PD-1和PD-L1表达与慢性HBV感染者肝脏病变程度的相关性

目的研究程序性死亡分子-1(programmed death-1,PD-1)及其主要配体(programmed death-1ligand,PD-L1)表达与肝脏病变程度的关系,并探讨其临床意义。方法不同临床类型慢性HBV感染者137例,含慢性乙型肝炎(CHB)患者102例,活动性肝硬化(LC)患者25例,慢加急性肝衰竭(ACLF)患者10例。其中,102例CHB患者中有22例行肝组织活检,炎症分级为G1～G4,纤维化程度分为S0～S4。健康对照10例。取新鲜血液分离外周血单个核细胞(PBMCs),流式细胞仪检测PBMCs表面PD-1和PD-L1阳性细胞比例以及人白细胞抗原(HLA)型别;对HLA-A2阳性者的PBMCs加入重组HBcAg体外培养7d,加入PE标记的HBV抗原表位肽-五聚体复合体(含HBV抗原表位肽为HB-cAg18-27),流式细胞仪计数CD8+T细胞数以及CD8和PD-1双阳性细胞。ELISA检测PBMCs培养上清液中IFN-γ。比较CHB、LC、ACLF三组间以及不同肝组织病变程度患者间PD-1、PD-L1、CD8+细胞数量和PD-1表达水平、IFN-γ水平的差异。结果在CHB、LC和ACLF组PD-1在PBMCs表面的表达阳性率分别为(55.4±16.5)%、(54.3±21.9)%、(45.5±26.5)%,与健康对照组比较,P值分别为0.000、0.001、0.071;三组PD-L1表达阳性率分别为(41.7±17.7)%、(33.4±17.9)%、(26.6±11.8)%,和对照组比较,P值分别为0.006、0.252、0.854。62例HLA-A2阳性患者的CD8+T细胞比例分别为(1.6±1.0)%、(3.1±1.6)%和(3.3±1.7)%,LC组和ACLF组明显高于CHB组(P0.05);PD-1阳性细胞占CD8+T细胞的比例各组间比较无统计学差异;三组PBMCs培养上清液中IFN-γ含量比较无统计学差异。不同肝组织炎症和纤维化程度的CHB患者间PD-1、PD-L1表达虽无明显统计学差异,但PD-L1在炎症G1和纤维化S0的患者中的表达较其他组患者高。结论慢性HBV感染者PD-1和PD-L1表达明显上调。肝脏病变程度与PD-1表达水平无关,但与HBV特异性CD8+T细胞数量及PD-L1表达相关。

The expressions of PD-1 and PD-L1 and the correlation with the degree of liver damage in HBV chronic infection

Objective To study the expression of programmed death-1 （ PD-1 ） and programmed death-1 ligand （PD-L1） on PBMCs in chronic HBV patients with different clinical types and pathological changes. Methods All of 137 chronic HBV infection subjects including 102 with chronic hepatitis B （CHB）, 25 with liver cirrhosis （LC） and 10 with acute on chronic liver failure （ACLF） were enrolled in this study. Histology features of 22 CHB patients have been desmonstrated by liver biopsy. PBMCs were isolated from fresh blood samples. HBV-specific T cells were expanded in vitro in 64 patients with HLA-A2 positive. Flow eytometry was used to detect the HLA-A2 type, the expression of PD-1 and PD-L1 on PBMCs and PD-1 on HBV specific CD8 ^± T ceils. IFN-γ was assayed by ELISA. Results In groups of CHB, LC and ACLF, PD-1 expression on PBMCs were （55.4±16.5）%, （54.3±21.9）% and （ 45.5±26.5）%, compared with control, P values were 0. 000, 0. 001 and 0. 071, respectively; PD-L1 were （41.7 ± 17.7） % , （33.4 ± 17.9） % and （26.6± 11.8 ） % , compared with control, P values were 0. 006, 0. 252 and 0. 854, respectively. Sixty four cases were HLA-A2 positive. The proportions of HBV-specific T cells were （1.6 ±1.0）%, （3.1 ±1.6）% and （3.3 ±1.7）%, respectively. In CHB, LC and ACLF subjects of HLA-A2 positive, significant differences were found between LC and control, ACLF and control （P 〈 0.05 ） ; There were no significant differences in PD-1 expression on HBV-specifie cells and IFN-γ levels in cell culture medium. PD-1 expressions on PBMCs were similar in CHB patients with different degrees of liver pathological changes, but PD-L1 expressions were higher in G1 and SO patients than that in other patients. Conclusions PD-1 and PD-L1 expressions were up-regulated in patients with HBV chronic infection. The degree of liver damage may be correlated with HBV-specifie cells and PD-L1 expression but not correlated with PD-1 expression.