Assistant Professor Stony Brook University, United States
Introduction: CAR-T therapy is a promising immunotherapy that reprograms a patient's T-cells to target cancer. Im-portantly, the average age of incidence for many of the cancers that CAR-T therapy is indicated for (DLBCL, follicular lymphoma, etc.)1 is over 65 years old. This becomes an issue as older patient’s T-cells are known to be less sensitive to activation signals and less func-tional2, which reduces the efficacy of CAR-T therapy. Reduced sensitivity to activation also reduces their ability to proliferate. Another critical issue for CAR-T therapy is T-Cell exhaustion, characterized by tran-scriptional changes which reduce cytokine production and induce upregulation of exhaustion receptors. The proteins 𝜷-Catenin and T-BET play an important role in T-cell activation and exhaustion signaling. Strate-gies that increase 𝜷-Catenin and T-BET would help improve the health of cells and likely increase the effi-cacy of CAR-T Therapy. The Chan/Rubin lab has stud-ied cell mechanosensitivity and the effect of mechani-cal signals such as Low Intensity Vibration (LIV) in a variety of cell types. In this pilot study the we aim to leverage cell mechanosensitivity to improve cell pro-liferation while minimizing T-Cell exhaustion signal-ing pathways.
Materials and
Methods: Low Intensity Vibration (LIV) was used to mechanically stimulate human primary T-Cells. LIV is characterized as a low magnitude signal with a frequency between 10-150Hz delivered via sinusoidal wave form. T-Cells were treated with IL-2 (10ng/mL), CD3+/CD28+ DynaBeads (1:2, DynaBeads:Cells), and the experimental group was exposed to LIV stimulation (0.7 g; g = 9.8m/s^2, 30Hz for 1hr, 2x daily with a 2hr rest period) for 5 days. Human primary T-cells were isolated from whole blood collected in the Stony Brook Blood Bank. T-Cells were then plated and treated with LIV for five days. On days 3 and 5 cell number was analyzed. ELISA assays were performed to quantify changes in 𝜷-Catenin concentration between groups. Real Time rtPCR was performed with both GAPDH and TBP-43 as endogenous controls to determine transcriptional changes in T-BET expression.
Results, Conclusions, and Discussions: By Day 5 LIV had improved T-Cell numbers by a 57% in the elderly sample (p < 0.005, Fig 1). Notably, LIV was more effective in elderly patients’ cells compared to young cells where there was no significant increase in proliferation by day 5. In general, however the younger patients’ cells saw a more growth than older cell counterparts. Exhaustion is a well characterized phenotype of T-Cells that reduces T-Cell activation sensitivity. Improvements in \beta-Catenin expression can improve activation signaling. Similarly, improvements in T-BET signaling reduce inhibitory receptor PD-1 expression. \mathbit{\beta}-Catenin expression showed differences by day 5, LIV treated samples saw an overall increase in expression by 21% (p < 0.005) compared to the controls. A crucial intermediary for T-Cell activation. Furthermore, T-BET expression was conserved or marginally (0.7%, p< 0.005) improved in LIV samples, an important regulator of T-Cell exhaustion receptors, conserved levels of T-BET expression indicate conservation of PD-1 expression. CAR-T manufacturers often have trouble expanding CAR-T cells from elderly patients to clinically relevant numbers due to decreased sensitivity of elderly cells. Many attempts to improve T-Cell number do so at the cost of functionality (Reductions in sensitivity, memory and issues of exhaustion) which is especially relevant in the age group for most CAR-T treatable cancers. This presents a significant issue for the manu-facturing of CAR-T treatments that could be improved upon by LIV. LIV was able to improve T-Cell number in older patients by 57% while also retaining T-BET expression and improving 𝜷-Catenin expression. This indicates an improvement or retention of functionality while also increasing the number of viable cells. LIV may represent a method to non-invasively stimulate elderly patients’ T-Cells to return them to a growth rate of a younger T-Cell without generating exhaustion.
Acknowledgements (Optional): National Institutes of Health REACH initiative, The Manufacturing and Technology Resource Consortium, and the New York Manufacturing Extension Partner-ship Center, and the Technology Accelerator Fund
