Enhanced and Sustained T Cell Activation, Proliferation, and Cytotoxicity in Response to Fluid Shear Stress

Introduction: Adoptive T cell transfer (ACT) has transformed cancer treatment by leveraging a patient's immune system to fight tumors, with remarkable successes in treating hematological cancers. However, the effectiveness of ACT in solid tumors is compromised by inadequate ex vivo T cell activation, which is crucial for in vivo persistence and cytotoxicity. Traditional methods for activating T cells, such as CD3/CD28 antibody-coated beads (αCD3/CD28), are not only costly but also demonstrate limited efficacy in activating T cells derived from immunocompromised patients, highlighting the need for more effective activation strategies.
The work presented here utilizes mechanical forces, specifically fluid shear stress (FSS), to modulate T cell function. Mechanosensitive ion channels (MSCs), such as Piezo1, are critical transducers of mechanical signals into biochemical cues within T cells. Activation of MSCs by FSS leads to calcium influx and a cascade of cellular responses, as calcium serves as an essential second messenger in numerous cellular processes including T cell activation, proliferation, and cytotoxic activity. By integrating mechanical stimuli with biochemical signals, this approach aims to enhance the functional capabilities of T cells, offering a promising avenue to overcome current limitations in T cell-based therapies for solid tumors.

Materials and

Methods: In this study, primary human T cells were isolated from whole blood samples obtained from healthy adult donors via Ficoll Paque density gradient centrifugation. T cell populations were further purified through negative selection with either a Pan or CD8+ T Cell isolation kit. The isolated T cells were either activated with αCD3/CD28 or left unstimulated to serve as control samples. For mechanical stimulation experiments, the T cells were exposed to FSS at a magnitude of 5 dyn/cm² for 1 hour using cone-and-plate viscometers, an instrument designed to deliver a constant and uniform FSS. To assess the role of MSCs in T cell activation, some samples were treated with the MSC-specific inhibitor GsMTX-4 prior to FSS exposure. Following activation treatments, T cells were cultured and measured at various timepoints to determine T cell function dynamics (Immediately post-activation and 1, 2, 3, 5, 7, 10 days post-activation). T cell viability and activation were evaluated using flow cytometry to measure expression of activation markers (CD69, CD25), proliferation (Ki67+, VPD450), and cytotoxicity indicators (CD107a, granzyme-B, perforin). Intracellular calcium levels were monitored using the Fluo-4 AM dye, and protein expression related to activation pathways (phospho-NF-κB, NFATc1, and phospho-c-Fos) were evaluated in flow cytometry and confocal microscopy. Moreover, proinflamatory cytokine production and secretion were evaluated via intracellular cytokine staining in flow cytometry and ELISA, respectively, for key effector molecules like IFN-γ, TNF-α, and IL-2.

Results, Conclusions, and Discussions: In this study, we investigated the impact of constant and uniform mechanical stimulation on primary human T cell activation and function, in the context of developing novel strategies to improve ex vivo activation for a broad class of T cell-based applications such as ACT. We sought to map out a 10-day kinetic profile of T cell response to FSS in the presence and absence of αCD3/CD28, due to the dynamic nature of this process and the clinical relevance of T cell longevity for ACT.
We found that T cell exposure to FSS at 5 dyn/cm² for 1 hr in cone-and-plate viscometers synergistically enhanced the effects of traditional biochemical stimuli (αCD3/CD28), leading to a heightened proinflammatory phenotype in primary human T cells. Over the course of 10 days, T cells exposed to αCD3/CD28 and FSS were observed to exhibit a significantly increased and sustained expression of activation markers, CD69 and CD25, and increased signaling of T cell activation pathways, NF-κB, NFAT, and AP-1, suggesting the potential for prolonged T cell efficacy in therapeutic applications (Fig. 1A-B). Furthermore, the extent of cell division and active proliferation was measured in flow cytometry and confocal imaging utilizing proliferation dye (VPD450) and Ki67 staining. Our results indicate that FSS stimulation alone increases T cell proliferation (via VPD450) in an MSC-dependent manner, whereas this increase was eliminated with the treatment of GsMTX-4 (Fig. 1C). Furthermore, the combination of αCD3/CD28 and FSS significantly enhanced Ki67-DAPI colocalization evaluated via confocal imaging (Fig. 1D). Since some ACT therapies utilize only cytotoxic CD8+ T cells, we performed an additional T cell purification with a CD8+ T cell isolation kit and observed that FSS markedly increased the production of CD107a, granzyme-B (Fig. 1E), and perforin (Fig. 1F) in CD8+ T cells, enhancing their ability to target and kill tumor cells effectively. This was also accompanied by an increase in the secretion of key pro-inflammatory cytokines, IFN-γ, TNF-α, and IL-2 (Figure 1G). Our findings highlight the major role that mechanotransduction plays in influencing T cell function (Fig. 1H), offering significant implications for ex vivo T cell manufacturing strategies.

Acknowledgements (Optional):