乙型肝炎病毒特异性CD8＋T淋巴细胞表达T淋巴细胞免疫球蛋白黏蛋白分子-3和程序性死亡受体-1的水平及其与肝脏炎症活动度的相关性

Correlation between programmed death-1 and T-cell immunoglobulin mucin-3 co-expression on hepatitis Bvirus-specific CD8^＋ T cells and liver inflammatory activities in patients with chronic hepatitis B virusinfection

目的评价慢性HBV感染者外周血单个核细胞（PBMC）和HBV特异性CD8^＋T淋巴细胞表达T淋巴细胞免疫球蛋白黏蛋白分子（Tim）-3和程序性死亡受体（PD）-1的水平及其与肝脏炎症活动度的相关性。方法中山大学附属第三医院感染科住院和门诊就诊的160例慢性HBV感染者，包括慢性肝炎轻中度63例，重度31例，慢加急性肝功能衰竭（ACLF）55例，非活动性HBsAg携带者11例。另选取20名健康体检者作为对照组。实时定量PCR检测HBVDNA水平；HBV血清学标志物采用ELISA法检测；流式细胞仪检测人白细胞抗原（HLA）A2、外周血中Tim3和PD-1阳性的PBMC和HBV特异性CD8^＋T淋巴细胞数量。比较不同临床类型患者Tim3和PD-1阳性和共阳性的PBMC和HBV特异性CD8^＋T淋巴细胞数量的差异及其与常用评估炎症活动度指标的相关性。正态分布的计量资料的组间比较采用方差分析，非正态分布的计量资料采用KruskalWallis检验；Spearman相关分析用于评估Tim-3／PD-1表达水平与炎症活动度指标的相关性。结果160例患者外周血PBMC中Tim-3／PD-1阳性比例在ACLF组（0．25％）、慢性肝炎重度（0．24％）和轻中度组（0．13％），明显高于健康对照组（0．03％，P值分别为0．0049，0．0025和0．0006），Tim-3／PD-1阴性比例则低于健康对照组（P=0.0000），而非活动性HBsAg携带者组（0．10％）与健康对照组（0．03％）间差异无统计学意义（P=0．28）。160例患者中有78例为HLA-A2阳性，HBV特异性CD8^＋T淋巴细胞Tim-3／PD-阳性比例在ACI．F组和慢性肝炎重度组分别为68．72％±17．21％和59．66％±19．25％，明显高于非活动性HBsAg携带者组的33．93％±10．80％（P值分别为0．0000和0．0054）；Tim-3／PD-1阴性比例ACLF组为2．80％，低于非活动性HBsAg携带者组的27．24％（P=0．0004）。HBV特异性CD8^＋T淋巴细胞的Tim-3／PD-1阳性比例与ALT（r=0．26，P=0．022）、AST（r=0．28，P=0．012）及TBil（r=0．36，P=0．001）呈正相关。HBV特异性CD8^＋T淋巴细胞的Tim3／PD-1阳性比例与Alb呈负相关（r=-0．35，P=0．002），在ACLF患者中与凝血酶原国际标准化比率（INR，r=0．34，P=0．045）及终末期肝病模型（MELD）分值（r=0．43，P=0．027）呈正相关。结论慢性乙型肝炎患者外周血PBMC和HBV特异性CD8^＋T淋巴细胞Tim-3和PD-1共表达水平明显上调，且共表达水平与肝脏炎症活动度有一定的相关性，提示Tim-3和PD-1可能参与慢性乙型肝炎活动和肝功能衰竭的发生机制。

Objective To study programmed death 1 （PD-1） and T-cell immunoglobulin mucin- 3 （Tim-3） co expression on peripheral blood mononuclear cells （PBMC） and hepatitis B virus （HBV）- specific CD8- T ceils in patients with chronic HBV infection and its correlation with liver inflammatory aclivities. Methods One hundred and sixty subjects with chronic HBV infection who visited the outpatient and inpatient Department of Infectious Diseases in the Third Affiliated Hospital of Sun YaP sen University were enrolled, including 63 cases of mild or moderate CHB （MCHB）, 31 of severe CHB （SCHB）, 55 of acute-on-chronic liver failure （ACLF） and 11 inactive HBsAg carriers. Twenty healthy subjects were enrolled as controls. Real time quantitative polymerase chain reaction （qRT- PCR） was used to detect HBV DNA, enzyme-linked immunosorbent assay （ELISA） to measure HBV serological markers, and flow cytometry to detect human leukocyte antigen （HLA）-A2 and determine the expression of PD-1 and Tim-3 on PBMC and HBV-specific CD8^＋ T cells. Cell counts of Tim 3^＋ , PD-1＋ , and Tim-3^＋/PD-1^＋ PBMC and HBV specific CD8^＋ T ceils were compared among different groups, and their correlation with commonly used inflammatory activity indicators were studied. Analysis of variance and Kruskal-Wallis test were used for measurement data with gaussian distribution and skewed distribution, respectively. Spearman correlation analysis was used to assess the association between Tim 3/PD-1 expression and inflammatory activity indicators. Results The frequency of Tim-3＋/PD-1＋ PBMC was 0.25% in ACLF group （P=0. 0049）, 0.24% in SCHB group （P=0. 0025） and 0.13% in MCHB group （P=0. 0006）, which were significantly higher than that in control group （0. 03%）, and the frequency of Tim-3 /PD-1 PBMC in the three groups were significantly lower than that in control group （P=0. 0000）, but the differences between HBsAg carriers （0.10%） and controls （0.03%） were not statistically significant （P=0. 28）. Among 160 subjects, 78 were HLA-A2 positive. The frequency of Tim-3＋/PD-1＋ HBV specific CD8＋ T cells was 68.72%±17.21% in ACLF group, and 59.66% ±19.25% in SCHB group, which were significantly higher than that in HBsAg carrier group （33. 93%±10. 80%, P= 0. 0000, P=0. 0054）. The frequency of Tim 8 /PD-1- HBV-specific CD8^＋ T cells in ACLF group was 2. 80%, which was significantly lower than that in HBsAg carrier group （27.24%, P = 0. 0004）. The frequency of Tim 3＋ /PD-1^＋ HBV-specific CD8＋ T ceils was positively correlated with alanine aminotransferase （r=0.26, P = 0. 022）, aspartate aminotransferase （r=0. 28, P= 0. 012） and total biliruhin levels （ r=0.36, P 0. 001）; and inversely correlated with alhumin level （r =0. 35, P= 0. 002） in serum. Furthermore, it was positively correlated with international normalized ratio （INR, r= 0. 34, P= 0. 045 ） and model for end stage liver disease score （r= 0.43, P=0. 027） in ACLF group. Conclusions Co-expressions of Tim-3 and PD-1 on PBMC and HBV-specific CD8＋ T cells are significantly up regulated in patients with CHB, and correlated with liver inflammatory activities. These findings indicate that Tim-3 and PD-1 co-expression may involve in disease activity and liver failure in CHB.