Department of Medicine, Laboratory of Translational Immunology, Division of Immunology and Allergy, Faculty of Medicine, Geneva University Hospitals, Geneva, Switzerland.
Department of Physical Chemistry, Faculty of Science, University of Geneva, Geneva, Switzerland.
Sci Rep. 2024 Oct 15;14(1):24199. doi: 10.1038/s41598-024-75217-3.
Natural Killer (NK) cells are pivotal in immune responses to viral infections, malignancies, autoimmune diseases, and transplantation. Assessment of NK cell adhesion, migration, and cytotoxicity is fundamental for in vitro studies. We propose a novel live-cell tracking method that addresses these three major aspects of NK cell function using human NK cells and primary porcine aortic endothelial cells (PAECs) in two-dimensional (2D) static assays and an in-house cylindrical 3D microfluidic system. The results showed a significant increase of NK cytotoxicity against pTNF-activated PAECs, with apoptotic cell death observed in the majority of dead cells, while no difference was observed in the conventional Delfia assay. Computed analysis of NK cell trajectories revealed distinct migratory behaviors, including trajectory length, diameter, average speed, and arrest coefficient. In 3D microfluidic experiments, NK cell attachment to pTNF-activated PAECs substantially increased, accompanied by more dead PAECs compared to control conditions. NK cell trajectories showed versatile migration in various directions and interactions with PAECs. This study uniquely demonstrates NK attachment and killing in a 3D system that mimics blood vessel conditions. Our microscope method offers sensitive single-cell level results, addressing diverse aspects of NK functions. It is adaptable for studying other immune and target cells, providing insights into various biological questions.
自然杀伤 (NK) 细胞在病毒感染、恶性肿瘤、自身免疫性疾病和移植的免疫反应中起着关键作用。评估 NK 细胞的黏附、迁移和细胞毒性对于体外研究至关重要。我们提出了一种新的活细胞跟踪方法，该方法使用人 NK 细胞和原代猪主动脉内皮细胞 (PAEC) 在二维 (2D) 静态测定和内部圆柱形 3D 微流控系统中，解决了 NK 细胞功能的这三个主要方面。结果表明，NK 对 pTNF 激活的 PAEC 的细胞毒性显著增加，大多数死亡细胞观察到凋亡性细胞死亡，而在传统的 Delfia 测定中未观察到差异。对 NK 细胞轨迹的计算分析显示出不同的迁移行为，包括轨迹长度、直径、平均速度和阻滞系数。在 3D 微流控实验中，与对照条件相比，NK 细胞与 pTNF 激活的 PAEC 的附着显著增加，同时死亡的 PAEC 也更多。NK 细胞轨迹表现出各种方向的灵活迁移，并与 PAEC 相互作用。这项研究独特地证明了 3D 系统中 NK 的附着和杀伤，该系统模拟了血管条件。我们的显微镜方法提供了敏感的单细胞水平结果，解决了 NK 功能的多个方面。它适用于研究其他免疫和靶细胞，为各种生物学问题提供了深入的见解。
