HBV感染者外周血自然杀伤样B淋巴细胞、自然杀伤细胞、B淋巴细胞亚群的变化及临床意义

杨晓飞; 申焕君; 黄高铂; 连建奇; 张野

doi:10.3969/j.issn.1001-5256.2022.07.008

HBV感染者外周血自然杀伤样B淋巴细胞、自然杀伤细胞、B淋巴细胞亚群的变化及临床意义

DOI: 10.3969/j.issn.1001-5256.2022.07.008

1.
空军军医大学第二附属医院(第四军医大学唐都医院) 传染病科，西安 710038
2.
西安交通大学第一附属医院肝胆外科，西安 710061

基金项目:

国家自然科学基金 (31370856);

国家自然科学基金 (81671555)

伦理学声明：本研究方案于2015年5月21日经由空军军医大学第二附属医院(第四军医大学唐都医院)伦理委员会批准，批号：TDLL-201505-013，所有入组受试者均签署知情同意书。

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：杨晓飞、申焕君、黄高铂负责实验操作和论文撰写；杨晓飞、连建奇、张野负责数据分析；连建奇、张野负责入组患者、课题设计、指导论文撰写并最后定稿。

详细信息

通信作者:
张野，zhangyefmmu@hotmail.com

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出版历程
- 收稿日期: 2021-11-24
- 录用日期: 2022-02-09
- 出版日期: 2022-07-20

Changes and clinical significance of natural killer-like B cells, natural killer cells, and B cells in peripheral blood of patients with hepatitis B virus infection

1.
Department of Infectious Diseases, The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University, Xi'an 710038, China
2.
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China

Research funding:

National Natural Science Foundation of China (31370856);

National Natural Science Foundation of China (81671555)

More Information

Corresponding author: ZHANG Ye, zhangyefmmu@hotmail.com(ORCID: 0000-0002-2573-5471)

摘要

摘要: 目的观察HBV感染者自然杀伤样B淋巴细胞(NKB淋巴细胞)、NK细胞和B淋巴细胞亚群的变化，分析其与临床指标的相关性。方法选取2017年1月—2018年12月在唐都医院就诊的急性乙型肝炎(AHB)患者15例、慢性乙型肝炎(CHB)患者30例、无症状HBV携带者(ASC)29例和对照者12例。采集外周血，分离血浆和外周血单个核细胞。酶联免疫吸附试验检测血浆IL-18和IFNγ水平。酶联斑点吸附试验检测HBV特异性CD8⁺T淋巴细胞分泌IFNγ水平。流式细胞术检测CD3^-CD19⁺CD56⁺CD16⁺NKB淋巴细胞、不同NK细胞亚群(CD3^-CD19^-CD56^highCD16^-NK细胞、CD3^-CD19^-CD56⁺CD16⁺NK细胞、CD3^-CD19^-CD56^-CD16⁺NK细胞)、CD3^-CD19⁺B淋巴细胞比例，分析与病毒复制、肝脏炎症指标的相关性。符合正态分布的计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。不符合正态分布的计量资料多组间比较采用Kruskal-Wallis H检验。相关性分析采用Pearson相关性检验。结果 CD3^-CD19⁺CD16⁺CD56⁺NKB淋巴细胞占淋巴细胞的比例在AHB患者、CHB患者、ASC和对照者之间的差异有统计学意义(F=16.42，P＜0.000 1)，CHB患者NKB淋巴细胞比例[(0.79±0.13)%]显著低于AHB患者[(0.94±0.15)%]、ASC[(1.02±0.12)%]和对照者[(1.11±0.27)%](P值均＜0.001)。血浆IL-18水平在AHB患者、CHB患者、ASC和对照者之间的差异有统计学意义(F=5.733，P=0.001)，CHB患者IL-18水平[(259.30±70.09)pg/mL]显著低于AHB患者[(336.00±103.00)pg/mL]和对照者[(319.30±64.80)pg/mL](P值均＜0.05)，ASC IL-18水平[(258.60±59.82)pg/mL]亦显著低于AHB患者和对照者(P值均＜0.01)，但血浆IL-18在CHB患者和ASC之间的差异无统计学意义(P=0.965)。CD3^-CD19^-CD56^highCD16^-NK细胞、CD3^-CD19^-CD56⁺CD16⁺NK细胞、CD3^-CD19^-CD56^-CD16⁺NK细胞以及B淋巴细胞比例在4组之间的差异均无统计学意义(P值均＞0.05)。CHB患者血浆IFNγ水平显著低于AHB患者、ASC者和对照者(P值均＜0.01)。NKB淋巴细胞比例和IL-18与HBV DNA定量、ALT水平均无显著相关性(P值均＞0.05)。CHB患者中NKB淋巴细胞占淋巴细胞的比例与血浆IL-18水平呈正相关(r=0.432，P=0.017)，与病毒特异性CD8⁺T淋巴细胞分泌IFNγ水平呈正相关(r=0.493，P=0.006)。结论 NKB淋巴细胞和IL-18可能参与HBV感染慢性化，与慢性HBV感染自然史相关。
- 乙型肝炎病毒 /
- 自然杀伤T细胞 /
- B-淋巴细胞
Abstract: Objective To investigate the changes of natural killer-like B (NKB) cells, natural killer (NK) cells, and B cells and their correlation with clinical indices in patients with hepatitis B virus (HBV) infection. Methods A total of 15 patients with acute hepatitis B (AHB), 30 patients with chronic hepatitis B (CHB), 29 asymptomatic HBV carriers (ASCs), and 12 controls who attended Tangdu Hospital from January 2017 and December 2018 were enrolled. Peripheral blood samples were collected, and plasma and peripheral blood mononuclear cells (PBMCs) were isolated. ELISA was used to measure the plasma levels of interleukin-18 (IL-18) and interferon-γ (IFNγ), and enzyme-linked immunospot assay was used to measure the level of IFNγ secreted by HBV-specific CD8⁺ T cells; flow cytometry was used to measure the percentages of CD3^-CD19⁺CD56⁺CD16⁺ NKB cells, different NK cell subsets (including CD3^-CD19^-CD56^highCD16^- NK cells, CD3^-CD19^-CD56⁺CD16⁺ NK cells, and CD3^-CD19^-CD56^-CD16⁺ NK cells), and their correlation with viral replication and liver inflammation markers was analyzed. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups; non-normally distributed continuous data were expressed as M(P₂₅-P₇₅), and the Kruskal-Wallis H test was used for comparison between multiple groups; the Pearson correlation test was used for correlation analysis. Results There was a significant difference in the percentage of CD3^-CD19⁺CD16⁺CD56⁺ NKB cells between the AHB patients, CHB patients, ASCs, and controls (F = 16.42, P < 0.000 1), and the CHB patients had a significantly lower percentage of NKB cells than the AHB patients, ASCs, and controls (0.79%±0.13% vs 0.94%±0.15%/1.02%±0.12%/1.11%±0.27%, all P < 0.001). There was a significant difference in plasma IL-18 level between the AHB patients, CHB patients, ASCs, and controls (F = 5.733, P = 0.001); the CHB patients had a significantly lower IL-18 level than the AHB patients and the controls (259.30±70.09 pg/mL vs 336.00±103.00 pg/mL and 319.30±64.80 pg/mL, both P < 0.05), and ASCs had a significantly lower IL-18 level (258.60±59.82 pg/mL) than the AHB patients and the controls (both P < 0.01), while there was no significant difference in plasma IL-18 level between the CHB patients and ASCs (P = 0.965). There were no significant differences in the percentage of CD3^-CD19^-CD56^highCD16^- NK cells, CD3^-CD19^-CD56⁺CD16⁺ NK cells, CD3^-CD19^-CD56^-CD16⁺ NK cells, and B cells between the four groups (all P > 0.05). The CHB patients had a significantly lower plasma IFNγ level than the AHB patients, ASCs, and controls (all P < 0.01). The percentage of NKB cells and the level of IL-18 were not significantly correlated with HBV DNA quantification or alanine aminotransferase level (all P > 0.05). In the CHB patients, the percentage of NKB cells was positively correlated with plasma IL-18 level (r = 0.432, P = 0.017) and the level of IFNγ secreted by HBV-specific CD8⁺ T cells (r =0.493, P=0.006). Conclusion NKB cells and IL-18 might be involved in the chronicity of HBV infection and is associated with the natural history of chronic HBV infection.
- Hepatitis B virus /
- Natural Killer T-Cells /
- B-Lymphocytes

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图 1 外周血NKB淋巴细胞、NK细胞、B淋巴细胞的流式分析策略

Figure 1. Flow cytometry analysis strategy for peripheral NKB cells, NK cells, and B cells

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图 2 HBV感染者和对照者外周血NKB淋巴细胞、NK细胞、B淋巴细胞比例和IL-18、IFNγ水平比较

注：a，CD3^-CD19⁺CD16⁺CD56⁺NKB淋巴细胞；b，CD3^-CD19^-CD56^highCD16^- NK细胞；c，CD3^-CD19^-CD56⁺CD16⁺NK细胞；d，CD3^-CD19^-CD56^-CD16⁺ NK细胞；e，B淋巴细胞；f，IL-18；g，IFNγ。

Figure 2. Comparison of peripheral NKB cells, NK cells, and B cells percentage and IL-18, IFNγlevel among HBV infected patients and controls

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图 3 CHB患者中NKB淋巴细胞与其他指标相关性分析

注：a，NKB淋巴细胞比例与血浆IL-18水平相关性分析；b，NKB淋巴细胞比例与血浆IFNγ水平相关性分析；c，NKB淋巴细胞比例与病毒特异性CD8⁺T淋巴细胞分泌IFNγ水平相关性分析。

Figure 3. Correlation analysis of NKB cells and other index in CHB patients

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表 1 入组受试者一般资料比较

Table 1. Comparison of clinical data of enrolled subjects

项目	AHB患者 (n=15)	CHB患者 (n=30)	ASC (n=29)	对照者 (n=12)	统计值	P值
男/女(例)	13/2	23/7	22/7	10/2	χ²=0.429	0.286
年龄(岁)	33.50±8.54	32.20±8.86	26.70±8.43	31.20±3.74	F=3.311	0.024
ALT(U/L)	384(250~822)	114(81~172)	32(24~39)	16(9~31)¹⁾	H=22.160	＜0.000 1
HBV DNA (log₁₀IU/mL)	4.24±1.44	7.85±1.38	7.60±0.79		F=51.070	＜0.000 1
HBeAg阳性	11	21	29
抗-HBe阳性	4	9	0
注：1)此结果为10例对照者的结果，2例对照者未行ALT检测。

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