Zhou Z, Liu S, Li J, Chen M, Lin H, Chen Y, Chen W, Lin J, Zhou H, Zheng Q
Laboratory of Immuno-Oncology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou 350014, China.
School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350100, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Oct 20;44(10):1926-1936. doi: 10.12122/j.issn.1673-4254.2024.10.11.
To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.
The modified IL-21-CCL19 NKP30 CAR-T cells expressing IL-21 and CCL19 fusion gene was constructed based on NKP30 CAR-T cells and stimulated with CD3CD28 antibodies and IL-2. The immunophenotype and migration of the cells in the presence of IL-21 were investigated using flow cytometry and migration experiments. Lactate dehydrogenase (LDH) release and sphere formation assays were used to assess the killing and infiltration capabilities of CAR-T cells, and the secretion levels of IFN-γ, IL-21 and CCL19 were determined with enzyme-linked immunospot assay (ELISPOT) and ELISA. A zebrafish model bearing HCG-27 cell xenograft was established by microinjection of the tumor cells into the yolk sac followed 24 h later by injection of the immune cells at the same site, and the fluorescence signals were captured using a fluorescent microscopy.
The NKP30 ligand B7H6, which was almost undetectable in normal tissues and blood cells, was highly expressed (over 90%) in lung cancer cells. Compared with NKP30 CAR-T cells and conventional T cells, IL-21-CCL19 NKP30 CAR-T cells exhibited stronger proliferative and migration capabilities with the formation of central memory T cells. The reduced expressions of CTLA4 and PD1 in the constructed cells resulted in enhanced killing efficiency against lung cancer cells accompanied by significantly increased production of IFN-γ, IL-21 and CCL19. In the zebrafish models, CAR-T cells exhibited stronger cytotoxicity and proliferative abilities than typical T cells, but these differences were not statistically significant between the two CAR-T cells.
Modification of NKP30 CAR-T cells with IL-21 and CCL19 facilitates their access into solid tumors for more effective tumor cell killing while producing a large number of memory T cells.
研究用白细胞介素-21（IL-21）和CC趋化因子配体19（CCL19）修饰是否能增强自然杀伤细胞蛋白30（NKP30）嵌合抗原受体T细胞（CAR-T细胞）在肺癌中的杀伤及肿瘤浸润效率。
基于NKP30 CAR-T细胞构建表达IL-21和CCL19融合基因的修饰型IL-21-CCL19 NKP30 CAR-T细胞，并用抗CD3CD28抗体和IL-2进行刺激。采用流式细胞术和迁移实验研究细胞在IL-21存在下的免疫表型和迁移情况。用乳酸脱氢酶（LDH）释放实验和球形成实验评估CAR-T细胞的杀伤和浸润能力，并用酶联免疫斑点分析（ELISPOT）和酶联免疫吸附测定（ELISA）测定干扰素-γ（IFN-γ）、IL-21和CCL19的分泌水平。通过将肿瘤细胞显微注射到卵黄囊中建立携带人绒毛膜癌27（HCG-27）细胞异种移植的斑马鱼模型，24小时后在同一部位注射免疫细胞，并用荧光显微镜捕获荧光信号。
NKP30配体B7H6在正常组织和血细胞中几乎检测不到，但在肺癌细胞中高表达（超过90%）。与NKP30 CAR-T细胞和传统T细胞相比，IL-21-CCL19 NKP30 CAR-T细胞表现出更强的增殖和迁移能力，并形成了中央记忆T细胞。构建细胞中细胞毒性T淋巴细胞相关抗原4（CTLA4）和程序性死亡蛋白1（PD1）表达降低，导致对肺癌细胞的杀伤效率增强，同时IFN-γ、IL-21和CCL19的产生显著增加。在斑马鱼模型中，CAR-T细胞比典型T细胞表现出更强的细胞毒性和增殖能力，但两种CAR-T细胞之间的这些差异无统计学意义。
用IL-21和CCL19修饰NKP30 CAR-T细胞有助于它们进入实体瘤，以更有效地杀伤肿瘤细胞，同时产生大量记忆T细胞。
