State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Tianjin Key Laboratory of Cell Therapy for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Tianjin, China; Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Tianjin Key Laboratory of Cell Therapy for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Tianjin, China; Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
Cytotherapy. 2023 Oct;25(10):1080-1090. doi: 10.1016/j.jcyt.2023.07.002. Epub 2023 Jul 29.
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.
在鉴定自然杀伤 (NK) 细胞作为对抗恶性转化细胞的潜在效应细胞几十年后，越来越多的研究表明，NK 细胞除了 T 淋巴细胞外，也是癌症免疫治疗中免疫细胞的理想选择。最近的研究在造血干细胞移植与异体 NK 细胞输注联合治疗恶性肿瘤方面取得了突破。然而，患者 NK 细胞的寿命短是主要障碍，限制了它们的疗效。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。众所周知，NK 细胞使用“缺失自我”机制来裂解靶细胞，并通过广泛的激活、共刺激和抑制受体发挥其功能。我们之前的研究表明，共刺激受体 CD244（2B4）之一可以改善嵌合抗原受体 NK 细胞的功能。然而，2B4 如何参与 NK 细胞的功能的潜在机制仍需要进一步研究。总的来说，我们构建了表达 2B4 配体 CD48 的饲养细胞，以研究 2B4-CD48 轴在 NK 细胞中的功能，同时探索新生成的饲养细胞是否能改善体外扩增的 NK 细胞的功能。
首先，构建了过表达 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克隆。这些广泛使用的饲养细胞（K562-41BBL-mbIL-21）被命名为基础饲养细胞。在此基础上，过表达 CD48，命名为 CD48 饲养细胞。然后，利用基因修饰的饲养细胞从外周血或脐带血中扩增原代 NK 细胞。进行体外实验比较不同饲养细胞刺激的 NK 细胞的增殖能力、细胞毒性、存活和激活/抑制表型。通过尾静脉注射 NK 细胞将 K562 细胞注入裸鼠皮下，检测不同饲养系统 NK 细胞在体内的持久性和功能。
与基础饲养细胞相比，CD48 饲养细胞可通过激活 p-ERK/BCL2 信号通路促进外周血和脐带血来源的原代 NK 细胞的增殖，并减少 NK 细胞凋亡，在体内外均不影响整体表型。此外，通过 CD48 饲养细胞扩增的 NK 细胞具有更强的抗肿瘤能力和浸润肿瘤微环境的能力。
在这项临床前研究中，2B4-CD48 轴的结合可以通过 p-ERK/BCL2 信号通路抑制 NK 细胞的凋亡，从而提高治疗效率。
