New insights to B cell tolerance involving the mechanosensitive ion channel Piezo1

Abstract

B cell tolerance is critical for preventing autoimmunity, yet the mechanisms by which B cells discriminate self from non-self antigens remain incompletely understood. While early findings emphasize the role of classical antigen-mediated BCR signaling strength by varying antigen formats, emerging evidence highlights the importance of mechanical cues during antigen recognition. This review explores how mechanosensitive ion channels, particularly Piezo1, contribute to B cell activation and tolerance by integrating physical forces at the immune synapse. We discuss how membrane-bound and particulate antigens induce mechanotransduction through Piezo1, promoting enhanced B cell responses by extracellular calcium influx. Additionally, we consider the differential roles of Piezo1 in various physiological contexts, including shear stress, tissue migration, and substrate stiffness. Understanding mechanosensor- mediated signaling in coordination with other pathways such as antigen recognition, T cell help, or cytokine signaling expands our knowledge of B cell biology and introduces a new paradigm for modulating humoral immunity in health and disease.

INTRODUCTION

Upon infection by pathogens, B cells, along with other immune cells, govern the humoral response by producing neutralizing antibodies while retaining memory B cells for the future (1). However, in autoimmune diseases, B cell responses against self are critical yet hard to overcome. To prevent such unwanted immune activation, central and peripheral tolerance mechanisms come into play to ultimately remove the auto-reactive clones of both T and B cells (2-4), features well described in the text-book level. However, B cells are more loosely regulated and thus, leaving 20-30% self-reactive anergic clones in the secondary lymphoid compartments (3). Hence, exploration of the behavior and strategies employed by B cells to control the magnitude of activation in response to self and non-self antigens is crucial in the field of vaccinology and disease conditions. In this review, we characterize the updated details behind how B cells maintain or break tolerance in response to antigen interaction, focusing on the role of novel players, markedly mechanosensors, in orchestrating effective immune-mediate protection without inducing autoimmune responses.

B CELL TOLERANCE: HOW DOES IT WORK?

Nature of BCR signaling

Antigen-mediated cross-linking of B cell receptor (BCR) initiates the early phosphorylation cascade, followed by subsequent intracellular calcium responses (4). Further signal transduction activates the key transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), nuclear factor of activated T cells (NFAT), AKT serine-threonine kinase (AKT), extracellular signal-regulated kinase (ERK), activator protein 1 (AP-1), MYC proto-oncogene (MYC), and so on (5, 6). Interestingly, although resting B cells rely on tonic BCR signals for survival (7), constitutive NF-κB signals often result in the formation of autoreactive germinal centers (GCs) (8). These observations highlight the importance of tightly regulated B cell signaling to remain below or rise above the activation threshold in order to balance between survival and prevention of autoimmunity.

During development, central tolerance removes the potentially risky autoreactive clones from the repertoire (2, 9). In fact, during this process, B cells discriminate between self and non-self antigens based on their BCR signaling strength (10). Thus, the nature of the antigen, including affinity, avidity, and format, became a significant topic of interest. Goodnow and Nemazee each published a back-to-back paper (11, 12) showing that membrane-bound self-antigen (HEL; MHCI antigen K^b) display removes the antigen-specific B cell clones (MD4: HEL-specific BCR mouse model; 3-83 Ig-Tg: MHCI antigen K^b-specific BCR mouse model) during developmental stages. In contrast, presentation of soluble HEL during development does not delete the HEL-specific B cell clones but rather renders them silent (10, 13), establishing an anergic mouse model. In this case, due to the weaker signals provided by soluble antigens, B cells were able to avoid clonal deletion yet display phenotypes (downregulation of IgM and elevated traces of BCR signaling during development) exhibited by naturally occurring anergic B cells. Although initial interpretations of these studies primarily focused on how the magnitude of self-reactivity correlates with clonal deletion during B cell development, such findings also pose a more fundamental question of B cell biology: why and how do membrane-bound and soluble antigens differentially activate B cells?

B cell tolerance mechanisms are not limited to developmental stages of B cells. When mature naïve B cells encounter antigens, B cells go through multiple checkpoints to decide the stop-or-go for activation or tolerance (4). Here, accumulating evidence suggest that the discrimination between membrane and soluble antigens, as well as other features involved in self-non-self discrimination, plays a critical role.

Tolerance mechanisms of naïve B cells

Deviating from the traditional ‘all or none’ view of B cell tolerance, exquisite studies have offered new insights that B cell tolerance may be acting in multiple layers during the various phases of B cell activation, well-described in our previous review (14). Briefly, from antigen recognition to BCR signal transduction and downstream activation of key transcription factors, B cells set an activation threshold to become fully activated when various requirements are met. As B cells respond differently to soluble and particulate or membrane-associated antigens to promote distinct signaling patterns, it is important to highlight the nature of BCR signaling in different antigen contexts.

In response to soluble (and low valency) antigens, B cells are moderately activated, often quantified by transient intracellular calcium influx (15-18). This outcome fails to fully activate the NF-κB and NFAT pathways which are essential for survival, proliferation, and differentiation (19). Subsequently, B cells activated by soluble antigens undergo apoptosis, a phenomenon called activation-induced cell death (AICD) (20). These activated B cells require ‘signal two’ from antigen-specific T helper cells for additional survival signals. The requirement for T cell help acts as a check-point to avoid any chances of self-reactive clones from becoming fully activated and/or being recruited to the GC.

However, a recent study by Zikherman group has demonstrated that multivalent particulate antigens may be recognized as danger signals by B cells, inducing highly amplified phosphorylation cascades and robust calcium signaling patterns, ultimately activating the NF-κB pathway even in the absence of T cell help (19). As most pathogenic antigens from viruses or bacteria exhibit multivalent epitope display (21-23), this finding suggests that viral pathogens may maximally activate B cells in a T cell-independent manner for rapid and robust immune responses.

Less has been elucidated in the membrane-bound antigen context. For now, the general viewpoint categorizes membrane-bound antigens with the particulate antigens where BCR signaling is significantly enhanced, although in different mechanisms (19, 24-26). Particulate antigens evade the Lck/Yes-related novel tyrosine kinase (LYN)-mediated negative feedback loop to maximize their signaling capacity (19) whereas membrane-bound antigens go through a more intricate ‘spreading and contraction’ process first described by Batista group (26). During this process, cell-cell (B cell-antigen presenting cell) interaction triggers the formation of the immune synapse (IS), antigen-BCR microclusters, and central supramolecular activation cluster (cSMAC) where antigens are centralized for efficient extraction (26, 27). Molecular details are well described in the following reviews (28, 29). In short, these distinct features lower the activation threshold to enhance B cell sensitivity to low antigen conditions, ultimately driving a qualitatively advanced B cell response to more physiologically relevant contexts (29). Yet, these pieces of puzzles are not clear enough to picture the full nature of BCR signaling. New topics have been introduced by researchers, especially focusing on the immune synapse formation to membrane-bound antigens.

PIEZO1 AS A KEY PLAYER IN B CELLS

Piezo1 as a novel player in immune responses

Piezo1 is a mechanosensitive ion channel that transduces mechanical stimuli into cellular signals by allowing the influx of cations, particularly calcium ions, in response to mechanical force (30, 31). Originally identified in a neuroblastoma cell line (32), Piezo1 has been a hot topic in various cell types due to its wide range of expression. Now, Piezo1, along with other mechanosensors, has emerged as a key player in immune regulation (33). Recent studies have demonstrated that Piezo1 is expressed in a range of immune cells, including macrophages, dendritic cells, neutrophils, T cells, and B cells (33). In these populations, Piezo1 influences various immune functions such as cytokine production, cell migration, phagocytosis, and metabolic adaptation, particularly under conditions of mechanical stress or inflammation (33). Its ability to integrate physical cues with immunological signaling pathways highlights Piezo1 as a crucial mediator of immune cell function in various contexts. Here, we outline the general mechanism of Piezo1 activation in B cell biology and further discuss the biological meaning of recent findings.

Mechanosensing the immune synapse

Early studies on the IS formation in membrane-bound antigen context have demonstrated how pulling forces exerted on the antigen-BCR interface enables B cells to discriminate antigen affinity (34). During the spreading phase, ‘passive force’ is produced by membrane tensions and cell spreading across the planar lipid bilayer (PLB) platform. However, when B cells start to contract, antigens gather at the cSMAC and are then pulled by B cells for antigen extraction. These cytoskeleton-mediated pulling exerts a much stronger ‘active force’ (34, 35). It has been implicated that these pulling force also has a role in testing the affinity of the antigen. Notably, membrane force has been viewed as a necessary condition for B cell spreading and contraction, confirmed by differential B cell behavior to varying grades of substrate stiffness (36). In this regard, recent studies have added new insights to this topic with accumulating evidence of mechanosensitive ion channels playing major roles in B cell activation and possibly contributing to B cell tolerance.

This brings us back to the concept of self-non-self antigen discrimination. Early mouse studies have demonstrated how antigen affinity, avidity, and format mediate central and peripheral tolerance. The take home message here was that, somehow, membrane-bound antigens hyperactivate B cells compared to soluble antigens. In fact, foreign antigens are known to be captured by follicular dendritic cells (FDCs) and presented directly on their surface for B cell recognition (37). However, the molecular basis of BCR-antigen interaction in this context remains unclear.

Recently, Kwak and colleagues have highlighted the role of Piezo1 during B cell responses to membrane-associated antigens, followed by reports addressing other mechanosensitive ion channels such as TRPV-family proteins (38-40). In fact, Piezo1 is expressed at considerable levels in various subsets of murine and human B cells (41). Kwak and colleagues highlighted the major role of Piezo1-dependent calcium influx during the formation of the IS which ultimately affects the key features of B cell activation. Interestingly, inhibition of Piezo1 with an antagonist significantly impairs the B cell response to membrane-bound antigens but not soluble antigens (Fig. 1). Also, Piezo1-knock down B cells failed to properly ‘spread and contract’ on membrane-bound antigens exhibiting diminished intracellular calcium dynamics. As the initial proof-of-concept study has clearly demonstrated the activation of Piezo1 upon membrane-bound antigen stimulation, it remains to be investigated whether Piezo1-mediated calcium influx plays critical functional roles during B cell activation and/or humoral immune responses to immunization or infection settings. As calcium signaling pathway has been valued for playing major roles in transducing various downstream pathways (42), Piezo1-dependent calcium mobilization during BCR signaling is a key aspect to be covered.

Fig. 1.

Differential activation of Piezo1 by membrane-bound/particulate antigens versus soluble antigens. Based on findings by Kwak et al. (38) and Torgbor et al. (44), Piezo1 is activated in response to membrane-bound or particulate antigens, as reflected by antigen-induced calcium dynamics. Inhibition with the Piezo1 antagonist markedly reduces calcium responses under these conditions. By contrast, soluble antigens elicit comparable calcium responses regardless of Piezo1 antagonist treatment.

In a similar sense, Liu and colleagues have highlighted the functional role of TRPV2 (39), another mechanosensitive ion channel highly expressed in murine B cells. Similarly, formation of the IS was impaired followed by decreased levels of BCR signaling, B cell activation, and antibody production. Interestingly, expression level of TRPV2 correlated with SLE progression. This finding highlights the potential risk of having unneeded expression and activation of such mechanosensitive ion channels. On the other hand, Ardem Patapoutian, the discoverer of Piezo1, reported that one in three Africans carries a gain-of-function (GOF) variant of Piezo1 (43). In this case, Piezo1-GOF variant conferred protection against malarial infection by the inherent defense mechanism of Piezo1-GOF red blood cells (RBCs). However, the role of Piezo1 in the immune cells remain widely unknown. Mechanistic studies not only in B cells but also in other immune compartments could offer valuable insights into both innate and adaptive immune responses, as well as their roles in disease contexts.

Mechanosensing soluble particulate antigens

Recently, Pierce and colleagues reported that virus-like particles (VLPs) also activate Piezo1 for enhanced B cell responses (44) (Fig. 1). Considering the size of VLPs which ranges around 40-50 nm, it is remarkable that small-scale immunogens may exert confined microdomain membrane tension force strong enough to open up the Piezo1 ion channel. As previously explained, growing evidence point to the fact that particulate antigens may be recognized as a danger signal (19) for reasons that are yet ambiguous. Here, Piezo1 is described as a crucial factor during B cell activation, as in the membrane-bound antigen context. Strikingly, when particulate HEL antigens were introduced to the sHEL:IgHEL anergic mouse model, B cell anergy was reversed (45). This phenomenon is analogous to studies by Goodnow and Nemazee previously described, highlighting the ability of particulate and membrane-bound antigens to induce robust B cell responses leading to clonal deletion or B cell activation, respectively. Although Piezo1 may not be the sole factor, current evidence suggest that mechanosensitive ion channels are indispensable during initial BCR responses in both soluble and membrane-associated antigen contexts.

Notably, biophysical assays have further shown that activation of BCR is dependent on mechanical force (46, 47). By using the NP-TGT molecules with predefined force, Liu and colleagues demonstrated that BCR microcluster formation and phosphorylation level of Spleen tyrosine kinase (SYK) increases with stronger mechanical forces (46). Interestingly, the threshold of mechanical force required for BCR signaling varied among different isotypes. In particular, IgG, due to its functional cytoplasmic tail (with phosphorylation sites), has low force requirement to initiate BCR signaling (46). As IgG-expressing B cells are mostly class-switched plasma cells or memory B cells, it can be logically inferred that low activation threshold of such subsets allow our immune system to quickly respond to various antigens regardless of their mechanical force. Also, as IgM and IgD (isotypes mostly expressed in naïve B cells) possess extremely short non-functional cytoplasmic tails with 3-4 amino acids (48), these isotypes lack the ability to lower their threshold requirement for mechanical force. Thus, naïve B cells may rely on additional mechanosensors such as Piezo1 to discriminate mechanical force provided by antigen substrates. Furthermore, B cell activation assays using this system revealed that full activation of IgM and IgD isotypes were induced by > 50 pN force (46), which is the force required for Piezo1 activation (49). Future investigations are required to determine whether Piezo1 activation proceeds or follows BCR activation, as their interplay is not yet fully understood.

However, despite these advances, the mechanistic role of Piezo1 in B cell tolerance remains to be fully elucidated. Current findings suggest plausible links between Piezo1-dependent calcium influx and tolerance outcomes such as anergy or deletion, but direct experimental evidence is still limited. Given that mechanotransduction can shape both activation and tolerance pathways, future studies will be essential to dissect how Piezo1 integrates with established BCR signaling networks to influence B cell fate decisions. Such efforts are expected to clarify whether Piezo1 functions as a critical determinant of tolerance checkpoints or acts as a modulatory factor in the broader landscape of B cell regulation.

Calcium signaling determines B cell fate

So far, this review has discussed the molecular basis by which membrane-bound or particulate antigens act as key determinants in self and non-self discrimination, particularly through the engagement of mechanosensors. Low valency soluble antigens provide weak BCR signaling and fail to activate mechanosensitive ion channels due to lack of membrane tension. On the other hand, particulate or membrane-associated antigens induce stronger BCR signaling (50) and synergistically activates the mechanosensitive ion channels (38, 39, 44) for additional calcium influx. Thus, it could be implicated that these extra source of calcium, together with strong BCR signaling, help B cells reach the activation threshold to break tolerance (Fig. 2). In fact, the NF-κB pathway fails to activate upon antigen stimulation in calcium-depleted conditions, leading to increased rate of AICD (51). These findings are in line with B cell dependency on T cell help (via CD40L-induced NF-κB signaling) for enhanced survival (4).

Fig. 2.

B cell tolerance during development and immune responses. (A) When B cells encounter cognate soluble self-antigens during development (mimicked by MD4:ML5 mouse model), the BCR signaling strength remains moderate giving rise to an anergic population. In the case of low-affinity soluble antigens, immature B cells remain unresponsive via clonal ignorance and differentiate into mature B cells. However, presenting self-antigens in the membrane-bound form results in clonal deletion due to excessive B cell activation combined with Piezo1 activation, contributing to the breakdown of tolerance. (B) When mature B cells encounter (foreign) antigens for immune responses, soluble antigens provide weak BCR signaling and fail to open up Piezo1. However, particulate or membrane-bound antigens induce stronger BCR activation with Piezo1 activation, synergistically driving robust B cell activation.

The calmodulin-NFAT axis is more strictly regulated by intracellular calcium concentrations (42, 52). In B cells, however, in vitro stimulation via antibody-mediated BCR cross-linking often fails to generate a sustained and robust calcium influx which is required to activate NFAT-dependent transcriptional programs. As a result, studies often resort to artificial agents such as ionomycin or thapsigargin, which bypass physiological signaling pathways to induce maximal calcium mobilization. While these pharmacological tools have been invaluable for dissecting calcium-dependent pathways, they do not faithfully recapitulate the dynamics of calcium signaling during antigen recognition. As we highlight the key role of mechanosensitive ion channels during the initial phase of BCR signaling, we anticipate that these ion channels such as Piezo1 may serve as critical modulators of early BCR signaling. Future investigations into Piezo1-mediated calcium influx may offer more physiologically relevant insights into how calcium thresholds are achieved to modulate intracellular pathways and ultimately contribute to B cell tolerance.

T cell studies have demonstrated that TCR signaling, in conjunction with calcium influx, activates AP-1 and NFAT. Under such stimulation conditions, AP-1 and NFAT form heterodimers that drive a transcriptionally distinct program. In contrast, NFAT activation in the absence of AP-1 has been shown to promotes T cell anergy and exhaustion (53-55), clearly demonstrated by a study incorporating an engineered NFAT mutant lacking the AP-1 dimerization domain, which consequently induced T cell exhaustion (53). These findings suggest that B cells, closely related to T cells, may exhibit similar behavior to different combinations of BCR and calcium signaling patterns. The initial insights from T cell studies broaden our understanding of transcriptional regulation from a merely additive to a potentially synergistic model. Based on these groundwork, it is plausible that the diverse signals received by B cells, such as BCR signaling, T cell help, cytokines, integrin signaling, and additional co-receptor pathways, may cooperate with Piezo1-mediated pathways to orchestrate unique transcriptomic programs.

Various sources of mechanical force may initiate differential patterns of cellular signaling

Although we mostly focus on membrane force generated by B cells during the formation of IS, many other sources of mechanical force have been found to activate Piezo1 in other tissue environments. For example, in vascular studies, fluid shear stress created by blood flow is a critical source of mechanical force that opens up Piezo1 for ion uptake (56-58). In this case, force is constantly exerted leading to sustained or oscillatory signaling. Such calcium signaling patterns are known to activate the NFAT pathway (42). B cells may also experience cell crowding, compression, or even osmotic swelling which all can activate Piezo1. Yet, in vitro treatment of chemical-based agonists for Piezo1 activation generalizes these diversities which may be a significant limitation of current mechanobiology studies. To mimic physiological conditions, efforts have been made by many groups utilizing PLBs, laser-induced shockwaves (LIS), or the flow chamber system.

In particular, Pan and colleagues utilized the HEK 293T cell line to study the biophysical response of Piezo1 to LIS pulses or a chemical agonist (Yoda1) (59). Interestingly, transient (single LIS pulse or low dose Yoda1) activation of Piezo1 activated focal adhesion kinase (FAK) whereas sustained (multiple LIS or high dose Yoda1) activation of Piezo1 inhibited FAK, hinting that varying patterns of mechanical stimuli may promote differential signaling patterns. Directly applying these findings to B cell mechanobiology, transient mechanical stimuli such as cellular spreading and contraction may activate Piezo1 in a manner that promotes FAK activation, thereby facilitating dynamic actin remodeling during antigen engagement. In contrast, circulating peripheral B cells continuously exposed to shear stress in the bloodstream may experience sustained or oscillating Piezo1 activation, leading to FAK inactivation as a means of restraining inappropriate activation under homeostatic conditions. However, under B cell contexts, activation of Piezo1 is often associated with BCR signaling especially during the early events of B cell activation. Thus, studies incorporating HEK 293T cell lines may significantly differ in terms of intrinsic cellular behavior or lack of BCR compartments. These findings require further validation with primary B cells within specific activation contexts.

A more B cell relevant example comes from macrophage studies, where Atcha and colleagues demonstrated that macrophages sense substrate stiffness via Piezo1 to differentially polarize their responses: stiff substrates promote an M1-like pro-inflammatory phenotype, whereas soft environments favor an M2-like anti-inflammatory state (60). Like macrophages, B cells are also sensitive to substrate stiffness which results in differential BCR signaling patterns (36). Although our discussion has centered on whether Piezo1 is activated during B cell stimulation, it is plausible that once activated, Piezo1 may elicit a bimodal response depending on substrate stiffness, either promoting activation or reinforcing tolerance depending on the mechanical context.

As B cells navigate through diverse environments such as bloodstream, tissue migration, influx and efflux from secondary lymphoid organs, and cell-cell interaction, Piezo1 is expected to play diverse roles depending on the subset or physiological localization of B cells (Fig. 3). This review focuses on the influence of Piezo1 on intracellular signaling dynamics which ultimately connects to B cell fate decision via tolerance mechanisms. However, as Piezo1 is broadly expressed across various B cell subsets, further efforts are required to fully grasp its role in B cell function.

Fig. 3.

Various sources of mechanical force encountered by B cells across physiological contexts. During its life cycle, B cells experience various environments which exert different types of mechanical forces. Each of these environments are thought to provide different patterns of mechanical forces. For example, shear stress given by bloodstream is known to exert sustained, oscillating mechanical force. On the other hand, B cell migration to tissues results in a transient compression and squeezing force while B cells transmigrate into the tissues. During antigen recognition by antigen-presenting cells, B cells form the immune synapse which stretches the membrane apart. Such mechanical forces are sufficient to activate Piezo1 which may result in differential patterns of B cell responses.

FURTHER CLINICAL PERSPECTIVES

Since its discovery, Piezo1 has drawn increasing attention for its associations with a broad spectrum of human diseases. Both heightened and reduced Piezo1 activity have been implicated in pathology, underscoring its dual role in human diseases. Overexpression or gain-of-function mutations can drive conditions such as xerocytosis, altered iron metabolism, or psoriasis (61-63), while reduced expression or loss-of-function states are linked to multiple sclerosis, osteoarthritis susceptibility, and vascular dysfunction in SLE (64-66). These findings support the notion of a ‘Goldilocks effect,’ where Piezo1 activity must be maintained within a narrow range, too much or too little can be detrimental. Such nature makes Piezo1 a compelling yet challenging target for therapeutic intervention.

Recently, Goon and colleagues developed a new analog of Yoda1, Yaddle1, with enhanced solubility and structural conformation with Piezo1 (67). To demonstrate its potential as an adjuvant candidate, they tested this analog on a human T cell and successfully triggered a robust calcium influx. Nevertheless, these findings represent only an initial step, and substantial validation remains for further translational applications.

Moving forward, translating this knowledge into clinical applications will require extensive efforts including discovery and optimization of small molecules, validation of their efficacy, and development of reliable, cell type-specific delivery strategies. Given Piezo1’s broad expression across tissues, therapeutic approaches must be carefully optimized to achieve precision without off-target effects.

CONCLUSION

Since the discovery of the immune synapse formation to membrane-associated antigens, B cell research has made significant progress in uncovering the molecular and physical nature of the BCR–antigen interface. Furthermore, BCR signaling integrates not only ligand-receptor-mediated biochemical interactions but also mechanosensors, translating physical cues like membrane tension into immune outcomes. Mechanosensitive ion channels, markedly Piezo1, act as key mediator of this mechanotransduction, enabling B cells to discriminate between soluble (self) antigens and particulate or membrane-bound (non-self) antigens. While B cells are likely exposed to diverse sources of mechanical stress throughout their life cycle, this aspect remains largely unknown. Further investigation will be key to uncovering how mechanosensors like Piezo1 contribute to B cell function and fate.