In principle, adoptive cell therapy (ACT) requires T cell activation, which is governed by three signals (Figure 2B) (Huppa and Mark, 2003): 1) an essential stimulation signal by specific antigen combined with the TCR/CD3 complex for initial activation; 2) the co-stimulatory signal by CD28 for triggering T cell proliferation; and 3) autocrine or paracrine cytokines for T cell differentiation. Optimized conditions are required for each step to achieve the best therapeutic outcome (Waldman et al., 2020). While T cell activation has been known as a purely biochemical process, recent evidence suggests that mechanical forces can significantly enhance T cell activation by causing allosteric effects in the TCR/CD3 complex, which acts as a mechanosensor for T cells (Liu et al., 2014). The TCR can bear 12–19 pN of force in the time of encountering with antigen peptide-bound major histocompatibility complex (pMHC) (Liu et al., 2016; Ma et al., 2019), contributing to the specific sensitivity response of TCR triggering, and the discrimination to agonist quality and affinity (Wu et al., 2019). The simplified pattern of force-dependent TCR signaling is shown in Figure 3.

The TCR plays a critical role as a mechanosensitive receptor of T cell within the early stage of triggering activation, relying on the intracellular actin cytoskeleton for proper functions (Hu et al., 2016; Butte et al., 2017). Through the TCR, T cells are able to sense external mechanical properties such as substrate stiffness and spatial arrangement of ligands (Zhu et al., 2019). Interestingly, TCR exhibits a biphasic response to substrate stiffness, which is distinct from other well-known mechanosensitive receptors like integrin (Wahl et al., 2019; Yuan et al., 2021). Upon mechanical activation of the TCR, cytoskeletal forces are generated via polymerization and rearrangement of actin, and retrograde actin flow mediated by myosin-II (Hui et al., 2015; Hu and Butte, 2016), allowing for the connection between T cells and their environment on account of cytoskeletal forces transmitting to antigen-presenting cells (APCs) or target cells via the transmembrane domain of TCR (Judokusumo et al., 2012; Colin-York et al., 2019). While inhibition of the cytoskeletal forces leads to restraint of T cell activation occurrences, such as sharp reduction of interleukin-2 (IL-2) production. Additionally, recent studies have shown that the exertion of mechanical forces against cancer cells by cytotoxic T lymphocytes (CTLs) through immunological synapse, promote killing to cancer cells via enhancing pore formation induced by the perforin protein into the membrane of cancer cells (Basu et al., 2016). Furthermore, the membrane protrusions of CTLs at F-actin-rich regions have been demonstrated to be essential for synaptic mechanical force output and efficient cytotoxicity against target cells (Huse, 2019; Jin et al., 2019). T cell mechanical signals are force-dependent triggered by TCR/CD3 complexes upon binding to pMHCs or anti-CD3 antibodies (Hu et al., 2016). Recently, Tang’s group developed a force-triggered system that is responsive to cellular force of T cell for drugs delivery, releasing the drug upon TCR triggered (Lei and Tang, 2020). These anticancer drugs delivery design based on T cell mechanical signal has been shown to significantly potentiate the destruction to cancer cells both in vitro and vivo, demonstrating the potential for T cell mechanical force that can coordinate to controlled drugs delivery by precisely releasing of desired molecules for enhanced therapeutic efficacy. These evidences indicate that mechanical forces are crucial for not only T cell activation, but also for T cell mediated cytotoxicity.

Integrins play a critical role in regulating cell proliferation, adhesion, and migration by transmitting mechanical and chemical signals between extracellular environment and the cell interior (Saraswathibhatla et al., 2023). Upon binding to the ligands in extracellular matrix (ECM), force generation promotes integrin activation and integrin clusters forming, which can allow translation of mechanical force to the cytoskeleton via a series of mechanical sensitive adaptor proteins (Kechagia et al., 2019). These effects induce the rearrangements of actin cytoskeleton and the formation of focal adhesions, such as actin bundling and stress fibres. These rearrangements, in turn, activates intracellular signalling pathways (Cosgrove et al., 2016). Integrins also have vital function in cell migration, with integrin-mediated molecular clutch mechanisms proposed to explain the relationship between cell migration speeds and ECM rigidity in haptotaxis and durotaxis (Bangasser et al., 2017; Isomursu et al., 2022). Recent studies have identified integrin signaling as a key driver of multiple cell functions, including stem cell differentiation (Zhang C et al., 2021; Zhang et al., 2022), tumor initiation, epithelial plasticity, metastatic reactivation, and immune-targeted therapies, etc. (Pang et al., 2023). Integrins are also as force sensors that actively modulate T cells effector functions (Comrie et al., 2015b). For instance, in response to high substrate rigidity, integrin lymphocyte function-associated antigen-1 (LFA-1) undergoes a conformational change which can improve the affinity with immobilized intercellular adhesion molecule-1 (ICAM-1), facilitating T cell adhesion and priming (Li et al., 2009). This process occurs during dendritic cells (DCs) maturation (Comrie et al., 2015a). Recent studies show that LFA-1/ICAM-1 interactions can facilitate close and physically active contact between the CLTs and target cells, resulting in enhanced degradation and cytotoxic secretion, thus contributing to the potency and specificity of killing target cells (Wang et al., 2022).

Recent research has shown that RGD immobilized on stiff substrates enhances integrin-mediated signaling from the ECM, facilitating F-actin assembly and actomyosin contractility, leading to cytoskeletal tension, and nuclear localization of Yea-associated protein (YAP), as a mechanotransducer based on mechanical signals of extracellular matrix (Vining and David, 2017). Similar stiffness-mediated cellular localization of YAP has exposed in T cells (Elosegui-Artola et al., 2017). Phosphorylated YAP plays a role in modulating T cell activation based on matrix stiffness by regulating the binding affinity between IQGAP1 (Ras GTPase-activating-like protein) and NFAT-1 (nuclear factor of activated T cells-1), affecting the T cell activation and metabolism by promoting IL-2 secretion (Meng et al., 2020). YAP stabilizes interactions of IQGAP1 and NFAT-1 in the cytoplasm by suppressing NFAT-1 from nuclear localization in response to low ECM rigidity via T cell adhesion to RGD that is immobilized on hydrogels, attenuating metabolic reprogramming and T cell activation. In contrast, on high ECM rigidity, most cytoplasmic YAP translocates into the nucleus, thus promoting NFAT-1 transportation into the nuclear and upregulating proliferation and metabolism. Therefore, YAP mediates the restriction of T cell responses, once the mechanical environment transitions from high rigidity (inflammation) to low rigidity (baseline) (Figure 4). This stiffness-dependent suppression also preserves healthy tissues from attacking by T cell-mediated autoimmunity. Overall, this “mechanical checkpoint” behavior-mediated the special mechanical properties of T cells may present a novel anchor for engineering optimal T cell effector responses.

T cells upregulate the expression of co-inhibitory receptors, also called as immune checkpoints, on their membrane when exposed to the tumor microenvironment. These immunosuppressive receptors regulate T cell immunity negatively, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and mucin domain-3 (TIM-3), etc., which can reduce T cell activity and inhibit their ability to proliferate and produce cytokines (Wherry and Kurachi, 2015). These inhibitory signals present significant challenges for ACT (Curdy et al., 2019). Recent findings suggest a possible association between immune checkpoints and mechanotransduction. Salaita et al. reported a DNA probe that can preserve the mechanical information generated by T cells and reveal the role of mechanical force in modulating the immune checkpoint. Additionally, their research revealed that T cells exert force on anti-PD-1 antibody, through the PD-1 receptor (Ma et al., 2019).

Immune checkpoint inhibitors (ICIs) can conquer the inhibitory signals to re-activate exhausted T cells (Rumpret et al., 2020). Monoclonal antibodies (mAbs) targeting PD-1/PD-L1 (Programmed cell death 1 ligand 1) have remarkably innovated cancer therapy through immune checkpoint blockade. Chen et al. developed an ultra-stable bio-membrane force probe and benchmarked the dissociation kinetics of three clinically approved PD-1 blockade mAbs (An et al., 2020), which correlated well with the objective response rates in cancer clinical treatment. This platform potentiates the screening, optimization, and clinical selection of therapeutic antibodies in the future. Moreover, a recent study revealed that cancer cells deplete CD80 and attenuate T cell activation by utilizing force-dependent trans-endocytosis. Forces induced by cancer cells are transmitted through the binding of CTLA-4 and CD80 (Park et al., 2020). The generation of inhibiting force in cancer cells could facilitate the activating capacity of APCs to T cells (Oh et al., 2020; Peng et al., 2020). This may inspire a new strategy to rescue anti-tumor immunity through inhibiting force-generation. However, further research is required to explore the mechanics of force-responded immune checkpoints.

Mechanotransduction refers to the conversion of mechanical signals into biochemical signals that activate cellular pathways and regulate cell function. T cells, in particular, heavily rely on rearrangement in their cytoskeletal structures in response to mechanical cues for functions such as activation, immunological synapse formation, immune reactivity, and migration. Understanding mechanosensing pathways and actin function in T cells could offer the potential to predict and guide clinical applications. As researchers sheds more light on nuclear actin (Irvine et al., 2021), there is growing interest in modulating T cell activation and differentiation as well as enhancing or restraining the immune response through considering actin dynamics.

The mechanical properties of tissues play a critical role in modulating immune function, and the diseased tissues tend to suffer from diverse ECM properties in contrast to the healthy tissues. For instance, in vivo mouse models, the stiffness of sentinel lymph nodes in inflamed tissue becomes up to 40 kPa, which is stiffer than normal lymph nodes (only about 4 kPa) (Meng et al., 2020). Such changes in mechanics of lymph node tissue have been demonstrated to affect T cell activation, with T cells exhibiting increased activation on stiffer ECM, which is mediated by YAP mechanosensing (Figure 4). To better understand the impact of mechanical properties on T cell activation and differentiation, researchers have been employed 2D hydrogels with varying stiffness (Yuan et al., 2021). These studies have shown that TCR and LFA-1have differential contributions to T cell spreading on different stiffness, with TCR regulating a biphasic response, and LFA-1 showing a monotonous response upon co-engagement. These results indicate that T cells distinguish from different substrate stiffnesses in vivo and adjust their behaviors correspondingly.

Efforts have been devoted to leverage the stiffness responsiveness of T cells to improve cancer immunotherapy. For instance, researchers have designed an artificial T cell stimulating matrix (aTM) hydrogel based on ECM materials with mutable stiffness and T cell stimulation signals. The aTM has been shown to potentiate mechanically sensitive TCR signaling, resulting in a rapid and robust expansion of T cells and effective immunotherapy (Hickey et al., 2019). This approach not only holds immediate immunotherapeutic applications, but also provides a roadmap for the future engineering ECM-simulate materials for therapeutic T cell activation. Overall, these findings underscore the significance of understanding the impact of tissue mechanics on immune function and suggest that engineering tissue-simulate materials can offer a promising strategy for therapeutic immune modulation.

Additional studies have revealed the cellular rigidity acts a significant role in modulating T cell responses through cell-cell interactions. As the major professional APCs, DCs are particularly effective in priming T cell responses. Actin rearrangements in both DCs and T cells characterize the DC-T cell immunological synapse (Leithner et al., 2021). Actin rearrangements through Wiskott-Aldrich syndrome protein (WASp) and Hem1 containing WASp-family verprolin-homologous protein (WAVE) complexes promote the appearance of podosomes on the DC side, which are thought to increase rigidity and promote maintenance of cell contact, thereby enhancing T cell priming and activation (Figure 5A) (Malinova et al., 2015).

The malignant and progression of tumors have been found to be associated with increased cancer cells rigidity. Researches have shown that cancer cells cultured on stiff substrates are more vulnerable to cytotoxicity of CTLs, and T cells exhibit greater activity coculturing with the target cells which are grown on stiffer substrates, indicating that the biomechanics of target cells can affect T cell immunotherapies (Basu et al., 2016). Furthermore, increasing cellular rigidity of cancer cells through overexpression of the myocardin related transcription factor (MRTF) have been found to reduce their metastatic colonization and enhance responsiveness to CTL-mediated killing (Figure 5B) (Tello-Lafoz et al., 2021).

Similarly, Tang et al. demonstrated that cholesterol depletion-induced cancer-cell stiffening increased T cell cytotoxicity and improved the efficacy of adoptive T cell therapy against solid tumors in mice. This was due to increased T cell forces caused by the constantly amass of filamentous actin in immune synapse (Figure 5B) (Lei et al., 2021). However, softening cell prevents cytolytic T cell killing of tumor-repopulating cells (TRC) (Liu et al., 2021). The softness of TRCs prevents the adequate generation of contractile force for perforin pore formation, as the interaction of perforin with non-muscle myosin heavy-chain 9 in soft TRCs results in reduction of F-actin. Consequently, perforin released by the TRC softness is unable to create membrane pores, leading to attenuated cytotoxicity and increased immune evasion. This process of T cells sensing biomechanical vulnerability is called “mechanical immunosurveillance”, which reveals a potential role of biomechanical properties in T cell immunity and provide a primary direction for mechanical immune engineering of T cells.

Optimizing T cell activation and expansion in vitro is an important therapeutic approach toward for enhancing immune response to specific antigens (Eggermont et al., 2014). As such, the development of engineered surfaces for optimizing the activation and expansion of T cells are crucial (Babensee et al., 2015). The geometry of artificial antigen-presenting cells (aAPCs) acts a key role in modulating the effective binding of ligands with T cell membrane by triggering more contact area and frequency. For example, it has been shown to promote higher proliferation of cytotoxic T cells in vivo and ex vitro by stimulated with ellipsoidal aAPCs, compared to their spherical counterparts, even at similar antigen density (Meyer et al., 2015).

Another promising approach for T cells expansion involves attaching T cell ligands to the artificial substrates with high surface area. For instance, bundled carbon nanotubes can provide a high density of surface stimulation, promoting generation of abundant cytotoxic T cells (Fadel et al., 2014). In this case, the proliferation of cytotoxic T cells was significantly improved by controlling the release of IL-2 from these nanotubes. Additionally, the nanoparticles with stimulating antibodies to CD3 and CD28, have shown promising in efficient TCR activation and expansion of T cells (Mi et al., 2018). Overall, these findings demonstrate the critical role of aAPC geometry in governing the contact surface area between aAPCs and T cells to enhance cancer immunotherapy. Therefore, the development of aAPCs with optimized geometry and surface area of contact holds great promise for improving T cell activation and expansion.

Additionally, the spatial arrangement of TCR complexes is critical for TCR triggering, as has been evidenced (Mossman et al., 2005; Cai et al., 2018). The spatial arrangement of TCRs is the confirmation of their response to stimulus signals (Dustin and Groves, 2012; Goyette et al., 2019). To investigate the connection between transverse ligand spacing and T cell activation, researchers have applied ligand nanopatterning techniques. For instance, Spatz’s group found that the antibodies to CD3 had a transverse spacing of less than 60 nm for optimal T cell activation, but the transverse spacing of pMHC was up to 150 nm, indicating that a spatial requirement is being for TCR priming (Deeg et al., 2013; Matic et al., 2013).

The performance of artificial substrates loading with nanopatterned antibodies and optimized transverse spacing have surpassed traditional dynabeads, indicating that ligand patterning may be a useful tool for engineering more efficient aAPCs for in vitro expansion of T cell (Guasch et al., 2018). This is supported by the Kinetic-Segregation (KS) model, which mentions the limitation of TCR ligands at the nanoscale is essential to the triggering of T cell activation (Choudhuri et al., 2005). Additionally, in the KS model, the phosphorylation and dephosphorylation of zet-chain-associated protein kinase of 70 (Zap-70), an key kinase of TCR signal downstream, are equilibrated by lymphocyte-specific protein tyrosine kinase (LCK) and CD45, respectively, at the resting state (Davis and van der Merwe, 2006). Thus, upon TCRs/pMHCs engagement, the intermembrane distance is limited to approximately 13 nm, which physically excludes the proximity of bulky CD45, leading to elevated phosphorylation of Zap-70 and T cell triggering (Carbone et al., 2017). Notably, the transverse spacing under 50 nm (the actual spacing was 40 nm) rescued the inhibition of T cell triggering, even with the nano-pedestal pattern, indicating that the physical separation of CD45 from the TCR-ligand complexes needs the transverse TCR clustering with less than 50 nm spacing to trigger T cell responses (Figure 6A) (Cai et al., 2018). This model demonstrates a vital parameter for engineering aAPCs to enhance T cell behavior. The TCR bending mechanosignal (TBM) model propose that localized T cell membrane bending at the nanoscale makes constant binding between pMHC and TCR to formulate small complexes which distribute among large surface molecules (CD45) and adhesion molecules (LFA-1) on T cells (Figure 6B) (Al-Aghbar et al., 2022). The TBM model suggests that immunomodulators capable of altering the softness of T cell membranes, such as cholesterol-derived and lipid-soluble compounds which alter lipid composition under aging and pathological conditions, and could lead to new designs for adoptive T cell therapy.

The approaches currently used for activating and expanding T cells ex vivo, which take into consideration the T cell behaviors to the surface mechanical properties of materials, such as stiffness, topography and ligand presentation, as summarized in Table 2.