IL-6对肝细胞癌患者CD8<sup>+</sup>T淋巴细胞程序性死亡受体1表达和功能的影响

薛松; 黄高铂; 杨晓飞; 张野; 李彧

doi:10.3969/j.issn.1001-5256.2021.10.015

IL-6对肝细胞癌患者CD8⁺T淋巴细胞程序性死亡受体1表达和功能的影响

DOI: 10.3969/j.issn.1001-5256.2021.10.015

薛松¹,
黄高铂²,
杨晓飞³,
张野³,
李彧^4, ,

1.
西安市人民医院泌尿外科，西安 710000
2.
西安交通大学第一附属医院肝胆外科，西安 710061
3.
空军军医大学第二附属医院(第四军医大学唐都医院)传染科，西安 710038
4.
陕西省人民医院感染性疾病科，西安 710068

基金项目:

西安市科技计划项目 (20YXYJ0009〔11〕)

详细信息

通信作者:
李彧，drlee2810@126.com

薛松和黄高铂对本文贡献相同，同为第一作者

中图分类号: R735.7
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出版历程
- 收稿日期: 2021-02-26
- 录用日期: 2021-03-19
- 出版日期: 2021-10-20

Influence of interleukin-6 on the expression and function of programmed death-1 in CD8⁺ T cells from patients with hepatocellular carcinoma

1.
Urinary Surgery, Xi'an People's Hospital, Xi'an 710000, China
2.
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
3.
Department of Infectious Diseases, The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University, Xi'an 710038, China
4.
Department of Infectious Diseases, Shaanxi Provincial People's Hospital, Xi'an 710068, China

Research funding:

Xi'an Municipal Science and Technology Planning Project (20YXYJ0009〔11〕)

摘要

摘要: 目的观察血浆IL-6及程序性死亡受体(PD-1)在肝细胞癌(HCC)患者外周血CD8⁺T淋巴细胞中的表达，评估IL-6对HCC患者CD8⁺T淋巴细胞中PD-1表达和功能的影响。方法纳入2019年1月—2019年9月期间在陕西省人民医院或空军军医大学第二附属医院(第四军医大学唐都医院)就诊的HCC患者44例(HCC组)，同时纳入年龄和性别匹配的健康对照者19例(HC组)，采集外周血，分离血浆和外周血单个核细胞，分选CD8⁺T淋巴细胞，ELISA法检测血浆IL-6水平，流式细胞术检测PD-1在CD8⁺T淋巴细胞中的表达水平。使用IL-6中和抗体刺激分选的CD8⁺T淋巴细胞24 h，CCK-8法检测细胞增殖，ELISA法检测培养上清IFNγ和TNFα水平，实时定量PCR法检测穿孔素、颗粒酶B和颗粒溶素mRNA相对表达量，Western blot法检测STAT3和Src磷酸化水平。计量资料两组间比较采用t检验或配对t检验；计数资料组间比较采用χ²检验。结果 HCC组患者血浆IL-6水平较HC组显著升高[(99.67±20.92)pg/mL vs (81.05±16.76)pg/mL，t=3.427，P=0.001 1]。虽然CD3⁺CD8⁺T淋巴细胞比例在HCC组和HC组患者之间的差异无统计学意义(P＞0.05)，但PD-1⁺CD8⁺细胞比例在HCC组患者中显著升高(3.79%±1.36% vs 2.20%±0.47%，t=5.335，P＜0.000 1)。使用IL-6中和抗体抑制HCC组患者CD8⁺T淋巴细胞的IL-6虽不影响细胞增殖，但可降低PD-1表达(2.67%±0.91% vs 3.33%±1.12%，t=2.177，P=0.035)，增加IFNγ分泌[(13.50±3.82)pg/mL vs (10.82±1.37)pg/mL，t=3.170，P=0.002 8]，穿孔素和颗粒酶B mRNA相对表达量亦显著升高(t值分别为6.161、14.140，P值均＜0.000 1)，同时伴有磷酸化STAT3水平降低(P＜0.000 1)。结论抗人IL-6中和抗体可能通过增加穿孔素和颗粒酶B水平、增强细胞因子分泌以及抑制PD-1表达增强HCC患者CD8⁺T淋巴细胞的功能。
- 癌，肝细胞 /
- CD8阳性T淋巴细胞 /
- 程序性细胞死亡受体1 /
- 白细胞介素6
Abstract: Objective To investigate the influence of interleukin-6(IL-6) on the expression and function of programmed death-1(PD-1) in patients with hepatocellular carcinoma (HCC) by measuring the plasma level of IL-6 and the expression of PD-1 in peripheral blood CD8⁺ T cells from HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital or The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University from January to September 2019 were enrolled as HCC group, and 19 healthy controls, matched for age and sex, were enrolled as HC group. Peripheral blood was collected, and plasma and peripheral blood mononuclear cells were isolated to separate CD8⁺ T cells. ELISA was used to measure the plasma level of IL-6, and flow cytometry was used to measure the expression level of PD-1 in CD8⁺ T cells. The separated CD8⁺ T cells were stimulated with anti-IL-6 neutralizing antibody for 24 hours; CCK-8 assay was used to measure cell proliferation, ELISA was used to measure the levels of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in supernatant, real-time PCR was used to measure the relative mRNA expression levels of perforin, granzyme B, and granulysin, and Western blot was used to measure the phosphorylation levels of STAT3 and Src. The t-test or the paired t-test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between groups. Results Compared with the HC group, the HCC group had a significant increase in the plasma level of IL-6 (99.67±20.92 pg/mL vs 81.05±16.76 pg/mL, t=3.427, P=0.001 1). There was no significant difference in the percentage of CD3⁺CD8⁺ T cells between the HCC group and the HC group, while there was a significant increase in the percentage of PD-1⁺CD8⁺ cells in HCC patients (3.79%±1.36% vs 2.20%±0.47%, t=5.335, P < 0.000 1). In the patients with HCC, although anti-IL-6 neutralizing antibody for inhibiting IL-6 in CD8⁺ T cells did not affect cell proliferation, it downregulated the expression of PD-1 (2.67%±0.91% vs 3.33%±1.12%, t=2.177, P=0.035) and increased the secretion of IFNγ (13.50±3.82 pg/mL vs 10.82±1.37 pg/mL, t=3.170, P=0.002 8), and there were also significant increases in the relative mRNA expression levels of perforin and granzyme B (t=6.161 and 14.140, both P < 0.000 1) and a significant reduction in the level of phosphorylated STAT3 (P < 0.000 1). Conclusion Anti-IL-6 neutralizing antibody can enhance the function of CD8⁺ T cells in HCC patients possibly by increasing the levels of perforin and granzyme B, improving the secretion of cytokines, and inhibiting the expression of PD-1.
- Carcinoma, Hepatocellular /
- CD8-Positive T-Lymphocytes /
- Programmed Cell Death 1 Receptor /
- Interleukin-6

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图 1 HC组和HCC组血浆IL-6水平比较

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图 2 HC组和HCC组CD8⁺T淋巴细胞中PD-1表达的比较

注：a，典型流式散点图；b，CD3⁺CD8⁺T淋巴细胞比例；c，PD-1⁺CD8⁺细胞比例。

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图 3 HCC患者外周血分选的CD8⁺T淋巴细胞在抗人IL-6中和抗体处理后细胞增殖、PD-1表达以及分泌细胞因子的变化

注：a，细胞增殖；b，PD-1⁺CD8⁺细胞比例；c，IFNγ水平；d，TNFα水平。

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图 4 HCC患者外周血分选的CD8⁺T淋巴细胞在抗人IL-6中和抗体处理后穿孔素、颗粒酶B和颗粒溶素mRNA以及STAT3和Src蛋白磷酸化的变化

注：a，穿孔素mRNA；b，颗粒酶B mRNA；c，颗粒溶素mRNA；d，总STAT3、磷酸化STAT3、总Src、磷酸化Src Western blot检测；e，条带灰度比较。

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表 1 实时定量PCR引物序列

基因名称	上游引物(5′-3′)	下游引物(5′-3′)
穿孔素	CGCCTACCTCAGGCTTATCTC	CCTCGACAGTCAGGCAGTC
颗粒酶B	TGGGGGACCCAGAGATTAAAA	TTTCGTCCATAGGAGACAATGC
颗粒溶素	ACTGAAGATGGTGGATAAGCC	GCCCTGGGTAACTCTAGACTG
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGG

下载: 导出CSV

表 2 入组志愿者一般资料

资料	HC组 (n=19)	HCC组 (n=44)	统计值	P值
男/女(例)	11/8	28/16	χ²=0.728	0.537
年龄(岁)	54.2±7.9	58.7±12.1	t=1.486	0.142
AFP(ng/mL)	6.1±1.8	871.2±243.0	t=15.444	＜0.000 1
HBV/HCV感染史(例)	0	38	-	-

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