E.D. Butcher Chair of Bioengineering, Associate Vice President for Research Rice University, United States
Introduction: : Immune cells express several ion channels, such as Piezo1, which recognize different forms and/or magnitudes of mechanical cues, leading to an influx of ions such as calcium. Intracellular calcium triggers multiple signaling pathways that drive immune cell activation. Researchers have employed various mechanical stimuli to activate immune cells, including compressive forces, tensile stretch, and substrate stiffness1,2. B cells, key regulators of the adaptive immune system, recognize soluble or membrane-presented antigens through B cell receptors (BCRs), leading to activation and antibody production. Although limited research has been conducted on the mechanoactivation of B cells, emerging evidence suggests that mechanical forces play a significant role in their activation. We hypothesize that mechanical forces induce calcium influx in B cells, triggering a cascade of events leading to their activation and differentiation. As a result, we aim to develop a gentle yet effective immunotherapy that can target deep tissues, including solid tumors and immune organs, to activate B cells.
Materials and
Methods: : PBMCs were isolated from human blood, and B cells subsequently purified from the isolated PBMCs. Freshly isolated B cells were stained with a calcium-sensitive dye and subjected to mechanical stimulation via either fluid shear stress2 (applied using a VIAFLO 96 multichannel pipetting device equipped with 22G needles, generating forces up to 290 dyn/cm²) or ultrasound forces (frequency and intensity up to 1.1 MHz and 1.5 MPa, respectively), in the presence or absence of the mechanosensitive ion channel inhibitor GsMTX4. Calcium influx was then assessed using flow cytometry. Mechanically stimulated B cells were then cultured and harvested at specified time points using various biological methods including flow cytometry and confocal microscopy analysis to evaluate B cells activation.
Results, Conclusions, and Discussions:: Results and
Discussion: Confocal microscopy confirmed robust expression of Piezo1 in B cells, as shown in Fig 1a. Calcium influx assays performed on freshly isolated primary B cells subjected to mechanical stimulation demonstrated that ultrasound induced a significant increase in intracellular calcium levels (Fig. 1b). Shear stress induced a limited calcium influx, which was significantly lower than that observed under ultrasound conditions. Live-dead cell assays indicated that the viability of mechanically stimulated B cells was comparable to that of untreated control cells, confirming that the applied mechanical forces did not adversely affect cell viability. Furthermore, flow cytometry analysis revealed a marked upregulation of the activation marker CD69 in B cells following mechanical stimulation (Fig 1c). While these results support the hypothesis that mechanical forces induce calcium influx and activate B cells further analysis needs to be performed to fully determine the role of Piezo1-mediated calcium influx. Extensive experiments and analysis have been performed to determine the precise mechanisms by which B cells are activated.
Conclusions: Mechanosensors such as Piezo1 play a critical role in sensing mechanical forces to initiate calcium influx and immune cell activation. B cells have been reported to respond to some mechanical stimuli such as substrate stiffness. We specifically hypothesized that our novel mechanical stimulation method would induce calcium influx in B cells and trigger their activation. Our results demonstrate that this approach successfully elicits calcium influx and leads to CD69 overexpression-a hallmark of B cell activation-supporting the mechanistic link between mechanical forces and B cells activation via mechanosensitive pathways.
