First, K562 cells overexpressing 4-1BBL and membrane-bound IL-21 (mbIL-21) were constructed (K562-41BBL-mbIL-21) and were sorted to generate the single clone. These widely used feeder cells (K562-41BBL-mbIL-21) were named as Basic Feeder hereinafter. Based on the Basic feeder, CD48 was overexpressed and named as CD48 Feeder. Then, the genetically modified feeder cells were used to expand primary NK cells from peripheral blood or umbilical cord blood. In vitro experiments were performed to compare proliferation ability, cytotoxicity, survival and activation/inhibition phenotypes of NK cells stimulated via different feeder cells. K562 cells were injected into nude mice subcutaneously with tail vein injection of NK cells from different feeder system for the detection of NK in vivo persistence and function.
Compared with Basic Feeders, CD48 Feeders can promote the proliferation of primary NK cells from peripheral blood and umbilical cord blood and reduce NK cell apoptosis by activating the p-ERK/BCL2 pathway both in vitro and in vivo without affecting overall phenotypes. Furthermore, NK cells expanded via CD48 Feeders showed stronger anti-tumor capability and infiltration ability into the tumor microenvironment.
In this preclinical study, the engagement of the 2B4-CD48 axis can inhibit the apoptosis of NK cells through the p-ERK/BCL2 signal pathway, leading to an improvement in therapeutic efficiency.
方法:首先,构建过表达4-1BBL和膜结合IL-21(mbIL-21)的K562细胞(K562-41BBL-mbIL-21),并进行分选以产生单个克隆。这些广泛使用的饲养细胞(K562-41BBL-mbIL-21)在下文中被命名为基本饲养细胞。基于基本馈线,CD48过表达并命名为CD48给料机。然后,基因修饰的饲养细胞用于扩增外周血或脐带血的原代NK细胞.进行体外实验以比较增殖能力,细胞毒性,通过不同饲养细胞刺激的NK细胞的存活和活化/抑制表型。将来自不同饲养系统的NK细胞尾静脉注射K562细胞,皮下注射到裸鼠体内,以检测NK在体内的持久性和功能。
结果:与基本馈线相比,CD48饲养者可以通过激活p-ERK/BCL2途径在体外和体内促进外周血和脐带血的原代NK细胞的增殖,并减少NK细胞的凋亡,而不影响整体表型。此外,通过CD48Feeders扩增的NK细胞显示出更强的抗肿瘤能力和向肿瘤微环境的浸润能力。
结论:在这项临床前研究中,参与2B4-CD48轴可通过p-ERK/BCL2信号通路抑制NK细胞凋亡,导致治疗效率的提高。