligands elicit more robust responses compared with dispersed ones (36). Studies have found that the appearance of high-density ligand aggregation could promote a high degree of TCR aggregation, enabling the accumulation of force and distribution of tension among ligand-receptor pairs for more effective T cell activation (37). Ligand spacing is also important both for TCR stimulation and costimulatory receptor costimulation during T cell activation. Wide ligand spacing leads to weaker force transmission, resulting in low cell spreading and weak T cell activation (38). By tuning the horizontal distance between ligands, recent studies have found that a small ligand spacing of 16–60 nm maximizes TCR aggregation and signaling (24,25), and a relatively large spacings between TCR ligands and costimulatory ligands are optimal for T cell activation (25). Also, a study found that the intermembrane spacing between T cell and antigen-presenting cell critically regulates CD45 exclusion and transmission of mechanical force, and thus T cell activation (37). Moreover, ligand mobility on the cell membrane increases the stability of TCR clusters by allowing the formation and movement of TCR clusters (39). Overall, ligand geometric arrangement and mobility influence the force threshold and the efficiency of force transmission during T cell activation, which are integral in triggering mechanotransduction of T cells.

Cytoskeleton remodeling and synaptic force dynamics during immunological synapse formation

Subsequent T cell immune responses after TCR activation are also heavily governed by mechanotransduction. After antigen recognition through immunoreceptors, activated T cells form a highly organized structure termed “immunological synapse,” involving both a physical handshake and a simultaneous bilateral transfer of soluble biochemical signals between the T cell and the cancerous cell (40). T cell cytotoxicity is co-operated by tension generated by an intense remodeling of the actin cytoskeleton and membrane tension (41). During engagement with antigens, the ERM (ezrin-radixin-moesin) proteins that link the plasma membrane to the actin cytoskeleton deactivated, resulting in more deformational cell membrane and low membrane tension, facilitating ligation of T cell and antigen-presenting cells (42). As immunological synapse matures, TCR and adhesion molecules that are linked directly to the cytoskeleton are recruited toward the center of the T cell-target cell interface (43), triggering mechanotransduction signaling cascades that activate small GTPases, such as Rac1, Cdc42, and RhoA, which regulate robust retrograde flow of F-actin and enhance actomyosin contractility (44,45,46). This cytoskeletal remodeling and mechanical force exertion motivates the spreading of T cells across the activating surface, driving membrane topological changes, hallmarking the biophysical trigger for secretion of cytolytic perforin and granzymes essential for target eradication (47,48). Alterations in this mechanoresponse may greatly compromise T cell antitumor cytotoxicity.

Cell adhesive molecules and ion channels regulate T cell mechanosensing

Integrins are principal transmembrane adhesion receptors on T cells binding to the extracellular matrix and ligands expressed on neighbor cells that can initiate various signaling cascades regulating T cell motility, proliferation, differentiation, and mechanotransduction. Multiple α (e.g., α_L, α_4/5/6) and β integrin subunits (e.g., β_1/2/3/7) are expressed during T cell development (49). Lymphocyte function-associated antigen 1 (LFA-1), or integrin α_Lβ₂, is most discussed for its importance in TCR activation, triggering T cell actomyosin cytoskeleton remodeling and effector responses. Binding of LFA-1 on T cells to intercellular adhesion molecule 1 (ICAM1) expressed on antigen-presenting cells would create an inside-out mechanical force for T cell adhesion and stabilize the immunological synapse formation, providing the prolonged mechanosignals required for the effective activation (26). Integrins take part in mechanosensing of T cells to substrate stiffness, initiate F-actin polymerization, formation of lamellipodia, and mediate their migration during immune responses (50). Other integrins such as integrin α₄β₁ (VLA-4), which binds VCAM-1 and integrin α4β7, are often expressed in activated T cells and are important for T cell trafficking (51,52). CD44-mediated adhesion signaling also contributes to T cell mechanosensing, invasive migration, an ex vivo expansion of T cells, via a cytoskeleton-dependent pathway (53,54).

The mechanical forces experienced by T cells are sensed by mechanosensitive ion channels on the cell membrane, triggering various downstream signaling pathways crucial for T cell activation, cytokine production, and cytotoxic functions. Among them, Piezo1 ion channels have been commonly found as mechanosensors in various cell types (55,56,57), including immune cells in the innate and adaptive immune (58,59,60,61). It was well established that Piezo1 is highly sensitive to changes in membrane curvature and membrane tension (62,63). In T cells, Piezo1 channels regulate TCR activation (64), chemotactic migration (65), and mechanosensing to external mechanical cures such as shear stress (66). Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension. Interestingly, Pang et al. demonstrated that continuous activation of Piezo1 could lead to T cell exhaustion and blocking Piezo1 ion channels in cytotoxic T cells can enhance their antitumor responses in vitro and in vivo (67). The contradiction with previous findings on the essential role of Piezo1 for T cell activation highlights the possible side effect correlated with prolonged Piezo1-mediated activities (67). While research is limited, TRPV4 and Orai1 are other channels that have been described in T cell mechanosensation (68,69). These mechanosensitive channels influence the cytoskeletal dynamics in a Ca²⁺-dependent manner (70), therefore regulating the force and function outcomes of T cells.

Downstream mechanoimmunological regulators

Cytoskeleton activities of T cells during immunological synapse formation are regulated via various mechanosignaling pathways (71). Rho and Rac GTPases such as RhoA and Rac1/2 are the key cytoskeleton signal transducers for T cell adhesion, activation, and migration (44,45,46). Li et al. showed that chemical activation Frizzled receptors trigger the Wnt noncanonical signaling pathway and enhance T cell traction force via RhoA/ROCK signaling, thus upregulating T cell activation-related genes and surface markers (72). Govendir et al. reported that the cytoskeletal forces direct synapse membrane curvature in a biphasic manner, with Rho-associated protein kinase ROCK mediating myosin II activities during the first phase and dynein-mediated lytic granule release in the second phase (48). The reorganization of plasma membrane actin at the immunological synapse was also regulated by actin-associated proteins WAVE2 and cofilin (73).

Alterations in cytoskeletal dynamics subsequently influence downstream gene regulators that activate or inhibit transcriptions of genes essential for further T cell mechanoresponse and antitumor functions. For example, YAP/TAZ mechanosensitive transcriptional coactivators were found to respond to cytoskeletal tension and substrate stiffness (74). Lebid et al. reported upregulation of YAP in activated T cells as an immunosuppressive event to prevent their excessive activation and cytotoxic activities, while deletion of YAP significantly enhances inflammatory cytokine production and suppressed tumor growth in vitro (75). Piezo1-dependent activation of YAP was found to promote osteogenic differentiation of valve interstitial cells in calcific aortic valve disease (76) and promote tumor metastasis in various cancers (77,78,79). In other pathways, the same study from Pang et al. revealed the downregulation of transcriptional factor GRHL3 reducing expression of the E3 ubiquitin ligase during F-actin arrangement and augment traction force generation in T cells (67). Zhang et al. further showed that the transcription factor Osr2 is selectively induced in activated cytotoxic T cells in response to mechanical stress in the tumor microenvironment through the Piezo1/CaMKII/CREB/Osr2 signaling axis, suppressing cytotoxic gene expression (e.g., Ifng and Prf1) and promoting T cell exhaustion (80).

In summary, fine-tuning mechanotransduction in T cells by precise control over ligand geometric arrangement, mobility, and force transmission in the immunoreceptor-ligand complex, direct activation or inhibition of upstream mechanosensitive ions channels and adhesion molecules, as well as downstream cytoskeletal signaling and transcriptional regulators, is a promising approach to boost T cell antitumor function toward effective cancer immunotherapy.

Nanoengineered surface with fine-tuned ligand presentation and spatial organization for optimizing TCR activation

The structure and spatial organization of ligands targeting TCR and costimulatory receptors in T cells set the stage for mechanosensing mechanisms involved in triggering T cell immune function. Factors to consider for surface presentation of ligands includes ligand density, spacing, geometrical arrangement, and ligand mobility to better mimic natural ligation between the immunoreceptor and the ligand (Fig. 3). As the critical dimensions of these immunoreceptor-ligand bonds are in a range of a few to tens of nanometers, finding ways to precisely fine-tune these parameters at a nanometer scale has become a pivotal aspect in biofunctionalized surface design and engineering.

Figure 3.

Nanoengineered surface with fine-tuned ligand presentation and spatial organization for optimizing TCR activation. Reproduced with permission from (24). Copyright 2018, Springer Nature.

Lateral spacing and density of ligands set the stage for T cell activation

Various nanoengineering techniques such as nanolithography, nanoarrays, and DNA-based nanostructures have been employed for precise control of spatial distribution of ligands, aiding to find the optimal configurations that maximize TCR force transmission for T cell activation. Cai et al. reported nanofabricated glass surfaces using electron beam lithography with a precise control of both lateral and axial ligand positioning in three dimensions (3D) to study the effect of ligand spatial distribution on TCR triggering (24). They nanofabricated a single-molecule array platform by independent control of interparticle spacing and clustering pattern of gold nanoparticles (10 nm in diameter) on a glass surface with a single CD3 antibody molecule functionalized on each nanoparticle. This nanoarray allows to decouple lateral spacing (40–120 nm), cluster size (240–1440 nm diameter), the number, density (30–100 μm⁻²), and geometric distribution of ligands in each TCR cluster. TCR activation requires enough number of ligands in a cluster, thus the cluster size influences TCR ligation. Using the nanofabricated array decorated with nanoparticle-bound TCR ligands, Cai et al. found that clusters of as few as 3 ligands were able to activate TCR and a cluster of 37 ligands was optimal for TCR activation, and further increasing cluster size correlated with a relative decrease TCR activation efficiency. Moreover, Cai et al. reported that close packing of engaged TCR in clusters with smaller lateral ligand spacing (40 nm) and higher ligand density (100 μm⁻²) were more optimal for TCR activation, whereas spacing plays a far more dominant role in TCR triggering than does density (24). In another study, Matic et al. introduced a nanoarray platform to present CD3 antibodies immobilized on gold nanoparticles embedded in a PEG matrix with adjustable particle spacing (81). The group employed block copolymer micellar nanolithography, an advanced nanopatterning technique that utilizes the self-assembly properties of block copolymers (e.g., polystyrene-block-poly(2-vinylpyridine)) to create nanoscale patterns on surfaces. They found that the CD3-coated nanoarrays with 60 nm interparticle distance can best trigger CD69 upregulation, and costimulation with soluble CD28 antibodies was essential for eliciting cellular responses such as IL-2 secretion and T cell proliferation. Meanwhile, increasing interparticle distance to between 60 and 100 nm declined T cell responses, emphasizing the importance of controlled lateral spacing in designing ligand presentation.

To fabricate a 3D substrate with greater biocompatibility and resistance to bending, researchers have turned to DNA-based technologies such as DNA origami scaffolds. In agreement with the Cai et al. and Matic et al. studies, other studies using DNA-based surface engineering approaches also found that smaller ligand spacing and high-density ligand aggregation could promote a high degree of TCR aggregation, which would effectively activate downstream signaling pathways (25,37,81). For example, Sun et al. nanopatterned CD3ε antibodies simulating TCR and anti-CD28 antibodies simulating costimulatory ligands onto a rectangular DNA origami to interrogate the roles of TCR ligand spacing, the local copy number of TCR ligands, and the spacing between TCR ligands and costimulatory ligands on T cell activation (25). Their method involved a two-step process: first, antibodies were conjugated to single DNA strands using a cross-linker; then, these antibody-DNA conjugates were hybridized with preassembled single-stranded DNA origami structures at specific binding sites. By adjusting the ligand spacing between 16 and 96 nm, their results proved that locally concentrated TCR ligands with a smaller ligand spacing (16 nm) benefitted T cell activation, and a spacing of 38 nm, rather than 16 nm, between TCR ligands and costimulatory ligands was optimal for T cell activation. Consistent with the Cai et al. study, the group also found that T cell tyrosine phosphorylation level and proliferation maximize with three copy numbers of CD3ε ligands and then reached a plateau with increasing ligand density, indicating that even a few numbers of clustered high-affinity peptide-MHC complexes could initiate trigger TCR activation. Employing a similar DNA origami nanofabrication, Fang et al. showed that decorating DNA origami flat sheets with programmed death-1 ligand (PD-1L), an immunoinhibitory molecule targeting the PD1 receptor, with a minimum of 2 ligands spaced 200 nm apart, can suppress T cell activation (27). This finding indicates that spatial organization of ligands is crucial not only for TCR and costimulatory signaling, but also for other immunomodulatory receptors on the T cell surface.

Axial distance of the immunoreceptor-ligand interface determines activation threshold

As described in the kinetics-segregation model, the axial distance in the contact zone between T cells and antigen-presenting cells is a critical factor in T cell activation, where close proximity facilitates TCR-ligand binding and exclusion of CD45, an inhibitory enzyme that dephosphorylates signaling molecules (82,83). To validate this, Cai et al. designed 3D nanoparticle arrays with glass-etched pedestals to elevate each nanoparticle by 10 nm. This increased the axial distance between TCR and ligands from 13 to 23 nm, allowing the group to study the effects of axial distance on TCR activation (24). They found a more distinct lateral interreceptor spacing threshold of <50 nm for TCR triggering and a less-effective CD45 exclusion at 23 nm spacing compared with 13 nm. In another study, Du et al. utilized tetrahedral DNA nanostructures with different sizes by DNA self-assembly technology and anchored the DNA nanojunctions to cell membranes by cholesterol tags to shorten the intermembrane spacing between T cells and antigen-presenting cells down to 10 nm to maximize transmission of mechanical force for T cell activation (37). They found that DNA nanojunctions of a smaller axial distance (∼9.8 nm) enhanced T cell activation markers and phosphorylation signal intensity. Overall, T cell activation peaks at an axial distance of approximately 10–13 nm, with activation efficiency decreasing as this distance increases.

Dynamic ligand presentation and ligand mobility enhance TCR activation

While static ligand presentation remains the common approach in modulating TCR activation, dynamic ligand presentation systems with tunable ligand binding can offer a more physiologically relevant model that can adapt to the evolving needs of T cells throughout their activation and effector phases. These systems allow for controlled, tunable, and reversible interactions, closely resembling the dynamic nature of the cellular microenvironment. Wei et al. previously employed noncovalent host-guest polymer linkers to synthesize a mechanically dynamic matrix that induces recruitment and differentiation of human mesenchymal stem cells (28). This innovative method integrates a “host-guest macromer” system with 1) a host molecule (e.g., cyclodextrins), 2) a guest molecule (e.g., adamantane) that interact with the host, and 3) a polymer linker (e.g., hyaluronic acid) to connect the host-guest complexes to the hydrogel substrate. This system offers various advantages over static ligand presentation platforms, allowing for controlled, tunable, and reversible interactions by modifying the chemical structure of the host or guest molecule, mimicking the natural flexibility of the biological receptor-ligand interaction. The same host-guest system could be applied to design a biomimetic interface targeting TCR ligands and CAR ligands with tunable receptor force (31), where the force threshold could be determined by varying configuration of the synthesized guest molecules (84). A potential challenge in this approach is difficulty in selecting a host-guest pair that can reflect accurately the activation force in TCR-MHC and CAR-antigen complexes, while having a wide range of rupture forces for fine-tuning the mechanosensitive signaling in the TCRs. For example, TCRs require a precise range of forces (12–20 pN) for optimal activation (31), while for CARs, due to their recombinant antibody binding mechanism, need much higher forces (60–100 pN) (85). Utilizing systems with tunable rupture force, a ligand presentation platform can be designed for TCRs with distinct force requirements, as well as response to different ligands varying in binding affinity and the required mechanical force threshold.

Improving mobility of ligands on surface-engineered substrates can be very useful in giving ligands the ability to move or rearrange themselves on surfaces, thus better mimicking the mobility of ligands observed during stochastic membrane fluctuations (86). High-mobility ligands can effectively promote the centripetal movement of immunoreceptor microclusters, which enhances the formation of central supramolecular activation clusters, thus contributing to the intensity and duration of T cell activation (87,88,89). Dynamic modifications through, for example, the formation of self-assembled monolayers on surfaces (90) or supported lipid bilayers has been integrated into a nanoengineered platform (39,91). Substrate-based dynamic ligand presentation can also potentially be achieved via cell-response shape memory polymers, a class of smart “biomaterials” that can memorize a specific shape through covalent or physical cross-linking (92). As exerting compression is a hallmark of T cell cytotoxicity (43), shape memory polymers can be designed with a compatible stiffness to cancer cells and deform in response to mechanical force exerted by T cells. This membrane curvature change will induce ligand organization on the material surface and regulate T cell responses in a spatiotemporal manner.

Overall, by optimizing the spatial arrangement and mobility of ligands, combined with reversible molecular strategies for dynamic ligand presentation, the described nanoengineered platforms can better mimic natural ligation between immunoreceptor and ligand in a controlled, tunable, and reversible manner, promoting more efficient T cell activation and the corresponding signaling mechanisms including mechanotransduction.

Surface decoration of mechanomodulatory ligands for fine-tuning T cell mechanotransduction

Recent advancements in surface engineering have led to the development of sophisticated mechanochemical stimulatory platforms that can effectively modulate T cell mechanotransducive pathways, leveraging the unique properties of bioactive immunomodulatory materials such as synthetic peptides, receptors, and small molecules. Surface decoration of specific ligands on biomaterial-based platform, with a proper design principle, can mimic and enhance the natural mechanical and chemical cues that T cells encounter during activation and effector functions. Previous research successfully designed immunomodulatory molecules, most commonly cytokines, as cargo that can be decorated on the immune cell surface via cell backpacking (93,94) and metabolic labeling (95,96). Yet, presentation of immunomodulators on biomaterial platforms such as 2D/3D scaffolds, nano/microfabricated structures, and biofunctionalized particles offers a more efficient, localized, and precise approached compared with those in soluble form (97). Biomaterial-based approaches such as 2D scaffolds and 3D hydrogels is more biomimicking and able to replicate the complex spatial organization of ligands in the extracellular matrix (98,99), while particle-based approaches are utilized for their flexibility in biofunctionalization, size and shape design, and ease of delivery to cells in vitro and in vivo (100,101,102). Surface decoration of receptors, peptides, and small molecules on these platforms has shown great potential to efficiently activate or inhibit mechanosensitive receptors, adhesion molecules, mechanosensitive channels on cell membrane, as well as downstream mechanosensitive pathways regulating cytoskeletal dynamics and transcriptional regulators in T cells (Fig. 4; see Table 1 for a detailed summary).

Figure 4.

Surface engineering of mechanomodulatory ligands for fine-tuning T cell mechanotransduction toward efficient immunotherapy.

Table 1.

Immunomodulators for fine-tuning mechanotransduction

Ligand	Type	Target	Mechanotransduction pathways	Decoration platform	Ref.
RGD	peptide	integrin α5β1, α5β1 αVβ3/5/6	cell adhesion, focal adhesion assembly, activate Rac1-dependent cytoskeletal remodeling	gold nanoparticles, hydrogel, multisegmented nanorods	(105,106,108)
LDV	peptide	integrin α4β1		self-assembling peptide	(109)
Foxy5	synthetic peptide	Frizzled (Fz) receptors	activate noncanonical Wnt signaling, RhoA/ROCK signaling	PDMS, hyaluronic acid hydrogel	(72,111,112)
Jagged-1	synthetic peptide	Notch receptors	activate Notch signaling and subsequent canonical Wnt-β-catenin signaling	hyaluronic acid hydrogel	(113)
Cad	synthetic peptide	N-cadherin	cell-cell-mediated signaling	hyaluronic acid hydrogel	(114,115)
Yoda1/2	small molecules	Piezo1	activate Piezo1 channel	potential for immobilization on flexible substrates	(129,130,131)
Jedi1/2	small molecules	Piezo1	activate Piezo1 channel	potential for immobilization on flexible substrates	(129)
Margaric acid	fatty acids	Piezo1	inhibit Piezo1 activation	potential for lipid-based nanoparticles	(127)
Polyunsaturated fatty acids	fatty acids	Piezo1	modulate Piezo1 inactivation	potential for lipid-based nanoparticles	(127)
Gsmtx4	peptide	Piezo1	inhibit Piezo1 channel (nonselective)	potential for surface decoration	(132)
P10581	synthetic peptide	Piezo1			(133)
Tβ4	peptide	actin	inhibit actin polymerization	potential for surface decoration	(123)
PF-06446846	small molecule	PCSK9	activate the PI3K/AKT/mTOR pathway, metabolic reprogramming	nanoparticles in injectable hydrogel	(153)

Targeting adhesion molecule-based mechanotransduction

One of the most common strategies to modulate T cell mechanotransduction is using surface-decorated materials to target cell adhesion molecules. To date, ligand presentation methods targeting immune cells largely rely on Arg-Gly-Asp sequencing (RGD) binding to integrin α₅β₁ or α_Vβ_3/5/6, and Leu-Asp-Val (LDV) sequence binding to integrin α₄β₁ or VLA-4 to promote cell adhesion, focal adhesion assembly, and cytoskeleton organization (103,104). RGD-based dynamic ligand presentation methods have been applied previously in immunomodulation (105,106). Wang et al. pioneered the use of a catalyst (aniline) to mediate the dynamic presentation of RGD to mesenchymal stem cells (106). In their study, aniline-RGD motifs were conjugated to gold nanoparticles, which were tethered to a glass substrate via hydrazone bond. Aniline catalyzes the formation of breaking of hydrazone bonds, enhancing their dynamic nature and enabling the dynamic presentation of the RGD motifs. This catalyst-mediated RGD presentation regulates mechanosensation via Rac1-dependent cytoskeletal remodeling, enhancing stem cell metabolism and osteogenic differentiation.

Integrin ligand binding also depends on the presence of divalent cations such as Ca²⁺ and Mg²⁺, in whose presence integrins are usually in a low-affinity binding state (107). Kang et al. designed biomaterials with convertible cell-adhesive surface properties via ligand-cation dynamic coordination to regulate polarization of macrophages (105). In brief, the group utilized reversible Mg²⁺-bisphosphonate conjugated to RGD nanoparticles to promote attachment of macrophages and M2-type polarization. Addition of EDTA can chelate Mg²⁺, inducing detachment of macrophages, thereby offering a nontoxic and reversible method to direct macrophage attachment. For multiligand presentation of immunomodulators, multisegmented nanorods of 1–4 μm in length and 300 nm in diameter fabricated from nickel and gold were used to copresent RGD peptide and mannose in the respective metabolic section, allowing for heterogeneous surface chemistries (108). Presentation of different ligands, mannose carbohydrate ligand targeting dendritic cells, and RGD peptide ligand targeting T cells on distinct segments of the nanorod created a bridging system that can bring these cells into closer proximity, enhancing the efficiency of antigen presentation and subsequent T cell activation. Although less common in surface decoration, the LDV sequence is emerging as a targeting ligand of interest in T cell mechanomodulation. Jie et al. developed a multifunctional immune cell matrix scaffold fabricated from a self-assembling peptide FEFEFKFK (FEFK) covalently conjugated with LDV to accelerate CAR T cell proliferation via integrin-mediated mechanotransduction (109). The group has shown that a controlled molar ratio of LDV-FEFK ligand presentation (optimized ratio of 4:1), along with the presence of the immune checkpoint inhibitor PD-1L, on the nanofibrous hydrogel significantly improves CAR T cell phenotypes and their infiltration into the tumor site.

Besides adhesion molecules, mechanotransduction of T cells could also be regulated via mechanosensitive receptors and the downstream cytoskeletal signaling pathways. Synthetic peptides, artificially created short chains of amino acids that mimic fragments of larger proteins, or naturally occurring peptides, offer an interesting approach with precise control over the peptide sequence, ease of modification, biomimicry, and ease of conjugation to various biomaterials for T cell activation and expansion (110). For example, Li et al. employed a surface functionalization of Foxy5 (formyl-Met-Asp-Gly-Cys-Glu-Leu), a formylated hexapeptide derived from the sequence of the Wnt5a ligand, on elastic polydimethylsiloxane (PDMS) to enhance activation of CD8⁺ cytotoxic T cells (72). The presentation of Wnt5-mimetic peptide triggers noncanonical Wnt signaling and its downstream RhoA/ROCK signaling, supporting actomyosin contraction and the retrograde flow of F-actin during formation of immunological synapses between T cells and target cells. This amplification of synaptic force promotes the transcription of TCR activation-related cytokines and kinases, enhancing T cell cytotoxicity. Another study by Li et al. showed the possibility of Foxy5 on a methacrylate hyaluronic acid hydrogel activates noncanonical Wnt signaling in stem cells through the RhoA-ROCK pathway (111). This activation leads to increased calcium influx and enhanced actomyosin contractility, promoting osteogenic lineage commitment in stem cells. Interestingly, the Foxy5 peptide can be copresented with other immunomodulatory ligands via synthetic macromolecule-based nanomaterials. Specifically, Chen et al. developed a bottom-up method to prepare single-polymeric chain-based nanogels that allows for bioconjugation of multiple ligands with varying chemical structures by using different monomers and intrachain cross-linkers (112). They found that copresentation of Foxy5 and RGD provides a synergistic effect on different mechanosignaling pathways in stem cells.

Although choices of synthetic peptides for fine-tuning mechanotransduction in T cells remain limited, peptides have been successfully applied in other cell types and could be explored. Pioneer work in regenerative medicine has demonstrated the potential of hydrogel-mediated presentation of mechanosensitive motifs (113,114,115). For example, Jagged-1 mimetic ligands decorated on hyaluronic acid hydrogels have been shown to trigger mechanotransduction cascades that activate Notch signaling and subsequent canonical Wnt-β-catenin signaling, synergistically enhancing osteogenesis (113). In macrophages, Notch signaling via Wnt affects their polarization and remodeling of the tumor microenvironment (116,117). In T cells, activation of Notch signaling enhances antitumor responses and the memory capacity of cytotoxic T cells, as demonstrated in both in vitro and in vivo studies (118,119). Notably, recent advancements in next-generation synthetic Notch-CAR T cells have illuminated the mechanosensitive nature of the Notch pathway (120). Upon antigen recognition, mechanical forces trigger the cleavage of the intracellular domain of the synthetic Notch receptor, activating the transcription factor that initiates the expression of the CAR constructs (121). Thus, the surface presentation of Jagged-1 holds significant potential to enhance the Notch-mediated mechanotransduction and antitumor responses in T cells. N-Cadherin mimetic peptides (Cad) functionalized onto hyaluronic acid hydrogel backbones successfully promoted osteogenesis and chondrogenesis in mesenchymal stem cells through cell-cell-mediated signaling (114,115). N-Cadherin controls spindle alignment in murine T cells (122), and the interplay of Piezo1, integrin, and cadherin were implicated in mechanosensation of glioblastoma cancer cells (56). Therefore, the Cad peptide remains an interesting mechanostimulatory candidate to explore in T cells. In addition, Lee et al. showed that stimulation of soluble thymosin β (Tβ4), an actin sequestering peptide, regulates actin polymerization and increased cytotoxicity of natural killer cells (123). Manipulation of actin polymerization by Tβ4 could potentially be utilized for directed migration and homing of T cells at tumor sites.

Targeting mechanosensitive ion channels

To date, strategies for targeting mechanosensitive channels, especially Piezo1, through ligand presentation are limited, with minimal research exploring this direction. Various current studies have revealed the roles of mechanosensitive ion channels, especially Piezo1, in immune cell mechanotransduction. Previous studies have shown that mechanical or chemical regulation of Piezo1 induces a calcium-dependent activation of TCR, extensively influencing the cytoskeletal dynamics, and therefore may substantially regulate the force and function outcomes of CAR T cells (124,125). Small molecules such as Yoda1/2 and Jedi1/2 have been used to chemically activate Piezo1, while GsMTx-4 peptide, P10581 (GsMTx4-based short synthetic peptide), margaric acid, and polyunsaturated fatty acids have been employed to inhibit Piezo1 activation and modulate its inactivation (126,127,128,129,130,131,132,133). Although these compounds interact with Piezo1, they were not designed as targeting ligands nor decorated on substrates. Although Piezo1 channels have been identified as a key regulator of mechanotransduction in T cells, there has been limited application of Piezo1-targeting ligands for surface decoration in T cell-based therapies, despite growing evidence that mechanosensitive pathways can enhance immune cell activation. Therefore, exploring new mechanomodulatory ligands, including those targeting Piezo1 and other mechanosensors, is critically needed to fully harness the potential of mechanical cues in optimizing T cell immunotherapy.

Since Piezo1 channels play both mechanostimulatory and inhibitory roles among cell types, decorating these small molecules and peptides on biomaterial-based substrates could fine-tune Piezo1 in a more localized and controlled manner (58,59,67,132,134,135). For example, as margaric acid and polyunsaturated fatty acids are lipids, they could be presented on lipid-based nanoparticles to modulate Piezo1 signaling pathways (136). The surface of the nanoparticles can be functionalized by targeting ligands such as RGD or LDV to enhance their specificity for T cells, improving the localization of Piezo1 modulation (137,138). Another approach to target Piezo1 mechanosensation involves combining ligand-decorated strategies with external stimulations. Luu et al. utilized RGD-integrin interactions to conjugate lipid microbubbles to the membrane of vascular smooth muscle cells and applied ultrasound stimulation (55). This strategy effectively activated Piezo1 channels, inducing intracellular calcium influx and regulating mechanosensitivity of vascular smooth muscle cells. Similarly, Pan et al. employed RGD-tagged microbubbles to engage with integrins on T cells as a platform to mechanically activate Piezo1 channels, converting activated Piezo1 into transcriptional NFAT activities (15).

An alternative strategy could involve immobilizing Piezo1-activating ligands (like Yoda1/2 or Jedi1/2) on nanoengineered and smart biomaterial-based substrates. Cells grown on these substrates could experience mechanical stimulation through substrate modification, while the immobilized ligands ensure specific targeting of mechanotransduction. Tabdanov et al. engineered a “2.5D” nanotextured polyacrylamide gel surfaces that can enhance T cell 3D migration in the tumor microenvironment (139). Submillimeter-thick hard PDMS stamps functionalized with ICAM1 were used to nanoprint the substrate surface with defined geometric patterns including ridges and grooves to regulate spreading and protrusion of T cells. These topographical cues induce local changes in membrane curvature and tension, mediating RhoA activities and cortical contractility. As Piezo1 is the direct membrane tension sensor, nanopatterning and micropatterning surfaces can fine-tune the sensitivity of Piezo1 channels. In addition, surface presentation in combination with "smart" biomaterials, such as piezoelectric scaffolds, offers promising multimodal stimulation approaches. These methods, although not widely explored, utilize mechanical cues with electrical stimulation to create noninvasive and controllable effects on T cell activation and function. Piezoelectric materials can convert electrical stimulation into mechanical deformation and vice versa. Das et al. and Vinikoor et al. successfully employed a piezoelectric poly(L) lactic scaffold and hydrogel (PLLA) stimulated with low-frequency ultrasound to induce mechanotransduction of fibroblasts epithelial cells, and adipose-derived stem cells via a calcium-dependent pathway (140,141,142). Although not investigated in piezoelectric properties, Piezo1 channels were found to contribute to ultrasound modulation and calcium-induced transcriptional activities in Jurkat T cells and neurons (15,143). In response to ultrasound stimulation, immunomodulators decorated on piezoelectric platforms thus can trigger T cell multifaceted mechanotransduction involving multiple mechanosignaling pathways cooperating to amplify the force needed to exert anticancer responses.

Strategies to modulate T cell response in vivo has been applied previously to antitumor therapy via injectable hydrogel and biofunctionalized particle delivery, yet strategies to target T cell mechanotransduction is very limited because of the difficulties in activating a certain mechanosignaling pathway in a controlled and localized manner (144,145). The ability to mechanically manipulate T cell behaviors in vivo is thus a concern to address for clinical applications. To address this drawback, controlled targeting of Piezo1 signaling by only applying mechanical stimulation when T cell enter the tumor sites can reduce the on-target off-tumor effect observed in T cells and CAR T cells targeting solid tumor in vivo (146). This electromechanosensitive pathway is also a new mechanotransduction mechanism that could further be explored in the future for fine-tuning mechanosignaling of T cells.

Targeting cellular metabolism

Targeting mechanotransduction via enhancing cellular metabolism offers an interesting approach in immunomodulation of T cells. Metabolism, the sum of biochemical processes that produce or consume energy, plays an important role in T cell immunity (147). T cell metabolic programming significantly increased upon antigen engagement to promote T cell proliferation, effector responses, and differentiation into memory phenotype (148,149). The cross talk between cellular metabolism and mechanotransducive signaling has emerged as a topic of interest in recent years, has emerged as an important area of study, highlighting how the mechanical properties of the cell’s microenvironment can regulate metabolism, and how specific metabolic traits can influence a cell’s mechanical behavior. For example, Hu et al. discovered that the actin cytoskeleton regulates glucose metabolism by sequestering the glycolytic enzyme aldolase, which is released during actin turnover mediated by Rac1, enhancing glycolysis via downstream of PI3K activity (150). Wang et al. identified energy landscapes as the biophysical foundation of cellular adaptation to mechanical cues, showing how energy-driven changes in cytoskeletal dynamics enable cells to sense and respond to mechanical stimulation, such as ultrasound tweezers (151). Notably, there is growing evidence that T cell metabolic reprogramming is linked to YAP mechanosensation (9), and E-cadherin-mediated mechanotransduction in T cells is mediated by force-activated AMPK-mTOR pathways (152). For surface presentation of ligands, Zhang et al. designed an injectable hydrogel that can be constructed at tumor sites using oxidized sodium alginate-alginate-modified tumor cell membrane vesicles, in combination with surface-decorated nanoparticles as a gelator (153). Specifically, the small-molecule PF-06446846, a protein convertase subtilisin/kexin type 9 inhibitor, was formulated into nanoparticles of 195 nm using a nanoprecipitation method. Integration of PF-06446846-coated nanoparticles promoted T cell mitochondrial biogenesis, and reduced hypoxia with axitinib can reverse T cell exhaustion via the PI3K/AKT/mTOR pathway. Although not widely explored, utilizing the cross-bridge of metabolism and mechanotransduction in T cells can expand the horizons of immunomodulatory ligand selection for surface decoration strategies.

Conclusion and perspective

To date, ligand-based surface engineering strategies for fine-tuning T cell mechanotransduction remain an underexplored direction in immunotherapy. The previous sections in this review summarize the current efforts in decorating immunomodulatory ligands on nanoengineered and biomaterial-based platforms, as well as highlighting the tremendous potential for further improvements. The future of mechanoimmunoengineering for T cell modulation should focus on two key aspects: exploration of new mechanomodulatory ligands targeting T cells and improving biomimicry of surface decoration platforms regarding the selection of biomaterials and surface engineering techniques.

High-throughput screening of synthetic peptides can be applied to identify more ligands that are relevant to T cell mechanotransduction. Recent advancements in synthetic biology have opened avenues to enhance control over mechanical responses of T cells via designing artificial mechanosensitive receptors that can be genetically engineered into T cells and CAR T cells. For example, an abbreviated Piezo1 protein that is significantly smaller in size compared with the full-length Piezo1 proteins, but meticulously preserves the critical mechanosensitive function of Piezo1, could be synthesized in T cells of dysfunctional mechanophenotype to optimize CAR T cell mechanosensitivity and improve their ability to exert and transduce mechanical forces, leading to more effective and durable anticancer responses. Exploring new mechanosignaling pathways in T cells also broadens the horizon of mechanostimulatory ligand selection. Large-scale transcriptomic and proteomic analyses such as bulk-RNA sequencing, single-cell RNA sequencing, and DNA nanotechnologies can identify the alterations in mechanoregulatory pathways and the enriched genes associated with T cell cytotoxicity. Further deciphering the interplay between mechanical signaling pathways and traditional immunological signaling cascades, such as cellular metabolism and cytokine secretion, may reveal synergistic targets for disease intervention.

Surface decoration approaches also require further refinement. Nanoengineered techniques, including nanolithography, nanoarrays, and DNA origami can provide precise spatial control of ligand presentation, yet the substrates are mostly limited to 2D. While hydrogel-based substrates offer 3D homing of cells with tunable mechanical properties, controlled patterning remains technically challenging. Further advanced surface modification techniques that can combine both the precise control over spatial organization of ligands and the biocompatibility of 3D hydrogels are in need. Developing systems for dynamic ligand presentation based on reversible noncovalent binding chemistry that respond to cellular cues or external stimuli could offer spatiotemporal control over TCR activation and downstream mechanoresponse. Multivalent ligands that simultaneously present both mechanomodulators and adhesion binding motifs represent an exciting frontier in this field. Furthermore, reversible molecular strategies can also introduce bioactive molecules on biomaterial surfaces, mimicking dynamic bioligands in the T cell microenvironment. Various bioactive ligands (antibodies, peptides, cell-binding small molecules) can be dynamically linked to surfaces using reversible molecular interactions. External stimuli (light, temperature, pH, electrical, enzymatic) can trigger changes in these reversible molecular linkers, altering bioligand accessibility and cell behaviors.

Translating these ligand decoration approaches into clinical applications presents both challenges and opportunities for the mechanoimmunoengineering field. Comprehensive preclinical studies are necessary to ensure the safety and efficacy of engineered surfaces on T cell function in vivo. The choice of biomaterial platform needs to ensure proper biocompatibility and biodegradability when transplanted into patients. In CAR T cell therapy, monitoring long-term effects of ligand-decorating strategies on patient responses, notably cytokine release syndrome and early CAR T cell exhaustion due to robust chronic activation. Developing cost-effective, scalable manufacturing processes for mechanically engineered T cells or materials is crucial for clinical viability. This is a current challenge in nanoengineering methods that often require expensive equipment, complex techniques, and highly specialized personnel.

The principles of mechanomodulatory ligand decoration have potential applications beyond cytotoxic anticancer responses of T cells. These approaches could be applied to modulate T cell development, differentiation, homing, and migration. In the broader context of T cell immunotherapy, modulation of T cell mechanobiology can also benefit therapeutic intervention in autoimmune diseases, chronic infections, and vaccine development. For instance, engineering surfaces that promote regulatory T cell function could induce tolerance in organ transplantation, while optimizing mechanical activation could enhance T cell responses against chronic infections such as HIV, hepatitis C, or SARS-CoV-2 infection. Applications of ligand decoration have been and can further be improved and modified for targeting other immune cells. Exposing macrophages to mechanical cues or mechanostimulatory ligands can induce their polarization into either proinflammatory or antiinflammatory phenotypes in appropriate scenarios. Enhancing the mechanical environment of dendritic cells could improve antigen presentation and dynamic interaction with T cells. Investigating how mechanical forces influence B cell activation and antibody production in the lymph nodes and lymphatic tissues could lead to improvements in vaccine efficacy. In conclusion, the convergence of biophysics, biomaterials, mechanobiology, and immunology is pushing the frontiers of T cell immunology research, giving confidence and hope for the understanding and personalized application of T cell immunotherapy.

Acknowledgments

This work was supported by the National Institutes of Health (R35GM133646) and the American Heart Association (24PRE1241970, 24IAUST1187087). All figures were created with BioRender.com.

Declaration of interests

The authors declare no competing interests.

Editor: Jeremiah Zartman.