白细胞介素33在肝细胞癌患者中的表达及其对CD8<sup>+</sup>T淋巴细胞功能的调节作用

王海鹏; 刘屹; 李东辉; 沈光辉

doi:10.3969/j.issn.1001-5256.2022.01.018

白细胞介素33在肝细胞癌患者中的表达及其对CD8⁺T淋巴细胞功能的调节作用

DOI: 10.3969/j.issn.1001-5256.2022.01.018

1.
陕西省人民医院肿瘤内科，西安 710068
2.
陕西中医药大学附属医院肿瘤医院三病区，陕西咸阳 712000

基金项目:

西安市科技计划项目 (2019114613YX011S f 035(3));

西安交通大学基本业务项目 (XZY012019112);

国家自然科学基金 (81402012)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：王海鹏、李东辉参与研究数据的分析解释，并起草文章；刘屹参与实验设计及修改文章关键内容；沈光辉对研究的思路或设计有关键贡献，并参与修改文章。

详细信息

通信作者:
沈光辉，guangxian1982@126.com

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出版历程
- 收稿日期: 2021-06-19
- 录用日期: 2021-07-29
- 出版日期: 2022-01-20

Expression of interleukin-33 in hepatocellular carcinoma patients and its role in regulating CD8⁺ T cell function

1.
Department of Medical Oncology, Shaanxi Provincial People's Hospital, Xi'an 710068, China
2.
Third Ward of Oncology, The Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712000, China

Research funding:

Scientific Plan Program of Xi’an (2019114613YX011S f 035(3));

Basic Program of Xi’an Jiaotong University (XZY012019112);

National Natural Science Foundation of China (81402012)

摘要

摘要: 目的观察肝细胞癌(HCC)患者外周血白细胞介素33(IL-33)的变化，探讨IL-33对HCC患者CD8⁺T淋巴细胞的调节作用和可能机制。方法选择2019年4月—2020年1月陕西省人民医院收治的44例HCC患者和20例健康对照者为研究对象。采集外周血，分离血浆和外周血单个核细胞(PBMC)，酶联免疫吸附试验检测血浆IL-33及其受体肿瘤抑制素2(ST2)的水平，实时定量PCR法检测PBMC中IL-33和ST2 mRNA相对表达量。纯化CD8⁺T淋巴细胞，使用重组IL-33刺激培养，CCK-8法检测细胞增殖，酶联斑点吸附试验检测穿孔素和颗粒酶B分泌，流式细胞术检测程序性死亡受体-1(PD-1)、淋巴细胞活化基因-3(LAG-3)和细胞毒性T淋巴细胞相关蛋白4(CTLA-4)表达，比较IL-33刺激前后细胞增殖、毒性分子分泌、免疫检查点分子的变化。CD8⁺T淋巴细胞与HepG2细胞共培养，通过检测乳酸脱氢酶表达计算CD8⁺T淋巴细胞诱导HepG2细胞死亡比例，比较IL-33刺激前后CD8⁺T淋巴细胞杀伤功能的变化。计量资料两组间比较采用t检验或配对t检验，相关性分析采用Pearson分析法。结果 HCC组血浆IL-33水平低于对照组[(269.80±63.08) pg/mL vs (339.50±64.43) pg/mL，t=4.072，P＜0.001]，HCC组PBMC中IL-33 mRNA相对表达量低于对照组(1.07±0.14 vs 2.45±0.87，t=10.250，P＜0.001)。血浆ST2水平和PBMC中ST2 mRNA相对表达量在HCC组和对照组之间的差异均无统计学意义(P值均＞0.05)。CD8⁺T淋巴细胞比例与血浆IL-33、ST2水平均无明显相关性(P值均＞0.05)。HCC组CD8⁺T淋巴细胞分泌穿孔素、颗粒酶B的水平均低于对照组(P值均＜0.05)，而HCC组CD8⁺T淋巴细胞中PD-1、LAG-3、CTLA-4阳性细胞比例均高于对照组(P值均＜0.05)。重组IL-33刺激对两组CD8⁺T淋巴细胞增殖和免疫检查点分子表达均无显著影响(P值均＞0.05)，但可促进穿孔素、颗粒酶B的分泌(P值均＜0.05)。HCC组CD8⁺T淋巴细胞杀伤活性较对照组降低(P＜0.05)，重组IL-33刺激可提升CD8⁺T淋巴细胞杀伤功能，主要表现为诱导HepG2细胞死亡比例升高(P＜0.05)，IFNγ和TNFα分泌水平增加(P值均＜0.05)。结论 HCC患者血浆IL-33水平降低。IL-33可通过促进穿孔素和颗粒酶B的分泌增强HCC患者CD8⁺T淋巴细胞杀伤活性，为HCC治疗提供新的靶点。
- 癌，肝细胞 /
- 白细胞介素33 /
- CD8阳性T淋巴细胞
Abstract: Objective To investigate the change in interleukin-33 (IL-33) in the peripheral blood of hepatocellular carcinoma (HCC) patients and the role and potential mechanism of IL-33 in regulating CD8⁺ T cell function in HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital from April 2019 to January 2020 and 20 healthy controls were enrolled. Peripheral blood was collected, and plasma and peripheral blood mononucleated cells (PBMCs) were isolated; ELISA was used to measure the plasma levels of IL-33 and its receptor ST2, and quantitative real-time PCR was used to measure the relative mRNA expression levels of IL-33 and ST2 in PBMCs. CD8⁺ T cells were purified and stimulated with recombinant IL-33; CCK-8 assay was used to assess cell proliferation, enzyme-linked immunospot assay was used to measure the secretion of perforin and granzyme B, and flow cytometry was used to measure the expression of PD-1, LAG-3, and CTLA-4; changes in cell proliferation, secretion of cytotoxic molecules, and immune checkpoint molecules after IL-33 stimulation were compared. CD8⁺ T cells were co-cultured with HepG2 cells; the expression of lactate dehydrogenase was measured to calculate the proportion of dead HepG2 cells induced by CD8⁺ T cells, and the change in the killing function of CD8⁺ T cells after IL-33 stimulation was compared. The t-test or the paired t-test was used for comparison of continuous data between two groups, and a Pearson correlation analysis was performed. Results Compared with the control group, the HCC group had significantly lower plasma level of IL-33 (269.80±63.08 pg/ml vs 339.50±64.43 pg/ml, t=4.072, P < 0.001) and relative mRNA expression level of IL-33 in PBMCs (1.07±0.14 vs 2.45±0.87, t=10.250, P < 0.001). There were no significant differences in the plasma level of ST2 and the relative mRNA expression level of ST2 in PBMCs between the HCC group and the control group (P > 0.05). The proportion of CD8⁺ T cells was not correlated with the plasma level of IL-33 or ST2 (both P > 0.05). Compared with the control group, the HCC group had significantly lower levels of perforin and granzyme B (both P < 0.05) and a significantly higher proportion of CD8⁺ T cells with positive PD-1, LAG-3, and CTLA-4 (P < 0.05). Stimulation with recombinant IL-33 did not affect the proliferation of CD8⁺ T cells or the expression of immune checkpoint molecules (P > 0.05), but it promoted the secretion of perforin and granzyme B (P < 0.05). Compared with the control group, the HCC group had a significant reduction in the killing activity of CD8⁺ T cells (P < 0.05), and stimulation with recombinant IL-33 enhanced the killing function of CD8⁺ T cells, which was mainly reflected in the increases in the proportion of dead HepG2 cells (P < 0.05) and the secretion of IFNγ and TNFα (P < 0.05). Conclusion There is a reduction in the plasma level of IL-33 in HCC patients. IL-33 can enhance the killing activity of CD8⁺ T cells by promoting the secretion of perforin and granzyme B, which provides a new target for the treatment of HCC.
- Carcinoma, Hepatocellular /
- Interleukin-33 /
- CD8-Positive T-Lymphocytes

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图 1 CD3⁺CD8⁺T淋巴细胞典型流式分析图

注：a, HCC组; b，对照组。

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图 2 CD3⁺CD8⁺T淋巴细胞与IL-33、ST2水平相关性分析

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图 3 两组CD8⁺T淋巴细胞无IL-33刺激和经IL-33刺激后PD-1、LAG-3和CTLA-4表达的典型流式分析图

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表 1 两组一般资料比较

指标	HCC组 (n=44)	对照组 (n=20)	统计值	P值
男/女(例)	28/16	13/7	χ²=0.644	0.497
年龄(岁)	49.5±13.4	46.2±11.8	t=0.946	0.348
AFP(ng/mL)	778.1±129.8	5.9±0.7	t=26.489	＜0.001

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表 2 两组IL-33和ST2水平比较

指标	HCC组(n=44)	对照组(n=20)	t值	P值
CD3⁺CD8⁺T淋巴细胞(%)	27.82±6.97	26.80±10.31	0.468	0.641
血浆IL-33(pg/mL)	269.80±63.08	339.50±64.43	4.072	＜0.001
IL-33 mRNA	1.07±0.14	2.45±0.87	10.250	＜0.001
血浆ST2(pg/mL)	82.58±10.92	83.50±4.94	0.359	0.721
ST2 mRNA	1.09±0.14	1.07±0.10	0.432	0.667

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表 3 两组无IL-33刺激和经IL-33刺激后CD8⁺T淋巴细胞增殖、毒性分子分泌和免疫检查点分子表达的比较

指标	HCC组(n=28)				对照组(n=11)
指标	无IL-33刺激	经IL-33刺激	t值	P值	无IL-33刺激	经IL-33刺激	t值	P值
细胞增殖(×10⁵个)	1.24±0.08	1.29±0.19	1.077	0.286	1.20±0.13	1.25±0.24	0.581	0.568
穿孔素(×10⁵个)	64.21±9.83¹⁾	111.60±36.90²⁾	6.563	＜0.001	74.55±8.45	160.90±49.25	5.733	＜0.001
颗粒酶B(×10⁵个)	38.82±14.05¹⁾	51.32±19.25²⁾	2.775	0.008	92.92±29.44	130.40±46.97	2.436	0.023
PD-1⁺(%)	7.55±3.53¹⁾	7.63±2.92²⁾	0.095	0.925	1.98±0.16	2.17±0.35	1.646	0.116
LAG-3⁺(%)	3.30±1.01¹⁾	3.63±1.07²⁾	1.207	0.233	2.51±0.64	2.82±0.65	1.140	0.268
CTLA-4⁺(%)	7.85±1.00¹⁾	7.86±0.73²⁾	0.014	0.989	4.15±1.13	4.86±0.97	1.600	0.125
注：与对照组无IL-33刺激比较，1) P＜0.05；与对照组经IL-33刺激比较，2)P＜0.05。

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表 4 无IL-33刺激和经IL-33刺激后CD8⁺T淋巴细胞杀伤功能比较

指标	HCC组(n=11)				对照组(n=6)
指标	无IL-33刺激	经IL-33刺激	t值	P值	无IL-33刺激	经IL-33刺激	t值	P值
细胞死亡比例(%)	14.90±4.27	20.86±3.73	3.482	0.002	21.97±5.72	30.30±4.24	2.866	0.017
IFNγ (pg/mL)	29.27±6.08¹⁾	38.36±9.75²⁾	2.624	0.016	38.33±10.09	56.00±7.18	3.493	0.006
TNFα (pg/mL)	807.8±130.5¹⁾	1276±368.2²⁾	3.978	＜0.001	1147±264.0	2200±706.2	3.421	0.007
注：与对照组无IL-33刺激比较，1) P＜0.05；与对照组经IL-33刺激比较，2)P＜0.05。

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