Palamarchuk Anastasia I, Kovalenko Elena I, Streltsova Maria A
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Biomedicines. 2024 Mar 14;12(3):650. doi: 10.3390/biomedicines12030650.
The NK cell exhaustion state evolving during extensive and prolonged cultivation is still one of the limitations of NK cell approaches. In this research, we transduced NK cells with the and genes. hTERT overexpression can prevent the functional exhaustion of NK cells during long-term cultivation, but, still, the therapeutic use of such cells is unsafe without irradiation. To overcome this obstacle, we additionally transduced NK cells with the transgene that enables the rapid elimination of modified cells. We compared the proliferative and functional activities of the hTERT- and/or iCasp9-modified NK cells, determined their exhaustion state and monitored the levels of EOMES and T-BET, the main NK cell transcription factors. The and genes were shown to affect the EOMES and T-BET levels differently in the NK cells. The EOMEST-BET phenotype characterized the functionally active NK cells during two months of culture upon stimulation with IL2 and K562-mbIL21 feeder cells, which induced the greatest expansion rates of the NK cells, independently of the transgene type. On the other hand, under cytokine stimulation, the hTERT-iCasp9-NK cells displayed improved proliferation over NK cells modified with iCasp9 alone and showed an increased proliferation rate compared to the untransduced NK cells under stimulation with IL2 and IL15, which was accompanied by reduced immune checkpoint molecule expression. The individual changes in the EOMES and T-BET levels strictly corresponded to the NK cell functional activity, the surface levels of activating and inhibitory receptors along with the expansion rate and expression levels of pro-survival and pro-apoptotic genes.
在广泛且长期培养过程中自然出现的NK细胞耗竭状态仍是NK细胞疗法的局限之一。在本研究中，我们用 和 基因转导NK细胞。hTERT的过表达能够防止NK细胞在长期培养过程中发生功能耗竭，但是，未经辐照的这类细胞用于治疗仍不安全。为克服这一障碍，我们额外用 转基因转导NK细胞，该转基因能够快速清除修饰细胞。我们比较了hTERT和/或iCasp9修饰的NK细胞的增殖和功能活性，确定它们的耗竭状态，并监测主要NK细胞转录因子EOMES和T-BET的水平。结果显示， 和 基因对NK细胞中EOMES和T-BET水平的影响不同。在用IL2和K562-mbIL21饲养细胞刺激培养的两个月期间，EOMES/T-BET表型表征功能活跃的NK细胞，这两种饲养细胞能诱导NK细胞实现最大程度的扩增，且与转基因类型无关。另一方面，在细胞因子刺激下，hTERT-iCasp9-NK细胞相比于仅用iCasp9修饰的NK细胞增殖能力增强，并且与未转导的NK细胞相比，在IL2和IL15刺激下增殖速率增加，同时免疫检查点分子表达降低。EOMES和T-BET水平的个体变化与NK细胞的功能活性、激活和抑制性受体的表面水平以及促生存和促凋亡基因的扩增速率和表达水平严格对应。
