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慢性乙型肝炎临床治愈者的血清sPD-1和sPD-L1水平及临床特点
DOI: 10.3969/j.issn.1001-5256.2023.01.008
伦理学声明：本研究于2021年1月13日由北京大学第一医院伦理委员会批准，伦理审查编号：2020科研363。
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：谭宁负责数据收集，统计分析，撰写文章；刘建湘负责资料分析，拟定写作思路，撰写文章；谭宁和刘建湘贡献等同，为共同第一作者。亢倩、潘家莉、韩一凡、陈宏宇参与收集数据和分析；徐小元负责指导撰写文章并最后定稿。
Serum levels of soluble programmed death-1 and soluble programmed death-ligand 1 in chronic hepatitis B patients with clinical cure and their clinical features
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摘要:
目的 对慢性乙型肝炎(CHB)临床治愈者的血清可溶性程序性死亡蛋白-1(sPD-1)和程序性死亡蛋白配体-1(sPD-L1)进行分析，并通过流式细胞技术分析与体内PD-1+淋巴细胞的相关性，探究CHB临床治愈者体内病毒特异性免疫的恢复情况。 方法 纳入2022年1月—5月在北京大学第一医院门诊诊断的CHB临床治愈者26例、CHB初治患者26例和健康对照者26例，收集临床数据和外周血样本。使用ELISA法对血清sPD-1和sPD-L1进行检测。使用流式细胞技术分析外周血淋巴细胞PD-1分布。将CHB临床治愈者分别与CHB初治患者和健康对照者进行比较。非正态分布计量资料3组间比较采用Kruskal-Wallis H检验，计数资料3组间比较采用χ2检验。使用Pearson相关或Spearman相关进行两个连续变量之间的相关性分析。 结果 CHB临床治愈者26例平均抗病毒治疗时间约8.33年，抗病毒治疗药物均为恩替卡韦。临床治愈者的sPD-1和sPD-L1水平显著高于健康对照者(P值均 < 0.05)，PD-1+细胞/淋巴细胞、PD-1+CD8+T淋巴细胞/淋巴细胞显著低于CHB初治患者(P值均 < 0.05)。CHB初治患者血清sPD-1、sPD-L1与HBsAg水平均呈中等程度负相关(r值分别为-0.524、-0.583，P值均 < 0.05)。血清sPD-1、sPD-L1水平和PD-1+CD8+ T淋巴细胞/淋巴细胞具有中等程度的正相关(r值分别为0.535、0.419，P值均 < 0.05)。CHB临床治愈者血清sPD-1和sPD-L1水平和年龄、性别、ALT、T淋巴细胞/淋巴细胞、CD8+T淋巴细胞/淋巴细胞、PD-1+细胞/淋巴细胞、PD-1+CD8+T淋巴细胞/淋巴细胞均无相关性(P值均 > 0.05)。 结论 CHB初治患者血清sPD-1、sPD-L1水平主要与外周血耗竭型CD8+T淋巴细胞有关。CHB临床治愈者血清sPD-1、sPD-L1水平与外周血耗竭型CD8+T淋巴细胞相关性不显著。 -
关键词:
- 乙型肝炎, 慢性 /
- 可溶性程序性死亡蛋白-1 /
- CD8阳性T淋巴细胞
Abstract:Objective To investigate the serum levels of soluble programmed death-1 (sPD-1) and soluble programmed death-ligand 1 (sPD-L1) in chronic hepatitis B (CHB) patients with clinical cure, the correlation between programmed death-1 (PD-1) and lymphocytes by flow cytometry, and the recovery of hepatitis B virus (HBV)-specific immunity. Methods A total of 26 CHB patients with clinical cure, 26 treatment-naïve CHB patients, and 26 healthy controls who were diagnosed at the outpatient service of Peking University First Hospital from January to May of 2022 were enrolled, and related clinical data and peripheral blood samples were collected. ELISA was used to measure the serum levels of sPD-1 and sPD-L1, and flow cytometry was used to measure the expression of PD-1 in peripheral blood lymphocytes. CHB patients with clinical cure were compared with the treatment-naïve CHB patients and the healthy controls. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between three groups, and the chi-square test was used for comparison of categorical data between groups. The Pearson correlation analysis or the Spearman correlation analysis was used to investigate the correlation between two continuous variables. Results For the 26 CHB patients with clinical cure, the mean time of antiviral therapy was 8.33 years, with entecavir as the antiviral drug. The CHB patients with clinical cure had significantly higher levels of sPD-1 and sPD-L1 than the healthy controls (P < 0.05) and significantly lower percentages of PD-1+ cells/lymphocytes and PD-1+CD8+ T cells/lymphocytes than the treatment-naïve CHB patients (P < 0.05). In the treatment-naïve CHB patients, the serum levels of sPD-1 and sPD-L1 were moderately negatively correlated with HBsAg level (r=-0.524 and -0.583, both P < 0.05). The serum levels of sPD-1 and sPD-L1 were moderately positively correlated with PD-1+CD8+ T cells/lymphocytes (r=0.535 and 0.419, both P < 0.05). In the CHB patients with clinical cure, the serum levels of sPD-1 and sPD-L1 were not correlated with age, sex, alanine aminotransferase, T cells/lymphocytes, CD8+ T cells/lymphocytes, PD-1+ T cells/lymphocytes or PD-1+CD8+ T cells/lymphocytes (all P > 0.05). Conclusion The serum levels of sPD-1 and sPD-L1 in treatment-naïve CHB patients are mainly associated with exhausted CD8+ T cells in peripheral blood, while there is no significant correlation between serum sPD-1/sPD-L1 and exhausted CD8+ T cells in peripheral blood in CHB patients with clinical cure. -
表 1 CHB患者和健康对照者临床特点分析
Table 1. The clinical characteristics of CHB patients and healthy controls
指标 CHB初治患者(n=26) CHB临床治愈者(n=26) 健康对照者(n=26) χ2值 P值 年龄(岁) 49.0(40.5~54.0) 51.5(46.5~63.0) 53.0(34.0~62.0) 2.073 0.355 男/女(例) 20/6 15/11 13/13 4.225 0.121 ALT(U/L) 22.00(17.75~35.50) 19.00(14.00~27.00) 17.00(14.50~24.50) 3.194 0.203 sPD-1(log10 pg/mL) 2.84(2.58~2.99) 2.45(2.13~3.19) 2.17(1.32~2.42)1) 16.000 < 0.001 sPD-L1 (log10 pg/mL) 1.52(1.36~1.83) 1.72(1.33~2.39) 0.18 (0~1.11)1) 28.420 < 0.001 T淋巴细胞/淋巴细胞(%) 73.15(61.18~77.70) 72.50(63.80~83.20) 70.10(61.65~78.20) 0.516 0.772 CD8+T淋巴细胞/淋巴细胞(%) 28.55(26.15~37.50) 29.10(20.25~36.15) 31.95(27.48~36.75) 1.422 0.491 PD-1+细胞/淋巴细胞(%) 29.70(25.70~36.30)1) 20.00(17.90~30.85) 22.35(17.83~27.93) 10.910 0.004 PD-1+CD8+ T淋巴细胞/淋巴细胞(%) 12.00(9.35~15.35)1) 6.45(6.07~10.94) 8.81(8.08~11.14) 8.097 0.018 注：与CHB临床治愈者相比，1)P < 0.05。 
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表 2 健康对照者血清sPD-1和sPD-L1与各指标的相关性
Table 2. The correlation analysis between sPD-1/sPD-L1 and other factors in healthy controls
指标 sPD-1 sPD-L1 r值 P值 r值 P值 年龄 0.321 0.498 0.464 0.372 性别 -0.104 0.222 0.056 0.786 ALT -0.193 0.603 0.311 0.450 T淋巴细胞/淋巴细胞 0.367 0.336 -0.238 0.582 CD8+T淋巴细胞/淋巴细胞 0.286 0.501 0.200 0.714 PD-1+细胞/淋巴细胞 0.550 0.133 -0.024 0.977 PD-1+CD8+ T淋巴细胞/淋巴细胞 0.595 0.132 0.429 0.419
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表 3 CHB初治患者血清sPD-1和sPD-L1与各指标的相关性
Table 3. The correlation analysis between sPD-1/sPD-L1 and other factors in treatment-naïve CHB patients
指标 sPD-1 sPD-L1 r值 P值 r值 P值 年龄 -0.190 0.353 -0.146 0.478 性别 -0.183 0.360 -0.275 0.166 ALT -0.027 0.894 -0.154 0.444 HBsAg -0.524 0.032 -0.583 0.016 HBV DNA 0.255 0.199 0.219 0.273 HBeAg 0.042 0.836 -0.021 0.918 T淋巴细胞/淋巴细胞 0.002 0.990 0.093 0.645 CD8+T淋巴细胞/淋巴细胞 0.154 0.473 0.150 0.486 PD-1+细胞/淋巴细胞 0.350 0.073 0.283 0.152 PD-1+CD8+ T淋巴细胞/淋巴细胞 0.535 0.007 0.419 0.041
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表 4 临床治愈CHB患者血清sPD-1和sPD-L1与各指标的相关性分析
Table 4. The correlation analysis betweensPD-1/sPD-L1 and other factors in CHB patients with clinical cure
指标 sPD-1 sPD-L1 r值 P值 r值 P值 年龄 0.014 0.973 0.215 0.524 性别 -0.258 0.133 -0.169 0.393 ALT 0.282 0.402 0.055 0.881 T淋巴细胞/淋巴细胞 -0.109 0.755 -0.065 0.850 CD8+T淋巴细胞/淋巴细胞 0.042 0.918 0.176 0.632 PD-1+细胞/淋巴细胞 0.300 0.371 0.264 0.435 PD-1+CD8+ T淋巴细胞/淋巴细胞 0.552 0.105 0.309 0.387
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