Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. This study introduces a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. We hypothesize that utilizing high-voltage pulsed electric fields may be an ideal method to activate and expand CTLs due to their non-thermal cell death mechanism.

Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes (1250, 2500 V/cm) and pulse widths (1, 5, and 10 µs), or irreversible electroporation (IRE) at 1250 V/cm. The treated tumor cells were subsequently cocultured with CD4+ and CD8+ T cells along with antigen-presenting cells.

We show that tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. Our results suggest that CTLs are more effectively expanded when pulsed with HFIRE conditions that induce significant cell death (longer pulse widths and higher voltages). Activated CD8+ T cells demonstrate cytotoxicity to untreated tumor cells suggesting effector function of the activated CTLs. The activated CTLs produced with our technique could be used for clinical applications with the goal of targeting and eliminating the tumor.

细胞毒性 T 淋巴细胞 (CTL) 的扩增是几乎所有癌症免疫治疗方法中的关键步骤。目前用于扩增肿瘤反应性 CTL 的技术存在主要局限性。本研究介绍了一种使用高压脉冲电场有效生产和扩增肿瘤激活 CTL 的新方法。我们假设利用高压脉冲电场可能是激活和扩展 CTL 的理想方法，因为它们的非热细胞 死亡机制。

对肿瘤细胞进行具有各种电场强度（1250、2500 V/cm）和脉冲宽度（1、5 和 10 µs）的高频不可逆电穿孔 (HFIRE)，或 1250 V/cm 的不可逆电穿孔 (IRE) . 经处理的肿瘤细胞随后与 CD4+ 和 CD8+ T 细胞以及抗原呈递细胞共培养。

我们表明，当暴露于处理过的肿瘤细胞时，可以产生和扩增肿瘤激活的 CTL。我们的结果表明，当使用诱导显着细胞死亡的 HFIRE 条件（更长的脉冲宽度和更高的电压）进行脉冲时，CTL 会更有效地扩展。激活的 CD8+ T 细胞对未处理的肿瘤细胞表现出细胞毒性，表明激活的 CTL 具有效应器功能。用我们的技术生产的活化 CTL 可用于临床应用，目标是靶向和消除肿瘤。