IL-21和CCL19修饰可提高NKP30 CAR-T细胞对肺癌的杀伤效率并促进其肿瘤浸润  

Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer

作　　者：周智锋[1,2,3] 柳硕岩 李洁羽[1,2,3] 陈明秋[5] 林辉 陈宇杰[4] 陈伟杰[4] 林军鹏 周航 郑庆丰 ZHOU Zhifeng;LIU Shuoyan;LI Jieyu;CHEN Mingqiu;LIN Hui;CHEN Yujie;CHEN Weijie;LIN Junpeng;ZHOU Hang;ZHENG Qinfeng(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;Fujian Key Laboratory of Translational Cancer Medicine,Fuzhou 350014,China;Department of Thoracic Surgery,Clinical Oncology School of Fujian Medical University,Fujian Cancer Hospital,Fuzhou 350014,China;Department of Thoracic Oncology Radiation,Clinical Oncology School of Fujian Medical University,Fujian Cancer Hospital,Fuzhou 350014,China)

机构地区：[1]福建医科大学肿瘤临床医学院//福建省肿瘤医院肿瘤免疫研究室,福建福州350014 [2]福建医科大学基础医学院,福建福州350100 [3]福建省肿瘤转化医学重点实验室,福建福州350014 [4]福建医科大学肿瘤临床医学院//福建省肿瘤医院胸部外科,福建福州350014 [5]福建医科大学肿瘤临床医学院//福建省肿瘤医院胸部肿瘤放疗科,福建福州350014

出　　处：《南方医科大学学报》2024年第10期1926-1936,共11页Journal of Southern Medical University

基　　金：国家自然科学基金(82173051);福建省自然科学基金(2022J01426);福建省科技创新联合资金项目(2023Y9407);福建省肿瘤医院院内课题(2023YN07)。

摘　　要：目的探讨细胞因子IL-21和趋化因子CCL19修饰的NKP30 CAR-T细胞是否增强对肺癌的杀伤和浸润作用。方法在NKP30 CAR基础上融合基因IL-21和CCL19构建IL-21-CCL19 NKP30 CAR;CAR-T细胞的培养使用CD3CD28单抗及细胞因子IL-2刺激;流式细胞术检测免疫细胞表型;迁移实验检测IL-21对免疫细胞的迁移作用;乳酸脱氢酶(LDH)及成球实验检测CAR-T细胞的杀伤及浸润能力;酶联免疫斑点技术(ELISPOT)检测IFN-γ的分泌数量;ELISA检测IL-21及CCL19的分泌情况;体内实验中,将肿瘤细胞显微注射到斑马鱼卵黄囊,构建斑马鱼移植瘤模型,24 h后将免疫细胞注射至同样部位,体式荧光显微镜拍摄荧光。结果NKP30配体(B7H6)在正常组织及血液细胞不表达,在肺癌细胞上高表达(90%以上)。IL-21-CCL19 NKP30 CAR-T细胞与NKP30 CAR-T细胞和常规T细胞相比,具有更强的增殖能力、迁移能力及中心记忆T细胞的形成(P<0.001),免疫抑制分子CTLA4与PD1显著降低(P<0.005),对肺癌细胞具有更强的杀伤能力(P<0.001),伴随IFN-γ数量明显增加(P<0.001)。IL-21-CCL19 CAR-T细胞杀伤肺癌细胞中产生大量细胞因子IL‑21(3152.33±526.74 pg/mL)和趋化因子CCL19(1853±211.95 pg/mL)。体内实验中,CAR-T细胞和普通T细胞比较,具有较强的杀伤能力和增殖能力,但2种CAR-T细胞无明显差异(P>0.05)。结论IL-21-CCL19 NKP30 CAR-T细胞更容易浸润到肿瘤内部,有效杀伤肿瘤细胞,同时产生更多的记忆T细胞。Objective To investigate whether modification with IL-21 and CCL19 enhances killing and tumor-infiltrating efficiency of NKP30 CAR-T cells in lung cancer.Methods 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.Results 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.Conclusion 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.

关 键 词：肺癌 NKP30 嵌合抗原受体基因修饰T淋巴细胞 IL-21 CCL19 

分 类 号：R734.2[医药卫生—肿瘤]