Abstract：
Decades after the identification of natural killer (NK) cells as potential effector cells against malignantly transformed cells, an increasing amount of research suggests that NK cells are a prospective choice of immunocytes for cancer immunotherapy in addition to T lymphocytes for cancer immunotherapy. Recent studies have led to a breakthrough in the combination of hematopoietic stem-cell transplantation with allogeneic NK cells infusion for the treatment of malignant tumors. However, the short lifespan of NK cells in patients is the major impediment, limiting their efficacy. Therefore, prolonging the survival of NK cells will promote the application of NK-cell immunotherapy. As we have known, NK cells use a \"missing-self\" mechanism to lyse target cells and exert their functions through a wide array of activating, co-stimulatory and inhibitory receptors. Our previous study has suggested that CD244 (2B4), one of the co-stimulatory receptors, can improve the function of chimeric antigen receptor NK cells. However, the underlying mechanism of how 2B4 engages in the function of NK cells requires further investigation. Overall, we established a feeder cell with the expression of CD48, the ligand of 2B4, to investigate the function of 2B4-CD48 axis in NK cells, and meanwhile, to explore whether the newly generated feeder cell can improve the function of ex vivo-expanded NK cells.
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.
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.
摘要：
目的：自然杀伤（NK）细胞被鉴定为对抗恶性转化细胞的潜在效应细胞，越来越多的研究表明,除了用于癌症免疫治疗的T淋巴细胞外,NK细胞也是用于癌症免疫治疗的免疫细胞的前瞻性选择.最近的研究在造血干细胞移植与同种异体NK细胞输注相结合治疗恶性肿瘤方面取得了突破。然而,患者NK细胞的短寿命是主要障碍，限制其功效。因此,延长NK细胞的存活时间将促进NK细胞免疫治疗的应用。正如我们所知道的,NK细胞使用“自身缺失”机制来裂解靶细胞，并通过广泛的激活来发挥其功能，共刺激和抑制受体。我们之前的研究表明，CD244(2B4)，一种共刺激受体,能提高嵌合抗原受体NK细胞的功能。然而,2B4如何参与NK细胞功能的潜在机制需要进一步研究.总的来说,我们建立了一个表达CD48的饲养细胞，CD48是2B4的配体，以研究2B4-CD48轴在NK细胞中的功能，同时,探讨新产生的饲养细胞是否可以改善离体扩增的NK细胞的功能。
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。
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。
