慢性乙型肝炎病毒感染者T淋巴细胞和NK细胞T淋巴细胞免疫球蛋白黏蛋白分子3表达与肝脏炎症和纤维化相关性分析

Correlation of Tim-3 expression on T lymphocytes and natural killer cells with hepatic inflammation and hepatic fibrosis in chronic hepatitis B patients

目的探究慢性乙型肝炎病毒(HBV)感染者T淋巴细胞及自然杀伤(NK)细胞T淋巴细胞免疫球蛋白黏蛋白分子3(Tim-3)表达及其在评估肝纤维化程度中的潜在价值。方法选取2016年6月1日至2018年6月1日于中山大学附属第三医院感染性疾病科门诊就诊的320例慢性HBV感染者,将其分成免疫耐受期组(31例),免疫活动期组(184例),非活动期组(48例)和灰色区组(57例);同时纳入17例健康志愿者作为健康对照组。分离每例研究对象外周血单个核细胞并采用流式细胞仪分别检测CD3+T淋巴细胞及其亚群(CD4+和CD8+T淋巴细胞)以及NK细胞及其亚群(NK-bright和NK-dim细胞)Tim-3表达频率和平均荧光强度(MFI)。收集患者临床资料并计算天冬氨酸转氨酶与血小板计数比值指数(APRI)。采用Kruskal-Wallis H检验进行多组间非正态分布计量资料比较,组间两两比较采用Mann-Whitney U检验。计数资料采用例(百分数)表示,采用卡方检验进行比较。相关性分析采用Spearman秩相关。采用受试者工作特征曲线(ROC)分析CD3+T淋巴细胞与NK细胞Tim-3表达比值在评估慢性乙型肝炎病毒(HBV)感染者肝纤维化进展中的预测价值。P<0.05为差异具有统计学意义。结果免疫耐受期、免疫活动期、非活动期、灰色区和健康对照组年龄、天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、白蛋白(Alb)、总胆红素(TBil)和肝脏硬度差异均有统计学差异(H=12.40、169.70、210.70、25.17、24.21和86.50,P值均<0.05)。免疫耐受期、免疫活动期、非活动期和灰色区组APRI评分、乙型肝炎e抗原(HBeAg)阳性患者比例、乙型肝炎表面抗原(HBsAg)和HBV-DNA差异均有统计学差异(H=89.45、118.00和14.81,χ^(2)=148.20,P值均<0.05)。免疫耐受期、免疫活动期、非活动期、灰色区及健康对照组CD3+、CD4+和CD8+T淋巴细胞以及NK细胞、NK-bright和NK-dim细胞Tim-3表达频率及MFI差异均有统计学意义(H=13.57、51.55、8.58、44.25、20.32、47.96和12.45、33.69、4.96、32.47、10.63、30.46,P值均<0.05)。慢性HBV感染者CD3+、CD4+和CD8+T淋巴细胞Tim-3表达频率和MFI与ALT和AST水平均呈正相关(r=0.2134、0.4733、0.2090、0.4333、0.1771、0.4417、0.1780、0.3956、0.2618、0.4671、0.2614和0.4326,P值均<0.05);CD8+T淋巴细胞Tim-3表达频率和MFI以及CD3+和CD4+T淋巴细胞Tim-3 MFI与TBil水平均呈正相关(r=0.1342、0.2635、0.2739和0.2526,P值均<0.05)。慢性HBV感染者NK及NK-dim细胞Tim-3表达频率和MFI与ALT、AST和TBil水平均呈负相关(r=-0.2671、-0.4093、-0.2451、-0.4099、-0.1807、-0.1823、-0.2733、-0.4224、-0.2576、-0.4206、-0.1798和-0.1946,P均<0.05);NK-bright细胞Tim-3 MFI与ALT、AST和TBil水平均呈负相关(r=-0.3775、-0.3562和-0.1633,P值均<0.05);CD3+、CD4+和CD8+T淋巴细胞Tim-3表达频率和MFI与肝脏硬度值均呈正相关(r=0.1789、0.3896、0.1518、0.3521、0.2117和0.3579,P值均<0.05);CD4+和CD8+T淋巴细胞Tim-3表达频率和MFI以及CD3+T淋巴细胞MFI与APRI评分均呈正相关(r=0.1487、0.2604、0.2296、0.4858和0.2853,P值均<0.05);NK和NK-dim细胞Tim-3表达频率和MFI以及NK-bright Tim-3 MFI与肝脏硬度值均呈负相关(r=-0.2686、-0.3975、-0.2852、-0.3991和-0.3531,P值均<0.05)。NK和NK-dim细胞Tim-3表达频率和MFI以及NK-bright细胞Tim-3 MFI与APRI评分均呈负相关(r=-0.3589、-0.4158、-0.3591、-0.4108和-0.3966,P值均<0.05)。CD3+T淋巴细胞与NK细胞Tim-3表达比值预测慢性HBV感染者肝脏纤维化ROC曲线下面积为0.783(95%CI:0.723~0.843,P<0.05)。当截断值=0.612,敏感度为61.9%,特异度为99.3%。结论慢性HBV感染者T淋巴细胞和NK细胞Tim-3表达与肝脏炎症及纤维化的相关性表现为相反的特征性,Tim-3分子在T淋巴细胞和NK细胞上表达比值对评估慢性HBV感染肝纤维化具有一定价值。

Objective To explore the correlation of the expression of lymphocyte immunoglobulin-mucin domain 3(Tim-3)on T lymphocytes and natural killer(NK)cells with hepatic inflammation and hepatic fibrosis in patients with chronic hepatitis B virus(HBV)infection.Methods A total of 320 patients of chronic HBV infection who visited the Infectious Diseases Department in the Third Affiliated Hospital of Sun Yat-sen University from June 2016 to June 2018 were enrolled.The patients were divided into four groups:immune tolerant group(IT,n=31),immune active group(IA,n=184),inactive carriers group(IC,n=48),and gray zone group(GZ,n=57).And 17 healthy controls(HC group)were included at the same time.Peripheral blood mononuclear cells were separated and the frequency and mean fluorescence intensity(MFI)of Tim-3 on T cells(CD3+,CD4+and CD8+T cells)and NK cells(NK,NK-bright and NK-dim cells)were detected by flow cytometry.The clinical data of patients were collected and aspartate aminotransferase-to-platelet ratio index(APRI)score was calculated.Kruskal-Wallis H test was used for comparing the data of non-normal distribution among groups,and Mann Whitney U test was used for the comparison between two groups.Enumeration data were expressed as cases(percentage)and compared by the Chi-square test.Spearman rank correlation was used for correlation analysis.Receiver operating characteristic curve(ROC)was used to analyze the predictive value of Tim-3 expression on T cells and NK cells in evaluating liver fibrosis in patients with chronic HBV infection.P<0.05 was considered statistically significant.Results Significant differences were found in the age,aspartate aminotransferase(AST),alanine aminotransferase(ALT),albumin(Alb),total bilirubin(TBil)and liver stiffness measurement(LSM)among IT,IA,IC,GZ and HC groups(H=12.40,169.70,210.70,25.17,24.21 and 86.5,all P<0.05).And the differences in APRI score,proportion of HBeAg-positive patients,HBsAg and HBV-DNA among the IT group,IA group,IC group,GZ group were also significant(H=89.45,118.00 and 14.81,χ^(2)=148.20,all P<0.05).The frequency and MFI of Tim-3 on CD3+,CD4+and CD8+T cells,NK cells,NK-bright and NK-dim cells among the IT group,IA group,IC group,GZ group and the HC group were significantly different(H=13.57,51.55,8.58,44.25,20.32,47.96 and 12.45,33.69,4.96,32.47,10.63,30.46,all P<0.05).Both of the frequency and MFI of Tim-3 on CD3+,CD4+and CD8+T cells were positively correlated with ALT and AST levels in patients with chronic HBV infection(r=0.2134,0.4733,0.2090,0.4333,0.1771,0.4417,0.1780,0.3956,0.2618,0.4671,0.2614 and 0.4326,all P<0.05).While the frequency and MFI of Tim-3 on CD8+T cells and MFI on CD3+and CD4+T cells were also positively correlated with TBil levels(r=0.1342,0.2635,0.2739 and 0.2526,all P<0.05).The frequency and MFI of Tim-3 on NK and NK-dim cells were negatively correlated with the levels of ALT,AST and TBil(r=-0.2671,-0.4093,-0.2451,-0.4099,-0.1807,-0.1823,-0.2733,-0.4224,-0.2576,-0.4206,-0.1798 and-0.1946,all P<0.05).The MFI of Tim-3 on NK-bright cells was also negatively correlated with ALT,AST and TBil(r=-0.3775,-0.3562 and-0.1633,all P<0.05).Both of the frequency and MFI of Tim-3 on CD3+,CD4+and CD8+T cells were positively correlated with liver fibrosis(r=0.1789,0.3896,0.1518,0.3521,0.2117 and 0.3579,all P<0.05).Both of the frequency and MFI of Tim-3 on CD4+and CD8+T cells and the MFI of Tim-3 on CD3+T cells were positively correlated with APRI score(r=0.1487,0.2604,0.2296,0.4858 and 0.2853,all P<0.05).The expression frequency and MFI of Tim-3 on NK and NK-dim cells and MFI of Tim-3 on NK-bright cells were negatively correlated with LSM(r=-0.2686,-0.3975,-0.2852,-0.3991 and-0.3531,all P<0.05).The expression frequency and MFI of Tim-3 on NK and NK-dim cells and MFI of Tim-3 on NK-bright were negatively correlated with APRI score(r=-0.3589,-0.4158,-0.3591,-0.4108 and-0.3966,all P<0.05).The ratio of Tim-3 expression on CD3+T cells to that on NK cells was shown to be able to predict liver fibrosis in chronic HBV infected patients and the area under the ROC curve was 0.783(95%CI:0.723~0.843,P<0.05),and when the cut-off value was 0.612,the sensitivity was 61.9%,and the specificity was 99.3%.Conclusion The relationship of Tim-3 expression on T cells with liver inflammation and fibrosis is opposite to that on NK cells in patients with chronic HBV infection,indicating that the ratio of Tim-3 expression on T cells to that on NK cells may be valuable in evaluating liver fibrosis in patients.