Department of General Pediatrics, Hematology and Oncology, University Children's Hospital Tübingen, 72076 Tübingen, Germany.
MaxCyte Inc., Rockville, MD 20850, USA.
Int J Mol Sci. 2023 Nov 8;24(22):16065. doi: 10.3390/ijms242216065.
Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout , , , and . The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that and knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.
自然杀伤 (NK) 细胞免疫疗法已成为治疗各种癌症类型（包括白血病）的新方法。近年来，在临床前和临床环境中，对 T 细胞和 NK 细胞抑制性信号通路的调节已成为广泛研究的课题。然而，为了优化抗白血病活性，特别是针对基于 NK 细胞的免疫疗法，仍需要进一步的研究。本研究的核心科学问题是通过抑制抑制性受体来增强扩增和激活的 NK 细胞的细胞毒性的潜力。为了解决这个问题，我们使用 CRISPR-Cas9 系统靶向 NK 细胞中的三个不同的抑制性信号通路。具体来说，我们研究了 A2AR 在代谢嘌呤能信号通路中的作用、CBLB 作为 NK 细胞中的细胞内调节剂、以及表面受体 NKG2A 和 CD96 在增强 NK 细胞抗白血病功效中的作用。在 NK 细胞成功扩增后，我们用 Cas9+sgRNA RNP 转染它们以敲除 、 、 、和 。对所有四个靶点的插入缺失频率的分析表明，在扩增的 NK 细胞中，敲除效率良好，导致蛋白表达减少，这一点通过流式细胞术和 Western blot 分析得到了证实。我们的体外杀伤实验表明， 和 敲除仅导致 NK 细胞对 AML 和 B-ALL 细胞的细胞毒性略有改善。此外， 敲除 NK 细胞的抗白血病活性没有显著增强，并且 A2AR 的阻断也没有导致杀伤效率的显著提高。总之，我们的研究结果表明，基于 CRISPR-Cas9 的免疫检查点敲除策略可能不足以有效地增强扩增（和激活）NK 细胞的抗白血病功能，同时也需要强大的细胞激活信号，例如通过转基因嵌合抗原受体表达来实现。
