治疗后慢性乙型肝炎病毒感染者中低病毒血症人群外周血淋巴细胞状态分析

Analysis of peripheral blood lymphocyte status in low-level-viremia patients with chronic hepatitis B virus infection after nucleos(t)ide analogue treatment

目的评估治疗后慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者中低病毒血症人群外周血T、B、NK淋巴细胞情况,为解决低病毒血症提供思路。方法回顾性收集344例治疗后慢性HBV感染者的基本情况、生化、凝血、HBV DNA、外周血淋巴细胞计数、T淋巴细胞PD1及CD28、NK淋巴细胞穿孔素及颗粒酶B等数据,分为低病毒血症(low-level viremia,LLV)组与完全病毒学应答(complete virological response,CVR)组,比较两组外周血淋巴细胞计数、T淋巴细胞PD1及CD28表达、NK淋巴细胞穿孔素及颗粒酶B表达,倾向性评分匹配分析,验证结果的准确性。结果对162例LLV者和182例CVR者进行疾病诊断、谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、白蛋白(albumin,ALB)水平比较,差异无统计学意义(P>0.05),性别和年龄差异有统计学意义(P<0.05);两组外周血CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞计数和比例,以及CD4^(+)/CD8^(+)比值差异无统计学意义(P>0.05),LLV组外周血CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞PD1和CD28表达高于CVR组,差异有统计学意义(P<0.05);LLV组外周血CD19^(+)B淋巴细胞计数高于CVR组,差异无统计学意义(P>0.05),LLV组外周血CD19^(+)B淋巴细胞比例高于CVR组,差异有统计学意义(P<0.05);LLV组外周血CD16^(+)CD56^(+)NK淋巴细胞计数及穿孔素表达低于CVR组,差异无统计学意义(P>0.05),LLV组外周血CD16^(+)CD56^(+)NK淋巴细胞比例及颗粒酶B低于CVR组,差异有统计学意义(P<0.05)。倾向性评分匹配后108例LLV者和108例CVR者,基本情况差异无统计学意义(P>0.05);外周血T淋巴细胞亚群中,LLV组CD4^(+)T淋巴细胞比例与CD4^(+)/CD8^(+)比值高于CVR组,CD8^(+)淋巴细胞比例低于CVR组,差异有统计学意义(P<0.05),且CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞PD1和CD28表达仍高于CVR组,差异有统计学意义(P<0.05);与CVR组比较,LLV组外周血CD19^(+)B淋巴细胞计数及比例差异无统计学意义(P>0.05),CD16^(+)CD56^(+)NK淋巴细胞计数、比例及穿孔素表达差异无统计学意义(P>0.05),CD16^(+)CD56^(+)NK淋巴细胞颗粒酶B表达降低,差异有统计学意义(P<0.05)。结论T淋巴细胞数量及功能异常和NK淋巴细胞功能下降可能是治疗后慢性HBV感染者发生LLV的免疫因素,因此未来LLV治疗除可调整核苷(酸)类似物,也可从T、NK淋巴细胞着手。

Objective To evaluate the status of T,B and NK lymphocytes in peripheral blood of patients with chronic hepatitis B virus(HBV)infection and low-level viremia after nucleos(t)ide analogue(NA)treatment and to provide ideas for solving low-level viremia.Methods This retrospective study involved 344 patients with chronic HBV infection who had been treated with NAs.They were divided into two groups:low-level viremia group(LLV group)and complete virological response group(CVR group).Clinical data including basic information,biochemistry and coagulation test results,HBV DNA,peripheral blood lymphocyte counts,PD1 and CD28 expression by T lymphocytes,and perforin and granzyme B expression by NK lymphocytes were collected and compared between the two groups.Propensity matching analysis was performed to verify the accuracy of the results.Results Among the 344 cases,162 were in the LLV group and 182 in the CVR group.There were no significant differences in disease diagnosis,alanine aminotransferase(ALT),aspartate aminotransferase(AST)or albumin(ALB)level between the two groups(P>0.05),but the differences in gender and age were statistically significant(P<0.05).The differences in the counts and percentages of peripheral blood CD3^(+),CD4^(+)and CD8^(+)T lymphocyte and CD4^(+)/CD8^(+)ratios between the two groups were not statistically significant(P>0.05),but the expression of PD1 and CD28 by peripheral blood CD3^(+),CD4^(+)and CD8^(+)T lymphocytes was higher in the LLV group than in the CVR group(P<0.05).The count of peripheral blood CD19^(+)B lymphocytes in the LLV group was higher than that in the CVR group(P>0.05),and the percentage of peripheral blood CD19^(+)B lymphocytes was also higher in the LLV group(P<0.05).The count of peripheral blood CD16^(+)CD56^(+)NK lymphocytes and the expression of perforin in the LLV group were lower than those in the CVR group(P>0.05).The percentage of peripheral blood CD16^(+)CD56^(+)NK lymphocytes and the expression of granzyme B in the LLV group were lower than those in the CVR group(P<0.05).After propensity score matching,108 cases in the LLV group and 108 cases in the CVR group showed no significant differences in basic information(P>0.05);the percentage of CD4^(+)T lymphocytes and CD4^(+)/CD8^(+)ratio in peripheral blood T lymphocyte subsets were higher in the LLV group than in the CVR group,while the percentage of CD8^(+)lymphocytes was lower in the LLV group(P<0.05);the expression of PD1 and CD28 by CD3^(+),CD4^(+)and CD8^(+)T lymphocytes remained higher in the LLV group(P<0.05);the differences in the counts and percentages of peripheral blood CD19^(+)B lymphocytes as well as CD16^(+)CD56^(+)NK lymphocytes between the two groups were not statistically significant(P>0.05);no significant difference in the expression of perforin by CD16^(+)CD56^(+)NK lymphocytes was found between the two groups(P>0.05),and the expression of granzyme B remained lower in the LLV group(P<0.05).Conclusions Abnormal number and function of T lymphocytes and decreased function of NK lymphocytes might be related to the development of LLV in patients with chronic HBV infection after treatment.Therefore,in addition to adjusting NAs,targeting of T and NK lymphocytes might also be a feasible measure for future LLV treatment.