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

doi:10.12122/j.issn.1673-4254.2024.10.11

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1926-1936.doi: 10.12122/j.issn.1673-4254.2024.10.11

• • 上一篇

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

周智锋¹^,²^,³(), 柳硕岩⁴, 李洁羽¹^,²^,³, 陈明秋⁵, 林辉⁴, 陈宇杰⁴, 陈伟杰⁴, 林军鹏⁴, 周航⁴, 郑庆丰⁴()

^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

收稿日期:2024-01-11 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 郑庆丰 E-mail:zzf2004312 @fjzlhospital.com;zhqf@msn.com
作者简介:周智锋，硕士，副主任技师，硕士生导师，E-mail: zzf2004312 @fjzlhospital.com
基金资助:
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)

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

Zhifeng ZHOU¹^,²^,³(), Shuoyan LIU⁴, Jieyu LI¹^,²^,³, Mingqiu CHEN⁵, Hui LIN⁴, Yujie CHEN⁴, Weijie CHEN⁴, Junpeng LIN⁴, Hang ZHOU⁴, Qinfeng ZHENG⁴()

^1.Laboratory of Immuno-Oncology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou 350014, China
^2.School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350100, China
^3.Fujian Key Laboratory of Translational Cancer Medicine, Fuzhou 350014, China
^4.Department of Thoracic Surgery, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou 350014, China
^5.Department of Thoracic Oncology Radiation, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou 350014, China

Received:2024-01-11 Online:2024-10-20 Published:2024-10-31
Contact: Qinfeng ZHENG E-mail:zzf2004312 @fjzlhospital.com;zhqf@msn.com
Supported by:
National Natural Science Foundation of China(82173051)

摘要/Abstract

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。

关键词: 肺癌, NKP30, 嵌合抗原受体基因修饰T淋巴细胞, IL-21, CCL19

Abstract:

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.

周智锋, 柳硕岩, 李洁羽, 陈明秋, 林辉, 陈宇杰, 陈伟杰, 林军鹏, 周航, 郑庆丰. 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[J]. 南方医科大学学报, 2024, 44(10): 1926-1936.

Zhifeng ZHOU, Shuoyan LIU, Jieyu LI, Mingqiu CHEN, Hui LIN, Yujie CHEN, Weijie CHEN, Junpeng LIN, Hang ZHOU, Qinfeng ZHENG. Modification with IL-21 and CCL19 enhances killing efficiency and tumor infiltration of NKP30 CAR-T cells in lung cancer[J]. Journal of Southern Medical University, 2024, 44(10): 1926-1936.

图/表 7

图1 NKP30配体(B7H6)在细胞上的表达

Fig.1 Expression of the NKP30 ligand B7H6 on the cells. A: Analysis of the Human Proteome Map database showing negative B7H6 expression in normal tissues. B: B7H6 is expressed in most of the tumor tissues. C: B7H6 is highly expressed in different lung cancer cell lines.

图 2 CAR的构建和体外转染T细胞

Fig.2 Construction of CAR and transfection of T cells. A: Schematic diagram of NKP30 CAR and IL-21-CCL19 NKP30 CAR. B: Fluorescence appeared in T cells 72 h after transfection (Original magnification: ×100). C: Results of CAR virus transfection in peripheral blood mononuclear cells from 3 donors, in which both Conv.CAR and 21×19 CAR achieved over 50% transfection after 7 days. D: Transfection efficiency of CAR-T cells on CD4+ and CD8+ T cells at different time points.

图3 21×19 CAR-T促进T细胞增殖与迁移

Fig.3 21×19 CAR-T promotes T cell proliferation and migration. A: Histograms of the numbers of cell divisions. The numbers in the donut charts represent percentages of each gated fraction in the cultured cells. B: Number of cells in each group on the 5th and 7th day. C: Anti-IL-21R monoclonal antibody down-regulates the formation of memory cells in 21×19 CAR-T group. D: Flow cytometry for analyzing chemotactic ability of the cells toward T cells in each group. E: Anti-CCR7 monoclonal antibody down-regulates the chemotactic ability of cells in 21×19 CAR-T group. **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA with multiple comparisons.

图4 体外检测培养的CAR-T 细胞表型

Fig.4 Phenotype of cultured CAR-T cells. 21×19 CAR-T cells expressed significantly higher levels of activation markers CD3/CD25 and central memory markers CD62L/CD45RO with lower CTLA4 and PD1 expressions than other cells. The red box indicates the positive region. *P<0.05, **P<0.01.

图5 21×19 CAR 增强体外杀伤活性

Fig.5 21×19 CAR show enhanced cytotoxic activity in vitro. A: LDH assay for assessing cytotoxic activity of immune cells from each group co-cultured with lung cancer cells at effector-to-target (E:T) ratios of 1:1, 5:1, 10:1, and 20:1. B: Immune cells from each group co-cultured with the target tumor cell spheroids at different time points (E:T=1:1). Green fluorescence represents the effector cells, and red fluorescence represent the tumor cells (×100). C: ELISPOT for measuring the amount of IFN-γ produced by the effector cells in response to lung cancer cells. **P<0.01, ****P<0.0001.

图6 21×19 CAR-T 能有效分泌IL-21及CCL19细胞因子

Fig.6 21×19 CAR-T cells can effectively secrete IL-21 and CCL19 cytokines. A, B: Compared with Mock cells and conventional CAR-T cells, 21×19 CAR-T cells secreted a large amount of IL-21 (A) and CCL-19 (B). C: Both CAR-T and 21×19 CAR-T cells produce higher IL-6 level than cells in the Mock group, there is no significant difference among the 3 groups. ***P<0.001, ****P<0.0001.

图7 CAR-T细胞对斑马鱼体内肺癌细胞的清除

Fig. 7 CAR-T cells eliminate lung cancer cells in zebrafish. A: Fluorescence microscopy of the tumor-bearing zebrafish at 0 and 24 hpi time points (×30). The tumor cells A549 were stained with red fluorescent Dil, and the effector cells carry green fluorescent protein (GFP). B: Tumor volume increases in the zebrafish models 24 h after PBS injection. C, D: No significant area expansion of T cells and increased tumor volume in the Mock group after 24 h. E, F: In the conv. group, the area of CAR-T cells shows partial expansion with reduced tumor volume after 24 hours. G: In the 21×19 CAR-T group, the area of CAR-T cells significantly expands and the tumor volume is significantly reduced after 24 h. *P<0.05, **P<0.01.

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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

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