Mechanical communication and function regulation of immune cells.

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Learn more: PMC Disclaimer | PMC Copyright Notice Fundam Res . 2024 Apr 12;6(2):672–684. doi: 10.1016/j.fmre.2024.04.008 Search in PMC Search in PubMed View in NLM Catalog Add to search Mechanical communication and function regulation of immune cells Chen Yang Chen Yang a School of Mechanical Engineering & Automation, Beihang University, Beijing 100191, China b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China Find articles by Chen Yang a, b , Ruipei Xie Ruipei Xie b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China Find articles by Ruipei Xie b , Ting Cao Ting Cao c The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Find articles by Ting Cao c , Yiyu Zhang Yiyu Zhang b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China Find articles by Yiyu Zhang b , Xiaochen Wang Xiaochen Wang b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China d Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325000, China Find articles by Xiaochen Wang b, d, ⁎ , Ye Xu Ye Xu a School of Mechanical Engineering & Automation, Beihang University, Beijing 100191, China Find articles by Ye Xu a, ⁎ , Qihui Fan Qihui Fan b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China Find articles by Qihui Fan b, ⁎ , Fangfu Ye Fangfu Ye b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China d Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325000, China Find articles by Fangfu Ye b, d, ⁎ Author information Article notes Copyright and License information a School of Mechanical Engineering & Automation, Beihang University, Beijing 100191, China b Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China c The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China d Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325000, China ⁎ Corresponding authors. [email protected] [email protected] [email protected] [email protected] Received 2023 Sep 28; Revised 2024 Feb 7; Accepted 2024 Apr 3; Collection date 2026 Mar. © 2024 The Authors. Publishing Services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PMC Copyright notice PMCID: PMC13069623  PMID: 41971825 Abstract As the pivotal components of the immune system, immune cells can migrate rapidly and with flexibility to fulfill their critical functions. The cytoskeleton and mechano-sensitive proteins anchored within them play a crucial role in mechano-transduction, facilitating the conversion of mechanical signals into responses within the immune cells when they encounter external stimuli. In recent years, an increasing number of studies have revealed that mechanical signals play important roles in the immune response. This article reviews advanced techniques for analyzing cellular-level mechanical forces and provides insights into the current understanding of mechanical signals in the immune system. It describes how mechano-sensitive proteins collaborate to mediate mechano-transduction and examines the profound impact of mechanical signals on immune cell functions. This review highlights the growing importance of mechano-biology in the immune system and offers a glimpse of exciting future developments in this emerging field. Keywords: Mechano-transduction, Micro-environment, Mechanical cytoskeleton, Mechano-sensitive proteins, Immune cells 1. Introduction The life activities of cells are strictly controlled by extracellular and intracellular signals. In the immune system, signal transmission is the key foundation of activation and dynamic regulation of the immune response. The loss or interruption of signaling leads to immune dysfunction, which is associated with immune-related illness. In the past decades, it has been demonstrated that many immune cell functions, including cell growth, proliferation, differentiation, migration, and activation, are mediated by bio-chemical signals [ [1] , [2] , [3] , [4] , [5] , [6] ]. Various studies have revealed how bio-chemical signals are transmitted inwards and outwards. The involved bio-chemical signaling pathways include but are not limited to the initial activation by the combination of ligands and cell receptors, downstream spreading of messengers, dynamic feedback of relative effectors, and genic regulation by nuclei. However, the importance of physical signals in cell communication has not been adequately emphasized compared to traditional bio-chemical signals. As patrollers, immune cells adhere to blood vessels, squeeze into tissues, migrate through the interstitial matrix, and form dynamic contacting surfaces with target cells. These processes are tightly related to physical rules. Various types of immune cells, such as lymphocytes, monocytes, macrophages, and neutrophils, are co-stimulated by both bio-chemical and mechanical signals during patrolling [ [7] , [8] , [9] , [10] ]. In addition to traditional bio-chemical signals, mechanical stimuli ( e.g. , flow shear, stiffness, and topography) from the extracellular environment can significantly influence immune cell behaviors [ 10 , 11 ]. The cortical cytoskeleton, which is now recognized as the mechano-transduction bridge, allows immune cells to sense physical changes in the micro-environment through cytoskeleton-anchored mechano-sensitive proteins, such as integrin, selectin, cadherin, and T/B cell receptor (TCR/BCR) [ 7 , 12 ]. On the other hand, myosin Ⅱ, which is a type of molecular motor, can also dynamically re-organize the actin filaments, which generate myosin-based cytoskeletal forces and transmit mechanical signals to the extracellular environment [ [13] , [14] , [15] , [16] , [17] ]. Immune cells translate physical stimuli into bio-chemical signals through a series of mechano-transduction signaling networks and exert cellular forces on the micro-environment, thus setting up a mechanical communication mode that plays a crucial role in the immune system. As research findings in this field continue to accumulate, mechano-transduction has emerged as an attractive research topic. A growing number of studies have explored numerous fundamental biological processes that are regulated by mechanical signals, such as correlated cell migration [ 18 ], cardiac beating [ 19 , 20 ], vascular development [ [21] , [22] , [23] ], blood homeostasis [ 24 , 25 ], proprioception [ 26 , 27 ], and many immune activities [ [7] , [8] , [9] , [10] , 13 , 16 , 28 , 29 ]. In the immune system, leukocytes are flexible and adaptively adjust their coping strategies when maintaining tissue homeostasis or fighting against infection and tumors based on mechanical signal guidance, thus widely influencing physiological and pathological processes [ 28 , [30] , [31] , [32] ]. Moreover, recent studies have shown that mechano-sensitive ion channels, including Piezo and transient receptor potential (TRP) families [ 20 , [33] , [34] , [35] , [36] , [37] ], can serve as drug targets for related illnesses in clinical treatment [ 38 , 39 ], especially Piezo1, which is one of the primary factors in immune response regulation [ 36 , 38 ]. Based on the importance of mechano-transduction in mediating immune functions, recent progress in mechano-biology of the immune system is reviewed, highlighting the current understanding on how immune cells communicate with micro-environment via mechanical signals and how mechanical signals regulate immune cell functions. 2. Forms of mechanical communication 2.1. Force exertion Immune cells can mechanically communicate with the micro-environment in various forms. Dynamically re-organizing their cortical cytoskeleton is the fundamental approach for cells to exert mechanical forces against the environment. Depending on source forms, cytoskeletal forces can be further divided into two types: actin polymerization and myosin-based contraction. Actin polymerization is a fundamental method for immune cells to exert cytoskeletal forces ( Fig. 1a ). In particular, globular actins (G-actins) are recruited to the nucleation sites by nucleators, such as actin-related protein 2/3 (ARP2/3) complex, forming branched actin arrays. After the initial formation of a branched actin filament network, formins mediate the subsequent actin filament elongation [ 40 , 41 ]. The new filamentous actins (F-actins) can cause a local cell membrane bulge, generating cellular forces against the environment. It has been widely observed that actin polymerization-based cell protrusions and synapses can greatly deform the extracellular matrix (ECM) or contacting cell surfaces [ 13 , 42 , 43 ]. For example, when in contact with target cells, T lymphocytes spread their immunological synapses on target cell surfaces, thereby enhancing their membrane tension [ 13 , 43 ]. Conversely, retrograde actin flow derived from membrane tension can also contribute to force generation. Fig. 1. Open in a new tab Forms of mechano-transduction . Immune cells can exert cellular forces against micro-environment via (a) dynamically re-organized cytoskeleton based on actin polymerization, and (b) myosin contractility. (c) Immune cells sense external mechanical stimuli through mechano-transduction, which can be initiated by mechanosensitive ion-channels. (d) Some bi-directional signaling proteins, typically TCRs, activated by actomyosin forces and external counterforces, undergo conformational changes thus triggering downstream signaling. A recent study demonstrated that the re-organized actin architecture based on dynamic actin polymerization can induce counterforces promoting cell migration in tiny channels. Neither myosin-dependent contractility nor cell adhesion is required to trigger motility [ 44 ]. Actin polymerization-based protrusion jams the interstitial matrix pores, while the increasing membrane tension pushes back F-actins centripetally, causing continuous retrograde actin flow. This subsequently induces uneven friction at the leading protrusion and rear, enabling the cell to move forward [ 45 ]. In addition to forces generated by actin polymerization, myosin-based contractility is another way for immune cells to exert cytoskeletal forces ( Fig. 1b ). Myosin Ⅱ is a motor protein that broadly exists in the F-actin network. By driving antiparallel F-actins, myosin Ⅱ can mechanically alter cytoskeletal architecture, exerting cytoskeletal forces outward through ligand and cytoskeleton-anchored protein junctions [ 15 , 46 , 47 ]. Myosin-based contractility in the immune system has been observed among various types of leukocytes [ [13] , [14] , [15] , [16] , [17] ]. Typically, B lymphocytes discriminate antigen affinities according to myosin-based mechanical contraction [ 16 ]. Actin polymerization and myosin-based contraction enable immune cells to exert cytoskeletal forces against their environment, and cooperative regulation of actin dynamics by these two forces is usually tightly coupled during force exertion. For example, the collaboration of contractile and protrusive forces is reflected in cell motility. Initially, a lamellipodium is assembled by polymerizing a new F-actin array at the front cell edge, which is then immobilized to the substrate via integrins. Subsequently, continuous actin polymerization at the leading cell edge increases the local membrane tension, triggering retrograde actin flow and resulting in counterforces from the substrate. Finally, myosin-based contraction pulls back the F-actin structures at the cell edge, enhancing the retrograde actin flow speed and counterforce strength, thus propelling the cell forward [ 48 , 49 ]. Force coupling can also be found in target cell killing of cytotoxic T lymphocytes [ 13 ]. 2.2. Force sensation Immune cells receive mechanical signals and transfer mechanical stimuli into bio-chemical signals via bio-sensors, typically ion channels ( Fig. 1c ). For instance, Piezo family members undergo conformational changes under mechanical stimuli, leading to the local ion flux increase and downstream signaling activation, thereby endowing immune cells with the ability to sense external mechanical signals [ 36 , 38 ]. In addition, TRP family members are another type of mechano-sensitive ion channels that can cooperatively mediate immune cell mechano-sensing [ 50 , 51 ]. Unlike the TRP family, which consists of various subtypes, the mechano-sensitive Piezo family only comprises two paralogs, including Piezo1 and Piezo2 [ 52 ]. Despite the small size of the Piezo family, its members undertake many important biological functions in vertebrates [ [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [53] , [54] , [55] , [56] ], and Piezo1/Piezo2 dysfunction results in genetic diseases [ 57 ]. Since the Piezo family plays a pivotal role in mediating physiological and pathological processes, Piezo1 structure has been recently resolved using cryo-electron microscopy [ 33 , 58 ]. Piezo1 is determined to be a protein with three-bladed, propeller-like trimeric architecture that can be further divided into a central pore module and three peripheral modules. The central pore module, which comprises the C-terminal extracellular domain (CED), outer and inner helices (OH and IH), and C-terminal intracellular domain (CTD), functions as a pathway for ion conduction. The peripheral modules, including transmembrane blades, intracellular beams, and anchors, are regarded as mechanical sensors. In contrast to other ion channels, the transmembrane regions of Piezo1 are characterized by considerable curvature, which is an important structural foundation for sensing membrane tension changes. Experimentally, the dramatic conformational changes of transmembrane blades under external forces further support the mechano-sensitivity of Piezo1 [ 33 ]. The structure of Piezo2 has also been explored using cryo-electron microscopy [ 34 ]. Similar to Piezo1, the non-planar, concave, trimeric structure of Piezo2 indicates its mechano-sensitive nature. Consistent with the force-dependent Piezo family activation, recent studies on the TRP family have revealed that TRPs use helical spring structures to activate ion conduction under mechanical loads [ 59 , 60 ]. The current understanding of how immune cells sense external stimuli via mechano-transduction has been enhanced significantly since the structures of mechano-sensitive ion channels have been resolved. In addition to the Piezo and TRP families, other mechano-sensitive ion channels, such as OSCA (hyperosmolality-induced Ca 2+ increase), TMEM63 (transmembrane protein 63), transmembrane channel-like TMC1/TMC2, Twik-related K + channel (TREK), and Twik-related arachidonic acid-stimulated K + channel (TRAAK), cooperatively mediate cellular mechano-sensing processes [ 61 ]. Although direct evidence for the regulation of immune responses by some of these mechano-sensitive ion channels is lacking, they have been shown to sense various external stimuli, such as stretch, flow shear, pressure, gravity, temperature, and sound. These channels undergo conformational changes to shift the concentration of related ion flux, thereby converting the mechanical stimuli into bio-chemical signals [ [61] , [62] , [63] , [64] , [65] , [66] ]. 2.3. Coupling of force exertion and sensation Apart from mechano-sensitive ion channels, immune cells can also sense physical forces via various cytoskeleton-anchored proteins, such as integrins and talins. These bi-directional signaling proteins serve as supporting anchors for force exertion and enable immune cells to sense mechanical stimuli by undergoing conformational changes. However, in contrast to mechano-sensitive ion channels mentioned above, activation of these bi-directional signaling proteins usually requires actomyosin forces. Hence, force sensation in these proteins is always accompanied by force exertion. Typically, this coupling can be reflected in the dynamical interfaces of focal adhesions between the cells and the matrix [ [67] , [68] , [69] , [70] , [71] , [72] , [73] ]. On one side, focal adhesions firmly fix cells in substrates, providing physical fulcrums for force exertion. On the other side, focal adhesion proteins, such as integrins and talins, can sense the contractile forces originated from the cytoskeleton. Specifically, focal adhesion maturation requires myosin-based contractile forces, which can induce conformational changes in integrins and talins and reveal cryptic talin sites for vinculin binding to enhance focal adhesion stability [ 73 ]. In addition to cytoskeletal contractile forces, integrins and talins can also sense the external force stimuli, which are required for focal adhesion maturation. Without enough counterforces derived from the substrates, cells usually fail to form focal adhesions, resulting in cell apoptosis [ 74 ]. Consistent with the fact that cells need counterforces and contractile forces to form focal adhesions, studies on integrins and talins confirm that single talin or integrin molecules can be mechanically activated by external forces [ [75] , [76] , [77] ]. This stretch-related activation demonstrates the complex crosstalk between force exertion and sensation during cell-matrix mechanical communications. In the immune system, immune cells, such as macrophages, prefer to form podosomes for fast migration instead of focal adhesions [ 10 ]. Mechano-sensitive proteins, such as integrins and talins, are also found in these adhesion-like structures for mechano-transduction [ 78 ]. Interestingly, a prior study revealed the checks and balances between integrin and Piezo1 expression in macrophages. The result strongly demonstrated the crosstalk between mechano-sensitive integrins and ion channels during mechanical communications, further indicating the complexity of the mechanical signaling network [ 79 ]. The mechano-sensitive characteristics of BCRs were explored in a study based on DNA tension gauge tethers [ 80 ]. It was found that BCRs cannot trigger downstream signaling when immobilizing antigens to the DNA duplex with a low rupture force threshold. At the same time, BCRs induce robust B lymphocyte activation while immobilizing antigens to the DNA duplex with a high rupture force threshold. These findings clearly demonstrated that B lymphocytes are capable of sensing mechanical stimuli via BCR-antigen bonding for efficient activation. Furthermore, an in vitro experiment determined that B lymphocytes require myosin-based contractile forces to discriminate antigen affinities, since low-affinity or individual BCR-antigen bonds tend to be ruptured under myosin-based contractility [ 16 ]. The coupling of force exertion and sensation in BCR-antigen bonding promotes the precision and efficiency of the immune system and plays important roles in regulating mechano-transduction. Force exertion and sensation coupling is further reflected in mechano-sensitive catch bonds, which require actomyosin forces and external counterforces to trigger conformational changes ( Fig. 1d ). This enables immune cells to sense mechanical stimuli experienced by these catch bonds and activate downstream signaling. Generally, most bio-molecular interactions behave as slip bonds, which have a shortened lifetime under mechanical loads. Some bio-molecular linkages can have a prolonged lifetime under mechanical loads and are defined as catch bonds [ 81 ]. For example, integrin-matrix bonding in focal adhesions is just one type of a catch bond, as the peak affinity of this linkage is only achieved under applied forces [ 9 , 73 ]. These bi-directional signaling catch bonds are significant in mechano-transduction and play decisive roles in regulating the immune response. For example, T lymphocytes can form catch bonds when in contact with antigen-presenting cell (APC) surfaces for antigen recognition. TCRs form strengthened catch bonds with high-affinity antigens upon mechanical loads, triggering robust downstream signaling. Only shorter-lived slip bonds form with low-affinity antigens to avoid unnecessary activation [ 82 ]. Force-dependent TCR activation dramatically enhances the discriminatory ability of T cells. TCRs can not only form bi-molecular catch bonds with peptide major histocompatibility complexes (pMHCs) to select high-affinity antigens [ 83 ], but can also form cooperative TCR-pMHC-CD8 tri-molecular catch bonds [ 84 ]. Similar to integrins, which experience conformational changes under applied forces, data have shown that mechanical forces applied to TCR-pMHC bonding result in the increased complex length, indicating the mechano-sensitive TCR characteristics [ 85 ]. Interestingly, a study combining steered molecular dynamic stimulation, magnetic tweezers, bio-membrane force probe, and functional assays determined that the increased TCR-pMHC complex length is due to the conformational change in pMHC rather than that in TCR. The applied forces allosterically strengthen the TCR-pMHC bonding via MHC extension and rotation in agonist pMHC recognition and dissociate the connection between TCR and pMHC via linkage disruption in antagonist pMHC recognition [ 83 ]. Thus, the conformational MHC change promotes the cascade propagation of conformational changes from CDR loop to CD3, thus activating downstream signaling [ 83 ]. The bi-directional signaling of TCR-pMHC catch bonds cooperatively mediates the mechano-sensing of T cells with other mechano-sensitive proteins and pivotally regulates the activation of adaptive immunity. Since a conformational MHC change is so decisive in agonist-specific TCR-pMHC catch bonds for mechano-transduction, cancer-associated MHC mutations may attenuate the discriminatory ability of T lymphocytes by reinforcing initial MHC conformation [ 86 ]. In addition to intergins and TCRs, selectins also form catch bonds during leukocyte rolling under blood flow. P-selectin-PSGL-1 (P-selectin glycoprotein ligand-1) bonding between leukocytes and endothelial cells shows low affinity without flow shear. These interactions are strengthened under optimal flow shear, leading to the conformational changes in P-selectins to enhance the adhesion of leukocytes to endothelial walls [ 87 ]. To date, it has been widely observed that catch bonds participate in many bio-molecular immune cell linkages involving TCR, integrins, selectins, and immunoglobulin superfamily (IgSF) [ 9 , [88] , [89] , [90] ]. In summary, using cooperative regulation of mechano-sensitive proteins, including cell adhesion molecules (CAMs), ion-channels, TCR/BCR, and actin/myosin, immune cells construct a mechanically communicative network that dominates immune response functioning. 3. Mechano-biology measurement techniques Mechano-transduction events generally occur at cell-cell and cell-ECM interactions. Sensitive measuring techniques are required to quantify cellular forces during mechano-transduction processes in order to discover a subtle relevance between mechanical forces and immune activities. Several methods have been developed to measure cellular and molecular forces [ 74 , 91 , 92 ], thus greatly advancing our understanding of mechano-regulated immunity. 3.1. Traction force microscopy Silicone rubber sheet is an early approach used to measure cellular forces. This thin elastic substrate (approximately 1 µm) can be distorted and wrinkled by cells, allowing cellular forces to be measured. Additionally, a glass micro-needle is used to distort a substrate to the same extent as the cell. The cellular force can be indirectly quantified based on the lateral micro-needle bending [ 93 ]. Although this original approach does not have a high precision, it reveals a completely new way of force quantification and visualization, inspiring further developments and serving as an experimental basis for traction force microscopy (TFM). The first generation of TFM is implemented using an improved thin elastic silicon substrate embedded with tracing particles, which makes it possible to draw cellular force distribution maps [ 94 ]. In comparison to the silicone substrate, TFM is more flexible and precise as it uses elastic materials embedded with fluorescent beads. Cellular forces can be quantified according to bead displacements, which can then be converted into force maps based on the gel's Young's modulus [ 95 ]. Bead displacement resolution is generally at micron level. TFM was utilized to explore the directional migration of macrophages to cancer cells via mechano-transduction [ 31 ]. Specifically, a quasi-three-dimensional (3D) in vitro culture model was constructed based on a collagen ECM substrate embedded with bead markers (diameter ∼0.8 µm). ECM deformation fields derived from continuous contractions of cancer cells seeded on the collagen gel were quantified based on local bead displacements. Benefiting from cellular force quantification by TFM, it was demonstrated that the ECM-transmitted mechanical signals induced by contractile forces of cancer cells activate the mechano-sensitive ion channels (MSCs)/Ca 2+ signaling pathway in macrophages, serving as physical cues for guiding the precise macrophage tracing to cancer cells [ 31 ]. 3.2. Pillar arrays A pillar array is a variation of the TFM technique that offers several advantages. Micro-pillars can be fabricated in different sizes or from various materials, resulting in their disparate physical properties, such as rigidity. Similar to the traditional TFM technique, which obtains cellular forces from bead displacements, a pillar array calculates cellular forces by measuring micro-pillar deflection [ 96 , 97 ]. In contrast to the hydrogels used in traditional TFM, micro-pillars are separate from each other, enabling precise local force characterization of each cell part based on the deflection of individual micro-pillars. The observation efficiency can be further improved with the addition of fluorescent makers or gold nano-particles [ 92 ]. The pillar array deflection can be measured at the nano-scale. Basu et al. designed a polydimethylsiloxane (PDMS)-based micro-pillar system (pillars are 0.7 µm in diameter, 6 µm in height, 2 µm in spacing) to evaluate dynamic forces in the plane of cytotoxic T lymphocyte (CTL) immunological synapses during immune killing [ 13 ]. Converting pillar deflections into force vectors indicated that CTLs can exert asymmetric synaptic forces against the target cells (up to 0.4 nN locally). Follow-up micro-pillar experiments further revealed that the degranulation zone is spatiotemporally correlated with force exertion at the immunological synapses due to the increased membrane tension [ 13 ]. Recently, photo-luminescent pillar arrays have been developed based on the InGaN/GaN multiple quantum wells. The internal potentials of nano-pillars can be dynamically re-distributed by cellular forces, affecting the local photoluminescence intensities. Combined with confocal laser microcopy, real-time mapping of force distribution can thus by achieved [ 98 ]. 3.3. Atomic force microscopy Atomic force microscopy (AFM) is a technique used to detect molecular forces or physical properties of materials. This method involves a calibrated micro-manipulated cantilever connected with a protein-coated tip. By connecting this tip with substrate ligands and subsequently pulling the tip back, the bond strength can be determined based on the tip deflection [ 99 ]. AFM is commonly used in immunological studies to measure the molecular forces and interactions of specific bindings [ 100 ]. Hu et al. investigated mechanical TCR responses by introducing a ligand-coated (pMHC or anti-CD3) functionalized AFM cantilever and unveiled a mechanical–chemical feedback loop during T lymphocyte activation [ 101 ]. Additionally, a cantilever can be directly pushed onto the test sample surface. The physical sample properties, such as topography and stiffness, can be obtained based on cantilever tip deflection. In this mode, scientists employ AFM as a physical characterization tool for immune cells ( e.g. , cytoskeleton analysis). Combining AFM-based mechano-imaging of cell biophysical features with a deep learning algorithm, Wu et al. accurately predicted the phenotype of macrophages without conventional biomarkers [ 102 ]. Since the macrophage phenotype is correlated with functional polarization, biophysical features may serve as innovative indicators for clinical diagnostics. Recently, an advanced AFM technique based on algorithmic optimization, termed localization AFM (LAFM), further improved the resolution limited by the cantilever tip size [ 103 ]. The LAFM surface structure resolution can reach Ångstrom level. 3.4. Optical trapping Dielectric particles that are micron-sized or smaller can be captured using an optical trap by focused laser beams due to the lateral or vertical restoring force. The dielectric particle movement in the optical trap can be conveniently controlled by adjusting the focal position of the optical trap. Linkage strength can be calculated based on the dielectric particle displacement from the optical trap focal point by tethering the dielectric particle to the surface of a cell via bio-molecular bonding [ 104 ]. This approach offers a force resolution of < 1 pN. Feng et al. used an optical trapping device equipped with a single pMHC-functionalized particle (diameter: 1.09 µm) to show that T lymphocytes can be reliably activated with limited pMHC numbers under mechanical shear load of approximately 10 pN [ 105 ]. Since mechanical forces have a crucial impact on the activation and function of T lymphocytes during immune response, the unveiled mechano-features of TCRs may contribute to chimeric antigen receptor design optimization in clinical immunotherapy. 3.5. Magnetic tweezers Magnetic tweezers utilize gradient magnetic fields to apply adjustable forces to magnetic objects. For example, a protein-coated magnetic sphere can be attached to ligands on the cell surface. By controlling the movement of a sphere through magnetic force, linkage strength between target molecules can be measured based on the sphere's displacement and magnetic field intensity [ 74 ]. The displacement resolution is generally at the nanometer scale. Force response of a talin domain with distinct chaperones was investigated based on single-molecule magnetic tweezers. This study demonstrated that adhesion protein stability can be modulated by chaperones by tuning their folding mechanics (unfoldases lower unfolding force from ∼10 pN to ∼7 pN, while foldases shift it up to ∼15 pN) [ 106 ]. These findings can deepen the current understanding of talin mechanics in the context of complex interactions with cytoplasmic proteins. 3.6. Bio-membrane force probe Bio-membrane force probe (BFP) makes use of red blood cells (RBCs) or artificial lipid bilayer vesicles. The sensitive bio-transducer consists of three parts: a micro-pipette, a bio-membrane structure, and a ligand bead. The bio-membrane structure (RBCs/artificial lipid bilayer vesicles) is initially immobilized to the head of a micro-pipette, serving as a spring for force quantification. Then, a ligand bead is firmly attached to the opposite side of bio-membrane structure. By pushing or withdrawing the bead, the bio-membrane structure is distorted just like spring. Hence, forces can be derived from the rigidity of bio-membrane structure and ligand bead displacement. Additionally, the sensitivity of this bio-transducer can be adjusted by regulating bio-membrane structure rigidity [ 107 ]. The force resolution of this approach can be < 1 pN. Typically, Liu et al. and others exploited BFP, which comprised a pMHC-coated bead and a micropipette-aspirated RBC as a pico-force transducer, to characterize single-bond lifetimes of the TCR-pMHC complex upon varied mechanical loads. Their results indicated a force-enhanced agonist-specific catch bond between TCR-pMHC interactions, thus having important implications for lymphocyte-based immunotherapy [ 82 , 83 ]. 3.7. Fluorescence resonance energy transfer The fluorescence resonance energy transfer (FRET) technique significantly improves the observation of molecular forces on temporal and spatial scales. In this technique, a pair of FRET donor and receptor molecules is arranged in a folded peptide sequence, which can be extended by external forces. When an increased force is applied to the bio-molecular bond, the folded protein extends, causing the separation of the donor and receptor and leading to fluorescence signal attenuation. Therefore, force can be measured by characterizing FRET efficiency [ 108 ]. The FRET technique is capable of detecting molecular forces at pico scale. Chang et al. adopted a FRET-based molecular force sensor to quantify the amplitude and distribution of the mechanical load experienced by individual integrins in a living cell. FRET/force measurements showed that the majority of integrins bear forces of 1–7 pN, while a fraction of those experiencing higher forces (> 7 pN) is enriched within adhesions [ 109 ]. 3.8. DNA tension gauge tethers This approach is implemented by immobilizing a selected ligand to a short DNA duplex. The rupture force of each DNA duplex can be customized by modulating its sequence, length, or binding site with a ligand. As a result, the linkage strength between the ligand and the cell receptor can be deduced based on the customized rupture force [ 110 ]. One research study introduced a series of DNA-based tension gauge tethers with rupture thresholds ranging from 12 pN to 56 pN to characterize the mechano-sensitivity and activation thresholds of isotype-switched IgG- and IgE-BCR (< 12 pN) or IgM-BCR (> 50 pN) [ 80 ]. A pair of fluorophores is introduced in the second generation of DNA tension gauge tethers. Similar to the FRET technique, the fluorophore pair is separated once the external force exceeded the rupture threshold, causing the change in fluorescence intensity. This development greatly improves force measurement efficiency on temporal and spatial scales [ 111 , 112 ]. For example, Liu et al. developed a modified fluorophore-labeled DNA tension probe and obtained the first pico-newton tension maps of a single TCR-pMHC interaction during T lymphocyte activation [ 112 ]. This work revealed that naïve T lymphocytes produce ∼12–19 pN forces through TCRs during antigen recognition, which is in alignment with previous results showing that ∼10–20 pN mechanical forces are required to enhance the TCR-pMHC bond lifetime for agonists [ 82 ]. DNA tension gauge tethers can measure pico-newton forces. In summary, rapid developments and remarkable refinements in mechano-biology measurement techniques pave a new avenue for immunological research. These advanced biophysics tools enable scientists to address more challenging problems of immunity and investigate innovative force-related immuno-mechanisms that could not be effectively explored in the past. 4. Immune system mechano-regulation Immune response can be categorized into two main components: the innate immune response and the adaptive immune response. Recent research has clearly demonstrated that both innate and adaptive immunities are mechano-regulated by physical stimuli. 4.1. Innate immunity Innate immunity is governed by professional phagocytes, natural killer cells, and complement proteins. Professional phagocytes, such as macrophages and neutrophils, engulf and digest invading microorganisms or abnormal cells. Natural killer cells can directly kill infected cells, while complement proteins enhance phagocytosis and trigger inflammation. As the second barrier against pathogens after skin and mucosa, the innate immunity function is strongly correlated with mechanical signals. 4.1.1. Phagocytosis Macrophages have strong powers of recognition and phagocytosis, and sufficient evidence shows that these abilities are shaped by mechanical signals. When hunting Escherichia coli , CD48 in macrophage filopodia can form specific catch bonds with fimbrin adhesion (FimH) of E. coli . Macrophages tightly hook E. coli via CD48-FimH bonding, thus avoiding their escape. Then, macrophages extend lamellipodia between E. coli and the substrate, mechanically shoveling E. coli off the substrate for phagocytosis ( Fig. 2a ) [ 88 ]. The participation of mechanical forces in this hook-and-shovel mechanism promotes macrophage efficiency. However, the physical shapes of pathogens or waste to be cleaned in vivo are varied. Fig. 2. Open in a new tab Mechano-regulation of immune response . (a) Phagocytosis: Macrophage clears E. Coli via hook and shovel mechanism (ⅰ); Cytoskeleton dynamics are tightly associated with the physical properties of prey during phagocytic cup formation (ⅱ). (b) Migration: The catch bond via PSGL-1 and P-selectin interaction regulates the leukocytic rolling under flow shear (ⅰ); Reconstructed ECM induced by cancer cell contractility guides the directional migration of macrophages (ⅱ). (c) Polarization: The polarization of macrophages is significantly affected by various physical factors. (d) Cytotoxicity: Cytotoxic T lymphocytes exert synaptic forces to enhance the membrane tension of abnormal cells, thus promoting the efficiency of perforin for pore-forming. (e) Activation: TCR-pMHC linkage contributes to the efficient discrimination between antagonist and agonist (ⅰ); B lymphocytes use cytoskeletal forces to discriminate antigen affinity for necessary activation (ⅱ). In addition to bio-chemical signals, the recognition by macrophages has a great relevance in physical matching between cell membrane surface features ( e.g. , membrane ruffles) and prey geometries [ 113 ]. After recognition, macrophages activate phagocytosis to maintain homeostasis. Compared to softer spherical particles, harder spherical particles exhibit greatly enhanced cellular uptake by macrophages [ 114 ]. Moreover, macrophages prefer to internalize spherical particles with a smaller size. Conversely, harder rod-like particles are detrimental to the internalization by macrophages [ 115 ]. This indicates that particle size, hardness, and shape have combinatorial effects on phagocytosis. The differences in phagocytic response caused by different physical properties of prey are tightly associated with mechanical cytoskeleton dynamics during phagocytic cup formation ( Fig. 2a ) [ 116 ]. As efficacy of drug delivery vehicles targeting macrophages can be tightly associated with phagocytic capacity, optimization of drug capsule physical properties might contribute to a more effective treatment. Substrate stiffness also remarkably influences macrophage phagocytosis. Macrophages exhibit the highest phagocytic capability when cultured on a substrate with a medium stiffness (88 kPa). They show a lower phagocytic ability when cultured on a softer substrate (11 kPa). Moreover, cells cultured on a hard substrate (323 kPa) show significantly impaired phagocytic capability. This stiffness-regulated phagocytosis is associated with macrophage migrating strategy and phenotypic tendency on substrates with different stiffness characteristics [ 117 ]. Furthermore, other findings suggest that ECM stiffness signals can activate mechano-sensitive ion channels, which cooperatively mediate macrophage phagocytosis [ 50 ]. 4.1.2. Migration In addition, macrophage migration is also regulated by mechanical signals. It is reported that interstitial flow can enhance macrophage motility via focal adhesion kinase (FAK)/Akt (also known as protein kinase B) phosphorylation in a 3D matrix [ 118 ]. Significantly, enhanced macrophage motility can potentially promote cancer cell invasion mechanically, as more motile macrophages can open more tunnels for tumor exit [ 119 ]. In addition, macrophages migrate upstream against interstitial flow. It was proposed that localized integrin activation at upstream sides induces FAK/Akt phosphorylation, contributing to the phenomenon of upstream migration [ 118 ]. Blood flow has a key role in regulating monocyte/neutrophil rolling ( Fig. 2b ). As mentioned above, the flow shear does not only allosterically strengthen the bonding between selectins/integrins and their ligands [ 87 , 90 , 120 , 121 ], but also up-regulates the expression of relative CAMs to enhance the interactions between monocytes/neutrophils and endothelial cells [ 122 ]. After a tight attachment to endothelial walls, immune cells, such as monocytes and neutrophils, extravasate through the vessel walls via paracellular or transcellular route [ 123 ]. This transmigration process requires strong mechanical forces and it is vitally important for immune cells to mechanically squeeze their bodies through tiny spaces [ 124 ]. Moreover, it has been directly observed that the efficiency of macrophage infiltration into embryonic tissue is correlated with ectodermal cell focal adhesion strength, as focal adhesions serve as mechanical barriers for blocking macrophage invasion. A reduction in adhesion strength enables macrophages to squeeze through these narrow spaces more easily [ 125 ]. Recent work by Yang et al. [ 31 ] utilizing a micro-manipulation system revealed that far-reaching mechanical signals generated by cancer cell contractions can guide the directional migration of macrophages via mechano-sensitive ion channels in a fibrous collagen matrix, while chemical signals are neither required nor sufficient in this targeting process, which is in accordance with a prior study ( Fig. 2b ) [ 28 ]. As ECM physical remodeling in tumors (such as matrix stiffening, deformation, and fiber alignment) has been widely recognized [ 126 ], this novel mechanotaxis of macrophages targeting cancer cells can be vital in exploring immune-therapy, including inspiring and improving the development of 3D in vitro models for drug screening or providing potential mechano-related molecular targets for drug development. Stiffness can also affect macrophage migration mode. On a soft substrate, macrophages exhibit Rho-A kinase (ROCK)-dependent amoeboid migration, while on a hard substrate, they prefer podosome-dependent mesenchymal migration [ 117 ]. 4.1.3. Activation and maturation Stimulatory immune receptors, such as natural killer group 2 member D (NKG2D), can recognize various ligands to trigger a distinct immune response [ 127 , 128 ]. A research study based on a single-molecule BFP technique determined that NKG2D receptors discriminate diverse ligands in a force-dependent manner. It was found that the interactions between NKG2D and MICA/MICB (MHC class I polypeptide-related sequence A/B) and their lifetimes can be augmented through allosteric change in ligands via optimal mechanical stress, while the interactions between NKG2D and UL16 binding protein cannot [ 129 ]. Similar to TCR-pMHC linkage mechano-sensitivity, force-enhanced NKG2D-MICA/MICB linkage exhibits the catch bond behavior, demonstrating mechano-regulated natural killer activation. As the primary effector lymphocytes for attacking circulating tumor cells (CTCs) in vasculature, natural killers show improved cytotoxicity under flow shear. Fluid shear stress enhances CTC death rate via shear-induced NKG2D-MICA signal activation [ 130 ]. As powerful APCs, dendritic cells (DCs) bridge the innate and adaptive immunity and are crucial in physiological and pathological processes [ 131 ]. By quantifying the expression level of biomarkers (co-stimulatory and MHC molecules), a prior study showed that interstitial fluid pressure can significantly affect DC activation and maturation. Both mature and immature DCs exposed to elevated pressure upregulate the expression of biomarkers (CD80+, CD86+, CD83+, CD40+, and MHC-I/II+ for mature DCs; CD80+, CD86+, CD83+, and MHC-II+ for immature DCs) [ 132 ]. Complementally, a recent study indicated that interstitial fluid shear can promote the expression of CD86 and MHC I in bone marrow dendritic cells (BMDCs) [ 133 ]. However, pressure-induced phenotypic maturation was observed to be associated with weakened phagocytosis [ 132 ], suggesting that elevated interstitial fluid level in a tumor microenvironment (TME) [ 134 ] may lead to functionally aberrant maturation and impaired immune response of DCs against tumors. Investigating the novel mechano-sensing mechanism of DCs in interstitial fluid may contribute to the improvement of clinical immunotherapy strategies, such as potential drug targets. Cyclic strain has also been found to influence DC activation. For cells seeded on varied protein-coated surfaces, including laminin, collagen, and fibrinogen, mechanical strain (3%, 1 Hz) significantly upregulates the expression of CD86, CD40, and MHC-II in comparison to the no strain controls [ 135 ]. Another study utilized MICA-functionalized nanowires to investigate the impact of substrate topography on natural killer function. It was found that nanowires trigger a more robust immune response compared to flat surfaces, though cells seeded on nanowires are exposed to less MICA than those cultured on a flat MICA surface (∼30-fold). The study proposed that enhanced activation was probably attributed to highly concentrated MICA and mobility of these clusters on nanowires, which resulted in the high NKG2D agglomeration at the interface [ 136 ]. Since the manner in which natural killers sense ECM topography remains elusive, further studies are required to determine the exact mechanism for substrate topography in the natural killer activation. Substrate surface properties take part in mediating DC activation and function. When cells are cultured on titanium implants with varied topography and surface energy, roughness and high surface energy promote a non-inflammatory immature DC phenotype [ 137 ]. Moreover, micro-patterned substrates dictate the spatial organization and dissolution of DC podosomes via suppression of pro-inflammatory lipid prostaglandin E 2 -induced Ras homologous A (Rho-A) activation, as well as phagocytosis against bacteria [ 138 ]. Interestingly, DC activation is correlated with antigen geometry and size. In the context of vaccine delivery, previous research showed that spherical nano-sized particles can boost the DC phagocytosis and maturation marker (CD83, CD86) expression in comparison to micro-sized or elongated nano-sized particles [ 139 ]. In addition to topography, substrate stiffness also has a significant influence on natural killer activation. By tuning the stiffness of MICA-coated nanowire-based surfaces (shear moduli of 2.5 kPa, 500 Pa, and 100 Pa), a recent study illustrated that a softer substrate can greatly promote the natural killer activation compared to a harder substrate [ 140 ]. However, in the context of planar surfaces coated with antibodies against natural cytotoxicity-triggering receptor 3 (NCR3) and lymphocyte function-associated antigen-1 (LFA-1), elevated substrate stiffness (Young's moduli of 142 kPa, 33 kPa, and 1 kPa) facilitates lytic granule secretion of natural killers [ 141 ]. Complementary to this result, the bell-shaped behavior of natural killer activation was observed in a stiffness range of 30 kPa to 3 MPa (peaks at 150 kPa, MICA-coated surface) [ 142 ]. As these controversial results may be due to the varied range of substrate stiffness, different surface topography, and even diverse ligand coating, more comprehensive research is needed to address the relevance between substrate stiffness and immune response of natural killers. In addition, DC activation and function can also be mechano-mediated by substrate stiffness. A comprehensive study highlighting the impact of extracellular stiffness on BMDCs showed that substrate stiffness can significantly regulate BMDC pro-inflammatory function, activation, and metabolism [ 143 ]. Specifically, a stiffer PDMS-based hydrogel substrate with an elastic modulus of 50 kPa markedly facilitates pro-inflammatory cytokine secretion, co-stimulatory and MHC molecule expression, phagocytosis capacity, and tumor clearance, and boosts glucose metabolism, such as glycolysis gene expression, metabolic flux, and glucose uptake, in comparison to a substrate with an elastic modulus of 2 kPa. A mechanistic workup determined that stiffness-induced DC alterations are controlled by downstream Hippo-signaling mediator Yes-associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif, as well as the MSCs/Ca 2+ pathway [ 143 ]. Target cell stiffness can also alter the immune response of natural killers. Friedman et al. synthesized anti-NCR3/anti-LFA-1-coated cell-sized beads with varied stiffness properties (9–254 kPa) and discovered that natural killers show decreased degranulation against soft beads due to the formation of unstable synapses (kinapses) caused by impaired polarization of the microtubule-organizing center and lytic granules [ 141 ]. Notably, tumor cells, especially the metastatic type, are usually softer than healthy cells [ 144 ]. As this mechanical property of cancer cells may inhibit the activation of natural killers in vivo , sensitizing natural killer cell to soft tumor cells or stiffening tumor cells may be an effective way to improve natural killer function in cancer-immunotherapy. 4.1.4. Polarization Over the past few decades, macrophage polarization upon various mechanical stimuli has been explored ( Fig. 2c ). Both hydrostatic pressure and interstitial flow have a significant impact on macrophage polarization. It was found that cyclical hydrostatic pressure in the lung initiates mechanical activation of Piezo1, leading to the inflammatory response of macrophages [ 36 ]. Interestingly, compared to newly recruited monocytes/macrophages, Piezo1 in alveolar macrophages became mechanically desensitized to prevent chronic inflammation [ 36 ]. Interstitial fluid pressure is remarkably elevated in tumors [ 134 ]. A high level of interstitial flow can mechanically promote macrophages to polarize into M2 phenotype via β1-integrin, Src/Akt/FAK, and STAT3/6 (signal transducer and activator of transcription 3/6) signaling pathway [ 118 ]. In addition to macrophages, recent studies have shown that neutrophil activation is also regulated by flow shear [ 90 , 145 ]. Macrophage polarization is strongly correlated with the topography and spatial structures of the extracellular environment. A study on the foreign body reaction based on porous implants observed that compared to externalized cells, macrophages in implant pores and on the outer implant surfaces show a polarized shift towards M1 phenotype [ 146 ]. Moreover, macrophages cultured on rough surfaces increase their secretion of pro-inflammatory cytokine TNF-α (tumor necrosis factor-α) than that cultured on smooth surfaces [ 147 ]. Consistent with these results, in vivo experiments in mice have shown that TiO 2 implants with rougher surfaces promote the polarized shift of macrophages towards M1 phenotype [ 148 ]. Currently, implant devices are widely applied in clinical therapy. Thus, understanding the role of topography/geometry in mechano-immunity may benefit the proper activation of desirable immune responses by modifying medical implant materials with optimized physical properties. Macrophages can adopt flexible shapes to adapt to a dynamic environment [ 149 , 150 ]. It was determined that confining spaces or changing geometries of macrophages influences their expression of pro-inflammatory/pro-healing cytokines [ 151 , 152 ]. Shape-related macrophage polarization is regulated by the cytoskeleton, and cytoskeletal contractility inhibition eliminates shape-related polarization [ 152 ]. The role of matrix stiffness in regulating macrophage polarization was also explored. Macrophages prefer to polarize into a pro-inflammatory phenotype in a stiff matrix, while macrophages in a soft matrix shift to have an anti-inflammatory phenotype [ 117 , 153 ]. Essentially, stiffness-induced M2 polarization in a soft matrix is based on the ROCK signaling pathway and is not affected by ECM components, such as collagen, fibronectin, vitronectin, and laminin [ 117 ]. Another study showed that TRP also plays a crucial role in matrix stiffness-induced macrophage polarization. It was determined that a stiffer matrix promotes the M1 phenotype in a TRPV4-dependent manner [ 154 ]. In addition to matrix stiffness, cyclic mechanical strain was observed to promote the inflammatory cytokine secretion in macrophages [ 155 , 156 ]. However, a later study reported that bone marrow-derived macrophages (BMDM) exposed to cyclic mechanical strain changed the expression of related proteins (F480+/iNOS−/Fizz1+), suggesting that mechanical strain promoted macrophages to polarize towards the M2 phenotype [ 157 ]. These controversial results are probably due to the differences in form, strength, and frequency of mechanical strain or even macrophage type. Further studies are required to address the role of mechanical strain in macrophage polarization. 4.2. Adaptive immunity Adaptive immunity consists of humoral immunity, which involves antibody production by B cells, and cellular immunity, which involves T cell activation. Unlike innate immunity, adaptive immunity exhibits high specificity in recognizing and targeting specific pathogens. Recent studies provided evidence that mechanical signals play a role in mediating lymphocyte function in adaptive immunity [ 158 , 159 ]. 4.2.1. Cytotoxicity Cytotoxic T lymphocytes spread immunological synapses over target cell surfaces and secrete cytotoxic proteases for abnormal cell killing. During the killing process, cytotoxic T lymphocytes mechanically increase target cell tension by exerting synaptic forces, boosting perforin efficiency for pore formation ( Fig. 2d ) [ 13 , 43 ]. Recently, researchers have found that cytotoxic lymphocytes can search characteristic biophysical vulnerabilities in tumors. It was proposed that cancer cells overexpressing myocardin-related transcription factors (MRTFs) enhance lymphocyte activation and cytotoxicity by triggering mechanical immune-surveillance [ 160 ]. In clinical trials, the MRTF overexpression does not only enhance metastatic tumor colonization, but also stiffens cancer cell cytoskeleton, making cells more sensitive to lymphocyte-mediated lysis. Consistent with these results, strong MRTF signaling boosts the therapy efficiency of immune checkpoint blockade in vivo [ 160 ]. 4.2.2. Motility In addition to lymphocytic activation, some studies have also explored the impact of mechanical signals on other lymphocyte functions. It is now clear that lymphocyte motility is also accompanied by mechanical signaling. Similar to monocyte rolling under blood flow, the catch bond linkages between lymphocytic integrins/selectins and their corresponding ligands are mechanically strengthened via conformational changes under flow shear, promoting T cell adhesion to high endothelial venules [ 159 , 161 ]. Another study further reported that T cell migration is potentiated by matrix stiffness. Culturing human primary CD4 + T cells on poly-acrylamide (PA) gels coated with intercellular cell adhesion molecule-1 (ICAM-1) demonstrates that CD4 + T cells exhibit higher instantaneous velocity and migrate over longer distance on stiffer PA gels compared to softer PA gels, revealing stiffness-sensing via LFA-1/ICAM-1 catch bonds [ 162 ]. Moreover, it was proposed that the stiffness of a substrate with TCR ligand surface-coating can affect the arrest signals (stop the migration) via TCR/CD3 triggering in T cell migration [ 162 ]. 4.2.3. Activation Adaptive immunity activation is associated with mechanical signals. During antigen recognition, lymphocytes can form catch bonds with APCs or target cells via LFA-1/ICAM-1 connecting. Cytoskeletal force-induced conformational changes in LFA-1 enhance the bonding affinity, reinforcing the communication between lymphocytes and APCs/target cells for following activation [ 89 ]. Furthermore, LFA-1/ICAM-1 bonding facilitates actomyosin forces, which in turn modulate the assembly of actin downstream of TCRs [ 163 ]. After discovering that a combination of TCRs with high-affinity pMHCs is enhanced upon applied mechanical forces ( Fig. 2e ) [ 83 ], a recent study reported that cytoskeletal actin dynamics primarily contribute to the activation of T lymphocytes [ 164 ]. The mechanical forces induce conformational changes in TCR-related proteins, such as CD3, thus triggering downstream signaling for T cell activation [ 165 ]. Similar to T cells, B cells also use mechanical forces to discriminate antigen affinity for activation as mentioned above ( Fig. 2e ) [ 16 ]. Interestingly, activated lymphocytes also provide feedback for APC communication. It has been reported that activated T cells spread larger immunological synapses than naïve T cells on APC surfaces by softening the cytoskeleton, revealing the crosstalk between cytoskeletal mechanical properties and lymphocytic activation [ 166 ]. Dendritic cells actively strengthen the rigidity of their cortical cytoskeleton during maturation. It has been observed that maturation-dependent DC stiffening augments T lymphocyte activation as increasing cytoskeleton stiffness lowers the agonist dose needed for T lymphocyte activation. This indicates the important role of mechanical signals in regulating lymphocytic activation [ 167 ]. Recently, a study utilizing oscillatory forces generated by an orbital shaker further showed that the frequency of external forces contributes to T cell activation, as roughly a double-strength antigenic signal was observed under dynamic oscillations compared to that in conventional static environment [ 168 ]. In addition to mechanical force frequency, lymphocytes can sense matrix stiffness, which also plays an important role in mediating lymphocyte activation. It has been reported that the activation of T cells cultured on a stiffer substrate is augmented compared to those cultured on a softer substrate [ 169 , 170 ]. It was thus proposed that matrix mechanics regulate T cell activation in a YAP-dependent manner [ 170 ]. During clinical tumor therapy, immune cells, such as T cells and macrophages, are genetically engineered to fight against cancer cells. A mechano-immunology review of chimeric antigen receptor T cell (CAR-T) comprehensively discussed the significance of mechanical signals in mechano-regulating CAR-T activation [ 171 ]. As CAR-Ts show exciting promise against tumors in clinical cancer-immunotherapy, all of these findings do not only deepen our current understanding of mechanical communication between lymphocytes and target cells (including molecular interactions and corresponding signaling mechanism), but also shed light on the development of efficient next-generation CAR-Ts. 5. Concluding remarks The field of mechano-transduction is currently a prominent area of research. Over the past few decades, numerous theoretical and experimental studies have provided evidence that mechanical signals play a crucial role in regulating the fundamental functions of immune cells. These studies have also revealed the complex mechanism through which these mechanical signals mediate immuno-functioning. These exciting findings include identifying various mechano-sensitive proteins, revealing their work modes, and exploring the corresponding transmission networks of mechanical signaling. However, there are still deficiencies and uncertainties in this body of research. First, although a significant number of downstream bio-chemical factors involved in mechano-transduction have been identified, the complete mechano-transduction pathways remain elusive. The currently identified and defined mechano-sensitive proteins represent only a fraction of the whole. Due to the complexity and diversity of biological molecules, further research is required for identification of new mechano-sensitive proteins. Moreover, mechano-sensitive proteins, such as intergins, selectins, TCRs/BCRs, and mechano-sensitive ion channels, are activated via conformational changes under applied mechanical loads, which subsequently initiate downstream signaling. However, it remains a topic for future investigation to explore the possibility of additional forms of mechanical activation or force-related binding kinetics. Second, scientists have shown that physical cues, such as matrix stiffness, spatial environmental structures, surface topography, flow shear, and mechanical stretch, can activate immune cell mechano-transduction, thus controlling the immuno-functioning. However, these physical cues cannot present a complete picture of mechanical signals. Certain mechanical signals, such as force frequency, have received limited attention in research studies. While some investigations have introduced oscillator or cyclic mechanical stimulation to explore the effects of force frequency on mechano-transduction, it is important to note that directly showing the regulatory role of force frequency on mechano-responsiveness of the immune system is challenging. This is primarily due to the presence of complex variables and interferences in experiments. Third, there have been numerous studies on mechano-transduction to date, but the majority of them are based on in vitro systems due to the higher complexity and lower observability of an in vivo system. Although there are certain commonalities and referential values between the in vivo and in vitro systems, the complex cytokines and physical environments in vivo are significantly distinctive compared to those in vitro. Moreover, immune cells in vitro and in vivo exhibit dramatic differences, which may vary among different organs or tissues in vivo . The mechano-responsiveness of immune cells to mechanical stimuli may differ between the in vitro and in vivo microenvironments. Since even a minimal deviation can lead to significant divergence, how to validate the mechano-responsiveness of immunity in vivo becomes a critical and fundamental step towards translating theoretical understanding into clinical practice. In the realm of clinical trials, the field of cancer immuno-therapy, which aims to reactivate the inherent immunity against tumors and holds great potential [ 172 , 173 ], could potentially benefit from the rapid development of mechano-immunology. In a complex TME, the interactions between immune and cancer cells affect the fate of both sides. Although various studies have shown that the bio-chemical signals play crucial roles in mediating the communication between cancer and immune cells, thus regulating subsequent cell behaviors [ 1 , 3 , 4 , 174 ], how immune cells precisely target and trace cancer cells via environmental cues remains unclear. Due to the limitation of bio-chemical signals (rapid attenuation during diffusion) [ 175 , 176 ], they may not be sufficient for immune cells to precisely locate individual cancer cells. In a TME, immune cells are exposed to intense mechanical stimuli, such as a stiffer matrix, re-aligned fibers, and continuous ECM strain. In this case, other signaling mechanisms likely also participate in this targeting process. In recent years, the significance of ECM in mechanical transmission was gradually explored. As one of the most abundant ECM components with a fibrous structure and non-linear viscoelasticity, collagen has been widely studied [ [177] , [178] , [179] , [180] ]. It does not only function as a mechanical support for tissues, but also offers the potential for mechanical communication among cells, which share the same matrix. It was shown that invasive cancer cells can mechanically remodel ECM via dynamic cell contractions [ [181] , [182] , [183] ]. ECM-transmitting mechanical signals promote cell-cell communications [ 181 , 182 ] and cancer cell spread/invasion [ [184] , [185] , [186] , [187] ]. Based on the excellent mechanical transmission characteristics of collagen, ECM can provide a compensatory mechanical communication mode for cross-space and long-distance signal communication between immune and cancer cells. A recent study based on a 3D in vitro system discovered that continuous ECM strain, which is generated by cell contractions, can guide the directional macrophage migration [ 28 ]. This study also pointed out that the chemotaxis is neither sufficient nor required to attract remote macrophages to migrate towards fibroblasts. Both T lymphocytes and macrophages need to establish a full contact with cancer cells to execute their killing function when participating in immune resistance against cancer cells. Exploring how immune cells are attracted by cancer cells and comprehensively revealing their signaling mechanism during cancer cell targeting and tracking can help to artificially regulate the body to enhance its immune response. Furthermore, immune rejection occurs during the transplantation of organs and other artificial devices. By understanding the importance of mechanical forces in the recognition and activation of lymphocytic antigens, it becomes feasible to recode transplanted organ MHCs to reduce the affinity with lymphocytic receptors. In the case of artificial devices, considering that immune cells can have varying reactions depending on material hardness, physical structures, and surface topography, the optimization of physical material parameters to achieve the optimal compatibility state will be a key focus in future research. Declaration of competing interest The authors declare that they have no conflicts of interest in this work. Acknowledgments This work was supported by the National Key Research and Development Program of China (2020YFA0908200), The National Natural Science Foundation of China (12325405, 12074407, 12090054, 12072010, T2221001), Strategic Priority Research Program of Chinese Academy of Sciences (XDB33000000) and the Youth Innovation Promotion Association of CAS (2021007). Biography Fangfu Ye received his Ph.D. in Physics in 2007 from the University of Pennsylvania, and later worked as a postdoctoral research associate at the Liquid Crystal Institute of Kent State University, the Department of Physics of the University of Illinois at Urbana-Champaign, and the School of Physics of Georgia Institute of Technology, successively. He joined the Institute of Physics, Chinese Academy of Sciences in 2013, with research interests in interdisciplinary areas between physics and biology, including cell migration, extracellular matrix-cell interaction, and development of organoids. 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