Leveraging T cell mechanotransduction for enhanced and sustained ex vivo activation

Introduction:: Adoptive T cell transfer (ACT) is a promising form of immunotherapy that has shown remarkable clinical success in treating hematological cancers. The process involves isolating T cells from the patient, activating and expanding them outside the body, and then reinfusing them back into the patient to enhance their immune system's ability to combat the disease. However, ACT's efficacy in treating solid tumors is limited due to poor T cell cytotoxicity and persistence in vivo, which can be attributed to the lack of adequate T cell activation prior to reinfusion. One common approach for T cell activation is the addition of anti-CD3/CD28 antibody coated magnetic beads (Dynabeads); however, this method is expensive, low-throughput, and often insufficient in activating exhausted cancer patient T cells. Since T cell activation is reliant on physical and chemical signals in their environment, it is essential to understand the mechanisms by which optimal T cell activation can be achieved ex vivo to ensure their persistence in vivo. Piezo1, a mechanosensitive ion channel, transduces physical stimuli into biochemical signals, where cell membrane deformation caused by fluid shear stress (FSS) leads to calcium influx, ultimately affecting T cell activation, proliferation, differentiation, and cytotoxic effector functions. In this study, we demonstrate that primary T cells exposed to FSS ex vivo in cone-and-plate viscometers display a remarkable increase in T cell activation, giving rise to an inexpensive and high-throughput approach for ex vivo T cell activation for ACT therapies.




Materials and Methods:: Primary T cells were isolated from whole blood samples from healthy donors. T cells were activated with or without Dynabeads and subjected to constant fluid shear stress (FSS) at a rate of 5.0 dyn/cm² for 1 hr using cone-and-plate viscometers (Brookfield). Following activation, T cells were cultured and evaluated at multiple timepoints ranging from 0 hr - 7 days. Indicators of T cell activation, such as the phosphorylation of nuclear factor kappa beta (phos-NF-κB) and expression of CD69 and CD25 were quantified using flow cytometry. T cell subpopulations were identified using CD4 and CD8 markers in flow cytometry prior to activation and over time. To gain further insight into FSS's impact on T cell function, the secretion of proinflammatory cytokines, including interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2), were evaluated in flow cytometry. Intracellular calcium levels via Fluor-4, NFkB phosphorylation, and the colocalization of the T cell nucleus with transcription factors involved in activation and proliferation, such as nuclear factor of activated T cells (NFAT) and Ki67, were quantified using a Zeiss LSM900 confocal microscope. T cell Piezo1 expression was evaluated in flow cytometry and confocal microscopy prior to activation.




Results, Conclusions, and Discussions:: In this study, we found that primary T cell activation was significantly enhanced and sustained over time by using both ex vivo Dynabead and FSS activation, as compared to Dynabead activation alone. We observed an increase in activation markers (phos-NF-κB, CD69, CD25) in flow cytometry, with sustained activation for up to 7 days (Fig. 1A). Furthermore, T cells activated with FSS in combination with Dynabeads led to an increased secretion of proinflammatory cytokines (IFN-γ, TNF-α, and IL-2) when compared to T cells activated with Dynabeads alone (Fig. 1B). While FSS alone did not significantly upregulate activation markers, the proinflammatory cytokine secretion was greater in T cells treated with only FSS. Confocal microscopy further corroborated enhanced T cell activation, as demonstrated by increased fluorescence intensity of phosphorylated NF-κB. Interestingly, T cells exposed to FSS exhibited decreased actin fluorescence intensity, whereas Dynabead treatment led to a significant increase in T cell area (Fig. 1C, 1D). We also quantified Piezo1 expression before and after FSS exposure and found a strong negative correlation between Piezo1 expression and phos-NF-κB 48 hours post-FSS (Fig. 1E). This relationship between Piezo1 and the capacity for NF-κB phosphorylation is likely attributed to increased Piezo1 expression and subsequent increase in calcium influx leading to activation-induced cell death (AICD). This study demonstrates that ex vivo mechanical stimulation of Piezo1 enhances primary T cell activation and function in a Piezo1-dependent manner, with implications for potential therapeutic interventions for a variety of diseases that are characteristic of impaired immune cell function and of altered blood flow velocity. Further implications of this work suggest new approaches that utilize FSS to significantly augment T cell activation, enabling an inexpensive and high-throughput methodology for ex vivo immune cell activation in the context of adoptive T cell immunotherapies.




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